Abstract
Exosite 1 on thrombin mediates low affinity binding to sites on the NH2 termini of the alpha- and beta-chains of fibrin. A subpopulation of fibrin molecules (gammaA/gamma'-fibrin) has an alternate COOH terminus of the normal gamma-chain (gammaA/gammaA-fibrin) that binds thrombin with high affinity. To determine the roles of exosites 1 and 2 in the high affinity interaction of thrombin with gammaA/gamma'-fibrin, binding studies were done with thrombin variants and exosite 1- or 2-directed ligands. alpha-Thrombin bound gammaA/gamma'-fibrin via high and low affinity binding sites. A peptide analog of the COOH terminus of the gamma'-chain that binds alpha-thrombin via exosite 2 blocked the high affinity binding of alpha-thrombin to gammaA/gamma'-fibrin, suggesting that the interaction of alpha-thrombin with the gamma'-chain is exosite 2-mediated. In support of this concept, (a) gamma-thrombin, which lacks a functional exosite 1, bound to gammaA/gamma'-fibrin, but not to gammaA/gammaA-fibrin; (b) thrombin R93A/R97A/R101A, an exosite 2-defective variant, bound only to gammaA/gamma'-fibrin via low affinity sites; and (c) exosite 2-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. However, several lines of evidence indicate that exosite 1 contributes to the high affinity interaction of thrombin with gammaA/gamma'-fibrin. First, the affinity of gamma-thrombin for gammaA/gamma'-fibrin was lower than that of alpha-thrombin. Second, removal of a low affinity binding site on the beta-chain of gammaA/gamma'-fibrin reduced its affinity for alpha-thrombin. Third, exosite 1-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. Taken together, these data suggest that, although exosite 2 mediates the interaction of thrombin with the gamma'-chain of gammaA/gamma'-fibrin, simultaneous ligation of exosite 1 by low affinity binding sites is essential for the high affinity interaction of thrombin with gammaA/gamma'-fibrin.
Highlights
Thrombin is a serine protease that plays an essential role in hemostasis, effecting procoagulant, anticoagulant, and profibrinolytic responses
To confirm the role of the ␥Ј-chain in the high affinity interaction of ␣-IIa with fibrin, we examined the influence of the ␥Ј-peptide on 125I-FPR-␣-IIa binding to ␥A/␥Ј-fibrin. 2 M ␥A/␥Јfibrinogen was clotted in the presence of 125I-FPR-␣-IIa (0 – 8 M) in the absence and presence of 50 M ␥Ј-peptide (Fig. 5)
These findings suggest that removal of a low affinity binding site on ␥A/␥Ј-fibrin has profound effects on the high affinity interaction of thrombin
Summary
Reagents—Human ␣-thrombin (␣-IIa),1 ␥-thrombin (␥-IIa), and fibrinogen were obtained from Enzyme Research Laboratories Inc. (South Bend, IN). A 22-amino acid peptide analog of the NH2-terminal domain (residues 54 –75) of heparin cofactor II (heparin cofactor II-(54 –75), H-Gly-Glu-Glu-Asp-Asp-Asp-Tyr-LeuAsp-Leu-Glu-Lys-Ile-Phe-Ala-Glu-Asp-Asp-Asp-Tyr-Ile-Asp-OH) [29] that binds to exosite 1 on thrombin was synthesized by Chiron Mimotopes Peptide Systems (San Diego). Preparation of Des-B1– 42-␥A/␥A- and Des-B1– 42-␥A/␥Ј-Fibrinogen—␥A/␥A- or ␥A/␥Ј-fibrinogen (8.4 M; in TBS containing 12 mM EDTA) was incubated with 2.3 M protease III fraction from C. atrox venom for 90 min at 23 °C. Active site-blocked 125I-␣-IIa was prepared by incubating 125I-␣-IIa with a 10-fold molar excess of FPRck, followed by dialysis against TBS. Trace amounts of 125I-FPR-␣-IIa were combined with FPR-␣-IIa
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