Mechanism of action of thrombin on fibrinogen: NMR evidence for a .beta.-bend at or near fibrinogen a.alpha. Gly(P5)-Gly(P4)

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Abstract
The following synthetic linear A.alpha. fibrinogen-like peptides were studied by NMR spectroscopy in aqueous solution: Ac-Asp(P10)-Phe(P9)-Leu-Ala-Glu-Gly(P5)-Gly(P4)-Gly(P3)-Val-Arg(P1)-Gly-(P1'')-Pro-Arg(P3'')-Val-NHCH3 (F-8), Ac-Phe-Leu-Ala-Glu-Gly-Gly(P4)-Gly(P3)-Val-Arg-Gly-Pro-NHCH3 (F-6), Ac-leu-Ala-Glu-Gly-Gly(P4)-Gly(P3)-Val-Arg-Gly-Pro-NHCH3 (F-7) and Ac-Gly-Gly(P4)-Gly-(P3)-Val-Arg-Gly-Pro-NHCH3 (F-9). The temperature dependence of the amide proton chemical shift is smaller by .apprx. 22% for the Gly(P3) amide proton in F-9, F-6 and F-8, and is similarly smaller for the Gly(P4) amide proton in F-6 and F-8, but not F-9, relative to other amide protons in these peptides. The exchange rates with solvent water for the Gly(P3) amide proton in each of these 4 peptides were determined by solvent spin saturation transfer experiments. The exchange rate constant for the Gly(P3)amide proton of F-8 was half that of the rate constant determined for this proton in F-9, F-7 and F-6. In conjunction with previously reported data for the rate of hydrolysis of the Arg(P1).sbd.Gly(P1'') bond by thrombin, these results suggest that there is a .beta.-bend at Gly(P5)-Gly(P4), possibly stabilized by salt links between Asp(P10) and Arg(P3''), and between phosphorylated Ser(P14) and Arg(P7''), which brings Phe(P9) close to the hydrolyzable Arg.sbd.Gly bonds. This explanation accounts for the importance of Phe(P9) for thrombin action, for diminished activity of several abnormal fibrinogens that have amino acid substitutions at positions P10 and P3'', and for the enhanced activity of fibrinogen with Ser(P14) phosphorylated.