vonWillebrand factor (vWf) is involved in attachment of platelets to subendothelium following vasuclar injury. Deficiency or structural defects in the protein result in a bleeding disorder called von Willebrand disease. vWf is synthesized by endothelial cells first as a large precursor polypeptide chain (pro-vWf) which then undergoes cleavage and multimerization by disulfide bond formation. The multimers can be secreted from the endothelial cells either constitutively or through a requlated pathway from storage granules called Weibel-Palade bodies. Only the largest and biologically most active multimers are released from the Weibel-Palade bodies after stimulation. The major aims of the proposed research are: a) to investigate the mechanisms of vWf multimerization. Free sulfhydryls will be localized on the pro- vWf subunit and the possibility of multimerization by direct oxidation of the free sulfhydrys will be evaluated. We will clarify the role of acidic pH in the Trans Golgi apparatus in the multimerization process and investigate the function of the very large prosequence of vWf. Both the low pH conditions and the presence of the propeptide appear to be necessary for the successful multimerization of the protein. b) to study the storage and secretion of vWf by endothelial cells. We will search for the recognition element that directs the protein into secretory granules and localize it on the pro-vWf subunit. We will examine if the concentration of vWf in the secretory granules promotes multimerization and if this process coincides with the formation of tubular structures such as those seen in Weibel-Palade bodies. The partioning of vWf between the constitutive and regulated pathways of secretion and the polarity of secretion (basolateral:apical) from endothelial cells will be investigated. c) to define the interactions of vWf with extracellular matrix. During the first stages of hemostasis, vWf links the platelet glycoprotein 1b to an unknown noncollagenous component of the subendothelium. We will identify the binding site for vWf in the extracellular matrix (the in vitro model od subendothelium) and characterize the structure and possible covalent interactions of vWf in the extracellular matrix. Specific procedures in this proposal include: metabolic labelling of cultured human umbilical vein endothelial cells, weak base inhibition of multimerization, isolation of Golgi apparatus and Weibel-Palade bodies rich fractions, in vitro assay for multimerization of dimeric vWf, induction of vWf release from Weibel-Palade bodies by Ca++ ionophore, derivatization of vWf with a bifunctional radioactive crosslinker and expression of full and partial length c-DNA for vWf in AtT-20 secretory cells. The understanding of basic cellular processes involving vWf should provide explanations for molecular defects in von Willebrand disease and for the role of vWf in hemostatic plugs and atherosclerotic lesions.
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