Localization of the Thrombin-binding Domain on Prothrombin Fragment 2

Patricia C.Y Liaw,James C Fredenburgh,Alan R Stafford,Alexander Tulinsky,Richard C Austin,Jeffrey I Weitz

doi:10.1074/jbc.273.15.8932

Patricia C.Y Liaw, James C Fredenburgh + Show 4 more

Open Access

https://doi.org/10.1074/jbc.273.15.8932

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Abstract

Co-crystallographic studies have shown that the interaction of human prothrombin fragment 2 (F2) with thrombin involves the formation of salt bridges between the kringle inner loop of F2 and anion-binding exosite II of thrombin. When F2 binds to thrombin, it has been shown to evoke conformational changes at the active site and at exosite I of the enzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin-binding domain on F2. F2, rF2-(1-116), rF2-(55-116), and sF2-(63-116), all of which contain the kringle inner loop (residues 64-93) and the acidic COOH-terminal connecting peptide (residues 94-116), bind to thrombin-agarose. In contrast, analogues of the kringle inner loop, sF2-(63-90), or the COOH-terminal connecting peptide, sF2-(92-116), do not bind. Thus, contrary to predictions from the crystal structure, the COOH-terminal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2-(63-116) bind saturably to fluorescently labeled active site-blocked thrombin with Kd values of 4.1 and 51.3 microM, respectively. The affinity of sF2-(63-116) for thrombin increases about 5-fold (Kd = 10 microM) when Val at position 78 is substituted with Glu. F2 and sF2-(63-116) bind to exosite II on thrombin because both reduce the heparin-catalyzed rate of thrombin inhibition by antithrombin approximately 4-fold. In contrast, only F2 slows the uncatalyzed rate of thrombin inactivation by antithrombin. Like F2, sF2-(63-116) induces allosteric changes in the active site and exosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently labeled hirudin54-65 from active site-blocked thrombin, respectively. Both peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion, reflecting their effects on the active site and/or exosite I. These studies provide further insight into the regions of F2 that evoke functional changes in thrombin.

Highlights

Verted to the serine protease thrombin in the final stages of the blood coagulation cascade
The fragment 2 (F2) domain has been shown to interact with factor Va (5), recent studies indicate that F2-factor Va interactions are not required for factor Va to enhance the catalytic efficiency of factor Xa within the prothrombinase complex (6)
Purification of F2 Peptides—F2 is a 116-amino acid prothrombin activation fragment that consists of a 14-residue interkringle peptide, a 79-residue kringle, and a 25-residue acidic COOH-terminal kringlecatalytic domain-connecting peptide (Fig. 1, panel A)

Summary

The abbreviations used are

F1, prothrombin fragment 1; F2, prothrombin fragment 2; sF2, synthetic F2 peptides; rF2, recombinant F2 peptides; AT, antithrombin; ANS, anilinonaphthalene-6-sulfonic acid; FPR, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone; FITC, fluorescein 5-isothiocyanate; ATA-FPR, N-((acetylthio)acetyl)-D-PhePro-Arg-CH2Cl; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction. The sequences of the primers used to PCR amplify recombinant F2 peptides are shown along with the corresponding amino acid residues of the coding regions of F2 to which the primers hybridize. 5Ј-GGAGTACTAGTAACCCTGGC-3Ј ing analogue encompassing the kringle inner loop and the COOH-terminal connecting peptide, with F2 in terms of their ability to modulate thrombin function

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION