196 results on '"Mark Roest"'
Search Results
2. Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood
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Sandra Konrath, Reiner K. Mailer, Manu Beerens, Hanna Englert, Maike Frye, Piotr Kuta, Roger J. S. Preston, Coen Maas, Lynn M. Butler, Mark Roest, Bas de Laat, and Thomas Renné
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thrombin generation ,plasma contact system ,factor XII ,polyphosphate ,CAT assay ,diagnostics ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12−/−) and FXI-deficient (F11−/−) mice. Moreover, reconstitution of blood from F12−/− mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_Δ12 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development.
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- 2022
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3. Von Willebrand factor, ADAMTS13 and mortality in dialysis patients
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Gurbey Ocak, Mark Roest, Marianne C. Verhaar, Maarten B. Rookmaaker, Peter J. Blankestijn, Willem Jan W. Bos, Rob Fijnheer, Nathalie C. Péquériaux, and Friedo W. Dekker
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Dialysis ,Von Willebrand Factor ,ADAMTS13 ,Mortality ,Epidemiology ,Diseases of the genitourinary system. Urology ,RC870-923 - Abstract
Abstract Background Von Willebrand Factor (VWF) multimers are cleaved into smaller and less coagulant forms by the metalloprotease ADAMTS13. The aim of this study was to investigate the association between VWF and ADAMTS13 and mortality in dialysis patients. Methods We prospectively followed 956 dialysis patients. VWF levels and ADAMTS13 activity were measured. Cox proportional hazard analyses were used to calculate hazard ratios (HRs) with 95 % confidence intervals (CIs) to investigate the association between quartiles of VWF levels and ADAMTS13 activity and all-cause mortality. HRs were adjusted for age, sex, body mass index, cardiovascular disease, dialysis modality, primary kidney disease, use of antithrombotic medication, systolic blood pressure, albumin, C-reactive protein and residual GFR. Results Of the 956 dialysis patients, 288 dialysis patients died within three years (mortality rate 151 per 1000 person-years). The highest quartile of VWF as compared with lower levels of VWF was associated with a 1.4-fold (95 %CI 1.1–1.8) increased mortality risk after adjustment. The lowest quartile of ADAMTS13 activity as compared with other quartiles was associated with a 1.3-fold (95 %CI 1.0-1.7) increased mortality risk after adjustment. The combination of the highest VWF quartile and lowest ADAMTS13 quartile was associated with a 2.0-fold (95 %CI 1.3-3.0) increased mortality risk as compared with the combination of the lowest VWF quartile and highest ADAMTS13 quartile. Conclusions High VWF levels and low ADAMTS13 activity were associated with increased mortality risks in dialysis patients.
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- 2021
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4. Population-wide persistent hemostatic changes after vaccination with ChAdOx1-S
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Bas de Laat, Hendrik Stragier, Romy de Laat-Kremers, Marisa Ninivaggi, Dieter Mesotten, Steven Thiessen, Kristien Van Pelt, Mark Roest, Joris Penders, Pascal Vanelderen, Dana Huskens, Raf De Jongh, Margot Vander Laenen, Tom Fivez, Hugo ten Cate, Rene Heylen, Line Heylen, and Deborah Steensels
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ChAdOx1-S ,COVID-19 ,thrombin generation ,vaccination ,hemostasis ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
Various vaccines were developed to reduce the spread of the Severe Acute Respiratory Syndrome Cov-2 (SARS-CoV-2) virus. Quickly after the start of vaccination, reports emerged that anti-SARS-CoV-2 vaccines, including ChAdOx1-S, could be associated with an increased risk of thrombosis. We investigated the hemostatic changes after ChAdOx1-S vaccination in 631 health care workers. Blood samples were collected 32 days on average after the second ChAdOx1-S vaccination, to evaluate hemostatic markers such as D-dimer, fibrinogen, α2-macroglobulin, FVIII and thrombin generation. Endothelial function was assessed by measuring Von Willebrand Factor (VWF) and active VWF. IL-6 and IL-10 were measured to study the activation of the immune system. Additionally, SARS-CoV-2 anti-nucleoside and anti-spike protein antibody titers were determined. Prothrombin and fibrinogen levels were significantly reduced after vaccination (−7.5% and −16.9%, p < 0.0001). Significantly more vaccinated subjects were outside the normal range compared to controls for prothrombin (42.1% vs. 26.4%, p = 0.026) and antithrombin (23.9% vs. 3.6%, p = 0.0010). Thrombin generation indicated a more procoagulant profile, characterized by a significantly shortened lag time (−11.3%, p < 0.0001) and time-to-peak (−13.0% and p < 0.0001) and an increased peak height (32.6%, p = 0.0015) in vaccinated subjects compared to unvaccinated controls. Increased VWF (+39.5%, p < 0.0001) and active VWF levels (+24.1 %, p < 0.0001) pointed toward endothelial activation, and IL-10 levels were significantly increased (9.29 pg/mL vs. 2.43 pg/mL, p = 0.032). The persistent increase of IL-10 indicates that the immune system remains active after ChAdOx1-S vaccination. This could trigger a pathophysiological mechanism causing an increased thrombin generation profile and vascular endothelial activation, which could subsequently result in and increased risk of thrombotic events.
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- 2022
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5. Patients With Multiple Myeloma Have a Disbalanced Whole Blood Thrombin Generation Profile
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Li Li, Mark Roest, Yaqiu Sang, Jasper A. Remijn, Rob Fijnheer, Karel Smit, Dana Huskens, Jun Wan, Bas de Laat, and Joke Konings
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blood cells ,multiple myeloma ,platelet function ,thrombin generation ,thrombosis ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
BackgroundMultiple myeloma (MM) is associated with a high prevalence of bleeding and an increased risk of thrombo-embolism. MM patients have reduced platelet- and red blood cell (RBC) numbers in blood, which may indicate that the paradoxical hemostasis profile is a consequence of a disturbed platelet and RBC homeostasis.ObjectivesTo get better insight in the disbalanced hemostasis of MM patients.MethodsWe conducted a case-control study on the whole blood (WB) coagulation profiles of 21 MM patients and 21 controls. We measured thrombin generation (TG) in WB and platelet poor plasma (PPP) of MM patients and controls.ResultsIn WB-TG, we observed that the median time to the thrombin Peak was 52% longer in MM patients than in controls, while the median endogenous thrombin potential until the Peak (ETPp) was 39% higher in MM-patients than in controls. In line with these findings, the levels of platelets, RBCs, white blood cells and agonist induced platelet activation were decreased in MM patients compared to controls. The plasma TG experiments showed no differences between MM-patients and controls.ConclusionPatients with MM have a disturbed blood cell metabolism and a disbalanced WB-TG profile. This disbalance may explain the paradoxically high prevalence of bleeding symptoms in MM patients vs. an increased thrombosis risk. There was no disturbance observed in plasma TG, indicating that blood cells are the major determinants for the disbalanced hemostasis in MM patients.
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- 2022
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6. Beta-2-glycoprotein I as a biomarker for sepsis in critically ill patients in the intensive care unit: a prospective cohort study
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Irene T. Schrijver, Hans Kemperman, Mark Roest, Jozef Kesecioglu, and Dylan W. de Lange
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B2GPI ,Biomarkers ,Sepsis ,Critical care ,ICU ,SIRS ,Medical emergencies. Critical care. Intensive care. First aid ,RC86-88.9 - Published
- 2020
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7. Functional changes in hemostasis during asexual and sexual parasitemia in a controlled human malaria infection.
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Shengshi Huang, Wouter van der Heijden, Isaie J Reuling, Jun Wan, Qiuting Yan, Romy M W de Laat-Kremers, Andre J Van der Ven, Philip G de Groot, Matthew McCall, Robert W Sauerwein, Teun Bousema, Mark Roest, Marisa Ninivaggi, Quirijn de Mast, and Bas de Laat
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Medicine ,Science - Abstract
Decreased platelet count is an early phenomenon in asexual Plasmodium falciparum parasitemia, but its association with acute or long-term functional changes in platelets and coagulation is unknown. Moreover, the impact of gametocytemia on platelets and coagulation remains unclear. We investigated the changes in platelet number and function during early asexual parasitemia, gametocytemia and convalescence in 16 individuals participating in a controlled human malaria infection study, and studied its relationship with changes in total and active von Willebrand factor levels (VWF) and the coagulation system. Platelet activation and reactivity were determined by flow cytometry, and the coagulation system was assessed using different representative assays including antigen assays, activity assays and global functional assays. Platelet count was decreased during asexual blood stage infection but normalized during gametocytemia. Platelet P-selectin expression was slightly increased during asexual parasitemia, gametocytemia and at day 64. In contrast, platelet reactivity to different agonists remained unchanged, except a marked decrease in reactivity to low dose collagen-related peptide-XL. Thrombin generation and antigen assays did not show a clear activation of the coagulation during asexual parasitemia, whereas total and active VWF levels were markedly increased. During gametocytemia and on day 64, the endogenous thrombin potential, thrombin peak and velocity index were increased and prothrombin conversion and plasma prothrombin levels were decreased. We conclude that the decreased platelet count during asexual parasitemia is associated with increased active VWF levels (i.e. endothelial activation), but not platelet hyperreactivity or hypercoagulability, and that the increased platelet clearance in asexual parasitemia could cause spontaneous VWF-platelet complexes formation.
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- 2022
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8. Reversible Platelet Integrin αIIbβ3 Activation and Thrombus Instability
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Jinmi Zou, Frauke Swieringa, Bas de Laat, Philip G. de Groot, Mark Roest, and Johan W. M. Heemskerk
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ADP ,collagen ,fibrinogen ,integrin ,platelets ,thrombin ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Integrin αIIbβ3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbβ3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbβ3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbβ3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents.
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- 2022
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9. Long-term platelet priming after glycoprotein VI stimulation in comparison to Protease-Activating Receptor (PAR) stimulation.
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Jinmi Zou, Jiayu Wu, Mark Roest, and Johan W M Heemskerk
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Medicine ,Science - Abstract
Platelets can respond to multiple antagonists and agonists, implying that their activation state is a consequence of past exposure to these substances. While platelets are often considered as one-time responsive cells, they likely can respond to sequential application of inhibitors and stimuli. We hypothesized that the ability of platelets to sequentially respond depends on the time and type of repeated agonist application. The present proof-of-concept data show that iloprost (cAMP elevation), tirofiban (integrin αIIbβ3 blocker) and Syk kinase inhibition subacutely modulated platelet aggregation, i.e. halted this process even when applied after agonist. In comparison to thrombin-activated receptor (PAR) stimulation, glycoprotein VI (GPVI) stimulation was less sensitive to time-dependent blockage of aggregation, with Syk inhibition as an exception. Furthermore, cytosolic Ca2+ measurements indicated that, when compared to PAR, prior GPVI stimulation induced a more persistent, priming activation state of platelets that influenced the response to a next agent. Overall, these data point to an unexpected priming memory of activated platelets in subacutely responding to another inhibitor or stimulus, with a higher versatility and faster offset after PAR stimulation than after GPVI stimulation.
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- 2021
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10. Active von Willebrand Factor in patients with a bleeding diathesis
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Lisa N. van der Vorm, Dana Huskens, Lisa Florin, Pieter De Kesel, Mark Roest, Bas de Laat, and Katrien M.J. Devreese
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Active von willebrand factor ,Bleeding diathesis ,von willebrand disease ,Platelets ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
Introduction: Increased levels of circulating von Willebrand Factor (VWF) in its active, platelet-binding conformation have been implicated in the pathogenesis of several thrombotic conditions as well as bleeding conditions characterized by severe thrombocytopenia. However, it is unclear whether the proportion of activated VWF in the circulation also plays a role in patients with mild to moderate bleeding without thrombocytopenia. Methods: Citrated plasma samples were collected from 145 patients with a bleeding diathesis with unknown cause. Active VWF levels were measured with an in-house developed ELISA assay. In addition, VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) and (flow-cytometric) platelet-VWF binding (Plt:VWF) were determined. Results: Active VWF levels were on average mildly, but not significantly, lowered in patients with a bleeding diathesis compared to the reference interval (especially in individuals with non-O blood groups). Active VWF was not significantly different for subjects with (median 107.4%, IQR 18.3) versus without (median 111.1%, IQR 32.3%) an increased bleeding score, nor between subjects with suspected VWD (median 104%, IQR 20.6%) versus other suspected causes of bleeding diathesis (median 111.7%, IQR 33.3%). Conclusion: In this clinically heterogeneous population of patients with a mild bleeding phenotype, quantification of active VWF levels does not have added diagnostic value to VWF:Ag and VWF activity assays in the diagnosis of unexplained bleeding disorders.
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- 2020
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11. Platelet Activation Mechanisms and Consequences of Immune Thrombocytopenia
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Siyu Sun, Rolf T. Urbanus, Hugo ten Cate, Philip G. de Groot, Bas de Laat, Johan W. M. Heemskerk, and Mark Roest
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autoimmune disorders ,immune thrombocytopenia ,platelet ,thrombosis ,auto-antibodies ,Cytology ,QH573-671 - Abstract
Autoimmune disorders are often associated with low platelet count or thrombocytopenia. In immune-induced thrombocytopenia (IIT), a common mechanism is increased platelet activity, which can have an increased risk of thrombosis. In addition, or alternatively, auto-antibodies suppress platelet formation or augment platelet clearance. Effects of the auto-antibodies are linked to the unique structural and functional characteristics of platelets. Conversely, prior platelet activation may contribute to the innate and adaptive immune responses. Extensive interplay between platelets, coagulation and complement activation processes may aggravate the pathology. Here, we present an overview of the reported molecular causes and consequences of IIT in the most common forms of autoimmune disorders. These include idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), drug-induced thrombocytopenia (DITP), heparin-induced thrombocytopenia (HIT), COVID-19 vaccine-induced thrombosis with thrombocytopenia (VITT), thrombotic thrombocytopenia purpura (TTP), and hemolysis, the elevated liver enzymes and low platelet (HELLP) syndrome. We focus on the platelet receptors that bind auto-antibodies, the immune complexes, damage-associated molecular patterns (DAMPs) and complement factors. In addition, we review how circulating platelets serve as a reservoir of immunomodulatory molecules. By this update on the molecular mechanisms and the roles of platelets in the pathogenesis of autoimmune diseases, we highlight platelet-based pathways that can predispose for thrombocytopenia and are linked thrombotic or bleeding events.
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- 2021
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12. Myeloperoxidase can differentiate between sepsis and non-infectious SIRS and predicts mortality in intensive care patients with SIRS
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Irene T. Schrijver, Hans Kemperman, Mark Roest, Jozef Kesecioglu, and Dylan W. de Lange
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Myeloperoxidase ,Sepsis ,Critical care ,Biomarkers ,Mortality ,SIRS ,Medical emergencies. Critical care. Intensive care. First aid ,RC86-88.9 - Abstract
Abstract Background Systemic inflammatory response syndrome (SIRS) is a clinical syndrome following inflammation. Clinically, it is difficult to distinguish SIRS following an infection, i.e., sepsis, from non-infectious SIRS. Myeloperoxidase is a hemeprotein stored in the neutrophil azurophilic granules and is one of the main pillars of neutrophil attack. Therefore, we hypothesized that myeloperoxidase can differentiate between sepsis and non-infectious SIRS in patients with systemic inflammatory response syndrome in the intensive care unit (ICU). Methods An observational single-center cohort study was conducted measuring myeloperoxidase in patients with SIRS in the first 48 h after admission. The outcomes were established using predefined definitions. Thirty-day mortality was retrospectively assessed. Results We found significantly higher levels of myeloperoxidase in patients with sepsis and septic shock compared to patients without sepsis (60 ng/ml versus 43 ng/ml, P = 0.002). Myeloperoxidase levels were related to 30-day mortality (P = 0.032), and high MPO levels on top of a high APACHE IV score further increased mortality risk. Conclusions We show that myeloperoxidase is a potentially novel biomarker for sepsis in the ICU. Myeloperoxidase could eventually help in diagnosing sepsis and predicting mortality. However, more research is necessary to confirm our results.
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- 2017
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13. The effect of P2Y12 inhibition on platelet activation assessed with aggregation- and flow cytometry-based assays
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Tesse C. Leunissen, Peter Paul Wisman, Thijs C. van Holten, Philip G. de Groot, Suzanne J. Korporaal, Arnold C. Koekman, Frans L. Moll, Martin Teraa, Marianne C. Verhaar, Gert Jan de Borst, Rolf T. Urbanus, and Mark Roest
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platelet aggregation ,platelet aggregation inhibitor ,platelet function test ,platelet membrane glycoprotein iib ,p-selectin ,Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Patients on P2Y12 inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing, might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, the response to ADP does not necessarily reflect the effect of P2Y12 inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity toward other physiologically relevant agonists could provide more insight in the efficacy of P2Y12 inhibitors. The effect of in vitro and in vivo P2Y12 inhibition on αIIbβ3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha, and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y12 inhibitors was compared to VerifyNow results. P2Y12 inhibitors caused strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen-related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y12 inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y12-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed with flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition. Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response identifies different patients as nonresponders to P2Y12 inhibitors, compared to only ADP-induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.
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- 2017
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14. Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor.
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Lisa N van der Vorm, Li Li, Dana Huskens, Walid Chayouâ, Hilde Kelchtermans, Philip G de Groot, Mark Roest, Jasper A Remijn, and Bas de Laat
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Medicine ,Science - Abstract
BackgroundInteraction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF A1 domain, enabling platelet glycoprotein Ibα binding. Quantification of this ''active" conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.MethodsWe developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF A1 domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.ResultsIntra- and inter-assay CVs were between 2.4-7.2% and 4.1-9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103±3%. The assay was linear in the range of 90.1-424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6-154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF:GP1bM binding.ConclusionsWe developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.
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- 2019
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15. Thrombin generation and platelet activation in cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy - A prospective cohort study.
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Sven Van Poucke, Dana Huskens, Kurt Van der Speeten, Mark Roest, Bart Lauwereins, Ming-Hua Zheng, Seppe Dehaene, Joris Penders, Abraham Marcus, and Marcus Lancé
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Medicine ,Science - Abstract
BACKGROUND AND OBJECTIVES:Cytoreductive surgery (CRS) with hyperthermic intraperitoneal peroperative chemotherapy (HIPEC), indicated for patients with peritoneal metastases from digestive or gynecological malignancies alike, demonstrates a considerable impact on hemostatic metabolism, both on platelet and on coagulation level. The potential hemostatic interference in CRS and HIPEC is phase dependent. The hypothesis of this prospective cohort study is that the procedure exposed an increased thrombotic risk, resulting in a faster and increased thrombin generation and hyper platelet function. METHODS:This study explores the combined use of ROTEM (rotational thromboelastometry), PACT (platelet activation test) and CAT (thrombin generation test) assays during CRS and HIPEC with a follow-up of 7 days postoperative in 27 patients with confirmed histological diagnosis of peritoneal disease. RESULTS:Platelet reactivity (relative to before incision values) to CRP (collagen-related peptide) (p value 0.02) and TRAP (thrombin receptor activator peptide) (p value 0.048) seems to be slightly reduced during CRS and HIPEC with regard to αIIbβ3 activation, while P-selectin expression is not affected. During surgery, CAT demonstrates that, the LT (lagtime) (p value 0.0003) and TTP (time-to-thrombin peak) values (p value 0.002) decrease while and the TP (thrombin peak) (p value 0.004) and ETP (endogenous thrombin potential) (p value 0.02) increase. Subsequently, after surgery, the LT and TTP increase and ETP and TP decrease in time. ROTEM EXTEM (extrinsic) MCF (maximum clot firmness) (p value 0.005), INTEM (intrinsic) MCF (p value 0.003) and FIBTEM (fibrinogen) MCF (p value
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- 2018
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16. Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test.
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Dana Huskens, Yaqiu Sang, Joke Konings, Lisa van der Vorm, Bas de Laat, Hilde Kelchtermans, and Mark Roest
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Medicine ,Science - Abstract
Platelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals.Platelet function tests with 3 agonists and antibodies against P-selectin, activated αIIbβ3 and glycoprotein Ib (GPIb), were prepared and stored at -20°C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig κ particles were included to validate stability of the test and to standardize results. Reference intervals were determined.Blood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37°C resulted in stable results (%CV
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- 2018
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17. Soluble P-selectin as a Biomarker for Infection and Survival in Patients With a Systemic Inflammatory Response Syndrome on the Intensive Care Unit
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Irene T Schrijver, Hans Kemperman, Mark Roest, Jozef Kesecioglu, and Dylan W de Lange
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Medicine (General) ,R5-920 - Abstract
Aims: This study investigated the ability of soluble platelet selectin (sP-selectin) to identify infection and predict 30-day mortality in patients with a systemic inflammatory response syndrome (SIRS) on the intensive care unit. Methods: Soluble platelet selectin levels were measured daily in the first 48 hours in patients presenting with SIRS. The outcome, proven infection, was established using predefined definitions. The 30-day mortality was retrospectively assessed. Results: In a total of 313 patients with SIRS, sP-selectin levels were measured. Of these, 114 patients had proven infection on admission or developing during their intensive care unit (ICU) stay. Patients with proven infection had moderately higher levels of sP-selectin (147 ng/mL; interquartile range [IQR], 93.4-203 ng/mL) compared with noninfected patients (143.8 ng/mL; IQR, 89.6-194.7 ng/mL). This difference was not statistically significant ( P = .072). However, in patients who were not admitted for infection (n = 235), sP-selectin levels were significantly related to the subsequent development of infection ( P = .013). Soluble platelet selectin levels were particularly high in patients with abdominal sepsis and skin infections. Higher sP-selectin levels were associated with higher mortality (although not statistically significant, P = .08). Conclusions: This study shows that in patients with SIRS not admitted for infection, sP-selectin levels are significantly related to the subsequent development of infection. Furthermore, patients with higher sP-selectin levels in the first 2 days of admission had higher 30-day mortality, although this association is not statistically significant. Therefore, we conclude that sP-selectin is a potential future biomarker for both mortality and infection in patients with SIRS, but more research is needed to confirm its prognostic role.
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- 2017
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18. Application of an optimized flow cytometry-based quantification of Platelet Activation (PACT): Monitoring platelet activation in platelet concentrates.
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Cécile H Kicken, Mark Roest, Yvonne M C Henskens, Bas de Laat, and Dana Huskens
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Medicine ,Science - Abstract
BACKGROUND:Previous studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage. STUDY DESIGN AND METHODS:The optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference. RESULTS:Both PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis. CONCLUSION:PACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor-, time- and cost efficiency.
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- 2017
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19. Baseline Platelet Activation and Reactivity in Patients with Critical Limb Ischemia.
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Peter Paul Wisman, Martin Teraa, Gert Jan de Borst, Marianne C Verhaar, Mark Roest, and Frans L Moll
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Medicine ,Science - Abstract
Patients with critical limb ischemia (CLI) have a high risk to develop cardiovascular events (CVE). We hypothesized that in CLI patients platelets would display increased baseline activation and reactivity.We investigated baseline platelet activation and platelet reactivity in patients with CLI.In this study baseline platelet activation and platelet reactivity in response to stimulation of all major platelet activation pathways were determined in 20 CLI patients (11 using aspirin and 9 using vitamin K-antagonists) included in the Juventas-trial (clinicaltrials.gov NCT00371371) and in 17 healthy controls. Platelet activation was quantified with flow cytometric measurement of platelet P-selectin expression and fibrinogen binding.CLI patients not using aspirin showed higher baseline platelet activation compared to healthy controls. Maximal reactivity to stimulation of the collagen and thrombin activation pathway was decreased in CLI patients compared to healthy controls. In line, attenuated platelet reactivity to stimulation of multiple activation pathways was associated with several traditional risk factors for cardiovascular disease.Baseline platelet activation was increased in CLI patients, whereas the reactivity of circulating platelets to several stimulatory agents is decreased. Reactivity of platelets was inversely correlated with cardiovascular risk factors.
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- 2015
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20. Indications that paraoxonase-1 contributes to plasma high density lipoprotein levels in familial hypercholesterolemia
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Thomas M. van Himbergen, Mark Roest, Jacqueline de Graaf, Eugène H.J.M. Jansen, Hiroaki Hattori, John J.P. Kastelein, Hieronymus A.M. Voorbij, Anton F.H. Stalenhoef, and Lambertus J.H. van Tits
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atherosclerosis ,antioxidants ,polymorphisms ,Biochemistry ,QD415-436 - Abstract
HDL-associated paraoxonase type 1 (PON1) can protect LDL and HDL against oxidative modification in vitro and therefore may protect against cardiovascular disease. We investigated the effects of PON1 levels, activity, and genetic variation on high density lipoprotein-cholesterol (HDL-C) levels, circulating oxidized LDL (OxLDL), subclinical inflammation [high-sensitive C-reactive protein (Hs-CRP)], and carotid atherosclerosis. PON1 genotypes (L55M, Q192R, −107C/T, −162A/G, −824G/A, and −907G/C) were determined in 302 patients with familial hypercholesterolemia. PON1 activity was monitored by the hydrolysis rate of paraoxon, diazoxon, and phenyl acetate. PON1 levels, OxLDL, and Hs-CRP were determined using an immunoassay. The genetic variants of PON1 that were associated with high levels and activity of the enzyme were associated with higher HDL-C levels (P values for trend: 0.008, 0.020, 0.042, and 0.037 for L55M, Q192R, −107C/T, and −907G/C, respectively). In addition to the PON1 genotype, there was also a positive correlation between PON1 levels and activity and HDL-C (PON1 levels: r = 0.37, P < 0.001; paraoxonase activity: r = 0.23, P = 0.01; diazoxonase activity: r = 0.29, P < 0.001; arylesterase activity: r = 0.19, P = 0.03).Our observations support the hypothesis that both PON1 levels and activity preserve HDL-C in plasma.
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- 2005
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21. Endocannabinoids control platelet activation and limit aggregate formation under flow.
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Valentina De Angelis, Arnold C Koekman, Cees Weeterings, Mark Roest, Philip G de Groot, Eszter Herczenik, and Coen Maas
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Medicine ,Science - Abstract
BACKGROUND:The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function. OBJECTIVES:Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo. METHODS AND RESULTS:We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa. CONCLUSIONS:Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function may prove beneficial in the search for new antithrombotic therapies.
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- 2014
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22. Platelet activation determines angiopoietin-1 and VEGF levels in malaria: implications for their use as biomarkers.
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Judith Brouwers, Rintis Noviyanti, Rob Fijnheer, Philip G de Groot, Leily Trianty, Siti Mudaliana, Mark Roest, Din Syafruddin, Andre van der Ven, and Quirijn de Mast
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Medicine ,Science - Abstract
INTRODUCTION: The angiogenic proteins angiopoietin (Ang)-1, Ang-2 and vascular endothelial growth factor (VEGF) are regulators of endothelial inflammation and integrity. Since platelets store large amounts of Ang-1 and VEGF, measurement of circulation levels of these proteins is sensitive to platelet number, in vivo platelet activation and inadvertent platelet activation during blood processing. We studied plasma Ang-1, Ang-2 and VEGF levels in malaria patients, taking the necessary precautions to avoid ex vivo platelet activation, and related plasma levels to platelet count and the soluble platelet activation markers P-selectin and CXCL7. METHODS: Plasma levels of Ang-1, Ang-2, VEGF, P-selectin and CXCL7 were measured in CTAD plasma, minimizing ex vivo platelet activation, in 27 patients with febrile Plasmodium falciparum malaria at presentation and day 2 and 5 of treatment and in 25 healthy controls. RESULTS: Levels of Ang-1, Ang-2 and VEGF were higher at day 0 in malaria patients compared to healthy controls. Ang-2 levels, which is a marker of endothelial activation, decreased after start of antimalarial treatment. In contrast, Ang-1 and VEGF plasma levels increased and this corresponded with the increase in platelet number. Soluble P-selectin and CXCL7 levels followed the same trend as Ang-1 and VEGF levels. Plasma levels of these four proteins correlated strongly in malaria patients, but only moderately in controls. CONCLUSION: In contrast to previous studies, we found elevated plasma levels of Ang-1 and VEGF in patients with malaria resulting from in vivo platelet activation. Ang-1 release from platelets may be important to dampen the disturbing effects of Ang-2 on the endothelium. Evaluation of plasma levels of these angiogenic proteins requires close adherence to a stringent protocol to minimize ex vivo platelet activation.
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- 2014
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23. Increased platelet reactivity is associated with circulating platelet-monocyte complexes and macrophages in human atherosclerotic plaques.
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Bert Rutten, Claudia Tersteeg, Joyce E P Vrijenhoek, Thijs C van Holten, Ellen H A M Elsenberg, Elske M Mak-Nienhuis, Gert Jan de Borst, J Wouter Jukema, Nico H J Pijls, Johannes Waltenberger, Anton Jan van Zonneveld, Frans L Moll, Elizabeth McClellan, Andrew Stubbs, Gerard Pasterkamp, Imo Hoefer, Philip G de Groot, and Mark Roest
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Medicine ,Science - Abstract
Platelet reactivity, platelet binding to monocytes and monocyte infiltration play a detrimental role in atherosclerotic plaque progression. We investigated whether platelet reactivity was associated with levels of circulating platelet-monocyte complexes (PMCs) and macrophages in human atherosclerotic carotid plaques.Platelet reactivity was determined by measuring platelet P-selectin expression after platelet stimulation with increasing concentrations of adenosine diphosphate (ADP), in two independent cohorts: the Circulating Cells cohort (n = 244) and the Athero-Express cohort (n = 91). Levels of PMCs were assessed by flow cytometry in blood samples of patients who were scheduled for percutaneous coronary intervention (Circulating Cells cohort). Monocyte infiltration was semi-quantitatively determined by histological examination of atherosclerotic carotid plaques collected during carotid endarterectomy (Athero-Express cohort).We found increased platelet reactivity in patients with high PMCs as compared to patients with low PMCs (median (interquartile range): 4153 (1585-11267) area under the curve (AUC) vs. 9633 (3580-21565) AUC, P
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- 2014
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24. The relationship between fractional flow reserve, platelet reactivity and platelet leukocyte complexes in stable coronary artery disease.
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Jan-Willem E M Sels, Bert Rutten, Thijs C van Holten, Marieke A K Hillaert, Johannes Waltenberger, Nico H J Pijls, Gerard Pasterkamp, Philip G de Groot, and Mark Roest
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Medicine ,Science - Abstract
BACKGROUND: The presence of stenoses that significantly impair blood flow and cause myocardial ischemia negatively affects prognosis of patients with stable coronary artery disease. Altered platelet reactivity has been associated with impaired prognosis of stable coronary artery disease. Platelets are activated and form complexes with leukocytes in response to microshear gradients caused by friction forces on the arterial wall or flow separation. We hypothesized that the presence of significantly flow-limiting stenoses is associated with altered platelet reactivity and formation of platelet-leukocyte complexes. METHODS: One hundred patients with stable angina were studied. Hemodynamic significance of all coronary stenoses was assessed with Fractional Flow Reserve (FFR). Patients were classified FFR-positive (at least one lesion with FFR≤0.75) or FFR-negative (all lesions FFR>0.80). Whole blood samples were stimulated with increasing concentrations of ADP, TRAP, CRP and Iloprost with substimulatory ADP. Expression of P-selectin as platelet activation marker and platelet-leukocyte complexes were measured by flowcytometry. Patients were stratified on clopidogrel use. FFR positive and negative patient groups were compared on platelet reactivity and platelet-leukocyte complexes. RESULTS: Platelet reactivity between FFR-positive patients and FFR-negative patients did not differ. A significantly lower percentage of circulating platelet-neutrophil complexes in FFR-positive patients and a similar non-significant decrease in percentage of circulating platelet-monocyte complexes in FFR-positive patients was observed. CONCLUSION: The presence of hemodynamically significant coronary stenoses does not alter platelet reactivity but is associated with reduced platelet-neutrophil complexes in peripheral blood of patients with stable coronary artery disease.
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- 2013
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25. Circulating biomarkers for predicting cardiovascular disease risk; a systematic review and comprehensive overview of meta-analyses.
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Thijs C van Holten, Leonie F Waanders, Philip G de Groot, Joost Vissers, Imo E Hoefer, Gerard Pasterkamp, Menno W J Prins, and Mark Roest
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Medicine ,Science - Abstract
BACKGROUND: Cardiovascular disease is one of the major causes of death worldwide. Assessing the risk for cardiovascular disease is an important aspect in clinical decision making and setting a therapeutic strategy, and the use of serological biomarkers may improve this. Despite an overwhelming number of studies and meta-analyses on biomarkers and cardiovascular disease, there are no comprehensive studies comparing the relevance of each biomarker. We performed a systematic review of meta-analyses on levels of serological biomarkers for atherothrombosis to compare the relevance of the most commonly studied biomarkers. METHODS AND FINDINGS: Medline and Embase were screened on search terms that were related to "arterial ischemic events" and "meta-analyses". The meta-analyses were sorted by patient groups without pre-existing cardiovascular disease, with cardiovascular disease and heterogeneous groups concerning general populations, groups with and without cardiovascular disease, or miscellaneous. These were subsequently sorted by end-point for cardiovascular disease or stroke and summarized in tables. We have identified 85 relevant full text articles, with 214 meta-analyses. Markers for primary cardiovascular events include, from high to low result: C-reactive protein, fibrinogen, cholesterol, apolipoprotein B, the apolipoprotein A/apolipoprotein B ratio, high density lipoprotein, and vitamin D. Markers for secondary cardiovascular events include, from high to low result: cardiac troponins I and T, C-reactive protein, serum creatinine, and cystatin C. For primary stroke, fibrinogen and serum uric acid are strong risk markers. Limitations reside in that there is no acknowledged search strategy for prognostic studies or meta-analyses. CONCLUSIONS: For primary cardiovascular events, markers with strong predictive potential are mainly associated with lipids. For secondary cardiovascular events, markers are more associated with ischemia. Fibrinogen is a strong predictor for primary stroke.
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- 2013
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26. Up-regulation of platelet activation in hemophilia A
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Esther R. van Bladel, Mark Roest, Philip G. de Groot, and Roger E.G. Schutgens
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Background Platelets are an underappreciated factor in the classification of the bleeding tendency of patients with hemophilia. In this cross-sectional study, we investigated platelet activation status and responsiveness in relation to residual factor VIII activity and, within the group with severe hemophilia (
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- 2011
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27. Maternal TLR4 and NOD2 gene variants, pro-inflammatory phenotype and susceptibility to early-onset preeclampsia and HELLP syndrome.
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Bas B van Rijn, Arie Franx, Eric A P Steegers, Christianne J M de Groot, Rogier M Bertina, Gerard Pasterkamp, Hieronymus A M Voorbij, Hein W Bruinse, and Mark Roest
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Medicine ,Science - Abstract
BACKGROUND: Altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome. We examined whether allelic variants of the innate immune receptors Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 2 (NOD2), that impair the inflammatory response to endotoxin, are related to preeclampsia and HELLP syndrome. METHODS AND FINDINGS: We determined five common mutations in TLR4 (D299G and T399I) and NOD2 (R702W, G908R and L1007fs) in 340 primiparous women with a history of early-onset preeclampsia, of whom 177 women developed HELLP syndrome and in 113 women with a history of only uneventful pregnancies as controls. In addition, we assessed plasma levels of pro-inflammatory biomarkers C-reactive protein, interleukin-6, soluble intercellular adhesion molecule-1, fibrinogen and von Willebrand factor in a subset of 214 women included at least six months after delivery. After adjustment for maternal age and chronic hypertension, attenuating allelic variants of TLR4 were more common in women with a history of early-onset preeclampsia than in controls (OR 2.9 [95% CI 1.2-6.7]). Highest frequencies for TLR4 variants were observed in women who developed HELLP syndrome (adjusted OR 4.1 [95% CI 1.7-9.8]). In addition, high levels of interleukin-6 and fibrinogen were associated with a history of early-onset preeclampsia. Combined positivity for any of the TLR4 and NOD2 allelic variants and high levels of interleukin-6 was 6.9-fold more common in women with a history of early-onset preeclampsia (95% CI 2.1-23.2) compared to controls. CONCLUSIONS: We observed an association of common TLR4 and NOD2 gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and HELLP syndrome. These findings suggest involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy.
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- 2008
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28. Hyperresponsive Platelets and a Reduced Platelet Granule Release Capacity Are Associated with Severity and Mortality in COVID-19 Patients
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Fadel Muhammad Garishah, Dana Huskens, Setyo Gundi Pramudo, Dessy Andriani, Mila Astrilia, Rizky Akbar Sentosa, André J. A. M. van der Ven, Bas de Laat, Muhammad Hussein Gasem, Quirijn de Mast, and Mark Roest
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Blood Platelets ,C-Reactive Protein ,Adenosine Triphosphate ,lnfectious Diseases and Global Health Radboud Institute for Health Sciences [Radboudumc 4] ,SARS-CoV-2 ,Critical Illness ,Humans ,COVID-19 ,Hematology ,Thrombocytopenia ,Retrospective Studies - Abstract
Background Coronavirus disease 2019 (COVID-19) is often associated with mild thrombocytopenia and increased platelet reactivity. Objective The aim of the current study was to investigate the adenosine triphosphate (ATP) release kinetics of platelets in hospitalized SARS-CoV-2-infected patients. Methods We studied time-dependent platelet activation in whole blood by monitoring the ATP release kinetics upon stimulation with a PAR1 receptor agonist in 41 hospitalized critically ill COVID-19 patients, 47 hospitalized noncritically ill COVID-19 patients, and 30 healthy controls. Results Our study demonstrated that platelets of critically ill COVID-19 patients were hyper-responsive with a shorter platelet response time (PRT) and a reduced platelet granule release capacity (GRC), probably due to chronic activation. The median PRT of COVID-19 patients admitted to the critical care unit was 10 and 7 seconds shorter than the median PRT in healthy controls and noncritical COVID-19 patients, respectively. Both PRT and GRC were also associated with D-dimer (Spearman r [r s] = −0.51, p Conclusion Using an accessible agonist-induced platelet granule ATP release assay, we show that platelet hyper-responsiveness and reduced platelet GRC in COVID-19 patients were associated with critical illness and mortality.
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- 2022
29. High-throughput assessment identifying major platelet Ca2+ entry pathways via tyrosine kinase-linked and G protein-coupled receptors
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Hilaire Yam Fung Cheung, Jinmi Zou, Chukiat Tantiwong, Delia I. Fernandez, Jingnan Huang, Robert Ahrends, Mark Roest, Rachel Cavill, Jon Gibbins, Johan W.M. Heemskerk, Biochemie, RS: Carim - B04 Clinical thrombosis and Haemostasis, RS: Carim - B03 Cell biochemistry of thrombosis and haemostasis, Dept. of Advanced Computing Sciences, RS: FSE DACS, and RS: FSE DACS Mathematics Centre Maastricht
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Physiology ,Cell Biology ,Molecular Biology - Abstract
In platelets, elevated cytosolic Ca 2+ is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca 2+ response consists of Ca 2+ mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca 2+ entry pathways. Several channels can contribute to the Ca 2+ entry, but their relative contribution is unclear upon stimulation of ITAM-linked receptors such as glycoprotein VI (GPVI) and G-protein coupled receptors such as the protease-activated receptors (PAR) for thrombin. We employed a 96-well plate high-throughput assay with Fura-2-loaded human platelets to perform parallel [Ca 2+] i measurements in the presence of EGTA or CaCl 2. Per agonist condition, this resulted in sets of EGTA, CaCl 2 and Ca 2+ entry ratio curves, defined by six parameters, reflecting different Ca 2+ ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca 2+ entry ratio of 3-7. Strikingly, in combination with Ca 2+-ATPase inhibition by thapsigargin, the maximal Ca 2+ entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca 2+ entry. By pharmacological blockage of specific Ca 2+ channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca 2+ entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na +/Ca 2+ exchange (NCE) > P2× 1 (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca 2+ carriers regulating GPVI- and PAR-induced Ca 2+ entry in human platelets.
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- 2023
30. Flow Cytometry Based Platelet Reactivity Testing to Predict the Occurrence of Per-operative Solid Microemboli During Carotid Endarterectomy
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Aarent R.T. Brand, Tesse C. Leunissen, Daniel van Vriesland, Gerard Pasterkamp, Mark Roest, Suzanne J.A. Korporaal, Rolf T. Urbanus, and Gert J. de Borst
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Adenosine Diphosphate ,Stroke ,Endarterectomy, Carotid ,Intracranial Embolism ,Ultrasonography, Doppler, Transcranial ,Embolism ,Humans ,Carotid Stenosis ,Surgery ,Flow Cytometry ,Cardiology and Cardiovascular Medicine - Abstract
Peri-operative antiplatelet therapy (APT) aims to prevent thrombotic events such as stroke. High platelet reactivity ,despite the use use of APT, increases the risk of thrombotic events. Transcranial Doppler imaging (TCD) is used to detect peri-operative microembolic signals (MES) during carotid endarterectomy (CEA). Peri-operative MES are associated with an increased risk of procedural stroke and new silent lesions on diffusion weighted magnetic resonance imaging following surgery. The main components of TCD detected MES are platelet aggregates, and therefore patients displaying multiple MES during surgery could have benefited from more stringent APT. This study investigated whether the use of flow cytometry based platelet reactivity measurements were correlated with the incidence of pre-operative MES and thereby in the future suitable to predict patients at increased risk of peri-operative thrombotic events.Bilateral TCD with MES detection was performed in 197 patients undergoing CEA. Platelet reactivity was assessed with a flow cytometry based platelet reactivity assay measuring platelet response in whole blood. High on treatment platelet reactivity status was assessed for all patients. The secondary outcome was major adverse cardiovascular events (MACE) within one year.In total, 197 patients were included, 49 had peri-operative MES. The platelet response to adenosine diphosphate (ADP) correlated with MES (p = .021), and high on treatment platelet reactivity after adenosine diphosphate stimulation was associated with MACE (OR 2.34, 95% confidence interval 1.126 - 4.890, p = .023).Pre-operative platelet reactivity determined by flow cytometry after ADP stimulation correlated with the occurrence of intra-operative MES and post-operative MACE. Clopidogrel treatment showed the most substantial effect on reducing MES frequency and platelet reactivity measured by flow cytometry.
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- 2022
31. Targeted SERPIN (TaSER): A dual‐action antithrombotic agent that targets platelets for SERPIN delivery
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Minka Zivkovic, Mark Roest, Thomas Renné, Chantal C. Clark, Arjan D. Barendrecht, Coen Maas, Hinde El Otmani, Simone M. Smits, Wariya Sanrattana, Nadine D van Kleef, and Steven de Maat
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Blood Platelets ,biology ,Chemistry ,Hematology ,Serpin ,Fibrin ,Cell biology ,Tissue factor ,Platelet Adhesiveness ,Thrombin ,Fibrinolytic Agents ,Von Willebrand factor ,von Willebrand Factor ,Antithrombotic ,medicine ,biology.protein ,Humans ,Platelet ,Platelet activation ,Serpins ,circulatory and respiratory physiology ,medicine.drug - Abstract
BACKGROUND Occlusive thrombi are not homogeneous in composition. The core of a thrombus is rich in activated platelets and fibrin while the outer shell contains resting platelets. This core is inaccessible to plasma proteins. We produced a fusion protein (targeted SERPIN-TaSER), consisting of a function-blocking VH H against glycoprotein Ibα (GPIbα) and a thrombin-inhibiting serine protease inhibitor (SERPIN; α1-antitrypsin 355 AIAR358 ) to interfere with platelet-driven thrombin formation. AIM To evaluate the antithrombotic properties of TaSER. METHODS Besides TaSER, we generated three analogous control variants with either a wild-type antitrypsin subunit, a non-targeting control VH H, or their combination. We investigated TaSER and controls in protease activity assays, (platelet-dependent) thrombin generation assays, and by western blotting. The effects of TaSER on platelet activation and von Willebrand factor (VWF) binding were studied by fluorescence-activated cell sorting, in agglutination studies, and in ATP secretion experiments. We studied the influence of TaSER in whole blood (1) on platelet adhesion on VWF, (2) aggregate formation on collagen, and (3) thrombus formation (after recalcification) on collagen and tissue factor. RESULTS TaSER binds platelets and inhibits thrombin activity on the platelet surface. It blocks VWF binding and disassembles platelet agglutinates. TaSER delays tissue factor-triggered thrombin generation and ATP secretion in platelet-rich plasma in a targeted manner. In flow studies, TaSER interferes with platelet adhesion and aggregate formation due to GPIbα blockade and limits thrombus formation due to targeted inhibition of platelet-dependent thrombin activity. CONCLUSION The synergy between the individual properties of TaSER makes it a highly effective antithrombotic agent with possible clinical implications.
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- 2022
32. A novel mouse whole blood thrombin generation assay sensitive to FXI- and FIX-mediated amplification of coagulation
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Jun Wan, Pansakorn Tanratana, Mark Roest, Andras Gruber, Rafal Pawlinski, Alisa S. Wolberg, Nigel Mackman, and Steven P. Grover
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Hematology - Abstract
Thrombin generation (TG) assays serve as a valuable tool to study the amplifying roles of intrinsic pathway factors in human coagulation and provide functional insights into the increased bleeding observed in individuals deficient in factors (F) XI, IX, or VIII. Mice are used extensively in hemostasis research owing to the availability of coagulation factor–deficient mice. However, phenotypic differences between mouse and human TG have become apparent. In this study, we describe a novel, calibrated mouse whole blood (WB) TG assay used to assess the amplifying roles of intrinsic pathway factors in mouse coagulation. WB- and plasma-TG was triggered with either silica or tissue factor (TF) in samples from wild-type mice and mice deficient for FXII, FXI, or FIX. Expectedly, silica-triggered WB-TG and platelet-poor plasma (PPP)-TG were significantly reduced by deficiencies for FXII, FXI, or FIX. FXII deficiency had no effect on WB-TG or PPP-TG when triggered with TF. However, FXI deficiency resulted in significantly reduced WB-TG triggered by low concentrations of TF but had no effect on TF-triggered PPP-TG. FIX deficiency profoundly reduced WB-TG when triggered by low or high concentrations of TF whereas TG in PPP or platelet-rich plasma was only moderately reduced under these conditions. In conclusion, we have developed a novel mouse WB-TG assay with enhanced sensitivity to FXI- and FIX-dependent amplification of coagulation compared with an established plasma-TG assay. The enhanced sensitivity of WB-TG to FXI and FIX-dependent amplification of coagulation suggests an important role of blood cells in this process.
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- 2022
33. Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood
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Sandra, Konrath, Reiner K, Mailer, Manu, Beerens, Hanna, Englert, Maike, Frye, Piotr, Kuta, Roger J S, Preston, Coen, Maas, Lynn M, Butler, Mark, Roest, Bas, de Laat, and Thomas, Renné
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Cardiology and Cardiovascular Medicine - Abstract
Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12−/−) and FXI-deficient (F11−/−) mice. Moreover, reconstitution of blood from F12−/− mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_Δ12 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development.
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- 2022
34. Flow cytometric analysis of platelet function to detect high on-treatment residual platelet reactivity in patients on dual antiplatelet therapy
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Mark Roest, Lisa Florin, Dana Huskens, Bas de Laat, Katrien Devreese, Li Li, Biochemie, and RS: Carim - B01 Blood proteins & engineering
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Blood Platelets ,medicine.medical_specialty ,Platelet Aggregation ,Platelet Function Tests ,Clinical Biochemistry ,Residual ,Flow cytometry ,Platelet reactivity ,Internal medicine ,medicine ,Humans ,Platelet ,In patient ,RISK ,medicine.diagnostic_test ,business.industry ,flow cytometry ,Biochemistry (medical) ,Hematology ,General Medicine ,dual antiplatelet therapy ,Flow (mathematics) ,LTA ,Cardiology ,business ,Function (biology) ,Platelet Aggregation Inhibitors - Published
- 2022
35. Platelet Activation via Glycoprotein VI Initiates Thrombin Generation: A Potential Role for Platelet-Derived Factor IX?
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Li Li, Mark Roest, Joost C. M. Meijers, Bas de Laat, Rolf T. Urbanus, Philip G. de Groot, Dana Huskens, Experimental Vascular Medicine, Vascular Medicine, ACS - Pulmonary hypertension & thrombosis, Biochemie, and RS: Carim - B01 Blood proteins & engineering
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Blood Platelets ,Thrombin ,Platelet Membrane Glycoproteins ,Hematology ,coagulation factors ,Platelet Activation ,Blood Coagulation Factors ,Factor IX ,Humans ,Collagen ,coagulation ,activated platelets ,collagen receptor ,Glycoproteins - Abstract
Collagen triggers coagulation via activation of factor (F) XII. In a platelet-rich environment, collagen can also trigger coagulation independently of FXII. We studied a novel mechanism of coagulation initiation via collagen-dependent platelet activation using thrombin generation (TG) in platelet-rich plasma. Collagen-induced coagulation is minimally affected by active-site inactivated FVIIa, anti-FVII antibodies, or FXIIa inhibition (corn trypsin inhibitor). Activation of platelets via specific glycoprotein (GP) VI agonists initiates TG, FX activation, and fibrin formation. To determine the platelet-derived trigger of coagulation, we systematically reconstituted factor-deficient plasmas with washed platelets. TG triggered by GPVI-activated platelets was significantly affected in FIX- and FVIII-deficient plasma but not in FVII- and FXII-deficient plasma. In a purified system composed of FX and FVIII, we observed that absence of FIX was compensated by GPVI-activated platelets, which could be inhibited by an anti-FIX antibody, suggesting FIXa activity from activated platelets. Furthermore, with the addition of FVIII in FIX-deficient plasma, TG induced by GPVI-activated platelets was restored, and was inhibited by the anti-FIX antibody. In conclusion, GPVI-activated platelets initiate TG, probably via platelet-derived FIXa activity.
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- 2022
36. Efficacy of pro- and anticoagulant strategies in plasma of patients undergoing hepatobiliary surgery
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Nigel Heaton, Bente P. van den Boom, Andreas Prachalias, Zoka Milan, William Bernal, Fraser Dunsire, Jelle Adelmeijer, Anju Phoolchund, Ton Lisman, Sarah Bos, Tsai-Wing Ow, Mark Roest, and Groningen Institute for Organ Transplantation (GIOT)
- Subjects
anticoagulants ,medicine.medical_specialty ,medicine.drug_class ,Low molecular weight heparin ,030204 cardiovascular system & hematology ,Gastroenterology ,Dabigatran ,Plasma ,03 medical and health sciences ,hepatectomy ,0302 clinical medicine ,Internal medicine ,medicine ,Humans ,Rivaroxaban ,liver transplantation ,biology ,Heparin ,business.industry ,HAEMOSTASIS ,Anticoagulant ,Original Articles ,Hematology ,Heparin, Low-Molecular-Weight ,thrombin ,Prothrombin complex concentrate ,Blood Coagulation Factors ,Pharmaceutical Preparations ,Recombinant factor VIIa ,biology.protein ,Original Article ,Fresh frozen plasma ,business ,medicine.drug - Abstract
BACKGROUND: In vitro efficacy of pro- and antihemostatic drugs is profoundly different in patients with compensated cirrhosis and in those who have cirrhosis and are critically ill.OBJECTIVES: Here we assessed the efficacy of pro- and anticoagulant drugs in plasma of patients undergoing hepato-pancreato-biliary (HPB) surgery, which is associated with unique hemostatic changes.METHODS: We performed in vitro analyses on blood samples of 60 patients undergoing HPB surgery and liver transplantation: 20 orthotopic liver transplantations, 20 partial hepatectomies, and 20 pylorus-preserving pancreaticoduodenectomies. We performed thrombin generation experiments before and after in vitro addition of fresh frozen plasma (FFP), prothrombin complex concentrate (PCC), recombinant factor VIIa (rFVIIa), low molecular weight heparin (LMWH), unfractionated heparin, dabigatran, and rivaroxaban.RESULTS: We showed that patients undergoing HPB surgery are in a hypercoagulable state by thrombin generation testing. FFP and rFVIIa had minimal effects on thrombin generation, whereas PCC had a more pronounced procoagulant effect in patients compared with controls. Dabigatran showed a more pronounced anticoagulant effect in patients compared with controls, whereas rivaroxaban and LMWH had a decreased anticoagulant effect in patients.CONCLUSION: We demonstrate profoundly altered in vitro efficacy of commonly used anticoagulants, in patients undergoing HPB surgery compared with healthy controls, which may have implications for anticoagulant dosing in the early postoperative period. In the correction of perioperative bleeding complications, PCCs appear much more potent than FFP or rFVIIa, and PCCs may require conservative dosing and caution in use in patients undergoing HPB surgery.
- Published
- 2020
37. Flow cytometric analysis of platelet function to improve the recognition of thrombocytopathy
- Author
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Lisa Florin, Dana Huskens, Katrien Devreese, Bas de Laat, Mark Roest, Pieter M. De Kesel, Li Li, Biochemie, and RS: Carim - B01 Blood proteins & engineering
- Subjects
Blood Platelets ,medicine.medical_specialty ,Platelet Aggregation ,Platelet Function Tests ,BLEEDING ASSESSMENT-TOOL ,030204 cardiovascular system & hematology ,DIAGNOSIS ,Gastroenterology ,VALIDATION ,Platelet function defect ,Flow cytometry ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Thrombocytopathy ,Medicine and Health Sciences ,medicine ,Humans ,Platelet ,Platelet activation ,Whole blood ,Hematology ,medicine.diagnostic_test ,FUNCTION DISORDERS ,business.industry ,Gold standard (test) ,Platelet Activation ,medicine.disease ,THROMBUS FORMATION ,Bleeding diathesis ,stomatognathic diseases ,030220 oncology & carcinogenesis ,Aggregometry ,lipids (amino acids, peptides, and proteins) ,Blood Platelet Disorders ,GLYCOPROTEIN-VI ,CONSENSUS ,business ,Platelet Aggregation Inhibitors - Abstract
Introduction: Light transmission aggregometry (LTA) is the gold standard for diagnosing bleeding disorders. Although LTA is laborious, requires large volumes of blood and is relatively insensitive to small changes in platelet function, there is still no competing alternative approach to replace LTA for the diagnosis of platelet bleeding disorders.Materials and methods: This study investigates the correlation between flow cytometry-based whole blood platelet activation test (WB-PACT) and LTA and whether WB-PACT is of additional value for the identification of bleeding disorders. In total, 161 patients with suspected bleeding diathesis were tested.Results: A correlation of 0.41 between LTA and WB-PACT was found, and there was agreement between tests in 62% of cases (kappa = 0.23). The WB-PACT is of additional value to LTA to detect platelet function disorders (PFD) as 10 patients with elevated bleeding score (BS) were detected with WB-PACT, 4 with LTA and 7 patients were positive with both tests. Interestingly, in contrast to LTA, WB-PACT has an additional option to detect VWF disfunctions.Conclusion: WB-PACT may have added value for the routine diagnostic work-up in patients who need to have platelet function tested.
- Published
- 2020
38. A novel assay for studying the involvement of blood cells in whole blood thrombin generation
- Author
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Mark Roest, Bas de Laat, Hilde Kelchtermans, Qiuting Yan, Joke Konings, Jun Wan, Romy Kremers, Biochemie, and RS: Carim - B01 Blood proteins & engineering
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Blood Platelets ,Reference range ,030204 cardiovascular system & hematology ,Hematocrit ,Andrology ,03 medical and health sciences ,0302 clinical medicine ,Thrombin ,medicine ,platelet activation ,Humans ,Platelet ,Platelet activation ,Blood Coagulation ,Coagulation Disorder ,Whole blood ,medicine.diagnostic_test ,PLASMA ,Chemistry ,HAEMOSTASIS ,Reproducibility of Results ,Original Articles ,Hematology ,thrombin ,Coagulation ,erythrocytes ,Original Article ,blood coagulation disorders ,blood cells ,medicine.drug - Abstract
Background Fluorogenic thrombin generation (TG) assays are commonly used to determine global coagulation phenotype in plasma. Whole blood (WB)-TG assays reach one step closer to physiology by involving the intrinsic blood cells, but erythrocytes cause variable quenching of the fluorescence signals, hampering its routine application.Objective To develop a new assay for continuous WB-TG measurement.Methods In the new WB-TG assay, the erythrocyte-caused distortion of signal was solved by continuously mixing the sample during the measurement. The assay was validated by evaluating the reproducibility and comparing with the paper-based WB-TG assay. Reconstituted human blood and WB from 119 healthy donors was tested to explore the influences of hematocrit and platelet count on TG.Results This novel WB-TG assay showed good reproducibility while being less affected by contact activation compared with the previous paper-based assay. Reconstitution experiments showed that the lag time of TG was shortened by the addition of platelets but not erythrocytes. Increasing hematocrit strongly augmented the peak thrombin, even in the presence of high platelet counts. The lag time and peak of WB-TG of 119 healthy donors were positively related to erythrocyte count after adjusting for age, sex, and oral contraceptive use with multiple linear regression analyses. The reference range and interindividual variation of WB-TG were determined in the healthy cohort.Conclusions A novel WB-TG assay was developed, which is a straightforward tool to measure the involvement of platelets and erythrocytes in TG and may assist the research of blood cell-associated coagulation disorders.
- Published
- 2020
39. Whole blood thrombin generation profiles of patients with cirrhosis explored with a near patient assay
- Author
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Mark Roest, Roopen Arya, Joke Konings, Lara N. Roberts, Vishal C. Patel, Jun Wan, Wasiliki Hendrix, Ton Lisman, Omar Barbouti, Liane Rabinowich, Bas de Laat, Tsai-Wing Ow, William Bernal, Biochemie, RS: Carim - B01 Blood proteins & engineering, and Groningen Institute for Organ Transplantation (GIOT)
- Subjects
Liver Cirrhosis ,medicine.medical_specialty ,Cirrhosis ,COAGULATION ,030204 cardiovascular system & hematology ,Hematocrit ,Thrombomodulin ,Gastroenterology ,DISEASE ,ACUTE DECOMPENSATION ,03 medical and health sciences ,0302 clinical medicine ,Thrombin ,Internal medicine ,medicine ,Coagulation testing ,Humans ,HEMOSTASIS ,Blood Coagulation ,thrombosis ,Platelet-poor plasma ,Whole blood ,COAGULOPATHY ,medicine.diagnostic_test ,business.industry ,HAEMOSTASIS ,THROMBOMODULIN ,chronic liver disease ,Original Articles ,Hematology ,medicine.disease ,bleeding ,HYPERCOAGULABLE STATE ,Hemostasis ,thrombin generation ,PROCOAGULANT IMBALANCE ,PROTEIN-C ,Original Article ,ACUTE LIVER-INJURY ,Blood Coagulation Tests ,business ,medicine.drug - Abstract
Background and Aims Patients with cirrhosis have a rebalanced hemostasis, often with normal or elevated thrombin-generating (TG) capacity in plasma. Whole blood (WB) TG allows faster determination and, importantly, includes the influence of all circulating blood cells. We aimed to study the TG profile of patients with cirrhosis in WB and in platelet poor plasma.Methods Thrombin-generating capacity in WB and plasma were assessed with a near-patient WB-TG assay and the calibrated automated thrombinography assay, respectively. TG assays were tested in presence and absence of thrombomodulin. Conventional coagulation tests were also performed.Results Thirty-four patients with cirrhosis and twenty-two controls were analyzed. Compared with controls, patients had substantially deranged results in conventional coagulation tests. Comparable WB-TG capacity (endogenous thrombin potential until peak, ETPp) but significantly lower peak thrombin were found in patients, and these results persisted when thrombomodulin was present. TG of the patients was more resistant to thrombomodulin than controls in both WB and plasma, although the inhibitory effect of thrombomodulin was drastically weaker in WB than in plasma. The peak of WB-TG in patients correlated moderately with their hematocrit and platelet count. Significant correlations were found between TG results in WB and plasma.Conclusions The WB-TG assay shows a normal to hypocoagulable state in patients with cirrhosis with a decreased anticoagulant activity of TM compared to plasma-TG. The clinical value of this assay needs further validation.
- Published
- 2020
40. Differences in thrombin and plasmin generation potential between East African and Western European adults: The role of genetic and non-genetic factors
- Author
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Godfrey S. Temba, Nadira Vadaq, Jun Wan, Vesla Kullaya, Dana Huskens, Tal Pecht, Martin Jaeger, Collins K. Boahen, Vasiliki Matzaraki, Wieteke Broeders, Leo A.B. Joosten, Sultana M.H. Faradz, Gibson Kibiki, Saskia Middeldorp, Duccio Cavalieri, Paolo Lionetti, Philip G. de Groot, Joachim L. Schultze, Mihai G. Netea, Vinod Kumar, Bas de Laat, Blandina T. Mmbaga, Andre J. van der Ven, Mark Roest, Quirijn de Mast, Groningen Institute for Gastro Intestinal Genetics and Immunology (3GI), Biochemie, RS: Carim - B01 Blood proteins & engineering, Vascular Medicine, ACS - Pulmonary hypertension & thrombosis, and ARD - Amsterdam Reproduction and Development
- Subjects
Adult ,metabolism [Thrombin] ,genetic association studies ,Vascular damage Radboud Institute for Health Sciences [Radboudumc 16] ,LOCI ,lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,COAGULATION ,Black People ,Tanzania ,White People ,genetics [Inflammation] ,parasitic diseases ,Humans ,Fibrinolysin ,ddc:610 ,genetics [Blood Coagulation] ,Blood Coagulation ,METAANALYSIS ,Netherlands ,plasmin ,RISK ,Inflammation ,VENOUS THROMBOEMBOLISM ,Thrombin ,Vascular damage Radboud Institute for Molecular Life Sciences [Radboudumc 16] ,ASSOCIATION ,Hematology ,metabolism [Fibrinolysin] ,SEASONAL-VARIATIONS ,thrombin ,lnfectious Diseases and Global Health Radboud Institute for Health Sciences [Radboudumc 4] ,CARDIOVASCULAR-DISEASE ,inflammation ,OBESITY ,ethnicity ,metabolome ,Blood Coagulation Tests - Abstract
Contains fulltext : 251902.pdf (Publisher’s version ) (Open Access) BACKGROUND: Geographic variability in coagulation across populations and their determinants are poorly understood. OBJECTIVE: To compare thrombin (TG) and plasmin (PG) generation parameters between healthy Tanzanian and Dutch individuals, and to study associations with inflammation and different genetic, host and environmental factors. METHODS: TG and PG parameters were measured in 313 Tanzanians of African descent living in Tanzania and 392 Dutch of European descent living in the Netherlands and related to results of a dietary questionnaire, circulating inflammatory markers, genotyping, and plasma metabolomics. RESULTS: Tanzanians exhibited an enhanced TG and PG capacity, compared to Dutch participants. A higher proportion of Tanzanians had a TG value in the upper quartile with a PG value in the lower/middle quartile, suggesting a relative pro-coagulant state. Tanzanians also displayed an increased normalized thrombomodulin sensitivity ratio, suggesting reduced sensitivity to protein C. In Tanzanians, PG parameters (lag time and TTP) were associated with seasonality and food-derived plasma metabolites. The Tanzanians had higher concentrations of pro-inflammatory cytokines, which correlated strongly with TG and PG parameters. There was limited overlap in genetic variation associated with TG and PG parameters between the two cohorts. Pathway analysis of genetic variants in the Tanzanian cohort revealed multiple immune pathways that were enriched with TG and PG traits, confirming the importance of co-regulation between coagulation and inflammation. CONCLUSIONS: Tanzanians have an enhanced TG and PG potential compared to Dutch individuals, which may relate to differences in inflammation, genetics and diet. These observations highlight the importance of better understanding of the geographic variability in coagulation across populations.
- Published
- 2022
41. A Synthetic Triple Helical Collagen Peptide as a New Agonist for Flow Cytometric Measurement of GPVI-Specific Platelet Activation
- Author
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Yaqiu Sang, Mark Roest, Bas de Laat, Dana Huskens, Kanin Wichapong, Gerry A. F. Nicolaes, Promovendi CD, Biochemie, RS: Carim - B01 Blood proteins & engineering, RS: CARIM - R1.01 - Blood proteins & engineering, and RS: CARIM - R1 - Thrombosis and haemostasis
- Subjects
0301 basic medicine ,Agonist ,Adult ,Blood Platelets ,Male ,medicine.drug_class ,synthetic triple helical collagen peptide ,Peptide ,Platelet Glycoprotein GPIIb-IIIa Complex ,Platelet Membrane Glycoproteins ,030204 cardiovascular system & hematology ,Protein Engineering ,Flow cytometry ,03 medical and health sciences ,Young Adult ,0302 clinical medicine ,Platelet Adhesiveness ,Protein Domains ,cross-linked collagen-related peptide ,medicine ,platelet activation ,Humans ,Platelet ,Platelet activation ,Cysteine ,Receptor ,Aged ,chemistry.chemical_classification ,Binding Sites ,medicine.diagnostic_test ,Chemistry ,Activator (genetics) ,Hematology ,Middle Aged ,Flow Cytometry ,Healthy Volunteers ,P-Selectin ,030104 developmental biology ,Biophysics ,Female ,VI ,Collagen ,GPVI ,Carrier Proteins ,Peptides - Abstract
Synthetic cross-linked collagen-related peptide (CRP-XL) is a glycoprotein VI (GPVI) receptor activator for platelet activation. This triple helical peptide, widely used in platelet function tests, is synthesized and cross-linked through cysteine residues at its N-terminus and C-terminus. Currently, there is only one laboratory, which is capable to produce this valuable peptide for clinical applications. In an attempt to provide a standardized alternative for CRP-XL, we developed a synthetic triple helical collagen peptide (STH-CP) with the same primary sequence as CRP-XL (GPC-(GPO)10-GPCG-amide)3, which was both on the C-terminus and on the N-terminus fixed on a scaffold with a binding side for each of the three peptides. The performance of STH-CP on platelet function was studied using flow cytometry and compared with CRP-XL. We found that platelet activation pattern in response to STH-CP and CRP-XL is similar, although the STH-CP requires sixfold higher concentrations to activate platelets to the same state. The intra-assay percent coefficient of variation of STH-CP and CRP-XL were both Our findings show that this new STH-CP is a stable and potent platelet GPVI agonist which can induce the same reproducible platelet activation as CRP-XL and that STH-CP can be considered as a good alternative for CRP-XL.
- Published
- 2019
42. Platelet Activation Mechanisms and Consequences of Immune Thrombocytopenia
- Author
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Bas de Laat, Hugo ten Cate, Rolf T. Urbanus, Mark Roest, Johan W. M. Heemskerk, Philip G. de Groot, and Siyu Sun
- Subjects
VON-WILLEBRAND-FACTOR ,QH301-705.5 ,ANTIPHOSPHOLIPID SYNDROME ,Review ,ELEVATED LIVER-ENZYMES ,Models, Biological ,autoimmune disorders ,Immune system ,Antiphospholipid syndrome ,hemic and lymphatic diseases ,auto-antibodies ,medicine ,INTRAVENOUS IMMUNOGLOBULIN ,Animals ,Humans ,Platelet ,Platelet activation ,SYSTEMIC-LUPUS-ERYTHEMATOSUS ,Biology (General) ,thrombosis ,platelet ,Purpura, Thrombocytopenic, Idiopathic ,business.industry ,Autoantibody ,GLYCOPROTEIN IB-ALPHA ,General Medicine ,medicine.disease ,Platelet Activation ,Thrombocytopenic purpura ,Complement system ,Coagulation ,immune thrombocytopenia ,FC-GAMMA-RIIA ,Immunology ,HEPARIN-INDUCED THROMBOCYTOPENIA ,ANTIPLATELET AUTOANTIBODIES ,business ,COMPLEMENT ACTIVATION ,Signal Transduction - Abstract
Autoimmune disorders are often associated with low platelet count or thrombocytopenia. In immune-induced thrombocytopenia (IIT), a common mechanism is increased platelet activity, which can have an increased risk of thrombosis. In addition, or alternatively, auto-antibodies suppress platelet formation or augment platelet clearance. Effects of the auto-antibodies are linked to the unique structural and functional characteristics of platelets. Conversely, prior platelet activation may contribute to the innate and adaptive immune responses. Extensive interplay between platelets, coagulation and complement activation processes may aggravate the pathology. Here, we present an overview of the reported molecular causes and consequences of IIT in the most common forms of autoimmune disorders. These include idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), drug-induced thrombocytopenia (DITP), heparin-induced thrombocytopenia (HIT), COVID-19 vaccine-induced thrombosis with thrombocytopenia (VITT), thrombotic thrombocytopenia purpura (TTP), and hemolysis, the elevated liver enzymes and low platelet (HELLP) syndrome. We focus on the platelet receptors that bind auto-antibodies, the immune complexes, damage-associated molecular patterns (DAMPs) and complement factors. In addition, we review how circulating platelets serve as a reservoir of immunomodulatory molecules. By this update on the molecular mechanisms and the roles of platelets in the pathogenesis of autoimmune diseases, we highlight platelet-based pathways that can predispose for thrombocytopenia and are linked thrombotic or bleeding events.
- Published
- 2021
43. Development of a digital twin of the temperature distribution in datacenters through fusion of CFD and real time temperature measurements
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Eric Terry and Mark Roest
- Published
- 2021
44. Kallikrein augments the anticoagulant function of the protein C system in thrombin generation
- Author
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Nadira Vadaq, Joke Konings, Quirijn de Mast, Philip G. de Groot, Jun Wan, Leo A. B. Joosten, Martin Jaeger, Vinod Kumar, André J. A. M. van der Ven, Mihai G. Netea, Bas de Laat, Mark Roest, Groningen Institute for Gastro Intestinal Genetics and Immunology (3GI), Biochemie, RS: Carim - B04 Clinical thrombosis and Haemostasis, and RS: Carim - B01 Blood proteins & engineering
- Subjects
VENOUS THROMBOSIS ,genetic association ,Thrombomodulin ,lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,Tissue factor ,Thrombin ,medicine ,kallikrein ,Humans ,APOLIPOPROTEIN-A-IV ,GENOME-WIDE ASSOCIATION ,thrombosis ,RISK ,IDENTIFICATION ,biology ,Chemistry ,Factor V ,Prekallikrein ,FACTOR-XIA ,Anticoagulants ,Hematology ,Kallikrein ,Prostate-Specific Antigen ,Molecular biology ,lnfectious Diseases and Global Health Radboud Institute for Health Sciences [Radboudumc 4] ,FACTOR-V ,Coagulation ,biology.protein ,INACTIVATION ,Kallikreins ,Blood Coagulation Tests ,PLASMA KALLIKREIN ,Protein C ,HUMAN FACTOR-VIII ,circulatory and respiratory physiology ,medicine.drug - Abstract
Contains fulltext : 245643.pdf (Publisher’s version ) (Open Access) BACKGROUND: Genetics play a significant role in coagulation phenotype and venous thromboembolism risk. Resistance to the anticoagulant activated protein C (APC) is an established risk for thrombosis. Herein, we explored the genetic determinants of thrombin generation (TG) and thrombomodulin (TM)-modulated TG using plasma from the Human Functional Genomics Project. METHODS: Calibrated TG was measured both in absence and presence of TM using tissue factor as trigger. Genetic determinants of TG parameters and protein C pathway function were assessed using genome-wide single-nucleotide polymorphism (SNP) genotyping. Plasma samples were supplemented with purified apolipoprotein A-IV, prekallikrein, or kallikrein to test their influence on the anticoagulant function of TM and APC in TG. RESULTS: Thrombin generation data from 392 individuals were analyzed. Genotyping showed that the KLKB1 gene (top SNP: rs4241819) on chromosome 4 was associated with the normalized sensitivity ratio of endogenous thrombin potential to TM at genome-wide level (nETP-TMsr, P = 4.27 × 10(-8) ). In vitro supplementation of kallikrein, but not prekallikrein or apolipoprotein A-IV, into plasma dose-dependently augmented the anticoagulant effect of TM and APC in TG. Variations of rs4241819 was not associated with the plasma concentration of prekallikrein. Association between rs4241819 and nETP-TMsr was absent when TG was measured in presence of a contact pathway inhibitor corn trypsin inhibitor. CONCLUSIONS: Our results suggest that kallikrein plays a role in the regulation of the anticoagulant protein C pathway in TG, which may provide a novel mechanism for the previously observed association between the KLKB1 gene and venous thrombosis.
- Published
- 2021
45. Circulating cell-free DNA levels increment with worsening cirrhosis severity and associate with platelet exhaustion and mortality
- Author
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Marilena Stamouli, Jelle Adelmeijer, Dana Huskens, Savannah Rivera, Kevin O’Reilly, Elena Palma, Neil Youngson, William Bernal, Shilpa Chokshi, Mark Roest, Ton Lisman, and Vishal Patel
- Subjects
Hepatology - Published
- 2022
46. The anticoagulant effect of dabigatran is reflected in the lag time and time-to-peak, but not in the endogenous thrombin potential or peak, of thrombin generation
- Author
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Jonathan Douxfils, Mark Roest, Saartje Bloemen, Suzanne Zwaveling, Romy Kremers, François Mullier, UCL - SSS/IREC/MONT - Pôle Mont Godinne, UCL - (MGD) Laboratoire de biologie clinique, Biochemie, RS: CARIM - R1.01 - Blood proteins & engineering, Promovendi CD, and RS: CARIM - R1.04 - Clinical thrombosis and haemostasis
- Subjects
Thrombin generation ,ALWAYS ,Thrombin/analysis ,medicine.drug_class ,Idarucizumab ,COAGULATION ,SUFFICIENT ,030204 cardiovascular system & hematology ,Pharmacology ,Antithrombins ,SUSTAINED REVERSAL ,WARFARIN ,Dabigatran ,03 medical and health sciences ,0302 clinical medicine ,Thrombin ,Endogenous Thrombin Potential ,medicine ,Dabigatran/blood ,MANAGEMENT ,Humans ,Blood Coagulation Tests/methods ,030212 general & internal medicine ,THROMBOGRAM ,Blood Coagulation ,PLASMA ,Chemistry ,Antithrombins/blood ,Anticoagulant ,Hematology ,EX-VIVO ,Coagulation ,Blood Coagulation/drug effects ,Calibration ,Blood Coagulation Tests ,Ex vivo ,medicine.drug - Abstract
Introduction: Calibrated automated thrombinography (CAT) is a sensitive method to assess coagulation. Dabigatran inhibits both free thrombin and the alpha(2)macroglobulin (alpha M-2)-thrombin complex, which results in an erroneously increased peak and endogenous thrombin potential (ETP) without affecting lag time and time-to-peak. The aim of this study was to elucidate the artefacts in CAT when dabigatran is present.Materials and methods: Thrombin generation (TG) was measured in vitro by using CAT in the presence or absence of 6 mu M idarucizumab in plasma spiked with dabigatran. Additionally, ex vivo measurements were performed in plasmas of 63 patients using dabigatran in the presence and absence of idarucizumab.Results: The in vitro experiments confirmed that the ETP, peak and velocity index were artificially increased. This was mainly due to the inhibition of the calibrator by dabigatran and partly due to CAT algorithms. The calibration artefact could be resolved by adding idarucizumab to the calibrator well. However, the second, mathematical artefact remains when dabigatran is present in the TG well. These findings were corroborated by ex vivo experiments i.e. the lag time and time-to-peak were significantly reduced in patients upon addition of idarucizumab, but the ETP and peak were not significantly affected. The velocity index did change significantly, since this is a combination of time-dependent factors and the peak.Conclusions: The peak, ETP and velocity index do not represent the anticoagulant effect of dabigatran on TG measured with CAT. The lag time and time-to-peak, however, do reflect the effect of dabigatran.
- Published
- 2018
47. Added Value of Blood Cells in Thrombin Generation Testing
- Author
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Joke Konings, Jun Wan, Bas de Laat, Mark Roest, and Tilman M. Hackeng
- Subjects
0301 basic medicine ,Blood Platelets ,Erythrocytes ,Time Factors ,BLEEDING PHENOTYPE ,030204 cardiovascular system & hematology ,Pharmacology ,PLATELET-RICH PLASMA ,ACTIVATED PROTEIN-C ,03 medical and health sciences ,Tissue factor ,0302 clinical medicine ,Thrombin ,Predictive Value of Tests ,medicine ,WHOLE-BLOOD ,Leukocytes ,Humans ,Platelet ,Platelet activation ,CANCER-CELLS ,Blood Coagulation ,Coagulation Disorder ,thrombosis ,Whole blood ,VENOUS THROMBOEMBOLISM ,Blood Cells ,Chemistry ,CALIBRATED AUTOMATED THROMBOGRAM ,whole blood ,Hematology ,Neoplastic Cells, Circulating ,030104 developmental biology ,Coagulation ,TISSUE FACTOR ,FACTOR-V ,Platelet-rich plasma ,thrombin generation ,RISK-FACTORS ,haemorrhage ,Blood Coagulation Tests ,Biomarkers ,medicine.drug - Abstract
The capacity of blood to form thrombin is a critical determinant of coagulability. Plasma thrombin generation (TG), a test that probes the capacity of plasma to form thrombin, has improved our knowledge of the coagulation system and shows promising utility in coagulation management. Although plasma TG gives comprehensive insights into the function of pro- and anticoagulation drivers, it does not measure the role of blood cells in TG. In this literature review, we discuss currently available continuous TG tests that can reflect the involvement of blood cells in coagulation, in particular the fluorogenic assays that allow continuous measurement in platelet-rich plasma and whole blood. We also provide an overview about the influence of blood cells on blood coagulation, with emphasis on the direct influence of blood cells on TG. Platelets accelerate the initiation and velocity of TG by phosphatidylserine exposure, granule content release and surface receptor interaction with coagulation proteins. Erythrocytes are also major providers of phosphatidylserine, and erythrocyte membranes trigger contact activation. Furthermore, leukocytes and cancer cells may be important players in cell-mediated coagulation because, under certain conditions, they express tissue factor, release procoagulant components and can induce platelet activation. We argue that testing TG in the presence of blood cells may be useful to distinguish blood cell–related coagulation disorders. However, it should also be noted that these blood cell–dependent TG assays are not clinically validated. Further standardization and validation studies are needed to explore their clinical usefulness.
- Published
- 2021
48. Nonredundant Roles of Platelet Glycoprotein VI and Integrin αIIbβ3 in Fibrin-Mediated Microthrombus Formation
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Paola E. J. van der Meijden, Gina Perrella, Jingnan Huang, Mark Roest, Floor C J I Heubel-Moenen, Isabella Provenzale, Richard W. Farndale, Robert A. S. Ariëns, Steve P. Watson, Frauke Swieringa, Martine Jandrot-Perrus, Mark R Thomas, Johan W. M. Heemskerk, Biochemie, RS: Carim - B03 Cell biochemistry of thrombosis and haemostasis, MUMC+: MA Hematologie (9), Interne Geneeskunde, RS: Carim - B04 Clinical thrombosis and Haemostasis, and RS: Carim - B01 Blood proteins & engineering
- Subjects
Male ,0301 basic medicine ,ADHESION ,030204 cardiovascular system & hematology ,0302 clinical medicine ,fibrin ,blood platelet ,biology ,Chemistry ,Microfluidic Analytical Techniques ,Ligand (biochemistry) ,thrombin ,3. Good health ,Cell biology ,platelet aggregation ,Female ,GPVI ,Cardiology and Cardiovascular Medicine ,Thrombasthenia ,KEY ROLE ,medicine.drug ,Blood Platelets ,VON-WILLEBRAND-FACTOR ,Integrin ,microfluidics ,Platelet Glycoprotein GPIIb-IIIa Complex ,Platelet Membrane Glycoproteins ,Fibrin ,03 medical and health sciences ,Tissue factor ,Platelet Adhesiveness ,PROCOAGULANT ACTIVITY ,Thrombin ,Von Willebrand factor ,KINASE ,medicine ,Humans ,Syk Kinase ,Calcium Signaling ,Thrombus ,Blood Coagulation ,RECEPTOR ,Thrombosis ,medicine.disease ,COLLAGEN ,THROMBUS FORMATION ,030104 developmental biology ,TISSUE FACTOR ,Case-Control Studies ,biology.protein - Abstract
Objective: Fibrin is considered to strengthen thrombus formation via integrin αIIbβ3, but recent findings indicate that fibrin can also act as ligand for platelet glycoprotein VI. Approach and Results: To investigate the thrombus-forming potential of fibrin and the roles of platelet receptors herein, we generated a range of immobilized fibrin surfaces, some of which were cross-linked with factor XIIIa and contained VWF-BP (von Willebrand factor-binding peptide). Multicolor microfluidics assays with whole-blood flowed at high shear rate (1000 s −1 ) indicated that the fibrin surfaces, regardless of the presence of factor XIIIa or VWF-BP, supported platelet adhesion and activation (P-selectin expression), but only microthrombi were formed consisting of bilayers of platelets. Fibrinogen surfaces produced similar microthrombi. Markedly, tiggering of coagulation with tissue factor or blocking of thrombin no more than moderately affected the fibrin-induced microthrombus formation. Absence of αIIbβ3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca 2+ signaling and aggregation, while inhibition of Syk further reduced these responses. In platelet suspension, glycoprotein VI blockage or Syk inhibition prevented fibrin-induced platelet aggregation. Microthrombi on fibrin surfaces triggered only minimal thrombin generation, in spite of thrombin binding to the fibrin fibers. Conclusions: Together, these results indicate that fibrin fibers, regardless of their way of formation, act as a consolidating surface in microthrombus formation via nonredundant roles of platelet glycoprotein VI and integrin αIIbβ3 through signaling via Syk and low-level Ca 2+ rises.
- Published
- 2021
49. Plasmatic Coagulation Capacity Correlates With Inflammation and Abacavir Use During Chronic HIV Infection
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Wouter A van der Heijden, Mihai G. Netea, Jun Wan, Lisa Van de Wijer, Mark Roest, Martin Jaeger, Quirijn de Mast, Philip G. de Groot, André J. A. M. van der Ven, and RS: Carim - B01 Blood proteins & engineering
- Subjects
Male ,Thrombomodulin ,lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,Lipopolysaccharide Receptors ,HIV Infections ,DISEASE ,Plasma ,ANTIRETROVIRAL THERAPY ,MARKERS ,Abacavir ,Pharmacology (medical) ,Prospective Studies ,RISK ,virus diseases ,ASSOCIATION ,Middle Aged ,C-Reactive Protein ,Infectious Diseases ,Coagulation ,thrombin generation ,Biomarker (medicine) ,Female ,medicine.symptom ,medicine.drug ,Adult ,Cart ,Anti-HIV Agents ,Inflammation ,Fibrin Fibrinogen Degradation Products ,All institutes and research themes of the Radboud University Medical Center ,PLATELET REACTIVITY ,D-dimer ,medicine ,Humans ,coagulation ,Blood Coagulation ,thrombosis ,business.industry ,MORTALITY ,abacavir ,Dideoxynucleosides ,INDIVIDUALS ,Regimen ,lnfectious Diseases and Global Health Radboud Institute for Health Sciences [Radboudumc 4] ,Cross-Sectional Studies ,MYOCARDIAL-INFARCTION ,inflammation ,Immunology ,business ,Biomarkers ,Ex vivo - Abstract
Background: D-dimer concentrations in people living with HIV (PLHIV) on combination antiretroviral therapy (cART) are increased and have been linked to mortality. D-dimer is a biomarker of in vivo coagulation. In contrast to reports on D-dimer, data on coagulation capacity in PLHIV are conflicting. In this study, we assessed the effect of cART and inflammation on coagulation capacity. Setting: We explored coagulation capacity using calibrated thrombin generation (TG) and linked this to persistent inflammation and cART in a cross-sectional study including PLHIV with viral suppression and uninfected controls. Methods: We used multivariate analyses to identify independent factors influencing in vivo coagulation (D-dimer) and ex vivo coagulation capacity (TG). Results: Among 208 PLHIV, 94 (45%) were on an abacavir-containing regimen. D-dimer levels (219.1 vs 170.5 ng/mL, P = 0.001) and inflammatory makers (sCD14, sCD163, and high-sensitive C-reactive protein) were increased in PLHIV compared with those in controls (n = 56). PLHIV experienced lower TG (reflected by endogenous thrombin potential [ETP]) when compared with controls, after correction for age, sex, and antiretroviral therapy. Abacavir use was independently associated with increased ETP. Prothrombin concentrations were strongly associated with ETP and lower in PLHIV on a non-abacavir-containing regimen compared with those in controls, suggesting consumption as a possible mechanism for HIV-associated reduction in TG. D-dimer concentrations were associated with inflammation, but not TG. Conclusions: Abacavir use was associated with increased TG and could serve as an additional factor in the reported increase in thrombotic events during abacavir use. Increased exposure to triggers that propagate coagulation, such as inflammation, likely underlie increased D-dimer concentrations found in most PLHIV.
- Published
- 2021
50. Acute exacerbations of COPD induce a prothrombotic state through platelet-monocyte complexes, endothelial activation and increased thrombin generation
- Author
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Philip G. de Groot, Jasper A. Remijn, Dana Huskens, Sami O. Simons, Lisa N. van der Vorm, Janine J. J. Hulstein, Hugo ten Cate, Bas de Laat, Mark Roest, and Li Li
- Subjects
medicine.medical_specialty ,biology ,Exacerbation ,Endothelium ,business.industry ,Convalescence ,media_common.quotation_subject ,Systemic inflammation ,Gastroenterology ,Endothelial activation ,medicine.anatomical_structure ,Von Willebrand factor ,Internal medicine ,medicine ,biology.protein ,Platelet ,Platelet activation ,medicine.symptom ,business ,media_common - Abstract
Background: Patients with chronic obstructive pulmonary disease (COPD) are at increased risk for cardiovascular events, particularly following an acute exacerbation (AE-COPD). Aims: This prospective cohort study aimed to determine the effects of an AE-COPD on platelet activation, the endothelium and plasmatic coagulation, and its association with systemic inflammation. Methods: Fifty-two patients were included. Blood samples at admission, day 3 of treatment and at convalescence were collected. Platelet-monocyte complex (PMC) formation, monocyte Mac-1 expression and platelet (re)activity (P-selectin expression, GPIIbIIIa activation) were measured by flow cytometry. Von Willebrand Factor (VWF) and thrombin generation (TG) were determined as measures of endothelial activation and plasmatic coagulation, respectively. Results: Exacerbations were associated with increased PMCs (MFI 31.3 vs 23.8, p=0.004) and Mac-1 (MFI 38.2 vs 34.8, p=0.006) compared to convalescence, but not with changes in platelet (re)activity. VWF (antigen, activity, active fraction) and TG were all significantly higher during AE-COPD compared to convalescence. PMCs, Mac-1, VWF and TG were positively associated with systemic inflammation (CRP). Moreover, platelet hyperreactivity on admission was associated with an increased risk for exacerbation relapse. Conclusion: In conclusion, acute exacerbations induce an inflammation-associated prothrombotic state, characterized by increased PMCs, endothelial activation and plasmatic coagulation. Our findings provide direction for future studies on biomarkers predicting the risk of relapse and cardiovascular events.
- Published
- 2020
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