Coagulation-induced shedding of platelet glycoprotein VI mediated by factor Xa

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3912-3920 ◽  
Author(s):  
Mohammad Al-Tamimi ◽  
George Grigoriadis ◽  
Huy Tran ◽  
Eldho Paul ◽  
Patricia Servadei ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Tissue Factor ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Selective Inhibitor ◽  
Thrombin Inhibitors ◽  
Platelet Glycoprotein ◽  
Ectodomain Shedding ◽  
Glycoprotein Vi ◽  
Collagen Receptor

Abstract This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinase-mediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n = 36, P < .0001), and 14.6 ng/mL (95% confidence interval, 7.9-27.1 ng/mL) in healthy subjects (n = 25, P = .002). In conclusion, coagulation-induced GPVI shedding via FXa down-regulates GPVI under procoagulant conditions. FXa inhibitors have an unexpected role in preventing GPVI down-regulation.

Effects of Dabigatran, a Direct Thrombin Inhibitor, as Compared to the Direct Factor Xa Inhibitors, Rivaroxaban and Apixaban, on Tissue Factor-Induced Human Platelet Aggregation in Platelet Rich Plasma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1884-1884 ◽  
Author(s):  
Joanne van Ryn ◽  
Monika Kink-Eiband ◽  
Norbert Hauel ◽  
Henning Priepke ◽  
Wolfgang Wienen
Keyword(s):  
Platelet Aggregation ◽  
Tissue Factor ◽  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Factor Xa Inhibitors ◽  
Direct Thrombin Inhibitors ◽  
Direct Factor ◽  
Direct Factor Xa Inhibitors

Abstract Direct thrombin inhibitors (DTIs) have been shown to be very potent inhibitors of platelet function when platelets are activated with thrombin. This action does not occur by direct binding of the DTI to the platelet PAR-1/-4 receptor, but indirectly, by reducing thrombin concentrations and thereby reducing the interactions of thrombin with its receptor on the platelet. It was hypothesized that both thrombin and factor Xa inhibitors could inhibit platelet aggregation, if the stimulus to initiate aggregation was higher in the cascade than factor Xa, such as tissue factor. Thus, dabigatran, a DTI, and the direct factor Xa inhibitors, rivaroxaban and apixaban were tested. Free flowing whole blood (60 ml) was obtained from an antecubital vein using an 18 gauge needle from healthy human volunteers. Blood was collected in tubes containing 3.13% sodium citrate (1 in 10 dilution with whole blood). Blood was centrifuged at 200x g to obtain platelet rich plasma (PRP). Samples (300 μL PRP) were placed in a 6-channel aggregometer, equilibrated for 5 min at 37°C and calibrated with PPP from same individual (0–1 Volts). Photometric tracings were continuously digitally recorded over 5 min following the addition of tissue factor and curves were evaluated as AUC over this time interval. Each PRP sample was incubated with 2 mg/ml Pefabloc®FG (Gly-Pro-Arg-Pro) to prevent fibrin polymerisation, 5 mM CaCl2 and increasing concentrations of dabigatran or factor Xa inhibitor. Tissue factor stimulus (range, 5–27 μl of 10 ml Innovin solution) was tailored for each individual, so that the minimum concentration that resulted in maximum aggregation was used. As positive controls, aggregation was also performed after stimulating with ADP (10 μM), collagen (2 μg/ml), TRAP (20 μM) or ecarin (0.1 U/ml). All substances inhibited tissue factor-induced platelet aggregation in a concentration-dependent manner. Dabigatran was the most potent inhibitor of platelet aggregation among the substances tested, with an IC50 of 35 nM, rivaroxaban and then apixaban followed, with IC50s of 312 and 817 nM, respectively. All substances had no effect on platelet aggregation induced by ADP, collagen and TRAP. Dabigatran was a potent inhibitor of ecarin-induced platelet aggregation, while the factor Xa inhibitors had no effect, as expected from their mechanism of action. Thus, these studies demonstrate that both direct thrombin inhibitors (by inhibiting thrombin) and direct factor Xa inhibitors (by preventing thrombin generation) indirectly inhibit platelet aggregation, though dabigatran was more potent than rivaroxaban and apixaban under these experimental conditions. Thus, these substances may not only be effective in venous/stasis thrombotic episodes where fibrin formation plays an important role, but may also be effective in more platelet dominant, arterial thrombosis settings.

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.

Snake venom metalloproteinases, crotarhagin and alborhagin, induce ectodomain shedding of the platelet collagen receptor, glycoprotein VI

2007 ◽  
Vol 98 (12) ◽  
pp. 1285-1290 ◽  
Author(s):  
Lakshmi Wijeyewickrema ◽  
Elizabeth Gardiner ◽  
Masaaki Moroi ◽  
Michael Berndt ◽  
Robert Andrews
Keyword(s):  
Snake Venom ◽  
Kinase Inhibitors ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Maximal Activity ◽  
Ectodomain Shedding ◽  
Glycoprotein Vi ◽  
Syk Inhibitor ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryGlycoprotein (GP)VI, that binds collagen, together with GPIb- IX-V which binds von Willebrand factor, forms an adheso-signalling complex on platelets that initiates thrombus formation in haemostasis and thrombosis. In this study, we show that two snake venom metalloproteinases, crotarhagin and alborhagin, induce ectodomain shedding of GPVI by a mechanism that involves activation of endogenous platelet metalloproteinases. Alborhagin is a viper venom metalloproteinase from Trimeresurus albolabris, while crotarhagin is a previously undescribed toxin from the rattlesnake Crotalus horridus horridus (~60-kDa non-reduced and reduced). Like alborhagin, crotarhagin induces aggregation in human platelet-rich plasma (maximal activity, ~0.3 μg/ ml). Aggregation of washed platelets was inhibited by soluble GPVI ectodomain expressed as an Fc-fusion protein, confirming crotarhagin targeted GPVI. Treating washed platelets with crotarhagin or alborhagin resulted in time-dependent loss of surface GPVI and the appearance of an ~55-kDa soluble GPVI fragment in supernatants. Crotarhagin also induced shedding in GPVItransfected RBL-2H3 cells. Crotarhagin-induced shedding was metalloproteinase-dependent (inhibited by EDTA), but also blocked by inhibitors of GPVI signalling (Src kinase inhibitors, PP1 or PP2, or Syk inhibitor, piceatannol), indicating shedding required GPVI-dependent platelet activation. Together, the data suggest that the rattlesnake metalloproteinase, crotarhagin, and the viper toxin alborhagin, induce GPVI shedding by a mechanism involving activation of endogenous platelet metalloproteinases rather than direct cleavage of GPVI.

scholarly journals Nerve Growth Factor Inhibits Metalloproteinase-Disintegrins and Blocks Ectodomain Shedding of Platelet Glycoprotein VI

2010 ◽  
Vol 285 (16) ◽  
pp. 11793-11799 ◽  
Author(s):  
Lakshmi C. Wijeyewickrema ◽  
Elizabeth E. Gardiner ◽  
Elsa L. Gladigau ◽  
Michael C. Berndt ◽  
Robert K. Andrews
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Platelet Glycoprotein ◽  
Ectodomain Shedding ◽  
Nerve Growth ◽  
Glycoprotein Vi

Human Platelet Glycoprotein VI: Identification of Residues Involved in the Binding to Collagen.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1550-1550
Author(s):  
Martine Jandrot-Perrus ◽  
Christelle Lecut ◽  
Veronique Arocas ◽  
Hans Ulrichts ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Platelet Glycoprotein ◽  
Directed Mutagenesis ◽  
Time Sharing ◽  
Binding Assays ◽  
Glycoprotein Vi ◽  
Linear Peptide ◽  
Relative Homology ◽  
Collagen Receptor ◽  
Competition Assays

Abstract Glycoprotein VI (GPVI), an essential platelet collagen receptor, belongs to the immunoglobulin (Ig) superfamily and presents two Ig-like loops in its extracellular domain (EC1, EC2). Little is known about the structures involved in GPVI binding to collagen and to its specific ligands, convulxin and collagen related peptides (CRP). Our aim was to characterize these sites using different approaches: competition assays, monoclonal antibodies (MoAb) directed against the GPVI extracellular par, phage display, molecular modelling and site-directed mutagenesis. In binding assays using soluble or cell-expressed human GPVI, we observed that: (i) collagen, CRP and convulxin, competed with one another for the binding to GPVI; (ii) GPVI interaction with either ligand is inhibited by at least two MoAb directed to different independent epitopes, and (iii) MoAb displayed selective inhibitory properties on GPVI interaction with each ligand. These observations indicate that the binding sites on GPVI for the diverse ligands seem to be distinct, exhibiting specific features but at the same time sharing common elements. MoAb 9O12 is a potent inhibitor of GPVI binding to collagen. Its epitope was mapped by screening a 15 mer linear peptide library. This allowed the identification of a motif of 7 residues of which 5 are conserved and that presented a relative homology with the GPVI 30–40 sequence. In a computer model, these residues appeared in close vicinity on a surface loop exposed at the surface of the molecule and accessible to ligands. Site-directed mutagenesis revealed that Val34 and Leu36 are critical for GPVI interaction with collagen and CRP. This study allowed the identification of residues within a loop on GPVI EC1 that might be part of a collagen/CRP-binding site.

Human Atheromatous Plaques Stimulate Thrombus Formation by Activating Platelet Glycoprotein VI.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2623-2623
Author(s):  
Armin J. Reininger ◽  
Richard Brandl ◽  
Sandra Penz ◽  
Pankaj Goyal ◽  
Tamer Rabie ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Initial Step ◽  
Dense Granule ◽  
Platelet Glycoprotein ◽  
Atherosclerotic Plaques ◽  
Glycoprotein Vi ◽  
Atheromatous Plaques ◽  
Dense Granule Secretion

Abstract Lipid-rich atherosclerotic plaques are vulnerable, and upon disruption trigger intraarterial thrombus formation. Tissue factor activating blood coagulation is viewed as the major prothrombotic stimulus within the plaque. We isolated lipid-rich atheromatous plaques from 50 patients with carotid artery stenosis and identified morphologically diverse collagenous structures within in the plaques. They stimulated platelet adhesion, dense granule secretion and aggregation, and triggered thrombus formation in hirudin-anticoagulated blood under arterial flow conditions. Even in fully anticoagulated flowing blood, i.e. in the absence of tissue factor-mediated coagulation, plaques were able to activate platelets. Thrombus formation was more rapid and stable when blood was anticoagulated with a low concentration of heparin, but, although fibrin was detectable within the thrombus, the initial step was always single platelet adhesion and not fibrin formation. In contrast, absence or inhibition of the platelet collagen receptor glycoprotein VI prevented platelet adhesion to atheromatous plaques and thrombus formation. We thus identified platelet glycoprotein VI as being essential and sufficient to mediate plaque-induced thrombus formation. Our study suggests a novel anti-thrombotic strategy to prevent and treat atherothrombosis in patients with vulnerable atherosclerotic plaques. Figure Figure

Oral Anti-Factor Xa and Factor IIa Agent Mediated Inhibition Of Tissue-Factor Mediated Generation Of Thrombin In Prothrombin Complex Concentrates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4810-4810
Author(s):  
Daneyal Syed ◽  
Debra Hoppensteadt ◽  
Daniel Kahn ◽  
Job Harenberg ◽  
Jawed Fareed
Keyword(s):  
Tissue Factor ◽  
Oral Anticoagulant ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Onset Time ◽  
Thrombin Inhibitors ◽  
Bleeding Complications ◽  
Prothrombin Complex Concentrates ◽  
Inhibition Of Thrombin

Introduction Several oral anti-factor IIa and factor Xa agents have recently been developed. These include the thrombin inhibitors Ximelagatran/Melagatran (M) and Dabigatran Etexilate/Dabigatran (D), which require endogenous conversion to the active agents D and M. The factor Xa inhibitors, Rivaroxaban (R) and Apixaban (A), are anti-Xa agents that do not require any endogenous activation. Ximelagatran was withdrawn from the market due to adverse reactions. Dabigatran, Rivaroxaban, and Apixaban are approved for various clinical indications. Antagonism of the anticoagulant effect may be required in bleeding complications. Contradictory results were reported for the efficacy of various prothrombin complex concentrates (PCCs) with these new oral anticoagulants (NOACs). The purpose of this study was to determine the differences in the thrombin generation inhibitory profiles of the newer oral anticoagulant agents. Methods Commercially available PCCs namely Octaplex and Beriplex, were used as a source of Factors II, VII, IX and X. To investigate the effect of each of these agents, a working solution of 1U/ml of both PCCs were supplemented in a graded concentration of 0-1250ng/ml with M, D, R and A. Thrombin generation studies were carried out using a thromboplastin activator (RC High, Technoclone Vienna, Austria). Total thrombin generated was measured in terms of nM’s. The IC-50 for each agent was calculated individually. The time course of thrombin generation was also measured following the kinetic profiles and AUC. Results Dabigatran and Melagatran produced relatively weaker inhibition of thrombin generation with the IC-50 values ranging from 410-110ng/ml in Beriplex and 350-1120ng/ml in Octaplex. Both Rivaroxaban and Apixaban produced strong inhibition of thrombin generation, with the IC-50 ranging from 58-62ng/ml in Octaplex; whereas, in Beriplex these values ranged from 48-50ng/ml. The onset time for thrombin generation and total thrombin formation was concentration dependent. The kinetics of thrombin generation with A and R were distinct from D and M. At concentrations below 310ng/ml the total amount of thrombin generated was comparable to the control; however, its formation was delayed. In both systems, D exhibited the weakest thrombin generation inhibitory potential. While the onset time of thrombin generation was delayed at concentrations below 310ng/ml the levels were comparable to or higher than the control. Discussion This data suggests that PCC’s such as Octaplex and Beriplex can be used to generate thrombin and it’s inhibition by new oral anticoagulant drugs. Octaplex generates much higher amount of thrombin than Beriplex at equivalent units. These results also show that in comparison to the oral anti-Xa agents, the oral anti-IIa agents are relatively weaker inhibitors of thrombin generation. These studies also suggest that the differential inhibition of the generation of thrombin through tissue factor by the anti-Xa and IIa agents may contribute to the potential neutralization profile of PCC’s for these drugs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels

Blood ◽  
2012 ◽  
Vol 119 (18) ◽  
pp. 4311-4320 ◽  
Author(s):  
Mohammad Al-Tamimi ◽  
Chee Wee Tan ◽  
Jianlin Qiao ◽  
Gabrielle J. Pennings ◽  
Ashkan Javadzadegan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Intracellular Signaling ◽  
Platelet Rich Plasma ◽  
Coronary Vessels ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Shear Rates ◽  
Glycoprotein Vi ◽  
Artery Disease ◽  
The Stability

Abstract Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds−1) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of αIIbβ3, GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds−1 (peak shear, 19 224 seconds−1). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

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