Pharmacological characteristics of solid‐phase von Willebrand factor in human platelets

The pharmacological characteristics of solid‐phase von Willebrand factor (svWF), a novel platelet agonist, were studied. Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase‐contrast microscopy, flow cytometry and zymography. Incubation of platelets with svWF (0.2 – 1.2 μg ml−1) resulted in their adhesion to the ligand, while co‐incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation. 6B4 inhibitory anti‐glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N‐Acetyl‐Pen‐Arg‐Gly‐Asp‐Cys, an antagonist of GPIIb/IIIa. Platelet adhesion stimulated with svWF was associated with a concentration‐dependent increase in expression of GPIb, but not of GPIIb/IIIa. In contrast, collagen (0.5 – 10.0 μg ml−1) caused down‐regulation of GPIb and up‐regulation of GPIIb/IIIa in platelets. Solid‐phase vWF (1.2 μg ml−1) resulted in the release of MMP‐2 from platelets. Inhibition of MMP‐2 with phenanthroline (10 μM), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF. In contrast, human recombinant MMP‐2 potentiated both the effects of svWF on adhesion and up‐regulation of GPIb. Platelet adhesion and aggregation stimulated with svWF were reduced by S‐nitroso‐n‐acetyl‐penicillamine, an NO donor, and prostacyclin. Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively. Mechanisms of activation of GPIb by svWF involve the release of MMP‐2, and are regulated by NO and prostacyclin. British Journal of Pharmacology (2001) 134, 1013–1020; doi:10.1038/sj.bjp.0704345