Heparin binding to protein C inhibitor.

Protein C inhibitor is a plasma protein whose ability to inhibit activated protein C, thrombin, and other enzymes is stimulated by heparin. These studies were undertaken to further understand how heparin binds to protein C inhibitor and how it accelerates proteinase inhibition. The region of protein C inhibitor from residues 264-283 was identified as the heparin-binding site. This differs from the putative heparin-binding site in the related proteins antithrombin and heparin cofactor. The glycosaminoglycan specificity of protein C inhibitor was relatively broad, including heparin and heparan sulfate, but not dermatan sulfate. Non-sulfated and non-carboxylated polyanions also enhanced proteinase inhibition by protein C inhibitor. Heparin accelerated inhibition of alpha-thrombin, gamma T-thrombin, activated protein C, factor Xa, urokinase, and chymotrypsin, but not plasma kallikrein. The ability of glycosaminoglycans to accelerate proteinase inhibition appeared to depend on the formation of a ternary complex of inhibitor, proteinase, and glycosaminoglycan. The optimum heparin concentration for maximal rate stimulation varied from 10 to 100 micrograms/ml and was related to the apparent affinity of the proteinase for heparin. There was no obvious relationship between heparin affinity and maximum inhibition rate or degree of rate enhancement. The affinity of the resultant protein C inhibitor-proteinase complex was also not related to inhibition rate enhancement, and the results showed that decreased heparin affinity of the complex is not an important part of the catalytic mechanism of heparin. The importance of protein C inhibitor as a regulator of the protein C system may depend on the relatively large increase in heparin-enhanced inhibition rate for activated protein C compared to other proteinases.

Protein C inhibitor is a plasma protein whose ability to inhibit activated protein C, thrombin, and other enzymes is stimulated by heparin. These studies were undertaken to further understand how heparin binds to protein C inhibitor and how it accelerates proteinase inhibition. The region of protein C inhibitor from residues 264-283 was identified as the heparin-binding site. This differs from the putative heparin-binding site in the related proteins antithrombin and heparin cofactor. The glycosaminoglycan specificity of protein C inhibitor was relatively broad, including heparin and heparan sulfate, but not dermatan sulfate. Non-sulfated and non-carboxylated polyanions also enhanced proteinase inhibition by protein C inhibitor. Heparin accelerated inhibition of a-thrombin, TT-thrombin, activated protein C, factor Xa, urokinase, and chymotrypsin, but not plasma kallikrein. The ability of glycosaminoglycans to accelerate proteinase inhibition appeared to depend on the formation of a ternary complex of inhibitor, proteinase, and glycosaminoglycan. The optimum heparin concentration for maximal rate stimulation varied from 10 to 100 pg/ml and was related to the apparent affinity of the proteinase for heparin. There was no obvious relationship between heparin affinity and maximum inhibition rate or degree of rate enhancement. The affinity of the resultant protein C inhibitor-proteinase complex was also not related to inhibition rate enhancement, and the results showed that decreased heparin affinity of the complex is not ap important part of the catalytic mechanism of heparin. The importance of protein C inhibitor as a regulator of the protein C system may depend on the relatively large increase in heparin-enhanced inhibition rate for activated protein C compared to other proteinases.
Hemostasis requires a balance between procoagulant and anticoagulant forces. Among the anticoagulant mechanisms is the protein C system. Thrombin generated during coagulation binds to thrombomodulin on vessel walls and the thrombin-thrombomodulin complex activates the zymogen protein C. Activated protein C, with its cofactor protein S, proteolytically inactivates coagulant factors V and VI11 (1). The importance of the protein C system is demonstrated by the incidence of thrombosis in individuals who lack protein C (2) or protein S (3). The protein C system is believed to be * This work was supported in part by Research Grant HL-06350 from the National Institutes of Health. 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 18 U.S.C. Section 1734 solely to indicate this fact.
$ T O whom correspondence and reprint requests should be addressed The University of North Carolina, Div. of Hematology, Campus Box 7035, Chapel Hill, NC 27599-7035. Fax: 919-966-7639. regulated by a plasma glycoprotein named protein C inhibitor, also known as plasminogen activator inhibitor-3 (4). Three other major plasma proteins, az-macroglobulin, az-antiplasmin, and a'-proteinase inhibitor inhibit activated protein C (5) but might be effective only by virtue of their relatively high concentration in plasma. Protein C inhibitor reacts with the active site of activated protein C to form an essentially irreversible complex (6). Interestingly, protein C inhibitor also inhibits thrombin, the final proteinase of the coagulation pathway, as well as other procoagulant enzymes. This broad target proteinase specificity of protein C inhibitor presents a problem in understanding the physiological importance of protein C inhibitor as a regulator of the protein C system. Direct evidence for the involvement of protein C inhibitor is lacking, as an inhibitor deficiency has yet to be documented.
Protein C inhibitor is a member of the serine proteinase inhibitor (serpin)' superfamily of proteins, whose prototype is a'-proteinase inhibitor (7). Protein C inhibitor can be further classified as a heparin-binding serpin, along with the proteinase inhibitors antithrombin (historically known as antithrombin 111) and heparin cofactor (also called heparin cofactor 11). Heparin and some other glycosaminoglycans act to increase the rate of proteinase inhibition by these three plasma inhibitors, in some cases as much as several thousandfold ($). The mechansim whereby heparin catalyzes proteinase inhibition is a subject of much study, especially due to the widespread use of heparin as a therapeutic anticoagulant. The ability of heparin to accelerate the inhibition of activated protein C by protein C inhibitor, thereby favoring coagulation, is at odds with the anticoagulant effect of heparin therapy. As a first step toward understanding this apparent contradiction and in order to gather insight into the physiological importance of protein C inhibitor, a series of studies was undertaken. The work presented here describes the heparinbinding site of protein C inhibitor, the polyanion specificity of protein C inhibitor, and the mechanism whereby heparin accelerates proteinase inhibition. Some of these results have appeared previously in abstract form (9). The following report compares protein C inhibitor to antithrombin and heparin cofactor (10).

Heparin
Binding to Protein C Inhibitor ochem, bovine chymotrypsin from Cooper Biomedical, and neutrophil elastase from Elastin Products (Pacific, MO). The following proteinase substrates were used Chromozym TH (tosyl-Gly-Pro-Arg-p-nitroanilide) for thrombin from Boehringer Mannheim, Spectrozyme PCa (Lys(Cbo)-Pro-Arg-p-nitroanilide) for activated protein C and Spectrozyme FXa (MeO-CO-CHG-Gly-Arg-p-nitroanilide) for factor Xa from American Diagnostica, S-2444 (Glu-Gly-Arg-p-nitroanilide) for urokinase and S-2302 (Pro-Phe-Arg-p-nitroanilide) for kallikrein from KabiVitrum, and Suc-Ala-Ala-Pro-Phe-p-nitroanilide for chymotrypsin from Sigma. The following were purchased from Sigma: bovine serum albumin, polybrene (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide), bovine heparan sulfate, bovine chondroitin sulfate A, shark chondroitin sulfate C, fucoidan (a sulfated polymer of fucose from a marine alga), phosvitin (a phosphoserinecontaining glycoprotein from egg yolk), sulfatides, and tetrapolyphosphate. Unfractionated heparin was from Diosynth (Oss, the Netherlands). Low molecular weight (M, 5500) heparin was from Calbiochem. Dermatan sulfate was purchased from Calbiochem and treated with nitrous acid to remove contaminating heparin (16). DSPG I1 (a dermatan sulfate proteoglycan from bovine skin) was the gift of Dr. Lawrence Rosenberg, Montefiore Hospital, Bronx, NY. Heparin fractions with low and high affinity for antithrombin were the gift of Dr. Ingemar Bjork, Swedish University of Agricultural Sciences Uppsala, Sweden. Chemically depolymerized heparin (average molecular weight 5000, 3500, and 2500) was the gift of Dr. Charles Griffin, Miami University, OH. Fmoc-amino acids were from MilliGen. Heparin-Sepharose was from Pharmacia LKB Biotechnology Inc. and heparin-agarose was prepared as described (12). Peptide Synthesis-Peptides were assembled from Fmoc amino acids using a Milligen pepsynthesizer according to the reported cDNA sequence of protein C inhibitor (17). In peptide 1-16, tyrosine was added to the carboxyl terminus, and in peptide 80-93, Phe-92 was substituted with tyrosine to facilitate spectrophotometric quantitation of the peptides. Purity of the peptides was verified by reversephase HPLC, and when necessary, further purification was accomplished by HPLC. Heparin affinity of peptides was measured using a 1-ml column of heparin-agarose in 20 mM HEPES, 0.1% PEG 8000, pH 7.4, with a linear salt gradient.
Peptide Competition Assays-Peptides (0.1-100 FM) in 20 mM HEPES, 150 mM NaCl, 0.1% PEG, pH 7.4, were added to assay mixtures containing 1 pg/ml heparin, 2 mg/ml bovine serum albumin, and 50 or 100 nM protein C inhibitor in the same buffer at 25 "C. Reactions were started with the addition of 5 nM thrombin or 10 nM activated protein C. After incubation, remaining proteinase activity was measured using 0.15 mM chromogenic substrate with 2 mg/ml Polybrene. Substrate hydrolysis was linearly related to proteinase concentration. Control experiments verified that none of the peptides affected either the ability of proteinase to hydrolyze substrate or the ability of protein C inhibitor to inhibit proteinases in the absence of heparin.
Polyanion Acceleration of Protein C Inhibitor Activity-Polyanions were dissolved in 20 mM HEPES, 150 mM NaC1, 0.1% PEG, pH 7.4, and various concentrations (0.1-1000 pg/ml) were added to 60 nM protein C inhibitor and 2 mg/ml bovine serum albumin in the same buffer at 25 "C. Reactions were started with the addition of 5 nM thrombin or activated protein C, incubated for various times, and remaining proteinase activity was measured. Control experiments in the absence of protein C inhibitor verified that none of the polyanions directly affected the ability of proteinase to hydrolyze substrate. Second order proteinase inhibition rate constants were calculated as -In a/t [protein C inhibitor] where a is the fractional proteinase activity remaining relative to the uninhibited control, t is the time of incubation, and [protein C inhibitor] is the inhibitor concentration, 60 nM. The inhibition rate increase was calculated by dividing the maximum inhibition rate in the presence of polyanion by the rate in the absence of polyanion, and the optimum polyanion concentration was the concentration that produced the maximum inhibition rate.
Heparin Acceleration of Proteinase Inhibition-Heparin (0.1-1000 pg/ml) was added to 50 nM protein C inhibitor in 20 mM HEPES, 150 mM NaC1, 0.1% PEG, pH 7.4, containing 2 mg/ml bovine serum albumin at 25 "C. Reactions were started by adding 5 nM proteinase (a-thrombin, yT-thrombin, activated protein C, factor Xa, urokinase, plasma kallikrein, or chymotrypsin). Incubation times varied among proteinases and remaining proteinase activity was determined by substrate hydrolysis. Proteinase inhibition rate constants were calculated as described above.
Heparin Affinity Chromatography-Experiments were performed using a Pharmacia fast protein liquid chromatography system and a 5-ml column of heparin-Sepharose in 20 mM HEPES, 10 mM NaC1, 0.1% PEG, pH 7.4. Samples of protein C inhibitor, proteinase, and protein C inhibitor-proteinase complexes were eluted with a 1 ml/ min linear salt gradient from 10 mM to 1.2 M NaCl; 0.25-ml fractions were collected. Proteins were detected by absorbance at 280 nm and by reactivity with a rabbit polyclonal antiserum to protein C inhibitor (to detect protein C inhibitor and protein C inhibitor-proteinase complexes) and by chromogenic substrate hydrolysis (to detect proteinases and protein C inhibitor-proteinase complexes; an extended incubation time was required to detect proteinase activity in complexes). Results were plotted and the salt concentration corresponding to peak elution was determined. The mean and standard deviation were calculated from multiple runs (2-10) of each sample. Protein C inhibitor-proteinase complexes were prepared by incubating proteinase with a slight excess of protein C inhibitor for a time previously determined to allow complete reaction, at least five times the halflife of the reaction. Inactivation of protein C inhibitor by neutrophil elastase was followed by the loss of thrombin inhibition activity.

RESULTS
Identification of the Heparin-binding Site of Protein C Inhibitor-Because heparin is a negatively charged glycosaminoglycan, it is expected to bind to basic residues of protein C inhibitor. Three regions were identified as potential heparinbinding sites: residues 1-11, 82-90, and 266-278. Residues 266-278 were previously identified as a heparin-binding site by homology to a consensus glycosaminoglycan recognition site (18). Peptide 1-11 also follows the consensus sequence. Sequences corresponding to the three regions of protein C inhibitor (1-16,80-93, and 264-283, and the 264-283 random sequence) are shown in Fig. 1, along with helical-wheel projections of these sequences (19). The projections show that the selected sequences can form amphipathic helices with basic and uncharged residues on opposite faces of the helix.
To test whether the selected sequences represent the heparin-binding site of protein C inhibitor, the three peptides were synthesized and their ability to bind heparin was tested in proteinase inhibition assays. The peptides were added to systems containing protein C inhibitor, proteinase, and heparin; with the premise that heparin-binding peptides would compete with protein C inhibitor for heparin, thus decreasing hibitor. Three regions rich in basic residues were selected and peptides corresponding to these regions were synthesized. The sequences of the peptides are shown along with helical wheel diagrams of the sequences (assuming 3.6 residues/helical turn). In peptide 1-16, a tyrosine residue not found in native protein C inhibitor was added to the carboxyl terminus. In peptide 80-93, Phe-92 was replaced with tyrosine in the synthetic peptide. The random sequence peptide was constructed using the same residues as peptide 264-283, but in random order. The first and last residues of peptide 264-283 and the random sequence peptide are not visible in the helical wheel diagrams.
the rate of the proteinase inhibition reaction. The results revealed that of the three peptides, only peptide 264-283 competed with protein C inhibitor for binding to heparin, as shown by the increase in proteinase activity with increasing peptide concentration (Fig. 2). This effect was observed for both thrombin and activated protein C. A peptide comprised of the same residues as peptide 264-283, but in random sequence, did not compete as effectively, suggesting that the three-dimensional distribution of charged residues rather than the number of charged residues was responsible for its heparin binding function. As further confirmation of peptides as models for the heparin binding region in proteins, peptide 264-283 interfered with heparin-catalyzed inhibition of thrombin by two other heparin-binding inhibitors, antithrombin and heparin cofactor, and peptides corresponding to the putative antithrombin heparin-binding site (Ala-124-Leu-140, Ref. 10 ) competed for heparin binding in the protein C inhibitor assay (results not shown). Furthermore, peptide 264-283 bound to immobilized heparin and was eluted with 800 mM NaC1, indicating even higher affinity than native protein C inhibitor (elution at approximately 500 mM NaC1) The random sequence peptide bound less tightly (elution at 380 mM NaC1, Ref 10).
Polyanwn Specificity of Protein C Inhibitor-In addition to heparin, some other glycosaminoglycans have been found to accelerate proteinase inhibition by protein C inhibitor (20,21). To further assess the specificity of protein C inhibitor, we tested the ability of various polyanions to catalyze protein C inhibitor inhibition of thrombin and activated protein C.
Each polyanion was tested at various concentrations in order to determine the maximum degree of rate enhancement and the polyanion concentration at which that occurred. The results are presented in Table I  Dermatan sulfate showed no activity with thrombin and very low activity with activated protein C; a highly purified dermatan sulfate preparation (DSPG 11) showed no activity with either proteinase. Chondroitin sulfate C did not accelerate inhibition. Interestingly, fucoidan (a sulfated polymer of fucose from a marine alga) was relatively effective, and phosvitin (a phosphoserine-containing glycoprotein from egg yolk) was also active. Sulfatides and tetrapolyphosphate did not affect the rate of proteinase inhibition by protein C inhibitor (not shown). A number of heparin fractions accelerated inhibition of thrombin and activated protein C by protein C inhibitor (Table I). Protein C inhibitor clearly differs from antithrombin in responding equally well to heparin with high or low affinity for antithrombin. Protein C inhibitor exhibited some specificity for the size of the heparin molecule, as greater concentrations of the smaller heparin fractions were required for maximum stimulation of the inhibition reaction.
Mechanism of Heparin Acceleration of Inhibition-The mechanism whereby heparin accelerates proteinase inhibition by protein C inhibitor could depend on heparin binding to the inhibitor, to the proteinase, or to both proteins. Examination of the effect of increasing concentrations of heparin on the rate of inhibition of six different proteinases (a-thrombin, Y~thrombin, activated protein C, factor Xa, urokinase, and chymotrypsin, Fig. 3) reveals a bell-shaped curve that is consistent with a ternary complex model for heparin action (22)(23)(24). According to this model, heparin binds both inhibitor and proteinase to bring the reactants into closer proximity, and the rate of reaction increases as heparin concentration increases. When heparin concentrations increase beyond a certain point, the proteinase and inhibitor are more likely to bind to separate heparin molecules, thus decreasing the catalytic efficiency of the glycosaminoglycan. If heparin binding to only one of the two reactants were important, then heparin would exhibit a saturation phenomenon in these experiments.
The optimum heparin concentration as well as the relative which the maximum proteinase inhibition rate occurs.
to the rate in the absence of polyanion.
The y axis shows the relative inhibition rate, the ratio of the rate constant at a particular heparin concentration to the rate constant in the absence of heparin. increase in inhibition rate varied among different proteinases. All experiments were performed with identical concentrations of protein C inhibitor and proteinase in order to rule out protein concentration-dependent effects on heparin optimum or maximum rate. Interestingly, the rate of inhibition of factor Xa was diminished at low heparin concentrations, and at optimum heparin the rate was only slightly greater than the rate in the absence of glycosaminoglycan. In addition, increasing concentrations of heparin progressively decreased the rate of inhibition of plasma kallikrein by protein C inhibitor (Fig.  3). These two cases suggest that the catalytic effect of heparin on protein C inhibitor reactivity is not a simple phenomenon, but might involve additional effects of heparin on the particular inhibitor-proteinase pair.
The failure of attempts to demonstrate the importance of simultaneous binding of both inhibitor and proteinase to heparin using a kinetics approach (25, 26) was most likely due to the fact that heparin elicited a relatively mild rate increase. This meant that rate enhancement was not detectable under the required conditions, where proteinase and inhibitor concentrations must saturate a small amount of heparin.
Heparin affinity chromatography was used to further assess the contribution of heparin binding to protein C inhibitor and various proteinases before and after the inhibition reaction. Protein C inhibitor and five proteinases (a-thrombin, activated protein C, factor Xa, urokinase, and chymotrypsin) bound to immobilized heparin and were eluted by salt concentrations greater than physiological (290-640 mM NaC1, Table  11). Therefore, in inhibition assays (containing 150 mM NaC1) heparin binding would occur. Table I1 demonstrates the inverse relationship between the apparent heparin affinity of a particular proteinase and the heparin concentration required for maximum inhibition rate enhancement; for example, hep- Chymotrypsin 2 9 0 f 28 100 203 3.0 a Heparin affinity is given as the salt concentration required for peak elution from immobilized heparin. This value is an indication of heparin affinity, not a true affinity constant.
The optimum heparin concentration is the concentration at which the maximum inhibition rate occurs.
The rate increase is calculated as the ratio of the maximum rate to the rate in the absence of heparin.

Heparin affinity of protein C inhibitor and protein C inhibitorproteinase complexes
Inhibitor-proteinase complex Salt conc. at peak elution" The salt concentration required for peak elution from immobilized 'Protein C inhibitor affinity was measured in the absence of heparin. proteinase.
arin was most efficient at catalyzing the inhibition of thrombin, which had the highest heparin affinity. There is no obvious relationship between heparin affinity and the maximum rate of inhibition or the degree of rate enhancement. Kallikrein, which exhibited no inhibition rate enhancement, did bind to immobilized heparin, eluting a t 305 mM NaCl. The salt concentration at which a protein elutes from heparin is an indication of heparin affinity and is useful for comparative purposes, but is not identical to an affinity constant, which requires more rigorous measurement. Table I11 describes the apparent heparin affinity of protein C inhibitor following reaction with four proteinases. The reaction between protein C inhibitor and its target proteinases results in a stable bimolecular complex (6). Complexes containing protein C inhibitor and one of four proteinases were applied to immobilized heparin. The protein C inhibitorthrombin complex eluted at higher salt concentration than protein C inhibitor alone, most likely due to the contribution of thrombin, whose affinity for heparin was even higher. The protein C inhibitor-factor Xa complex eluted at essentially the same salt concentration as protein C inhibitor alone, although the affinity of factor Xa itself was lower than that of protein C inhibitor. The other complexes, protein C inhibitor-activated protein C and protein C inhibitor-urokinase, showed heparin affinities intermediate to those of protein C inhibitor and the individual proteinase. It is obvious from these results that there is no uniform decrease in apparent heparin affinity of protein C inhibitor following reaction with proteinase. Nor does the change in heparin affinity correlate with the degree of inhibition rate enhancement caused by heparin (listed in Table 11). Protein C inhibitor inactivated with neutrophil elastase, presumably at or near the reactive site, eluted from heparin-Sepharose at 530 f 14 mM NaC1.

DISCUSSION
These studies were undertaken in order to better understand how heparin binds to protein C inhibitor and how this is important for the physiological function of protein C inhibitor. The results focus on three areas: identification of the putative heparin-binding site of protein C inhibitor, the glycosaminoglycan specificity of protein C inhibitor, and the mechanism whereby heparin accelerates proteinase inhibition. A region of the protein C inhibitor molecule, residues 264-283, was identified as the heparin-binding site. This sequence can be represented as an amphipathic helix, with basic and uncharged residues on opposite faces of the helix, which is a property of other heparin-binding peptides (18). A synthetic peptide corresponding to this region of protein C inhibitor bound to heparin and interfered in heparin-catalyzed proteinase inhibition. This sequence, located in the H helix of protein C inhibitor (7), clearly differs from the heparin-binding sites of the related proteins antithrombin and heparin cofactor, which have been assigned primarily to the D helix on the basis of chemical modification experiments and natural mutations (7). The present results also differ from a recent report implicating the amino terminus of protein C inhibitor (the A+ helix) in heparin binding (27). Failure to detect heparin binding of synthetic peptide 1-16 in the present experiments could be due to the assumption that the protein C inhibitor heparin-binding site is simply a linear sequence of residues and that synthetic peptides would adopt the same structure (and therefore function) as the sequences in the native protein. It is also possible that the monoclonal antibody used in the previous study, which bound to helix A+, might interfere with heparin binding to the H helix; this is certainly a possibility if the A+ and the H helices together form a heparin-binding site, as was suggested (27).
Acceleration of protein C inhibitor proteinase inhibition by a variety of glycosaminoglycans (especially heparin, heparan sulfate, fucoidan, and low molecular weight heparin) and other polyanions (phosvitin) is consistent with a relatively nonspecific heparin-binding site in protein C inhibitor. This is in contrast to antithrombin, which exhibits narrow specificity for heparin and heparan sulfate (28). Protein C inhibitor more closely resembles heparin cofactor, which allows an even wider variety of polyanions to accelerate thrombin inhibition (29-31). Previous studies noted the ability of several sulfated glycosaminoglycans to enhance protein C inhibitor activity, although no effect of heparan sulfate was detected (20, 21). The ability of fucoidan (which contains no carboxyl groups) and phosvitin (which contains no sulfate groups) to enhance proteinase inhibition by protein C inhibitor is further evidence for a relatively nonspecific glycosaminoglycan-binding site in protein C inhibitor. Interestingly, the "permissive" glycosaminoglycan-binding site of protein C inhibitor did not accommodate dermatan sulfate well; this glycosaminoglycan is the most effective at increasing the inhibition rate of heparin cofactor (32). The identity of the glycosaminoglycan that accelerates thrombin or activated protein C inhibition by protein C inhibitor in vivo remains a mystery, although heparan sulfate, which lines vessel walls and is believed to be the primary antithrombin-activating glycosaminoglycan (28), is a candidate. Protein C inhibitor, however, did not distinguish heparin of high or low affinity for antithrombin. The low activity of chondroitin sulfate A measured in this study could be due to heparan sulfate contamination, as this glycosaminoglycan was not treated with nitrous acid to destroy traces of heparin (16). The present study is at odds with a recent report that dermatan sulfate from various sources accelerated inhibition of urokinase by protein C inhibitor (33).* The chemical heterogeneity and difficult purification of glycosaminoglycans from different tissues and species could contribute to the discordant findings.
The degree of acceleration of activated protein C or thrombin inhibition by protein C inhibitor varied with the polyanion. In any case, the increase in rate is relatively mild when protein C inhibitor is compared to antithrombin or heparin cofactor (IO), although the increase in rate appears to depend on the particular reaction conditions, as different results have been reported for the heparin enhancement of protein C inhibitor activity (11, 34). The experiments in the present study were performed with constant concentrations of proteins in order to facilitate comparisons among polyanions. The concentration of a polyanion required for maximum rate enhancement also varied; this most likely reflects the affinity of the glycosaminoglycan for protein C inhibitor and the proteinases. Among the polyanions tested, the optimum concentrations varied in parallel for thrombin and activated protein C inhibition; this suggests a specific interaction of the polyanions with the inhibitor.
The effect of various concentrations of heparin on the rate of inhibition of five proteinases (thrombin, factor Xa, activated protein C, urokinase, and chymotrypsin) by protein C inhibitor was consistent with the ternary complex model (22) in which both proteinase and inhibitor bind to the glycosaminoglycan. This phenomenon has been observed previously for protein C inhibitor (20, 21, 34) and is well-characterized for the related inhibitors antithrombin and heparin cofactor. Most of the polyanions that stimulated proteinase inhibition in the present study and in other reports appear to follow the same mechanism. In no case was a saturation effect observed, although it is difficult to completely rule out this possibility in cases where stimulation is weak and polyanion concentrations cannot be increased further.
This work represents a further attempt to correlate the mechanism of heparin action with heparin binding properties of the proteins involved. The heparin concentration required for maximum inhibition rate enhancement is a function of the apparent affinity of the proteinase for heparin, but there appears to be no general rule governing the magnitude of the maximum rate or the rate increase. The rate of inhibition of factor Xa was actually decreased at low concentrations of heparin, and the rate of inhibition of plasma kallikrein progressively decreased with increasing heparin. One explanation for these results is that in the case of thrombin, activated protein C, urokinase, or chymotrypsin, the primary role of heparin is to bind both inhibitor and proteinase to bring them into close proximity for more rapid reaction than in the absence of heparin. However, in the case of factor Xa and kallikrein, other factors such as ionic interactions, conformational changes, or steric hindrance might partially or completely offset the rate enhancement due to the proximity effect. Different heparin-enhanced rate constants have been reported previously (6, 11, 34); some of the differences are most likely due to reaction conditions, particularly temperature and protein concentration, as well as the source of heparin. The concentrations of inhibitor and proteinase have been shown to influence the optimum heparin concentration and maximum rate enhancement (26). In the present study experiments with different proteinases were performed with the same protein concentrations to eliminate other variables.
It has been reported that the inhibition of activated protein We found that dermatan sulfate that had not been treated with nitrous acid to destroy contaminating heparin did accelerate urokinase inhibition by protein C inhibitor, but acid-treated dermatan sulfate had no effect on the urokinase inhibition rate.

Heparin
Binding to Protein C Inhibitor C by aprotinin is enhanced by heparin (35); this suggests that heparin might directly affect the catalytic properties of activated protein C. Inhibition of activated protein C by protein C inhibitor is accelerated by heparin to a much greater degree than for any other proteinase tested, although no effect of heparin on hydrolysis of a small peptide substrate by activated protein C was detected in the present study.
It has been proposed that part of the ability of heparin to dramatically accelerate thrombin inhibition by antithrombin is due to the decreased heparin affinity of the resultant antithrombin-thrombin inhibitory complex, which allows heparin to efficiently dissociate from the proteins in order to participate in additional rounds of catalysis (36). (Native antithrombin eluted from heparin-Sepharose at 925 mM NaC1, while the antithrombin-thrombin complex and antithrombin inactivated at the reactive site by neutrophil elastase eluted at 430 mM NaCl, in agreement with a previous report (36); results not shown.) This phenomenon is probably less important for protein C inhibitor inhibition of proteinases, as there was no consistent correlation of maximum inhibition rate or rate enhancement with changes in apparent heparin affinity of different protein C inhibitor-proteinase complexes. The absence of a change in heparin affinity of the protein C inhibitor-activated protein C complex has been previously noted (37).
In conclusion, protein C inhibitor clearly belongs to the group of serpins whose reactivity toward certain proteinases is stimulated by heparin. (A comparison of three of these proteins is contained in the following paper, Ref. 10.) The mechanism of heparin acceleration of proteinase inhibition by protein C inhibitor was consistent with a ternary complex model, but there were indications that the exact mechansim is a more complicated function of the particular proteinase involved, as shown by the large rate enhancement for activated protein C inhibition relative to other proteinases. The question remains how protein C inhibitor can regulate the anticoagulant protein C system when it is also an inhibitor of procoagulant enzymes. So far there is little evidence for a protein C system-specific glycosaminoglycan that might preferentially accelerate activated protein C inhibition, since all of the polyanions tested in the present study stimulate inhibition of both thrombin and activated protein C. It is also possible that additional factors, such as the relative concentrations of activated protein C, thrombin, and other enzymes or their localization in. uiuo, are critical for determining the physiological effectiveness of protein C inhibitor.