Inhibition of Meizothrombin and Meizothrombin(desF1) by Heparin Cofactor II

Abstract

Meizothrombin and meizothrombin(desF1) are intermediates formed during the conversion of prothrombin to thrombin by factor Xa, factor Va, phospholipids, and Ca 2؉ (prothrombinase). These intermediates are active toward synthetic peptide substrates but have limited ability to interact with platelets or macromolecular substrates such as fibrinogen. Meizothrombin and meizothrombin(desF1) activate protein C, however, and may exert primarily an anticoagulant effect. In this study, we investigated the inhibition of meizothrombin and meizothrombin(desF1) by two glycosaminoglycandependent protease inhibitors, heparin cofactor II (HCII) and antithrombin (AT). Purified recombinant meizothrombin and meizothrombin(desF1) were inhibited by HCII in the presence of dermatan sulfate with maximal second-order rate constants of 8 ؋ 10 6 M ؊1 ⅐min ؊1 and 1.8 ؋ 10 M؊1 ⅐min ؊1, respectively, but were inhibited less than one-tenth as fast by AT in the presence of heparin. Similarly, the products of the prothrombinase reaction were inhibited in situ more effectively by HCII than by AT. When HCII and dermatan sulfate were present continuously during the prothrombinase reaction, meizothrombin was trapped as a sodium dodecyl sulfate-stable complex with HCII and no amidolytic activity could be detected with a thrombin substrate. Our findings indicate that HCII is an effective inhibitor of meizothrombin and meizothrombin(desF1) and, therefore, might regulate the anticoagulant activity of these proteases.Thrombin is a key enzyme in several biological processes, including blood coagulation, wound healing, and inflammation (1). Factor Xa converts human prothrombin to thrombin by cleavage of the peptide bonds following Arg-271 and Arg-320 (2). The order in which these bonds are cleaved depends on assembly of the prothrombinase complex. In the presence of factor Xa and Ca 2ϩ , prothrombin is first cleaved after Arg-271, giving rise to fragment 1⅐2 and prethrombin 2 (3). When factor Xa and its cofactor, factor Va, are assembled on a membrane surface in the presence of Ca 2ϩ , factor Xa first cleaves after Arg-320, giving rise to meizothrombin (4, 5). Cleavage of the second factor Xa-sensitive bond in either prethrombin 2 or meizothrombin yields thrombin. Meizothrombin has been shown recently to be a major intermediate formed during coagulation of whole blood in vitro (6).Meizothrombin retains the N-terminal ␥-carboxyglutamic acid domain of prothrombin, which enables it to bind to membrane phospholipids in the presence of Ca 2ϩ . Cleavage of the thrombin-sensitive peptide bond following Arg-155 in meizothrombin removes the ␥-carboxyglutamic acid domain and generates meizothrombin(desF1) (7). In contrast to prothrombin or prethrombin 2, meizothrombin and meizothrombin(desF1) hydrolyze synthetic peptide substrates at rates comparable with that of thrombin (8 -10). Meizothrombin and meizothrombin(desF1) are much less active with respect to fibrinogen clotting, platelet activation, and activation of the thrombinactivable fibrinolysis inhibitor (9 -11...

HD1, a Thrombin-directed Aptamer, Binds Exosite 1 on Prothrombin with High Affinity and Inhibits Its Activation by Prothrombinase

“…Previous structural (39) and functional (40,41) data indicate that F2 binds exosite 2. Consequently, exosite 2 should only be accessible on thrombin and pre2, derivatives lacking F2.…”

Effects of Activation Peptide Bond Cleavage and Fragment 2 Interactions on the Pathway of Exosite I Expression during Activation of Human Prethrombin 1 to Thrombin

“…Removal of F2 from the proteinase domain thus does not affect the affinity of exosite I for the specific peptide ligand. Previous studies indicated no detectable binding of thrombomodulin to exosite I on human Pre 2 (31), whereas fluorescein-labeled hirudin [53][54][55][56][57][58][59][60][61][62][63][64] (hirugen) bound with the same high affinity to bovine Pre 2 as to bovine thrombin (32). The source of this difference is not known but may be due to intrinsic differences in the affinities for human and bovine Pro species (26).…”

“…Both MzT and MzT(-F1), however, bind thrombomodulin with affinity comparable to thrombin and exhibit comparable activity in protein C activation (53,55), which are also exosite I-dependent processes. Similarly, MzT and MzT(-F1) react with the exosite I-dependent inhibitors, hirudin (55) and heparin cofactor II (56), and MzT(-F1) binds an exosite I-specific antibody (31). The low activity of MzT and MzT(-F1) in factor V activation may reflect the participation of exosite II, which is inaccessible on MzT and MzT(-F1), in addition to exosite I (19 -21).…”

“…In studies of the bovine proteins, however, binding of fluorescein-labeled hirudin [53][54][55][56][57][58][59][60][61][62][63][64] (hirugen) to Pro and Pre 1 was undetectable, whereas Pre 2 bound the peptide with similar affinity as thrombin (32). MzT and MzT-(-F1) both bound the peptide with 2-to 8-fold higher affinity than Pre 2, suggesting that either peptide bond cleavage in Pro results in activation of exosite I (32).…”

Molecular Mapping of the Thrombin-Heparin Cofactor II Complex

“…The role of thrombin exosite-1 has been partially characterized using dysthrombin Quick 1 (R62C) (50), ␥ T -and ⑀-thrombins (27,30,51), exosite-1 mutants R68E and R70E (5), and 10 basic exosite-1 residues changed to Gln (38). In the current study, we measured the inhibition of 22 different Ala-scanned exosite-1 thrombin mutants.…”