. Normal hemostasis is maintained by a highly, regulated complex mechanism involving plasma, platelets, and the endothelial lining of blood vessels. Von Willebrand factor is an important regulatory protein that promotes the adhesion of platelets to the vessel wall after injury. Plasma von Willebrand factor binds to the platelet membrane glycoprotein, GPlb to initiate this process although other adhesion and cohesion receptors may augment this response and recruit other activated platelets to this site of injury. The physiologic stimulus for the binding of von Willebrand factor to GPlb has not been elucidated. Several observations by our laboratory and other investigators help us formulate an hypothesis concerning the nature of this interaction. Circulating von Willebrand factor does not bind to circulating platelets unless either the von Willebrand factor or the platelet is activated. Asialo-von Willebrand factor will bind spontaneously to GPlb. Porcine and bovine von Willebrand factor will also bind spontaneously to human platelets without an agonist. The antibiotic, ristocetin, will induce von Willebrand factor binding to platelets and is the laboratory reagent used to study this function. Fragments of von Willebrand factor from certain regions of the molecule will bind to GPlb without added ristocetin. Hereditary defects of platelets (platelet-type von Willebrand's disease) or von Willebrand factor (type IIb von Willebrand's disease) will bind to each other without an added stimulus. This project formulates a hypothetical model that involves these observations and new knowledge of how the antibiotic, ristocetin, exerts its effect on von Willebrand factor. From this model, mutants of von Willebrand factor will be designed to study selective removal of sugar moieties, ristocetin binding sites and differential structure in porcine and bovine von Willebrand factor molecules of the normal function of human von Willebrand factor. Additional studies will be carried out to elucidate the role that vW AgII, the large 100 kD propolypeptide of von Willebrand factor, plays in the multimerization of biologically function von Willebrand factor multimers. Since ABO blood type affects vWf concentration, but not the concentration of the propolypeptide, studies are designed to differentiate an accelerated clearance of vWf in patients who are blood group O or alternatively, a decrease in cellular synthesis of this important regulatory protein. Techniques will include DNA sequencing, site-directed mutagenesis, and monoclonal antibodies to direct the studies that will test the hypotheses put forth. These studies will impact on clinical diagnosis, the expression of clinical symptoms, and characterize the normal physiologic and pathologic events that regulate bleeding and thrombosis.
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