Proteolysis of factor Va by factor Xa and activated protein C

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.     

Mechanism of inhibition of activated protein C by protein C inhibitor

Protein C inhibitor isolated from human plasma inhibited thrombin, factor Xa, trypsin and chymotrypsin as well as activated protein C, but had very little effect on urokinase and plasmin. The inhibition constants (K1) of protein C inhibitor for activated protein C, thrombin and factor Xa were 5.6 X 10(-8) M, 6.7 X 10(-8) M and 3.1 X 10(-7) M, respectively. The second-order rate constant for inhibition of activated protein C by the inhibitor increased about 30-fold in the presence of an optimal heparin concentration (5-10 units/ml). The inhibition of activated protein C by plasma protein C inhibitor was also accelerated by heparin. When activated protein C (Mr = 62,000) was incubated with protein C inhibitor (Mr = 57,000), enzyme-inhibitor complexes with apparent Mr = 102,000 and 88,000 were observed in the nonreduced and the reduced samples, respectively, on SDS-polyacrylamide gel electrophoresis. In addition to these complexes, a band of unbound enzyme and a band with Mr = 54,000 were detected. When 125I-labeled protein C inhibitor was exposed to activated protein C, the inhibitor band was converted to bands with apparent Mr = 102,000 and 54,000 in the nonreduced samples, as determined by autoradiography after gel electrophoresis in SDS. The band with Mr = 54,000 also appeared when the inhibitor reacted with other serine proteases. The activated protein C was released from the inactive complex by treatment with 1 M ammonia or hydroxylamine. This phenomenon was found by SDS-polyacrylamide gel electrophoresis to represent the dissociation of the enzyme-inhibitor complex by ammonia or hydroxylamine into the free enzyme and the proteolytically modified inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A low molecular weight platelet inhibitor of factor XIa: purification, characterization, and possible role in blood coagulation.

A low molecular weight platelet inhibitor of factor XIa (PIXI) has been purified 250-fold from releasates of washed and stimulated human platelets. Molecular weight estimates of 8400 and 8500 were determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, although a second band of Mr 5000 was present upon electrophoresis. The inhibitor does not appear to be one of the platelet-specific, heparin-binding proteins, since it neither bound to nor was affected by heparin. An amount of PIXI which inhibited by 50% factor XIa cleavage of the chromogenic substrate S2366 (Pyr-Glu-Pro-Arg-pNA-2H2O) only slightly inhibited (5-9%) factor XIIa, plasma kallikrein, plasmin, and activated protein C and did not inhibit factor Xa, thrombin, tPA, or trypsin, suggesting specificity for factor XIa. Kinetic analyses of the effect of PIXI on factor XIa activity demonstrated mixed-type, noncompetitive inhibition of S2366 cleavage and of factor IX activation with Ki's of 7 x 10(-8) and 3.8 x 10(-9) M, respectively. Immunoblot analysis showed that PIXI is not the inhibitory domain of protease nexin II, a potent inhibitor of factor XIa also secreted from platelets. Amino acid analysis showed that PIXI has no cysteine residues and, therefore, is not a Kunitz-type inhibitor. PIXI can prevent stable complex formation between alpha 1-protease inhibitor and factor XIa light chain as demonstrated by SDS-polyacrylamide gel electrophoresis. The inhibition by PIXI of factor XIa-catalyzed activation of factor IX and its capacity to prevent factor XIa inactivation by alpha 1-protease inhibitor, combined with the specificity of PIXI for factor XIa among serine proteases found in blood, suggest a role for PIXI in the regulation of intrinsic coagulation.     

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)     

A new and simple isolation procedure for human protein C inhibitor. Evidence for a second inhibitor for activated protein C present in human plasma

Human plasma contains an inhibitor of activated protein C (APC) which is termed according to its function protein C inhibitor (PCI). High purification of functionally active PCI with a yield of 18% is achieved by an improved procedure consisting of 4 steps: precipitation by rivanol, fractionation with ammonium sulfate, ion-exchange chromatography on DEAE Sephacel and chromatography on dextran sulfate Sepharose. This purification results in the isolation of a homogeneous PCI which migrates in immunoelectrophoresis with the beta-globulins of human plasma and in SDS PAGE as one single band at Mr = 57,000 both under reducing and nonreducing conditions. The specific activity of the highly purified PCI was determined to be 226 units/mg, 1 unit being equivalent to the activity of 1 ml fresh human citrated plasma. PCI forms complexes with 1:1 stoichiometry (Ki: 1.4 x 10(-8) M) resulting in a loss of the amidolytic activity of APC as measured on Tos-Glu-Pro-Arg-pNA (S 2366). The inhibition rate of APC by PCI (k: 7.5 x 10(5) M-1 min-1) is significantly increased in the presence of 5 i.u./ml heparin (kH: 2.2 x 10(7) M-1 min-1). PCI also blocks the amidolytic activities of urokinase plasminogen activator (u-PA), thrombin and factor Xa on their chromogenic substrates in a heparin-dependent manner. According to the Ki-values measured for these reactions PCI is a noncompetitive inhibitor of these proteases. The Ki-values calculated do not differ significantly from those obtained for the inhibition of APC by PCI. Immunodepleted PCI-deficient plasma still contains an inhibitory activity against APC which, however, only slowly inactivates the amidolytic activity of APC and in a time and concentration-dependent manner. Addition of heparin has no influence on the inhibition rate. This finding suggests the existence of a second, heparin-independent PCI present in human plasma.     

Proteolytic activation of tissue plasminogen activator by plasma and tissue enzymes

Tissue kallikrein and factor Xa were found to activate tissue plasminogen activator (t-PA) at a rate comparable with that of plasmin. During the activation reaction, the single-chain molecule was converted into a two-chain form. A slight t-PA activating activity was also found in plasma kallikrein. Other activated coagulation factors, factor XIIa, factor XIa, factor IXa, factor VIIa, thrombin and activated protein C had no effect on t-PA activation. t-PA was also activated by a tissue kallikrein-like enzyme that was isolated from the culture medium of melanoma cells. These results indicate that tissue kallikrein and factor Xa may participate in the extrinsic pathway of human fibrinolysis.     

The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin.

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.     

The synthesis of arginylfluoroalkanes, their inhibition of trypsin and blood-coagulation serine proteinases and their anticoagulant activity.

Seven arginylfluoroalkanes ('arginine fluoroalkyl ketones') were synthesized by using a modified Dakin-West procedure. The structure of benzoyl-Arg-CF2CF3 was analysed by 19F-n.m.r. spectroscopy and m.s. and the compound was shown to exist primarily as a hydrate or cyclic carbinolamine. Arginylfluoroalkanes are good inhibitors of blood-coagulation serine proteinases and were found to be slow-binding inhibitors for bovine trypsin with Ki values of 0.2-56 microM. Benzoyl-Arg-CF2CF3 was the best inhibitor for bovine thrombin and human Factor XIa, and inhibited thrombin and Factor XIa competitively with Ki values of 13 microM and 62 microM respectively. The best inhibitor for pig pancreatic kallikrein was p-toluoyl-Arg-CF3, with a Ki value of 35 microM. Benzoyl-Arg-CF3 and benzoyl-Arg-CF2CF3 inhibited human plasma kallikrein competitively, with Ki values of 50 microM. None of the seven arginylfluoroalkanes was a good inhibitor of human factor Xa or of Factor XIIa. The arginylfluoroalkanes were tested in the prothrombin time (PT) and activated partial thromboplastin time (APTT) coagulant assays. Two fluoroketones, benzoyl-Arg-CF2CF3 and 1-naphthoyl-Arg-CF3, had significant anticoagulant activity. Benzoyl-Arg-CF2CF3 was found to prolong the PT 1.8-fold at 120 microM and to prolong the APTT 2.4-fold at 90 microM, whereas 1-naphthoyl-Arg-CF3 only prolonged the APTT 1.7-fold at 100 microM.     

Purification of a proteinase inhibitor from bovine serum with C1-inhibitor activity

This report describes the purification of a novel proteinase inhibitor from bovine serum. This protein was purified to apparent homogeneity employing affinity binding to sulfated dextran and precipitation by ammonium sulfate, followed by sequential chromatography on DEAE-cellulose, heparin-Sepharose and Sephacryl S-200. Quantitative enzyme-linked immunosorbent assays revealed that the concentration of this inhibitor is approximately 3 microM in bovine serum. The inhibitor is a single polypeptide chain with an estimated Mr of 83,000 as determined by SDS-polyacrylamide gel electrophoresis. An aspartic acid was found at the amino terminus of the protein; N-terminal amino acid sequence data indicated that there was no significant homology with other reported amino acid sequences. This bovine inhibitor covalently complexed the human proteinases C1-r, C1-s, factor XIIa and plasma kallikrein, which are also complexed and inactivated by human C1-inhibitor. In addition, the bovine inhibitor complexed and inactivated bovine chymotrypsin, a feature which functionally distinguishes it from human C1-inhibitor. Although the bovine inhibitor appears functionally very similar to C1-inhibitor, we found no evidence for structural homology with the human counterpart.     

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.     

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.     

The protease specificity of heparin cofactor II. Inhibition of thrombin generated during coagulation

125I-labeled heparin cofactor II (HCII) was mixed with plasma and coagulation was initiated by addition of CaCl2, phospholipids, and kaolin or tissue factor. In the presence of 67 micrograms/ml of dermatan sulfate, radioactivity was detected in a band which corresponded to the thrombin-HCII complex (Mr = 96,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No other complexes were observed. The thrombin-HCII complex was undetectable when 5 units/ml of heparin was present or when prothrombin-deficient plasma was used. In experiments with purified proteases, HCII did not significantly inhibit coagulation factors VIIa, IXa, Xa, XIa, XIIa, kallikrein, activated protein C, plasmin, urokinase, tissue plasminogen activator, leukocyte elastase, the gamma-subunit of nerve growth factor, and the epidermal growth factor-binding protein. HCII inhibited leukocyte cathepsin G slowly, with a rate constant of 8 X 10(4) M-1 min-1 in the presence of dermatan sulfate. These results indicate that the protease specificity of HCII is more restricted than that of other plasma protease inhibitors and suggest that the anticoagulant effect of dermatan sulfate is due solely to inhibition of thrombin by HCII.     

Isolation and characterization of an inhibitor of factor XIIa from bovine plasma

An inhibitor of factor XIIa has been purified to homogeneity from bovine plasma. The purification steps included precipitation of contaminating proteins with polyethylene glycol and chromatography on DEAE-cellulose, Affi-Gel blue, and immobilized wheat germ lectin. The apparent molecular weight of the XIIa inhibitor (called INH1) was 85,000, reduced, and 92,000, nonreduced, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The extinction coefficient (E0.1%(280)) of INH1 is 1.3, and the protein contains 17.7% carbohydrate. Purified antibody to INH1 raised in either rabbits or chickens formed a precipitin line of identity with purified INH1 and a component of bovine plasma, but there was no reaction with purified human inhibitors or with any component of human plasma. INH1 inhibits bovine and human XIIa, bovine and human C1-esterase, and human kallikrein, but does not inhibit bovine kallikrein, bovine trypsin, human plasmin, or human thrombin. This activity is similar to that of C1-inhibitor but different from antithrombin III, alpha 2-antiplasmin, or alpha 1-protease inhibitor. INH1 at a physiological concentration (0.47 microM) causes rapid inactivation of XIIa. The two molecules react in a 1:1 stoichiometry with a second-order rate constant of 1.23 X 10(6) M-1 min-1.     

Interaction of type 1 plasminogen activator inhibitor with the enzymes of the contact activation system

The interaction between type 1 plasminogen activator inhibitor (PAI-1), a serine protease inhibitor, and the three serine proteases generated during contact activation of plasma was studied using functional and immunologic approaches. Incubation of Factor XIIa, Factor XIa, and plasma kallikrein with either purified PAI-1 or platelet-derived PAI-1 resulted in the formation of sodium dodecyl sulfate-stable complexes as revealed by immunoblotting techniques. Functional assays indicated that Factor XIa and, to a lesser extent, Factor XIIa and plasma kallikrein neutralized the ability of purified PAI-1 to bind to immobilized tissue-type plasminogen activator (t-PA). Immunoblotting demonstrated that these enzymes also neutralized the ability of PAI-1 to form complexes with fluid-phase t-PA. Clot lysis assays employing purified proteins and 125I-fibrinogen were used to investigate the profibrinolytic effect of these contact activation enzymes. At enzyme concentrations that did not result in direct activation of plasminogen, only Factor XIa was capable of stimulating the lysis of clots supplemented with both t-PA and PAI-1. As a consequence of their interactions with PAI-1, the amidolytic activity of Factor XIIa, Factor XIa, and plasma kallikrein was neutralized by this inhibitor in a time-dependent and concentration-dependent manner. Minimum values estimated for the apparent second-order rate constant of inhibition were 1.6 x 10(4), 2.1 x 10(5), and 6.0 x 10(4) M-1 s-1 for Factor XIIa, Factor XIa, and plasma kallikrein, respectively. These data define new reactions between coagulation and fibrinolysis proteins and suggest that a major mechanism for stimulation of the intrinsic fibrinolytic pathway may involve neutralization of PAI-1 by Factor XIa.     

Monoclonal antibodies to human protein C: effects on the biological activity of activated protein C and the thrombin-catalyzed activation of protein C1

Thirteen monoclonal antibodies designated as MFC-1 to MFC-13 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C. Studies were made to determine where the antibodies bound to the molecule of protein C and whether they affected the biological actions of protein C. By using the immunoblotting technique, six of these antibodies were shown to bind to the light chain of protein C, and five to the heavy chain of protein C and also activated protein C. The remaining two antibodies bound to neither the light chain nor the heavy chain, though both antibodies bound to the intact protein C. Antibodies specific for the light chain did not bind to the gamma-carboxyglutamic acid-domain. Two of the antibodies specific for the heavy chain (MFC-13 and -1) inhibited the amidolytic activity of activated protein C. The MFC-13 also inhibited the activity of bovine activated protein C, but not that of human Factor IXa, Factor Xa, or thrombin. In addition to these two antibodies, another one for the heavy chain (MFC-10) and two antibodies for the light chain (MFC-9 and -11) inhibited the inactivation of Factor Va by human activated protein C. One of the antibodies which inhibited the enzyme activity (MFC-1) blocked the inhibition of activated protein C by protein C inhibitor. Another one for the heavy chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. Based on these results, we have established the positions of some monoclonal antibody-binding sites on the protein C molecule.     

Sulfated, low molecular weight lignins inhibit a select group of heparin-binding serine proteases

Sulfated low molecular weight lignins (LMWLs), designed as oligomeric mimetics of low molecular weight heparins (LMWHs), have been found to bind in exosite II of thrombin. To assess whether sulfated LMWLs recognize other heparin-binding proteins, we studied their effect on serine proteases of the coagulation, inflammatory and digestive systems. Using chromogenic substrate hydrolysis assay, sulfated LMWLs were found to potently inhibit coagulation factor XIa and human leukocyte elastase, moderately inhibit cathepsin G and not inhibit coagulation factors VIIa, IXa, and XIIa, plasma kallikrein, activated protein C, trypsin, and chymotrypsin. Competition studies show that UFH competes with sulfated LMWLs for binding to factors Xa and XIa. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold.     

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.     

Inhibition of proteases in coagulation, kinin-forming and complement systems by alpha2-plasmin inhibitor.

Inhibitory activities of alpha2-plasmin inhibitor against various proteases were investigated. The inhibitor promptly inhibited the esterolytic activity of alpha-chymotrypsin and progressively inhibited the esterolytic or amidolytic activities of bovine plasma kallikrein, bovine thrombin and bovine activated factor X. Heparin had no effect on the reaction of the inhibitor with thrombin or activated factor X. However, the inhibitor had no effect on the activities of human C-1-esterase, papain and snake venom kininogenase. On the basis of its rapid inhibition of kallikrein, alpha2-plasmin inhibitor is considered to exert some regulating effect on kallikrein activity in plasma.     

Mechanism-based isocoumarin inhibitors for serine proteases: use of active site structure and substrate specificity in inhibitor design

Isocoumarins are potent mechanism-based heterocyclic irreversible inhibitors for a variety of serine proteases. Most serine proteases are inhibited by the general serine protease inhibitor 3,4-dichloroisocoumarin, whereas isocoumarins containing hydrophobic 7-acylamino groups are potent inhibitors for human leukocyte elastase and those containing 7-alkylureidogroups are inhibitors for procine pancreatic elastase. Isocoumarins containing basic side chains that resemble arginine are potent inhibitors for trypsin-like enzymes. A number of 3-alkoxy-4-chloro-7-guanidinoisocoumarins are potent inhibitors of bovine thrombin, human factor Xa, human factor XIa, human factor XIIa, human plasma kallikrein, porcine pancreatic kallikrein, and bovine trypsin. Another cathionic derivative, 4-chloro-3-(2-isothiureidoethoxy) isocoumarin, is less reactive toward many of these enzymes but is an extremely potent inhibitor of human plasma kallikrein. Several guanidinoisocoumarins have been tested as anticoagulants in human plasma and are effective at prolonging the prothrombin time. The mechanism of inhibition by this class of heterocyclic inactivators involves formation of an acyl enzyme by reaction of the active site serine with the isocoumarin carbonyl group. Isocoumarins with 7-amino or 7-guanidino groups will then decompose further to quinone imine methide intermediates, which react further with an active site residue (probably His-57) to form stable inhibited enzyme derivatives. Isocoumarins should be useful in further investigations of the physiological function of serine proteases and may have future therapeutic utility for the treatment of emphysema and coagulation disorders.