In vitro multimerization of von Willebrand factor is triggered by low pH. Importance of the propolypeptide and free sulfhydryls.

Large von Willebrand factor (vWf) multimers are assembled by the formation of disulfide bonds between dimers in trans Golgi and post-Golgi compartments. We were able to reproduce this process in a cell-free system using purified vWf dimers. The multimers formed in vitro extended to 5 x 10(6) Da and were similar in size distribution to those secreted constitutively by endothelial cells in culture. Multimerization occurred only at acidic pH with an optimum at pH 5.8 and needed the continued presence of an acidic pH for it to proceed. Pro-vWf dimers multimerized, whereas mature dimers failed to assemble into large multimers. Multimerization required the propolypeptide to be a contiguous part of pro-vWf subunits since free propolypeptide did not promote multimerization of mature dimers. In addition, multimers formed in the presence of both pro-vWf and mature vWf dimers incorporated only pro-vWf dimers. Two out of six available monoclonal antibodies to the prosequence inhibited multimerization. Multimerization was also inhibited by chemical blocking of free sulfhydryl(s). The free sulfhydryl(s) were localized to the mature region of the pro-vWf by examination of the mature subunit and the propolypeptide derived by proteolytic cleavage of pro-vWf subunits.