Identification of a sequence of human activated protein C (residues ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 01991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 36, Issue of December 25. pp. 24514-24519, 1991 Printed in U.S.A.

Identification of a Sequence ofHuman Activated Protein C (Residues 390-404) Essential for Its Anticoagulant Activity* (Received for publication, June 28, 1991)

Rolf M. Mesters, Richard A. HoughtenS, and JohnH. Griffins From the Committee on Vascular Biology and the Department of Molecular and ExperimentalMedicine, The Scripps Research Institute and theSTorrey Pines Institute for Molecular Studies, La Jolla, California 92037

Activated protein C (APC)exerts itsphysiologic an- serine protease (1).When activated in blood (2, 3), activated ticoagulant role by proteolytic inactivation of the blood protein C (APC), in conjunction with its cofactor protein S, coagulation cofactors V a and VIIIa. To identify the inhibits blood coagulation by proteolytic inactivation of the regions on the surface that mediate anticoagulant ac- blood coagulation cofactors Va and VIIIa (4-8). The importivity, 26 synthetic peptides were prepared representtant role of protein C as an anticoagulantin vivo is shown by ing 90%of the human proteinC heavy chain primary the fact that heterozygous PC deficiency in some families is structure and tested for their ability to inhibit APC anticoagulant activity. Peptide-(390-404)specifically associated with venous thromboembolism (9),while homozygous PC deficiency or acquired inhibitors of PC have been inhibited APC activity in activated partial thromboplastin time andXa- 1-stage coagulation assaysin nor- associatedwith severe and generalized thrombotic disease (10-12). The functionalimportance of theGla-containing mal, in protein S-depleted and Factor VIII-deficient plasma with50%inhibition at 5 p M peptide. Polyclonal region (13-15) and the epidermal growth factor-like regions antibodies raised against this peptide and immunoaf- (16-21) for the biological activity of APC has been described, finity-purified on a protein C-Sepharose column inhib- and potential binding sites of bovine APC on bovine factor Va and on human factor VIIIa have been reported (22). In ited APC anticoagulant activity in activated partial thromboplastin time and Xa-1-stage assays in normal, this study, we identify a sequence in human PC essentialfor protein S-depleted, and Factor VIII-deficient plasma its anticoagulant activityby using overlapping synthetic pepwith half-maximal inhibitionat 30 nM anti-(390-404) tides and anti-peptide antibodies. antibody. Neither the peptide-(390-404) nor the anti(390-404) antibodies inhibited APC amidolytic activEXPERIMENTAL PROCEDURES AND RESULTS’ ity or thereaction of APC with recombinant [Arg3“] DISCUSSION al-antitrypsin. Furthermore, ina purified system,peptide-(390-404) inhibited APC-catalyzed inactivation Activated protein C, a vitamin K-dependent serine proof Factor Va in the presenceas well as in the absence of phospholipids with 50%inhibition at 4 PM peptide. tease, is an important anticoagulant regulator of the blood These data suggest that theregion containing residues coagulation pathway by inactivating the blood coagulation we have identified 390-404 in APC is essential for anticoagulant activity cofactors Va and VIIIa (1-12). In this study a sequence in the heavy chain of human APC thatis essential and is available to interact with antibodies or with other proteins suchas the macromolecular substrates for exerting its anticoagulant activity by using overlapping synthetic peptides and a specific anti-peptide antibody. The Factors V a or VIIIa. PC peptide-(390-404), containingthe sequenceYGVYTK VSRYLDWIH, potently inhibited APC anticoagulant activity in coagulation assays with half-maximal inhibition at 5 p M Protein C (PC)’ is a vitamin K-dependent zymogen of a peptide (Table I, Fig. 1).Three other peptides, two sharing * This work was done during the tenure of a fellowship (to R. M. partial sequence with peptide-(390-404), required highercon50%of APCanticoagulant M.) from the Deutsche Forschungsgemeinschaft (FederalRepublic of centrations in order to inhibit Germany). This study was supported in part by Grants HL-31950 activity under the conditions tested peptide-(396-410) with and Mol-RR00833 from the National Institutesof Health. The costs of 70 p ~peptide-(384-398) ; with ICs0of 425 p ~peptide; of publication of this article were defrayed in part by the payment of (317-331) with ICso of 500 p~ (Table I). page charges. This article must therefore be hereby marked “aduerThe effect of the most potent inhibitor of APC anticoagutisement” inaccordance with 18U.S.C. Section 1734 solely to indicate lant activity, peptide-(390-404),seemed to be specific for this fact. To whom correspondence should be addressed Dept.of Molecu- APC,sincethepeptideinhibitedAPCactivityinAPTT lar and ExperimentalMedicine, BCR-5, The Scripps Research Insti- assays in normal as well as protein S-depleted plasmas and tute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554- in Xa-1-stage assays in normalwell as as FactorVIII-deficient 8220; Fax: 619-554-6732. significant plasmas (Fig. 1) and since this peptide had no The abbreviationsused are: PC, proteinC; APC, activated protein effect on these coagulation assays in theabsence of APC (up C; APTT, activated partial thromboplastin time; Gla, y-carboxyglutamic acid NHP, normal human plasma; PSDP, protein S-depleted to 240 p~ tested). The specificity of the peptide was furthermore supported by the fact that peptide-(404-390) with the plasma; S-2238, H-o-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride; S-2366, L-pyroglutamyl-L-prolyl-L-arginine-p-reverse sequence of peptide-(390-404) failed to inhibit APC nitroanilide hydrochloride; BSA, bovine serum albumin; TBS, Trisbuffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; KLH, keyhole limpethemocyanin;SPDP, N succinimidyl-3-(2-pyridyldithio)propionate;ELISA, enzyme-linked immunosorbent assay; HPLC, high performance liquid chromatography.

Portions of thispaper(including“ExperimentalProcedures,” “Results,” Figs. 1-4, and Tables I-IV) are presented in miniprint a t the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that available is from Waverly Press.

24514

APC Sequence Essential

for Anticoagulant Activity

anticoagulant activity (Table I, Fig. 1).The observed dose dependence of peptide-(390-404) was similar using either APTT or Xa-1-stage assays in normal, PSDP, or Factor VIIIdeficient plasmas. This suggests that the inhibitory effect of this peptide is not dependent on the presence of protein S and suggests therefore that theinteraction between APC and PS is not disturbed by the peptide. Furthermore, peptide-(390-404) as well as peptide-(317331) did not affect the amidolytic activity of APC towards the chromogenic substrate S-2366 (Table IV) or the inhibition of APC by recombinant [Ar~s]al-antitrypsin.These data indicate that peptide-(390-404) and peptide-(317-331) do not exert their inhibition of APC anticoagulant activity by blocking regions close to the active site of the enzyme that are involved in cleavage of a small substrate like S-2366 or in recognition of the recombinant mutant al-antitrypsin. Polyclonal antibodies raised against the most potent inhibitory peptide-(390-404) were immunoaffinity-purified on a PC-Sepharose column indicating that at least parts of the region represented by this peptide in PC are exposed and available for interaction with antibodies at thesolvent accessible surface of PC. Antibodies produced against small synthetic peptides are frequently capable of recognizing native proteins (53,54).The immunoaffinity-purified anti-(390-404) antibody recognized PC as well as APC with an apparent dissociation constant ( K d )of0.34nM. Anti-(390-404) antibodyrecognizedspecifically the sequence of peptide-(390404) but did not significantly bind to peptides-(404-390), -(384-398), or -(396-410) (Table 111). Thus, the epitope for anti-(390-404) required the peptide sequence comprising residues 390-404. Anti-(390-404) antibody appeared highly specific for APC and PC, although the sequence of residues 390404 in PC shows a high degree of homology to Factor IX, Factor X, and prothrombin (Table 11) since anti-(390-404) antibody did not significantly recognize these immobilized proteins. The specificity was further emphasized by the finding that fluid phase PC andpeptide-(390-404), but not Factor IX, Factor X, prothrombin, or peptide-(404-390), were capable of competing with the binding of anti-(390-404) antibody to immobilized PC. This observation also suggests that peptide-(390-404) in fluid phase represents an epitope capable of competing with the native protein for its recognition by anti(390-404) antibody. The fact that half-maximal inhibition of anti-(390-404) antibody binding to immobilized PC occurred at about 9.2 nM PC and 4.4 p M peptide-(390-404) might furthermore suggest that approximately 1 out of 500 peptide molecules at any time is in a conformation that enables the peptide to compete with native PC. The specificity of the described anti-peptide antibody agrees well with earlier reports concerning the specificity of anti-peptide antibodies (33). Similar to peptide-(390-404), the anti-(390-404) antibody potently inhibited APC anticoagulant activity in APTT assays in normal plasma and PSDP as well as in Xa-1-stage assays in normal and Factor VIII-deficient plasmas with 50% inhibition at -30 nM antibody in each assay. The inhibition of APC by anti-(390-404) antibody was specific since nonimmune rabbit IgG showed no effect on APC anticoagulant activity and the anti-(390-404) antibody had no effect on the coagulation assays in the absence of APC. The fact that the dose response for anti-(390-404) antibody for inhibition of APC in normal human plasma was almost identical to that observed in PSDP suggests that the antibody does not interfere with the interaction of APC with its cofactor, protein s. Thus, it is unlikely that the region of residues 390-404 is involved in APC interactions with protein S.

24515

The assembly of APC with its substrates, Factors Va and VIIIa, on a phospholipid surface is presumably mediated by the Factor Va light chain and by the Gla-domain of APC in the presence of calcium (13-15, 41, 55, 56). The observation that peptide-(390-404) inhibited APC-catalyzed inactivation of purified Factor Va in the presence as well as in the absence of phospholipids excludes the possibility that peptide-(390404) inhibits binding of APC or its substrate Factor Va to phospholipids. Therefore, we suggest that theregion of residue 390-404 represents an exosite on APC essential for its anticoagulant activity and for the recognition of its macromolecular substrate Factor Va and possibly Factor VIIIa. This exosite is not involved in primary substrate recognition near the active site, since neither peptide-(390-404) nor the anti(390-404) antibody inhibited the amidolytic activity of APC or the reaction of APC with the serpin [Ar$5s]al-antitryp~in. Furthermore, thisexosite is not essential for APC interactions with protein S since identical results were obtained in APC anticoagulant activity assays in the absence or presence of protein S. When APC cleaves a peptide bond in the heavy chain of Factor Va, this exosite might associate with the Va light chain, or vice versa when the light chain of Va is cleaved by APC. This hypothesized exosite would presumably be located where the homologous residues 225-239of chymotrypsin are found in the three-dimensional x-ray crystallographic model of chymotrypsinogen. This site is distant from the hydrophobic substrate binding pocket of the catalytic domain. The region of residues 225-239 in chymotrypsinogen (homologous to 390-404 in PC) is partially buried with residues 225-229 forming a @-strand with residues 214-219, while residues 230-239 form an amphipathic a-helix that is on the surface and thatcould potentially mediate the binding of the homologous enzyme APC to its macromolecular substrates, Factors Va and VIIIa. The C-terminal helix of APC is unlikely to be involvedin primary interactionswith smaller substrates based on the chymotrypsin model. This inference is very reasonable because of the sequence homology observed for the heavy chain of APC and other serine proteases (45, 51, 60) and because this class of enzymes hasa common threedimensional structure allowing inferences not requiring high resolution structures about structure-function relationships of APC using models based on the structure of homologous enzymes (57-60). In the region of Factor IX that is homologous to residues 390-404 in APC (Table 11) two pointmutations that are responsible for the phenotypic display of hemophilia B have been reported, namely Factor &,,, Beach, Ile397to Thr (61, 62), Factor I X V ~Ile397 ~ to ~ Thr ~ ~(63), ~ and ~ ~Factor , IXAngers, Gly396to Arg (64), that potentially affect the hypothesized exosite. For the homologous regionin Factor VIIa, a synthetic peptide comprising residues 376-390of Factor VIIa (Table 11) was reported that inhibited the Factor VIIa-tissue factormediated conversion of Factor X to Factor Xa (65). These findings suggest the general importance of the region of APC containing residues 390-404 and of the homologous regions in the vitamin K-dependent serine proteases Factor IXa, Factor VIIa, Factor Xa, and a-thrombin for recognition of their macromolecular substrates or cofactors. Acknowledgments-We are grateful to Dr. AndrLs Gruber forpreparation of APC, to Dr. Hernan Cuervo for synthesis and analysis of peptides, and to Jim Roberts and Benjamin Gutierrez forimmunizing and bleeding rabbits. We also would like to thank Dr. Rainer Bischoff and Transgene for the recombinant [Arg358]al-antitrypsin,Drs. Guido Tans and Jan Rosing for providing the purified Factor Va, and Drs. Mary Jo Heeb, Josk Fernandez and Zaverio Ruggeri fortheir helpful discussions.

24516

APC Sequence Essential for Alnticougulant Actiuity

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APC Sequence Essential for Anticoagulant Activity

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APC Sequence Essential

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