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Heparin Binding to Protein C Inhibitor

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Heparin Binding to Protein C Inhibitor THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, No. 13, Issue of May 5, pp. 8789-8794, 1992 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A. Heparin Binding toProtein C Inhibitor* (Received for publication, December 11, 1991) Charlotte W. PrattS and FrankC. Church From the DeDartment of Pathology and The Center for Thrombosis and Hemostasis, The University of North Carolina School of Medicine, C&el Hill, North Ca&na 27599 Protein C inhibitor isa plasma protein whose ability regulated by a plasma glycoprotein named protein C inhibitor, to inhibit activated protein C, thrombin, and other also known as plasminogen activator inhibitor-3 (4). Three enzymes is stimulated by heparin. These studies were other major plasma proteins, az-macroglobulin, az-antiplas- undertaken to further understand how heparin binds min, and a’-proteinase inhibitor inhibit activated protein C to protein C inhibitor andhow it accelerates proteinase (5) but might be effective only by virtue of their relatively inhibition. The region of protein C inhibitor from res- high concentration in plasma. Protein C inhibitor reacts with idues 264-283 was identified as the heparin-binding the active site of activated protein C to form an essentially site. This differs from the putative heparin-binding irreversible complex (6). Interestingly, protein C inhibitor site in the related proteins antithrombin and heparin also inhibits thrombin, the final proteinase of the coagulation cofactor. The glycosaminoglycan specificity of protein pathway, as well as other procoagulant enzymes. This broad C inhibitor was relatively broad,including heparin and heparan sulfate, but not dermatan sulfate. Non-sul- target proteinase specificity of protein C inhibitor presents a fated and non-carboxylated polyanions also enhanced problem in understanding the physiological importance of proteinase inhibition by protein C inhibitor. Heparin protein C inhibitor as a regulator of the protein C system. accelerated inhibition of a-thrombin, TT-thrombin, ac- Direct evidence for the involvement of protein C inhibitor is tivated protein C, factor Xa, urokinase, and chymo- lacking, as an inhibitor deficiency has yet to be documented. trypsin, but not plasma kallikrein. The abilityof gly- Protein C inhibitor is a member of the serine proteinase cosaminoglycans to accelerateproteinase inhibition inhibitor (serpin)’ superfamily of proteins, whose prototype appeared to depend on the formationof a ternary com- is a’-proteinase inhibitor (7). Protein C inhibitor can be plex of inhibitor, proteinase, and glycosaminoglycan. further classified as a heparin-binding serpin, along with the The optimum heparin concentration for maximal rate proteinaseinhibitors antithrombin (historically known as stimulation varied from 10 to 100 pg/ml and was re- antithrombin 111) and heparin cofactor (also called heparin lated to the apparent affinity of the proteinase for cofactor 11). Heparin and some other glycosaminoglycans act heparin. There was no obvious relationship between to increase the rate of proteinase inhibition by these three heparin affinity and maximum inhibition rate or de- plasma inhibitors, insome cases as much as several thousand- gree of rate enhancement. The affinityof the resultant fold ($). The mechansim whereby heparin catalyzes protein- protein C inhibitor-proteinase complex was also not ase inhibition is a subject of much study, especially dueto the related toinhibition rate enhancement, and the results widespread use of heparin as a therapeuticanticoagulant. The showed that decreased heparin affinityof the complex ability of heparin to accelerate the inhibition of activated is not ap important part of the catalytic mechanism of protein C by protein C inhibitor, therebyfavoring coagulation, heparin. The importance of protein C inhibitor as a regulator of the protein C system may depend on the is at odds with the anticoagulant effect of heparin therapy. relatively large increase in heparin-enhanced inhibi- As a first steptoward understanding this apparentcontradic- tion rate for activated protein C compared to other tion and in order to gather insight into the physiological proteinases. importance of protein C inhibitor, a series of studies was undertaken. The work presented here describes the heparin- binding site of protein C inhibitor, the polyanion specificity of protein C inhibitor, and the mechanism whereby heparin Hemostasis requires a balance between procoagulant and accelerates proteinase inhibition. Some of these results have anticoagulant forces. Among the anticoagulant mechanisms appeared previously in abstract form (9). The following report is the protein C system. Thrombin generated during coagu- compares protein C inhibitor to antithrombin and heparin lation binds to thrombomodulin on vesselwalls andthe cofactor (10). thrombin-thrombomodulin complex activates the zymogen protein C. Activated protein C, with its cofactor protein S, EXPERIMENTALPROCEDURES (1). proteolytically inactivates coagulant factors V and VI11 Materiab-Human protein C inhibitor was purified as previously The importance of the protein C system is demonstrated by described (ll), as were antithrombin and heparin cofactor (12). All the incidence of thrombosis in individuals who lack protein C proteinases wereof human origin, with the exception of bovine (2) or protein S (3). The protein C system is believed to be chymotrypsin. a-Thrombin was purified as described (13) or con- verted to ?,-thrombin (14). Protein C was prepared as a by-product * This work was supported in part by Research Grant HL-06350 of the factor X preparation (15), and further purified and activated from the National Institutes of Health. The costs of publication of by incubating it with thrombin, then passing the mixture over im- this article were defrayed in part by the payment of page charges. mobilized antithrombin to remove thrombin and traces of factor Xa. This article must therefore be hereby marked “advertisement” in Urokinase was purchased from Sigma, plasma kallikrein from Calbi- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $TO whom correspondence and reprint requests should be ad- The abbreviations used are: serpin, serine proteinase inhibitor; dressedThe University of North Carolina, Div. of Hematology, HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid; PEG, Campus Box 7035, Chapel Hill, NC 27599-7035. Fax: 919-966-7639. polyethylene glycol; HPLC, high performance liquid chromatography. 8789 This is an Open Access article under the CC BY license. 8790 Protein to Binding Heparin C Inhibitor ochem, bovine chymotrypsin from Cooper Biomedical, and neutrophil 5-ml column of heparin-Sepharose in 20 mM HEPES, 10 mM NaC1, elastase from Elastin Products (Pacific, MO). The following protein- 0.1% PEG, pH 7.4. Samples of protein C inhibitor, proteinase, and ase substrates were used Chromozym TH (tosyl-Gly-Pro-Arg-p-ni- protein C inhibitor-proteinase complexes were eluted with a 1 ml/ troanilide) for thrombin from Boehringer Mannheim, Spectrozyme min linear salt gradient from 10 mM to 1.2 M NaCl; 0.25-ml fractions PCa (Lys(Cbo)-Pro-Arg-p-nitroanilide)for activated protein C and were collected. Proteins were detected by absorbance at 280 nm and Spectrozyme FXa (MeO-CO-CHG-Gly-Arg-p-nitroanilide)for factor by reactivity with a rabbitpolyclonal antiserum to protein Cinhibitor Xa from American Diagnostica, S-2444 (Glu-Gly-Arg-p-nitroanilide) (to detect protein Cinhibitor and proteinC inhibitor-proteinase for urokinase and S-2302 (Pro-Phe-Arg-p-nitroanilide) for kallikrein complexes) and by chromogenic substrate hydrolysis (to detect pro- from KabiVitrum, and Suc-Ala-Ala-Pro-Phe-p-nitroanilidefor chy- teinases and protein C inhibitor-proteinase complexes; an extended motrypsin from Sigma. The following were purchased from Sigma: incubation time was required to detect proteinase activity in com- bovine serum albumin, polybrene (1,5-dimethyl-1,5-diazaundecame- plexes). Results were plotted and the saltconcentration correspond- thylene polymethobromide), bovine heparan sulfate, bovine chon- ing to peak elution was determined. The mean and standard deviation droitin sulfate A, shark chondroitin sulfate C, fucoidan (a sulfated were calculated from multiple runs (2-10) of each sample. Protein C polymer of fucose from a marine alga), phosvitin (a phosphoserine- inhibitor-proteinase complexes were prepared by incubating protein- containing glycoprotein from egg yolk), sulfatides, and tetrapoly- ase with a slight excess of protein C inhibitor for a time previously phosphate. Unfractionated heparin was from Diosynth (Oss, the determined to allow complete reaction, at least five times the half- Netherlands). Low molecular weight (M,5500) heparin was from life of the reaction. Inactivation of protein C inhibitor by neutrophil Calbiochem. Dermatan sulfate was purchased from Calbiochem and elastase was followedby the loss of thrombin inhibition activity. treated with nitrous acid to remove contaminatingheparin (16). DSPG I1 (a dermatan sulfate proteoglycan from bovine skin) was the RESULTS gift of Dr. Lawrence Rosenberg, Montefiore Hospital, Bronx, NY. Heparin fractions with low and high affinity for antithrombin were Identification of the Heparin-binding Site of Protein C In- the gift of Dr. Ingemar Bjork, Swedish University of Agricultural hibitor-Because heparin is a negatively charged glycosami- Sciences Uppsala, Sweden. Chemically depolymerized heparin (av- noglycan, it is expected
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