scholarly journals Effect of blood flow on thrombin generation is dependent on the nature of the thrombogenic surface

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2206-2213 ◽  
Author(s):  
A Diquelou ◽  
S Lemozy ◽  
D Dupouy ◽  
B Boneu ◽  
K Sakariassen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Shear Rate ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Type Iii Collagen ◽  
Fibrin Deposition ◽  
Positive Correlation ◽  
Shear Rates ◽  
Fibrin Formation

We have investigated the influence of blood flow on thrombin generation, fibrin formation, and fibrin deposition on procoagulant and nonprocoagulant surfaces. Nonanticoagulated human blood was drawn for 5 minutes directly from an antecubital vein over stimulated endothelial cells expressing tissue factor and over human type III collagen fibrils, positioned in parallel-plate perfusion chambers. The shear rates at these surfaces were 50, 650, and 2,600 s-1. Deposition of platelets and fibrin was measured by morphometry. Thrombin and fibrin formation was determined by measuring prothrombin fragments 1 + 2 (F 1 + 2), thrombin-antithrombin III complexes, (T-AT) and fibrinopeptide A (FPA) in blood effluent from the perfusion chamber at the end of the 5- minute perfusion period. On procoagulant endothelial cells, the thrombi were primarily composed of fibrin. The fibrin deposition (81%, 21%, and 2% at 50, 650, and 2,600 s-1, respectively) and plasma levels of F 1 + 2, T-AT and FPA were shear rate dependent and highest at 50 s-1. There was a positive correlation between F 1 + 2 and T-AT and the fibrin deposition (P < .01). In contrast, the collagen surface triggered primarily thrombi that were composed of platelets. The platelet thrombi and plasma levels of F 1 + 2 and T-AT were also dependent on the shear rate, but highest at 650 and 2,600 s-1. F 1 + 2 and T-AT reached the same level as observed with procoagulant endothelial cells at the higher shear rates. There was a positive correlation between F 1 + 2 and T-AT and the platelet thrombus formation (P < .05), confirming the predominant role of platelets in thrombin generation. Thus, thrombin formation is strongly influenced by the blood flow, and this effect depends on the composition of the thrombogenic surface.

scholarly journals Effect of blood flow on thrombin generation is dependent on the nature of the thrombogenic surface

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2206-2213 ◽  
Author(s):  
A Diquelou ◽  
S Lemozy ◽  
D Dupouy ◽  
B Boneu ◽  
K Sakariassen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Shear Rate ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Type Iii Collagen ◽  
Fibrin Deposition ◽  
Positive Correlation ◽  
Shear Rates ◽  
Fibrin Formation

Abstract We have investigated the influence of blood flow on thrombin generation, fibrin formation, and fibrin deposition on procoagulant and nonprocoagulant surfaces. Nonanticoagulated human blood was drawn for 5 minutes directly from an antecubital vein over stimulated endothelial cells expressing tissue factor and over human type III collagen fibrils, positioned in parallel-plate perfusion chambers. The shear rates at these surfaces were 50, 650, and 2,600 s-1. Deposition of platelets and fibrin was measured by morphometry. Thrombin and fibrin formation was determined by measuring prothrombin fragments 1 + 2 (F 1 + 2), thrombin-antithrombin III complexes, (T-AT) and fibrinopeptide A (FPA) in blood effluent from the perfusion chamber at the end of the 5- minute perfusion period. On procoagulant endothelial cells, the thrombi were primarily composed of fibrin. The fibrin deposition (81%, 21%, and 2% at 50, 650, and 2,600 s-1, respectively) and plasma levels of F 1 + 2, T-AT and FPA were shear rate dependent and highest at 50 s-1. There was a positive correlation between F 1 + 2 and T-AT and the fibrin deposition (P < .01). In contrast, the collagen surface triggered primarily thrombi that were composed of platelets. The platelet thrombi and plasma levels of F 1 + 2 and T-AT were also dependent on the shear rate, but highest at 650 and 2,600 s-1. F 1 + 2 and T-AT reached the same level as observed with procoagulant endothelial cells at the higher shear rates. There was a positive correlation between F 1 + 2 and T-AT and the platelet thrombus formation (P < .05), confirming the predominant role of platelets in thrombin generation. Thus, thrombin formation is strongly influenced by the blood flow, and this effect depends on the composition of the thrombogenic surface.

scholarly journals Procoagulant activity of endotoxin-treated human endothelial cells exposed to native human flowing blood

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 729-733 ◽  
Author(s):  
M Clozel ◽  
H Kuhn ◽  
HR Baumgartner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Shear Rate ◽  
Fibrin Deposition ◽  
Human Endothelial Cells ◽  
Cellular Activation ◽  
Shear Rates ◽  
Fibrin Formation ◽  
Cultured Endothelial Cells ◽  
Flowing Blood

Abstract It has been reported that cultured endothelial cells become procoagulant when exposed to endotoxin. This prompted us to investigate whether human endothelial cells treated with endotoxin could promote the generation of fibrin when exposed to human flowing blood. For this purpose we used a parallel-plate perfusion chamber in which confluent cultured endothelial cells from human umbilical veins were exposed for five minutes to directly drawn human nonanticoagulated blood, at wall shear rates of 100, 650, and 2600 sec-1. Fibrin deposition was assessed by morphometry. No fibrin deposition occurred on normal endothelial cells. In contrast, cells incubated with endotoxin for 4 or 18 hours induced fibrin deposition, but only at a shear rate of 100 sec-1. Since some extracellular matrix was exposed between the cells, we investigated whether extracellular matrix played a role in fibrin formation. When the endothelial cells incubated or not with endotoxin were removed by EDTA, the exposed extracellular matrix perfused with blood at 100 sec-1 supported platelet and fibrin deposition in both cases. This suggests that the effect of endotoxin on endothelial cells was not due to extracellular matrix alteration but only to cellular activation or secretion of procoagulant substances. We conclude that human endothelial cells treated with endotoxin can trigger fibrin formation and deposition at their surface when exposed to flowing blood at low shear rate.

scholarly journals Procoagulant activity of endotoxin-treated human endothelial cells exposed to native human flowing blood

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 729-733
Author(s):  
M Clozel ◽  
H Kuhn ◽  
HR Baumgartner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Shear Rate ◽  
Fibrin Deposition ◽  
Human Endothelial Cells ◽  
Cellular Activation ◽  
Shear Rates ◽  
Fibrin Formation ◽  
Cultured Endothelial Cells ◽  
Flowing Blood

It has been reported that cultured endothelial cells become procoagulant when exposed to endotoxin. This prompted us to investigate whether human endothelial cells treated with endotoxin could promote the generation of fibrin when exposed to human flowing blood. For this purpose we used a parallel-plate perfusion chamber in which confluent cultured endothelial cells from human umbilical veins were exposed for five minutes to directly drawn human nonanticoagulated blood, at wall shear rates of 100, 650, and 2600 sec-1. Fibrin deposition was assessed by morphometry. No fibrin deposition occurred on normal endothelial cells. In contrast, cells incubated with endotoxin for 4 or 18 hours induced fibrin deposition, but only at a shear rate of 100 sec-1. Since some extracellular matrix was exposed between the cells, we investigated whether extracellular matrix played a role in fibrin formation. When the endothelial cells incubated or not with endotoxin were removed by EDTA, the exposed extracellular matrix perfused with blood at 100 sec-1 supported platelet and fibrin deposition in both cases. This suggests that the effect of endotoxin on endothelial cells was not due to extracellular matrix alteration but only to cellular activation or secretion of procoagulant substances. We conclude that human endothelial cells treated with endotoxin can trigger fibrin formation and deposition at their surface when exposed to flowing blood at low shear rate.

Antithrombotic properties of pravastatin reducing intra-thrombus fibrin deposition under high shear blood flow conditions

2011 ◽  
Vol 105 (02) ◽  
pp. 313-320 ◽  
Author(s):  
Masaaki Hamada ◽  
Hideto Matsui ◽  
Tomohiro Mizuno ◽  
Yasuaki Shida ◽  
Masaaki Doi ◽  
...  
Keyword(s):  
Blood Flow ◽  
Shear Rate ◽  
Whole Blood ◽  
Cardiovascular Events ◽  
Molecular Mechanisms ◽  
Favourable Effect ◽  
High Shear ◽  
Fibrin Deposition ◽  
Flow Conditions ◽  
Shear Rates

SummaryIn addition to lowering cholesterol, the 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) have a range of pleiotropic effects that help reduce the risk of adverse cardiovascular events. We sought to understand the molecular mechanisms by which statins could exert anti-platelet actions under physiologic whole blood flow conditions. Using an in vitroperfusion chamber system, we examined the anti-platelet effects of pravastatin under whole blood flow conditions with high or low shear rates. We determined that pravastatin significantly suppressed platelet activation-dependent procoagulant activity, decreasing P-selectin membrane expression, tissue factor accumulation, and thrombin binding within platelet thrombi generated on a von Willebrand factor-surface under high shear rate conditions. These effects resulted in reductions of intra-thrombus fibrin deposition. These antithrombotic properties of pravastatin, which were comparable to those of atorvastatin, could be abrogated by mevalonate. Our experimental approach revealed a novel mechanism mediating the anti-platelet action of statins. Shear rate-dependent antithrombotic activity may explain the favourable effect of pravastatin on the reduction in cardiovascular events that typically occur in vivounder whole blood flow conditions with high shear rates.

Shear rate dependent margination of sphere-like, oblate-like and prolate-like micro-particles within blood flow

Soft Matter ◽  
2018 ◽  
Vol 14 (36) ◽  
pp. 7401-7419 ◽  
Author(s):  
Huilin Ye ◽  
Zhiqiang Shen ◽  
Ying Li
Keyword(s):  
Blood Flow ◽  
Shear Rate ◽  
Shape Effect ◽  
Shear Rates ◽  
Micro Particles ◽  
Rate Dependent ◽  
Wall Shear

The shape effect of micro-particles is examined by comparing the margination behaviors of sphere-like, oblate-like and prolate-like micro-particles under different wall shear rates in blood flow.

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

1991 ◽  
Vol 65 (05) ◽  
pp. 596-600 ◽  
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.

A Factor VII Deficiency Protects Against Lethal Endotoxemia in Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 694-694
Author(s):  
Haifeng Xu ◽  
Victoria A. Ploplis ◽  
David E. Joyce ◽  
Francis J. Castellino
Keyword(s):  
Thrombin Generation ◽  
Inflammatory Responses ◽  
Factor Vii ◽  
Fibrin Deposition ◽  
Protein Levels ◽  
Fibrin Formation ◽  
Early Stages ◽  
Lps Challenge ◽  
Fibrinolytic Potential

Abstract The formation of FVIIa:TF complex is a main contributor to the coagulopathy associated with acute bacterial infection and sepsis. In this study, the role of FVII has been investigated in a murine model of lethal endotoxemia induced by LPS using genetically altered mice expressing low FVII (FVIItTA/tTA). The results demonstrated that FVIItTA/tTA mice had reduced mortality, coagulation, and inflammatory responses compared to wild-type (WT) mice. The reduced thrombin generation in FVIItTA/tTA mice at early stages of post-LPS challenge correlated with less fibrin formation, which was characterized by diminished fibrin deposition in the liver and lower D-Dimer levels in the plasma. Fibrin deposition at late stages of endotoxemia was, however, similar between WT and FVIItTA/tTA mice. There was an apparent increased activation of the intrinsic coagulation pathway in FVIItTA/tTA mice as exhibited by an increased consumption of FXII and FXI zymogen in blood, which appeared to compensate for the diminished thrombin generation characteristic of the low FVII state. Fibrinolytic potential during endotoxemia remained the same between WT and FVIItTA/tTA mice. Reduced inflammatory responses were prominent in FVIItTA/tTA mice, as demonstrated by lower IL-6, MIP-2, and higher IL-10 plasma protein levels, which correlated with the total RNA expression levels in various tissues as determined by Quantitative RT-PCR. These reduced inflammatory responses could be directly due to less FVIIa:TF:PAR-2 signaling, and/or indirectly by less thrombin:PARs signaling in FVIItTA/tTA mice. Additionally, a slight increase in parenchymal organ bleeding was observed in FVIItTA/tTA mice challenged with LPS. Results from these studies indicate that low FVII benefits survival mainly through attenuation of coagulation and inflammatory responses as the result of potentially less coagulation protease signaling. Protection by mechanisms associated with regulation of fibrin formation appears to be limited to early stages of the disease.

Critical Role of Immobilized Factor VIII In Solid-Phase Blood Coagulation During Mural Thrombogenesis Under Whole Blood Flow Conditions

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2199-2199
Author(s):  
Masaaki Doi ◽  
Mitsuhiko Sugimoto ◽  
Hideto Matsui ◽  
Tomoko Matsumoto ◽  
Midori Shima
Keyword(s):  
Blood Flow ◽  
Shear Rate ◽  
Whole Blood ◽  
Solid Phase ◽  
Glass Plate ◽  
High Shear Rate ◽  
High Shear ◽  
Fibrin Deposition ◽  
Flow Conditions

Abstract Abstract 2199 Coagulation factor VIII (FVIII), lacking in hemophilic blood, plays an essential role in mechanisms of fibrin plug formation to arrest bleeding at sites of injured vessel walls. Physiologic activity of FVIII circulating in bloodstream (soluble FVIII; S-FVIII) could be extensively evaluated so far by classic plasma coagulation assays such as activated partial thromboplastin time. However, the in vivo functional relevance of FVIII bound to von Willebrand factor (VWF) which is immobilized in subendothelium (immobilized FVIII; I-FVIII) is more complex and remains to be addressed. Using an in vitro perfusion chamber system, we have therefore evaluated the function of I-FVIII in the process of mural thrombus generation under whole blood flow conditions. FVIII-free VWF was purified in the presence of 0.35 M CaCl2 from cryoprecipitate, and coated on a glass plate. Various concentrations (0 as a control, 0.1, 0.3, 1, or 3 U/ml) of recombinant FVIII (Kogenate FS provided by Bayer Pharmaceutical Co.) were reacted with the FVIII-free VWF-coated glass plate. After non-adherent proteins were washed out, the amount of FVIII immobilized to a glass surface via VWF (I-FVIII) was measured by ELISA-based assay using a peroxidase-conjugated anti-FVIII polyclonal antibody. Whole blood was then perfused over a glass plate described above in a parallel plate flow chamber with various shear rates, and the thrombus generation process on a glass surface was observed in real time by confocal laser scanning microscopy. The development of intra-thrombus fibrin deposition was assessed by immune-staining of thrombi with a fluorescence-labeled anti-fibrin specific monoclonal antibody (NYB-T2G1; Accurate Chem.), reflecting solid-phase blood coagulation reaction during mural thrombogenesis. In perfusion of control blood with a high shear rate (1500 s-1), the intra-thrombus fibrin deposition was found to increase as a function of I-FVIII, resulting in the 2.5-fold greater fibrin deposition at the plateau as compared to control thrombi generated in the absence of I-FVIII. This I-FVIII effect on intra-thrombus fibrin deposition was also confirmed in perfusion of synthetic hemophilic blood (S-FVIII activity < 1%) which was prepared by the incubation of control blood with an anti-FVIII human IgG (final inhibitor titer in synthetic blood; 5, 10, or 20 Bethesda U/ml). Indeed, I-FVIII normalized in a dose-dependent manner the reduced fibrin deposition (20-35% of normal control) within synthetic hemophilic thrombi generated in the absence of S-FVIII under a high shear rate condition. The improvement of impaired fibrin deposition by I-FVIII was unvarying regardless of the anti-FVIII inhibitor titer in synthetic hemophilic blood. In contrast, the direct addition of recombinant FVIII into synthetic hemophilic blood was poorly effective in this regard, due to the immediate neutralization of S-FVIII by an inhibitor involved in synthetic blood. Thus, these results clearly indicate that I-FVIII, independent of S-FVIII, does play a considerable role on the intra-thrombus fibrin-network formation in the process of mural thrombus generation under whole blood flow conditions with high shear rate, most relevant physiologically for the in vivo hemostasis and thrombosis. Our results might imply a possibility of novel strategic design targeting I-FVIII against hemophilic patients with a high titer anti-FVIII inhibitor. Disclosures: No relevant conflicts of interest to declare.

Biological Explanation of Clinically Observed Elevation of TFPI Plasma Levels After Treatment with TFPI-Antagonistic Aptamer BAX 499

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1104-1104 ◽  
Author(s):  
Michael Dockal ◽  
Robert Pachlinger ◽  
Rudolf Hartmann ◽  
Sabine Knappe ◽  
Benny Sorensen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Proteolytic Processing ◽  
Full Length ◽  
Model Systems ◽  
Cell Culture Supernatant ◽  
Clinical Samples ◽  
Dependent Manner ◽  
Molar Excess

Abstract Abstract 1104 BAX 499, an aptamer that binds with a high affinity to TFPI (KD ∼0.1 nM), blocks the anticoagulant action of TFPI in plasma and model systems. BAX 499 was recently tested in a phase 1 safety, tolerability and ex vivo efficacy study in hemophilia patients. The study was prematurely stopped due to an increased number of bleeding events. Baxter subsequently performed several analyses to investigate why clinical safety was inconsistent with preclinical observations. Analysis of plasma levels of full length TFPI (fl-TFPI) and BAX 499, of global hemostatic parameters via thrombin generation (CAT) and thromboelastography (ROTEM) demonstrated that BAX 499 induced a considerable increase in fl-TFPI plasma levels: over 25-fold at the highest dose (72 mg, SubQ). Pharmacodynamic assessment showed that high fl-TFPI concentrations substantially reduce thrombin generation even at excess BAX 499 plasma concentrations (∼1 μM). This strongly suggests that the BAX 499-induced rise in plasma fl-TFPI levels caused the increased bleeding. Assessment of biological mechanisms underlying the increase in fl-TFPI plasma levels showed that BAX 499 can potentially raise plasma levels by 1) increasing the synthesis of TFPI by endothelial cells, 2) affecting the distribution of TFPI which is bound to the endothelial surface, stored in platelets and circulated in plasma, 3) interfering with the metabolism of plasma TFPI, i.e. preventing the conversion of full length to truncated TFPI by proteases and 4) affecting TFPI clearance. BAX 499 treatment of Human Umbilical Vein Endothelial Cells (HUVECs) hardly affected TFPI mRNA production, indicating that BAX 499 has virtually no effect on TFPI synthesis at the transcriptional level. Treatment of HUVECs with BAX 499 stimulated release of TFPI to the cell culture supernatant in a dose dependent manner. FACS analysis showed that BAX 499 has a minimal influence on cell surface TFPI of endothelial cells but, like Heparin, is able to mobilize and release intracellularly-stored TFPI. TFPI released from HUVECs, however, is too low to explain the increased plasma fl-TFPI levels detected in clinical samples. Most TFPI circulating in blood is truncated (80–90%) and bound to lipoproteins. The Lys86-Gln90 region in TFPI is a hot spot for proteolytic cleavage by a wide variety of proteases such as elastase. Cleavage results in the simultaneous removal of TFPI Kunitz 1 domain and loss in activity. BAX 499 (1μM) caused a ∼4-fold decrease in elastase-catalyzed cleavage of TFPI, showing that BAX 499 can delay the proteolytic processing of full length TFPI. BAX 499 was tested in Biacore studies for the interference of fl- TFPI binding to low density lipoprotein receptor-related protein 1 (LRP1), involved in receptor-mediated endocytosis.BAX 499 (1 μM) strongly reduced TFPI binding to LRP1. Pharmacokinetic studies in mice showed that injected human fl-TFPI is rapidly cleared from their circulation and that administration of TFPI with a molar excess of a non-PEGylated variant of BAX 499 resulted in prolonged TFPI clearance. This effect was even more pronounced when TFPI was dosed with PEGylated BAX 499. In conclusion, the elevated TFPI plasma levels in hemophilia patients treated with aptamer BAX 499 can be explained as follows: BAX 499 releases intracellularly-stored TFPI, impacts its metabolism and prolongs the circulatory half life of fl-TFPI, most likely due to binding of BAX 499 to the Kunitz 3-C terminus domain of TFPI, a region required for fl-TFPI clearance. The net result of these effects is elevated plasma fl-TFPI, which even at a molar excess of BAX 499 retains anti-coagulant activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals GPVI Interaction with Polymerized Fibrin Boosts Thrombin Generation and Thrombus Growth

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4152-4152
Author(s):  
Martine Jandrot-Perrus ◽  
Elmina Mammadova-Bach ◽  
Veronique Ollivier ◽  
Stephane Loyau ◽  
Christian Gachet ◽  
...  
Keyword(s):  
Shear Rate ◽  
Tissue Factor ◽  
Platelet Activation ◽  
Thrombin Generation ◽  
Platelet Adhesion ◽  
Coagulation Cascade ◽  
Fibrin Polymerization ◽  
Shear Rates ◽  
Glycoprotein Vi ◽  
Deficient Patient

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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