Identification of structural domains in protein C involved in its

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, No. 2, Issue of January 15, pp. 703-706,1992 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A .

Communication Identification of Structural Domains in ProteinC Involved in Its Interaction with ThrombinThrombomodulin on the Surface of Endothelial Cells*

Protein C (PC),’ a vitamin K-dependent regulator of blood coagulation, is activated from a proenzyme to a serine proteinase by thrombin (IIa) incomplex with an endothelialcell membrane cofactor, thrombomodulin(TM). T M enhances the rateof PC activation by IIa more than 1000-fold. Binding of IIa to TM in the IIa-TM complex modulates the substrate specificity of IIa such that it nolonger cleaves fibrinogen to (Received for publication, August 30, 1991)form fibrin or activates platelets or factors V and VIII. T M thus converts IIa from a procoagulant to an anticoagulant Philip J. Hoggl, Ann-Kristin Ohling, and enzyme (1).T M is an integral membrane protein that consists Johan StenfloT of a COOH-terminal intracellular region, followed by a memFrom the Department of Clinical Chemistry, University of brane spanning region, an extracellular region with 0-linked Lund, Malmo General Hospital, 8-214 01 Malmo, Sweden carbohydrate side chains, and thereaftersix modules that are homologous to the EGFprecursor. The NHz-terminalregion The structural domains of protein C involved in its of T M is homologous to the asialoglycoprotein receptor (2) interaction with thrombin-thrombomodulin on the and other mammalian “lectin” domains, including the similar endothelial cell surface have been investigated using domain in ELAM-1, MEL-14, and GMP-140 (3-6). isolated intact domains of bovine protein C produced Recently, Kurosawaet al. (7) isolated a proteolyticfragment from controlled proteolytic digests of the protein. The fragments investigated include the y-carboxyglutamic from elastase digests of rabbit TM. The fragment consisted acid (G1a)-rich module,the two epidermal growth fac- of the six EGF-like modules and did not require detergents to tor (EGF)-like modules, and a fragment consisting of remain in solution. It interacted with both PC and IIa and the Gla and the two EGF-like modules. The effects of retained cofactor activity. It alsoappearedto be ableto these fragments on the catalytic efficiency (K, and interact specifically with the Gla region of PC. However, a V,,,) of activation of protein C by the endothelial cell fragment consisting of the fifth and sixth EGF-likemodules surface thrombin-thrombomodulin complex (IIa-TM) from T M lacked cofactor activity but retaineda high affinity have been evaluated in vitro using a stirred microcar- IIabindingsite (8, 9). Inaddition, Zushi et al. (10)have rier cell culture of bovine aortic endothelial cells and expressed deletion mutants of T M in COS-1 cells. Based on purified proteins. Neither the Gla nor the two EGF- studies of the mutants theysuggested that EGF-likemodules like modules alone had any discernible effect on pro- four to six had PC activatingcofactor activity. tein C activation. The intact Gla-EGF fragment, howWe have previously isolated and characterized proteolytic ever, inhibited protein C activation. The results are fragments of PC, andalso from factors IX and X, that consist consistent with a rapid equilibrium competitive inhi- of a single moduleor two or threemodules in combination, in bition model, in which the Gla-EGF fragment competes order to study structure-function relationshipsin the vitamin with protein C for binding to IIa-TM, and indicate that K-dependentplasmaproteins (11-18). We have now used the Gla-EGF fragment alone accounts for most of the fragments of bovine PC that consist of the Gla module, the binding energy of intact protein C for IIa-TM. In adtwo EGF-like modules, and theGla module linked to thetwo dition, arequirement for the Gla residues of protein C for binding is implied by the observation that heat- EGF-like modules in order to elucidate which part of PC decarboxylated Gla-EGF fragment was not an inhibi- interacts with the IIa-TMcomplex. The results demonstrate of the Gla and EGF-likemodules tor of protein C activation. In addition, chloromethyl that the fragment consisting ketone-inactivated activated protein C was found to accounts for most of the binding energy of intact PC for the bind to IIa-TM with the same affinity as protein C, IIa-TM complex on bovine aortic endothelial cells. suggesting that the changes which occur in protein C MATERIALSANDMETHODS upon activation do not affect that part of the protein responsible for binding to IIa-TM, that is the Gla-EGF Material-HEPES, p-nitrophenyl p’-guanidinobenzoate, heparin region. The Gla-EGF region from factor X also weakly (sodium salt, grade 11), and Oryuranus scutellutus (Taipan snake) inhibited the IIa-TM activation of protein C. venom were obtained from Sigma, D-Phe-Pro-Arg-chloromethyl ketone (PPACK) from Calbiochem, H-D-Ile-Pro-Arg-p-nitroanilide

*This investigation was supported by grants from the Swedish Medical Research Council (Project 44871), the Swedish Medical Society, Albert Pahlsson’s Foundation, and Kock’s Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by a Visiting Scientist Fellowship from the Swedish Medical Research Council. Present address: Dept. of Haematology, Prince of Wales Hospital, Randwick, N. S. W. 2031,Australia. 8 Present address: Dept. of Clinical Chemistry, Universityof Lund, University Hospital, S-22185Lund, Sweden. 1 To whom correspondence should be addressed.

The abbreviations used are: PC, bovine protein C; HEPES, N-(2hydroxyethyl)piperazine-N’-2-ethanesulfonicacid; PPACK, D-PhePro-Arg-chloromethylketone; S2288, H-D-Ile-Pro-Arg-p-nitroanilide; HBSS, Hanks’ balanced salt solution; DMEM, Dulbecco’s modified Eagle’s medium; S-MEM, minimum essential medium; FCS, fetal calf serum; PC-Gla, residues 1-41 of the light chain of PC; PCEGF, residues 42-143 of the light chain linked to 108-131 of the heavy chain of PC; PC-Gla-EGF, residues 1-143 of the light chain linked to 108-131 of the heavy chain of PC; PC,, activated protein C; PC.-CK, PC. inactivated with PPACK; PC-Gla-EGFDc, heat-decarboxylated PC-Gla-EGF; FX-Gla-EGF, light chain linked to residues 154-183 of the heavy chain of bovine factor X; prothrombin fragment 1, residues 1-155 of bovine prothrombin; BAEC, bovine aorta endothelial cells; TM, thrombomodulin; IIa-TM, endothelial cell surface thrombin-thrombomodulin complex; EGF, epidermal growth factor.

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Interaction of Protein C with Thrombin-Thrombomodulin

(S2288) from Kabi, and Cytodex 3 beads from Pharmacia LKB Biotechnology Inc. Hanks' balanced salt solution (HBSS), nutrient mixture Ham's F-12, Dulbecco's modified Eagle's medium(DMEM), minimum essential medium (S-MEM), fetal calf serum (FCS), and gentamicin were obtained from Gibco. All other chemicals were of reagent grade. Proteins-Bovine a-IIa was purified from prothrombin as described by Lundblad and co-workers (19) following activation with 0. scutellatus venom according to Owen and Jackson (20). Active site titration of the purified IIa with p-nitrophenyl p'-guanidinobenzoate (21), with corrections for turbidity (22), indicated that the enzyme was 92% active based on an Ai:,,, at 280 nm of 19.5 and M, of 37,400 (23). Human antithrombin 111 was purified according to Thaler and Schmer (24) and its concentration calculated using an Ai:,,, at 280 nm of 6.5 and M,of 58,000 (25). PC was purified from bovine plasma and activated with a-IIa as described previously (26, 12). The Gla region of PC (PC-Gla, residues 1-41of the light chain), the EGF homology region (PC-EGF, residues 42-143 of the light chain linked t o 108-131 of the heavy chain), and the combined Gla-EGF region (PC-Gla-EGF, residues 1-143 of the light chain linked to 108-131 of the heavy chain) were purified from controlled proteolytic digests of PC as described previously (11, 12). Activated PC (PC.) was inactivated with a 2-fold molar excess of PPACK for 5 hat 37 "C in 50 mM HEPES, 0.125 M NaCl, pH 7.4. The inactivated PC. (PC,-CK), which contained