Soluble Monomeric Von Willebrand Factor A1A2A3 (R1450E) Mutant Fragment Enhances Shear-Induced Platelet Aggregation and Platelet Adhesion to Fibrin(ogen) Via αIIbβ3 Under High Shear Stress.

Abstract 4025

Poster Board III-961

Recently, we described that the gain of function mutation R1450E in the A1 domain of von Willebrand factor (VWF) eliminates the formation of catch bond with glycoprotein (GP)Ibα, prolonging the bond lifetimes at low forces. Because those studies were performed with the mutant immobilized on a plastic surface, we further characterize the effect of this mutant on platelet function in solution and under shear stress. Both wild type (WT) and mutant A1A2A3 proteins were expressed in HEK293 cells and purified to homogeneity. The monomeric state of A1A2A3 proteins were assessed by gel filtration chromatography and neither of the proteins had formed dimers or any higher order aggregates. The recombinant A1A2A3 mutant bound spontaneously to GPIbα without the modulator ristocetin with a half-maximal binding observed at 65 ± 8 nM. This apparent dissociation constant was comparable to that of WT (50 ± 10 nM) in the presence of ristocetin. The mutant failed to induce spontaneous platelet aggregation under stirring conditions, and blocked 100% ristocetin-induced platelet agglutination (RIPA) at concentration of 250 nM. At the same concentration, the mutant increased shear-induced platelet aggregation (SIPA) at 500s^-1 and 5000s^-1 shear rates, reaching 42% and 66%, respectively, while SIPA did not exceed 18% in the presence of WT. The anti-αIIbβ3 antibody 7E3 blocked the effect of the mutant on SIPA. Blood was then incubated with the mutant (250 nM) and perfused over a surface coated with fibrin(ogen) at different shear rates. Blood containing WT resulted in <10% surface coverage by platelets after 1.5 minutes while platelets from blood containing the mutant rapidly bound covering 100% of the fibrin(ogen) surface area at 1500s^-1. At shear rate of 2500s^-1, surface coverage was 20% for the mutant and 0% for WT fragment. EDTA and antibodies 6D1 (GPIbα) and 10E5 (αIIbβ3) effectively blocked mutant-mediated platelet adhesion and thrombus formation under high shear rates. The addition of ristocetin (0.5 mg/ml) to whole blood prior perfusion reproduced the effect of the mutant. Here, we describe an A1A2A3 mutant that bound spontaneously to GPIbα but affected differently RIPA and SIPA. These results suggest that hydrodynamic forces directly act on the GPIbα-mutant A1A2A3 complex, regulating signaling. In addition, platelet activation induced by the binding of soluble mutant A1A2A3 or plasma VWF results in αIIbβ3-mediated platelet adhesion to fibrin(ogen) under high shear rates.