OPTIMIZATION OF A RECOMBINANT VONWILLEBRAND-FACTOR FRAGMENT AS AN ANTAGONIST OF THE PLATELET GLYCOPROTEIN-IB RECEPTOR

The binding of von Willebrand factor (vWF) to platelet glycoprotein (G P) lb receptor is one of the initial events in thrombus formation. Pre vious studies have shown that RG12986, a reduced and alkylated recombi nant fragment of vWF (Ser445-Val733), can inhibit binding of native vW F to GP lb and offers potential as an anti-thrombotic agent. We have n ow evaluated a series of deletion mutants of RG12986 and found that re duced and alkylated rvWF508-704 is close to the minimal sequence with optimal RG12986-like activity (IC50 for inhibition of GP Ib-dependent platelet aggregation in the absence of modulators: 0.022 muM +/- 0.01, n = 3) and that it too binds directly to GP Ib. Under in vitro condit ions, with no exogenous modulators present and in the absence of shear stress, oxidized rvWF508-704 (containing a disulfide bond between Cys 508 and Cys659) is approximately 5-fold less active than reduced and a lkylated rvWF508-704; the two fragments, however, display comparable a ctivity in the presence of the modulator botrocetin. The smaller rvWF5 08-704 fragment offers distinct advantages over RG12986. In particular , removal of non-active NH2 and COOH terminal sequences may reduce the risk of antigenicity and may contribute to rendering the molecule mos tly monomeric in solution, as opposed to the monomer-dimer equilibrium previously described for RG12986.