Optimization of a Recombinant von Willebrand Factor Fragment as an Antagonist of the Platelet Glycoprotein Ib Receptor

The binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) Ib receptor is one of the initial events in thrombus formation. Previous studies have shown that RG12986, a reduced and alkylated recombinant fragment of vWF (Ser 445 -Val 733 ), can inhibit binding of native vWF to GP Ib and offers potential as an anti-thrombotic agent. We have now evaluated a series of deletion mutants of RG12986 and found that reduced and alkylated rvWF 508–704 is close to the minimal sequence with optimal RG12986-like activity (IC 50 for inhibition of GP Ib-dependent platelet aggregation in the absence of modulators: 0.022 μ M±0.01, n=3) and that it too binds directly to GP Ib. Under in vitro conditions, with no exogenous modulators present and in the absence of shear stress, oxidized rvWF 508–704 (containing a disulfide bond between Cys 508 and Cys 659 ) is approximately 5-fold less active than reduced and alkylated rvWF 508–704 ; the two fragments, however, display comparable activity in the presence of the modulator botrocetin. The smaller rvWF 508–704 fragment offers distinct advantages over RG 12986. In particular, removal of non-active NH 2 and COOH terminal sequences may reduce the risk of antigenicity and may contribute to rendering the molecule mostly monomeric in solution, as opposed to the monomer-dimer equilibrium previously described for RG12986.