Formation of Procoagulant Platelet Derived Microparticles through Platelet GPIb-Interaction with Von Willebrand Factor under High Shear Stress.

Abstract

We describe here novel findings of the mechanism of initial platelet contact with immobilized von Willebrand factor (VWF) under high shear stress and how this leads to the formation of procoagulant platelet derived microparticles. In a parallel plate perfusion chamber whole blood was perfused over multimeric VWF or dimeric VWF A1 domain at shear rates between 2,000 s−1 and 40,000 s−1. Platelet attachment to VWF always occurred through glycoprotein Ibα receptors located in discrete adhesion points (DAPs), i.e. few limited membrane areas of 0.05 to 0.1 μm2 that arrested the platelets on the surface. The ongoing flow translocated such anchored platelets downstream, thus pulling membrane tethers from the intact and unstimulated platelet. Tethers could remain connected with the platelet body or be eventually severed, which occurred preferentially at shear rates above 6,000 s−1. Depending on the length of the severed membrane fragment they represented either isolated tethers or microparticles (arrowheads; see Figure below), the latter defined by a diameter of 50 to 100 nanometers. The shear rate threshold for microparticle formation was between 6,000 s−1 and 10,000 s−1. Immuno-fluorescence and immuno-electron microscopy showed glycoprotein Ibα clustered in DAPs of microparticles and tethers, i.e. the contact sites with the surface immobilized VWF. The microparticles also exhibited tissue factors on their surface and showed significant procoagulant activity measured by thrombin generation. We propose that after GPIbα anchoring to VWF in flowing blood passive mechanical pulling of membrane from platelets may generate platelet derived microparticles that can potentially support thrombogenesis. [Display omitted]