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Background Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and high levels may have an association with thrombosis. The aim of the current study was to investigate the association of TAFI antigen levels with pulmonary thromboembolism (PTE). Patients and Methods A case–control study was conducted with 29 patients with PTE and 17 age- and gender-matched control individuals. Plasma levels of TAFI were measured at the time of diagnosis, then at 3 and 6 months after the event. Results Initial TAFI levels (%) were higher in patients with PTE than in the control group (190,0 [65,0–250,0] vs 133,0 [83,0–153,0]; p = 0.003). TAFI levels significantly decreased at the third and sixth months after initial diagnosis (p 0.05). In the sixth month of treatment, patients with residual thrombosis were seen to have similar baseline levels and reductions of TAFI as patients without residual thrombosis (p > 0.05). Conclusion The result of this study suggests that high TAFI levels may have a role in the occurrence of PTE without impact on treatment outcome.

A method for determining the activity of РAI-1 is proposed, which allows determining the level of this inhibitor quickly and with high accuracy and can be used as a tool for diagnosing the development of cardiovascular diseases. Предложен способ определения активности РАІ-1, позволяющий быстро и с высокой точностью определить уровень этого ингибитора и применимый как инструмент диагностирования развития сердечно-сосудистых заболеваний. Запропонований спосiб визначення активностi РАI-1, який дозволяє швидко i з високою точнiстю визначити рiвень цього iнгiбiтора i може бути застосований як iнструмент дiагностування розвитку серцево-судинних захворювань.

BACKGROUND Hemorrhage is the leading cause of preventable trauma-related mortality and is frequently aggravated by acute traumatic coagulopathy (ATC). Viscoelastic tests such as rotational thromboelastometry (ROTEM) may improve identification and management of ATC. This study aimed to prospectively evaluate changes in ROTEM among combat casualties during the first 24 hours and compare the capabilities of our conventional clotting assay (international normalized ratio [INR], >1.2) to a proposed integrated ROTEM model (INR >1.2 with the addition of tissue factor pathway activation thromboelastometry [EXTEM] A5 ≤35 mm and/or EXTEM LI30

Adverse thromboembolic events following administration of the anti-fibrinolytic agent tranexamic acid (TA), used to prevent/treat excessive blood loss, are rare. We present the clinical course of two young patients (22 and 56 months) receiving busulfan/melphalan (Bu/Mel) high-dose chemotherapy with autologous hematopoietic stem cell transplantation (HSCT) to treat high-risk neuroblastoma, who developed hepatic sinusoidal obstruction syndrome (SOS) within 48 h after systemic TA treatment for a hemodynamically relevant hemorrhage. Defibrotide treatment resolved hepatic SOS, but the short time between TA administration and SOS onset suggests a causal association.

Measurement of Thrombin-activatable fibrinolysis inhibitor (TAFI) in human plasma is dependent on reproducible assays. To date, standards for measuring TAFI are frequently calibrated relative to pooled normal human plasma and arbitrarily assigned a potency of 100% TAFI, despite variation in TAFI concentrations between plasma pools. Alternatively, TAFI calibrators can be assigned a value in SI units but the approach used for value assignment is not consistent and furthermore, if purified TAFI is used to determine TAFI concentration in plasma, may be adversely affected by matrix effects. A TAFI plasma standard in mass units with traceability to the SI unit of mass is desirable. We report here the establishment of a quantitative mass spectrometry method for TAFI in plasma. Traceability is obtained by reference to calibrators that consist of blank plasma spiked with a defined amount of purified TAFI, value assigned by amino acid analysis. The calibrators are run alongside the samples, using the same preparation steps and conditions; an acetonitrile assisted tryptic digestion and multi-dimensional liquid chromatography (LC) separation followed by SRM-MS analysis. We measured the TAFI quantitatively in human plasma with reproducibility, reliability and precision, and demonstrated the applicability of this approach for value assigning a common reference standard.

BACKGROUND Because TAFI (thrombin-activatable fibrinolysis inhibitor) antigen varies widely among different populations, we performed this case-control study to explore the relationship between TAFI levels and stroke in a Chinese population. MATERIAL AND METHODS Our population-based case-control study included 217 stroke patients and 218 healthy controls. The plasma TAFI level was measured by immune turbidimetry. Univariate and multivariate logistic regression analyses were used to analyze the association between different TAFI levels and stroke and its subtypes. Restricted cubic spline (RCS) combined with logistic regression analysis were used to explore the dose-response relationship between TAFI levels and stroke. RESULTS The plasma TAFI levels of cases were much higher than in the control group (p=0.038) and this difference persisted even after adjustment (OR=2.2). In the elderly (aged over 60) and female subgroups, TAFI levels in stroke patients were higher than those in controls, and the results were also noted in ischemic stroke. The dose-response curve showed that, as a whole, with the increase of TAFI levels, the relative risk of stroke first increased and then decreased (p=0.0127). Similarly, in general, with the increase of TAFI levels, the curve showed that the relative risk of ischemic stroke first increased and then decreased (p=0.0110). CONCLUSIONS There was a definite correlation between TAFI levels and stroke in this Chinese population, and with the increase of TAFI levels, the relative risk of stroke or ischemic stroke first increased and then decreased.

The purpose of this study was to identify thrombin-activatable fibrinolysis inhibitor (TAFI) in saliva and to investigate the correlation between TAFI levels in saliva and plasma. Subjects included were healthy adults without diseases or medication that could affect coagulation. Samples of stimulated saliva and blood samples were obtained from 33 subjects. Levels of TAFI in saliva and plasma were analysed. The association between levels of TAFI in saliva and plasma was calculated using linear regression. Low levels of TAFIa/TAFIai were found in most saliva samples but only one sample had levels that were above the lower limit of detection of the assay used. TAFI (proenzyme) was not found in saliva, so no correlations could be calculated. In this study there was no indication that there is TAFI present in secreted saliva. Either TAFIa/TAFIai in saliva were much lower than in plasma and under the detection limit of the assay used, or there was no TAFIa/TAFIai in the saliva tested.

A large clot formed between the inside of the bony wall of the extraction socket and the immediate implant surface may have premature breakdown . Tranexamic acid (TXA) is fibrinolysis inhibitor and an analog of the amino acid lysine. In this paper the influence of tranexamic acid on the osseointegration of Ti-30Ta implants without primary stability was investigated. Were fabricated 32 implants of CP Titanium Grade 4 and 32 implants of Ti-30Ta alloy with dimensions of 2.1 x 2.8mm Ø. Bone defects of 2.5x3.2 mm Ø were created in right tibia of 64 Wistar male-rats using a small round bur. They were divided (n=16) into: Group I (CP-Ti machined implant), Group II (CP-Ti machined implant/ tranexamic acid), Group III (Ti-30Ta implant) and Group IV (Ti-30Ta implant/ tranexamic acid). The surgical defects of the Group II and Group IV were bathed with 20 ml of the tranexamic acid solution. The animals were euthanized at 45 days postoperative. In the right tibia of half each group (8 animals/8 tibiae) the maximum torque value necessary for manual removal of each implant was measured in Newton centimeters (Ncm). The right tibia of other half each group was subjected to non-decalcified histology processing (Stevenel's blue/Alizarin red). Data were analyzed statistically (Kruskal-Wallis Analyses) and demonstrated significant differences (P

Objective— The pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis. It remains to be elucidated whether TAFI is directly involved in the pathogenesis of CTEPH. We examined potential involvement of TAFI in the pathogenesis of CTEPH in humans. Approach and Results— We enrolled 68 consecutive patients undergoing right heart catheterization in our hospital, including those with CTEPH (n=27), those with pulmonary arterial hypertension (n=22), and controls (non–pulmonary hypertension, n=19). Whole blood clot lysis assay showed that the extent of clot remaining after 4 hours was significantly higher in CTEPH compared with pulmonary arterial hypertension or controls (41.9 versus 26.5 and 24.6%, both P P 1 (an inhibitor of activation of platelets). Importantly, plasma levels of TAFI were significantly correlated with the extent of clot remaining after 4 hours. In addition, the extent of clot remaining after 4 hours was improved with an activated TAFI inhibitor. Conclusions— These results indicate that plasma levels of TAFI are elevated in patients with CTEPH and are correlated with resistance to clot lysis in those patients.

In this issue of Blood, Wyseure et al identified that defective activation of thrombin-activatable fibrinolysis inhibitor (TAFI) drives joint bleeding in congenital but not acquired hemophilia A.(1)

The data in this paper is related to the research article entitled “Thrombin-activatable fibrinolysis inhibitor Thr325Ile polymorphism and plasma level in breast cancer: A pilot study” (Fawzy et al., 2015) [1]. Many emerging studies have begun to unravel the pathophysiologic role of the fibrinolytic system in breast cancer (BC) progression (Zorio et al., 2008) [2]. Activation of the fibrinolytic plasminogen/plasmin system results in degradation of protein barriers, thereby mediating cell migration essential for tumor growth, angiogenesis, and dissemination (Castellino and Ploplis, 2005) [3]. In the current study, in silico data analysis of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) gene and protein has been done. Data have been retrieved from several databases mentioned in details in the text. Determination and analysis of the structural and functional impact of TAFI and its expression could help elucidate the contribution of the TAFI pathway to acquired hemostatic dysfunction and will form the basis of potential therapeutic strategies to manipulate this pathway. An inhibition of TAFI (e.g. by FXI inhibitors) will offer the therapeutic possibilities to improve the decreased fibrinolysis and increase the efficiency of fibrinolytic therapy in thrombotic disorders including cancer.

OBJECTIVE: Level of injury (LOI) and the role of adipose tissue and its proinflammatory adipokines in cardiometabolic dysfunction following spinal cord injury (SCI) remains poorly understood. We aim to examine the influence of LOI on adipose tissue and its relationship to proinflammatory adipokines and cardiometabolic profiles following SCI. DESIGN: Cross sectional and correlational study. SETTING: Clinical hospital and academic setting. PARTICIPANTS: Forty-seven individuals with chronic motor complete SCI (age 43.8±11.5 y, BMI: 27.3±5.3) were classified as having tetraplegia (TSCI; n=12) or paraplegia (PSCI; n=35). INTERVENTION: Non applicable. OUTCOME MEASURES: Visceral (VAT) and subcutaneous (SAT) adipose tissue volumes were measured using magnetic resonance imaging. Proinflammatory adipokines (tumor neurosis factor-α, interleukin-6 (IL-6), plasminogen activatable inhibitor-1, thrombin-activatable fibrinolysis inhibitor, and high-sensitivity c-reactive protein) and cardiovascular, carbohydrate, and lipid profiles were assessed according to standard techniques. RESULTS: VAT volume was greater in TSCI versus PSCI (p=0.042); however, after covarying for age this significance was lost (p>0.05). IL-6 was significantly elevated in TSCI (p0.05). Systolic blood pressure and total cholesterol were significantly lower in TSCI (p

The thrombin-thrombomodulin (TM) complex activates thrombin-activable fibrinolysis inhibitor (TAFI) more efficiently than thrombin or plasmin alone. The exosite on TAFI required for its TM-dependent activation by thrombin has not been identified. Based on previous work by us and others, we generated TAFI variants with one or more of residues Lys 42, Lys 43, Lys 44 and Arg 12 within the activation peptide mutated to alanine. Mutation of one, two, or three Lys residues or the Arg residue alone decreased the catalytic efficiency of TAFI activation by thrombin-TM by 2.4-, 3.2-, 4.7-, and 15.0-fold, respectively, and increased the TAFI concentrations required for half-maximal prolongation of clot lysis times (K 1/2 ) by 3-, 4,- 15-, and 24-fold, respectively. Mutation of all four residues eliminated TM binding, decreased the catalytic efficiency of TAFI activation by 45.0-fold, increased the K 1/2 by 130-fold, and abolished antifibrinolytic activity in a clot lysis assay. When thrombin or plasmin was used as the activator, mutation of all four residues reduced the rate of activation by 1.1- and 4.0-fold compared with wild-type TAFI, respectively, suggesting that the mutation only impacted activation kinetics by thrombin-TM. Surface plasmon resonance data show that mutation of the four residues results in complete loss of binding, either in the presence or absence of thrombin. Together, our findings suggest that these four residues are critical for the interaction of TAFI with the thrombin-TM complex that modulates its antifibrinolytic activity.

AIM to investigate parameters of fibrinolysis in patients on long-term warfarin (W) therapy, and assess their relation to the risk of recurrent bleeding occurring at therapeutic international normalized ratio (INR). MATERIALS AND METHODS Our prospective study involved 78 W-naive patients (40 men, age 64.3+/-12.2 years). Follow up period was 5.6+/-3.4 months. INR was measured monthly; determination of coagulation parameters (D-dimer, fibrinogen, complex plasmin-2-antiplasmin [PAP] and thrombin-activatable fibrinolysis inhibitor [TAFI] was performed before and after at least 3 months of W therapy. RESULTS During follow-up bleedings occurred in 47 (60.3%) patients, 26 patients (33.3%) had recurrent bleedings at therapeutic INR and 21 patients (26.9%) had single bleeding. Mean time in therapeutic range (TTR) was >70.

Stroke is the second most common cause of death worldwide. Despite many advances in the field of vascular neurology, intravenous (IV) tissue plasminogen activator (tPA) remains the only approved treatment for acute ischemic stroke, even though complication risk such as hypofibrinolysis and reocclusion, are reported in as high as 25-34% of cases. The exact mechanisms of unsuccessful thrombolytic therapy are unclear. Some molecular events such as activation of intrinsic and extrinsic pathways C - reactive protein (CRP), thrombin-activatable fibrinolysis inhibitor (TAFI), plasminogen activator inhibitor-1 (PAI-1) are the main causes of failure. Moreover, release of thrombin after tPA therapy, tissue factor pathway inhibitor (TFPI) inhibition, and some of gene polymorphisms may play a crucial role in reocclusion. Due to the necessity for development of effective strategies to improve clinical efficacy of tPA therapy, we aimed to evaluate the possible mechanisms, which may be responsible for re-occlusive complications after tPA therapy.

A practical and chromatography-free multikilogram synthesis of a 3-isoxazolol containing antifibrinolytic agent, AZD6564, has been developed in eight steps and 7% overall yield starting from methyl 2-chloroisonicotinate. Highlights in the synthesis are a Negishi coupling and an enzymatic resolution of a racemic ester.

SummaryPatients with haemophilia A have seriously impaired thrombin generation due to an inherited deficiency of factor (F)VIII, making them form unstable fibrin clots that are unable to maintain haemostasis. Data on fibrin structure in haemophilia patients remain limited. Fibrin permeability, assessed by a flow measurement technique, was investigated in plasma from 20 patients with severe haemophilia A treated on demand, before and 30 minutes after FVIII injection. The results were correlated with concentrations of fibrinogen, FVIII and thrombin-activatable fibrinolysis inhibitor (TAFI), and global haemostatic markers: endogenous thrombin potential (ETP) and overall haemostatic potential (OHP). Fibrin structure was visualised using scanning electron microscopy (SEM). The permeability coefficient Ks decreased significantly after FVIII treatment. Ks correlated significantly with FVIII levels and dosage, and with ETP, OHP and levels of TAFI. SEM images revealed irregular, porous fibrin clots composed of thick and short fibers before FVIII treatment. The clots had recovered after FVIII replacement almost to levels in control samples, revealing compact fibrin with smaller intrinsic pores. To the best of our knowledge, this is the first description of fibrin porosity and structure before and after FVIII treatment of selected haemophilia patients. It seems that thrombin generation is the main determinant of fibrin structure in haemophilic plasma.

UNLABELLED Essentials Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for cardiovascular disorders. TAFI inhibitory nanobodies represent a promising step in developing profibrinolytic therapeutics. We have solved three crystal structures of TAFI in complex with inhibitory nanobodies. Nanobodies inhibit TAFI through distinct mechanisms and represent novel profibrinolytic leads. SUMMARY Background Thrombin-activatable fibrinolysis inhibitor (TAFI) is converted to activated TAFI (TAFIa) by thrombin, plasmin, or the thrombin-thrombomodulin complex (T/TM). TAFIa is antifibrinolytic, and high levels of TAFIa are associated with an increased risk for cardiovascular disorders. TAFI-inhibitory nanobodies represent a promising approach for developing profibrinolytic therapeutics. Objective To elucidate the molecular mechanisms of inhibition of TAFI activation and TAFIa activity by nanobodies with the use of X-ray crystallography and biochemical characterization. Methods and results We selected two nanobodies for cocrystallization with TAFI. VHH-a204 interferes with all TAFI activation modes, whereas VHH-i83 interferes with T/TM-mediated activation and also inhibits TAFIa activity. The 3.05-A-resolution crystal structure of TAFI-VHH-a204 reveals that the VHH-a204 epitope is localized to the catalytic moiety (CM) in close proximity to the TAFI activation site at Arg92, indicating that VHH-a204 inhibits TAFI activation by steric hindrance. The 2.85-A-resolution crystal structure of TAFI-VHH-i83 reveals that the VHH-i83 epitope is located close to the presumptive thrombomodulin-binding site in the activation peptide (AP). The structure and supporting biochemical assays suggest that VHH-i83 inhibits TAFIa by bridging the AP to the CM following TAFI activation. In addition, the 3.00-A-resolution crystal structure of the triple TAFI-VHH-a204-VHH-i83 complex demonstrates that the two nanobodies can simultaneously bind to TAFI. Conclusions This study provides detailed insights into the molecular mechanisms of TAFI inhibition, and reveals a novel mode of TAFIa inhibition. VHH-a204 and VHH-i83 merit further evaluation as potential profibrinolytic therapeutics.

Introduction: DS-1040 is a novel inhibitor of the activated thrombin-activatable fibrinolysis inhibitor (TAFIa) and is in clinical development for the treatment of acute ischemic stroke (AIS). The objective of this study was to investigate the effect of DS-1040 on cerebral blood flow (CBF) in a rat thromboembolic stroke model. Methods: The CBF was transcranially and continuously monitored during the experiment by laser Doppler flowmetry system using a probe fixed to the skull above the territory of the right middle cerebral artery (MCA) of an anesthetized male SHR/Izm rat. After recording basal CBF, non-autologous whole blood clot was injected into the internal carotid artery. We conducted a 3-arm study. Saline (control) and DS-1040 (3.0 mg/kg) were intravenously injected as a bolus 5 min after the clot injection. The dosage of DS-1040 was set to achieve full inhibition of TAFIa during the experiment. Recombinant tissue plasminogen activator (rt-PA, 7.0 mg/kg; positive control) was intravenously injected 5 min after the clot injection by 10% bolus and 90% infusion for 60 min. The CBF(%) was defined as percent of the CBF at each time point to the mean basal CBF and the area under the curve of the CBF(%) after the drug injection, AUC 5-110min , was calculated. Results: The CBF(%) decreased after the clot injection, indicating the clot embolized the MCA. A significant increase of the AUC 5-110min was observed in the rt-PA group (8416.2 %·min ± 642.4 %·min, mean ± SE) compared to the control group (3728.7 %·min ± 684.2 %·min), indicating the model is sensitive to tPA. DS-1040 also significantly increased the AUC 5-110min (6645.3 %·min ± 861.9 %·min) compared to the control group. Conclusions: DS-1040 restored CBF in a rat thromboembolic stroke model suggesting DS-1040 is expected to be beneficial for the treatment of patients with acute ischemic stroke. To the authors’ knowledge, this is the first report of an inhibitor of TAFIa improving the CBF in a thromboembolic stroke model.

Tanoglu, Alpaslan/0000-0002-7477-6640 WOS: 000380801100002 PubMed: 27432089 Objective Patients with ulcerative colitis (UC) are at an increased risk for thromboembolic events, particularly in patients with extensive and active disease. To date, a few studies have been published on the role of thrombin-activatable fibrinolysis inhibitor (TAFI) in UC. However, there are no reports in the literature investigating the effect of UC treatment on plasma TAFI levels. Methods The plasma TAFI antigen levels were quantitatively determined using ELISA kits for 20 UC patients at activation and remission, along with 17 healthy controls. The association between the TAFI levels and inflammatory markers was assessed to determine UC activation. To predict and determine the activation of UC, the Truelove-Witts index and the endoscopic activation index (EAI) were used for each subject. Results The plasma TAFI levels were higher in UC patients at activation of the disease compared with the remission state and in healthy controls. Spearman's correlation analyses revealed that the WBC (r: 0.586, p < 0.001), hsCRP (r: 0.593, p < 0.001) and EAI (r: 0.721, p < 0.001) were significantly correlated with the TAFI levels. The overall accuracy of TAFI in determining UC activation was 82.5% with a sensitivity, specificity, NPV and PPV of 80%, 85%, 81% and 84.2%, respectively (cut-off value: 156.2% and AUC: 0.879). Conclusion The present study demonstrates that the TAFI levels are elevated in the active state of UC. The assessment of TAFI levels in patients with UC in conjunction with other markers of inflammation may provide additional information for estimating UC activation and severity.

Hypertension is associated with fibrinolysis abnormality. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a novel molecule-linking coagulation and fibrinolysis. The aim of this study was to investigate the levels of TAFI in primary hypertensive patients and to compare the effects of amlodipine and ramipril on TAFI levels. The study was performed with 58 hypertensive subjects and 27 healthy volunteers. Biochemical and hematological parameters and TAFI levels were measured at baseline and after 1-month follow-up. TAFI concentrations increased in hypertensive patients compared with the controls (P = .030). Additionally, TAFI levels decreased with blood pressure control at 1-month follow-up (P = .026). There was no significant difference between TAFI levels in the amlodipine and ramipril groups at baseline. However, after 1-month follow-up, TAFI levels were decreased in the amlodipine group (P = .037) but not in the ramipril group. Our study is the first in the literature to determine increased TAFI levels in primary hypertension patients. In addition, we determined a decrease in TAFI levels in the amlodipine group after 1 month, but none in the ramipril group.

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples. ispartof: PLOS ONE vol:13 issue:5 ispartof: location:United States status: published

Thrombin-activatable fibrinolysis inhibitor (TAFI) provides an important molecular link between the coagulation and fibrinolytic systems. In this review, recent major advances in TAFI research, including the elucidation of crystal structures, the development of small inhibitors and the role of TAFI in systems other than hemostasis, are described and discussed.

Fibrinolysis is regulated by specific molecular interactions between its main components. Activation of plasminogen by tissue-type plasminogen activator (t-PA) is enhanced in the presence of fibrin or at the endothelial cell surface. Urokinase-type plasminogen activator (u-PA) binds to a specific cellular u-PA receptor (u-PAR), resulting in enhanced activation of cell-bound plasminogen. Inhibition of fibrinolysis occurs at the level of plasminogen activation or at the level of plasmin. Assembly of fibrinolytic components at the surface of fibrin results in fibrin degradation. Assembly at the surface of cells provides a mechanism for generation of localized cell-associated proteolytic activity. This review includes novel proteins such a thrombin-activatable fibrinolysis inhibitor (TAFI) and discusses new insights into molecular mechanisms obtained from the rapidly growing knowledge of crystal structures of proteins.

karadeniz, muammer/0000-0002-3345-5437 WOS: 000255035700001 PubMed: 18350479 Objective: We aimed to assess circulating thrombin activatable fibrinolysis inhibitor (TAFI) levels and carotid intima-media thickness (CIMT) in PCOS patients and control sujects. in this study we aimed to evaluate the relation between the levels of TAFI and homocysteine, high sensitive CRP (hsCRP), fibrinogen and CIMT in PCOS patients carrying a potential risk for developing CVD and diabetes and compared with age- and body mass index-matched controls. Research Design and Methods: We studied 68 PCOS patients and 26 healthy controls. We conducted an observational study examining non-invasive markers of early CV disease in women with PCOS including structural CIMT. Noninvasive markers of early CVD, CIMT were measured in PCOS patients and control subjects. Metabolic parameters included fasting insulin and glucose levels, lipid and androgen levels, TAFI levels, hsCRP. Results: Fasting glucose levels, prolactin, TSH, Total-cholesterol, LDL-cholesterol, triglyceride, estradiol, DHEA-S and age were similar in the two groups, whereas serum insulin, fibrinogen, hs-CRP, 17-OHP, free-testosterone, total testosterone, HCMA-IR, HDL were significantly elevated in PCOS patients in comparison to control subjects (p < 0.05). Plasma TAFI levels were similarly in PCOS patients compared with healthy controls. No difference was observed in the combined IMT among the studied groups. Conclusions: In our study, no significant difference in lipid parameters was determined between patients with PCOS and healthy controls. in our study, we did not observed any difference in CIMT measurements and TAFI levels between patients with PCOS and healthy controls that can be explained by their low ages and short duration of PCOS.

Aims and Objectives: The pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) remains to be elucidated. Since thrombin-activated fibrinolysis inhibitor (TAFI) inhibits fibrinolysis, we examined its potential involvement in the pathogenesis of CTEPH in mice and humans. Methods and Results: We enrolled consecutive 73 patients undergoing right heart catheterization, including those with CTEPH (n=29), those with pulmonary arterial hypertension (PAH, n=25) and controls (non-PH, n=19). Whole blood clot-lysis assay showed that thrombus reduction rate was significantly reduced in CTEPH patients compared with PAH patients or controls (58.1 vs. 73.5 or 75.1%, P 1 (an inhibitor of activation of platelets). Furthermore, plasma levels of TAFI were significantly higher in CTEPH patients than in PAH patients or controls (both P Conclusion: These results indicate that TAFI plays a crucial role in the pathogenesis of CTEPH.

Thrombin-activable fibrinolysis inhibitor (TAFI) is distinct from pancreatic procarboxypeptidase B in several ways. The enzymatic activity of TAFIa is unstable and decays with a half-life of a few minutes. During this study, we observed that (i) the isoelectric point (pI) of TAFI shifts dramatically from pH 5 toward pH 8 upon activation and (ii) TAFIa is significantly less soluble than TAFI. The structural bases for these observations were investigated by characterizing all post-translational modifications, including attached glycans and disulfide connectivity. The analyses revealed that all five potential N-glycosylation sites were utilized including Asn22, Asn51, Asn63, Asn86 (located in the activation peptide), and Asn219 (located in the catalytic domain). Asn219 was also found in an unglycosylated variant. Four of the glycans, Asn51, Asn63, Asn86, and Asn219 displayed microheterogeneity, while the glycan attached to Asn22 appeared to be homogeneous. In addition, bisecting GlcNAc attached to the trimannose core was detected, suggesting an origin other than the liver. Monosaccharide composition and LC-MS/MS analyses did not produce evidence for O glycosylation. TAFI contains eight cysteine residues, of which two, Cys69 and Cys383, are not involved in disulfides and contain free sulfhydryl groups. The remaining six cystines form disulfides, including Cys156-Cys169, Cys228-Cys252, and Cys243-Cys257. This pattern is homologous to pancreatic procarboxypeptidase B, and it is therefore unlikely that permutations in the cysteine connectivity are responsible for the enzymatic instability. LC-MS/MS analyses covering more than 90% of the TAFI amino acid sequence revealed no additional modifications. When these results are taken together, they suggest that the inherent instability of TAFIa is not caused by post-translational modifications. However, after activation, TAFIa loses 80% of the attached glycans, generating a large shift in pI and a propensity to precipitate. These changes are likely to significantly affect the properties of TAFIa as compared to TAFI.

Background and Purpose Thrombolytics-induced recanalization fails in a significant portion of patients with ischemic stroke, which is partly due to the resistance of clots to lysis by thrombolytic agents. The pretreatment level of endogenous fibrinolysis inhibitors may affect such thrombolysis failure. Methods We studied 43 stroke patients whose arterial recanalization had been evaluated by angiography, and whose blood had been obtained prior to the administration of thrombolytic agents. Plasma samples from 34 healthy volunteers were used as normal controls. Plasminogen activator inhibitor type 1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) levels were quantified using an enzyme-linked immunosorbent assay. Results Arteries were recanalized [Thrombolysis in Myocardial Infarction (TIMI) grade 2 or 3] in 30 patients, but not (TIMI grade 0 or 1) in the other 13. The plasma PAI-1 level was significantly higher in patients without recanalization (nonrecanalization) than in those with recanalization and in normal controls. The TAFI levels did not differ among the groups. Conclusions The pretreatment PAI-1 levels are increased in acute stroke patients with thrombolysis failure.

OBJECTIVES: Hypofibrinolysis has been proposed as a possible mechanism underlying the known risk of thrombosis observed in patients with inflammatory bowel diseases (IBD). Thrombin-activatable fibrinolysis inhibitor (TAFI) is a recently described inhibitor of fibrinolysis. Increased TAR plasma levels are associated with a risk for venous thrombosis. The objective was to evaluate TAR plasma levels and their possible correlations with clinical features and acute-phase reactants in IBD patients. METHODS: Eighty-one IBD patients (47 Crohn's disease and 34 ulcerative colitis) and 81 sex- and age-matched healthy controls were enrolled in the study; moreover, we studied 30 inflammatory controls (13 Reiter's syndrome, 4 Behget's syndrome, and 13 patients with newly diagnosed celiac disease). TAR plasma levels were assessed by means of a commercially available ELISA kit. Erythrocytes sedimentation rate, C-reactive protein, and alpha1-acid glycoprotein were measured as acute-phase reactants. Statistical analysis was performed by means of nonparametric tests and Fisher's exact test and chi(2) test for independence. RESULTS: Median TAR plasma levels were significantly higher in IBD patients (116.0%, range: 39.0-232.0%) and in inflammatory controls (176.0%, 50.0-435.0%) than in healthy controls (99.0%, 40.0-170.0%) (p < 0.05 and p < 0.001, respectively). TAR plasma levels higher than the 95th percentile of control values were significantly more frequent in IBD patients (19.7%) and in inflammatory controls (53.3%) than in healthy controls (4.9%) (p < 0.008 and p < 0.0001, respectively) and more frequent in clinically active IBD than in clinically quiescent IBD (31.4% vs 10.9%, p < 0.03). Finally, in IBD, significant correlations were observed between TAR plasma levels and erythrocytes sedimentation rate (p < 0.02), C-reactive protein (p < 0.001), and a1-acid glycoprotein (p < 0.05). CONCLUSIONS: TAR plasma levels are increased in IBD patients and correlate with acute-phase reactants. Increased TAR plasma levels might contribute to the prothrombotic state observed in IBD through the induction of hypofibrinolysis.

We report here, with respect to collagen production and the mechanical properties of a fibrin-based media equivalent (ME), on our efforts to optimize the culture conditions of neonatal SMCs entrapped in tubular fibrin gels. We examined several factors, including the concentration of fibrinolysis inhibitor, the cell source and initial number, the addition of TGF-beta and insulin to the culture medium, and the time in culture. We found that varying the concentration of epsilon-aminocaproic acid (ACA), an inhibitor of fibrinolysis, did not affect the collagen production, but that lower concentrations resulted in a compromised physical integrity of the ME. While use of neonatal SMCs yielded superior results over adult SMCs, a higher initial cell number did not improve results. The addition of 1 ng/mL of TGF-beta to the medium increased the collagen content fourfold and the ultimate tensile strength (UTS) and modulus approximately tenfold after 3 weeks, while the addition of both TGF-beta and insulin improved collagen content sixfold and UTS and modulus almost 20-fold. Additional TGF-beta (5 ng/mL) did not improve any of the properties measured, but additional time in culture did. Samples incubated for 6 weeks with TGF-beta and insulin contained about seven times the amount of collagen and had a three-times higher UTS and modulus than did samples incubated for only 3 weeks. When compared to collagen MEs, the fibrin MEs compacted to a greater extent and were both stronger and stiffer when cultured under the same conditions, having after 6 weeks a tensile modulus and ultimate tensile strength similar to those of rat abdominal aorta.

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates the fibrin cofactor function of tissue-type plasminogen activator-mediated plasmin formation and subsequently fibrin degradation. In the present study, we focused on the role of plasmin in the regulation of TAFIa activity. Upon incubation with plasmin, TAFIa activity was generated, which was unstable at 37 degrees C. Analysis of the cleavage pattern showed that TAFI was cleaved at Arg(92), releasing the activation peptide from the 35.8-kDa catalytic domain. The presence of the 35.8-kDa fragment paralleled the time course of generation and loss of TAFIa activity. This suggested that, in the presence of plasmin, TAFIa is probably inactivated by proteolysis rather than by conformational instability. TAFI was also cleaved at Arg(302), Lys(327), and Arg(330), resulting in a approximately 44.3-kDa fragment and several smaller fragments. The 44.3-kDa fragment is no longer activatable since it lacks part of the catalytic center. We concluded that plasmin can cleave at several sites in TAFI and that this contributes to the regulation of TAFI and TAFIa.

Globally the leading cause of long-term disability and mortality stems from cardiovascular diseases, which creates an enormous economic burden. Currently available treatments for intravascular thrombosis consist of a large repertoire of antithrombotic agents targeting coagulation and platelet function. However, the only agents available to enhance fibrinolysis are recombinant or modified forms of plasminogen activators. Their clinical use is limited by low efficacy, life-threatening side-effects (primarily caused by the high systemic dose required) and the inapplicability for prophylactic use. This review provides an update on the latest advances in targeting the antifibrinolytic proteins, plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor, and will highlight novel therapeutic avenues to enhance fibrinolysis.

BACKGROUND: Downregulation of fibrinolysis due to cleavage of C-terminal lysine residues from partially degraded fibrin is mainly exerted by the carboxypeptidase activity of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recently, some intrinsic carboxypeptidase activity (= zymogen activity) was reported for the proenzyme (TAFI), however, there is some discussion about its ability to cleave high molecular weight substrates. OBJECTIVE: We aimed to identify and characterize nanobodies towards mouse TAFI (mTAFI) that stimulate the zymogen activity and to test their effect in an in vitro clot lysis assay and an in vivo mouse thromboembolism model. METHODS AND RESULTS: Screening of a library of nanobodies towards mTAFI revealed one nanobody (VHH-mTAFI-i49) that significantly stimulates the zymogen activity of mTAFI from undetectable (

Thrombomodulin is a cofactor protein on vascular endothelial cells that inhibits the procoagulant functions of thrombin and enhances thrombin-catalyzed activation of anticoagulant protein C. Thrombomodulin also accelerates the proteolytic activation of a plasma procarboxypeptidase referred to as thrombin-activable fibrinolysis inhibitor (TAFI). In this study, we describe structures on recombinant membrane-bound thrombomodulin that are required for human TAFI activation. Deletion of the N-terminal lectin-like domain and epidermal growth factor (EGF)-like domains 1 and 2 had no effect on TAFI or protein C activation, whereas deletions including EGF-like domain 3 selectively abolished thrombomodulin cofactor activity for TAFI activation. Provided that thrombomodulin EGF-like domain 3 was present, TAFI competitively inhibited protein C activation catalyzed by the thrombin-thrombomodulin complex. A thrombomodulin construct lacking EGF-like domain 3 functioned normally as a cofactor for protein C activation but was insensitive to inhibition by TAFI. Thus, the anticoagulant and antifibrinolytic cofactor activities of thrombomodulin have distinct structural requirements: protein C binding to the thrombin-thrombomodulin complex requires EGF-like domain 4, whereas TAFI binding also requires EGF-like domain 3.

OBJECTIVES Activated thrombin-activatable fibrinolysis inhibitor is a coagulation factor in some thrombotic diseases. However, available data on whether thrombin-activatable fibrinolysis inhibitor is activated in islet transplant are limited. In this study, changes of plasma-activated thrombin-activatable fibrinolysis inhibitor levels in instant blood-mediated inflammatory reaction after islet transplant were assessed. MATERIALS AND METHODS Plasma concentrations of thrombin-antithrombin complex, D-dimer, C-peptide, and activated thrombin-activatable fibrinolysis inhibitor were assessed at 0 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, and 24 hours after an intraportal islet transplant using rats via an enzyme-linked immunosorbent assay, or solid-phase, 2-site chemiluminescent immunometric assay. We recovered the liver at 1 hour after the transplant for histologic examination. RESULTS Thrombin-antithrombin complex, C-peptide, and activated thrombin-activatable fibrinolysis inhibitor levels increased immediately after we stopped islet infusion, and their peak levels occurred at 1 hour after islet infusion. D-dimer levels increased continually after islet infusion was stopped, and peaked 24 hours after infusion. Histologic examination of the liver 1 hour after islet infusion revealed frequent portal venous thrombi, with entrapped islets. The entrapped islets showed a disrupted morphology. CONCLUSIONS Activated thrombin-activatable fibrinolysis inhibitor was generated and peaked 1 hour after islet transplant according with activating coagulation, indicating that thrombin-activatable fibrinolysis inhibitor is activated and accumulated at levels in instant blood-mediated inflammatory reaction was sufficient to affect fibrinolysis.

Summary Background Microparticles (MPs) are small membrane vesicles (0.1–1 μm) released from various cells after activation and/or apoptosis. There are limited data about their role in hemophilia A. Patients and Methods Blood samples were taken before and 30 min after FVIII injection in 18 patients with severe hemophilia A treated on demand. Flow-cytometric determination of total MPs (TMPs) using lactadherin, platelet MPs (PMPs) (CD42a), endothelial MPs (EMPs) (CD144) and leukocyte MPs (LMPs) (CD45) was performed. The results were compared with data on endogenous thrombin potential (ETP), overall hemostatic potential (OHP), fibrin gel permeability and thrombin-activatable fibrinolysis inhibitor (TAFI). Results and Conclusions TMPs and PMPs decreased after treatment (to 1015 ± 221 [SEM] and 602 ± 134 × 106 L−1) in comparison with values before treatment (2373 ± 618 and 1316 ± 331; P

Solulin is a soluble form of thrombomodulin that is resistant to proteolysis and oxidation. It has been shown to increase the clot lysis time in factor VIII (fVIII)–deficient plasma by an activated thrombin-activatable fibrinolysis inhibitor (TAFIa)–dependent mechanism. In the present study, blood was drawn from humans and dogs with hemophilia, and thromboelastography was used to measure tissue factor–initiated fibrin formation and tissue-plasminogen activator–induced fibrinolysis. The kinetics of TAFI and protein C activation by the thrombin-Solulin complex were determined to describe the relative extent of anticoagulation and antifibrinolysis. In severe hemophilia A, clot stability increased by > 4-fold in the presence of Solulin while minimally affecting clot lysis time. Patients receiving fVIII/fIX prophylaxis showed a similar trend of increased clot stability in the presence of Solulin. The catalytic efficiencies of TAFI and protein C activation by the thrombin-Solulin complex were determined to be 1.53 and 0.02/μM/s, respectively, explaining its preference for antifibrinolysis over anticoagulation at low concentrations. Finally, hemophilic dogs given Solulin had improved clot strength in thromboelastography assays. In conclusion, the antifibrinolytic properties of Solulin are exhibited in hemophilic human (in vitro) and dog (in vivo/ex vivo) blood at low concentrations. Our findings suggest the therapeutic utility of Solulin at a range of very low doses.

See also Sanglas L, Arolas JL, Valnickova Z, Aviles FX, Enghild JJ, Gomis-Ruth FX. Insights into the molecular inactivation mechanism of human activated thrombin-activatable fibrinolysis inhibitor. This issue, pp 1056–65.