LOCALIZATION OF THE CROSS-LINKING SITE OF GPRVVERHK IN THE GAMMA-CHAIN OF HUMAN FIBRINOGEN

The peptide alpha17-24-Lys (GPRVVERHK) corresponding to the N-terminus of the a chain of fibrin was synthesized and used to localize its binding site in the fibrinogen molecule. The peptide was radioiodinated, incubated with fibrinogen, cross-linked with a bifunctional reagent disuccinimidyl suberate and the resulting product was analyzed in several ways, including plasmin digestion. The binding of the radioactive peptide was mainly to the gamma-chain and was inhibited by unlabelled GPRVVERHK and GPRP. After plasmin digestion, the radioactivity was present in fragment D1 and also in its gamma-chain remnant, but not in fragments D2, D3 or E3. Fragment D1 cross-linked with iodinated GPRVVERHK was purified by affinity chromatography on immobilized anti-fragment D IgG, further digested with plasmin in the presence of EGTA and the peptides were fractionated by reverse-phase HPLC. The amino acid sequence analysis of the radioactive peak revealed the presence of two peptides, gamma357-373 and GPRVVERHK. It was concluded that the binding site for GPRVVERHK is in the sequence gamma357-373 which is present in fragment D1 but absent in fragments D2 and D3.