A modified Arg-Asp-Val (RDV) peptide derived during the synthesis of Arg-Glu-Asp-Val (REDV), a tetrapeptide derived from an alternatively spliced site in fibronectin, inhibits the binding of fibrinogen, fibronectin, von Willebrand factor and vitronectin to activated platelets.
Characterization of a side-product obtained during the synthesis of Arg-Glu-Asp-Val (REDV) with inhibitory activity in thrombin-activated platelet aggregation was carried out. The semipreparative column fractionation of REDV peptide was rechromatographed on an analytical HPLC column and revealed two peaks which were re-tested for inhibitory activity. Using amino acid analysis with sequencing and fast atom bombardment mass spectrometry (FABMS), the first peak was determined to be REDV with molecular mass of 517 Da, and the second peak was determined to be a modified RDV with a mass of 608 Da. The modified RDV peptide inhibited thrombin-induced platelet aggregation with an IC50 of 200 microM, and complete inhibition occurred at 600 microM. However, the REDV peptide did not inhibit platelet aggregation up to 1 mM concentration. The modified RDV peptide eluted platelet glycoprotein IIb-IIIa complex that had been bound to GRGDSP-agarose. These studies show that the modified RDV peptide interacts with the platelet glycoprotein IIb-IIIa complex. Based on the collision-induced dissociation (CID) mass spectral data analysis, the modified RDV peptide has been characterized to contain an N-terminus blocking group on the Arg residue. The origin of this blocking group is presumed to have originated from decomposition products of the phenylacetamidomethyl (PAM) resin used in the solid-phase synthesis of the target peptide Arg-Glu-Asp-Val.
Chen
C S
CS
Department of Medicine, University of Washington, Seattle.
Papayannopoulos
I A
IA
Timmons
S
S
Chou
S H
SH
Thiagarajan
P
P
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism*; D002846:Chromatography, Affinity; D005340:Fibrinogen / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification; D012326:RNA Splicing; D019096:Vitronectin; D014841:von Willebrand Factor / Q000378:metabolism*
D005353:Fibronectins; D006023:Glycoproteins; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D019096:Vitronectin; D014841:von Willebrand Factor; C071841:RDV peptide; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen