Serpins define a large protein family in which most members function as serine protease inhibitors. Here we report the results of a search for serpins in Drosophila melanogaster that are potentially required for oogenesis or embryogenesis. We cloned and sequenced ovarian cDNAs that encode six distinct proteins having extensive sequence similarity to mammalian serpins, including residues important in the serpin inhibition mechanism. One of these new serpins in recombinant form inactivates, and complexes with, trypsin-like proteases in vitro. To our knowledge, these results represent the first evidence for a serpin in Drosophila that functions as a serine protease inhibitor.z 2000 Federation of European Biochemical Societies.

Heparin cofactor II (HCII) inhibits thrombin by forming a stable 1:1 complex. Heparin and dermatan sulfate increase the rate of complex formation >/=1000-fold. Mutation of leucine 444 to arginine at the P1 position of recombinant HCII (rHCII) increases the rate of inhibition of thrombin approximately 100-fold in the absence of a glycosaminoglycan (Derechin, V. M., Blinder, M. A., and Tollefsen, D. M. (1990) J. Biol. Chem. 265, 5623-5628). We now report that heparin facilitates dissociation of the thrombin-rHCII(L444R) complex. In the presence of heparin, thrombin is inhibited rapidly and completely by a 35-fold molar excess of rHCII(L444R), but subsequently approximately 50% of the thrombin activity reappears with a t1/2 of approximately 20 min. At higher ratios of rHCII(L444R) to thrombin, the reappearance of thrombin activity is delayed and the final plateau of activity is decreased. Electrophoretic analysis indicates that proteolysis of excess rHCII(L444R) precedes the reappearance of thrombin activity. Addition of heparin at longer intervals after formation of the thrombin-rHCII(L444R) complex causes a progressive decrease in the thrombin plateau, suggesting that in the absence of heparin the complex is slowly converted to a non-dissociable form. By contrast to heparin, dermatan sulfate does not facilitate dissociation of the thrombin-rHCII(L444R) complex. Our findings indicate that the P1 residue of HCII affects not only the rate of inhibition of thrombin but also the stability of the resulting complex.

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...

Heparin cofactor II (HCII) inhibits thrombin rapidly in the presence of heparin or dermatan sulfate. The product of the inhibition reaction is a kinetically stable, 1:1 complex between the two proteins. We recently observed that heparin induces dissociation of complexes containing thrombin and the reactive site mutant HCII(L444R) to yield active thrombin and cleaved inhibitor (Han, J. -H., Van Deerlin, V. M. D., and Tollefsen, D. M. (1997) J. Biol. Chem. 272, 8243-8249). In the current study, we have investigated the mechanism by which heparin induces dissociation of the thrombin-HCII(L444R) complex. Heparin oligosaccharides >/=6 sugars in length induce dissociation, which suggests that dissociation does not depend on binding of a heparin molecule simultaneously to both proteins in the complex. Binding of heparin to HCII(L444R) in the complex also does not appear to be required, since the heparin dose response is unaltered for complexes containing the double mutant HCII(L444R/K173Q), which has decreased affinity for heparin. By contrast, binding of heparin to thrombin appears to be necessary and sufficient to induce dissociation. First, heparin fails to induce dissociation of complexes that contain thrombin(K236E), a variant with decreased heparin affinity. Second, a monoclonal IgG that interacts with the heparin-binding site of thrombin mimicks heparin in its ability to induce dissociation of the thrombin-HCII(L444R) complex. Finally, the complex of HCII(L444R) with thrombin(desPPW), which binds normally to heparin but lacks Pro60BPro60CTrp60D in an insertion loop ("60-loop") between the heparin-binding site and the catalytic site, does not dissociate in the presence of heparin. These results suggest that binding of heparin to thrombin induces an allosteric effect causing destabilization of the thrombin-HCII(L444R) complex and that the allosteric effect may be mediated by the 60-loop.