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SummaryPorcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) lb and, upon stirring (1500 rpm/min) at 37° C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP lb, obtained with anti GP lb monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP lb monoclonal antibody which does not inhibit the vWF binding to GP lb. An anti GP Ilb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP lb occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP lb, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxy-genase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

We have evaluated the properties of ␣-thrombin interaction with platelets within 1 min from exposure to the agonist, a time frame during which most induced activation responses are initiated and completed. Binding at 37°C was rapidly reversible and completely blocked by a monoclonal antibody, LJ-Ib10, previously shown to be directed against the ␣-thrombin interaction site on glycoprotein (GP) Ib␣. By 2-5 min, however, binding was no longer fully reversible and was only partially inhibited by the anti-GP Ib␣ antibody. Results were similar at room temperature (22-25°C), whereas the initial characteristics of ␣-thrombin interaction with platelets were preserved for at least 20 min at 4°C. Equilibrium binding isotherms obtained at the latter temperature were compatible with a two-site model, but the component ascribed to GP Ib␣, completely inhibited by LJ-Ib10, had "moderate" affinity (k d on the order of 10 ؊8 M) and relatively high capacity, rather than "high" affinity (k d on the order of 10 ؊10 M) and low capacity as currently thought. The parameters of ␣-thrombin binding to intact GP Ib␣ on platelets at 4°C corresponded closely to those measured with isolated GP Ib␣ fragments regardless of temperature. Blocking the ␣-thrombin-GP Ib␣ interaction caused partial inhibition of ATP release and prevented the association with platelets of measurable proteolytic activity. These results support the concept that GP Ib␣ contributes to the thrombogenic potential of ␣-thrombin.

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