PLATELET-AGGREGATION ON EXTRACELLULAR-MATRIX - EFFECT OF A RECOMBINANT GPIB-BINDING FRAGMENT OF VON-WILLEBRAND-FACTOR

Platelets in whole blood incubated on extracellular matrix (ECM) produ ced by bovine corneal endothelial cells under oscillatory flow conditi ons demonstrate extensive aggregate formation. Since both platelet-sub endothelium and platelet-platelet interactions are mediated by von Wil lebrand factor (vWF), we used this system to examine the effect of a r ecombinant GPIb-binding fragment of vWF (designated RG12986), comprisi ng residues 445-733 of the native vWF subunit, on platelet reactivity with ECM. The seven cysteines present in the RG12986 fragment were red uced and alkylated in order to achieve a monomeric conformation. The r ecombinant vWF fragment binds to unstimulated platelets in the absence of exogenous modulators. When added to platelet-rich plasma, it inhib its ristocetin-induced platelet agglutination. Binding of Cr-51-labele d platelets in reconstituted whole blood to ECM was inhibited by RG 12 986 in a dose dependent and saturable manner, with IC50 of 4 muM and m aximal inhibition (about 70%) at 6 muM. Scanning electron microscope ( SEM) analysis showed that addition of RG12986 to whole blood significa ntly inhibited platelet aggregation on ECM. The extent of inhibition o bserved with RG12986 at a final concentration of 4 muM was similar to that obtained with the cell adhesion peptide RGDS at the concentration of 0.1 mM. The ability of the RG12986 fragment to inhibit platelet ag gregation on ECM is in agreement with the concept that blockade of vWF -GPIb interaction may inhibit further events leading to activation of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex and subsequent thrombus formation.