The serpin protease nexin-1 regulates vascular smooth muscle cell adhesion, spreading, migration and response to thrombin.

BACKGROUND: Protease nexin-1 (PN-1) is an important physiological regulator of thrombin in the brain. PN-1 is also present in aortic smooth muscle cells and may thus participate in vascular biology. However, little is known about its function in the vessel wall. OBJECTIVES: In this study, we investigated the effect of PN-1 overexpression in smooth muscle cells (SMCs), on their sensitivity to thrombin, and their capacity for adhesion, spreading and migration. RESULTS: Two clones exhibiting a two- to threefold increase in PN-1 expression were selected and compared with untransfected and mock-transfected cells. Overexpression of PN-1 was observed to inhibit thrombin-induced cell responses as indicated by a twofold decrease in induction of PAI-1 expression, a decreased calcium mobilization in response to low thrombin concentrations and a twofold increase in the capacity to inhibit thrombin catalytic activity. Overexpression of PN-1 did not modify adhesion, spreading, and migration of SMCs on type I collagen. In contrast, SMCs overexpressing PN-1 exhibited a 40% reduction in adhesion, a 50% reduction in spreading and a complete absence of migration on vitronectin when compared with control SMCs. CONCLUSIONS: Our studies thus reveal that PN-1 is likely to play a critical role in regulating essential cell functions such as (i) thrombin-induced responses, which are dependent on its antiprotease activity, and (ii) adhesion, spreading, and migration, which are independent of its antiprotease activity and may be related to its interaction with other partners, such as vitronectin in the present case.

Lysine 114 of antithrombin is of crucial importance for the affinity and kinetics of heparin pentasaccharide binding.

Lys(114) of the plasma coagulation proteinase inhibitor, antithrombin, has been implicated in binding of the glycosaminoglycan activator, heparin, by previous mutagenesis studies and by the crystal structure of antithrombin in complex with the active pentasaccharide unit of heparin. In the present work, substitution of Lys(114) by Ala or Met was shown to decrease the affinity of antithrombin for heparin and the pentasaccharide by approximately 10(5)-fold at I 0.15, corresponding to a reduction in binding energy of approximately 50%. The decrease in affinity was due to the loss of two to three ionic interactions, consistent with Lys(114) and at least one other basic residue of the inhibitor binding cooperatively to heparin, as well as to substantial nonionic interactions. The mutation minimally affected the initial, weak binding of the two-step mechanism of pentasaccharide binding to antithrombin but appreciably (>40-fold) decreased the forward rate constant of the conformational change in the second step and greatly (>1000-fold) increased the reverse rate constant of this step. Lys(114) is thus of greater importance for the affinity of heparin binding than any of the other antithrombin residues investigated so far, viz. Arg(47), Lys(125), and Arg(129). It contributes more than Arg(47) and Arg(129) to increasing the rate of induction of the activating conformational change, a role presumably exerted by interactions with the nonreducing end trisaccharide unit of the heparin pentasaccharide. However, its major effect, also larger than that of these two residues, is in maintaining antithrombin in the activated state by interactions that most likely involve the reducing end disaccharide unit.

The region of antithrombin interacting with full-length heparin chains outside the high-affinity pentasaccharide sequence extends to Lys136 but not to Lys139.

The interaction of a well-defined pentasaccharide sequence of heparin with a specific binding site on antithrombin activates the inhibitor through a conformational change. This change increases the rate of antithrombin inhibition of factor Xa, whereas acceleration of thrombin inhibition requires binding of both inhibitor and proteinase to the same heparin chain. An extended heparin binding site of antithrombin outside the specific pentasaccharide site has been proposed to account for the higher affinity of the inhibitor for full-length heparin chains by interacting with saccharides adjacent to the pentasaccharide sequence. To resolve conflicting evidence regarding the roles of Lys136 and Lys139 in this extended site, we have mutated the two residues to Ala or Gln. Mutation of Lys136 decreased the antithrombin affinity for full-length heparin by at least 5-fold but minimally altered the affinity for the pentasaccharide. As a result, the full-length heparin and pentasaccharide affinities were comparable. The reduced affinity for full-length heparin was associated with the loss of one ionic interaction and was caused by both a lower overall association rate constant and a higher overall dissociation rate constant. In contrast, mutation of Lys139 affected neither full-length heparin nor pentasaccharide affinity. The rate constants for inhibition of thrombin and factor Xa by the complexes between antithrombin and full-length heparin or pentasaccharide were unaffected by both mutations, indicating that neither Lys136 nor Lys139 is involved in heparin activation of the inhibitor. Together, these results show that Lys136 forms part of the extended heparin binding site of antithrombin that participates in the binding of full-length heparin chains, whereas Lys139 is located outside this site.

The role of Arg46 and Arg47 of antithrombin in heparin binding.

Heparin greatly accelerates the reaction between antithrombin and its target proteinases, thrombin and factor Xa, by virtue of a specific pentasaccharide sequence of heparin binding to antithrombin. The binding occurs in two steps, an initial weak interaction inducing a conformational change of antithrombin that increases the affinity for heparin and activates the inhibitor. Arg46 and Arg47 of antithrombin have been implicated in heparin binding by studies of natural and recombinant variants and by the crystal structure of a pentasaccharide-antithrombin complex. We have mutated these two residues to Ala or His to determine their role in the heparin-binding mechanism. The dissociation constants for the binding of both full-length heparin and pentasaccharide to the R46A and R47H variants were increased 3-4-fold and 20-30-fold, respectively, at pH 7.4. Arg46 thus contributes only little to the binding, whereas Arg47 is of appreciable importance. The ionic strength dependence of the dissociation constant for pentasaccharide binding to the R47H variant showed that the decrease in affinity was due to the loss of both one charge interaction and nonionic interactions. Rapid-kinetics studies further revealed that the affinity loss was caused by both a somewhat lower forward rate constant and a greater reverse rate constant of the conformational change step, while the affinity of the initial binding step was unaffected. Arg47 is thus not involved in the initial weak binding of heparin to antithrombin but is important for the heparin-induced conformational change. These results are in agreement with a previously proposed model, in which an initial low-affinity binding of the nonreducing-end trisaccharide of the heparin pentasaccharide induces the antithrombin conformational change. This change positions Arg47 and other residues for optimal interaction with the reducing-end disaccharide, thereby locking the inhibitor in the activated state.

Inhibition of thrombin-catalyzed factor V activation by bothrojaracin.

We have previously identified and characterized a potent and specific thrombin inhibitor, isolated from Bothrops jararaca, named bothrojaracin. Bothrojaracin interacts with the two positively charged recognition sites of thrombin referred to as exosite 1 and exosite 2, whereas it does not interact with the thrombin active site. Consequently, bothrojaracin inhibits thrombin-induced fibrinogen to fibrin conversion and platelet activation, without inhibition of thrombin-catalyzed cleavage of small synthetic substrates. In the present study, we show that bothrojaracin exerts an anticoagulant effect in plasma, illustrated by the prolongation of the aPTT. Using purified proteins, we observed that the anticoagulant effect of bothrojaracin was not only due to the inhibition of fibrinogen to fibrin conversion, but in addition to the inhibition of factor V activation by thrombin. Bothrojaracin decreased the rate of thrombin-catalyzed proteolysis of factor V and concurrently the generation of factor Va cofactor activity measured in a prothrombinase assay. We compared the effect of bothrojaracin with that of ligands binding specifically exosite 1 (hirudin C-terminal peptide SH54-65) or exosite 2 (heparin, prothrombin fragment 2). SH54-65 delayed thrombin catalyzed factor V activation whereas heparin or prothrombin fragment 2 did not. The thrombin derivatives beta- and gamma-thrombin, which are defective in their exosite 1, but present with a normally exposed exosite 2, had a reduced capacity to activate factor V, which was not further impaired by the exosite 2 ligands, bothrojaracin, heparin or prothrombin fragment 2. Altogether, our results provide further insight into the anticoagulant effect of bothrojaracin showing that it is a potent inhibitor of the feedback activation of factor V by thrombin, and thus of the up-regulation of its own production by thrombin. Inhibition of thrombin-catalyzed factor V activation by bothrojaracin is mainly mediated through the interaction of the inhibitor with thrombin exosite 1, whereas contribution of the interaction with exosite 2 does not appear to play a direct role in factor V recognition by thrombin.

Molecular cloning and expression of bothrojaracin, a potent thrombin inhibitor from snake venom.

Bothrojaracin is a potent and selective thrombin inhibitor that has been isolated from the venom of Bothrops jararaca. It does not interact with the catalytic site of the enzyme but binds to both anion-binding exosites 1 and 2 resulting in a potent inhibition of thrombin activity towards fibrinogen and platelets [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C. & Bon, C. (1993) Biochemistry 32, 10794-108021. Bothrojaracin is a 27-kDa protein composed of two disulfide-linked polypeptide chains, A and B, of 15 kDa and 13 kDa, respectively. The sequences of A and B chains determined by molecular cloning exhibit a high degree of identity with other snake venom lectin-like proteins. In contrast to other ligands that interact with thrombin exosite 1, the amino acid sequence of bothrojaracin does not contain an acidic sequence similar to the C-terminal tail of hirudin. Expression of functional bothrojaracin was achieved in COS cells upon transfection with two pcDNA3 vectors containing the complete cDNAs. Recombinant bothrojaracin, which was secreted into the medium, was able to bind to and inhibit thrombin. When expressed alone, the B chain formed inactive dimers that were secreted into the culture medium. In contrast, no bothrojaracin-related protein was detected in conditioned media from cells transfected with the A chain.

Bothrojaracin: a potent two-site-directed thrombin inhibitor.

The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., & Bon, C. (1993) Biochemistry 32, 10794-10802], is a 27 kDa protein isolated from the venom of Bothrops jararaca that blocks several thrombin functions, including fibrinogen clotting, platelet activation, and fibrin and thrombomodulin binding, but does not interact with the catalytic site. In the present report, we show that the high affinity binding of alpha-thrombin to immobilized bothrojaracin (Kd = 0.6 nM) is inhibited by the C-terminal peptide of hirudin and that the gamma-cleavage within exosite 1 reduces the affinity of bothrojaracin for thrombin (Kd = 0.3 microM), indicating that bothrojaracin binding to exosite 1 is a major determinant of the thrombin-bothrojaracin interaction. In addition, we show that bothrojaracin decreases the rate of inhibition of alpha- and gamma-thrombin by the antithrombin III-heparin complex. Competition of bothrojaracin with heparin or prothrombin fragment 2 for binding to thrombin indicates that bothrojaracin not only binds exosite 1 but also binds exosite 2 or in close proximity. Bothrojaracin binds to the thrombin precursor, prothrombin. This interaction is calcium-independent and is prevented by heparin, suggesting that it is mediated by exosite 2. Bothrojaracin inhibits platelet activation induced by clot-bound thrombin and slowly dissociates thrombin from the fibrin clots. Altogether, our results indicate that the high affinity of bothrojaracin for thrombin is supported by a double-site interaction and results in an efficient inhibition of both soluble and clot-bound thrombin.