GPVI Interaction with Polymerized Fibrin Boosts Thrombin Generation and Thrombus Growth

Abstract Background: Fibrin, the end product of the coagulation cascade, consolidates the platelet plug at site of thrombosis: polymerized fibrin supports platelet adhesion under low and high shear rate conditions (Hantgan RR et al., Thromb Haemost 1992) and triggers platelet procoagulant activity (Beguin S et al., Blood 1999). These responses are largely independent of the integrin αIIβ3 and are carried by a yet ill-defined receptor. Platelet glycoprotein VI (GPVI) has a well-established key role in the initiation of thrombosis since it supports collagen-mediated platelet activation but it has recently been recognized to interact with other macromolecules such as fibronectin, vitronectin and laminins. We hypothesized GPVI could be the “missing” platelet receptor of fibrin. Aim of the study: to challenge the hypothesis that glycoprotein VI (GPVI) could be a functional fibrin receptor Methods: Thrombin generation was measured using calibrated automated thrombogram (CAT) in PRP from healthy volunteers, four GPVI-deficient patients and one patient with a fibrinogen deficiency. CAT was also performed on washed platelets mixed with prothrombin complex (FII, FVII, FIX, FX), antithrombin and fibrinogen. GPVI was blocked using the Fab of the monoclonal antibody 9O12. Fibrin polymerization was blocked using the GPRP peptide. GPVI binding to fibrin was measured in vitro using recombinant soluble GPVI (GPVI-Fc). Flow based adhesion assays were performed in capillary chambers coated with polymerized fibrin at variable shear rates and platelet morphological changes analyzed by scanning electron microscopy. The formation of fibrin-platelet thrombi was visualized by perfusing recalcified blood containing A647 fibrinogen in flow chambers (Vena8 Fluoro+ Cellix) coated with collagen and tissue factor. In a second step, the perfusion of hirudinated blood in which platelets were stained by A488-RAM1 allowed to visualize platelet recruitment by fibrin rich clots. Results: Thrombin generation triggered by tissue factor was impaired in the PRP of patients with a GPVI deficiency or in the presence of the Fab 9O12 as indicated by a respective decrease in the peak height of 45 and 25% as compared to controls. This effect was observed regardless the trigger of thrombin generation and required platelet activation. Measuring thrombin generation in a purified system showed that fibrinogen dose-dependently increased the thrombin peak by up to 150% at 3 mg/mL but the Fab 9O12 blunted this effect. Moreover, the Fab 9O12 had no effect on thrombin generation in the PRP of a fibrinogen-deficient patient confirming a GPVI/fibrin(ogen)interplay. Blocking fibrin polymerization by GPRP reduced the thrombin peak in normal PRP, in fibrinogen-supplemented PRP of the fibrinogen-deficient patient and in purified conditions. In contrast GPRP had no effect on the thrombin peak in normal PRP containing the Fab 9O12 and in the PRP of GPVI-deficient patients. The proof that GPVI specifically interacts with fibrin was obtained in a binding assay showing a dose-dependent binding of GPVI-Fc to fibrin polymers that was reversed by the Fab 9O12. Platelets adhered to polymerized fibrin resulting in platelet shape change and exposure of phosphatidylserine. Platelet adhesion on a fibrin network was observed at low (300 s-1) and high (1500 s-1) shear rates with the formation of small contractile thrombi. Adhesion was decreased by 62% for 9O12-treated platelets and by 43% with the blood of GPVI-deficient mice as compared to controls. Importantly, lack of GPVI or its blockade decreased stationary adhesion indicating that GPVI is required to stabilize the interactions between platelets and fibrin. Finally when hirudinated blood was perfused at a shear rate of 1500 s-1 onto preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 93%. Conclusions: Here we show for the first time that GPVI acts as a receptor for polymerized fibrin with two major functions: GPVI interaction with polymerized fibrin triggers (i) a new loop amplifying thrombin generation and (ii) platelet recruitment at the clot surface. These, so far, unrecognized properties of GPVI confer it a key role in the maturation of the thrombus by facilitating its growth and stabilization in addition to its well-known effect in the initiation of thrombus formation. Disclosures Jandrot-Perrus: Acticor Biotech: Other. Gachet:Acticor Biotech: Other.

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Upstream Thrombus Growth Impairs Downstream Thrombogenesis in Non-Anticoagulated Blood: Effect of Procoagulant Artery Subendothelium and Non-Procoagulant Collagen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648196 ◽

1991 ◽

Vol 65 (05) ◽

pp. 596-600 ◽

Cited By ~ 14

Author(s):

Kjell S Sakariassen ◽

Harvey J Weiss ◽

Hans R Baumgartner

Keyword(s):

Shear Rate ◽

Tissue Factor ◽

Platelet Adhesion ◽

Rabbit Aorta ◽

Axial Position ◽

Fibrillar Collagen ◽

Fibrin Deposition ◽

Clotting Factors ◽

Shear Rates ◽

Platelet Deposition

SummaryIn the present experiments we have investigated the influence of wall shear rate and axial position on platelet and fibrin deposition which results when flowing human non-anticoagulated blood is exposed to either non-procoagulant fibrillar collagen (human type III) or procoagulant subendothelium (rabbit aorta). Platelet adhesion, thrombus volume and fibrin deposition were morphometrically evaluated at axial positions of 1 and 13 mm following perfusions for 5 min at shear rates of 100, 650 and 2,600 s-1.An axially-dependent decrease of platelet adhesion (34-57%, p <0.01-0.05) and thrombus volume (57-80%, p <0.05) was observed on collagen at all shear rates. On subendothelium, an axially-dependent decrease was observed for platelet adhesion only at 100 s-1 (29% ; p <0.01) and for thrombus volume at shear rates of 650 s-1 and above (49-58%, p <0.01). Deposition of fibrin on subendothelium was axially decreased (16-42%, p <0.05) at all shear rates, while no significant axial differences were seen on collagen. However, substantially more fibrin was deposited on the subendothelium (p <0.05), and the upstream platelet adhesion and thrombus volume were lower than on collagen (p <0.05) at 100 s-1 and 650 s-1. The axially-dependent phenomena on the two surfaces are consistent with the concept of rapid-growing upstream thrombi which deplete the blood layer streaming adjacent to the surface of platelets, leading to decreased platelet deposition farther downstream. The observations suggest that deposition of fibrin is enhanced by subendothelial tissue factor, and that upstream depletion of clotting factors may lower the downstream deposition of fibrin, analogous to the depletion of platelets.

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Doxepin inhibits GPVI-dependent platelet Ca2+ signaling and collagen-dependent thrombus formation

AJP Cell Physiology ◽

10.1152/ajpcell.00262.2016 ◽

2017 ◽

Vol 312 (6) ◽

pp. C765-C774 ◽

Cited By ~ 2

Author(s):

Sascha Geue ◽

Britta Walker-Allgaier ◽

Daniela Eißler ◽

Roland Tegtmeyer ◽

Malte Schaub ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Tricyclic Antidepressant ◽

Thrombotic Occlusion ◽

Shear Rates ◽

Glycoprotein Vi ◽

Primary Hemostasis ◽

Intracellular Ca

Platelet adhesion, activation, and aggregation are essential for primary hemostasis, but are also critically involved in the development of acute arterial thrombotic occlusion. Stimulation of the collagen receptor glycoprotein VI (GPVI) leads to phospholipase Cγ2-dependent inositol triphosphate (IP3) production with subsequent platelet activation, due to increased intracellular Ca2+ concentration ([Ca2+]i). Although tricyclic antidepressants have been shown to potentially impair platelet activation, nothing is hitherto known about potential effects of the tricyclic antidepressant doxepin on platelet Ca2+ signaling and thrombus formation. As shown in the present study, doxepin significantly diminished the stimulatory effect of GPVI agonist collagen-related peptide (CRP) on intracellular Ca2+ release as well as subsequent extracellular Ca2+ influx. Doxepin was partially effective by impairment of CRP-dependent IP3 production. Moreover, doxepin abrogated CRP-induced platelet degranulation and integrin αIIbβ3 activation and aggregation. Finally, doxepin markedly blunted in vitro platelet adhesion to collagen and thrombus formation under high arterial shear rates (1,700−s). In conclusion, doxepin is a powerful inhibitor of GPVI-dependent platelet Ca2+ signaling, platelet activation, and thrombus formation.

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Prospective Assessment of Biomarkers of Hypercoagulability in Oncological Patients and Healthcare Workers Following Vaccination Against Sars-Cov-2 with the mRNA Vaccine. the Roadmap-COVID-19-Vaccin Study

Blood ◽

10.1182/blood-2021-146803 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3207-3207

Author(s):

Patrick Van Dreden ◽

Joseph Gligorov ◽

Evangelos Terpos ◽

Mathieu Jamelot ◽

Michele Sabbah ◽

...

Keyword(s):

Endothelial Cell ◽

Tissue Factor ◽

Platelet Activation ◽

Research Funding ◽

Thrombin Generation ◽

Cell Activation ◽

Control Group ◽

Clotting Time ◽

Endothelial Cell Activation ◽

Active Cancer

Abstract Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the 'cytokine storm' resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program. Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer. Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group. Results: All vaccinated subjects showed significantly increased levels of PF4 (71% >UNL, p<0.001), D-Dimers (74% >UNL, p<0.01), vWF (60% >UNL, p<0.01), FVIII (62% >UNL, p<0.01) and shorter Procoag-PPL clotting time (96% <LNL, p<0.001), as compared to controls. Thrombin generation showed significantly higher Peak (60% >UNL, p<0.01), ETP (38% >UNL, p<0.01) and MRI (66% >UNL, p<0.01) but no differences in lag-time in vaccinated subjects as compared to the control group. Vaccinated subjects did not show any increase at the levels of TFa, TFPI, TM and E-selectin in comparison with the control group. The studied biomarkers were not significantly different between the VOnco and VHcw groups. Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even in patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. Disclosures Gligorov: Roche-Genentech: Research Funding; Novartis: Research Funding; Onxeo: Research Funding; Daichi: Research Funding; MSD: Research Funding; Eisai: Research Funding; Genomic Heatlh: Research Funding; Ipsen: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding. Terpos: Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Beigene: Honoraria; Takeda: Honoraria.

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Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Blood ◽

10.1182/blood.v70.4.1214.bloodjournal7041214 ◽

1987 ◽

Vol 70 (4) ◽

pp. 1214-1217

Author(s):

E Fressinaud ◽

D Baruch ◽

C Rothschild ◽

HR Baumgartner ◽

D Meyer

Keyword(s):

Shear Rate ◽

Von Willebrand Factor ◽

Platelet Adhesion ◽

Von Willebrand Disease ◽

High Shear ◽

Shear Rates ◽

High Shear Rates ◽

Von Willebrand ◽

Willebrand Factor

Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.

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In vitro effects of human neutrophil cathepsin G on thrombin generation: Both acceleration and decreased potential

Thrombosis and Haemostasis ◽

10.1160/th09-10-0690 ◽

2010 ◽

Vol 104 (09) ◽

pp. 514-522 ◽

Cited By ~ 3

Author(s):

Thomas Lecompte ◽

Agnès Tournier ◽

Lise Morlon ◽

Monique Marchand-Arvier ◽

Claude Vigneron ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Time Course ◽

Anticoagulant Activity ◽

Cathepsin G ◽

Coagulation Cascade ◽

Clotting Factor ◽

Factor V ◽

Clotting Factors

SummaryCathepsin G (Cath G), a serine-protease found in neutrophils, has been reported to have effects that could either facilitate or impede coagulation. Thrombin generation (CAT method) was chosen to study its overall effect on the process, at a plasma concentration (240 nM) observed after neutrophil activation. Coagulation was triggered by tissue factor in the presence of platelets or phospholipid vesicles. To help identify potential targets of Cath G, plasma depleted of clotting factors or of inhibitors was used. Cath G induced a puzzling combination of two diverging effects of varying intensities depending on the phospholipid surface provided: accelerating the process under the three conditions (shortened clotting time by up to 30%), and impeding the process during the same thrombin generation time-course since thrombin peak and ETP (total thrombin potential) were decreased, up to 45% and 12%, respectively, suggestive of deficient prothrombinase. This is consistent with Cath G working on at least two targets in the coagulation cascade. Our data indicate that coagulation acceleration can be attributed neither to platelet activation and nor to activation of a clotting factor. When TFPI (tissue factor pathway inhibitor) was absent, no effect on lag time was observed and the anticoagulant activity of TFPI was decreased in the presence of Cath G. Consistent with the literature and the hypothesis of deficient prothrombinase, experiments using Russel’s Viper Venom indicate that the anticoagulant effect can be attributed to a deleterious effect on factor V. The clinical relevance of these findings deserves to be studied.

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Platelet adhesion to collagen types I through VIII under conditions of stasis and flow is mediated by GPIa/IIa (alpha 2 beta 1-integrin)

Blood ◽

10.1182/blood.v83.5.1244.1244 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1244-1250 ◽

Cited By ~ 174

Author(s):

EU Saelman ◽

HK Nieuwenhuis ◽

KM Hese ◽

PG de Groot ◽

HF Heijnen ◽

...

Keyword(s):

Shear Rate ◽

Platelet Adhesion ◽

Collagen Type ◽

Divalent Cations ◽

Collagen Types ◽

Shear Rates ◽

Rate Dependent ◽

Platelet Receptor ◽

Static Conditions ◽

Type V

Abstract Platelet adhesion to fibrillar collagens (types I, II, III, and V) and nonfibrillar collagens (types IV, VI, VII, and VIII) was investigated in the presence of physiologic concentrations of divalent cations under conditions of stasis and flow. Under static conditions, platelet adhesion was observed to collagen types I through VII but not to type VIII. Under flow conditions, platelet adhesion to collagen types I, II, III, and IV was almost independent of shear rates above 300/s. Collagen type V was nonadhesive. Platelet adhesion to collagen type VI was shear rate-dependent and optimal at a rate of 300/s. Collagen types VII and VIII showed minor reactivity and supported platelet adhesion only between shear rates 100 to 1,000/s. Monoclonal antibody (MoAb) 176D7, directed against platelet membrane glycoprotein Ia (GPIa; very late antigen [VLA]-alpha 2 subunit), completely inhibited platelet adhesion to all collagens tested, under conditions of both stasis and flow. Platelet adhesion to collagen type III at shear rate 1,600/s was only inhibited for 85%. The concentration of antibody required for complete inhibition of platelet adhesion was dependent on the shear rate and the reactivity of the collagen. An MoAb directed against GPIIa (VLA-beta subunit) partially inhibited platelet adhesion to collagen. These results show that GPIa-IIa is a major and universal platelet receptor for eight unique types of collagen.

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Recombinant Activated FVII (rFVIIa) Corrects Platelet Dysfunction in Hemophilic Patients: Study on Collagen and Tissue Factor Surfaces at High Shear Rates.

Blood ◽

10.1182/blood.v106.11.4034.4034 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4034-4034

Author(s):

Raul Tonda ◽

Ana M. Galan ◽

Irene Lopez-Vilchez ◽

Marcos Pino ◽

Antonio Ordinas ◽

...

Keyword(s):

Tissue Factor ◽

Cross Sections ◽

Platelet Adhesion ◽

Hemophilia A ◽

Severe Hemophilia ◽

Platelet Dysfunction ◽

Blood Samples ◽

Shear Rates ◽

High Shear Rates ◽

Exogenous Addition

Abstract Hemophilic patients suffer bleeding episodes despite having a normal bleeding time. A possible platelet dysfunction in these patients has not been deeply investigated. rFVIIa improves hemostasis of hemophilic patients, even in those who develop inhibitors. Clinical efficacy of this drug has been widely confirmed, though, its mechanism of action is not fully understood. We used the PFA-100® with specially devised cartridges whose membrane apertures were coated with collagen alone (COL) or collagen-tissue factor (COL-TF). Blood samples from normal donors or from a group of patients with severe hemophilia A, were anticoagulated with low molecular weight heparin (LMWH). We tested the ability of rFVIIa to shorten the closure times under the previous conditions. The structure of the hemostatic plugs formed on the membrane apertures were further analyzed using light microscopy on thin cross-sections. Closure times were statistically prolonged in blood samples from hemophilic patients tested with COL cartridges (255±22 s.vs.187±15 s in normal donors; p<0.05). Presence of TF in the apertures (COL-TF) caused a 20% shortening in closure times, both in normal donors and in hemophilic patients. Exogenous addition of 10 μg/ml rFVIIa to blood samples from hemophilic patients induced a further statistically significant reduction of closure times (p<0.05). This further reduction in closure times was not observed in blood samples drawn from normal individuals. Microscopical analysis of the plugs formed on the apertures showed that occlusive thrombi formed in the presence of TF are more compact and have higher occlusive capacity. Addition of FVIIa led to the formation of more organized platelet plugs which appeared further consolidated with fibrin strands within platelet masses. Patients with severe hemophilia showed platelet dysfunction that could be detected with the PFA-100® using specific cartridges. It is likely that the platelet dysfunction observed in these patients could be related to concurrent reductions in VWF that could affect platelet adhesion in these patients revealed at the very elevated shear rates used in the PFA-100®. Under these conditions, TF deposited onto the collagen-coated apertures proved to play a significant role in the initiation of hemostasis. rFVIIa improved the recruitment of platelets on COL-TF and contributed to a partial correction of the platelet dysfunction observed in patients with hemophilia A as further confirmed by the formation of more efficient aggregates in the PFA-100. In essence, rFVIIa circumvented a pre-existent platelet adhesion defect in hemophiliac patients. The pro-hemostatic action of rFVIIa was not observed in parallel studies with blood from healthy donors, indirectly suggesting a good safety profile for this agent when hemostasis is well preserved. PFA-100 could be considered as a possible monitoring system of FVIIa when hemostasis is impaired.

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Tissue Factor Firmly Immobilized on Surfaces Promotes Platelet Adhesion and Fibrin Formation in Studies with Circulating Human Blood: Importance of Shear Rate Conditions and FVIIa.

Blood ◽

10.1182/blood.v108.11.3925.3925 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3925-3925

Author(s):

Raul Tonda ◽

Irene Lopez-Vilchez ◽

Ana M. Galan ◽

Fulgencio Navalon ◽

Marcos Pino ◽

...

Keyword(s):

Tissue Factor ◽

Von Willebrand Factor ◽

Platelet Adhesion ◽

Statistical Significance ◽

Low Molecular Weight ◽

Shear Rates ◽

Fibrin Formation ◽

Closure Time ◽

Von Willebrand ◽

Willebrand Factor

Abstract While procoagulant activity of tissue factor (TF) has been widely investigated, its possible proadhesive properties towards platelets have not been studied in detail. We explored the interaction of platelets with human TF (hTF) firmly attached to a surface using anticoagulated blood with low molecular weight heparin (20 U/ml) at different shear rates. For studies at 250 s−1 and 600 s−1, TF adsorbed on a synthetic surface was exposed to circulating blood in flat perfusion devices. Deposition of platelets and fibrin formation were evaluated by morphometric, immunocytochemical and ultrastructural methods. For experiments at 5000 s−1, we used the PFA-100™ with experimental cartridges with collagen or collagen-hTF. Effect of rFVIIa was assessed in all experimental settings. Prothrombin fragment F1+2 levels were also measured. At 250 and 600 s−1 platelet interaction was 19.84±1.33% and 26.12±3.42% of the total surface respectively. Our inmunocytochemical results suggest that von Willebrand factor could mediate these interactions. Fibrin formation was significantly higher at 250 s−1 than at 600 s−1 (p<0.05). FVIIa tended to increase platelet deposition without reaching statistical significance, and raised fibrin formation and thrombin generation (p<0.05). Our At 5000 s−1, closure times in the PFA-100 were significantly shortened in the presence of hTF (154.09 ±14.69 s vs 191.45± 16.09 s with collagen alone; p<0.05). Addition of rFVIIa did not result in a further reduction of closure time. Our studies demonstrate that hTF is reactive for platelets. von Willebrand factor could mediate these interactions. Recombinant FVIIa enhances the procoagulant action of hTF at low and intermediate shear rates, but has no impact on the hemostatic performance at very elevated shear rates.

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The Effect of BIBT 986, a Novel Small Molecule Dual Inhibitor of Thrombin and Factor Xa, on Coagulation in a Model of Endotoxin Induced, Tissue Factor Triggered Coagulation Activation in Humans.

Blood ◽

10.1182/blood.v108.11.912.912 ◽

2006 ◽

Vol 108 (11) ◽

pp. 912-912 ◽

Cited By ~ 2

Author(s):

Bernd Jilma ◽

Judith M. Leitner ◽

Francesco Cardona ◽

Florian B. Mayr ◽

Christa Firbas ◽

...

Keyword(s):

Tissue Factor ◽

Platelet Activation ◽

Thrombin Generation ◽

Anticoagulant Activity ◽

Thrombin Time ◽

Coagulation Activation ◽

Dual Inhibitor ◽

Activation Markers ◽

Markers Of Inflammation ◽

Prolonged Aptt

Abstract Background: BIBT 986 is a novel potent anticoagulant that dually inhibits Factors Xa and IIa. We hypothesized that BIBT 986 would dose-dependently decrease endotoxin-induced, tissue factor triggered coagulation activation. Hence it was the aim of the study to compare with placebo the anticoagulant activity of three dosages of BIBT 986 on parameters of coagulation, platelet activation and inflammation and to examine the safety of BIBT 986 in this setting. Methods: This study was a prospective, randomized, double-blind, placebo-controlled, parallel-group dose escalation trial in 48 healthy male volunteers. Participants were randomised to receive bolus primed continuous infusions of one of the three doses of BIBT 986 or placebo. All of them received a bolus infusion of 2ng/kg body weight lipopolysaccharide (LPS). Results: BIBT dose-dependently increased anti-Xa activity, activated partial thromboplastin time (APTT), ecarin clotting time (ECT), thrombin time (TT) and the international normalisation ratio (INR). Importantly, BIBT 986 dose-dependently blocked the LPS-induced coagulation as assessed by the in vivo markers of thrombin generation and action: BIBT 986 doses that prolonged APTT by 25% were already effective. The BIBT dose that prolonged APTT by 100%, completely suppressed the increase in prothrombin fragment (F1+2), thrombin-antithrombin complexes (TAT) and D-dimer. BIBT 986 had no influence on activation markers of inflammation, fibrinolysis, endothelial or platelet activation. Conclusion: Infusion of BIBT 986 was safe and well tolerated. BIBT 986 specifically and dose-dependently blocked LPS-induced, tissue factor trigger coagulation. When compared to different anticoagulants tested previously in this standardized model, BIBT 986 was more effective in suppressing thrombin generation (F1+2 levels) than standard doses of danaparoid, dalteparin or lepirudin. BIBT 986 represents the first drug of a new class of dual FXa and FIIa inhibitors, and displays high potency.

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Revised Model for Platelet Adhesion to Collagen.

Blood ◽

10.1182/blood.v114.22.2999.2999 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2999-2999

Author(s):

Lucia Stefanini ◽

Moritz Stolla ◽

Sean F Maloney ◽

Timothy Daniel Ouellette ◽

Claire Roden ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Mapk Signaling ◽

Low Flow ◽

Flow Conditions ◽

Integrin Activation ◽

Shear Rates ◽

Slow Kinetics ◽

Rap1 Activation

Abstract Abstract 2999 Poster Board II-968 The Gi-coupled ADP receptor, P2Y12, is the target of clopidogrel bisulfate (Plavix), currently the most successful anti-platelet strategy used in the clinic. In a recent study, we have shown that the Ca2+-sensing nucleotide exchange factor, CalDAG-GEFI, and P2Y12 represent the major signaling pathways leading to Rap1 and integrin activation in platelets (Cifuni et al., 2008, Blood). In the present study, we have further evaluated the importance of CalDAG-GEFI signaling and Rap1 activation for various aspects of platelet activation, and we have compared thrombus formation of CalDAG-GEFI−/− and WT/clopidogrel platelets under static and flow conditions in vitro. Our studies establish a revised model for platelet activation by collagen. In platelets activated with threshold concentrations of GPVI agonists, CalDAG-GEFI serves as a highly sensitive response element to Ca2+ that allows for the rapid activation of Rap1. CalDAG-GEFI-mediated Rap1 activation triggers a first wave of integrin activation and ERK (MAPK) signaling, followed by TxA2 release. TxA2 provides crucial feedback for the activation of PKC and granule/ADP release. ADP in turn triggers the second, P2Y12-dependent wave of Rap1-mediated signaling events, leading to the sustained activation of integrins and further release of TxA2. Higher concentrations of GPVI agonists lead to the concomitant activation of CalDAG-GEFI and PKC, facilitating platelet aggregation independent of feedback by endogenous TxA2. Under physiological flow conditions, CalDAG-GEFI-dependent platelet activation (clopidogrel-treated WT platelets) allowed for the formation of small but unstable thrombi, which rapidly disintegrated at high shear rates. In contrast, CalDAG-GEFI−/− platelets (P2Y12-dependent platelet activation) in anticoagulated blood firmly adhered to the thrombogenic surface but failed to form thrombi, even at high concentrations of collagen. Addition of exogenous TxA2 to anticoagulated CalDAG-GEFI−/− blood did not restore thrombus formation under flow. However, small thrombi were observed with non-anticoagulated CalDAG-GEFI−/− blood perfused at venous but not arterial shear rates, suggesting that a) locally generated thrombin facilitates the recruitment of free flowing CalDAG-GEFI−/− platelets to already adherent platelets, and b) the slow kinetics of P2Y12-dependent Rap1 activation only supports thrombin-induced platelet-platelet cohesion at low shear conditions. In conclusion, our studies demonstrate that CalDAG-GEFI/Rap1 signaling plays a critical role for the first wave of integrin activation and TxA2 generation important for platelet adhesion to a thrombogenic surface. Signaling by P2Y12/Rap1 is essential for sustained platelet activation/thrombus stabilization and partially compensates for CalDAG-GEFI/Rap1-mediated platelet adhesion under low flow conditions. Disclosures: No relevant conflicts of interest to declare.

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