Structural studies of the functional heterogeneity of von Willebrand protein polymers

Studies of subunit chain size, disulfide bond arrangement, carbohydrate content, and pattern of tryptic degradation of von Willebrand protein polymers were undertaken in an attempt to explain their functional heterogeneity. Human von Willebrand protein purified from cryoprecipitate was separated by gel elution and sucrose gradient ultracentrifugation into groups of polymers of different size, ranging from a molecular weight greater than 10 × 106 to a minimum of 2.4 × 106. After disulfide bond reduction, all polymers showed a major band of 208,000 molecular weight with about 1% of the protein having lower molecular weights of 197,000, 174,000, and 154,000. Major and minor moieties were recovered from immunoprecipitates obtained with monospecific anti-von Willebrand protein antibody. The ristocetin cofactor activity of the different polymers showed increasing specific activity with increasing molecular weight, whether measured relative to protein concentration, von Willebrand antigen value or content of the 208,000 molecular weight subunit chain. This difference in specific activity was particularly evident when comparing groups of molecular weight greater than 10 × 106 with those of molecular weight less than 5 × 106. There was no difference in the content of the minor reduced bands in each polymer, no difference in carbohydrate concentration or susceptibility to neuraminidase or galactose oxidase, and no difference in the pattern of tryptic degradation or function of the 116,000 molecular weight tryptic remnant that retains ristocetin cofactor activity. The disulfide bond organization of the larger polymers appeared to differ from that of the smaller polymers inasmuch as partially reduced polymers obtained from the high specific activity group expressed more ristocetin cofactor activity than unreduced polymers of similar size present in the low specific activity group. Apparently, optimal interaction of the von Willebrand polymers with platelets is dictated not only by size but also by tertiary structure as shaped by disulfide bond organization.