VIEWS: 71 PAGES: 5 POSTED ON: 10/13/2011
THEJOURNAL BIOLOGICAL CHEMISTRY OF Val. 260, No. 6, Issue of March 25, pp. 3501-3505, 1985 8 1985 by The American Society of Bioiogical Chemists, Inc. Printed in U.S.A. The Protease Specificity of Heparin Cofactor11 INHIBITION OF THROMBINGENERATED DURING COAGULATION* (Received for publication, September 24,1984) M. Katherine A. Parkerg and Douglas Tollefsen$? From the Division of Hematology-Oncology, Departments of internal Medicine and Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110 '281-labeledheparincofactor I1 (HCII) was mixed amount of heparin or dermatan sulfate. was with plasma and coagulation initiated by addition Little information is available about the protease specificity of CaClz, phospholipids, and kaolin or tissue factor. In of HCII. In previous studies, HCII did not inhibit coagulation the presence of ggfml of dermatan 67 sulfate, radioac- factor Xa, plasmin, or trypsin (2, 4,6, 7). In contrast, ATIII tivity was detected in a band which corresponded to inhibits a broad range of proteolytic enzymes, including the the thrombin-HCII complex = 96,000)upon sodium coagulation factors thrombin, Xa, IXa, XIa, XIIa, and kalli- (M. gel dodecyl sulfate-polyacrylamide electrophoresis.No krein, and the fibrinolytic enzyme plasmin (9, 11).We have other complexes were observed. The thrombin-HCII now examined all of the proteases known to be involved in complex was undetectable when 5 units/ml of heparin coagulation and fibrinolysis, as well as several other extracel- was present or whenprothrombin-deficientplasma lular proteases, and have discovered that HCII is a relatively was used. In experiments with purified proteases, HCII did not significantly inhibit coagulation factors VIIa, specific inhibitor of thrombin. Previously we reported that IXa,Xa,XIa,XIIa, in kallikrein, activated protein C, ' " ~ - t ~ r o ~ badded to plasma containing dermatan sulfate plasmin, urokinase,tissue plasminogen activator, leu- becomes bound to HCII (5).We have now shown that throm- kocyte elastase, the r-subunit of nerve growth factor, bin generated in plasma during coagulation is inhibited by and the epidermal growth factor-binding protein. HCII HCII when dennatan sulfate is present, thus explaining the inhibited leukocyte cathepsin G slowly, with a rate anticoagu~ant effect of dermatan sulfate that has been ob- constant of 8 X lo4 I"1 min" in the presence of der- served i vitro (10, 12). n matan sulfate. These results indicate that the protease specificity of HCIIis more restricted than that of other EXPERIMENTALPROCEDURES plasma protease inhibitors and suggest that the anti- Materiok-Benzoyl-11-Glu-Gly-Arg-pnitroanilide (S-22221,pyro- coagulant effect of dermatan sulfate is due solely to Glu-Gly-Arg-p-nitroanilide(5-24441, Val-Leu-Lys-p-nitroanilide (S- by inhibition of thrombin HCII. 2251), Pro-Phe-Arg-p-nitroanilide(8-23023, and Phe-Pip-Arg-p-ni- troanilide (5-2238) were purchased from Helena Laboratories; succi- " from nyl-Ala-Ala-Pro-Phe-p-nitroanilide Vega Biochemicals; succi- nyl-A~a-Ala-Ala-p-nitroanilide from Sigma; and tosyl-Gly-Pro-Arg- Heparin cofactor I1 (HCII') is a65,600-dalton glycoprotein p-nitroanilide (Chromozyme TH) from Boehringer Mannheim. Hep- in human plasma which inhibits thrombinby forming a stable, arin from porcine intestinal mucosa was obtained from Abbott Lab- equimolar complex with the protease (1-7). Heparinand oratories. Porcine skin dermatan sulfate was obtained from Sigma and was treated with nitrous acid prior to use to remove contaminat- dermatan sulfate bindto HCII and thereby increase the rate ing heparin (5, 1 ) S ~ i ~ m [ ~ * ~ I ~ (16.8iCi/mg) was purchased 0. iod de of inhibition of thrombin -1000-fold (4,5, 8). Heparin also from Amersham. Iodogen was purchased from Pierce. Prothrombin- catalyzes the inhibition of thrombin and other proteases by deficient plasma containing -2% of the normal concentration of antithrombin I11 (ATIII) (9). In contrast, dermatan sulfate prothrombin was purchased from George King Biologicals. Normal specifically catalyzes the thrombin-HCII reaction but has no plasma was obtained from blood (4.5 ml)drawn into evacuated tubes appreciable effect on the activity of ATIII (5, 10). HCII is containing 0.5 mi of0.129 M buffered sodium citrate (Vacutainer #6418, Bectin-Dickinson). Activated partial thromboplastin reagent present in plasma at a concentration of -1.2 P M . ~A t this was obtained from Hyland Laboratories. Rabbit brain t h r o m ~ p l a s t i n concentration, thrombincould theoretically be inhibited with was obtained from Ortho Diagnostics and was reconstituted with a t I j 2approaching 50-100 ms in the presence of an optimal water according to the manufacturer. Human brain thromboplastin (13) was obtained from Dr. George Broze, Washington University. * This research was supported by grants from the National Insti- Rabbit brain cephalin was purchased from Sigma. Polybrene (1,5- tutes of Health (HL-27589) and the Monsanto Co. The costs of dimethyl-l,5-~azaundecamethyIenepolymethobromidef was ob- publication of this article were defrayed in part by the payment of tained from Aldrich. page charges. This article must therefore be hereby marked "adver- Proteins-Human HCII and thrombin were purified as previously to tisement" in accordance with 18U.S.C. Section 1734 solely indicate described (4). Human factor XIIa (14) was prepared by Dr. Allen this fact. Kaplan, State University of New York, Stony Brook. Human factor 4 Present address: Department of Internal Medicine, University of XIa (15) was obtained from Dr. Paul Bajaj, University of California, Texas Southwestern Medical School, Dallas, TX. San Diego. Human coagulation factors VIIa (13), IX, X, Xa (16), and 8 Recipient of National Institutes of Health Career Development activated protein C (17) were obtained from Drs. Hatem Salem, Award HL-01079. on George Broze, and Joseph Miletich, ~ a s h i n ~ University. Factor 'The abbreviations used are: HCII, heparin cofactor 11; ATIII, IX (69 FM)was converted to IXa by incubation with 40 nM factor antithrombin 111; TPA, tissue plasminogen activator; yNGF, the XIa for 2 h at 37 "cin buffer containing 5 mMCaC12, 0.05 M NaCl, pept,idase subunit of nerve growth factor; EGF-BP, epidermal growth 0.02 M Tris-HC1, pH 7. Tissue plasminogen activator derived from factor-binding protein, the peptidase subunit of epidermal growth cultured human melanoma cells (18) was obtained from Dr. DesirB factor; and SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel Collen, University of Leuven. Human urokinase was the product of electrophoresis. Winthrop Laboratories. Glu-plasminogen I1 waspurified from human * D. M. Tollefsen and C. A. Pestka, manuscript submitted. plasma by the method of Deutsch and Mertz (19) and the zymogen 3501 3502 of Protease Specificity HCII (1 p ~ was converted to plasminby incubation with 50 nM urokinase ) Assay for factor VIIa-Factor VIIa was incubated with HCII and for 30 min at 37 "c in buffer containing 8 mM lysine, 20% glycerol, heparin or dermatan sulfate in the presence of 5 mM CaClz and 1mg/ 0.8 M Tris-HC1, pH 9.6. Human leukocyte elastase (20) and cathepsin ml human brain thromboplastin in 0.15 M NaC1,0.02 M Tris-HC1, G (21, 22) were prepared by Dr. Robert Senior, Washington Univer- pH 7.4, for 15 min at 37 "C. Tritiated human factor X (4.8 X lo6 sity. The proteolytic subunits of murine nerve growth factor (-y-NGF) cpm/nmol) prepared by the method of Silverberg et al. (27) was then (23) and epidermal growth factor (EGF-BP) (24) were provided by added at a final concentration of 0.4 p ~ After 1 min, proteolysis of . Dr. Thomas Maciag, Meloy Laboratories. Human plasma kallikrein, 3H-factor X was terminated by addition of 50 mM EDTA, and the human fibrinogen, Bothrops atrox venom, and bovine serum albumin protein was precipitated with 5% trichloroacetic acid at 4 "C. The were purchased from Sigma. radioactivity of the supernatant solution containing thelabeled acti- Iodination of HCII-200 pl of HCII (-8 p~ in 0.15 M NaC1, 0.02 vation peptide of factor Xa was then determined. In control incuba- M Tris-HC1, pH 7.4) were incubated with 2 mCi of carrier-free NaIZ5I tions without HCII, the amount of 3H-peptide released was propor- for 10 min at 4 " C in a polypropylene tube coated with 100 pg of of tional to the concentration factor VII.. Iodogen according to themethod of Fraker and Speck (25). Unbound Assay for Factor IXa-Factor IXa, HCII, and heparin or dermatan Iz5Iwas removed by gel filtration on a 1 X 10-cm column of Sephadex sulfate were incubated for 15 min at 37 "C in 55pl of buffer containing (2-25. The final specific radioactivity was 1.2 X lo9 cpm/nmol of 0.15 M NaCI, 0.02 M Tris-HC1, pH 7.4, and 1 mg/ml bovine serum HCII. albumin. At the end of the incubation period, 45 pl of0.1 mg/ml Electrophoresis-SDS-PAGE was performed with 7.5% gels and Polybrene in the above buffer, 100 pl of citrate-anticoagulated normal the Laemmli buffer system (26) under nonreducing conditions. Au- plasma, 50 pl of rabbit brain cephalin, and 100 pl of 25 mM CaClZ toradiography was performed as described previously (3). Molecular were added sequentially at 15-5 intervals. The clotting time following weight standards obtained from BioRad included ovalbumin (M, = the addition of CaC& was determined with a Fibrometer (Bectin- 45,000), bovine serum albumin (M, 66,000), phosphorylase b (M, = = Dickinson). Samples (55 p l ) of factor IXa at concentrations of 15- 92,000), @-galactosidase(M, 116,000), and myosin (M, 200,000). = = 245 nM yielded clotting timeswhich decreased from 72 to 36 s. A plot Assays for Thrombin, Factors Xa, XIa,Kallikrein, Actiuated Protein of log clotting time versus log [factor IXa] was linear in this range. C, Plasmin, Urokinase, Cathepsin G, Elastase, y-NGF, and EGF-BP- Assay for Factor XIIa-Factor XIIa was assayed in the laboratory Reactions were carried out in 1.5-ml polypropylene microcentrifuge tubes at 37 "C. The enzyme, HCII, heparin, and dermatan sulfate of Dr. Allen Kaplan as previously described (14). were mixed at the final concentrations specified in Table I in buffer Assay for Tissue Plasminogen Actiuator-TPA, HCII, and heparin containing0.15 M NaCl, 0.02 M Tris-HC1, pH 7.4, and 1 mg/ml bovine or dermatan sulfatewere mixed with 0.9 mg/ml fibrinogen in 0.15 M serum albumin. Reactions were initiated by addition of the protease. NaC1,0.02 M Tris-HC1, pH 7.4. B. atrox venom (1 unit/ml final At specified times, protease activity was determined by addition of concentration) was added immediately to clot the fibrinogen. After a an equal volume of the appropriate chromogenic substrate in water. 60-min incubation at 37 "c,an equal volume of 0.6 mM $3-2444was Therate of hydrolysis of thesubstrate was determined froma added. Hydrolysis of the substrate was terminated after 3 min by continuous recording of the absorbance at 405 nm.Alternatively, addition of aceticacid (5% final concentration), the mixture was hydrolysis of the substratewas terminated after 3 min by addition of centrifugedfor 10 min in an Eppendorfmicrocentrifuge, and the 1/10 volume of 50% (v/v) acetic acid, the precipitated glycosamino- absorbance at 405 nm of the supernatant solution was determined. glycan was removed by centrifugation if necessary, and the absor- M Control experiments demonstrated (a) thatA 5 was proportional to bance of the supernatant solution was determined at 405 nm. In the the amount of TPA present in the absence of HCII; (b) that the absence of HCII, a standard curve of A A 4 0 5 versus protease concen- venom did not hydrolyze the substrate S-2444; and (c) that HCII did tration was linear for each proteaseintheconcentration range was not inhibit nor it degraded by the venom protease. In preliminary employed in the experiment. The following substrates were used 0.1 experiments, fibrinogen and B. atrox venom were omitted from con- mM Chromozym T H for thrombin, r-NGF, and EGF-BP mM S- 0.3 trol incubations without HCII; as previously reported (18), the rate 2222 for factor Xa; 3 mM S-2222 for factor XIa; 0.4 mM S-2302 for of substrate hydrolysis by TPA in the absence of fibrin was -30% of kallikrein; 0.2 mM S-2238 for activated protein C; 0.5 m M S-2251 for the rate obtainedin the presence of fibrin. plasmin; 0.6 mM $2444 for urokinase; 0.2 mM succinyl-Ala-Ala-Pro- Kinetic Analysis-The second-order rate constantfor inhibition of Phe-p-nitroanilide for cathepsin G; and 0.2 mM succinyl-Ala-Ala- a protease by HCII was estimated according to the equation: k = Ala-p-nitroanilide for elastase. ln([P]o/[P],)/t/[[HCII], in which [PIo= initial protease activity, [PIl I TABLE Inhibition of purified proteases by HCII Reactants were incubated at 37 "C at thefinal concentrations indicated in 0.15 M NaCl, 0.02 M Tris-HC1, and 1 mg/ml bovine serum albumin, pH 7.4. At the end of the incubation period, the remaining protease activity was determined as described under "Experimental Procedures." Activity remaining after incubation with Protease [Protease] [HCII] Incubation time [Heparin] 'Dermatan sulfate] HCII Heparin HCII + Dermatan HC1l + alone alone heparin alone sulfate nM min unitlrnl ccglml % of control' Thrombin 2.2 0.5 150 20 100 22 101 <2 100 <2 Factor Xa 11.6 200 30 1 100 109 NDb 94 ND 108 Factor VIIa 0.83 760 15 1 80 108 ND 110 ND 115 Factor IXa 69 1220 20 1 100 95 ND 106 ND 116 Factor XIa 2.8 100 15 1 100 105 103 106 79 83 Factor XIIa 4.3 50 15 1 100 100 101 92 98 95 Kallikrein 2.9 100 10 1 100 102 98 99 100 101 Protein Ca 83 760 10 1 100 98 98 99 47 50 Plasmin 290 750 30 1 100 100 101 84 87 98 Urokinase 540 830 30 1 100 107 108 104 109 106 TPA 55 760 60 0.5 400 102 51 56 95 95 Cathepsin G 730 1250 40 0.5 400 30 43 36 76 <2 Elastase 670 1780 60 0.5 400 56 45 50 95 57 y-NGF 100 760 10 1 100 106 105 103 91 91 EGF-BP 100 760 10 1 100 95 95 96 76 69 a Averages of duplicate determinations. ND, not determined. Protease Specificity of HCII 3503 = protease activity at time = t, and [HCII] = initial HCII concentra- ted did not contain labeled complexes regardless of whether tion, assuming pseudo-first orderconditions. heparin or dermatan sulfate was present (lanes B-D). These experiments indicate that thrombin generated in plasma by RESULTS activation of the intrinsic coagulation pathway is inhibited by Inhibition of Thrombin by HCII in Recalcified Plasma-We HCII in the presence of dermatan sulfate. have previously shown that thrombin combines with HCII to Inhibition of Thrombin by HCII in Plasma Activated by form a 96,000-dalton complex that is stable during SDS- Tissue Factor or Kaolin-In an attempt to detect complexes PAGE (3, 4 .To determine whether HCII inhibits thrombin ) of HCII with proteases other than thrombin, prothrombin- or other proteases as they are generated during coagulation, deficient plasma containing '251-HCII was incubated for 1 h tracer '251-HCII wasadded to citrate-anticoagulated plasma, at 37 "C with CaC12, phospholipids, and a source of tissue the mixture was warmed to 37 "C in a glass tube, and coagu- to factor (human brain thromboplastin) activate factor VI1 or lation was initiated by addition of CaC12.After 1h, the plasma kaolin (activated partial thromboplastin reagent) to activate was analyzed by SDS-PAGE and autoradiography (Fig. 1, factor XI1 and kallikrein. In neither case was any of the lZ5I- lanes E-G). Coagulation occurred in the absence of heparin HCII detected in complexes (Fig. 1, lanes Hand K). Identical or dermatan sulfate, and none of the lZ5I-HCII was detected results were obtained in the presence of 5 units/ml of heparin in higher molecular weight complexes (lune E). However, (lanes I and L). When incubations were repeated in the coagulation did not occur in the presence of67 pg/ml of presence of 67 pg/ml of dermatan sulfate, a trace amount of dermatan sulfate, and densitometry revealed that -13% of the label was present in a band corresponding to the throm- the label was present in a 96,000-dalton band (lane G) which bin-HCII complex (lanes J and M). No other complexes were co-migrated with the complex formed by incubation of an observed. When the exposure times of the autoradiographs in excess of purified thrombin with '251-HCII(luneA ) . Less than Fig. 1were extended from 1to 20 h to increase the sensitivity 1% the label was present inthe complex when prothrombin- of of the experiments, we observed numerous additional bands deficient plasma was substituted for normal plasma in the of representing as a whole 0.1% the total radioactivity present. incubation (not shown). Coagulation also did not occur in the Because there were no significant differences between the presence of 5 units/ml of heparin. In this case, none of the of additional bands and the pattern a gel containing '251-HCII '251-HCII wasdetected in the 96,000-dalton complex (lane F). alone (not shown), the bands were considered to represent trace contaminants inthe HCII preparation. This result is consistent with previous experiments which Inhibition of Purified Proteases by HCII-We assayed four- demonstrated that '251-thrombinis preferentially inhibited by teen purified proteases for activity after incubation with a ATIII in undiluted plasma at similar concentrations of hep- molar excess of HCII (Table I). The concentrations of pro- arin (3). Control incubations from which the CaC12 wasomit- tease and HCII were determined primarily according to the sensitivity of the assay for the protease. Incubation times were long in comparison to thet112for inhibition of thrombin 200 - by HCII (e.g. tl,2 = 8 s in the presence of 50 nM HCII and 0.5 unit/ml of heparin; Ref. 4). Under the conditions of each experiment, 20% inhibition of the protease would indicate a 116 - second-order rate constant 3 X lo5 M" min" (ie. 2000 times 1 for less than the rate constant inhibition of thrombin by HCII 921 in the presence of dermatan sulfate; see "Discussion"). Hep- arin or dermatan sulfate were present at concentrations pre- 66 - viously determined to accelerate the inhibition of thrombin by HCII (4,5). In addition, controls were performed to deter- mine the effects of heparin and dermatan sulfate alone on protease activity. As shown in Table I, 22% of thrombin 45 - activity remained after a 20-min incubation with 150 m HCII, while <2% activity remained in incubations that also M MW A B C D E F G H I J K L M included heparin or dermatan sulfate. The second-order rate Stds. constant calculated for inhibition of thrombin by HCII alone FIG. 1. IncorDorationof 12'I-HCII into comdexes in Dlasma inthis experiment was 5 X lo5 M" min", as previously during coagulation. Reagents were brought t o a final voiume of reported (4). Rate constants for inhibition of thrombin by 150 pl with 0.15 M NaC1, 0.02 M Tris-HC1, 1 mg/ml bovine serum HCII in the presence of heparin or dermatan sulfate could albumin, pH 7.4, and incubated for 1 h at 37 "C in glass (lunes B-G) not be determined accurately from the datain Table I, but in or polypropylene (lunes A and H-M) tubes. Each incubation con- both cases were >1.3 x lo6 M" min" (see "Discussion"). In tained 7.5 nM lZ5I-HCIIalong with the following additional reagents: did contrast, HCII not inhibit significantly coagulation factors lune A , 13 p~ thrombin; lane B, 25 p1 of normal plasma; lune C, same as I3 plus 5 units/ml heparin; lune D, same as B plus 67 pg/ml VIIa, IXa, Xa, XIa,XIIa, kallikrein, activated protein C, dermatan sulfate; lune E,25 pl of normal plasma and 6 mM CaCl2; plasmin, urokinase, TPA? or 7-NGF. Activated protein C lune F,same as E plus 5 units/ml heparin; lune G, same as E plus 67 and TPAwere moderately inhibited by dermatan sulfate and pg/ml dermatan sulfate; lune H , 25 pl of prothrombin-deficient heparin, respectively, but there was no further inhibition in plasma, 13 mM CaClZ, and 100 pl of human brain thromboplastin; either case when HCII was also present. Leukocyte elastase lune I, same as H plus 5 units/ml heparin; lane J , same as H plus 67 was partially inhibited during a 60-min incubation with 1.78 pg/ml dermatan sulfate; lune K, 25 p1 prothrombin-deficient plasma, 8 mM CaClZ,and 50 p1 of activated partial thromboplastin reagent; p~ HCII alone or with dermatan sulfate; in both cases the lune L,same asK plus 5 units/ml heparin; lune M , same as K plus 67 rate constants were 5 x lo3 M" min". Although elastase was pg/ml dermatan sulfate. At the end of the incubation period, 5-10 p1 moderately inhibited by heparin alone, heparin appeared to of each reaction mixture were subjected to SDS-PAGE. An autora- diograph of the gel exposed for 1 h at -70 "C is shown. The positions HCII was incubated with TPA alone (data not shown) or in the of molecular weight standards and of the 96,000-dalton thrombin- presence of fibrin as described under"Experimental Procedures" HCII complex (+) are indicated. (data in Table I). 3504 f i e1 Protease ~ ~ e ~ ~ of Hc1i t ~ protect the protease from further inhibition by HCII. EGF- other than thrombin, only leukocyte cathepsin G was inhib- B P was inhibitedpartially by dermatan sulfate and to a ited at a significant rate. However, the calculated second- slightly greater extent when both dermatan sulfate and HCII order rate constant for inhibition of cathepsin G by HCII in were present; in the latter case the calculated rate constant the presence of dermatan sulfate was -40,000-fold less than was 1 X io4 M" min-'. the rate constant reported for inhibition of cathepsin G by Inhibition of Cathepsin G by HCZZ-Because HCII appeared a1-antichymotrypsin (29). Therfore, inhibition of cathepsin to inhibit cathepsin G at a significant rate in the presence of G by HCII is unlikely to occur i n uiuo. In contrast, HCII dermatan sulfate (Table I), the time course of inhibition was inhibits thrombin with rate constants of 6.4 X lo8 M" min" studied in more detail (Fig. 2). In the absence of heparin or in the presence of 250 pg/ml of dermatan sulfate and 4.0 X dermatan sulfate, inhibition by 1.25 PM HCII occurred with 10' M" min" in the presence of 10 units/ml of heparin (5). a tllz= 24 min ( k = 1.4 X lo4M ' min-I). Although dermatan - Rate constants of this magnitude are characteristic of inhi- sulfate alone decreased cathepsin G activity 20-30% (Table bition reactions that arelikely to be "physiological" (30). I), it increased the rate of inhibition by HCII &fold (tIl2 4 = Our data indicate that the protease specificity of HCII is min; k = 8.4 x IO5 M" min"). In contrast, heparin decreased more restricted than thatof other plasma protease inhibitors, the activity of cathepsin G 50-60%, and it appeared to prevent including ATIII (9),al-proteinase inhibitor (30), a2-antiplas- min (31), and a2-macroglobulin (32), each of which can inhibit further inhibition of the protease by HCII. several of the proteases that we have tested. In addition, the DISCUSSION lack of inhibition of y-NGF, EGF-BP, plasmin, and urokinase distinguishes HCII from the cellular protease inhibitors The purpose of this investigation was to identify enzymes termed "protease nexins" (33). We have also found that, in that HCII can inhibit, and thereby to arrive at a hypothesis the presence of dermatan sulfate, HCII binds thrombin as it concerning the physiological function of HCII. We have tested is being generated in plasma during coagulation. Thus, inhi- various serine proteases, including all of thosecurrently bition of thrombin by HCII appears to explain the anticoag- known to be involved in coagulation and fibrinolysis (28), ulant activity of dermatan sulfate that has been observed in leukocyte cathepsin G (21) and elastase (ZO), and the pepti- vitro (lo, 12) and may also explain the antithrombotic effect dase subunits of nerve growth factor (23) (y-NGF) and epi- observed in uiuo after the administration of exogenous der- dermal growth factor (24) (EGF-BP).Of the enzymes tested matan sulfate (34). In addition, HCIImay inhibit other effects of thrombin, including platelet aggregation and secretion (35), chemotaxis (36), and mitogenesis (37), underappropriate 0.50 r circumstances. Rapid inhibition of thrombin by HCII i n vivo probably occurs only in the immediate vicinity of proteoglycans which contain oligosaccharide sequences that bind HCII (4,8). Sim- 04\ ,0 0.3oJi \ ilarly, ATIII requires specific oligosaccharide sequences for maximum activity (9). HCII and ATIII may becomeactivated in different environments,since there is evidence that differ- ent heparin molecules activate HCII and ATIII (38) and that dermatan sulfate only activates HCII (5). Dermatan sulfate comprises 60-70% of the glycosaminoglycans in the intima and media of large arteries (39), in addition to being present in skin, heart valves, and tendons (40). A small amount of dermatan sulfate is also synthesized by cultured endothelial cells (41). Whether the glycosaminoglycans present in these locations contain the proper sequences to active HCII remains 0 to be determined. z m 0.10 - Acknowledgments-We thank Dr. Allen Kaplan for performing the 0 w .. factor XIIa assays and Drs. Paul Bajaj, George Broze, DBsirB Collen, z 2 0.08 - Thomas Maciag, Joseph Miletich, Hatem Salem, and Robert Senior for providing proteases and otherreagents. U - REFERENCES 0.06 - 1. Briginshaw, G. F., and Shanberge, J. N. (1974) Arch. Biochem. Biophys. 161,683-690 r 2. Briginshaw, G . F., and Shanberge, J. X. (1974) Thromb. Res. 4, 463-477 10 20 30 40 3. Tollefsen, D.M., and Blank, M. K. (1981) J. Clin. Invest. 68, TIME (Min) 589-596 FIG. 2. Inhibition of leukocyte cathepsin G by HCII. Cathep- 4. Tollefsen, D. M., Majerus, D.W., and Blank, M. K. (1982) J. sin G (0.73 p ~ and HCII (1.25 p ~ were incubated for 1-40 min a t ) ) Rid. Chem. 257,2162-2169 37 "C with 0.5 unit/ml heparin (O), 100 pg/ml dermatan sulfate (A), 5. Tollefsen, D. M., Pestka, C. A., and Monafo, W. J. (1983)J. Biol. or no glycosaminoglycan (0)in 33 pl of0.15 M NaCl, 0.02 M Tris- Chem. 258,6713-6716 HC1, 1 mg/ml bovine serum albumin, pH 7.4. Then 167 pl of0.35 6. Griffith. M. J.. Carrawav, T., White, G. C., and Dombrose, F. A. mg/ml succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in water was added. (1983) mood 61,111-118 Hydrolysis of the substrate was stopped after 3 min by addition of 20 7. Wundenvald. P.. Schrenk. W. J.. and Port, H. (1982) Thromb. pl of 50% (v/v) acetic acid, the mixture was centrifuged for 10 min in Res. 25, 177-191 an Eppendorf microcentrifuge, and the absorbance of the supernatant 8. Griffith, M. J., and Marbet, G . A. (1983) Biochem. Biophys. Res. solution was determined at 405nm. In control experiments the Commun. 112,663-670 absorbance was directly p r o ~ ~ i o nto l theconcentration of enzyme a 9. Rosenberg, R. D. (1977) Semin. Hematol. 14,427-440 added in the absence of HCII and glycosaminoglycan. 10. Teien, A. X., Abildgaard, U., and Hook, M. (1976) Thromb. Res. Protease Specificity of HCII 3505 8,859-867 27. Silverberg, S. A., Nemerson, Y., and Zur, M. (1977)J. Biol. Chem. 11. Highsmith, R.F., and Rosenberg, R. D. (1974) J. Bioi Chem. 252,8481-8488 249,4335-4338 28. Jackson, C.M., and Nemerson, Y. (1980) Annu. Reu. Biochem. 12. Long, W. F., Williamson, F. B., Kindness, G., and Edward, M. 49,765-811 (1980) Thromb. Res. 1 8 , 493-503 29. Beatty, K., Bieth, J., and Travis, J. (1980) J . Biol. Chem. 255, 13. Broze, G. J., and Majerus, P. W. (1980) J . Bid. Chem. 255, 3931-3934 1242-1247 30. Travis, J., and Salvesen, G. S. (1983) Annu. Reu. Biochem. 52, 14. Silverberg, M., Dunn, J. T., Garen, L., and Kaplan, A. P. (1980) 655-709 J. Biol. Chem. 2 5 5 , 7281-7286 31. Saito, H., Goldsmith, G. H., Moroi, M., and Aoki, N. (1979) Proc. 15. Bajaj, S. P. (1982) J . Biol. Chem. 257,4127-4132 Natl. Acad. Sci. U. S. A. 76,2013-2017 16. Miletich, J. P., Broze, G. J., and Majerus, P. W. (1980) Anal. 32. Roberts, R. C., and Hall, P. K. (1983)Ann. N . Y. Acad. Sci. 421, Biochem. 105, 304-310 6 1-68 17. Salem, H. H., Broze, G. J., Miletich, J. P., and Majerus, P. W. 33. Knauer, D. J., Scaparro, K. M., and Cunningham, D. D. (1982) (1983) Proc. Natl. Acad. Sci. U. S A. 80, 1584-1588 . J. Biol. Chem. 257, 15098-15104 18. Rijken, D. C . , and Collen, D. (1981) J. Biol. Chem. 256, 7035- 34. Buchanan, M. R., Boneu, B., Ofosu, F., and Hirsh,J. (1985) Blood 7041 65,198-201 35. Sie, P., Fernandez, F., Caranobe, C., Gabaig, A. M., and Boneu, 19, Deutsch, D. G., and Mertz, E. T.(1970) Science 170,1095-1096 B. (1984) Thromb. Res. 35, 231-236 20. Baugh, R, J., and Travis, J. (1976) biochemist^ 15,836-841 36. Bar-Shavit, R., Kahn, A., Mudd, M. S., Wilner, G. D., Mann, K. ~ o ~. 21. Barrett, A. J. (1981) ~ e t ~ n z y m o80,561-565 t~ G., and Fenton, J. W., I1 (1984) B i o c h e m ~23,397-400 22. Senior, R. M., and Campbell, E. J. (1984) J. Immunol. 132, 37. Chen, L. B., and Buchanan, J. M. (1975) Pro;. Natl. Acad. Sci. 2547-2551 U. S. A. 72, 131-135 23. Greene, L. A,, Shooter, E. M., and Varon, S. (1969) B i o c ~ m 38. Hurst, R. E., Poon, M.-C., and Griffith, M. J. (1983) J . Clin. ~ ~ ~ 8,3735-3741 Invest. 72,1042-1045 24. Taylor, J. M., Mitchell, W. M., and Cohen, S. (1974) J. Biol. 39. Wight, T. N., and Ross, R. (1975) J. Cell Bioi 67,675-686 Chem. 249,2188-2194 40. Kumar, V., Berenson, G. S., Ruiz, H., Dalferes, E. R., and Strong, 25. Fraker, P. J., and Speck, J. C., Jr. (1978) Biochem. Biophys. Res. J. P. (1967) J. Atheroscler. Res. 7,583-590 Commun. 80, 849-857 41. Oohira, A., Wight, T. N., and Bornstein, P. (1983)J. Biol. Chem. 26. Laemmli, U. K. (1970) Nature (Land.)227, 680-685 258,2014-2021
"The Protease Specificity of Heparin Cofactor 11"