Revised Model for Platelet Adhesion to Collagen.

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.

Download Full-text

Anthocyanins Inhibit Platelet Activation and Attenuate Thrombus Growth In Both Human and Murine Thrombosis Models

Blood ◽

10.1182/blood.v116.21.3197.3197 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3197-3197 ◽

Cited By ~ 2

Author(s):

Yan Yang ◽

Zhenyin Shi ◽

Adili Reheman ◽

Wuxun Jin ◽

Conglei Li ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Intravital Microscopy ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Vessel Occlusion ◽

Shear Rates ◽

Perfusion Chamber ◽

Platelet Adhesion And Aggregation

Abstract Abstract 3197 Background: Thrombosis and cardiovascular diseases (CVDs) result from blood vessel occlusion by inappropriately activated platelets. They are the leading causes of morbidity and mortality worldwide. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies have demonstrated that anthocyanins are antioxidative and prevent inflammation (J Biol Chem. 2005; 280:36792-01; Arterioscler Thromb Vasc Biol. 2007; 27:519-24), which may indirectly affect platelet function. It has also been reported that anthocyanins affect platelet activities in whole blood and platelet rich plasma (PRP). However, the direct effects of anthocyanins on platelet function and thrombus formation have not been studied. Methods: Here we investigated the effects of anthocyanins on thrombosis using purified platelets as well as several thrombosis models in vitro and in vivo. Cyaniding-3-gulucoside (Cy-3-g) and delphinidin-3-glucoside (Dp-3-g), the two predominantly bioactive compounds of anthocyanin preparations, were prepared from Polyphenol AS Company in Norway. Purified gel-filtered platelets and PRP from healthy human volunteers and C57BL/6J mice were incubated at 37°C for 10 minutes with different concentrations (0.5μM, 5μM and 50μM) of Cy-3-g, Dp-3-g or PBS buffer as a control. Platelet aggregation was assessed by aggregometry using 5μM ADP, 10μg/ml collagen, or 100μM thrombin receptor activating peptide (TRAP; AYPGKF) as agonists. Platelet adhesion and aggregation were assessed in response to an immobilized collagen matrix in an ex vivo perfusion chamber at both high (1800 s-1) and low (600 s-1) shear rates. The expression of activated GPIIbIIIa was determined via PAC-1 monoclonal antibody in flow cytometry. Lastly, the effects of anthocyanins on thrombus formation in C57BL/6J mice were assessed using a FeCl3-induced intravital microscopy thrombosis model. Results: Both Cy-3-g and Dp-3-g significantly inhibited platelet aggregation induced by collagen and TRAP in gel-filtered platelets, and inhibited aggregation induced by ADP, TRAP and collagen in human and mouse PRP. These inhibitory functions were observed at Cy-3-g and Dp-3-g doses as low as 0.5μM. Cy-3-g and Dp-3-g also reduced the surface expression of activated GPIIbIIIa on resting human platelets in a dose-dependent manner. These compounds also markedly reduced platelet adhesion and aggregation in perfusion chamber assays at both low and high shear rates. Using intravital microscopy, we further demonstrated that Cy-3-g and Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for thrombus formation and vessel occlusion. Conclusions: our data clearly demonstrated for the first time that anthocyanin compounds directly inhibited platelet activation, adhesion and aggregation, as well as attenuated thrombus growth at both arterial and veinous shear stresses. These effects on platelets likely contribute to the protective effects of anthocyanins against thrombosis and CVDs. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CRGT Peptide In Cytoplasm Regulates Thrombus Formation Via Dissociating c-Src-β3 Interaction

Blood ◽

10.1182/blood.v122.21.3501.3501 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3501-3501

Author(s):

Jiansong Huang ◽

Xiaofeng Shi ◽

Wenda Xi ◽

Ping Liu ◽

Xiaodong Xi

Keyword(s):

Molecular Mechanisms ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Human Platelets ◽

Flow Conditions ◽

Cho Cell ◽

Integrin Αiibβ3 ◽

Shear Rates ◽

Platelet Adhesion And Aggregation

Abstract The RGT sequences of the integrin β3 tail directly and constitutively bind the inactive c-Src, regulating integrin αIIbβ3 signaling and platelet function. Previous work has shown that disrupting the interaction of c-Src with β3 via myristoylated RGT peptide or deletion of the RGT sequences in β3 selectively inhibits integrin αIIbβ3 outside-in signaling in platelets. However, the precise molecular mechanisms by which the Src-β3 association regulates integrin αIIbβ3 signaling need to be clarified. We found that active c-Src phosphoylated the Y747 and Y759 residues of β3 directly at the in vitro protein/protein level or in CHO cell models bearing Tac-β3 chimeras, which were devoid of the intact β3 signal transduction. Furthermore, data from mass spectrometry, [γ-32P] ATP incorporation assays and CHO cell/Tac-β3 chimeras demonstrated that the direct phosphorylation of Y747 and Y759 by active c-Src did not depend on the binding of c-Src to the RGT sequences of the β3 tail. To further investigate the biological functions of Src-β3 association in signal transduction we employed a cell-permeable and reduction-sensitive peptide (myr-AC∼CRGT), which disrupted the Src-β3 association in platelets independent of membrane-anchorage, and found that when platelets were stimulated by thrombin the c-Src activation and the phosphorylation of the tyrosine residues of the β3 tail were substantially inhibited by the presence of the peptide. These results suggest that one of the crucial biological functions of Src-β3 association is to serve as a “bridge” linking integrin signaling with the c-Src full activation and phosphorylation of the tyrosines of the β3 tail. To answer whether the RGT peptide binding to Src is able to alter the enzymatic activity of c-Src, we examined the Src-Csk association, the phosphorylation status of Y416 and Y527 of c-Src and the c-Src kinase catalytic activity. Results showed that myr-AC∼CRGT did not dissociate Csk from c-Src in resting platelets and the phosphorylation level of Y416 and Y527 of c-Src remained unaltered. Consistent data were also obtained from in vitro analysis of the c-Src kinase catalytic activity in the presence of CRGT peptide. These results suggest that myr-AC∼CRGT peptide per se does not fully activate c-Src. Myr-AC∼CRGT was also found to inhibit integrin αIIbβ3 outside-in signaling in human platelets. To examine the effect of the myr-AC∼CRGT on platelet adhesion and aggregation under flow conditions, we measured the platelet thrombus formation under different shear rates. Myr-AC∼CRGT did not affect the platelet adhesion at a wall shear rate of 125 s-1. The inability of myr-AC∼CRGT to affect platelet adhesion and aggregation remained at 500 s-1 shear rates. At 1,500 s-1, or 5,000 s-1 rates, myr-AC∼CRGT partially inhibited platelet adhesion and aggregation. These observations indicate that the Src-regulated outside-in signaling plays a pivotal role in the stable thrombus formation and the thrombus growth under flow conditions. The present study reveals novel insights into the molecular mechanisms by which c-Src regulates integrin αIIbβ3 signaling, particularly the phorsphorylation of the β3 cytoplasmic tyrosines, and provides first evidence in human platelets that the RGT peptide or derivatives regulate thrombus formation through dissociating the Src-β3 interaction. The data of this work allow us to anticipate that intracellular delivery of the RGT peptide or its analogues may have potential in the development of a new antithrombotic strategy where only the Src-β3 interaction is specifically interrupted so as to provide an effective inhibition on thrombosis together with a decent hemostasis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

FIBRONECTIN IS REQUIRED FOR PLATELET ADHESION AND FOR THROMBUS FORMATION ON SUBENDOTHELIUM AND COLLAGEN SURFACES

10.1055/s-0038-1643558 ◽

1987 ◽

Author(s):

E Bastida ◽

G Escolar ◽

R Castillo ◽

A Ordinas ◽

J J Sixma

Keyword(s):

Platelet Adhesion ◽

Thrombus Formation ◽

Fibrillar Collagen ◽

Flow Conditions ◽

Shear Rates ◽

Human Collagen ◽

Platelet Thrombus ◽

Computerized System ◽

Total Coverage

Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium.We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions.To do this we used two different perfusion models:1)the annular chamber with α -chymotrypsin-treated rabbit vessel segments and 2)the flat chamber with coverslips coated with fibrillar purified human collagen type III.Perfusates consisted of washed platelets, and washed red blood celIs,suspended in normal or FN-depleted plasma.Perfusions were carried out for 10 min at shear rates of 300 or 1300 sec™1 Platelet deposition and thrombus dimensions were morphometrically evaluated by a computerized system. We found that depletion of plasma FN significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (p < 0.01)(p < 0.01).The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN-depleted plasma.(p < 0.01). Addition of purified FN to FN-depleted perfusates restored all the values to those measured in the control perfusions.These results indicate that, in addition to supporting platelet adhesion to the subendothelium and to fibrillar collagen, FN contributes to platelet thrombus formation under flow conditions.

Download Full-text

Calin from Hirudo medicinalis, an inhibitor of von Willebrand factor binding to collagen under static and flow conditions

Blood ◽

10.1182/blood.v85.3.705.bloodjournal853705 ◽

1995 ◽

Vol 85 (3) ◽

pp. 705-711 ◽

Cited By ~ 37

Author(s):

J Harsfalvi ◽

JM Stassen ◽

MF Hoylaerts ◽

E Van Houtte ◽

RT Sawyer ◽

...

Keyword(s):

Shear Stress ◽

Platelet Adhesion ◽

Thrombus Formation ◽

High Shear ◽

High Shear Stress ◽

Flow Conditions ◽

Shear Rates ◽

Von Willebrand ◽

Willebrand Factor ◽

Low Shear

Calin from the saliva of the medicinal leech, Hirudo medicinalis, is a potent inhibitor of collagen mediated platelet adhesion and activation. In addition to inhibition of the direct platelet-collagen interaction, we presently demonstrate that binding of von Willebrand to coated collagen can be prevented by Calin, both under static and flow conditions in agreement with the occurrence of binding of Calin to collagen, confirmed by Biospecific Interaction Analysis. To define whether Calin acted by inhibiting the platelet-collagen or the platelet- von Willebrand factor (vWF)-collagen-mediated thrombus formation, platelet adhesion to different types of collagens was studied in a parallel-plate flow chamber perfused with whole blood at different shear rates. Calin dose-dependently prevented platelet adhesion to the different collagens tested both at high- and low-shear stress. The concentration of Calin needed to cause 50% inhibition of platelet adhesion at high-shear stress was some fivefold lower than that needed for inhibition of vWF-binding under similar conditions, implying that at high-shear stress, the effect of Calin on the direct platelet- collagen interactions, suffices to prevent thrombus formation. Platelet adhesion to extracellular matrix (ECM) of cultured human umbilical vein endothelial cells was only partially prevented by Calin, and even less so at a high-shear rather than a low-shear rate, whereas the platelet binding to coated vWF and fibrinogen were minimally affected at both shear rates. Thus, Calin interferes with both the direct platelet- collagen interaction and the vWF-collagen binding. Both effects may contribute to the inhibition of platelet adhesion in flowing conditions, although the former seems to predominate.

Download Full-text

Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces

Blood ◽

10.1182/blood.v70.5.1437.1437 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1437-1442 ◽

Cited By ~ 45

Author(s):

E Bastida ◽

G Escolar ◽

A Ordinas ◽

JJ Sixma

Keyword(s):

Platelet Adhesion ◽

Thrombus Formation ◽

Flow Conditions ◽

Shear Rates ◽

Platelet Aggregate ◽

Human Collagen ◽

Platelet Thrombus ◽

Computerized System ◽

Total Coverage

Abstract Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium. We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions. We used two different perfusion models: the annular chamber with alpha-chymotrypsin-treated rabbit vessel segments, and the flat chamber with coverslips coated with fibrillar purified human collagen type III. Perfusates consisted of washed platelets and washed RBCs, suspended in normal or FN-depleted plasma. Perfusions were carried out for ten minutes at shear rates of 300 or 1,300 s-1. Platelet deposition and thrombus dimensions were evaluated morphometrically by a computerized system. We found that depletion of plasma fibronectin significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (P less than .01) (P less than .01). The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN depleted plasma (P less than .01). Addition of purified FN to FN-depleted perfusates restored all values to those measured in the control perfusions. These results indicate that plasma FN is required for platelet aggregate and thrombus formation following adhesion under flow conditions.

Download Full-text

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.

Download Full-text

Effects of red blood cell concentration on hemostasis and thrombus formation in a primate model

Blood ◽

10.1182/blood.v75.11.2185.2185 ◽

1990 ◽

Vol 75 (11) ◽

pp. 2185-2193 ◽

Cited By ~ 43

Author(s):

Y Cadroy ◽

SR Hanson

Keyword(s):

Blood Cell ◽

Red Blood Cell ◽

Thrombus Formation ◽

Low Flow ◽

Flow Conditions ◽

Primate Model ◽

Shunt System ◽

Local Flow ◽

Shear Rates ◽

Venous Shunt

Abstract Because the effects of red blood cell (RBC) concentration on hemostasis and thrombus formation have not been studied experimentally under conditions of whole blood flow without anti-coagulation, normal baboons were bled or transfused to obtain three different groups: a low hematocrit (Ht) group (20% less than Ht less than 25%), a normal Ht group (35% less than Ht less than 40%), and a high Ht group (50% less than Ht less than 55%). Measurements of platelet count, bleeding time, platelet aggregation, fibrinogen level, and coagulation time (APTT) were equivalent to normal values in each group. Thrombus formation was induced using a device composed of collagen-coated tubing followed by two sequentially placed expansion chambers designed to exhibit flow recirculation and stasis. The device was exposed for up to 40 minutes in an arterio-venous shunt system. Wall shear rates in the tubular collagen segment were 100 seconds-1 and 500 to 750 seconds-1. The accumulation of 111In-platelets and 125I-fibrinogen/fibrin was measured radioisotopically; RBC incorporation was determined from measurements of total thrombus hemoglobin. Thrombus that formed on the collagen substrate was rich in platelets and poor in fibrin and RBCs. Under high flow conditions, thrombus composition showed no dependence on Ht. Surprisingly, under low flow conditions, platelet thrombus volume was negatively correlated with Ht (r = -.73, P = .005), and was increased by greater than twofold in the low Ht group as compared with the high Ht group. Thrombus that formed in the disturbed flow regions contained relatively few platelets but was rich in fibrin and RBCs. The predominant finding was a positive correlation between RBC incorporation and Ht at both high and low shear rates (r = .90, P = .00003; and r = .77, P = .002, respectively), with thrombus volume increasing three- to sixfold between the low and high Ht groups. Thus, in vivo variations in Ht ranging between 20% and 55% did not affect hemostasis, but were found either to promote or inhibit the net accumulation of thrombus, depending on local flow conditions.

Download Full-text

Effects of red blood cell concentration on hemostasis and thrombus formation in a primate model

Blood ◽

10.1182/blood.v75.11.2185.bloodjournal75112185 ◽

1990 ◽

Vol 75 (11) ◽

pp. 2185-2193

Author(s):

Y Cadroy ◽

SR Hanson

Keyword(s):

Blood Cell ◽

Red Blood Cell ◽

Thrombus Formation ◽

Low Flow ◽

Flow Conditions ◽

Primate Model ◽

Shunt System ◽

Local Flow ◽

Shear Rates ◽

Venous Shunt

Because the effects of red blood cell (RBC) concentration on hemostasis and thrombus formation have not been studied experimentally under conditions of whole blood flow without anti-coagulation, normal baboons were bled or transfused to obtain three different groups: a low hematocrit (Ht) group (20% less than Ht less than 25%), a normal Ht group (35% less than Ht less than 40%), and a high Ht group (50% less than Ht less than 55%). Measurements of platelet count, bleeding time, platelet aggregation, fibrinogen level, and coagulation time (APTT) were equivalent to normal values in each group. Thrombus formation was induced using a device composed of collagen-coated tubing followed by two sequentially placed expansion chambers designed to exhibit flow recirculation and stasis. The device was exposed for up to 40 minutes in an arterio-venous shunt system. Wall shear rates in the tubular collagen segment were 100 seconds-1 and 500 to 750 seconds-1. The accumulation of 111In-platelets and 125I-fibrinogen/fibrin was measured radioisotopically; RBC incorporation was determined from measurements of total thrombus hemoglobin. Thrombus that formed on the collagen substrate was rich in platelets and poor in fibrin and RBCs. Under high flow conditions, thrombus composition showed no dependence on Ht. Surprisingly, under low flow conditions, platelet thrombus volume was negatively correlated with Ht (r = -.73, P = .005), and was increased by greater than twofold in the low Ht group as compared with the high Ht group. Thrombus that formed in the disturbed flow regions contained relatively few platelets but was rich in fibrin and RBCs. The predominant finding was a positive correlation between RBC incorporation and Ht at both high and low shear rates (r = .90, P = .00003; and r = .77, P = .002, respectively), with thrombus volume increasing three- to sixfold between the low and high Ht groups. Thus, in vivo variations in Ht ranging between 20% and 55% did not affect hemostasis, but were found either to promote or inhibit the net accumulation of thrombus, depending on local flow conditions.

Download Full-text

Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces

Blood ◽

10.1182/blood.v70.5.1437.bloodjournal7051437 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1437-1442

Author(s):

E Bastida ◽

G Escolar ◽

A Ordinas ◽

JJ Sixma

Keyword(s):

Platelet Adhesion ◽

Thrombus Formation ◽

Flow Conditions ◽

Shear Rates ◽

Platelet Aggregate ◽

Human Collagen ◽

Platelet Thrombus ◽

Computerized System ◽

Total Coverage

Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium. We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions. We used two different perfusion models: the annular chamber with alpha-chymotrypsin-treated rabbit vessel segments, and the flat chamber with coverslips coated with fibrillar purified human collagen type III. Perfusates consisted of washed platelets and washed RBCs, suspended in normal or FN-depleted plasma. Perfusions were carried out for ten minutes at shear rates of 300 or 1,300 s-1. Platelet deposition and thrombus dimensions were evaluated morphometrically by a computerized system. We found that depletion of plasma fibronectin significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (P less than .01) (P less than .01). The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN depleted plasma (P less than .01). Addition of purified FN to FN-depleted perfusates restored all values to those measured in the control perfusions. These results indicate that plasma FN is required for platelet aggregate and thrombus formation following adhesion under flow conditions.

Download Full-text

Abstract 56: Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.56 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Jenya Zilberman-Rudenko ◽

Chantal Wiesenekker ◽

Asako Itakura ◽

Owen J McCarty

Keyword(s):

Shear Flow ◽

Platelet Activation ◽

Platelet Adhesion ◽

Ex Vivo ◽

Thrombus Formation ◽

Coagulation Factor ◽

Thrombin Inhibitor ◽

Dependent Manner ◽

Factor Xi ◽

Primate Model

Objective: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo . Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.

Download Full-text