281 results on '"Factor Xa"'
Search Results
2. Andexanet Alfa for Specific Anticoagulation Reversal in Patients with Acute Bleeding during Treatment with Edoxaban
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Alexander P, Benz, Lizhen, Xu, John W, Eikelboom, Saskia, Middeldorp, Truman J, Milling, Mark, Crowther, Patrick, Yue, Pamela, Conley, Genmin, Lu, Stuart J, Connolly, Vascular Medicine, ACS - Pulmonary hypertension & thrombosis, and ARD - Amsterdam Reproduction and Development
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Aged, 80 and over ,Male ,Pyridines ,Vascular damage Radboud Institute for Health Sciences [Radboudumc 16] ,Hemorrhage ,Thrombosis ,Hematology ,bleeding ,Anticoagulation Reversal ,Recombinant Proteins ,Thiazoles ,andexanet alfa ,Factor Xa ,edoxaban ,Humans ,Female ,reversal ,intracranial hemorrhage ,Factor Xa Inhibitors - Abstract
Background Andexanet alfa (andexanet) is approved for specific anticoagulation reversal in patients with life-threatening or uncontrolled bleeding during treatment with rivaroxaban or apixaban. There is limited experience with andexanet in patients with acute bleeding on edoxaban. Methods Patients with acute major bleeding within 18 hours of edoxaban intake were prospectively enrolled. Patients received a bolus and 2-hour follow-on infusion of andexanet. The co-primary efficacy outcomes were change in antifactor Xa activity and the percentage of patients achieving excellent or good hemostasis, 12 hours after andexanet treatment. Efficacy was analyzed in patients with confirmed major bleeding and baseline antifactor Xa activity ≥40 ng/mL. Safety was analyzed in all patients. Results Thirty-six patients (mean age: 82 years, 61.1% male and 91.7% with atrial fibrillation) with acute major bleeding on edoxaban received andexanet. The primary site of bleeding was intracranial in 29 patients (80.6%). In the efficacy population (n = 28), median antifactor Xa activity decreased from 121.1 (interquartile range [IQR]: 70.3–202.4) ng/mL at baseline to 24.0 (IQR: 77.7–83.7) ng/mL at the end of andexanet bolus (median decrease: 68.9%, 95% confidence interval [CI]: 56.1–77.7%). Excellent or good hemostasis at 12 hours was achieved in 78.6% (95% CI: 59.0–91.7%) of patients. Within 30 days, four patients (11.1%) experienced a thrombotic event and four others (11.1%) died. Conclusion In patients with acute major bleeding on edoxaban, andexanet significantly decreased antifactor Xa activity. Hemostatic efficacy was similar to that observed in patients with bleeding on rivaroxaban or apixaban. Thrombotic events occurred at a rate expected in such patients.
- Published
- 2022
3. Development of a Plasma-Based Assay to Measure the Susceptibility of Factor V to Inhibition by the C-Terminus of TFPIα
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Paolo Simioni, Tilman M. Hackeng, Peter van Doorn, Joost C. M. Meijers, Jan Rosing, Saskia Middeldorp, Elena Campello, Elisabetta Castoldi, RS: Carim - B01 Blood proteins & engineering, Biochemie, Vascular Medicine, ACS - Pulmonary hypertension & thrombosis, ARD - Amsterdam Reproduction and Development, and Experimental Vascular Medicine
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MECHANISM ,Heterozygote ,Lipoproteins ,Population ,PROTEIN-S ,Peptide ,FACTOR PATHWAY INHIBITOR ,Protein S ,CONTRIBUTES ,FACTOR-XA ,INITIATION ,Thrombin ,Tissue factor pathway inhibitor ,Prothrombinase ,medicine ,Factor V Leiden ,THROMBIN-CATALYZED ACTIVATION ,COFACTOR ,Humans ,education ,Blood Coagulation ,chemistry.chemical_classification ,education.field_of_study ,factor V ,biology ,Homozygote ,Factor V ,Hematology ,PROTHROMBINASE ,Blood Coagulation Disorders ,medicine.disease ,Molecular biology ,Peptide Fragments ,chemistry ,Factor Va ,factor V-short ,Factor Xa ,Mutation ,Proteolysis ,factor V Leiden ,biology.protein ,tissue factor pathway inhibitor ,INACTIVATION ,Blood Coagulation Tests ,medicine.drug - Abstract
Background Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPIα, while overexpression of FV splicing variants with increased affinity for TFPIα (FV-short) causes bleeding. Objective This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus. Materials and Methods FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPIα C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr). Results The TFPIr (0.25–0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28–0.31) to FVL heterozygotes (0.35, 95% CI 0.34–0.37) and homozygotes (0.39, 95% CI 0.37–0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-shortAmsterdam had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPIα. Conclusion We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPIα C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.
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- 2020
4. Knockdown and Knockout of Tissue Factor Pathway Inhibitor in Zebrafish
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Revathi Raman, Weam Fallatah, Ayah Al Qaryoute, Mia Ryon, and Pudur Jagadeeswaran
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Lipoproteins ,Factor Xa ,Homozygote ,Animals ,Hematology ,Factor VIIa ,Zebrafish ,Sequence Deletion - Abstract
Tissue factor pathway inhibitor (TFPI) is an anticoagulant that inhibits factor VIIa and Xa in the blood coagulation pathways. TFPI contains three Kunitz domains, K1, K2, and K3. K1 and K2 inhibit factor VIIa and Xa, respectively. However, the regulation of TFPI is poorly studied. Since zebrafish has become an alternate model to discover novel actors in hemostasis, we hypothesized that TFPI regulation could be studied using this model. As a first step, we confirmed the presence of tfpia in zebrafish using reverse transcription polymerase chain reaction. We then performed piggyback knockdowns of tfpia and found increased coagulation activity in tfpia knockdown. We then created a deletion mutation in tfpia locus using the CRISPR/Cas9 method. The tfpia homozygous deletion mutants showed increased coagulation activities similar to that found in tfpia knockdown. Taken together, our data suggest that tfpia is a negative regulator for zebrafish coagulation, and silencing it leads to thrombotic phenotype. Also, the zebrafish tfpia knockout model could be used for reversing this thrombotic phenotype to identify antithrombotic novel factors by the genome-wide piggyback knockdown method.
- Published
- 2021
5. Restart of Anticoagulant Therapy and Risk of Thrombosis, Rebleeding, and Death after Factor Xa Inhibitor Reversal in Major Bleeding Patients
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Peter Verhamme, Patrick Yue, Truman J. Milling, Deborah M. Siegal, Mark Crowther, Stuart J. Connolly, Saskia Middeldorp, Ben King, John W. Eikelboom, Jan Beyer-Westendorf, Lizhen Xu, Vascular Medicine, ACS - Pulmonary hypertension & thrombosis, and ARD - Amsterdam Reproduction and Development
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Male ,medicine.medical_specialty ,pulmonary embolism ,Time Factors ,medicine.drug_mechanism_of_action ,medicine.drug_class ,Factor Xa Inhibitor ,venous thromboembolism ,Vascular damage Radboud Institute for Health Sciences [Radboudumc 16] ,Hemorrhage ,030204 cardiovascular system & hematology ,Risk Assessment ,Drug Administration Schedule ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,Randomized controlled trial ,prevention ,law ,Recurrence ,Risk Factors ,Internal medicine ,medicine ,Humans ,Prospective Studies ,Stroke ,Aged ,Aged, 80 and over ,business.industry ,Proportional hazards model ,Anticoagulant ,Hazard ratio ,Thrombosis ,Hematology ,medicine.disease ,stroke ,Anticoagulation Reversal ,Confidence interval ,Recombinant Proteins ,Europe ,Treatment Outcome ,Anticoagulant Reversal Agents ,Factor Xa ,North America ,Female ,business ,030217 neurology & neurosurgery ,Factor Xa Inhibitors - Abstract
Background Lack of data on balancing bleeding and thrombosis risk causes uncertainty about restarting anticoagulants after major bleeding. Anticoagulant reversal trials offer prospectively gathered data after major bleeding with well-documented safety events and restarting behavior. Objectives To examine the relationship of restarting anticoagulation with thrombosis, rebleeding, and death. Methods This is a posthoc analysis of a prospective factor Xa inhibitor reversal study at 63 centers in North America and Europe. We compared outcomes of restarted patients with those not restarted using landmark and time-dependent Cox proportional hazards models. Outcomes included thrombotic and bleeding events and death and a composite of all three. Results Of 352 patients enrolled, oral anticoagulation was restarted in 100 (28%) during 30-day follow-up. Thirty-four (9.7%) had thrombotic events, 15 (4.3%) had bleeding events (after day 3), and 49 (14%) died. In the landmark analysis comparing patients restarted within 14 days to those not, restarting was associated with decreased thrombotic events (hazard ratio [HR] = 0.112; 95% confidence interval [CI]: 0.001–0.944; p = 0.043) and increased rebleeding (HR = 8.39; 95% CI: 1.13–62.29; p = 0.037). The time-dependent Cox model showed evidence for a reduction in a composite (thrombotic events, bleeding, and death) attempting to capture net benefit (HR = 0.384; 95% CI: 0.161–0.915; p = 0.031). Conclusion This analysis provides modest evidence that restarting anticoagulation in factor Xa inhibitor-associated major bleeding patients is correlated with reduced risk of thrombotic events and increased risk of rebleeding. There is low-level evidence of net benefit for restarting. A randomized trial of restarting would be appropriate.
- Published
- 2021
6. Performance of a Qualitative Point-of-Care Strip Test to Detect DOAC Exposure at the Emergency Department: A Cohort-Type Cross-Sectional Diagnostic Accuracy Study.
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Merrelaar AE, Bögl MS, Buchtele N, Merrelaar M, Herkner H, Schoergenhofer C, Harenberg J, Douxfils J, Siriez R, Jilma B, Spiel AO, and Schwameis M
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- Administration, Oral, Anticoagulants therapeutic use, Cross-Sectional Studies, Dabigatran therapeutic use, Emergencies, Emergency Service, Hospital, Factor Xa, Humans, Point-of-Care Systems, Point-of-Care Testing, Prospective Studies, Pyridones therapeutic use, Thrombin, Factor Xa Inhibitors therapeutic use, Rivaroxaban therapeutic use
- Abstract
An accurate point-of-care test for detecting effective anticoagulation by direct oral anticoagulants (DOACs) in emergencies is an unmet need. We investigated the accuracy of a urinary qualitative strip test (DOAC Dipstick) to detect relevant DOAC exposure in patients who presented to an emergency department. In this prospective single-center cohort-type cross-sectional study, adults on DOAC treatment were enrolled. We assessed clinical sensitivity and specificity of DOAC Dipstick factor Xa and thrombin inhibitor pads to detect DOAC plasma levels ≥30 ng/mL using urine samples as the testing matrix. Liquid chromatography coupled with tandem-mass spectrometry was used as the reference standard method for plasma and urine measurement of DOAC concentrations. Of 293 patients enrolled, 265 patients were included in the analysis, of whom 92 were treated with rivaroxaban, 65 with apixaban, 77 with edoxaban, and 31 with dabigatran. The clinical sensitivity and specificity of the dipstick on urine samples to detect ≥30 ng/mL dabigatran plasma levels were 100% (95% confidence interval [CI]: 87-100%) and 98% (95% CI: 95-99%), respectively. The sensitivity and specificity of the dipstick to detect ≥30 ng/mL factor Xa inhibitor plasma levels were 97% (95% CI: 94-99%) and 69% (95% CI: 56-79%), respectively. The DOAC Dipstick sensitively identified effective thrombin and factor Xa inhibition in a real-world cohort of patients presenting at an emergency department. Therefore, the dipstick might provide a valuable test to detect relevant DOAC exposure in emergencies, although further studies will be needed to confirm these findings., Competing Interests: J.H. is the CEO and founder of Doasense GmbH. J.D. is CEO of Qualiblood SA. All other authors declare no conflicts of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)
- Published
- 2022
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7. Accuracy of a Rapid Diagnostic Test for the Presence of Direct Oral Factor Xa or Thrombin Inhibitors in Urine-A Multicenter Trial
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Job, Harenberg, Jan, Beyer-Westendorf, Mark, Crowther, Jonathan, Douxfils, Ismail, Elalamy, Peter, Verhamme, Rupert, Bauersachs, Svetlana, Hetjens, Christel, Weiss, and Ulrich, Wolf
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0301 basic medicine ,Male ,medicine.drug_mechanism_of_action ,Pyridines ,030204 cardiovascular system & hematology ,Gastroenterology ,chemistry.chemical_compound ,0302 clinical medicine ,Rivaroxaban ,Edoxaban ,Tandem Mass Spectrometry ,dabigatran ,rivaroxaban ,Aged, 80 and over ,Hematology ,Middle Aged ,Dabigatran ,Factor Xa ,Apixaban ,point-of-care test ,Female ,medicine.drug ,medicine.medical_specialty ,Pyridones ,Point-of-Care Systems ,Factor Xa Inhibitor ,apixaban ,direct oral anticoagulants ,Sensitivity and Specificity ,Antithrombins ,03 medical and health sciences ,Thrombin ,Predictive Value of Tests ,Internal medicine ,medicine ,Humans ,Aged ,business.industry ,Reproducibility of Results ,Dipstick ,Thiazoles ,030104 developmental biology ,chemistry ,ROC Curve ,edoxaban ,Pyrazoles ,business ,Discovery and development of direct thrombin inhibitors ,Chromatography, Liquid ,Factor Xa Inhibitors - Abstract
The rapid determination of the presence of direct oral anticoagulants (DOACs) in a patient remains a major challenge in emergency medicine and for rapid medical treatment decisions. All DOACs are excreted into urine. A sensitive and specific point-of-care test has been developed to determine whether they are present in patient urine samples. This prospective multicenter study aimed to demonstrate at least 95% correct positive and negative predictive results for factor Xa and thrombin inhibitors in urine samples using DOAC Dipstick pads compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (NCT03182829). Nine hundred and fourteen subjects were included and 880 were evaluated per protocol (factor Xa inhibitors apixaban, edoxaban, and rivaroxaban: n = 451, thrombin inhibitor dabigatran: n = 429) at 18 centers. The sensitivity, specificity, accuracy, and predictive values and agreement between methods for determination of factor Xa inhibitors were at least noninferior to 95% with a 0.5% margin and of thrombin inhibitor superior to 97.5%. These results were compared with LC-MS/MS results in the intention-to-analyze cohort (all p
- Published
- 2019
8. Factor V Has Anticoagulant Activity in Plasma in the Presence of TFPI: Difference between FV1 and FV2
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Elisabetta Castoldi, Peter van Doorn, Connie Duckers, Paolo Simioni, Tilman M. Hackeng, Jan Rosing, Biochemie, Promovendi CD, and RS: CARIM - R1.01 - Blood proteins & engineering
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Male ,0301 basic medicine ,Glycosylation ,030204 cardiovascular system & hematology ,FACTOR PATHWAY INHIBITOR ,COFACTOR ACTIVITY ,FACTOR-XA ,chemistry.chemical_compound ,0302 clinical medicine ,Protein Isoforms ,RISK ,biology ,Chemistry ,Thrombin ,Factor V ,Hematology ,Middle Aged ,Phenotype ,Coagulation ,thrombin generation ,Factor Xa ,tissue factor pathway inhibitor ,Female ,Blood Coagulation Tests ,Antibody ,Adult ,Gene isoform ,VENOUS THROMBOSIS ,Adolescent ,Lipoproteins ,Phospholipid ,COAGULATION ,Thromboplastin ,ACTIVATED PROTEIN-C ,Young Adult ,03 medical and health sciences ,Tissue factor pathway inhibitor ,TISSUE FACTOR PATHWAY ,Prothrombinase ,R2 haplotype ,Humans ,Blood Coagulation ,phospholipids ,factor V ,PROTHROMBINASE ,Molecular biology ,ALPHA ,030104 developmental biology ,Haplotypes ,biology.protein ,Protein Processing, Post-Translational - Abstract
Background Activated factor V (FVa) is a potent procoagulant cofactor in the prothrombinase complex, whereas its precursor factor V (FV) stimulates the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-α (TFPIα), presumably by promoting TFPIα binding to phospholipids. Plasma FV comprises two glycosylation isoforms (FV1 and FV2) with low and high phospholipid-binding affinity, respectively. The FV1/FV2 ratio is increased in carriers of the FV R2 haplotype. Objective This article demonstrates the TFPIα-cofactor function of FV in plasma and compares FV1 and FV2. Materials and Methods Thrombin generation at low TF concentration was measured in FV-depleted plasma reconstituted with 0 to 100% FV, FV1 or FV2, and in 122 individuals genotyped for the R2 haplotype. The TFPIα-cofactor activities of FV1 and FV2 were also investigated in a model system of TFPIα-mediated FXa inhibition. Results In the FV titration, thrombin generation first increased (up to 5% FV) and then progressively decreased at higher FV concentrations. This anticoagulant effect of FV, which was also observed with FV2 but not with FV1, was largely abolished by anti-TFPIα antibodies, suggesting that it reflects TFPIα-cofactor activity of FV. In the model system of TFPIα-mediated FXa inhibition, FV2 was a more potent TFPIα-cofactor than FV1, in line with their respective phospholipid affinities. Accordingly, FV R2 carriers had higher thrombin generation than non-carriers, even after correction for demographics and plasma levels of coagulation factors and inhibitors. Conclusion FV (and particularly its FV2 isoform) contributes to the TFPIα-dependent down-regulation of thrombin generation in plasma triggered with low TF.
- Published
- 2018
9. Suppressive Role of Tissue Factor Pathway Inhibitor-alpha in Platelet-Dependent Fibrin Formation under Flow Is Restricted to Low Procoagulant Strength
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Marion A.H. Feijge, Roy Schrijver, Stella Thomassen, Judith M.E.M. Cosemans, Alan E. Mast, Kristien Winckers, Johan W. M. Heemskerk, Tilman M. Hackeng, Frauke Swieringa, Susan A. Maroney, Tom G. Mastenbroek, Biochemie, RS: CARIM - R1.03 - Cell biochemistry of thrombosis and haemostasis, MUMC+: MA Med Staf Artsass Interne Geneeskunde (9), and RS: CARIM - R1.01 - Blood proteins & engineering
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Male ,0301 basic medicine ,FACTOR VIIA ,PROTEIN-S ,030204 cardiovascular system & hematology ,Mice ,0302 clinical medicine ,fibrin clot formation ,Platelet ,BLOOD-COAGULATION ,biology ,Chemistry ,Hematology ,Thrombosis ,Healthy Volunteers ,EMBRYONIC-DEVELOPMENT ,Cell biology ,Perfusion ,Coagulation ,thrombin generation ,Factor Xa ,platelets ,Female ,Collagen ,KEY ROLE ,Blood Platelets ,Heterozygote ,VON-WILLEBRAND-FACTOR ,Lipoproteins ,haemophilia ,Hemophilia A ,Hemophilia B ,TFPI ,Article ,Fibrin ,Thromboplastin ,03 medical and health sciences ,Tissue factor ,Tissue factor pathway inhibitor ,Von Willebrand factor ,medicine ,Animals ,Humans ,Integrin Alpha-IIb/Beta-3 ,Blood Coagulation ,Crosses, Genetic ,mouse ,INTEGRIN ALPHA(IIB)BETA(3) ,Coagulants ,FACTOR-X ACTIVATION ,Anticoagulants ,medicine.disease ,Mice, Inbred C57BL ,THROMBUS FORMATION ,030104 developmental biology ,biology.protein - Abstract
Tissue factor pathway inhibitor-alpha (TFPI-α) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI-α thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI-α regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-α in plasma is a function of the local procoagulant strength—defined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI-α enhanced fibrin formation, extending from a platelet thrombus, under flow only at
- Published
- 2018
10. Passivating Injured Endothelium with Kinexins in Thrombolytic Therapy
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Shiaw-Pyng Wey, Yi-Ching Lu, Chao-Wei Huang, Chih-Jen Wen, Yunn-Hwa Ma, and Tze-Chein Wun
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Male ,Endothelium ,medicine.drug_class ,medicine.medical_treatment ,Factor VIIa ,030204 cardiovascular system & hematology ,Pharmacology ,Iodine Radioisotopes ,Rats, Sprague-Dawley ,03 medical and health sciences ,Tissue factor ,0302 clinical medicine ,In vivo ,Prothrombinase ,medicine ,Animals ,Humans ,Protease Inhibitors ,Thrombolytic Therapy ,Annexin A5 ,Blood Coagulation ,Abdominal Muscles ,Whole blood ,Spectroscopy, Near-Infrared ,business.industry ,Anticoagulant ,Anticoagulants ,Thrombosis ,Hematology ,Thrombolysis ,medicine.disease ,Rats ,Thrombelastography ,Perfusion ,Spectrometry, Fluorescence ,medicine.anatomical_structure ,Factor Xa ,Trypsin Inhibitor, Kunitz Soybean ,Peptides ,business ,030217 neurology & neurosurgery - Abstract
Without conjunctive administration of an anticoagulant, endothelial injury-induced thrombosis is resistant to thrombolysis and prone to re-thrombosis. We hypothesized that co-delivery of recombinant tissue plasminogen activator (rtPA) with annexin V–containing anticoagulants that specifically target the injured endothelium may passivate the thrombogenic elements of the vascular injury site and enhance rtPA-induced thrombolysis. In this study, the effects of conjunctive administration of Kinexins (Kunitz inhibitor–annexin V fusion proteins) with rtPA on thrombolysis were determined in vitro and in vivo. Thromboelastometry showed that both TAP-A (tick anticoagulant peptide–annexin V fusion protein; an inhibitor of factor Xa [FXa] and prothrombinase) and A-6L15 (annexin V-6L15 fusion protein; an inhibitor of tissue factor/FVIIa) exerted concentration-dependent (10–100 nM) effects on clot formation, with TAP-A being several folds more potent than A-6L15 in whole blood. Combination of TAP-A or A-6L15 with rtPA (1 μg/mL) led to decrease in lysis index, suggesting conjunctive enhancement of thrombolysis by combined use of rtPA with TAP-A or A-6L15. In a rat cremaster muscle preparation subjected to photochemical injury, conjunctive administration of rtPA and TAP-A significantly restored tissue perfusion to 56%, which is approximately two fold of that by rtPA or TAP-A alone. Near-infrared fluorescence images demonstrated local retention of a fluorescent A-6L15-S288 at the injury site, suggesting a targeting effect of the fusion protein. Pharmacokinetic analysis showed that 123I-labelled TAP-A and A-6L15 had initial distribution half-lives (T1/2α) of approximately 6 minutes and elimination half-lives (T1/2β) of approximately 2.3 hours. In conclusion, Kinexins were potentially useful adjunctive agents with rtPA thrombolytic therapy especially for thrombosis induced by endothelial injury.
- Published
- 2018
11. Activated Factor X-Based versus Thrombin-Based Antithrombin Testing in Thrombophilia Workup in the DOAC Era
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Jens Müller, Sara Reda, Bernd Pötzsch, Johannes Oldenburg, and Heiko Rühl
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Male ,Administration, Oral ,030204 cardiovascular system & hematology ,Gastroenterology ,0302 clinical medicine ,Rivaroxaban ,Thrombophilia ,Child ,Aged, 80 and over ,Antithrombin ,Thrombin ,Hematology ,Heparin ,Middle Aged ,Child, Preschool ,030220 oncology & carcinogenesis ,Factor Xa ,Activated factor X ,Population study ,Female ,Apixaban ,Blood Coagulation Tests ,medicine.drug ,Adult ,medicine.medical_specialty ,Adolescent ,Pyridones ,Antithrombin III ,Antithrombins ,Young Adult ,03 medical and health sciences ,Internal medicine ,medicine ,Humans ,Blood Coagulation ,Aged ,Dose-Response Relationship, Drug ,business.industry ,Anticoagulants ,Infant ,Heparin, Low-Molecular-Weight ,medicine.disease ,Mutation ,Pyrazoles ,business ,Factor Xa Inhibitors - Abstract
Antithrombin (AT) activity tests are used for diagnosing hereditary AT deficiency, a main genetic determinant of thrombophilia. They are either based on inhibition of thrombin (FIIa) or activated factor X (FXa). FXa-based assays have been suggested to be preferable to FIIa-based assays due to their higher sensitivity for certain AT deficiency causing mutations. To assess the performance of these two methods in a real-world scenario, 745 consecutively collected samples from patients referred to our institute during a 3-month period for thrombophilia testing were analysed. In samples from patients not receiving direct-acting oral anticoagulants or heparins (n = 485), both methods showed good agreement (r = 0.874, Bland–Altman limits of agreement 6.57%, −15.76%). While similar results were obtained in patients receiving low-molecular-weight heparin (LMWH, n = 76, r = 0.891, 4.09%, −14.35%), the agreement was lower in patients receiving rivaroxaban (n = 86, r = 0.570, 5.97%, −49.43%) and apixaban (n = 72, r = 0.735, 3.77%, −42.45%). Direct FXa inhibitors but not LMWH increased FXa-based assay results in a dose-dependent manner, while the FIIa-based test was unaffected. Both assay types were equally successful in detecting hereditary AT deficiency in our study population, as samples from 9 out of 10 patients with AT deficiency causing mutations were detected by each method. These data suggest that FXa-based AT testing can be preferred over FIIa-based methods only in the absence of direct FXa inhibitors. In patients receiving direct FXa inhibitors, AT activity testing should be performed using FIIa-based assays.
- Published
- 2018
12. Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens
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Tetsuhiro Soeda, Midori Shima, Tomoyuki Igawa, Keiji Nogami, Atsushi Muto, Kunihiro Hattori, Shinya Ishii, Takehisa Kitazawa, Keiko Esaki, Hiroyuki Tsunoda, Yoshiki Kawabe, and Tatsuhiko Tachibana
- Subjects
0301 basic medicine ,drug design ,Stereochemistry ,030204 cardiovascular system & hematology ,Antibodies, Monoclonal, Humanized ,Cofactor ,Factor IXa ,Antigen-Antibody Reactions ,Factor IX ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,Antibody Specificity ,Biomimetic Materials ,Epidermal growth factor ,Antibodies, Bispecific ,Humans ,Computer Simulation ,Surface plasmon resonance ,Ternary complex ,Factor VIIIa ,Emicizumab ,Binding Sites ,biology ,Chemistry ,Coagulation factors ,Models, Immunological ,Hematology ,Affinities ,030104 developmental biology ,Coagulation ,factor VIII ,Factor X ,Factor Xa ,biology.protein ,Coagulation and Fibrinolysis ,haemophilia therapy ,Factor Xa Inhibitors - Abstract
SummaryEmicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a manner similar to FVIIIa. However, details of the emicizumab–antigen interactions have not been reported so far. In this study, we first showed by surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa with moderate affinities (K D = 1.58, 1.52, 1.85, and 0.978 μM, respectively). We next showed by immunoblotting analysis that emicizumab recognised the antigens’ epidermal growth factor (EGF)-like domains. We then performed K D-based simulation of equilibrium states in plasma for quantitatively predicting the ways that emicizumab would interact with the antigens. The simulation predicted that only a small part of plasma FIX, FX, and emicizumab would form antigen-bridging FIX–emicizumab–FX ternary complex, of which concentration would form a bell-shaped relationship with emicizumab concentration. The bell-shaped concentration dependency was reproduced by plasma thrombin generation assays, suggesting that the plasma concentration of the ternary complex would correlate with emicizumab’s cofactor activity. The simulation also predicted that at 10.0–100 μg/ml of emicizumab–levels shown in a previous study to be clinically effective–the majority of plasma FIX, FX, and emicizumab would exist as monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar affinities at their EGF-like domains. The K D-based simulation predicted that the antigen-bridging ternary complex formed in circulating plasma would correlate with emicizumab’s cofactor activity, and the majority of FIX and FX would be free and available for other coagulation reactions.Institution where the work was carried out: Research Division, Chugai Pharmaceutical Co., Ltd.Supplementary Material to this article is available online at www.thrombosis-online.com.
- Published
- 2017
13. FVIIa Neutralization by a FXa Inhibitor: How It Could Dampen Tumorigenic Cancer Cell Phenotypes
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Bernd Engelmann
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Text mining ,Phenotype ,business.industry ,Chemistry ,Cancer cell ,Factor Xa ,Cancer research ,Hematology ,Factor VIIa ,business ,Neutralization - Published
- 2019
14. Converting the Distinct Heparins Sourced from Bovine or Porcine Mucosa into a Single Anticoagulant Drug
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Eduardo Vilanova, Bianca F. Glauser, Mariana S. Pereira, Adriana A. Piquet, Bruno C. Vairo, Kayene Vitória de A Micheli, Paulo A.S. Mourão, Paloma S. Santana, Stephan-Nicollas M. C. G. Oliveira, Ana M. F. Tovar, Gustavo R.C. Santos, and Nina V. Capillé
- Subjects
0301 basic medicine ,Drug ,Anions ,medicine.drug_class ,Swine ,media_common.quotation_subject ,Drug Compounding ,030204 cardiovascular system & hematology ,Pharmacology ,Neutralization ,03 medical and health sciences ,0302 clinical medicine ,Thromboembolism ,parasitic diseases ,medicine ,Potency ,Animals ,Humans ,Intestinal Mucosa ,media_common ,Anticoagulant drug ,biology ,medicine.diagnostic_test ,Chemistry ,Heparin ,Anticoagulant ,Anticoagulants ,Hematology ,Heparin, Low-Molecular-Weight ,Chromatography, Ion Exchange ,Protamine ,030104 developmental biology ,Therapeutic Equivalency ,Factor Xa ,biology.protein ,Cattle ,Partial Thromboplastin Time ,Prothrombin ,medicine.drug ,Partial thromboplastin time ,Protein Binding - Abstract
Unfractionated heparin (UFH) and their low-molecular-weight derivatives are sourced almost exclusively from porcine mucosa (HPI); however, a worldwide introduction of UFH from bovine mucosa (HBI) has been recommended to reinforce the currently unsteady supply chain of heparin products. Although HBI has different chemical composition and about half of the anticoagulant potency of HPI (∼100 and ∼180 international unit [IU]/mg, respectively), they have been employed as interchangeable UFHs in some countries since the 1990s. However, their use as a single drug provoked several bleeding incidents in Brazil, which precipitated the publication of the first monographs exclusive for HBI and HPI by the Brazilian Pharmacopoeia. Nevertheless, we succeed in producing with high-resolution anion-exchange chromatography a novel HBI derivative with anticoagulant potency (200 IU/mg), disaccharide composition (enriched in N,6-disulfated α-glucosamine) and safety profile (bleeding and heparin-induced thrombocytopaenia potentials and protamine neutralization) similar to those seen in the gold standard HPI. Therefore, we show that it is possible to equalize the composition and pharmacological characteristics of these distinct UFHs by employing an easily implementable improvement in the HBI manufacturing.
- Published
- 2019
15. Placental Pathological Findings following Adjusting Enoxaparin Dosage in Thrombophilic Women: Secondary Analysis of a Randomized Controlled Trial
- Author
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Raed Salim, Shabtai Romano, Israel Gavish, Gali Garmi, Noah Zafran, and Marina Okopnik
- Subjects
0301 basic medicine ,Adult ,medicine.medical_specialty ,Adolescent ,medicine.drug_class ,Placental Finding ,Placenta ,Pregnancy Complications, Cardiovascular ,Low molecular weight heparin ,030204 cardiovascular system & hematology ,Drug Administration Schedule ,law.invention ,03 medical and health sciences ,Young Adult ,0302 clinical medicine ,Randomized controlled trial ,law ,Pregnancy ,medicine ,Humans ,Thrombophilia ,Young adult ,Enoxaparin ,Pathological ,business.industry ,Obstetrics ,Incidence (epidemiology) ,Pregnancy Outcome ,Anticoagulants ,Hematology ,Heparin ,Venous Thromboembolism ,Heparin, Low-Molecular-Weight ,Middle Aged ,medicine.disease ,030104 developmental biology ,Data Interpretation, Statistical ,Factor Xa ,Female ,business ,medicine.drug ,Factor Xa Inhibitors - Abstract
Objective Randomized trials showed no improvement in pregnancy outcomes with the use of low molecular weight heparin (LMWH) to prevent placenta-mediated pregnancy complications (PMPCs) among thrombophilic women. However, the effect of treatment on placental findings was not examined. We aimed to examine the occurrence of placental vascular lesions in thrombophilic women treated with LMWH dose adjusted according to anti-factor Xa compared with a fixed dose. Study Design This study was a secondary analysis of a randomized trial designed to examine whether LMWH dose adjusted according to anti-factor Xa levels compared with a fixed dose would reduce the risk of PMPC. Eligible women were randomly allocated in a 1:1 ratio to either a fixed dose of 40 mg daily LMWH (fixed dose group) or adjusted dose according to anti-factor Xa levels (adjusted dose group). Placentas were examined by the same perinatal pathologist who was blinded to group allocation. The primary outcome for this analysis was the incidence of maternal placental vascular lesions. Results During the study period, 88 placentas were examined; 41 and 47 from the fixed and adjusted dose groups, respectively. Demographics, obstetrics and types of thrombophilias were similar between the groups. Maternal placental vascular lesions were observed in 23 (56.1%) and 21 (44.68%) placentas (p = 0.28) and foetal placental vascular lesions in 2 (4.88%) and 1 (2.13%) placentas (p = 0.59) in the fixed and adjusted groups, respectively. Conclusion Adjusted dose of enoxaparin according to anti-factor Xa levels compared with a fixed dose did not affect placental vascular lesions in thrombophilic women.
- Published
- 2019
16. Role of exosite binding modulators in the inhibition of Fxa by TFPI
- Author
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Stella Thomassen, Rudolf Hartmann, Sameera Peraramelli, Jan Rosing, Friedrich Scheiflinger, Tilman M. Hackeng, Alexandra C.A. Heinzmann, Michael Dockal, Promovendi CD, RS: CARIM - R1.01 - Blood proteins & engineering, and Biochemie
- Subjects
0301 basic medicine ,factor V/Va ,factor Xa ,Lipoproteins ,030204 cardiovascular system & hematology ,Pharmacology ,Bioinformatics ,Ligands ,Anticoagulant activity ,TFPI ,Protein S ,Catalysis ,03 medical and health sciences ,0302 clinical medicine ,Tissue factor pathway inhibitor ,In vivo ,Polysaccharides ,medicine ,Humans ,Blood Coagulation ,Volume concentration ,Phospholipids ,prothrombin ,biology ,Chemistry ,Heparin ,Exosites ,Thrombin ,Factor V ,Hematology ,Heparin, Low-Molecular-Weight ,Blood proteins ,Recombinant Proteins ,030104 developmental biology ,Coagulation ,Factor Va ,biology.protein ,medicine.drug ,Factor Xa Inhibitors ,Protein Binding - Abstract
SummaryTissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 μM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2–1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1–150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25–0.5 nM TFPI) inhibit FXa with a t1/2 between 3–15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.
- Published
- 2016
17. Phosphotidylserine exposure and neutrophil extracellular traps enhance procoagulant activity in patients with inflammatory bowel disease
- Author
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Zhipeng Yao, Hemant S. Thatte, Tao Li, Yu Si, Bo Yu, Ruishuang Ma, Shaohong Fang, Shufen Yang, Jialan Shi, Junjie Kou, Tao Jiang, Muhua Cao, Lirong Hao, Lu Zhao, Yayan Bi, Jin Zhou, Yan Zhang, Zhangxiu He, Zengxiang Dong, and Daxun Piao
- Subjects
Adult ,Male ,Phosphatidylserines ,030204 cardiovascular system & hematology ,Extracellular Traps ,Fibrin ,Flow cytometry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Thrombin ,Cell-Derived Microparticles ,Deoxyribonuclease I ,Humans ,Thrombophilia ,Medicine ,Platelet ,Blood Coagulation ,Cells, Cultured ,Aged ,Lactadherin ,biology ,medicine.diagnostic_test ,business.industry ,Hematology ,Phosphatidylserine ,Neutrophil extracellular traps ,Middle Aged ,Inflammatory Bowel Diseases ,Milk Proteins ,chemistry ,Factor Va ,Antigens, Surface ,Factor Xa ,Immunology ,biology.protein ,Female ,030211 gastroenterology & hepatology ,Tumor necrosis factor alpha ,business ,Protein Binding ,medicine.drug - Abstract
SummaryInflammatory bowel disease (IBD)-associated thromboembolic event often lacks precise aetiology. The aim of this study was to investigate the contribution of phosphatidylserine (PS) exposure and neutrophil extracellular traps (NETs) towards the hypercoagulable state in IBD. We demonstrated that the levels of PS exposed MPs and the sources of MP-origin, platelets, erythrocytes, leukocytes and cultured endothelial cells (ECs) were higher in IBD groups than in healthy controls using flow cytometry and confocal microscopy. Wright-Giemsa and immunofluorescence staining demonstrated that the elevated NETs were released by activated IBD neutrophils or by control neutrophils treated with IBD sera obtained from patients with the active disease. MPs and MP-origin cells in IBD groups, especially in active stage, markedly shortened coagulation time and had increased levels of fibrin, thrombin and FXa production as assessed by coagulation function assays. Importantly, we found that on stimulated ECs, PS rich membranes provided binding sites for FXa and FVa, promoting fibrin formation while TNF blockage or IgG depletion attenuated this effect. Treatment of control neutrophils with TNF and isolated IgG from PR3-ANCA-positive active IBD patients also resulted in the release of NETs. Blockade of PS with lactadherin prolonged coagulation time, decreased fibrin formation to control levels, and inhibited the procoagulant enzymes production in the MPs and MP-origin cells. NET cleavage by DNase I partly decreased PCA in IBD or stimulated neutrophils. Our study reveals a previously unrecognised link between hypercoagulable state and PS exposure or NETs, and may further explain the epidemiological association of thrombosis within IBD patients.
- Published
- 2016
18. Contribution of Factor VIII A2 Domain Residues 400-409 to a Factor X-Interactive Site in the Factor Xase Complex
- Author
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Shoko Furukawa, Midori Shima, Keiji Nogami, Masahiro Takeyama, and Kana Sasai
- Subjects
0301 basic medicine ,Models, Molecular ,Stereochemistry ,Peptide ,Cleavage (embryo) ,Hemophilia A ,Binding, Competitive ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Epitopes ,Structure-Activity Relationship ,law ,Antibody Specificity ,Catalytic Domain ,Structure–activity relationship ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Peptide sequence ,Autoantibodies ,Alanine ,chemistry.chemical_classification ,Factor VIII ,Factor X ,Hematology ,Peptide Fragments ,Neoplasm Proteins ,Cysteine Endopeptidases ,030104 developmental biology ,chemistry ,Factor Xa ,Mutation ,Recombinant DNA ,Tenase ,Protein Binding - Abstract
The link between factor (F)VIII and FX is essential for optimum activity of the tenase complex. The interactive site(s) in FVIII for FX remains to be completely clarified, however. We investigated the FVIII A2 domain-FX association that was speculated from inhibitory mechanism(s) by an anti-A2 autoantibody. SDS-PAGE demonstrated that the purified inhibitor IgG recognizing residues 373–562 blocked FXa cleavage at Arg372 in FVIII, and surface-plasmon resonance (SPR)-based assays showed that intact A2 subunit directly bound to FX (K d; 63 nM). The FVIII structure model indicated possible FX-binding site(s) in residues 400–429 in A2. One peptide corresponding to residues 400–409 competitively inhibited both the A2–FX binding and FVIIIa/FIXa-dependent FXa generation. Covalent cross-linking was observed between this peptide and FX following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. K408 and S409 were not evident in N-terminal sequence analysis of the cross-linked product, suggesting that two residues participated in cross-link formation. SPR-based assays using recombinant FVIII mutants with one or both residues substituted to alanine demonstrated that K408A and K408A/S409A had approximately fourfold high K d values of wild-type (WT-)FVIII. FXa cleavages at Arg372 in both mutants were significantly delayed, suggesting a contribution of K408 for FXa cleavage at Arg372. Furthermore, FXa generation assays with these mutants demonstrated that the K m values were 1.4- to 1.7-fold greater, and overall catalytic efficiency (k cat/K m) was 0.49- to 0.89-fold lower than with WT-FVIII, suggesting a significant contribution of K408 for FVIII–FX interaction in tenase assembly. We concluded that the K408 in the A2 domain provided an interactive-site for FX.
- Published
- 2018
19. Reversal of anticoagulants: an overview of current developments
- Author
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Thomas Thiele, Kathleen Selleng, and Andreas Greinacher
- Subjects
0301 basic medicine ,Vitamin K ,medicine.drug_mechanism_of_action ,Pyridines ,Administration, Oral ,Review ,030204 cardiovascular system & hematology ,Pharmacology ,Fondaparinux ,Hemostatics ,chemistry.chemical_compound ,0302 clinical medicine ,Rivaroxaban ,Edoxaban ,Infusions, Parenteral ,Protamines ,Clinical Trials as Topic ,Antibodies, Monoclonal ,Idarucizumab ,Hematology ,Recombinant Proteins ,Dabigatran ,Benzamides ,Factor Xa ,Intracranial Hemorrhages ,medicine.drug ,Pyridones ,Factor Xa Inhibitor ,Hemorrhage ,Antibodies, Monoclonal, Humanized ,Antithrombins ,03 medical and health sciences ,Polysaccharides ,medicine ,Animals ,Humans ,Heparin ,business.industry ,Anticoagulants ,Thrombosis ,Thiazoles ,030104 developmental biology ,chemistry ,Direct thrombin inhibitor ,Betrixaban ,Pyrazoles ,business ,Factor Xa Inhibitors - Abstract
SummarySeveral new anticoagulants have entered the clinical arena or are under clinical development. These drugs include indirect (fondaparinux) and direct oral factor Xa inhibitors (rivaroxaban, apixaban, edoxaban, betrixaban), and the direct thrombin inhibitor dabigatran. Especially the oral direct FXa and FIIa inhibitors overcome many of the shortcomings of heparins and vitamin K antagonists (VKAs). They are administered orally at a fixed dose; regular monitoring is not necessary; interaction with other drugs or nutrition occur less than with VKAs and they are at least as effective as VKAs for most indications tested. They are associated with about 50 % less intracranial bleeding than VKAs. Nevertheless, they are still associated with bleeding complications. Bleeding can occur spontaneously or as a result of trauma or urgent surgery. In such situations rapid reversal of the anticoagulant effect is highly desirable. For unfractionated heparin protamine, and for VKAs prothrombin complex concentrates are available as specific antidotes. Under clinical development are: for the direct and indirect FXa inhibitors a modified recombinant FXa (andexanet alpha), which lacks enzymatic activity; and for dabigatran a Fab fragment of a monoclonal antibody (idarucizumab). In addition a small molecule (aripazine) has entered phase I clinical trials, which seems to inhibit nearly all anticoagulants but VKAs and argatroban. This review summarises the current options and strategies in development to antagonise anticoagulants with a focus on the status of the development of antidotes for the oral direct FXa and FIIa inhibitors.
- Published
- 2015
20. Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa
- Author
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Hironao Wakabayashi, Philip J. Fay, and Jennifer Wintermute
- Subjects
0301 basic medicine ,Hot Temperature ,Proteolysis ,Protein subunit ,Kinetics ,Mutant ,030204 cardiovascular system & hematology ,Hemophilia A ,Protein Engineering ,Article ,03 medical and health sciences ,0302 clinical medicine ,Thrombin ,medicine ,Humans ,Protein Interaction Domains and Motifs ,Destabilisation ,Factor VIIIa ,medicine.diagnostic_test ,Protein Stability ,Chemistry ,Hematology ,Protein engineering ,Molecular biology ,Protein Structure, Tertiary ,Protein Subunits ,030104 developmental biology ,Factor Xa ,Mutation ,medicine.drug - Abstract
SummaryFVIIIa is labile due to the dissociation of A2 subunit. Previously, we introduced hydrophobic mutations at select A1/A2/A3 subunit interfaces yielding more stable FVIII(a) variants. Separately we showed that altering the sequence flanking the primary FXa cleavage site in FVIIIa (Arg336) yielded reduced rates of proteolytic inactivation of FVIIIa. In this study we prepared the FXa-cleavage resistant mutant (336(P4-P3’)562) combined with mutations of Ala108Ile, Asp519Val/ Glu665Val or Ala108Ile/Asp519Val/Glu665Val and examined the effects of these combinations relative to FVIII thermal stability, rates of FVIIIa decay and proteolytic inactivation of FVIIIa by FXa. Thermal decay rates for 336(P4-P3’)562/Ala108Ile, 336(P4-P3’)562/Asp519Val/ Glu665Val, and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ∼2– to 5-fold as compared with wild-type (WT) primarily reflecting the effects of the A domain interface mutations. FVIIIa decay rates for 336(P4-P3’)562/Asp519Val/Glu665Val and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ∼25 fold, indicating greater stability than the control Asp519Val/Glu665Val variant (∼14-fold). Interestingly, 336(P4-P3’)562/Asp519Val/Glu665Val and 336(P4-P3’)562/Ala108Ile/ Asp519Val/Glu665Val variants showed reduced FXa-inactivation rates compared with the 336(P4-P3’)562 control (∼4-fold), suggesting A2 subunit destabilisation is a component of proteolytic inactivation. Thrombin generation assays using the combination variants were similar to the Asp519Val/Glu665Val control. These results indicate that combining multiple gain-of-function FVIII mutations yields FVIII variants with increased stability relative to a single type of mutation.
- Published
- 2014
21. Thrombin selectively induces transcription of genes in human monocytes involved in inflammation and wound healing
- Author
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M. Lienhard Schmitz, Hector A. Cabrera-Fuentes, Victor Salazar, Gustavo Bruges, Gustavo Crespo, Mercedes López, Klaus T. Preissner, Heike Schneider, and Pierre-Antoine Deglesne
- Subjects
0301 basic medicine ,Transcription, Genetic ,MAP Kinase Signaling System ,Inflammation ,030204 cardiovascular system & hematology ,Biology ,Real-Time Polymerase Chain Reaction ,Thrombomodulin ,Monocytes ,Thromboplastin ,03 medical and health sciences ,Tissue factor ,0302 clinical medicine ,Thrombin ,Downregulation and upregulation ,Gene expression ,medicine ,Humans ,Receptor, PAR-1 ,Protease-activated receptor ,Blood Coagulation ,Cells, Cultured ,Oligonucleotide Array Sequence Analysis ,Feedback, Physiological ,Wound Healing ,Hematology ,Up-Regulation ,030104 developmental biology ,Gene Expression Regulation ,Factor Xa ,Cancer research ,medicine.symptom ,Wound healing ,medicine.drug - Abstract
SummaryThrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing.
- Published
- 2014
22. Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles
- Author
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Mary E. W. Collier, Camille Ettelaie, and Anthony Maraveyas
- Subjects
0301 basic medicine ,animal structures ,Filamins ,macromolecular substances ,030204 cardiovascular system & hematology ,Biology ,Protein Engineering ,Filamin ,Thromboplastin ,03 medical and health sciences ,Tissue factor ,0302 clinical medicine ,Western blot ,Cell-Derived Microparticles ,medicine ,Humans ,Receptor, PAR-2 ,Particle Size ,RNA, Small Interfering ,Cytoskeleton ,Cells, Cultured ,Sequence Deletion ,Secretory Pathway ,medicine.diagnostic_test ,Thrombin ,Endothelial Cells ,Hematology ,Transfection ,Molecular biology ,Microvesicles ,Protein Structure, Tertiary ,Cell biology ,030104 developmental biology ,Cytoplasm ,Factor Xa ,Phosphorylation - Abstract
SummaryWe previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90% suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation.
- Published
- 2014
23. Chromogenic assays for measurement of rivaroxaban from EDTA anticoagulated plasma samples
- Author
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Sandra Krämer, Job Harenberg, Martin Wehling, Christel Weiss, Christina Giese, Roland Krämer, and Shanshan Du
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Administration, Oral ,030204 cardiovascular system & hematology ,Pharmacology ,Sodium Citrate ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Rivaroxaban ,Sodium citrate ,Healthy volunteers ,Humans ,Medicine ,Citrates ,Blood Coagulation ,Edetic Acid ,Plasma samples ,business.industry ,Chromogenic ,Vascular biology ,Anticoagulants ,Optical Devices ,Hematology ,Healthy Volunteers ,030104 developmental biology ,Chromogenic Compounds ,chemistry ,Calibration ,Factor Xa ,Colorimetry ,business ,Blood Chemical Analysis ,Factor Xa Inhibitors ,medicine.drug - Abstract
Chromogenic assays for measurement of rivaroxaban from EDTA anticoagulated plasma samples
- Published
- 2015
24. Platelet protein S directly inhibits procoagulant activity on platelets and microparticles
- Author
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Nicole F. Davis, Erning Duan, Fabian Stavenuiter, Mary J. Heeb, and Andrew J. Gale
- Subjects
Blood Platelets ,0301 basic medicine ,Lipoproteins ,Blotting, Western ,Stimulation ,Factor VIIa ,030204 cardiovascular system & hematology ,Article ,Protein S ,Thromboplastin ,Cell-Derived Microparticles ,03 medical and health sciences ,0302 clinical medicine ,Thrombin ,medicine ,Humans ,Platelet ,Blood Coagulation ,biology ,Chemistry ,Hematology ,Antibodies, Neutralizing ,Molecular biology ,Blood proteins ,Kinetics ,030104 developmental biology ,Biochemistry ,Factor Xa ,biology.protein ,Protein C ,medicine.drug - Abstract
SummaryAnticoagulant plasma protein S (PS) is essential for maintaining haemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the “thrombin-sensitive region”, abolishing activated protein C (APC) cofactor activity. However, we showed that cleaved PS retains APC-independent anticoagulant activities (“PS-direct”). To investigate whether pltPS or pdPS exert PS-direct on platelets or platelet-shed microparticles, thrombin and factor (F)Xa generation on unstimulated or stimulated washed platelets and microparticles were measured. Western blotting revealed that pltPS and pdPS bound to washed, stimulated platelets and microparticles, and that pltPS had slower electrophoretic mobility than pdPS. Platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 2.6 ± 1.6-fold (p
- Published
- 2013
25. Ex vivo effects of low-dose rivaroxaban on specific coagulation assays and coagulation factor activities in patients under real life conditions
- Author
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Helen Mani, Gertrud Stratmann, Edelgard Lindhoff-Last, and Christian Hesse
- Subjects
Time Factors ,Arthroplasty, Replacement, Hip ,Morpholines ,Administration, Oral ,Enzyme-Linked Immunosorbent Assay ,Thiophenes ,030204 cardiovascular system & hematology ,Pharmacology ,Drug Administration Schedule ,03 medical and health sciences ,0302 clinical medicine ,Rivaroxaban ,Nephelometry and Turbidimetry ,Predictive Value of Tests ,Tandem Mass Spectrometry ,D-dimer ,medicine ,Von Willebrand disease ,Humans ,Thrombophilia ,False Positive Reactions ,030212 general & internal medicine ,Arthroplasty, Replacement, Knee ,Blood Coagulation ,Chromatography, High Pressure Liquid ,Clotting factor ,Chemistry ,Anticoagulants ,Reproducibility of Results ,Thrombosis ,Hematology ,Factor XIII ,medicine.disease ,Blood Coagulation Factors ,Treatment Outcome ,Chromogenic Compounds ,Coagulation ,Factor Xa ,Immunology ,Blood Coagulation Tests ,Biomarkers ,Protein C ,Factor Xa Inhibitors ,Tablets ,medicine.drug ,Blood sampling - Abstract
SummaryGlobal coagulation assays display variable effects at different concentrations of rivaroxaban. The aim of this study is to quantify the ex vivo effects of low-dose rivaroxaban on thrombophilia screening assays and coagulation factor activities based on the administration time, and to show how to mask possible interferences. Plasma samples from 40 patients receiving rivaroxaban 10 mg daily were investigated to measure activities of clotting factor II, V, VII, VIII, IX, XI, XII and XIII; protein C- and protein S-levels; lupus anticoagulants; anticardiolipin IgG and IgM; D-dimer, heparin-platelet factor 4 (HPF4) antibodies and screening tests for von Willebrand disease (VWD). Two hours after rivaroxaban administration, the activities of clotting factors were significantly decreased to different extents, except for factor XIII. Dilution of plasma samples resulted in neutralisation of these interferences. The chromogenic protein C activity assay was not affected by rivaroxaban. Depending on the timing of tablet intake in relation to blood sampling protein S activity was measured falsely high when a clotting assay was used. False-positive results for lupus anticoagulants were observed depending on the assay system used and the administration time of rivaroxaban. ELISA-based assays such as anticardiolipin IgG and IgM, D-dimer, HPF4-antibodies and the turbidimetric assays for VWD were not affected by rivaroxaban. Specific haemostasis clotting tests should be performed directly prior to rivaroxaban intake. Assay optimisation in the presence of rivaroxaban can be achieved by plasma dilution. Immunologic assays are not influenced by rivaroxaban, while chromogenic assays can be used, when they do not depend on factor Xa.
- Published
- 2013
26. Covalently linking heparin to antithrombin enhances prothrombinase inhibition on activated platelets
- Author
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Anthony K.C. Chan, Leslie R. Berry, Ivan Stevic, Howard H.W. Chan, and Ankush Chander
- Subjects
Blood Platelets ,0301 basic medicine ,Enzyme complex ,Pharmacology ,Fondaparinux ,Antithrombins ,Thromboplastin ,03 medical and health sciences ,0302 clinical medicine ,Nephelometry and Turbidimetry ,Prothrombinase ,medicine ,Humans ,Platelet ,Platelet activation ,Enzyme Inhibitors ,Blood Coagulation ,Heparin ,Chemistry ,Antithrombin ,Anticoagulants ,Hematology ,Flow Cytometry ,Platelet Activation ,030104 developmental biology ,Coagulation ,Factor Xa ,Immunology ,Protein Binding ,030215 immunology ,medicine.drug - Abstract
SummaryFactor (F)Xa within the prothrombinase complex is protected from inhibition by unfractionated heparin (UFH), enoxaparin and fondaparinux. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. We have also demonstrated that ATH is superior at inhibiting coagulation factors when assembled on artificial surfaces. The objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit FXa within the prothrombinase complex when the enzyme complex is assembled on the more native platelet system. Discontinuous inhibition assays were performed to determine final k 2-values for inhibition of FXa, FXa within the platelet-prothrombinase, or FXa within prothrombinase devoid of various components. Thrombin generation and plasma clotting was also assayed in the presence of resting/activated platelets ± inhibitors. Protection of FXa was not observed for ATH, whereas a moderate 60% protection was observed for AT+UFH. ATH inhibited platelet-prothrombinase ∼4-fold faster than AT+UFH. Relative to intact prothrombinase, rates for FXa inhibition by AT+UFH in prothrombinase complexes devoid of either prothrombin (II)/activated platelets/FVa were higher. However, inhibition by AT+UFH of prothrombinase devoid of FII yielded slightly lower rates compared to free FXa inhibition. Thrombin generation and plasma clotting was enhanced with activated platelets, while inhibition was better by ATH compared to AT+UFH, thus suggesting an overall enhanced anticoagulant activity of ATH against platelet-bound prothrombinase complexes.
- Published
- 2013
27. Contribution of factor VIII light-chain residues 2007–2016 to an activated protein C-interactive site
- Author
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Philip J. Fay, Hironao Wakabayashi, and Masahiro Takeyama
- Subjects
0301 basic medicine ,Proteolysis ,Blotting, Western ,Dot blot ,Enzyme-Linked Immunosorbent Assay ,030204 cardiovascular system & hematology ,Cleavage (embryo) ,Immunoglobulin light chain ,Models, Biological ,Article ,Cofactor ,03 medical and health sciences ,0302 clinical medicine ,Catalytic Domain ,Protein Interaction Mapping ,medicine ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Binding site ,Factor VIIIa ,chemistry.chemical_classification ,Binding Sites ,Factor VIII ,biology ,medicine.diagnostic_test ,Hematology ,Molecular biology ,Recombinant Proteins ,Enzyme Activation ,Kinetics ,Spectrometry, Fluorescence ,030104 developmental biology ,Enzyme ,Biochemistry ,chemistry ,Factor Xa ,Mutation ,Mutagenesis, Site-Directed ,biology.protein ,Protein C ,Protein Binding ,medicine.drug - Abstract
SummaryAlthough factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg336) and A2 subunits (Arg562), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilised active site-modified APC (DEGRAPC) (apparent K d ~270 nM and 1.0 μM, respectively). Fluid-phase binding studies using fluorescence indicated an estimated K d of ~590 nM for acrylodan-labelled LC binding to DEGR-APC. Furthermore, FVIII LC effectively competed with FVIIIa in blocking APC-catalysed cleavage at Arg336 (K i = 709 nM). A binding site previously identified near the C-terminal end of the A3 domain (residues 2007–2016) of FVIII LC was subjected to Ala-scanning mutagenesis. FXa generation assays and western and dot blotting were employed to assess the contribution of these residues to FVIIIa interactions with APC. Virtually all variants tested showed reductions in the rates of APC-catalysed inactivation of the cofactor and cleavage at the primary inactivation site (Arg336), with maximal reductions in inactivation rates (~3-fold relative to WT) and cleavage rates (~3 to ~9-fold relative to WT) observed for the Met2010Ala, Ser2011Ala, and Leu2013Ala variants. Titration of FVIIIa substrate concentration monitoring cleavage by a dot blot assay indicated that these variants also showed ~3-fold increases relative to WT while a double mutant (Met2010Ala/Ser2011Ala) showed a >4-fold increase in K m. These results show a contribution of a number of residues within the 2007–2016 sequence, and in particular residues Met2010, Ser2011, and Leu2013 to an APC-interactive site.
- Published
- 2013
28. The mild phenotype in severe hemophilia A with Arg1781His mutation is associated with enhanced binding affinity of factor VIII for factor X
- Author
-
Keiji Nogami, Midori Shima, Philip J. Fay, Koji Yada, and Hironao Wakabayashi
- Subjects
Adult ,Male ,0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,medicine.medical_specialty ,Genotype ,Haemophilia A ,030204 cardiovascular system & hematology ,Gene mutation ,Arginine ,Hemophilia A ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Thrombin ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Humans ,Histidine ,Whole blood ,Hemostasis ,Factor VIII ,Factor X ,Wild type ,Hematology ,medicine.disease ,Recombinant Proteins ,Thrombelastography ,Kinetics ,Phenotype ,030104 developmental biology ,Endocrinology ,Gene Expression Regulation ,chemistry ,Mutagenesis ,Factor Xa ,Mutation ,Immunology ,Protein Binding ,medicine.drug ,Tenase - Abstract
SummaryThe clinical severity in some patients with haemophilia A appears to be unrelated to the levels of factor (F)VIII activity (FVIII:C), but mechanisms are poorly understood. We have investigated a patient with a FVIII gene mutation at Arg1781 to His (R1781H) presenting with a mild phenotype despite FVIII:C of 0.9 IU/dl. Rotational thromboelastometry using the patient’s whole blood demonstrated that the clot time and clot firmness were comparable to those usually observed at FVIII:C 5–10 IU/dl. Thrombin and FXa assays using plasma samples also showed that the peak levels of thrombin formation and the initial rate of FXa generation were comparable to those observed at FVIII:C 5–10 IU/dl. The results suggested a significantly greater haemostatic potential in this individual than in those with severe phenotype. The addition of incremental amounts of FX to control plasma with FVIII:C 0.9 IU/dl in clot waveform analyses suggested that the enhanced functional tenase assembly might have been related to changes in association between FVIII and FX. To further investigate this mechanism, we prepared a stably expressed, recombinant, B-domainless FVIII R1781H mutant. Thrombin generation assays using mixtures of control plasma and FVIII revealed that the coagulation function observed with the R1781H mutant (0.9 IU/dl) was comparable to that seen with wild-type FVIII:C at ∼5 IU/dl. In addition, the R1781H mutant demonstrated an ∼1.9-fold decrease in K m for FX compared to wild type. These results indicated that relatively enhanced binding affinity of FVIII R1781H for FX appeared to moderate the severity of the haemophilia A phenotype.Note: An account of this work was presented, in part, at the 23rd Congress of the International Society of Thrombosis and Haemostasis, July 27, 2011, Kyoto, Japan.
- Published
- 2013
29. Apixaban pharmacodynamic activity in umbilical cord, paediatric, and adult plasma
- Author
-
Yu Chen Barrett, Charles Frost, Jessie Wang, Zhaoqing Wang, Robert Adamczyk, Eduardo Ramacciotti, and Robert J. Yetman
- Subjects
Adult ,Male ,medicine.medical_specialty ,Adolescent ,Pyridones ,030204 cardiovascular system & hematology ,Pharmacology ,Umbilical cord ,Gastroenterology ,03 medical and health sciences ,chemistry.chemical_compound ,Young Adult ,0302 clinical medicine ,Age groups ,Internal medicine ,medicine ,Humans ,030212 general & internal medicine ,Child ,Blood Coagulation ,Prothrombin time ,Plasma samples ,medicine.diagnostic_test ,Dose-Response Relationship, Drug ,business.industry ,Factor X ,Age Factors ,Infant, Newborn ,Infant ,Hematology ,Middle Aged ,Fetal Blood ,medicine.anatomical_structure ,chemistry ,Pharmacodynamics ,Child, Preschool ,Factor Xa ,Prothrombin Time ,Pyrazoles ,Apixaban ,Female ,business ,medicine.drug ,Factor Xa Inhibitors - Abstract
SummaryThe objective was to characterise apixaban pharmacodynamic (PD) activity in umbilical cord (UC), paediatric, and adult plasma. Plasma was obtained from blood samples from six UC donors, 70 paediatric (neonates [birth–≤1 month], infants [>1–≤6 months], toddlers [>6 months–≤2 years], young children [>2–≤6 years], children [>6–≤12 years], adolescents [>12–≤18 years]), and six adult (19–45 years) subjects. Plasma spiked with apixaban 0 (baseline), 30, or 110 ng/ml was analysed for anti-factor Xa activity, factor X levels, prothrombin time (PT), and modified PT (mPT). Apixaban had similar concentration-related effects on anti-factor Xa activity across groups (30 ng/ml: 0.223–0.295 IU/ml; 110 ng/ml: 1.212–1.474 IU/ml). Endogenous baseline factor X levels were 43%–68% lower in plasma from UC and subjects ≤6 months versus adults. Factor Xa inhibition (percentage change from baseline in apparent factor X levels) was similar for both apixaban concentrations across groups, except UC, neonate, and infant groups, which showed greater inhibition vs adults for apixaban 110 ng/ml. Baseline PT and mPT were similar across groups. Apixaban had no effect on PT at the concentrations tested. Apixaban 110 ng/ml prolonged mPT similarly across groups (44.4–53.2 s to 64.5–70.0 s); no prolongation was found with apixaban 30 ng/ml. Apixaban demonstrated consistent concentration-related effects on other PD endpoints in plasma samples from all age groups, except factor Xa inhibition.Supplementary Material to this article is available at www.thrombosis-online.com.
- Published
- 2016
30. Capillary blood samples for anti-Xa monitoring of heparin in paediatric patients
- Author
-
Sallyann Ridsdale, Victoria Martin, and Jeanette Payne
- Subjects
medicine.medical_specialty ,MEDLINE ,Gastroenterology ,03 medical and health sciences ,0302 clinical medicine ,Predictive Value of Tests ,Internal medicine ,medicine ,Humans ,Enoxaparin ,Child ,Blood Coagulation ,Paediatric patients ,Blood coagulation test ,Blood Specimen Collection ,business.industry ,Vascular biology ,Age Factors ,Infant, Newborn ,Infant ,Reproducibility of Results ,Hematology ,Heparin ,Venous Thromboembolism ,medicine.disease ,Thrombosis ,Surgery ,Capillaries ,030228 respiratory system ,030220 oncology & carcinogenesis ,Predictive value of tests ,Child, Preschool ,Factor Xa ,Blood Coagulation Tests ,Drug Monitoring ,business ,medicine.drug ,Factor Xa Inhibitors - Published
- 2016
31. Reduction of thrombus size in murine models of thrombosis following administration of recombinant α1-proteinase inhibitor mutant proteins
- Author
-
Varsha Bhakta, William P. Sheffield, Sharon Gataiance, and Louise J. Eltringham-Smith
- Subjects
Factor XIIa ,Ferric Compounds ,01 natural sciences ,law.invention ,Mice ,0302 clinical medicine ,law ,030212 general & internal medicine ,Heparin cofactor II ,biology ,Chemistry ,digestive, oral, and skin physiology ,Antithrombin ,Thrombin ,Hematology ,NG-Nitroarginine Methyl Ester ,Coagulation ,Factor Xa ,Injections, Intravenous ,Recombinant DNA ,medicine.drug ,Recombinant Fusion Proteins ,Hemorrhage ,Serpin ,Antithrombins ,Factor XIa ,Fibrin ,03 medical and health sciences ,Chlorides ,Fibrinolytic Agents ,medicine ,Animals ,0101 mathematics ,Dose-Response Relationship, Drug ,010102 general mathematics ,Fibrinogen ,Thrombosis ,Molecular biology ,Endotoxemia ,Endotoxins ,Disease Models, Animal ,Kinetics ,alpha 1-Antitrypsin ,Mutation ,biology.protein ,Carotid Artery Injuries ,Leukocyte Elastase ,Factor Xa Inhibitors ,Discovery and development of direct thrombin inhibitors - Abstract
SummaryThe variant serpin α1-PI M358R inhibits thrombin and other proteases such as activated protein C (APC) and factor XIa. We previously described recombinant proteins HAPI M358R (α1-PI M358R containing an N-terminal extension corresponding to residues 1–75 of heparin cofactor II) and HAPI RCL5 (HAPI M358R with F352-I356 and I360 substituted for the corresponding residues of antithrombin), with enhanced selectivity for thrombin over APC inhibition. We tested the hypotheses that these recombinant proteins would limit thrombosis in three mouse models, and that the HAPI chimeric proteins would be more effective than α1-PI M358R. Recombinant serpins were purified from Escherichia coli by nickel chelate and ion exchange affinity chromatography, and administered to mice intravenously. HAPI RCL5 reduced incorporation of radiolabelled fibrin(ogen) into thrombi in the ferric chloride-injured vena cava in a dose-dependent manner; HAPI M358R was less effective and α1-PI M358R was without effect. In a model of murine endotoxaemia, HAPI RCL5 was more effective than α1-PI M358R in reducing radiolabelled fibrin(ogen) deposition in heart and kidneys; immunohis-tochemistry of tissue sections showed lesser staining with anti-fibrin(ogen) antibodies with both treatments. In the ferric chloride-injured murine carotid artery, administration of both recombinant serpins was equally effective in lengthening the vessel’s time to occlusion. Our results show that the antithrombotic efficacy of the recombinant serpins correlates with their potency as thrombin inhibitors, since HAPI RCL5 inhibits thrombin, but not factors Xa, XIa, XIIa, or neutrophil elastase, more rapidly than α1-PI M358R.
- Published
- 2012
32. Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome
- Author
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Gary E. Gilbert, Jin Zhou, Chunying Yao, Fangfang Shi, Qin Wang, Chengyuan Yu, Rui Xie, Chunyan Gao, Rujuan Xie, and Jialan Shi
- Subjects
Adult ,Blood Platelets ,Male ,medicine.medical_specialty ,Erythrocytes ,030232 urology & nephrology ,Phosphatidylserines ,030204 cardiovascular system & hematology ,Glomerulonephritis, Membranous ,Thromboplastin ,Blood cell ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Internal medicine ,medicine ,Humans ,Platelet ,Particle Size ,Lactadherin ,Microscopy, Confocal ,Coagulants ,Nephrosis, Lipoid ,Glomerulonephritis ,Hematology ,Phosphatidylserine ,Middle Aged ,Milk Proteins ,medicine.disease ,Endothelial stem cell ,Endocrinology ,medicine.anatomical_structure ,Clotting time ,chemistry ,Coagulation ,Case-Control Studies ,Antigens, Surface ,Factor Xa ,Immunology ,Intercellular Signaling Peptides and Proteins ,Female ,Carrier Proteins - Abstract
SummaryRecent studies showed that an imbalance of prothrombotic and antithrombotic factors and impaired thrombolytic activity contribute to the thrombophilia of the nephrotic syndrome (NS). However, it is not clear whether blood cell injury and/or activation is involved in hypercoagulability in NS patients. Our objectives were to study the increase in microparticle (MP) release and phosphatidylserine (PS) exposure on the outer membrane of MP-origin cells in NS patients, and to evaluate their procoagulant activity (PCA). The subjects were patients with membranous nephropathy (MN), minimal change nephrotic syndrome (MCNS) and healthy controls. Analyses of MPs and PS exposure were performed using a flow cytometer. PCA was determined by clotting time and purified coagulation complex assays. We found that lactadherin+ MPs, which derived from red blood cells (RBC), platelet and endothelial cell, increased in NS patients. Moreover, PS exposure on RBCs and platelets in each NS group, especially in MN, are higher than that in controls. MP shedding and PS exposure of RBCs/platelets were highly procoagulant in NS patients. However, blockade of PS with lactadherin inhibited over 90% of PCA while an anti-tissue factor antibody had no significant inhibition effect. Our results demonstrate that the thrombophilic susceptibility of NS may be partly ascribed to MP release and PS exposure of RBCs, platelets and endothelial cells. Lactadherin is a sensitive probe for PS that has high anticoagulant activity.
- Published
- 2012
33. Structure and haemostatic effects of generic versions of enoxaparin available for clinical use in Brazil: Similarity to the original drug
- Author
-
Bianca F. Glauser, Mariana S. Pereira, Leonardo Paes Cinelli, Bruno C. Vairo, Stephan-Nicollas M. C. G. Oliveira, and Paulo A.S. Mourão
- Subjects
Male ,0301 basic medicine ,Drug ,medicine.medical_specialty ,Magnetic Resonance Spectroscopy ,Therapeutic equivalency ,media_common.quotation_subject ,Hemorrhage ,030204 cardiovascular system & hematology ,Pharmacology ,Anticoagulant activity ,Patents as Topic ,03 medical and health sciences ,0302 clinical medicine ,Molecular size ,Animals ,Drugs, Generic ,Humans ,Medicine ,Enoxaparin ,Rats, Wistar ,Intensive care medicine ,Blood Coagulation ,media_common ,Active ingredient ,business.industry ,Anticoagulants ,Thrombosis ,Hematology ,Rats ,Disease Models, Animal ,030104 developmental biology ,Therapeutic Equivalency ,Factor Xa ,Female ,Prothrombin ,business ,Patent system ,Brazil - Abstract
SummaryPatent protection for enoxaparin has expired. Generic preparations are developed and approved for clinical use in different countries. However, there is still skepticism about the possibility of making an exact copy of the original drug due to the complex processes involved in generating low-molecular-weight heparins. We have undertaken a careful analysis of generic versions of enoxaparin available for clinical use in Brazil. Thirty-three batches of active ingredient and 70 of the final pharmaceutical product were obtained from six different suppliers. They were analysed for their chemical composition, molecular size distribution, in vitro anticoagulant activity and pharmacological effects on animal models of experimental thrombosis and bleeding. Clearly, the generic versions of enoxaparin available for clinical use in Brazil are similar to the original drug. Only three out of 33 batches of active ingredient from one supplier showed differences in molecular size distribution, resulting from a low percentage of tetrasaccharide or the presence of a minor component eluted as monosaccharide. Three out of 70 batches of the final pharmaceutical products contained lower amounts of the active ingredient than that declared by the suppliers. Our results suggest that the generic versions of enoxaparin are a viable therapeutic option, but their use requires strict regulations to ensure accurate standards.
- Published
- 2012
34. Accurate determination of rivaroxaban levels requires different calibrator sets but not addition of antithrombin
- Author
-
Christian Hesse, Gabriele Rohde, Natalie Herth, Helen Mani, Gertrud Stratmann, Stephan Schwers, Edelgard Lindhoff-Last, and Elisabeth Perzborn
- Subjects
medicine.drug_mechanism_of_action ,Morpholines ,Factor Xa Inhibitor ,Analytical chemistry ,Thiophenes ,030204 cardiovascular system & hematology ,Antithrombins ,03 medical and health sciences ,0302 clinical medicine ,Rivaroxaban ,Tandem Mass Spectrometry ,medicine ,Humans ,030212 general & internal medicine ,Diagnostic Errors ,Chromatography, High Pressure Liquid ,High concentration ,Chromatography ,Chemistry ,Chromogenic ,Antithrombin ,Anticoagulants ,Hematology ,Linear relationship ,Calibration ,Factor Xa ,Drug Monitoring ,Ex vivo ,medicine.drug ,Blinded study - Abstract
SummaryRivaroxaban is a direct factor Xa inhibitor, which can be monitored by anti-factor Xa chromogenic assays. This ex vivo study evaluated different assays for accurate determination of rivaroxaban levels. Eighty plasma samples from patients receiving rivaroxaban (Xarelto®) 10 mg once daily and 20 plasma samples from healthy volunteers were investigated using one anti-factor Xa assay with the addition of exogenous antithrombin and two assays without the addition of antithrombin. Two different lyophilised rivaroxaban calibration sets were used for each assay (low concentration set: 0, 14.5, 59.6 and 97.1 ng/ml; high concentration set: 0, 48.3, 101.3, 194.2 and 433.3 ng/ml). Using a blinded study design, the rivaroxaban concentrations determined by the assays were compared with concentrations measured by HPLC-MS/MS. All assays showed a linear relationship between the rivaroxaban concentrations measured by HPLC-MS/MS and the optical density of the anti-FXa assays. However, the assay with the addition of exogenous anti-thrombin detected falsely high concentrations of rivaroxaban even in plasma samples from controls who had not taken rivaroxaban (intercept values using the high calibrator set and the low calibrator set: +26.49 ng/ml and +13.71 ng/ml, respectively). Plasma samples, initially determined by the high calibrator setting and containing rivaroxaban concentrations
- Published
- 2012
35. Heparin from bovine intestinal mucosa: Glycans with multiple sulfation patterns and anticoagulant effects
- Author
-
Stephan-Nicollas M. C. G. Oliveira, Ana M. F. Tovar, Gustavo R.C. Santos, Bruno C. Vairo, Nina V. Capillé, Paulo A.S. Mourão, and Roberto J. C. Fonseca
- Subjects
Male ,Glycosylation ,Magnetic Resonance Spectroscopy ,medicine.drug_class ,Swine ,Disaccharide ,Hemorrhage ,Disaccharides ,Thromboplastin ,Glycosaminoglycan ,chemistry.chemical_compound ,Structure-Activity Relationship ,Sulfation ,Intestinal mucosa ,Fibrinolytic Agents ,In vivo ,medicine ,Animals ,Humans ,Antithrombin Proteins ,Protamines ,Intestinal Mucosa ,Rats, Wistar ,Blood Coagulation ,Anion Exchange Resins ,Venous Thrombosis ,Heparinase ,Molecular Structure ,Chemistry ,Heparin ,Sulfates ,Anticoagulant ,Anticoagulants ,Heparin Antagonists ,Hematology ,Chromatography, Ion Exchange ,Rats ,Disease Models, Animal ,Biochemistry ,Heparin Lyase ,Factor Xa ,Cattle ,Female ,Partial Thromboplastin Time ,Prothrombin ,medicine.drug ,Factor Xa Inhibitors - Abstract
SummaryPharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide →4-α-IdoA2S-1 →4-α-GlcNS6S-1 →. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N-sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3-and 6-trisulfated a-glucosamine (lower proportions) and α-GlcNS-1→4-α-GlcA and α-IdoA2S-1→4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the a-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical he-parin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro. Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.
- Published
- 2012
36. Two missense mutations identified in venous thrombosis patients impair the inhibitory function of the protein Z dependent protease inhibitor
- Author
-
Paul Harper, Neil S. Van De Water, Paul Bernard Coughlin, Peter Browett, Laura Young, Paul Ockelford, Anita J Horvath, and Nigel P. Birch
- Subjects
Adult ,Male ,0301 basic medicine ,Protein Denaturation ,Genotype ,Protein Conformation ,Nonsense mutation ,Mutant ,Mutation, Missense ,Protein Z ,030204 cardiovascular system & hematology ,Biology ,medicine.disease_cause ,Risk Assessment ,Factor XIa ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Gene Frequency ,Risk Factors ,Enzyme Stability ,Odds Ratio ,medicine ,Humans ,Missense mutation ,Blood Coagulation ,Serpins ,Venous Thrombosis ,Mutation ,Chi-Square Distribution ,Hematology ,Middle Aged ,medicine.disease ,Molecular biology ,Recombinant Proteins ,Protease inhibitor (biology) ,Kinetics ,Venous thrombosis ,Phenotype ,030104 developmental biology ,Coagulation ,Case-Control Studies ,Factor Xa ,Female ,New Zealand ,medicine.drug - Abstract
SummaryProtein Z-dependent protease inhibitor (ZPI) is a plasma inhibitor of factor (F)Xa and FXIa. In an earlier study, five mutations were identified within the ZPI gene of venous thrombosis patients and healthy controls. Two of these were nonsense mutations and three were missense mutations in important regions of the protein. Here we report that two of these latter three mutations, F145L and Q384R, impair the inhibitory function of ZPI in vitro. Recombinant wild-type and mutant proteins were prepared; stability in response to thermal challenge was similar. Inhibition of FXa in the presence of the cofactor protein Z was reduced 68-fold by the Q384R mutant; inhibition of FXIa by the F145L mutant was reduced two- to three-fold compared to the wild-type ZPI. An analysis of all five ZPI mutations was undertaken in a cohort of venous thrombosis patients (n=550) compared to healthy controls (n=600). Overall, there was a modest increase in incidence of these mutations in the thrombosis group (odds ratio 2.0, 1.05–3.7, p=0.044). However, in contrast to W324X (nonsense mutation), the Q384R missense mutation and R88X nonsense mutation were evenly distributed in patients and controls; F145L was rare. The final mutation (S143Y) was also rare and did not significantly alter ZPI function in laboratory studies. The F145L and particularly the Q384R mutation impaired the function of the coagulation inhibitor ZPI; however, there was no convincing association between these mutations and venous thrombosis risk. The functional role for ZPI in vivo has yet to be clarified.
- Published
- 2012
37. Modelling and simulation of edoxaban exposure and response relationships in patients with atrial fibrillation
- Author
-
Robert P. Giugliano, Helen Kastrissios, Tomas S. Bocanegra, Simon Zhou, Michelle Green, Shashank Rohatagi, Indravadan Patel, Timothy J. Carrothers, Bruce E Dornseif, SaeHeum Song, Satoshi Kunitada, Minggao Shi, Jeanne Mendell, Daniel E. Salazar, Elliott M. Antman, and Masaya Tachibana
- Subjects
Male ,medicine.medical_specialty ,medicine.drug_mechanism_of_action ,Pyridines ,Factor Xa Inhibitor ,Administration, Oral ,Hemorrhage ,030204 cardiovascular system & hematology ,Models, Biological ,Risk Assessment ,030226 pharmacology & pharmacy ,03 medical and health sciences ,chemistry.chemical_compound ,Cmin ,0302 clinical medicine ,Pharmacokinetics ,Risk Factors ,Edoxaban ,Internal medicine ,Atrial Fibrillation ,Humans ,Medicine ,Computer Simulation ,Drug Interactions ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Blood Coagulation ,Stroke ,Models, Statistical ,Dose-Response Relationship, Drug ,business.industry ,Anticoagulants ,Atrial fibrillation ,Hematology ,medicine.disease ,Clinical trial ,Thiazoles ,Logistic Models ,Treatment Outcome ,Clinical Trials, Phase III as Topic ,chemistry ,Research Design ,Anesthesia ,Concomitant ,Factor Xa ,Female ,Kidney Diseases ,business ,Factor Xa Inhibitors - Abstract
SummaryEdoxaban is a novel, orally available, highly specific direct inhibitor of factor Xa and is currently being developed for the treatment and prevention of venous thromboembolism and prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation (NVAF). The objectives of the present analyses were to characterise edoxaban population pharmacokinetics (PPK) and identify potential intrinsic and extrinsic factors affecting variability in edoxaban exposure, determine if there are relationships between edoxaban pharmacokinetics or biomarkers and the risk of bleeding in patients with NVAF using an exposure-response model, and to use the PPK and exposure-response model to support dose selection for a phase III trial of edoxaban in patients with NVAF. PPK analysis of data from 1,281 edoxaban-dosed subjects with intrinsic factors such as renal impairment or NVAF and extrinsic factors such as concomitant medications revealed significant effects of renal impairment and concomitant strong P-glycoprotein (P-gp) inhibitors on the pharmacokinetics of edoxaban. Exposure-response analysis found that in patients with NVAF, the incidence of bleeding events increased significantly with increasing edoxaban exposure, with steady-state minimum concentration (Cmin,ss) showing the strongest association. Clinical trial simulations of bleeding incidence were used to select 30 mg and 60 mg once-daily edoxaban with 50% dose reductions for patients with moderate renal impairment or receiving concomitant strong P-gp inhibitors as the treatment regimens in the ENGAGE AF-TIMI 48 (NCT00781391) trial.The results of this study were previously presented at the 2009 International Society on Thrombosis and Haemostasis, July 2009, Boston, Massachusetts, USA.
- Published
- 2012
38. Oral direct factor Xa inhibition with edoxaban for thromboprophylaxis after elective total hip replacement
- Author
-
Alexander T. Cohen, Jeffrey I. Weitz, Gary E. Raskob, Tomas Bocanegra, David Puskas, Minggao Shi, and Bengt I. Eriksson
- Subjects
Dalteparin ,Male ,Time Factors ,Pyridines ,Arthroplasty, Replacement, Hip ,Administration, Oral ,law.invention ,chemistry.chemical_compound ,Randomized controlled trial ,Risk Factors ,Edoxaban ,law ,Medicine ,Venous Thrombosis ,medicine.diagnostic_test ,Anticoagulant ,Venous Thromboembolism ,Hematology ,Middle Aged ,Pulmonary embolism ,Europe ,Venous thrombosis ,Treatment Outcome ,Elective Surgical Procedures ,Anesthesia ,Factor Xa ,Female ,Canada ,Randomization ,medicine.drug_class ,Injections, Subcutaneous ,Venography ,Hemorrhage ,Risk Assessment ,Drug Administration Schedule ,Double-Blind Method ,Fibrinolytic Agents ,Humans ,cardiovascular diseases ,Aged ,business.industry ,Anticoagulants ,Phlebography ,medicine.disease ,United States ,Thiazoles ,Logistic Models ,chemistry ,Pulmonary Embolism ,business ,Fibrinolytic agent ,Factor Xa Inhibitors - Abstract
SummaryEdoxaban is a new oral direct factor Xa inhibitor. The purpose of this study was to evaluate the efficacy and safety of different doses of edoxaban for the prevention of venous thromboembolism (VTE) in patients undergoing elective total hip replacement. A total of 903 patients were randomised to oral edoxaban 15, 30, 60 or 90 mg once daily or subcutaneous dalteparin once daily (initial dose 2,500 IU, subsequent doses 5,000 IU). Both drugs were begun 6–8 hours postoperatively and continued for 7–10 days, when bilateral venography was performed. The primary efficacy endpoint was the incidence of total VTE, which included proximal and/or distal deep-vein thrombosis (DVT) by venography or symptomatic, objectively confirmed DVT or pulmonary embolism during the treatment period. The primary safety outcome was the incidence of the composite of major and clinically relevant non-major bleeding. All venograms and bleeding events were reviewed by a central independent adjudication committee blinded as to treatment allocation. Of the 903 patients randomised, 776 were evaluable for the primary efficacy analysis. The incidences of VTE were 28.2%, 21.2%, 15.2%, and 10.6% in patients receiving edoxaban 15, 30, 60 and 90 mg, respectively, compared with 43.8% in the dalteparin group (p
- Published
- 2010
39. Apixaban, a direct factor Xa inhibitor, inhibits tissue-factor induced human platelet aggregation in vitro: Comparison with direct inhibitors of factor VIIa, XIa and thrombin
- Author
-
Pancras C. Wong and Xiaosui Jiang
- Subjects
Time Factors ,Platelet Aggregation ,medicine.drug_mechanism_of_action ,Pyridines ,Pyridones ,Morpholines ,Factor Xa Inhibitor ,Factor VIIa ,Thiophenes ,Pharmacology ,Arginine ,Factor XIa ,Piperazines ,Argatroban ,Thromboplastin ,Tissue factor ,Thrombin ,Rivaroxaban ,medicine ,Humans ,Platelet ,Sulfonamides ,Dose-Response Relationship, Drug ,Chemistry ,Antithrombin ,Anticoagulants ,Hematology ,Dabigatran ,Adenosine Diphosphate ,Biochemistry ,Pipecolic Acids ,Hemostasis ,Factor Xa ,Azetidines ,Pyrazoles ,Benzimidazoles ,Apixaban ,Collagen ,Platelet Aggregation Inhibitors ,Factor Xa Inhibitors ,medicine.drug - Abstract
SummaryApixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late-stage clinical development. This study evaluated the in vitro effect of apixaban on human platelet aggregation induced by thrombin derived via the extrinsic pathway. Direct inhibitors of FXa (rivaroxaban), FVIIa (BMS-593214), thrombin (dabigatran, argatroban) and FXIa (BMS-262084) were included for comparison. Citrated human plateletsrich plasma (PRP) was treated with 50 μg/ml corn trypsin inhibitor (to block the contact factor pathway) and 3 mM H-Gly-Pro-Arg- Pro-OH-AcOH (to prevent fibrin polymerisation). Human tissue factor (TF) (Innovin®; dilution 1:1,000 to 1:1,500) plus 7.5 mM CaCl2 was added to PRP pre-incubated with vehicle or increasing concentrations of inhibitors. The TF-induced platelet aggregation was measured by optical aggregometry. TF produced 85 ± 3% aggregation of human platelets in the vehicle-treated group (n=10). Apixaban and other factor inhibitors, except the FXIa inhibitor, inhibited TF-induced platelet aggregation with IC50 (nM) values as follows: 4 ± 1 (apixaban), 8 ± 2 (rivaroxaban), 13 ± 1 (BMS-593214), 46 ± 1 (dabigatran) and 79 ± 1 (argatroban). BMS-262084 (IC50 = 2.8 nM vs. human FXIa) had no effect on TF-induced platelet aggregation at 10 μM. These inhibitors at 10 μM had no effect on platelet aggregation induced by ADP and collagen, as expected from their mechanism of action. This study demonstrates that inhibition of thrombin generation by blocking upstream proteases (FVIIa and FXa) in the blood coagulation cascade is as effective as direct thrombin inhibition in preventing TF-induced platelet aggregation. Under these experimental conditions, a FXIa inhibitor did not prevent TF-induced platelet aggregation.
- Published
- 2010
40. Age is a determinant factor for measures of concentration and effect in children requiring unfractionated heparin
- Author
-
Linda Johnston, Robyn Summerhayes, Vera Ignjatovic, Geoff Lane, Noel Cranswick, Paul Monagle, and Fiona Newall
- Subjects
Adult ,Male ,medicine.medical_specialty ,Adolescent ,medicine.drug_class ,Bolus (medicine) ,Reference Values ,Internal medicine ,medicine ,Humans ,Drug Dosage Calculations ,Child ,Evidence-Based Medicine ,Hematology ,medicine.diagnostic_test ,biology ,Heparin ,business.industry ,Anticoagulant ,Age Factors ,Infant, Newborn ,Infant ,Venous blood ,Protamine ,Blood proteins ,Endocrinology ,Child, Preschool ,Factor Xa ,Practice Guidelines as Topic ,Immunology ,biology.protein ,Female ,Partial Thromboplastin Time ,business ,Partial thromboplastin time ,medicine.drug - Abstract
SummaryPrevious studies investigating continuous unfractionated heparin (UFH) therapy report age-related differences in UFH response in children, as measured by APTT and anti-Xa assay. This study determined the age-related response following administration of a single UFH bolus of 75–100 IU/kg in children. Venous blood samples were collected from children (n=56) at 15, 30, 45 and 120 minutes post-UFH. Anti-Xa, anti-IIa, APTT, TCT and protamine titration were performed on all samples. Age-dependent differences in the effect and concentration of UFH were identified for the anti-Xa, anti-IIa and protamine titration as-says, respectively. In addition, a trend suggesting a proportional increase in anti-Xa and anti-IIa-mediated UFH effect with age was evident. Logistic regression demonstrated an increase in protamine titration of 0.6 IU/ml for every year of age in samples collected 15 minutes post-UFH. UFH-mediated anti-IIa activity was reduced compared to anti-Xa activity across childhood, with a two-fold increase in anti-Xa to anti-IIa ratio in infants less than one year of age compared to teenagers in the setting of high UFH concentrations. This study demonstrates that the previously reported age-dependent response to UFH occurs in the context of an age-dependent serum concentration of UFH. The trend toward increased UFH serum concentration and anticoagulant activity with age may be related to short-term differences in UFH binding to coagulant and competitive plasma proteins in vivo.
- Published
- 2010
41. Effect of apixaban, an oral and direct factor Xa inhibitor, on coagulation activity biomarkers following acute coronary syndrome
- Author
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Jessie Wang, John H. Alexander, Richard C. Becker, L K Newby, Lars Wallentin, H. Yang, Yuchen Barrett, Puneet Mohan, and Robert A. Harrington
- Subjects
Male ,Time Factors ,medicine.drug_mechanism_of_action ,Administration, Oral ,Phases of clinical research ,Gastroenterology ,Tandem Mass Spectrometry ,Odds Ratio ,Dose Reduced ,Aged, 80 and over ,medicine.diagnostic_test ,Thrombin ,Hematology ,Middle Aged ,Dose–response relationship ,Treatment Outcome ,Factor Xa ,Female ,Partial Thromboplastin Time ,Prothrombin ,Apixaban ,medicine.drug ,Partial thromboplastin time ,Adult ,medicine.medical_specialty ,Acute coronary syndrome ,Pyridones ,Factor Xa Inhibitor ,Down-Regulation ,Enzyme-Linked Immunosorbent Assay ,Placebo ,Fibrin Fibrinogen Degradation Products ,Double-Blind Method ,Fibrinolytic Agents ,Internal medicine ,medicine ,Humans ,International Normalized Ratio ,Acute Coronary Syndrome ,Blood Coagulation ,Aged ,Chi-Square Distribution ,Dose-Response Relationship, Drug ,business.industry ,Placebo Effect ,medicine.disease ,Peptide Fragments ,Endocrinology ,Pyrazoles ,business ,Biomarkers ,Chromatography, Liquid ,Factor Xa Inhibitors - Abstract
SummaryApixaban is an oral, direct factor Xa inhibitor under development for secondary prevention in acute coronary syndrome (ACS). Apixaban‘s effect on D-dimer and prothrombin fragment 1.2 (F1.2) (coagulation activity biomarkers ) was determined in a randomised, double-blinded, placebo-controlled, phase 2 study. Patients (n=1,715) with either ST-segment elevation or non-ST-segment elevation ACS received either placebo or apixaban 2.5 mg twice daily, 10 mg once daily, 10 mg twice daily or 20 mg once daily for six months. Samples were obtained at baseline (before study drug administration), week 3 and week 26. Apixaban plasma concentrations were measured directly by liquid chromatography/mass spectometry, and anti-Xa activity was determined using apixaban as a reference standard. D-dimer and F 1.2 were measured using ELISA-based methods. Most patients had elevated D-dimer and F1.2 levels at baseline. Both coagulation activity biomarkers decreased by week 3 in all treatment groups, but to a greater degree with apixaban than placebo (p
- Published
- 2010
42. Differential effects of TAK-442, a novel orally active direct factor Xa inhibitor, and ximelagatran, a thrombin inhibitor, on factor V-mediated feedback on coagulation cascade and bleeding
- Author
-
Noriko Konishi, Katsuhiko Hiroe, and Masaki Kawamura
- Subjects
Male ,Benzylamines ,medicine.medical_specialty ,Ximelagatran ,medicine.drug_mechanism_of_action ,Factor Xa Inhibitor ,Administration, Oral ,Hemorrhage ,Pyrimidinones ,Risk Assessment ,Antithrombins ,Thromboplastin ,Rats, Sprague-Dawley ,Thrombin ,Bleeding time ,Internal medicine ,medicine ,Animals ,Humans ,Sulfones ,Blood Coagulation ,Phospholipids ,Feedback, Physiological ,Venous Thrombosis ,Dose-Response Relationship, Drug ,biology ,medicine.diagnostic_test ,Chemistry ,Antithrombin ,Factor V ,Anticoagulants ,Hematology ,Rats ,Disease Models, Animal ,Endocrinology ,Coagulation ,Factor Xa ,Prothrombin Time ,biology.protein ,Azetidines ,Factor Xa Inhibitors ,medicine.drug ,Discovery and development of direct thrombin inhibitors - Abstract
SummaryThrombin amplifies the blood coagulation via factor V (FV)-mediated positive feedback loop. We hypothesised that factor Xa (FXa) inhibitors would interfere more gradually with this feedback activation loop than thrombin inhibitors, thereby achieving a better balance between haemostasis and prevention of thrombosis. In this study, we compared the effects of TAK-442, a novel FXa inhibitor, versus ximelagatran, a thrombin inhibitor, on FV-mediated positive feedback, venous thrombosis and bleeding. In normal plasma, TAK-442 delayed the onset of tissue factor-induced thrombin generation and prolonged prothrombin time (PT) with more gradual concentration-response curve than melagatran, the active form of ximelagatran. The effect of melagatran on the onset of thrombin generation decreased in an FVa-concentration-dependent manner in FV-deficient plasma supplemented with FVa. Furthermore, in FV-deficient plasma, the PT-prolonging potency of melagatran was markedly increased with a change in its concentration-response curve from steep to gradual. In the rat venous thrombosis model, TAK-442 (10 mg/kg, p.o.) prevented thrombus formation by 55% with 1.2 times prolongation of PT; a similar effect was observed in ximelagatran-treated (3 mg/kg, p.o.) rats. TAK-442 at 100 mg/kg prolonged PT by only 2.1 times with no change in bleeding time (BT), whereas ximelagatran at 10 mg/kg prolonged PT by 3.9 times and significantly increased BT. These results suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.
- Published
- 2010
43. Heparins from porcine and bovine intestinal mucosa: Are they similar drugs?
- Author
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Rafael S. Aquino, Gustavo R.C. Santos, Leonardo Paes Cinelli, Mariana S. Pereira, Roberto J. C. Fonseca, Bruno C. Vairo, and Paulo A.S. Mourão
- Subjects
Swine ,medicine.drug_class ,Hemorrhage ,Pharmacology ,Disaccharides ,Thromboplastin ,Sulfation ,Intestinal mucosa ,Antithrombotic ,medicine ,Animals ,Protamines ,Intestinal Mucosa ,Rats, Wistar ,Blood Coagulation ,Nuclear Magnetic Resonance, Biomolecular ,Venous Thrombosis ,biology ,Heparin ,business.industry ,Anticoagulant ,Hematology ,medicine.disease ,Thrombosis ,Protamine ,Rats ,Coagulation ,Factor Xa ,Immunology ,biology.protein ,Cattle ,business ,medicine.drug - Abstract
Increasing reports of bleeding and peri- or post-operative blood dyscrasias in Brazil were possibly associated with the use of heparin from bovine instead of porcine intestine. These two pharmaceutical grade heparins were analysed for potential differences. NMR analyses confirmed that porcine heparin is composed of mainly trisulfated disaccharides --4-alpha-IdoA2S-1--4-alpha-GlcNS6S-1--. Heparin from bovine intestine is also composed of highly 2-sulfated alpha-iduronic acid residues, but the sulfation of the alpha-glucosamine units vary significantly: approximately 50% are 6- and N -disulfated, as in porcine heparin, while approximately 36% are 6-desulfated and approximately 14% N -acetylated. These heparins differ significantly in their effects on coagulation, thrombosis and bleeding. Bovine heparin acts mostly through factor Xa. Compared to porcine heparin on a weight basis, bovine heparin exhibited approximately half of the anticoagulant and antithrombotic effects, but similar effect on bleeding. These two heparins also differ in their protamine neutralisation curves. The doses of heparin from bovine intestine required for effective antithrombotic protection and the production of adverse bleeding effects are closer than those for porcine heparin. This observation may explain the increasing bleeding observed among Brazilian patients. Our results suggest that these two types of heparin are not equivalent drugs.
- Published
- 2010
44. The direct thrombin inhibitor argatroban effectively prevents cardiac catheter thrombosis in vitro
- Author
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Uwe Raaz, Alexander Plehn, Marese Busshardt, Martin Russ, Anja Kaeberich, Henning Ebelt, Karl Werdan, Sebastian Schubert, Michael Buerke, Lars Maegdefessel, and Axel Schlitt
- Subjects
Blood Platelets ,Male ,Cardiac Catheterization ,Time Factors ,medicine.drug_class ,Antithrombin III ,Activated clotting time ,Arginine ,Fibrin ,Argatroban ,Humans ,Medicine ,Enoxaparin ,Thrombus ,Blood Coagulation ,Sulfonamides ,biology ,medicine.diagnostic_test ,Heparin ,business.industry ,Antithrombin ,Anticoagulant ,Thrombin ,Anticoagulants ,Thrombosis ,Hematology ,medicine.disease ,Direct thrombin inhibitor ,Pipecolic Acids ,Anesthesia ,Factor Xa ,biology.protein ,Stress, Mechanical ,business ,Factor Xa Inhibitors ,Peptide Hydrolases ,medicine.drug - Abstract
SummaryThe direct thrombin inhibitor argatroban offers some significant advantages over unfractionated heparin (UFH) and is recommended as an alternative anticoagulant during percutaneous coronary interventions (PCI). The impact of argatroban on cardiac catheter thrombosis – a severe potential complication of PCI – has not been systematically studied yet. The aim of the present study was to test in vitro the hypothesis that argatroban is equivalent to the more established anticoagulants UFH and enoxaparin in preventing catheter thrombus formation. Blood pretreated with the anticoagulants of interest was continuously circulated through a guiding catheter by using a roller pump for a maximum experimental period of 60 minutes. In an alternate model, coagulation was mechanically induced by a magnetic stirrer. Coagulation parameters, overall thrombus weight and electron microscopic features (deposits of platelets and fibrin on the catheter surface) were quantified as endpoints. Argatroban (administered as bolus or continuous in-fusion), UFH (bolus), and enoxaparin (bolus) significantly reduced catheter thrombus formation compared to untreated controls. Here, neither overall thrombus weight nor platelet/fibrin deposition was different among the specific anticoagulants. Declining ACT (activated clotting time) levels – which were found in the argatroban bolus group – could be prevented by continuous infusion. In magnetic stirrer-induced coagulation, thrombus weight was lower following bolus treatment with UFH and enoxaparin compared to argatroban. These data suggest that the potential for argatroban in preventing catheter thrombosis is comparable to that of UFH and enoxaparin. However, the anticoagula-tory efficacy varied, depending on the model of coagulation activation, which demonstrates the necessity for specific testing.
- Published
- 2010
45. Daunorubicin induces procoagulant activity of cultured endothelial cells through phosphatidylserine exposure and microparticles release
- Author
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Wen Li, Rui Xie, Gary E. Gilbert, Jinxiao Hou, Huibo Li, Yueyue Fu, Xue Yang, Jialan Shi, Jin Zhou, Chunyan Gao, and Xiushuai Dong
- Subjects
Endothelium ,Daunorubicin ,medicine.medical_treatment ,Immunology ,Phosphatidylserines ,Pharmacology ,Biochemistry ,Umbilical vein ,Thromboplastin ,Flow cytometry ,chemistry.chemical_compound ,Cell-Derived Microparticles ,Fibrinolysis ,medicine ,Humans ,Blood Coagulation ,Cells, Cultured ,Lactadherin ,Blood coagulation test ,Antibiotics, Antineoplastic ,Membrane Glycoproteins ,Dose-Response Relationship, Drug ,medicine.diagnostic_test ,business.industry ,Thrombin ,Anticoagulants ,Endothelial Cells ,Thrombosis ,Cell Biology ,Hematology ,Phosphatidylserine ,Flow Cytometry ,Milk Proteins ,Endothelial stem cell ,medicine.anatomical_structure ,Clotting time ,chemistry ,Factor Xa ,Blood Coagulation Tests ,business ,medicine.drug - Abstract
Abstract 5185 Administration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 μ M) for 24 h. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 μ M of daunorubicin or over. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2010
46. Direct inhibitors of coagulation proteins – the end of the heparin and low-molecular-weight heparin era for anticoagulant therapy?
- Author
-
Christoph Gerdes, Volker Laux, Elke Dittrich-Wengenroth, Anja Buchmüller, Elisabeth Perzborn, Georges von Degenfeld, Stefan Heitmeier, and Frank Misselwitz
- Subjects
medicine.medical_specialty ,Vitamin K ,medicine.drug_mechanism_of_action ,medicine.drug_class ,Antithrombin III ,Factor Xa Inhibitor ,Low molecular weight heparin ,Hemorrhage ,Pharmacology ,Dabigatran ,Postoperative Complications ,medicine ,Humans ,Thrombophilia ,Enzyme Inhibitors ,Clinical Trials as Topic ,Purpura, Thrombocytopenic, Idiopathic ,Rivaroxaban ,Heparin ,business.industry ,Anticoagulant ,Anticoagulants ,Factor V ,Venous Thromboembolism ,Hematology ,Heparin, Low-Molecular-Weight ,Blood Coagulation Factors ,Surgery ,Molecular Weight ,Direct thrombin inhibitor ,Drug Design ,Factor Xa ,business ,Factor Xa Inhibitors ,medicine.drug ,Discovery and development of direct thrombin inhibitors - Abstract
SummaryHeparins, either unfractionated or low-molecular-weight (UFH and LMWHs), and vitamin K antagonists (VKAs) are currently the anticoagulants of choice for the prevention of post-operative venous thromboembolism (VTE) and for the treatment of acute venous and arterial thromboembolism. While VKAs are widely used in the US, LMWHs are the standard of care in the EU. Although efficacious, these agents are associated with a number of drawbacks, such as the risk of heparin-induced thrombocytopenia, the need for frequent coagulation monitoring in the case of UFH and VKAs, and the parenteral mode of administration in the case of heparins, which can lead to problems associated with patient compliance. There is a need for new anticoagulants that overcome these limitations. Direct, small-molecule inhibitors of coagulation proteins targeting a single enzyme in the coagulation cascade – particularly thrombin or Factor Xa – have been developed in recent years. Two agents, the direct thrombin inhibitor dabigatran and the direct Factor Xa inhibitor rivaroxaban, have recently been approved in the EU and several other countries for the prevention of VTE after total hip or knee replacement surgery. Here we will review data that suggest that the antithrombin-independent mechanism of action of these agents, particularly that of direct Factor Xa inhibitors, leads to increased efficacy with similar safety profiles compared with the antithrombin-dependent heparins. Although the end of the heparins era is not to be expected, the new anticoagulants presented in this review potentially represent the future of anticoagulation.
- Published
- 2009
47. Anticoagulant activity of a sulfated galactan: Serpin-independent effect and specific interaction with factor Xa
- Author
-
Mariana S. Pereira, Alireza R. Rezaie, Robson Q. Monteiro, Paulo A.S. Mourão, Fábio R. Melo, Ricardo M. Rezende, Ivo M.B. Francischetti, and Bianca F. Glauser
- Subjects
In Vitro Techniques ,Serpin ,Galactans ,Article ,chemistry.chemical_compound ,Thrombin ,Prothrombinase ,medicine ,Humans ,Salivary Proteins and Peptides ,Blood Coagulation ,Serpins ,Heparin cofactor II ,Binding Sites ,Heparin ,Chemistry ,Antithrombin ,Anticoagulants ,Factor V ,Hematology ,Galactan ,Recombinant Proteins ,Neoplasm Proteins ,Cysteine Endopeptidases ,Coagulation ,Biochemistry ,Factor Xa ,Mutagenesis, Site-Directed ,Calcium ,Factor Xa Inhibitors ,medicine.drug - Abstract
SummaryAn algal sulfated galactan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant action is due to promoting thrombin and factor (F)Xa inhibition by antithrombin and heparin cofactor II. Here, we evaluated the anticoagulant effect of the algal sulfated galactan using serpin-free plasma. In contrast to heparin, the sulfated galactan is still able to prolong coagulation time and delay thrombin and FXa generation in serpin-free plasma. We further investigated this effect using purified blood coagulation proteins, discovering that sulfated galactan inhibits the intrinsic tenase and prothrombinase complexes, which are critical for FXa and thrombin generation, respectively. We also investigated the mechanism by which sulfated galactan promotes FXa inhibition by antithrombin using specific recombinant mutants of the protease. We show that sulfated galactan interacts with the heparin-binding exosite of FXa and Arg-236 and Lys-240 of this site are critical residues for this interaction, as observed for heparin. Thus, sulfated galactan and heparin have similar high-affinity and specificity for interaction with FXa, though they have differences in their chemical structures. Similar to heparin, the ability of sulfated galactan to potentiate FXa inhibition by antithrombin is calcium-dependent. However, in contrast to heparin, this effect is not entirely dependent on the conformation of the γ-carboxyglutamic acid-rich domain of the protease. In conclusion, sulfated galactan and heparin have some similar effects on blood coagulation, but also differ significantly at the molecular level. This sulfated galactan opens new perspective for the development of antithrombotic drugs.
- Published
- 2009
48. Prothrombin amino terminal region helps protect coagulation factor Va from proteolytic inactivation by activated protein C
- Author
-
Andrew J. Gale, Subramanian Yegneswaran, John H. Griffin, and Phuong M. Nguyen
- Subjects
Time Factors ,Proteolysis ,Plasma protein binding ,Binding, Competitive ,Article ,law.invention ,Thrombin ,Kringles ,law ,medicine ,Animals ,Humans ,Blood Coagulation ,Factor VIIIa ,Phospholipids ,Gla domain ,chemistry.chemical_classification ,Enzyme Precursors ,Oligopeptide ,medicine.diagnostic_test ,Chemistry ,Hematology ,Molecular biology ,Peptide Fragments ,Recombinant Proteins ,Protein Structure, Tertiary ,Enzyme ,Biochemistry ,Factor Va ,Factor Xa ,Mutation ,Recombinant DNA ,Prothrombin ,Protein C ,Protein Binding ,medicine.drug - Abstract
SummaryThe hypothesis that prothrombin (FII) protects coagulation factor Va (FVa) from proteolytic inactivation by activated protein C (APC) was tested using purified proteins. FII dose-dependently protected FVa from APC proteolysis under conditions where competition of proteins for binding to negatively-charged phospholipid surface was not relevant (i.e. either at high phospholipid vesicle concentrations or using soluble dicaproylphosphatidylserine at levels below its critical micellar concentration). Cleavages in FVa at both Arg506 and Arg306 by APC were inhibited by FII. FII did not alter the amidolytic activity of APC towards chromogenic oligopeptide substrates or inhibit FVIIIa inactivation by APC, implying that the FII-mediated protection of FVa from APC proteolysis was due to the ability of FII to inhibit protein-protein interactions between FVa and APC. FII also protected FVa from inactivation by Gla-domainless APC, ruling out a role for the APC Gla domain for these observations. To identify domains of FII responsible for the observed phenomenon, various forms or fragments of FII were employed. Biotin-PheProArg-CMK-inhibited meizothrombin and fII-fragment 12 protected FVa from proteolysis by APC. In contrast, no significant protection of FVa from APC cleavage was observed for Gladomainless-FII, prethrombin-1, prethrombin-2, FII fragment 1 or active site inhibited-thrombin (DEGR-thrombin). Overall, these data demonstrate that the Gla domain of FII linked to kringle 1 and 2 is necessary for the ability of FII to protect FVa from APC cleavage and support the general concept that assembly of the FII activation complex (FXaFVaFIIlipid surface) protects FVa from APC inactivation so that the procoagulant, thrombin generating pathway can act unhindered by APC. Only following FII activation and dissociation of the FII Gla domain fragments from the FII-ase complex, can APC inactivate FVa and down-regulate thrombin generation.
- Published
- 2009
49. Formation of tissue factor-factor VIIa-factor Xa complex induces activation of the mTOR pathway which regulates migration of human breast cancer cells
- Author
-
Michael E. Bromberg, Tracee S. Panetti, Shimei Zhu, and Xiaofeng Jiang
- Subjects
Cell signaling ,Morpholines ,Breast Neoplasms ,Factor VIIa ,Biology ,mTORC2 ,Thromboplastin ,Tissue factor ,Cell Movement ,Cell Line, Tumor ,Humans ,Receptor, PAR-2 ,Neoplasm Invasiveness ,Receptor, PAR-1 ,Phosphorylation ,Protein kinase A ,Protein Kinase Inhibitors ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Sirolimus ,Akt/PKB signaling pathway ,TOR Serine-Threonine Kinases ,RPTOR ,Ribosomal Protein S6 Kinases, 70-kDa ,Hematology ,Recombinant Proteins ,Cell biology ,Biochemistry ,Chromones ,Factor Xa ,Female ,Peptides ,Protein Kinases ,Proto-Oncogene Proteins c-akt - Abstract
SummaryTissue factor (TF) is a transmembrane glycoprotein that initiates blood coagulation when complexed with activated factor VII (FVIIa).TF is constitutively expressed in a variety of tumor cells and has been implicated in cellular signaling, angiogenesis, and tumor progression. Formation of TF-FVIIa complex and generation of downstream coagulation proteases, including activated factor X (FXa) and thrombin, initiate signaling by activation of protease-activated receptors (PARs).We have previously shown thatTFFVIIa-Xa complex formation promotes phosphorylation of p44/42 mitogen-activated protein kinase and Akt/protein kinase B in human breast cancer cells. In the present study, we show that formation of TF-FVIIa-FXa complex induces phosphorylation of mammalian target of rapamycin (mTOR) and p70 S6 kinase in a human breast cancer cell line, Adr-MCF-7. Activation of the mTOR pathway, which is probably mediated by PAR1 and/or PAR2, was associated with enhanced cell migration, a key step in the metastatic cascade. Inhibition of this pathway with the specific mTOR inhibitor, rapamycin, markedly decreased cell migration induced by formation of TFFVIIa-FXa complex. These studies suggest thatTFFVIIa-mediated signaling modulates mTOR pathway activation, which regulates in part breast cancer cell migration. Targeting the TF-mediated cell signaling pathway might represent a novel strategy for the treatment of breast cancer.
- Published
- 2008
50. An algal sulfated galactan has an unusual dual effect on venous thrombosis due to activation of factor XII and inhibition of the coagulation proteases
- Author
-
Paulo A.S. Mourão and Fábio R. Melo
- Subjects
Male ,Bleeding Time ,Hemorrhage ,Factor XIIa ,Pharmacology ,Galactans ,Antithrombins ,Thrombin ,Fibrinolytic Agents ,medicine ,Animals ,Humans ,Protease Inhibitors ,Rats, Wistar ,Blood Coagulation ,Venous Thrombosis ,Heparin cofactor II ,Factor XII ,Dose-Response Relationship, Drug ,Heparin ,Sulfates ,Chemistry ,Antithrombin ,Anticoagulants ,Thrombosis ,Hematology ,Factor XII activation ,medicine.disease ,Rats ,Molecular Weight ,Disease Models, Animal ,Venous thrombosis ,Coagulation ,Biochemistry ,Factor Xa ,Rhodophyta ,Heparin Cofactor II ,Female ,Blood Coagulation Tests ,Factor Xa Inhibitors ,medicine.drug - Abstract
SummarySulfated galactan from the red alga Botryocladia occidentalis has a potent anticoagulant activity,due to its ability to enhance thrombin and factor Xa inhibition by antithrombin and/or heparin cofactor II. It is less active than unfractionated heparin in arterial thrombosis,but in a venous thrombosis presents a dual effect,inhibiting thrombosis in low but not in high doses.This dual effect on venous thrombosis is a consequence of two actions, one that inhibits thrombin and factor Xa and one that induces factor XII activation. In order to dissociate these effects, we prepared derivatives of the sulfated galactan with low molecular weights. Two fractions that were similar in size to unfractionated heparin and low-molecular-weight heparin were obtained. As the molec- ular weight decreased, the ability to activate factor XII and to promote inhibition of coagulation proteases in the presence of antithrombin and heparin cofactor II diminished.At ∼5 kDa, the sulfated galactan fragment had no effect on factor XII activation, and showed the same effect as unfractionated heparin in a venous thrombosis model.The ∼5-kDa fragment is an antithrombotic with several advantages: i) It is as active as unfractionated heparin in venous thrombosis, but it has little activity in arterial thrombosis; ii) It inhibits venous thrombosis with very little anticoagulant effect; iii) It does not cause bleeding; and iv) It is not obtained from mammals.
- Published
- 2008
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