Analysis of the Involvement of the von Willebrand Factor–Glycoprotein Ib Interaction in Platelet Adhesion to a Collagen-Coated Surface Under Flow Conditions

The requisite initial reaction for in vivo thrombus formation in flowing blood is platelet adhesion to the exposed surface of the extracellular matrix. The contribution of von Willebrand factor (vWF ) in plasma and glycoprotein (GP) Ib on the platelet membrane to platelet adhesion has been well-documented. We have recently developed a procedure (the “flow adhesion assay”) for measuring platelet adhesion under flow conditions that allowed us to characterize platelet adhesion to a collagen-coated surface. Here, we apply our method to analyze platelet adhesion to a vWF-coated surface to determine how this might differ from adhesion to a collagen-coated surface. Platelet adhesion to the vWF-coated surface was monitored as the linear increase in the area occupied by adherent platelets. The fluorescence image showed that platelets adhering to the vWF surface were mainly single platelets, and if any were present, the platelet aggregates were small, this being the primary difference from the adhesion to a collagen surface, where adherent platelets were mostly in aggregates. The flow adhesion assay detected the movement of platelets on the vWF surface, suggesting the reversible binding of vWF with platelets. The velocity of the platelets increased at higher shear rates or at lower vWF densities on the surface. Treatment of the vWF-coated surface with the aggregating agent botrocetin before initiation of blood flow increased platelet adhesion while dramatically decreasing the velocity of platelet movement. The present observations on the adhesion of platelets to the vWF-pretreated collagen surface and measurements of the velocity of platelets moving on the collagen surface suggest that the first interaction on the collagen-coated surface is the binding of vWF molecules to the collagen surface. This small number of vWF molecules would serve to attract and slow platelets flowing near the surface. This would facilitate the actual adhesion to the collagen surface that is mainly generated by the interaction between platelet collagen receptors, including GP Ia/IIa and GP VI, with collagen.

Download Full-text

Analysis of the Involvement of the von Willebrand Factor–Glycoprotein Ib Interaction in Platelet Adhesion to a Collagen-Coated Surface Under Flow Conditions

Blood ◽

10.1182/blood.v90.11.4413 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4413-4424 ◽

Cited By ~ 53

Author(s):

Masaaki Moroi ◽

Stephanie M. Jung ◽

Shosaku Nomura ◽

Sadayoshi Sekiguchi ◽

Antonio Ordinas ◽

...

Keyword(s):

Von Willebrand Factor ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Initial Reaction ◽

Adhesion Assay ◽

Flow Conditions ◽

Coated Surface ◽

Von Willebrand ◽

Willebrand Factor

Abstract The requisite initial reaction for in vivo thrombus formation in flowing blood is platelet adhesion to the exposed surface of the extracellular matrix. The contribution of von Willebrand factor (vWF ) in plasma and glycoprotein (GP) Ib on the platelet membrane to platelet adhesion has been well-documented. We have recently developed a procedure (the “flow adhesion assay”) for measuring platelet adhesion under flow conditions that allowed us to characterize platelet adhesion to a collagen-coated surface. Here, we apply our method to analyze platelet adhesion to a vWF-coated surface to determine how this might differ from adhesion to a collagen-coated surface. Platelet adhesion to the vWF-coated surface was monitored as the linear increase in the area occupied by adherent platelets. The fluorescence image showed that platelets adhering to the vWF surface were mainly single platelets, and if any were present, the platelet aggregates were small, this being the primary difference from the adhesion to a collagen surface, where adherent platelets were mostly in aggregates. The flow adhesion assay detected the movement of platelets on the vWF surface, suggesting the reversible binding of vWF with platelets. The velocity of the platelets increased at higher shear rates or at lower vWF densities on the surface. Treatment of the vWF-coated surface with the aggregating agent botrocetin before initiation of blood flow increased platelet adhesion while dramatically decreasing the velocity of platelet movement. The present observations on the adhesion of platelets to the vWF-pretreated collagen surface and measurements of the velocity of platelets moving on the collagen surface suggest that the first interaction on the collagen-coated surface is the binding of vWF molecules to the collagen surface. This small number of vWF molecules would serve to attract and slow platelets flowing near the surface. This would facilitate the actual adhesion to the collagen surface that is mainly generated by the interaction between platelet collagen receptors, including GP Ia/IIa and GP VI, with collagen.

Download Full-text

Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice

Thrombosis and Haemostasis ◽

10.1055/s-0040-1702228 ◽

2020 ◽

Vol 120 (03) ◽

pp. 466-476

Author(s):

Sibgha Tahir ◽

Andreas H. Wagner ◽

Steffen Dietzel ◽

Hanna Mannell ◽

Joachim Pircher ◽

...

Keyword(s):

Von Willebrand Factor ◽

Knockout Mice ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Cd40 Ligand ◽

Inflammatory Conditions ◽

String Formation ◽

Von Willebrand ◽

Willebrand Factor

Abstract Background von Willebrand factor (vWF) plays an important role in platelet activation. CD40–CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo, whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes. Methods and Results The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo. Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas. Conclusion CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.

Download Full-text

Cytosolic Calcium Changes in a Process of Platelet Adhesion and Cohesion on a von Willebrand Factor-Coated Surface Under Flow Conditions

Blood ◽

10.1182/blood.v94.4.1149.416k18_1149_1155 ◽

1999 ◽

Vol 94 (4) ◽

pp. 1149-1155 ◽

Cited By ~ 1

Author(s):

Mitsuhiro Kuwahara ◽

Mitsuhiko Sugimoto ◽

Shizuko Tsuji ◽

Shigeki Miyata ◽

Akira Yoshioka

Keyword(s):

Von Willebrand Factor ◽

Platelet Adhesion ◽

Cytosolic Calcium ◽

Flow Chamber ◽

Flow Conditions ◽

Color Changes ◽

Coated Surface ◽

Von Willebrand ◽

Willebrand Factor ◽

Calcium Green

Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

Download Full-text

The Role of Platelet Adhesion Receptor GPIb α Far Exceeds That of Its Main Ligand von Willebrand Factor in Arterial Thrombosis.

Blood ◽

10.1182/blood.v108.11.1797.1797 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1797-1797 ◽

Cited By ~ 1

Author(s):

Wolfgang Bergmeier ◽

Crystal L. Piffath ◽

Tobias Goerge ◽

Stephen M. Cifuni ◽

Zaverio M. Ruggeri ◽

...

Keyword(s):

Von Willebrand Factor ◽

Arterial Thrombosis ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Interleukin 4 ◽

Arterial Thrombus ◽

A Site ◽

Von Willebrand ◽

Willebrand Factor

Abstract GPIbα binding to von Willebrand factor (VWF) exposed at a site of vascular injury is thought to be the first step in the formation of a hemostatic plug. However, our previous studies in VWF-deficient mice demonstrated delayed but not absent arterial thrombus formation suggesting that, under these conditions, GPIbα may bind other ligands or that a receptor other than GPIbα can mediate platelet adhesion. Here we studied thrombus formation in transgenic mice expressing GPIbα in which the extracellular domain was replaced by that of the human interleukin-4 receptor (IL4Rα/GPIbα-tg mice). Platelet adhesion to ferric chloride-treated mesenteric arterioles in IL4Rα/GPIbα-tg mice was virtually absent in contrast to avid adhesion in wild-type (WT) mice. As a consequence, arterial thrombus formation was completely inhibited in the mutant mice. Our studies further show that, when infused into WT recipient mice, IL4Rα/GPIbα-tg platelets or WT platelets lacking the 45 kD N-terminal domain of GPIbα failed to incorporate into growing arterial thrombi, even if the platelets were activated prior to infusion. Surprisingly, platelets lacking β3 integrins, which are unable to form thrombi on their own, incorporated efficiently into WT thrombi. Our studies provide in vivo evidence that GPIbα is absolutely required for recruitment of platelets to both exposed subendothelium and thrombi under arterial flow conditions. Thus, GPIbα contributes to arterial thrombosis by important adhesion mechanisms independent of the binding to VWF.

Download Full-text

KINETIC BEHAVIOUR OF VON WILLEBRAND FACTOR AND FIBRONECTIN EXPLAINS DEPENDENCE OF PLATELET ADHESION ON PHYSICAL PARAMETERS

10.1055/s-0038-1643632 ◽

1987 ◽

Author(s):

Hans H F I van Breugel ◽

Philip G de Groot ◽

Jan J Sixma

Keyword(s):

Von Willebrand Factor ◽

Platelet Adhesion ◽

Physical Parameters ◽

Flow Conditions ◽

Binding Studies ◽

Platelet Suspension ◽

Coated Surface ◽

Static Conditions ◽

Von Willebrand ◽

Willebrand Factor

To study the kinetics of the contribution of von Willebrand Factor (vWF) and fibronectin (FN) in platelet adhesion we developed a method with which we can perform binding studies of platelets to these purified proteins under static and flow conditions. Glass coverslips were incubated for one hour with vWF (50 (jg/ml) or FN (300 pg/ml) in saline and were perfused with washed platelets (resuspended in human albumin solution) in the flat perfusion chamber as developed by Sakariassen (J.Lab.Clin.Med. 102, 522-535, 1983). Static conditions were achieved by incubating the coated coverslips with the platelet suspension.In this system, adhesion of platelets to FN coated coverslips strongly decreased at shear rates above 300 /s. The adhesion to this surface could be inhibited with antibodies against platelet glycoprotein Ilbllla and against lb, under static and under flow conditions.Adhesion to vWF coated surfaces increased with increasing shear rate and ultimately reached a plateau at about 800 /s. Adhesion to a vWF coated surface could be totally inhibited by anti GP-Ib and only partially by GP-IIbllla.When after perfusion of a FN coated surface with platelets, the same surface was perfused with a platelet free perfusate, the coverage of platelets on this surface decreased. No decrease in platelet coverage was found when this experiment was performed with a vWF coated coverslip.From these results we conclude that platelets bind to FN at a high rate and with a low affinity, while they bind slowly but with a high affinity to vWF, probablyvia similar platelet receptors.

Download Full-text

Cytosolic Calcium Changes in a Process of Platelet Adhesion and Cohesion on a von Willebrand Factor-Coated Surface Under Flow Conditions

Blood ◽

10.1182/blood.v94.4.1149 ◽

1999 ◽

Vol 94 (4) ◽

pp. 1149-1155 ◽

Cited By ~ 37

Author(s):

Mitsuhiro Kuwahara ◽

Mitsuhiko Sugimoto ◽

Shizuko Tsuji ◽

Shigeki Miyata ◽

Akira Yoshioka

Keyword(s):

Von Willebrand Factor ◽

Platelet Adhesion ◽

Cytosolic Calcium ◽

Flow Chamber ◽

Flow Conditions ◽

Color Changes ◽

Coated Surface ◽

Von Willebrand ◽

Willebrand Factor ◽

Calcium Green

Abstract Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

Download Full-text

Von Willebrand factor accelerates platelet adhesion and thrombus formation on a collagen surface in platelet-reduced blood under flow conditions

Blood ◽

10.1182/blood-2004-05-1827 ◽

2005 ◽

Vol 105 (3) ◽

pp. 1078-1084 ◽

Cited By ~ 20

Author(s):

Kazuhiko Tomokiyo ◽

Yuichi Kamikubo ◽

Takako Hanada ◽

Tatsuya Araki ◽

Yasushi Nakatomi ◽

...

Keyword(s):

Von Willebrand Factor ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Platelet Glycoprotein ◽

Flow Conditions ◽

Platelet Aggregates ◽

Von Willebrand ◽

Willebrand Factor ◽

Ristocetin Cofactor ◽

Ristocetin Cofactor Activity

AbstractPlasma von Willebrand factor (VWF) has been identified as an indispensable factor for platelet adhesion and thrombus formation on a collagen surface under flow conditions. VWF binds to collagen and then tethers platelets to the collagen surface through interaction with platelet glycoprotein Ib and also contributes to the thrombus formation on the collagen surface. In the present study, we demonstrated that the addition of VWF/factor VIII complex or purified VWF (> 2 ristocetin cofactor activity units/mL) increased platelet adhesion to the collagen surface in platelet-reduced blood (∼ 5 × 104 platelets/μL) to the normal level. VWF had no stimulatory effect when it was allowed to bind to the collagen surface before blood flow was initiated. Addition of an excess of FITC (fluorescein-5-isothiocyanate)–abeled VWF to platelet-reduced blood under these flow conditions demonstrated that the VWF was mainly incorporated into the platelet aggregates. These results indicated that the supplemented VWF stimulates the platelet adhesion onto the collagen surface by enhancing platelet aggregation in the platelet-reduced condition. This also suggests a possibility that supplementation of VWF to individuals with thrombocytopenia might be effective for increasing their hemostatic potential.

Download Full-text

Levels of von Willebrand factor and ADAMTS13 determine clinical outcome after cardioversion for atrial fibrillation

Thrombosis and Haemostasis ◽

10.1160/th10-09-0615 ◽

2011 ◽

Vol 105 (03) ◽

pp. 435-443 ◽

Cited By ~ 12

Author(s):

Veronika Bruno ◽

Rudolf Jarai ◽

Susanne Gruber ◽

Thomas Höchtl ◽

Ivan Brozovic ◽

...

Keyword(s):

Atrial Fibrillation ◽

Von Willebrand Factor ◽

Platelet Adhesion ◽

Independent Predictor ◽

Thrombus Formation ◽

Critical Role ◽

Inverse Correlation ◽

Von Willebrand ◽

Rhythm Stability ◽

Willebrand Factor

SummaryVon Willebrand factor (vWF) plays an essential role in platelet adhesion and thrombus formation. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 antigen levels compared to controls. Little is known about vWF and ADAMTS13 in AF patients treated with cardioversion (CV). Thus we investigated the alterations of plasma vWF and ADAMTS13 after CV and evaluated the predictive value of these parameters for recurrence of AF. In this observational study we determined plasma levels of vWF and ADAMTS13 in 77 patients before and immediately after CV, as well as 24 hours (h) and six weeks thereafter, by means of commercially available assays. The vWF/ ADAMTS13-ratio was significantly elevated immediately after CV (p=0.02) and 24 h after CV (p=0.002) as compared to baseline levels. ADAMTS13, 24 h after CV, exhibited a significant association with recurrence of AF (HR: 0.97; p=0.037). Accordingly, tertiles of ADAMTS13 showed a stepwise inverse correlation with the risk of recurrent AF (HR: 0.50; p=0.009). After adjustment for confounders, ADAMTS13 remained significant as an independent predictor of recurrent AF (HR: 0.61; p=0.047). Similarly, the vWF/ADAMTS13-ratio, 24 h after CV, was associated with rhythm stability and remained an independent predictor of recurrent AF (HR: 1.88; p=0.028). The regulation of vWF and its cleaving protease ADAMTS13 after CV might play a critical role in producing a pro-thrombotic milieu immediately after CV for AF. Since ADAMTS13 plasma concentration and the vWF/ADAMTS13-ratio are independently associated with rhythm stability, these indexes might be used for prediction of recurrence of AF.

Download Full-text

Uremic platelets have a functional defect affecting the interaction of von Willebrand factor with glycoprotein IIb-IIIa

Blood ◽

10.1182/blood.v76.7.1336.1336 ◽

1990 ◽

Vol 76 (7) ◽

pp. 1336-1340 ◽

Cited By ~ 62

Author(s):

G Escolar ◽

A Cases ◽

E Bastida ◽

M Garrido ◽

J Lopez ◽

...

Keyword(s):

Von Willebrand Factor ◽

Platelet Adhesion ◽

Indirect Evidence ◽

Flow Conditions ◽

Experimental Conditions ◽

Shear Rates ◽

Functional Defect ◽

Uremic Patients ◽

Von Willebrand ◽

Willebrand Factor

Abstract Uremic patients have an impaired platelet function that has been related to membrane glycoprotein (GP) abnormalities. Using a perfusion system, we have studied the interaction of normal and uremic platelets with vessel subendothelium (SE) under flow conditions. Reconstituted blood containing washed platelets, purified von Willebrand factor (vWF) (1 U/mL), and normal washed red blood cells was exposed to de- endothelialized rabbit segments for 10 minutes at two different shear rates (800 and 1,600 seconds-1). In some experiments a monoclonal antibody to the GPIIb-IIIa complex (EDU3) was added to the perfusates. With normal platelets, the percentage of the vessel covered by platelets (%CS) was 23.1% +/- 3.7% at 800 seconds-1 and 30% +/- 4.3% at 1,600 seconds-1. Platelets were observed in contact or forming monolayers on vessel SE. EDU3 inhibited the spreading of normal platelets. The %CS (11.1% +/- 3.3%) was statistically decreased (P less than .01) and most of the platelets were observed in contact with the vessel surface. These data indicate that, under flow conditions, the interaction of vWF with GPIIb-IIIa can support the spreading of normal platelets in the absence of exogenous fibrinogen. Under the same experimental conditions, the interaction of uremic platelets with SE was markedly impaired at both shear rates studied (P less than .01 v normal platelets). The presence of EDU3 did not modify the interaction of uremic platelets. These results confirm the impairment of the platelet adhesion observed in uremic patients. Furthermore, they indicate the presence of a functional defect in the interaction of vWF with GPIIb-IIIa. The fact that perfusions with normal and uremic platelets in the presence of an antibody to the GPIIb-IIIa complex did not show any differences gives indirect evidence on a functionally normal interaction vWF/GPIb in uremic patients.

Download Full-text