Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor

The inhibition of kallikrein and factor XIa by protein C inhibitor (PCI) was studied. The method of Suzuki et al. [Suzuki, K., Nishioka, J., & Hashimoto, S. (1983) J. Biol. Chem. 258, 163-168] for the purification of PCI was modified in order to avoid the generation of proteolytic activity and subsequent inactivation of PCI. With the use of soybean trypsin inhibitor, an efficient inhibitor of kallikrein and factor XIa, the generation of proteolytic activity was avoided. The kinetics for the inactivation of activated protein C (APC), kallikrein, and factor XIa by PCI were determined. In the absence of heparin, no inactivation of APC was observed, in contrast to kallikrein and factor XIa, which are inhibited with second-order rate constants of (11 +/- 4) X 10(4) and (0.94 +/- 0.07) X 10(4) M-1 s-1, respectively. Addition of heparin potentiated the inhibition of APC [(1.2 +/- 0.2) X 10(4) M-1 s-1] and factor XIa [(9.1 +/- 0.7) X 10(4) M-1 s-1] by PCI, whereas the inhibition of kallikrein by PCI was unchanged [(10 +/- 1) X 10(4) M-1 s-1]. The second-order rate constants for the inhibition of kallikrein or factor XIa by PCI were similar to the second-order rate constants for the inhibition of their isolated light chains by PCI, indicating a minor role for the heavy chains of both molecules in the inactivation reactions. With sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and immunoblotting, complex formation of APC, kallikrein, and factor XIa with PCI could be demonstrated. APC and kallikrein formed 1:1 molar complexes with PCI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of tissue kallikrein by protein C inhibitor. Evidence for identity of protein C inhibitor with the kallikrein binding protein.

We studied the inhibition of tissue kallikrein by protein C inhibitor (PCI), a relatively unspecific heparin-dependent serine protease inhibitor present in plasma and urine. PCI inhibited the amidolytic activity (cleavage of H-D-valyl-L-leucyl-arginine-p-nitroaniline) of urinary kallikrein with an apparent second order rate constant of 2.3 x 10(4) M-1 s-1 and formed stable complexes (85 kDa) with urinary kallikrein as judged from silver-stained sodium dodecyl sulfate-polyacrylamide gels. Complex formation was time-dependent and was paralleled by a decrease in the intensity of the main PCI protein band (Mr = 57,000) and an increase in the intensity of the lower Mr (54,000) PCI form (cleaved inhibitor). Heparin interfered with the inhibition of tissue kallikrein by PCI and with the formation of tissue kallikrein-PCI complexes in a dose-dependent fashion and completely abolished PCI-tissue kallikrein interaction at 300 micrograms/ml. This is in contrast to findings on the interaction of PCI with all other target proteases studied so far (i.e. stimulation of inhibition by heparin) but is similar to the reaction pattern of 125I-labeled tissue kallikrein with so called kallikrein binding protein described in serum and other systems. To study a possible relationship between PCI and this kallikrein binding protein we incubated 125I-labeled urinary kallikrein in serum and in PCI-immunodepleted serum in the absence and presence of heparin and analyzed complex formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal serum, formed complexes co-migrated with complexes of purified PCI and 125I-kallikrein and were less intense in the presence of heparin. No complex formation at all was seen in PCI-depleted serum. Our data indicate that PCI may be a physiologically important endogenous inhibitor of tissue kallikrein and provide evidence that PCI may be identical to the previously described kallikrein binding protein.     

Basic residues in the 37-loop of activated protein C modulate inhibition by protein C inhibitor but not by alpha(1)-antitrypsin

The role of lysines 37-39 (chymotrypsin numbering) in the 37-loop of the serine protease activated protein C (APC) was studied by expressing acidic and neutral recombinant APC (rAPC) mutants. Activity of the APC mutants was assessed using human plasma and plasma-purified and recombinant derivatives of protein C inhibitor (PCI; also known as plasminogen activator inhibitor-3) and alpha(1)-antitrypsin, with and without heparin. The catalytic properties of the mutants to small peptidyl substrates were essentially the same as wild-type rAPC (wt-rAPC), yet their plasma anticoagulant activities were diminished. Analysis of the rAPC-protease inhibitor complexes formed after addition of wt-rAPC and mutants to plasma revealed no change in the inhibition pattern by alpha(1)-antitrypsin but a reduction in mutant complex formation by PCI in the presence of heparin. Using purified serpins, we found that inhibition rates of the mutants were the same as wt-rAPC with alpha(1)-antitrypsin; however, PCI (plasma-derived and recombinant forms) inhibition rates of the acidic mutants were slightly faster than that of wt-rAPC without heparin. By contrast, PCI-heparin inhibition rates of the mutants were not substantially accelerated compared to wt-rAPC. The mutants had reduced heparin-binding properties compared to wt-rAPC. Molecular modeling of the PCI-APC complex with heparin suggests that heparin may function not only to bridge PCI to APC, but also to alleviate putative non-optimal intermolecular interactions. Our results suggest that the basic residues of the 37-loop of APC are involved in macromolecular substrate interactions and in heparin binding, and they influence inhibition by PCI (with or without heparin) but not by alpha(1)-antitrypsin, two important blood plasma serpins.     

Inhibition of urokinase by protein C-inhibitor (PCI). Evidence for identity of PCI and plasminogen activator inhibitor 3

Human protein C-inhibitor (PCI) was isolated from human citrated plasma by combining rivanol precipitation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on dextran sulfate Sepharose. The purified PCI migrated with the beta-globulins and was free from protein contaminations as judged by immunoelectrophoresis. In SDS-PAGE under reducing and unreducing conditions PCI showed a single band at Mr = 57,000. The specific activity of the inhibitor was 226 units/mg. Surprisingly, the isolated PCI inhibited the amidolytic activity of urokinase (u-PA) on Glu-Gly-Arg-pNA (S-2444) in a time-dependent manner. Heparin, dextran sulfate and pentosanpolysulfate accelerated the reaction catalytically. PCI revealed itself as a non-competitive inhibitor of u-PA. The Ki-value was determined to be 7.9 x 10(-8)M. Inhibition of amidolytic activity was found to be associated with the formation of an 1:1 equimolar complex with a Mr of 110,000 as demonstrated by means of polyacrylamide gel electrophoresis and following Western blotting technique using polyclonal antibodies against u-PA and PCI. The specific activity of the isolated PCI of 226 units/mg, which approximates the theoretical value of pure PCI, indicates a highly purified preparation of PCI. The heparin-dependent inhibition of urokinase by this highly purified protein as well as comparison of the kinetic data and amino-acid composition of both PCI and the recently described plasminogen activator inhibitor (PAI) 3 give high evidence of identity of PCI and PAI-3.     

The heparin binding site of protein C inhibitor is protease-dependent

Protein C inhibitor (PCI) is a member of the serpin family of protease inhibitors with many biological functions and broad inhibitory specificity. Its major targets in blood are thrombin and activated protein C (APC), and the inhibition of both enzymes can be accelerated by glycosaminoglycans, including heparin. Acceleration of thrombin and APC inhibition by PCI requires that both protease and inhibitor bind to the same heparin chain to form a bridged Michaelis complex. However, the position of the heparin binding site of APC is opposite to that of thrombin, and formation of the bridged complexes must require either radical reorientation of the proteases relative to PCI or alternate heparin binding modes for PCI. In this study, we investigate how heparin bridges thrombin and APC to PCI by determining the effect of mutations in and around the putative heparin binding site of PCI. We found that heparin binds PCI in a linear fashion along helix H to bridge thrombin, consistent with our recent crystal structure (3B9F), but that it must rotate by approximately 60 degrees to engage Arg-229 to bridge APC. To gain insight into the possible modes of heparin binding to PCI, we solved a crystal structure of cleaved PCI bound to an octasaccharide heparin fragment to 1.55 angstroms resolution. The structure reveals a binding mode across the N terminus of helix H to engage Arg-229 and align the heparin binding site of APC. A molecular model for the heparin-bridged PCI.APC complex was built based on mutagenesis and structural data.     

Characterization of recombinant human protein C inhibitor expressed in Escherichia coli

The serine protease inhibitor (serpin) protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduction. The biochemical activity of PCI is not fully defined partly due to the lack of a convenient expression system for active rPCI. Using pET-15b plasmid, Ni(2+)-chelate and heparin-Sepharose affinity chromatography steps, we describe here the expression, purification and characterization of wild-type recombinant (wt-rPCI) and two inactive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in Escherichia coli. Wild-type rPCI, but not the two mutants, formed a stable bimolecular complex with thrombin, activated protein C and urokinase. In the absence of heparin, wt-rPCI-thrombin, -activated protein C, and -urokinase inhibition rates were 56.7, 3.4, and 2.3 x 10(4) M(-1) min(-1), respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 mug/mL heparin for each respective inhibition reaction. The stoichiometry of inhibition (SI) for wt-rPCI-thrombin was 2.0, which is comparable to plasma-derived PCI. The present report describes for the first time the expression and characterization of recombinant PCI in a bacterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rPCI for future structure-function studies.     

Protein C inhibitor. Purification from human plasma and characterization

Protein C inhibitor was isolated from human plasma using conventional chromatographic technique consisting of barium citrate adsorption, polyethylene glycol fractionation, DEAE-Sepharose CL-6B treatment, ammonium sulfate fractionation, dextran sulfate-agarose chromatography, gel filtration on ACA-44, and DEAE-Sephacel chromatography. The purified protein C inhibitor is a single polypeptide chain with an apparent Mr = 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor is heterogeneous in pI: six pIs exist between pH 7.4 and 8.6. The inhibitor was shown to be different from the already known plasma protease inhibitors by chemical and immunological analyses. It migrates to the late alpha 1-globulin region on agarose gel electrophoresis. The inhibitor reduced the amidolytic activity of activated protein C noncompetitively by forming a 1:1 molar complex with the enzyme, determined by the use of a fluorogenic substrate toward activated protein C (Boc-Leu-Ser-Thr-Arg-4-methylcoumaryl-7-amide). The inhibition constant (Ki) of the inhibitor against activated protein C was 5.8 x 10(-8) M. The inhibitor also blocked the prolongation of activated partial thromboplastin time by activated protein C. The immunoglobulin which was produced by the inhibitor completely removed the inhibitory activity present in normal human plasma against activated protein C. This suggests that the inhibitor which we have isolated is the only inhibitor in plasma against activated protein C.     

Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C

Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, alpha1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC.     

Immunological identity of heparin-dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3

Purified plasma and urinary protein C inhibitors (PCI) formed heparin-dependent complexes with activated protein C (APC) which were detected by immunoblotting after nondenaturing gel electrophoresis. Bands representing APC.PCI complexes were also seen on immunoblots after incubation of plasma with APC and heparin. The same immunoblot pattern of complexes was detected by three different methods: method A, monoclonal antibody to plasminogen activator inhibitor-3 (PAI-3, urinary urokinase inhibitor) + 125I-labeled anti-mouse IgG; method B, polyclonal antibodies to PCI + 125I-labeled purified plasma PCI; and method C, monoclonal antibody to protein C + 125I-protein C. Plasma depleted of PAI-3 by immunoadsorption with insolubilized monoclonal antibody to PAI-3 showed no detectable antigen or complexes with APC as visualized by methods A or B. This PAI-3-depleted plasma had less than 10% of the heparin-dependent inhibitory activity of normal plasma toward APC. Purified plasma PCI was fully reactive in an enzyme-linked immunoabsorbent assay for PAI-3, and plasma and urinary PCI inhibited urokinase activity in a heparin-dependent manner. These data indicate that heparin-dependent plasma and urinary PCI and PAI-3 are immunologically and functionally very similar if not identical. This observation identifies a new interrelation between the protein C anticoagulant and the fibrinolytic systems. In addition, plasma contains a heparin-independent inhibitor of APC which is not immunologically related to plasma PCI or to PAI-3.     

Competition of activated protein C and urokinase for a heparin-dependent inhibitor

Human urine contains a hitherto unrecognized heparin-dependent inhibitor of activated protein C (APC) (Mr approximately 50,000) that coelutes from heparin-Sepharose together with the only observed peak of urokinase inhibitory activity at a position (0.35 M NaCl) similar to that of plasma protein C (PC) inhibitor. Based on functional assays and immunoblot studies, urokinase and APC compete for this crude inhibitor in the absence or presence of heparin. These results suggest that the same heparin-dependent urinary inhibitor that is immunologically different from several known protease inhibitors is responsible for the observed inhibition of APC and urokinase. In the absence of heparin this inhibitor inhibits APC and urokinase with similar rates, and heparin enhances its inhibitory activity toward both enzymes with more pronounced stimulation of its PC inhibitory activity than its urokinase inhibitory activity. Half-maximal stimulation of inhibition of APC occurs at about 2 mU/ml and maximal stimulation (approximately 10-fold increase of the pseudo-first-order rate constant) at greater than or equal to 50 mU/ml of heparin. This is the first demonstration of competition between APC and urokinase for a heparin-dependent inhibitor. These results may therefore represent a new link between the two major antithrombotic pathways, the PC pathway and the fibrinolytic system.     

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urinary protein C inhibitor. Glycosaminoclycans synthesized by the epithelial kidney cell line TCL-598 enhance its interaction with urokinase.

Protein C inhibitor (PCI), also known as plasminogen activator inhibitor 3, inhibits a variety of serine proteases by forming sodium dodecyl sulfate-stable 1:1 complexes. In purified systems PCI is only a weak inhibitor of urokinase. Nevertheless, complexes between PCI and urokinase are found in appreciable amounts in native human urine. Since PCI activity is stimulated by heparin and other glycosaminoglycans, we investigated the presence of stimulating glycosaminoglycans on cells lining the urinary tract. We chose the epithelial kidney tumor cell line TCL-598 as a model and isolated metabolically labeled glycosaminoglycans. TCL-598 incorporated [35S] sulfate into high Mr components (Mr greater than 200,000 and approximately 75,000) as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of cell extracts; the Mr greater than 200,000 component bound specifically to PCI-Sepharose 4B and was eluted either with heparin (5 mg/ml) or with NaCl (2.0 M). Treatment of this PCI-binding material with chondroitinase ABC, but not with chondritinase AC or heparitinase, abolished binding to PCI-Sepharose, confirming the glycosaminoglycan nature of this material and suggesting the involvement of dermatan sulfate in binding. These glycosaminoglycans eluted from PCI-Sepharose stimulated urokinase inhibition by PCI in a dose-dependent way and enhanced complex formation of 125I-urokinase and PCI as did in control experiments dermatan sulfate from porcine skin and from bovine mucosa. Our results suggest that PCI activity might be regulated also in vivo by the presence or absence of stimulating glycosaminoglycans; dermatan sulfate-containing glycosaminoglycans associated with kidney cells might be responsible for stimulation of the urokinase inhibitory activity of PCI in the urinary tract; the type of glucosaminoclycans might furthermore regulate enzyme specificity of PCI.     

Reactive site mutants of recombinant protein C inhibitor

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C (APC). The residues F353-R354-S355 (P2-P1-P1′) constitute part of the reactive site loop of PCI with the R-S peptide bond being cleaved by the proteinase. Changing the reactive site P1 and P2 residues to those of either proteinase nexin-1, α₁-proteinase inhibitor or heparin cofactor II resulted in a decrease in inhibitory activity towards thrombin and APC. Changing the P2 residue F353 → P generated a rPCI which was a better thrombin inhibitor, but was 10-fold less active with APC. While these results support the concept that the P1 and P2 residues are important in the specificity of PCI, they suggest that the reactive site residues are not the only determinant of serpin specificity. Kinetic analysis of the rPCI variants was consistent with PCI operating by a mechanism similar to that proposed for other serpins. In this model an intermediary complex forms between inhibitor and proteinase that can proceed to either cleavage of the inhibitor as substrate or formation of an inactive complex.     

Interaction of heparin cofactor II with neutrophil elastase and cathepsin G

We investigated the interaction of the human plasma proteinase inhibitor heparin cofactor II (HC) with human neutrophil elastase and cathepsin G in order to examine 1) proteinase inhibition by HC, 2) inactivation of HC, and 3) the effect of glycosaminoglycans on inhibition and inactivation. We found that HC inhibited cathepsin G, but not elastase, with a rate constant of 6.0 x 10(6) M-1 min-1. Inhibition was stable, with a dissociation rate constant of 1.0 x 10(-3) min-1. Heparin and dermatan sulfate diminished inhibition slightly. Both neutrophil elastase and cathepsin G at catalytic concentrations destroyed the thrombin inhibition activity of HC. Inactivation was accompanied by a dramatic increase in heat stability, as occurs with other serine proteinase inhibitors. Proteolysis of HC (Mr 66,000) produced a species (Mr 58,000) that retained thrombin inhibition activity, and an inactive species of Mr 48,000. Amino acid sequence analysis led to the conclusion that both neutrophil elastase and cathepsin G cleave HC at Ile66, which does not affect HC activity, and at Val439, near the reactive site Leu444, which inactivates HC. Since cathepsin G is inhibited by HC and also inactivates HC, we conclude that cathepsin G participates in both reactions simultaneously so that small amounts of cathepsin G can inactivate a molar excess of HC. High concentrations of heparin and dermatan sulfate accelerated inactivation of HC by neutrophil proteinases, with heparin having a greater effect. Heparin and dermatan sulfate appeared to alter the pattern, and not just the rate, of proteolysis of HC. We conclude that while HC is an effective inhibitor of cathepsin G, it can be proteolyzed by neutrophil proteinases to generate first an active inhibitor and then an inactive molecule. This two-step mechanism might be important in the generation of chemotactic activity from the amino-terminal region of HC.     

Platelet plasminogen activator inhibitor: purification and characterization of interaction with plasminogen activators and activated protein C

Plasminogen activator inhibitor (PAI) was purified in active form from porcine platelets under nondenaturing conditions. The purified inhibitor (Mr 47,000) reacts with tissue-type plasminogen activator (t-PA), urokinase (UK), and activated protein C (APC) to yield both SDS-stable complexes and a modified PAI of slightly reduced molecular weight. The second-order rate constants for the inhibition of t-PA and UK by PAI are 3.5 X 10(7) and 3.4 X 10(7) M-1 s-1, respectively. Activated protein C reacts with PAI with a second-order rate constant of 1.1 X 10(4) M-1 s-1. This rate is not accelerated by protein S, phospholipid, and calcium, or heparin. It is concluded that (1) PAI can function as both inhibitor and substrate of its target proteases, (2) if APC promotes fibrinolysis via inactivation of PAI, then APC must be present in concentrations several orders of magnitude greater than t-PA, or the interaction of APC and PAI must be accelerated by presently unknown mechanisms, and (3) in the absence of heparin, platelet PAI is the most rapid inhibitor of APC yet described.     

Physiologic inhibition of human activated protein C by alpha 1-antitrypsin

The plasma antithrombotic enzyme activated protein C (APC) has two major plasma inhibitors. One is heparin-dependent, has been characterized, and is known as protein C inhibitor. The second inhibitor was isolated based on its heparin-independent ability to inhibit and complex with APC. The purified inhibitor had the amino acid composition and NH2 terminus of alpha 1-antitrypsin and reacted with monoclonal antibodies to alpha 1-antitrypsin. The inhibitor was greater than 95% pure alpha 1-antitrypsin as judged by electroimmunoassay, inactivation of trypsin, and electrophoresis in two gel systems. To identify the second major plasma inhibitor of APC, immunoblot studies of enzyme-inhibitor complexes were made to compare APC addition to normal plasma and to plasma deficient in protein C inhibitor or alpha 1-antitrypsin. The results showed that alpha 1-antitrypsin is the second major plasma APC inhibitor. Given the association rate constant of alpha 1-antitrypsin for APC of 10 M-1 s-1 and its plasma concentration of approximately 40 microM, it accounts for approximately half of the heparin-independent APC inhibitory activity of plasma. Based on immunoblot analysis plasmas of 15 patients with intravascular coagulation contained APC-alpha 1-antitrypsin complexes suggesting that this inhibition reaction occurs in vivo. Thus, alpha 1-antitrypsin is a major physiologic inhibitor of APC.     

Tracking structural features leading to resistance of activated protein C to alpha 1-antitrypsin

Activated protein C (APC) is a multi-modular anticoagulant serine protease, which degrades factor V/Va and factor VIIIa. Human APC (hAPC) is inhibited by human alpha 1-antitrypsin (AAT), while the bovine enzyme (bAPC) is fully resistant to this serpin. Structural features in the catalytic domains between the two species cause this difference, but detailed knowledge about the causal molecular difference is missing. To gain insight into the APC-AAT interaction and to create a human protein C resistant to AAT inhibition, we have used molecular modeling and site-directed mutagenesis. First, a structural model for bAPC based on the Gla-domainless X-ray structure of hAPC was built. Screening the molecular surface of the human and bovine APC enzymes suggested that a hAPC molecule resistant to AAT inhibition could be constructed by substituting only a few amino acids. We thus produced recombinant hAPC molecules with a single mutation (S173E, the numbering follows the chymotrypsinogen nomenclature), two mutations (E60aS/S61R) or a combination of all these substitutions (E60aS/S61R/S173E). Amidolytic and anticoagulant activities of the three mutant APC molecules were similar to those of wild-type hAPC. Inhibition of wild-type hAPC by AAT was characterized by a second-order rate constant (k2) of 2.71 M-1 s-1. The amino acid substitution at position 173 (S173E mutant) led to partial resistance to AAT (k2 = 0.84 M-1 s-1). The E60aS/S61R mutant displayed mild resistance to AAT inhibition (k2 = 1.70 M-1 s-1), whereas the E60aS/S61R/S173E mutant was inefficiently inactivated by AAT (k2 = 0.40 M-1 s-1). Inhibition of recombinant APC molecules by the serpin protein C inhibitor (PCI) in the presence and absence of heparin was also investigated.     

Basic residues in the 37-loop of activated protein C modulate inhibition by protein C inhibitor but not by α1-antitrypsin

The role of lysines 37–39 (chymotrypsin numbering) in the 37-loop of the serine protease activated protein C (APC) was studied by expressing acidic and neutral recombinant APC (rAPC) mutants. Activity of the APC mutants was assessed using human plasma and plasma-purified and recombinant derivatives of protein C inhibitor (PCI; also known as plasminogen activator inhibitor-3) and α₁-antitrypsin, with and without heparin. The catalytic properties of the mutants to small peptidyl substrates were essentially the same as wild-type rAPC (wt-rAPC), yet their plasma anticoagulant activities were diminished. Analysis of the rAPC-protease inhibitor complexes formed after addition of wt-rAPC and mutants to plasma revealed no change in the inhibition pattern by α₁-antitrypsin but a reduction in mutant complex formation by PCI in the presence of heparin. Using purified serpins, we found that inhibition rates of the mutants were the same as wt-rAPC with α₁-antitrypsin; however, PCI (plasma-derived and recombinant forms) inhibition rates of the acidic mutants were slightly faster than that of wt-rAPC without heparin. By contrast, PCI–heparin inhibition rates of the mutants were not substantially accelerated compared to wt-rAPC. The mutants had reduced heparin-binding properties compared to wt-rAPC. Molecular modeling of the PCI–APC complex with heparin suggests that heparin may function not only to bridge PCI to APC, but also to alleviate putative non-optimal intermolecular interactions. Our results suggest that the basic residues of the 37-loop of APC are involved in macromolecular substrate interactions and in heparin binding, and they influence inhibition by PCI (with or without heparin) but not by α₁-antitrypsin, two important blood plasma serpins.     

Interaction of thrombin with antithrombin, heparin cofactor II, and protein C inhibitor

-Thrombin is a trypsin-like serine proteinase involved in blood coagulation and wound repair processes. Thrombin interacts with many macromolecular substrates, cofactors, cell-surface receptors, and blood plasma inhibitors. The three-dimensional structure of human -thrombin shows multiple surface exosites for interactions with these macromolecules. We used these coordinates to probe the interaction of thrombin's active site and two exosites, anion-binding exosite-I and -II, with the blood plasma serine proteinase inhibitors (serpins) antithrombin (AT), heparin cofactor II (HC), and protein C inhibitor (PCI). Heparin, a widely used anticoagulant drug, accelerates the rate of thrombin inhibition by AT, PCI, and HC. Thrombin Quick II is a dysfunctional thrombin mutant with a Gly 226 Val substitution in the substrate specificity pocket. We found that thrombin Quick II was inhibited by HC, but not by AT or PCI. Molecular modeling studies suggest that the larger Val side chain protrudes into the specificity pocket, allowing room for the smaller P1 side chain of HC (Leu) but not the larger P1 side chain of AT and PCI (both with Arg). _T ^–-Thrombin and thrombin Quick I (Arg 67 Cys) are both altered in anion-binding exosite-I, yet bind to heparin-Sepharose and can be inhibited by AT, HC, and PCI in an essentially normal manner in the absence of heparin. In the presence of heparin, inhibition of these altered thrombins by HC is greatly reduced compared to both AT and PCI. -Thrombin with chemically modified lysines in both anion-binding exosite-I and -II has no heparin accelerated thrombin inhibition by either AT or HC. Thrombin lysine-modified in the presence of heparin has protected residues in anion-binding exosite-II and the loss of heparin-accelerated inhibition by HC is greater than that by AT. Collectively, these results suggest differences in serpin reactive site recognition by thrombin and a more complicated mechanism for heparin-accelerated inhibition by HC compared to either AT or PCI.Abbreviations used: AT, antithrombin; HC, heparin cofactor II; PCI, protein C inhibitor; serpin(s), serine proteinase inhibitor(s); FPRck, D-Phe-Pro-Arg-chloromethyl ketone; FPLck, D-Phe-Pro-Leu-chloromethyl ketone; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HNP, 20mM HEPES, 150mM NaCl, 0.1% (w/v) poly(ethyleneglycol) (M_r = 8000) buffer atpH 7.4; Unp-PLPT, unprotected pyridoxal 5phosphate modified-thrombin; HPPLPT, heparin-protected pyridoxal 5phosphate modifiedthrombin.     

The in situ inhibition of prothrombinase-formed human alpha-thrombin and meizothrombin(des F1) by antithrombin III and heparin

The inhibition of thrombin by antithrombin III (AT III) and heparin has been studied in pure systems to determine the kinetics of inhibition during human prothrombin activation. The present study shows that prothrombinase-catalyzed prothrombin activation resulted in the generation of thrombin and meizothrombin(des F1). In the absence of heparin the second-order rate constants of the inactivation of both thrombin and meizothrombin(des F1) formed in the reaction mixture appeared to be identical, k = 3.7 X 10(5) M-1 min-1. The rate constant of inhibition of purified thrombin was 6.5 X 10(5) M-1 min-1. In the presence of heparin the decay of the amidolytic activity was biexponential and could be modeled by a four-parameter equation to determine the pseudo first-order rate constants of inhibition as well as the composition of the reaction with respect to the levels of thrombin and meizothrombin(des F1). The ratio of thrombin over meizothrombin(des F1) varied with the initial prothrombin concentration. Heparin catalyzed the AT III inhibition of thrombin but not meizothrombin(des F1) formed during the prothrombin activation. Thrombin, generated by (Xa-Va-phospholipid-Ca2+) was inhibited by AT III/heparin more slowly than purified thrombin, and the saturation kinetics of the inhibition with respect to AT III differed from those found with purified thrombin.