title: Optimization of a recombinant von Willebrand factor fragment as an antagonist of the platelet glycoprotein Ib receptor.
abstract: 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 (Ser445-Val733), 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 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 microM +/- 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 rvWF508-704 (containing a disulfide bond between Cys508 and Cys659) is approximately 5-fold less active than reduced and alkylated rvWF508-704; the two fragments, however, display comparable activity in the presence of the modulator botrocetin. The smaller rvWF508-704 fragment offers distinct advantages over RG 12986. In particular, removal of non-active NH2 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.
authors.affiliation: Rhône-Poulenc Rorer, Central Research, King of Prussia, PA 19406.
authors.forename: C P
authors.initials: CP
authors.lastname: Prior
authors.forename: V
authors.initials: V
authors.lastname: Chu
authors.forename: B
authors.initials: B
authors.lastname: Cambou
authors.forename: J A
authors.initials: JA
authors.lastname: Dent
authors.forename: B
authors.initials: B
authors.lastname: Ebert
authors.forename: R
authors.initials: R
authors.lastname: Gore
authors.forename: J
authors.initials: J
authors.lastname: Holt
authors.forename: T
authors.initials: T
authors.lastname: Irish
authors.forename: T
authors.initials: T
authors.lastname: Lee
authors.forename: J
authors.initials: J
authors.lastname: Mitschelen
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006801:Humans; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011994:Recombinant Proteins; D017421:Sequence Analysis; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*
chemical_list: D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C081839:von Willebrand factor (445-733)