Vol. 268, No. 30, Issue of October 25, pp. 22531-22539,1993 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Effects of Ca2’ Binding on the ProteaseModule of Factor Xa and Its Interaction with FactorV a EVIDENCE FOR TWOGla-INDEPENDENTCa2+-BINDINGSITESIN

FACTOR Xa*

(Received for publication, March 3, 1993, and in revised form, June 2, 1993)

Egon PerssonS, Philip J. HoggQ,and JohanStenfloll From the Department of Clinical Chemistry, University of Lund, Malmo General Hospital, S-214 01 Malmo, Sweden

Prothrombinase, consisting of active factor X (factor Xa) The assembly of macromolecular complexes containing factorsXa and Va on suitable phospholipid surfaces and theactivated form of the cofactor, factor V (factor Va), a is crucial for rapid activation of prothrombin. We have suitable phospholipid surface, and calcium ions, is the macused quantitative affinity chromatography to charac- romolecular enzyme complex in the procoagulant cascade that terize the interaction between factor Va and intact has been studied in most detail (1,2). In the presence of Ca2+, factor Xa on the one hand and between factor Va and the phospholipid surface promotes enzyme-cofactor associafactor Xa lacking the y-carboxyglutamic acid (G1a)- tion lowering the apparent K,,, for prothrombin, whereas the containing module on the other. Thedissociation con- cofactor, factor Va, increases the rate (kcat) of factor Xastants were found to be 1.0 f 0.1 and 9.5 f 1.8 pM, catalyzed prothrombin activation (1-6). Complex assembly is respectively. There wasgood agreement between these believed to proceed via independent associations of factors dissociation constants and the concentrations of active site-inhibited factor Xa and Gla-domainless factor Xa Xa and Va with the membrane surface (5,7). The environthat caused half-maximal inhibition of prothrombin ment of the active site of factor Xa changes on complexation with factor Va, and theamidolytic activity toward low molecactivation. To investigatewhetherthenoncatalytic ular weight substrates is affected (6, 8 ) The active site of modules of factor Xa interacted directly with factor Va, intact modules were isolated from proteolytic di- factor Xa is not required for association with factor Va which gests of factor X and used as inhibitors of prothrombin permits the use of active site-modified species of factor Xa in studies of prothrombinase (9). The complete prothrombinase activation.Theinhibitory effect observed withthe isolated Gla modulein the absence of phospholipid was complex activates prothrombin approximately five orders of due to inhibition of the amidolytic activity of factor Xa magnitude faster than does factor Xa alone (3). rather than to an interaction with factorVa. The epiFactors VIIa, IXa,Xa,and activated proteinCsharea dermal growth factor-like modules did not inhibit pro- common modular structure (10). The y-carboxyglutamic acid thrombin activation. (Gla)’ module in each of the vitamin K-dependent plasma Using antibodies specific for calcium-dependent ep- proteins is essential for calcium and membrane binding (ll), itopes in the serine protease module of factor Xa we but it has also been implicated in the interactions of factors demonstrated that Ca2+binding to the Gla module al- IXa andVIIa with factor VIIIa and tissue factor, respectively, ters the conformation of the catalytic module. Half- and in the interaction of protein C with thrombin/thrombo8M Ca2+. Evi- modulin (12-16). Activation endows the serine protease modmaximal binding was observed at ~ 0 . m dence was also obtained for the presence of two Gla- ules with amidolytic activity and brings about a conformaindependent Ca2+-binding sites in factor Xa. One of tional transition that is crucial for factor Xa assembly with thesesites, located in theNHz-terminalepidermal growth factor-like module, was half-saturated at ~ 6 0factor Va to form the prothrombinase complex (17-21). The pM ca2+in intact factor Xa and at ~ 1 . 2 mM ca2+in protease module of factor IXa mediates binding to factor Gla-domainless factor Xa. This site appeared not to VIIIa (22), andthe protease module of factor VIIa appears to influence the conformation of the protease module. The contribute to the interaction with tissue factor (23, 24). The second site, which was half-saturated at -0.16 mM function of the epidermal growth factor (EGF)-like modules Ca2+,appeared to reside in the serine protease module in the enzyme-cofactor complexes is less clear, although the and to alter its conformation as judged by binding of The abbreviations used are: Gla, y-carboxyglutamic acid EGF, antibodies specific for calcium-dependent epitopes.

* This investigation was supported by Swedish Medical Research Council Projects 4212 and 4487, the Medical Faculty of Lund University, andthe Albert Pihlsson’s, Kock’s, Osterlund’s, Magnus Bergvall’s, and Knut andAlice Wallenberg’s Foundations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. $ Present address: Biopharmaceuticals Division, Novo Nordisk A/ S, Niels Steensens Vej 1, DK-2820 Gentofte, Denmark. J Present address: Dept. of Haematology, Prince of Wales Hospital, High Street, Randwick, New South Wales 2031, Australia. ‘1[ To whom correspondence should be addressed.

epidermal growth factor; DFP, diisopropyl fluorophosphate; PC, L-(Yphosphatidylcholine; PS, L-a-phosphatidyl-L-serine;DEGR-CK, dansylglutamylglycylarginyl chloromethyl ketone; pNA, p-nitroanilide; S-2238, H-D-Phe-L-pipecolyl-L-Arg-pNA; S-2288, H-D-Ile-LPro-L-Arg-pNA; S-2765, N-a-benzyloxycarbonyl-D-Arg-L-Gly-LArg-pNA; PEG, polyethylene glycol; PAGE, polyacrylamide gel electrophoresis; f x , bovine factor X; E-Gla, residues 1-44 of the light chain of bovine factor X; M-EGFN, residues 45-86 of the bovine factor X light chain; M-EGFC, residues 88-128 of the bovine factor X light chain; E-EGFNc, residues 45-140 of the light chain disulfidelinked to residues 154-171/183 of the heavy chain of bovine factor X; fX-GlaEGFN,residues 1-86 of the light chain of bovine factor X; EGlaEGFNc,the intact light chain of bovine factor X disulfide-linked to residues 154-183 of the heavy chain; RVV-V, the factor V activator from Russell’s viper venom; QAC, quantitative affinity chromatography; IgSPa, antibodies recognizing epitopes in the serine protease module of DEGR-factor Xa.

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Ca2+Effects on the Protease Module

Ca2+-binding site in the NH2-terminal EGF-like module is important for biological activity (25). The EGF-like modules of factor VIIa appear to interact with tissue factor (26), and the second EGF-like module in factor IXa hasbeen suggested to be involved in factor VIIIa binding (27). The dissociation constant ( K d ) for the binding of factor Xa to stimulated platelets(20,21,28) or to factor Va on synthetic phospholipid vesicles (3, 6, 18, 29, 30) is in the 0.1-1 nM range. The Kd for the interaction between factors Xa and Va in the absence of phospholipid has been determined to be 1 PM (31,32). Gla-domainless factor Xa has very low biological activity, and kinetic experiments suggest that it binds factor Va with lower affinity than does intact factor Xa, even in the absence of phospholipid (29, 33). The low biological activity after removal of the Gla module reflects a loss of membrane binding but may also be a consequence of reduced affinity for factor Va. We have extended previous studies (18) and investigated whether the isolated Gla or EGF-like modules of factor Xa inhibit prothrombin activation through a direct interaction with factor Va. The properties of intact and Gla-domainless DEGR-factor Xa were also studied and the K d values for the interactions between factor Va and intact or Gla-domainless DEGR-factor Xa were estimated from direct binding experiments. The different properties of the two forms of DEGRfactor Xa may be explained by Gla-dependent Ca2+-induced conformational changes inthe protease module of factor Xa. A second, Ca2+-inducedstructural transition in the catalytic module was observed in the Gla-domainless protein. It appeared to be a consequence of Ca2+binding to a site COOHterminal of the first EGF-like module, presumably located in the serine protease module. EXPERIMENTALPROCEDURES

Materials-Phenylmethylsulfonyl fluoride, p-nitrophenyl-p'-guanidinobenzoate, leupeptin, antipain, chymostatin, aprotinin, soybean trypsininhibitor, Russell's vipervenom,galactose oxidase(from Dactylium dendroides), neuraminidase (typeX from Clostridium perfringens),L-a-phosphatidylcholine(PC), L-a-phosphatidyl-L-serine (PS), Glu-specific endopeptidase fromStaphylococcus aureus V8, and ~OIY-L-GIU (molecularweight 2,000-15,000) were from Sigma and diisopropyl fluorophosphate (DFP)was from Fluka. CNBr-activated Sepharose 4B and agarose adipic acid hydrazide were from Pharmacia, dansyl-Glu-Gly-Arg chloromethyl ketone (DEGR-CK) from CalH-D-Phe-L-pipecolyl-L-Arg-pNA (Sbiochem, andthesubstrates 2238),H-D-Ile-L-Pro-L-Arg-pNA(S-2288), andN-a-benzyloxycarbonyl-D-Arg-L-Gly-L-Arg-pNA(S-2765) from Kabi Diagnostica. Other reagents were obtained from the sources listed in Refs. 34 and 35. Proteins-Bovine factor X, prothrombin, thrombin, and prethrombin 1 were isolated by standard procedures (36-38). Thrombin was assayed by active site titration(39). Intact and Gla-domainless factor Xa were isolated by established procedures (33, 40) and inactivated with a 5-fold molar excess of DEGR-CK for 15 min. Excess inhibitor was removed bygel filtration. The activesite-inhibitedproteins retained