Platelet surface glycoprotein Ib binding site of von Willebrand factor mediates platelet adhesion to collagen under flow conditions

A parallel plate flow chamber and an epifluorescence video microscopy system were used to investigate the role of the binding site for platelet surface glycoprotein Ib (GPIb) on monomers of von Willebrand factor (vWF) plasma polymeric protein, on platelet adhesion and the kinetics of platelet thrombus growth, during the perfusion of whole blood over collagen type I-coated glass slides under controlled flow. At a wall shear rate of 1500 s ¹, inhibition of platelet accumulation on the collagen surface by a F(ab)₂ fragment of a monoclonal antibody directed against the GPIb binding site of vWF indicated that insolubilization of vWF onto collagen, followed by binding of vWF to platelet surface GPIb, initiates the adhesion of platelets from flowing blood onto the collagen-coated slides. This was confirmed by using a recombinant fragment of vWF, produced in escherichia coli, containing amino acid residues 445 to 733 that include the platelet GPIb binding domain and the putative binding site for heparin. In a dose-dependent manner the vWF fragment inhibited platelet adhesion and accumulation from normal whole blood anticoagulated with either citrate or recombinant hirudin (a specific thrombin inhibitor): 60 mg/ml of the vWF fragment resulted in approximately 40% reduction and 200 mg/ml of the fragment in approximately 70% reduction of total platelet accumulation on collagencoated slides. The inhibitory activity of the vWF fragment was counteracted by therapeutic levels of porcine heparin (mol. wt. range approximately 3-30 kD; 0,.5-4 U/ml), which binds to the vWF fragment, but not by low molecular weight heparin (10 to 20 mg/ml, mol. wt. <6000), which apparently does not bind the vWF fragment.