ABNORMAL PROTEOLYTIC PROCESSING OF VON-WILLEBRAND-FACTOR ARG611CYS AND ARG611HIS

The structural and functional properties of plasma and platelet vWF we re studied in 8 patients (5 unrelated families) with vWD demonstrating a mutation at position 611 (R611C or R611H). Following reduction, ele ctrophoresis and immunoblotting with a polyclonal anti-reduced vWF ant ibody, abnormal proteolysis of vWF was demonstrated in plasma and to a lesser extent in platelets from all patients, leading to the formatio n of a unique 209 kDa fragment undetectable in control as well as in t ype 2A, 2B or 2N vWF. Immunoblotting with MoAbs to reduced vWF showed that the C-terminal end of the 209 kDa fragment was located beyond res idue 1744 of the subunit and that its N-terminus was between residues 523 and 1114. Multimeric analysis of patients vWF showed an abnormal p attern in both plasma and platelets, with a moderate decrease of the H MW multimers together with a significant increase of the lowest MW for ms. The specific sensitivity of vWF R611C and vWF R611H to proteolysis was further evidenced using V-8 protease. In all patient's samples th e enzyme produced a unique monomeric 80 kDa fragment, absent in V-8 di gested normal vWF, which overlapped the N-terminal part of the subunit . The functional analysis of vWF showed a markedly decreased affinity of mutated plasma vWF for platelet GPIb in the presence of ristocetin. Infusion of DDAVP in two of these patients did not lead to significan t platelet count change. It induced a limited increase of the HMW mult imers in plasma together with a poor correction of the vWF binding to platelet GPIb. In conclusion, our data demonstrate that in addition to a normal proteolysis, vWF mutated at position 611 undergoes a specifi c cleavage in plasma and platelets. In contrast to the increased prote olysis observed in type 2A and 2B patients' plasma, this additional cl eavage produced a unique 209 kDa species but maintained a HMW multimer -like structure of vWF R611C and R611H.