von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent platelets

Abstract

Summary. Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet-rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin-free planar fibrin layers and washed platelets to examine the relationship between platelet-fibrin interaction and exposure of coagulation-stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s À1 requires fibrin-bound VWF. ), but only a minority of the platelets (11 AE 8%) exposed PS. The essential role of VWF in this thrombin-induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 AE 23%) exposed PS and had peak values of [Ca 2þ ] i of about 0.6 mmol L À1 . Taken together, these results demonstrate that thrombin-induced exposure of PS and high calcium response on fibrin-adherent platelets depends on shear-or botrocetin-induced VWF-platelet interaction.Keywords: fibrin, platelet procoagulant activity, von Willebrand factor.Thrombus formation at an injured vessel wall involves platelet adhesion, platelet activation and fibrin formation. Previous studies have provided evidence that fibrin, besides physically entrapping platelets [1], may also be involved in signal pathways that result in the exposure of anionic phospholipids (PS) at the plasma membrane of platelets [2,3]. The presence of PS in the outer leaflet of the platelet plasma membrane is of physiological importance because of its critical role in maintaining thrombin generation at the surface of a developing thrombus [4]. Platelets adhere to fibrin by molecular mechanisms that vary with the surface shear rate [5,6]. At low shear rates, platelet adhesion is mediated by integrin a IIb b 3 whereas at high shear rates an interaction between platelet glycoprotein (GP) Ib and fibrin-bound von Willebrand factor (VWF) tethers the platelets as a prerequisite for an irreversible, integrin a IIb b 3 -mediated, adhesion to fibrin [7][8][9][10].Recently, conflicting reports appeared on the role for GPIb in the development of procoagulant platelets. Studies in plateletrich plasma revealed that VWF supports thrombin generation by two mechanisms: one that involves integrin a IIb b 3 but not fibrin and another one that depends on both fibrin and GPIb [3]. Other investigators studied the thrombin-dependent expression of procoagulant activity in suspensions of gel-filtered platelets and found that the GPIb binding site for thrombin, but not that for VWF, has a key role in the exposure of procoagulant phospholipids on the platelet surface, implying that VWF does not function in this response [11]. Earlier studies, however, indicated that PAR1 is the primary mediator of thrombin-stimulated platelet procoagulant activity, implicating that GPIb doe...

Von Willebrand factor C1C2 domain is involved in platelet adhesion to polymerized fibrin at high shear rate

“…The thrombin thus formed further activates platelets and stabilizes the growing thrombus by the formation of fibrin. Previous work from our laboratory 12,13 and others 14 suggests that in addition to its nonspecific trapping of platelets, fibrin could be actively involved in regulating thrombus growth. It is well accepted that under conditions of high shear stress VWF plays a critical role in the recruitment of circulating platelets to the fibrin network.…”

“…13 To find support for the notion that VWF adsorbs to fibrin from circulating plasma rather than is being trapped during clot formation, perfusion experiments were performed with blood and fibrin layers from wild-type and VWF-deficient mice at a shear rate of 1000 s Ϫ1 . During the perfusion of wild-type blood over wild-type fibrin, the number of platelets that made contact with the fibrin surface increased in time (Figure 9).…”

Thrombin-generating capacity in patients with von Willebrand's disease

“…at a low shear rate, whereas in the CAT method there is no stress at all. This is a major difference, because the role of VWF in the thrombin-induced procoagulant response of platelets requires at least a minimal shear, as shown by Briedé et al 24 Dörmann et al, 25 measuring thrombin generation on gel-filtered platelets in a system without shear stress, showed that VWF binding to GPIb was not essential to initiate platelet procoagulant activity. In the second place contact activation in the subsampling method is probably higher than in the automated method used in this study.…”

Covalently-Bound Heparin Makes Collagen Thromboresistant

“…For scanning electron microscopy (SEM) analysis, collagen sheets were treated as described. 16 SEM images were analyzed for platelet surface coverage using ImagePro software (Media Cybernetics). Collagen adherent platelets were incubated with 3 mmol/L CaCl 2 in Hepes buffer for 15 minutes.…”