This study used recombinant A1A2A3 tri-domain proteins to demonstrate that A domain association in von Willebrand factor (VWF) regulates the binding to platelet glycoprotein Ib␣ (GPIb␣). We performed comparative studies between wild type (WT) A1 domain and the R1450E variant that dissociates the tri-domain complex by destabilizing the A1 domain. Using urea denaturation and differential scanning calorimetry, we demonstrated the destabilization of the A1 domain structure concomitantly results in a reduced interaction among the three A domains. This dissociation results in spontaneous binding of R1450E to GPIb␣ without ristocetin with an apparent K D of 85 ؎ 34 nM, comparable with that of WT (36 ؎ 12 nM) with ristocetin. The mutant blocked 100% ristocetin-induced platelet agglutination, whereas WT failed to inhibit. The mutant enhanced shear-induced platelet aggregation at 500 and 5000 s ؊1 shear rates, reaching 42 and 66%, respectively. Shear-induced platelet aggregation did not exceed 18% in the presence of WT. A1A2A3 variants were added before perfusion over a fibrin(ogen)-coated surface. At 1500 s ؊1 , platelets from blood containing WT adhered <10% of the surface area, whereas the mutant induced platelets to rapidly bind, covering 100% of the fibrin(ogen) surface area. Comparable results were obtained with multimeric VWF when ristocetin (0.5 mg/ml) was added to blood before perfusion. EDTA or antibodies against GPIb␣ and ␣IIb␤3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of A domain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.Platelet adhesion at sites of vascular injury contributes to the arrest of bleeding as well as to the pathologic occlusion of diseased vessels under elevated shear stress. Under this high shear stress, the platelet-von Willebrand factor (VWF) 3 interaction is essential for platelet adhesion. The interaction between VWF and the exposed subendothelium permits the VWF to interact with circulating platelets via the receptor glycoprotein (GP)Ib/ IX/V complex (1). Mature VWF consists of a 2050-residue subunit that contains domains that are arranged in the order DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (cystine knot) (2-4). The VWF contains a triplicate repeat sequence or A domains in the central portion of the subunit. Binding sites for platelet GPIb␣, heparin, sulfatides, and collagen (5-9) are within the A1 domain, whereas its homologous A3 domain only binds to collagen, and the A2 domain contains the cleavage site for the metalloprotease ADAMTS-13 (10 -12). The functions of these A domains relevant to the biology of VWF have been characterized by individual recombinant expression of each of the A domains (9, 10, 13).The interaction between VWF and GPIb␣ occurs when the binding site for GPIb␣ in the A1 domain of VWF has been exposed by the influence of high fluid forces or when the multimeric protein has been immobilized (14). This activation can also be induced by nat...

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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. Disclosures: No relevant conflicts of interest to declare.