Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components and coagulation factors. It has been indicated that VWF crosslinks to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). The aim of our study was to investigate the mechanism of VWF incorporation into a fibrin network and thereby characterize the role of VWF in arterial thrombus growth.
We monitored the interactions of von Willebrand factor with fibrin(ogen) using different techniques and enzymes in purified test system at high shear stress. Applying surface techniques, ellipsometry and surface plasmon resonance, we real-time observed that VWF does not bind to either a fibrinogen monolayer or to a polymerized fibrin layer formed on the solid surface. However, we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin. Using either Arvin (cleaves FpA but not FpB) or protease III from snake venom Crotalus atrox (cleaves 42 amino acids, where FpB is located) we were able to show that VWF interacts with fibrin monomers. Furthermore, using transmittance measurements we observed that in the presence of VWF the density of the fibrin clot increases suggesting that VWF incorporates into fibrin network in solution. These findings indicate that incorporation of VWF into a fibrin network occurs via the E domain of fibrinogen where FpA and/or FpB were situated. Using a domain deletion mutant deltaC1C2-VWF we demonstrated the involvement of the C1C2 domain of VWF in the binding to fibrin monomers. Our results did not show any interaction of deltaC1C2-VWF with fibrin monolayer in the presence of thrombin. Application of the inhibitor K9-DON against factor XIIIa in the presence of calcium and thrombin slightly decreased the amount of VWF adsorbed on fibrin monolayer but not completely abolished it, illustrating that cross linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Additionally, under high shear conditions, we were able to show that platelets adhere to fibrin only if VWF had been incorporated. Addition of a GPIb blocking antibody almost completely abolished the adhesion of platelets to the fibrin monomers surface with bound VWF. These data provided evidence that the binding of platelets to a fibrin monomer layer under high shear rate is completely dependent on the VWF-GPIb interaction. All experiments were performed in the presence of abciximab suggesting that the GPIIb/IIIa receptor is not involved in this binding.
In conclusion, our experiments show that the C1C2 domain of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate. Additionally it may help to explain the observed phenotype in types I and III of von Willebrand Disease and the Bernard-Soulier Syndrome. Low amount of (functional) VWF or deficiency in GPIb-IX-V, here lead to disruption in thrombus formation and wound healing.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.