scholarly journals Rivaroxaban Effects Illustrate the Underestimated Importance of Activated Platelets in Thrombin Generation Assessed by Calibrated Automated Thrombography

2019 ◽  
Vol 8 (11) ◽  
pp. 1990 ◽  
Author(s):  
Stephanie Makhoul ◽  
Marina Panova-Noeva ◽  
Véronique Regnault ◽  
Wolfram Ruf ◽  
Philip Wenzel ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Underlying Mechanism ◽  
Second Phase ◽  
Factor X ◽  
Activated Platelets ◽  
Cat Assay ◽  
Tissue Factor Pathway ◽  
Coagulation Pathway ◽  
Potential Use

Background: The direct oral anticoagulant rivaroxaban inhibiting specifically activated factor X (FXa) causes delayed thrombin generation (TG) as measured by calibrated automated thrombography (CAT). The implications of these changes for assessing bleeding or residual prothrombotic risks of patients are unclear in the absence of a better understanding of the underlying mechanism. Methods: We compared platelet rich plasma (PRP) without or with prior collagen-induced platelet aggregation (agPRP) in the CAT assay to better characterize TG in the presence of rivaroxaban. Results: In the presence of rivaroxaban, TG curves in agPRP showed a distinct profile with a rapidly ascending phase followed with a protracted phase. Inhibition of tissue factor pathway inhibitor amplified the first phase of the curve which was also modulated by procoagulant phospholipids. Inhibition of FXIIa-dependent FXI activation revealed that aggregated platelets influenced the first phase by a combination of extrinsic and intrinsic coagulation pathway initiations. Thrombin-dependent amplification of TG (even prior collagen activation) was responsible for the second phase of the TG curve. Conclusions: AgPRP fully includes platelet ability to support TG and reveal distinct TG phases in the presence of direct FXa inhibitors highlighting its potential use in an anticoagulated setting.

Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Susan A Maroney ◽  
Rodney M Camire ◽  
Alan E Mast
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Platelet Rich Plasma ◽  
Anticoagulant Activity ◽  
Factor V Leiden ◽  
Factor Xa ◽  
Factor V ◽  
Activation Threshold ◽  
Tissue Factor Pathway

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.

scholarly journals von Willebrand factor binding to myosin assists in coagulation

2020 ◽  
Vol 4 (1) ◽  
pp. 174-180 ◽  
Author(s):  
Veronica H. Flood ◽  
Tricia L. Slobodianuk ◽  
Daniel Keesler ◽  
Hannah K. Lohmeier ◽  
Scot Fahs ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor V ◽  
Factor X ◽  
Skeletal Muscle Myosin ◽  
Myosin Binding ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factor V and factor X. We studied whether VWF can also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation. A myosin-binding assay was developed using skeletal muscle myosin to measure VWF binding, and plasma-derived and recombinant VWF containing molecular disruptions at key VWF sites were tested. Competition assays were performed using anti-VWF antibodies. FVIII binding to myosin was measured using a chromogenic FVIII substrate. Thrombin generation was measured using a fluorogenic substrate with and without myosin. Wild-type recombinant VWF and human plasma VWF from healthy controls bound myosin, whereas plasma lacking VWF exhibited no detectable myosin binding. Binding was multimer dependent and blocked by anti-VWF A1 domain antibodies or A1 domain VWF variants. The specific residues involved in myosin binding were similar, but not identical, to those required for collagen IV binding. FVIII did not bind myosin directly, but FVIII activity was detected when VWF and FVIII were bound to myosin. Myosin enhanced thrombin generation in platelet-poor plasma, although no difference was detected with the addition of myosin to platelet-rich plasma. Myosin may help to facilitate delivery of FVIII to sites of injury and indirectly accelerate thrombin generation by providing a surface for VWF binding in the setting of trauma and myosin exposure.

scholarly journals The Effect of Trace Amounts of Tissue Factor on Thrombin Generation in Platelet Rich Plasma, its Inhibition by Heparin

1989 ◽  
Vol 61 (01) ◽  
pp. 025-029 ◽  
Author(s):  
S Béguin ◽  
T Lindhout ◽  
H C Hemker
Keyword(s):  
Synergistic Effect ◽  
Human Brain ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Thrombin Inhibitor ◽  
Platelet Poor Plasma ◽  
Activated Platelets ◽  
Low Concentrations

SummaryAmounts of human brain thromboplastin that do not stimulate thrombin generation in platelet poor plasma, were shown to advance by about 4 min an explosive formation of thrombin that occurs after recalcification in the presence of blood platelets. This synergistic effect is inhibited by the specific thrombin inhibitor hirudin and mimicked by adding low concentrations (< 5 nM) of thrombin to platelet rich plasma. It is our conclusion that, small amounts of thrombin, generated under the influence of thromboplastin induced procoagulant activity in the blood platelets. This activity is most likely mainly due to procoagulant phospholipids. Heparin inhibits this effect and retards the explosive thrombin formation. It does not, however, diminish the peak amount of thrombin eventually formed, because heparin neutralizing material released from the activated platelets quenches the heparin effect.

Contribution of Platelets and Erythrocytes to Thrombin Generation in Patients with β-Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3820-3820
Author(s):  
Juana Valles ◽  
Marta Piñon ◽  
Maria A. Dasi ◽  
Antonio Moscardo ◽  
Maria P. Fuset ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Normal Subjects ◽  
Cell Types ◽  
Thalassemia Major ◽  
Phosphatidyl Serine ◽  
Activation Markers ◽  
Activated Platelets ◽  
Transfusion Requirements ◽  
Thalassemia Minor

Abstract Patients with β-thalassemia show an abnormally high proportion of red blood cells (RBCs) with exposed phosphatidyl serine (PS) molecules on the RBC membrane, which may contribute to thrombophilia in these patients (1). We have reported previously (2) that thromboxane A2 and free-arachidonate—substances released from activated platelets—induce PS exposure in a subpopulation of normal RBCs. The aims of the present study were first, to evaluate the presence of activated circulating platelets in patients with β-thalassemia, and second, to determine tissue factor (TF)-initiated thrombin generation in plasma and the influence of RBCs, platelets and their cooperation on this process in normal subjects and patients with β-thalassemia. Thrombin generation was assessed according to Hemker et al. (3) in 12 patients with β-thalassemia diagnosed by molecular biology, age 5–33 years and in 18 normal subjects. Two patients had thalassemia major (T-M) requiring blood transfusion every 3 weeks and the analysis were performed just prior transfusion. Two had thalassemia intermedia (T-I) with esplenectomy without transfusion requirements and eight had thalassemia minor (T-m). Thrombin generation was evaluated in platelet-poor plasma (PP), platelet-rich plasma (PRP), RBCs in the plasma (P-RBC) and in the presence of both cell types (PRP+RBC). The presence of activated platelets in circulation was evaluated in whole blood by flow cytometry. We monitored P-selectin (CD-62) and the activated αIIbβ3 receptor, defined as PAC-1 binding. The percentage of platelets exposing activation markers CD-62 (mean± SEM, 0.72± 0.17 vs. 4.03± 1.39) and PAC-1(0.75± 0.14 vs. 12.26± 6.47) were significantly higher in patients than in control subjects (P < 0.05). In normal subjects, the presence of erythrocytes in plasma (P-RBC) triggered a quicker thrombin generation and a significant elevation in the thrombin peak (32 ± 5 nM in PP vs. 86 ± 16 nM in P-RBC; P < 0.05), in the absence of cell lysis. These effects of RBCs were markedly higher in the patients with β-thalassemia, who had 2.5-fold higher thrombin generation at 3 min and higher peak thrombin levels (86 ± 16 nM in controls vs. 130 ± 15 nM in patients; P < 0.05). Thrombin generation in PRP was also higher in the patients with thalassemia at early times (3 min; 19 ± 3 nM in controls vs. 43 ± 8 nM in patients; P < 0.05). Importantly, the rate and peak of thrombin generation were significantly amplified by the presence of both cell types (PRP+RBCs), suggesting a biochemical cooperation between activated platelets and RBCs for thrombin generation. This was enhanced in patients with β-thalassemia, particularly those with T-I. In conclusion, chronic platelet activation in patients with β-thalassemia may influence TF-induced thrombin generation in platelets and erythrocytes. Thrombin generation appears to be a novel mode of platelet-erythrocyte cooperation in thrombogenesis.

Effect of Plasmatic Procoagulant Factors in Thrombin Generation Parameters in a Platelet-Poor Plasma Model System.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4021-4021
Author(s):  
Jogin R. Wu ◽  
Bing Bai
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Lag Time ◽  
Model System ◽  
Factor Vii ◽  
Factor X ◽  
Plasma Model ◽  
Cat Assay ◽  
Viii And Ix

Abstract The evolution of the thrombin generation (TG) concept and the recent technical advances in automated TG measurement have made its clinical utility practical but the clinical relevance of all parameters thrombin generation provided still remain unclear. In the current study we investigated effects of various procoagulant plasmatic factors in the TG parameters, such as rate, peak, lag time and area under the curve (AUC), in a platelet poor plasma model system using the calibrated automated thrombography (CAT). The plasma model system was made by mixing well-characterized commercial normal pooled human plasma with plasmas that are deficient in various procoagulant factors (factor VIII, IX, II, X, V and VII) to reach factor levels between &lt;1 % to 100% normal. Initially 5 pM of tissue factor and 4 μM of phospholipid were used and all TG parameters (lag time, peak, rate, and AUC) were compared with various levels of all procoagulant factors. Our result showed that the AUC is proportional to concentrations of factor II and X but not sensitive to factor VIII, IX and VII levels under such experimental conditions. Factor VII affects only the lag time and factor X affects both the lag time and the rate of thrombin generation. Factor II and X affect the peak as well. Factor VIII and IX affect both the rate and peak but not the AUC. Factor V plays very little role on affecting these parameters. Furthermore different concentrations of tissue factor (0.5 to 5 pM) were used to explore the sensitivity of TG for screening hemophiliac factor deficiencies. When ≤ 2pM of tissue factor and 4 μM of phospholipid were used the AUC showed three fold difference for factor VIII deficient plasma versus the normal plasma pool as compared with the less than 20% of difference in AUC (SD=15% for normal population) under the 5 pM concentration of tissue factor. Using the AUC and the rate of TG factor VIII and IX levels were well differentiated under such further diluted tissue factor concentrations (≤ 2 pM). Our study indicated that, under the standard CAT assay condition, 1) the lag time is mainly an indicator of extrinsic tenase reaction (factor VII, X); 2) the peak and rate reflect the intrinsic tenase reaction (factor X, IX, and VIII); and 3) the AUC and peak are largely affected by the prothrombinase factors (II, X) indicating the total free thrombin available for its overall plasmatic procoagulant activity. When proper concentration of triggering agent is used (≤ 2pM of tissue factor and 4 μM of phospholipid) TG becomes sensitive to all plasmatic procoagulant factors and CAT can be used for screening factor deficiencies in hemophiliac patients. A combination of these TG parameters can be used in an in-vitro screening assay to indicate the overall hemostasis balance in plasma under physiological conditions and an individual factor deficiency in related to hemostasis unbalance. Such further diluted triggering agent needs to be optimized for the CAT assay and a standardized large scale clinical retrospective study is needed to prove the utility of these parameters.

scholarly journals Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

Blood ◽  
2011 ◽  
Vol 118 (7) ◽  
pp. 1952-1961 ◽  
Author(s):  
Fabrizio Semeraro ◽  
Concetta T. Ammollo ◽  
James H. Morrissey ◽  
George L. Dale ◽  
Paul Friese ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Activation Process ◽  
Factor Xii ◽  
Contact Phase ◽  
Activated Platelets ◽  
Extracellular Histones ◽  
Dying Cells ◽  
Prothrombinase Activity

Abstract The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process.

A central role of GPIb-IX in the procoagulant function of platelets that is independent of the 45-kDa GPIbα N-terminal extracellular domain

Blood ◽  
2010 ◽  
Vol 116 (7) ◽  
pp. 1157-1164 ◽  
Author(s):  
Catherine Ravanat ◽  
Catherine Strassel ◽  
Béatrice Hechler ◽  
Simone Schuhler ◽  
Gaëtan Chicanne ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Phospholipid Composition ◽  
Extracellular Domain ◽  
Human Platelets ◽  
Normal Platelet ◽  
Activated Platelets ◽  
The Impact ◽  
Normal Calcium

Abstract Activated platelets become procoagulant and efficiently promote the conversion of prothrombin to thrombin. A role of the GPIb-V-IX complex has long been postulated in view of the decreased prothrombin consumption in Bernard-Soulier patients. We evaluated the impact of GPIb-V-IX deficiency and the requirement for the GPIbα extracellular domain. In GPIbβ−/− mice, thrombin generation was profoundly decreased in tissue factor– or collagen-related peptide (CRP)–activated platelet-rich plasma and in washed platelets supplemented with normal plasma or with FVa, FXa, and prothrombin. Phosphatidylserine (PS) exposure was similarly decreased in response to thrombin, CRP, or CRP + PAR4 peptide despite a normal platelet phospholipid composition. The hypothesis that these defects originate from lack of the GPIbα N-terminal domain was evaluated after its removal from normal mouse and human platelets with Nk protease or O-sialoglycoprotein endopeptidase. Unexpectedly, the treated platelets exhibited normal thrombin generation and PS exposure, indicating that GPIb-V-IX regulates procoagulant activity independently of its GPIbα-binding region. These results suggested a more general structuring role through intracellular cytoskeleton-anchoring portions regulating responses leading to PS exposure. This hypothesis was supported by the decreased calcium mobilization observed in GPIbβ−/− platelets in response to several agonists, some acting independently of GPIb, in contrast to the normal calcium responses in Nk protease–treated platelets.

scholarly journals Silica Nanoparticles Effects on Blood Coagulation Proteins and Platelets

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Volodymyr Gryshchuk ◽  
Natalya Galagan
Keyword(s):  
Silica Nanoparticles ◽  
Blood Coagulation ◽  
Platelet Rich Plasma ◽  
Drug Carriers ◽  
Human Platelets ◽  
Factor X ◽  
Activated Platelets ◽  
Haemostatic Agents ◽  
Coagulation Proteins

Interaction of nanoparticles with the blood coagulation is important prior to their using as the drug carriers or therapeutic agents. The aim of present work was studying of the primary effects of silica nanoparticles (SiNPs) on haemostasisin vitro. We studied the effect of SiNPs on blood coagulation directly estimating the activation of prothrombin and factor X and to verify any possible effect of SiNPs on human platelets. It was shown that SiNPs shortened coagulation time in APTT and PT tests and increased the activation of factor X induced by RVV possibly due to the sorption of intrinsic pathway factors on their surface. SiNPs inhibited the aggregation of platelet rich plasma induced by ADP but in the same time partially activated platelets as it was shown using flow cytometry. The possibility of SiNPs usage in nanomedicine is strongly dependant on their final concentration in bloodstream and the size of the particles that are used. However SiNPs are extremely promising as the haemostatic agents for preventing the blood loss after damage.

Effect of Sodium Arachidonate on Thrombin Generation through Platelet Activation – Inhibitory Effect of Aspirin

2000 ◽  
Vol 84 (12) ◽  
pp. 1109-1112 ◽  
Author(s):  
Alejandra Scazziota ◽  
Jorge Rouvier ◽  
Claudio Gonzalez ◽  
Raul Altman
Keyword(s):  
Beneficial Effect ◽  
Platelet Activation ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Lag Time ◽  
Inhibitory Effect ◽  
Prevention Of Thrombosis ◽  
Activated Platelets ◽  
The Difference

Summary Background. Sodium arachidonate was used in this study to determine its capacity to generate thrombin through platelet activation. Whether aspirin prevent this effect was also investigated. Methods and Results. Seventeen healthy volunteers without and after 160 mg/day aspirin intake for 3-5 days were studied. Lag-time and TG at basal condition and after platelet stimulation by sodium arachidonate (AA) were measured in normal non-aspirinated as well as “in vivo” aspirinated platelet rich plasma. (PRP). The lag-time was statistically significant shorter in non-aspirinated PRP activated with AA compared with non-activated PRP. This effect was inhibited by aspirin. In non-aspirinated PRP, there was an increase of TG at 4 and 6 min. incubation when platelets were activated with AA but the difference disappeared after 8 min. incubation, (84 ± 71; 148 ± 58 and 142 ± 92 nmol/L respectively) compared with non-aspirinated, non-activated platelets (16 ± 23; 55 ± 56 and 111 ± 76 nmol/L at 4, 6 and 8 min, p < 0.0001, p < 0.0001 and p = 0.292, respectively). The AUCo→22 min were 520.6 ± 545.5 in nonaspirinated, non-stimulated PRP and 808.9 ± 617, in non-aspirinated PRP activated with sodium arachidonate (p = 0.014). Aspirin administered in vivo produced a decrease of TG in PRP activated with AA. Conclusion. Platelet activated by AA trigged TG. This effect was inhibited by aspirin and could be an additional beneficial effect of aspirin in the prevention of thrombosis.

scholarly journals The Effect of Fibrin Clots and Clot-Bound Thrombin on the Development of Platelet Procoagulant Activity

1995 ◽  
Vol 74 (03) ◽  
pp. 962-968 ◽  
Author(s):  
Rachana Kumar ◽  
Suzette Béguin ◽  
H Coenraad Hemker
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Phosphatidyl Serine ◽  
Procoagulant Activity ◽  
Flip Flop ◽  
Activated Platelets ◽  
Absolute Requirement ◽  
Different Types ◽  
Rate Limiting ◽  
Fibrin Clots

SummaryWe tested different types of clot for their ability to provoke procoagulant activity in platelets: normal clots from platelet poor plasma (des AABB- or fibrin II clots), similar clots in which the adsorbed thrombin has been inhibited by hirudin, and clots obtained by the action of two snake venom enzymes that release only fibrinopeptide A (des AA- or fibrin I clots). Analogous clots from fibrinogen solutions were also tested.In platelet rich plasma (PRP), where platelet coagulant phospholipids (PCP) are rate limiting for thrombin generation, the addition of any type of clot enhances the generation of thrombin thus it induces the appearance of PCP. Clots containing active adsorbed thrombin are the most potent ones in this respect. Lactate dehydrogenase (LDH) levels do not increase in the course of the thrombin generation so the platelets are not damaged in the process. Non-centrifugable PCP could be demonstrated to appear during the process, so the production of procoagulant phospholipid microparticles must be part of the mechanism. Membrane transbilayer phosphatidyl serine movement (flip-flop) can not be demonstrated in PRP as the activated platelets are caught in the emerging clot.In order to demonstrate flip-flop, we tried to investigate the influence of clots on washed platelets. However, contrary to platelets in a plasma milieu, isolated platelets are damaged by fibrin clots, especially in the presence of thrombin, as can be judged from the appearance of LDHWe conclude that, in PRP, clots induce the appearance of PCP from platelets by vesiculation, possibly accompanied by flip-flop and that thrombin accelerates the process but is not an absolute requirement

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