Human Factor XIa Research Articles

Antithrombotic and hemostatic effects of a small molecule factor XIa inhibitor in rats

The effect of inhibiting activated blood coagulation factor XIa was determined in rat models of thrombosis and hemostasis. BMS-262084 is an irreversible and selective small molecule inhibitor of factor XIa with an IC 50 of 2.8 nM against human factor XIa. BMS-262084 doubled the activated thromboplastin time in human and rat plasma at 0.14 and 2.2 μM, respectively. Consistent with factor XIa inhibition, the prothrombin time was unaffected at up to 100 μM. BMS-262084 administered as an intravenous loading plus sustaining infusion was effective against FeCl 2-induced thrombosis in both the vena cava and carotid artery. Maximum thrombus weight reductions of 97 and 73%, respectively ( P < 0.05), were achieved at a pretreatment dose of 12 mg/kg + 12 mg/kg/h which increased the ex vivo activated thromboplastin time to 3.0 times control. This dose level also arrested growth of venous and arterial thrombi when administered after partial thrombus formation. BMS-262084 was most potent in FeCl 2-induced venous thrombosis, decreasing thrombus weight 38% ( P < 0.05) at a threshold dose of 0.2 mg/kg + 0.2 mg/kg/h. In contrast, doses of up to 24 mg/kg + 24 mg/kg/h had no effect on either tissue factor-induced venous thrombosis or the ex vivo prothrombin time. Doses of up to 24 mg/kg + 24 mg/kg/h also did not significantly prolong bleeding time provoked by either puncture of small mesenteric blood vessels, template incision of the renal cortex, or cuticle incision. These results demonstrate that pharmacologic inhibition of factor XIa achieves antithrombotic efficacy with minimal effects on provoked bleeding.

Design, Synthesis, and Biological Evaluation of Peptidomimetic Inhibitors of Factor XIa as Novel Anticoagulants

Human coagulation factor XIa (FXIa), a serine protease activated by site-specific cleavage of factor XI by thrombin, FXIIa, or autoactivation, is a critical enzyme in the amplification phase of the coagulation cascade. To investigate the potential of FXIa inhibitors as safe anticoagulants, a series of potent, selective peptidomimetic inhibitors of FXIa were designed and synthesized. Some of these inhibitors showed low nanomolar FXIa inhibitory activity with >1000-fold FXa selectivity and >100-fold thrombin selectivity. The X-ray structure of one of these inhibitors, 36, demonstrates its unique binding interactions with FXIa. Compound 32 caused a doubling of the activated partial thromboplastin time in human plasma at 2.4 microM and was efficacious in a rat model of venous thrombosis. These data suggest that factor XIa plays a significant role in venous thrombosis and may be a suitable target for the development of antithrombotic therapy.

Coumarin and Isocoumarin as Serine Protease Inhibitors

Serine proteases are attractive targets for the design of enzyme inhibitors since they are involved in the etiology of several diseases. Within the class of serine proteases, HLE is one of the most destructive enzymes in the body. It is implicated in the promotion or exacerbation of a number of diseases including pancreatitis, acute respiratory syndrome, rheumatoid arthritis, atherosclerosis, pulmonary emphysema, and cystic fibrosis. Thrombin, a trypsin-like serine protease, plays a dual role in thrombogenesis, including fibrin formation and platelet activation. As a result, thrombin constitutes one of the most widely studied targets for antithrombotic strategy. Numerous inhibitors of serine proteases have been reported during the past three decades. Among them, coumarin-type molecules displayed a high inhibitory potency towards various serine proteases. At that time, halomethyl dihydrocoumarins have been shown to behave as the first general suicide inhibitors of serine protease. These molecules inhibit several proteases such as human leucocyte elastase, porcine pancreatic elastase, thrombin, urokinase and human plasmin. Isocoumarins are very effective as mechanism-based inhibitors of serine proteases. Pharmacomodulation on the 3-alkoxy-4-chloroisocoumarins and the 3-alkoxy-7-amino-4-chloroisocoumarins led to strong inhibitors of numerous serine proteases such as HLE, human factor XIa and XIIa, thrombin, urokinase and kallikrein. Recently, a series of coumarins characterised by an alkyl, aryl ester, amide, thioester or ketone in the position 3 and an electrophilic chloromethyl moiety in the position 6 have been developed. These compounds were found to be high inhibitors of alpha-chymotrypin, HLE and human thrombin.

Characterization of Placental Bikunin, a Novel Human Serine Protease Inhibitor

We reported previously the cloning of a novel human serine protease inhibitor containing two Kunitz-like domains, designated as placental bikunin, and the subsequent purification of a natural counterpart from human placental tissue (Marlor, C. W., Delaria, K. A., Davis, G., Muller, D. K., Greve, J. M., and Tamburini, P. P. (1997) J. Biol. Chem. 272, 12202-12208). In this report, the 170 residue extracellular domain of placental bikunin (placental bikunin(1-170)) was expressed in baculovirus-infected Sf9 cells using its putative signal peptide. The resulting 21.3-kDa protein accumulated in the medium with the signal peptide removed and could be highly purified by sequential kallikrein-Sepharose and C18 reverse-phase chromatography. To provide insights as to the potential in vivo functions of this protein, we performed an extensive investigation of the inhibitory properties of recombinant placental bikunin(1-170) and both of its synthetically prepared Kunitz domains. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Placental bikunin(1-170) formed inhibitor-protease complexes with a 1:2 stoichiometry and strongly inhibited human plasmin (Ki = 0.1 nM), human tissue kallikrein (Ki = 0.1 nM), human plasma kallikrein (Ki = 0.3 nM) and human factor XIa (Ki = 6 nM). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (Ki = 1.6 microM), factor IXa (Ki = 206 nM), factor Xa (Ki = 364 nM), and factor XIIa (Ki = 430 nM). This specificity profile was to a large extent mimicked, albeit with reduced potency, by the individual Kunitz domains. As predicted from this in vitro specificity profile, recombinant placental bikunin(1-170) prolonged the clotting time in an activated partial thromboplastin time assay.

Clearance of human factor XIa–inhibitor complexes in rats

The serpins C1 esterase inhibitor (C1Inh), antithrombin (AT), alpha 1-antitrypsin (alpha 1AT) and alpha 2-antiplasmin (alpha 2AP) are known inhibitors of coagulation factor XIa (FXIa). Although initial studies suggested alpha 1AT to be the main inhibitor of FXIa, we recently demonstrated C1Inh to be a predominant inhibitor of FXIa in vitro in human plasma. The present study was performed to investigate the plasma elimination kinetics of preformed human FXIa-FXIa inhibitor complexes injected in rats. The amounts of complexes remaining in circulation were measured using enzyme-linked immunosorbent assays. The plasma half-life time of clearance (t1/2) was 98 min for FXIa-alpha 1AT complexes, whereas it was considerably shorter, i.e. 19, 18 and 15 min for FXIa-C1Inh, FXIa-alpha 2AP and FXIa-AT complexes, respectively. Thus, due to this different plasma t1/2, preferentially FXIa-alpha 1AT complexes may be detected in clinical samples. Furthermore, measuring FXIa-FXIa inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of FXIa in vivo.

Purification, characterization, and localization of follipsin, a novel serine proteinase from the fluid of porcine ovarian follicles.

Follipsin, an enzyme that accumulates in the follicular fluid of porcine ovaries during follicular maturation, was purified to apparent homogeneity. The purified enzyme consists of two different polypeptide chains having M(r) = 45,000 and 32,000 each, associated covalently. The enzyme activity was strongly inhibited by diisopropyl fluorophosphate, benzamidine, leupeptin, and antipain, indicating that follipsin is a serine proteinase. Using synthetic peptide substrates containing 4-methylcoumaryl-7-amide, follipsin was shown to preferentially hydrolyze Arg-X bonds but not Lys-X bonds. The NH2-terminal amino acid sequences of the 45- and 32-kDa polypeptides were highly homologous with those of the heavy and light chains, respectively, of human plasma kallikrein and human factor XIa. Immunological analyses and substrate specificity studies, together with other existing evidence, indicated that follipsin is distinct from kallikrein and factor XIa, thus being a novel type of serine proteinase. Follipsin is immunohistochemically localized in follicular fluid as well as in stroma cells of porcine ovaries. The results strongly suggest that follipsin originates from interstitial cells of the ovarian stroma.

A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning.

A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.

The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc-dependent binding to an anionic surface.

The histidine-glycine-rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin-forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 microM and 27 microM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M(r) was 7.3 kDa (range 6.2-8.1 kDa). Based on N-terminal sequencing, this polypeptide (1(2)), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 microM. The synthetic peptide HGLGHGH representing the N-terminal of the 1(2)) fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459-475) included within the 1(2) fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 microM in the absence and presence of added Zn2+ (30 microM). The specific binding of 125I-HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 microM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I-HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 microM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the 1(2) fragment was found to inhibit 50% of the 125I-HK binding to kaolin at a concentration of 380 microM. These results suggest that residues contained within the 1(2) fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.

Conversion of the Alzheimer's beta-amyloid precursor protein (APP) Kunitz domain into a potent human neutrophil elastase inhibitor.

Site-specific mutagenesis techniques have been used to construct active site variants of the Kunitz-type protease inhibitor domain present in the Alzheimer's beta-amyloid precursor protein (APP-KD). Striking alteration of its protease inhibitory properties were obtained when the putative P1 residue, arginine, was replaced with the small hydrophobic residue valine. The altered protein was no longer inhibitory toward bovine pancreatic trypsin, human Factor XIa, mouse epidermal growth factor-binding protein, or bovine chymotrypsin, all of which are strongly inhibited by the unaltered APP-KD (Sinha, S., Dovey, H. F., Seubert, P., Ward, P. J., Blacher, R. W., Blaber, M., Bradshaw, R. A., Arici, M., Mobley, W. C., and Lieberburg, I. (1990) J. Biol. Chem. 265, 8983-8985). Instead, the P1-Val-APP-KD was a potent inhibitor of human neutrophil elastase, with a Ki = 0.8 nM, as estimated by the inhibition of the activity of human neutrophil elastase measured using a chromogenic substrate. It also inhibited the degradation of insoluble elastin by the enzyme virtually stoichiometrically. Replacement of the P1' (Ala) and P2' (Met) residues of P1-Val-MKD with the corresponding residues (Ser, Ile) from alpha 1-proteinase inhibitor resulted in an inactive protein, underscoring the mechanistic differences between the serpins from the Kunitz-type protease inhibitor family. These results confirm the importance of the P1 arginine residue of APP-KD in determining inhibitory specificity, and are also the first time that a single amino acid replacement has been shown to generate a specific potent human neutrophil elastase inhibitor from a human KD sequence.

Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization.

Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.

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.

Expression and characterization of human factor IX and factor IX-factor X chimeras in mouse C127 cells.

Human blood clotting factor IX, and two chimeric molecules of factor IX, in which the first epidermal growth factor-like domain or both epidermal growth factor-like domains have been replaced by that of human factor X, have been expressed in mouse C127 cells. The recombinants have been purified using a metal ion-dependent monoclonal antibody specific for residues 1-42 of human factor IX. All recombinant molecules are activated normally by human factor XIa in the presence of calcium ion. Activation of the factor IX recombinants by factor VIIa-tissue factor appears to be normal for the epidermal growth factor-1 exchange but considerably reduced for the construction containing both epidermal growth factor-like domains of factor X. The analysis of gamma-carboxyglutamic acid residues reveals that all of the purified recombinants are almost fully carboxylated. The extent of aspartic acid hydroxylation at residue 64 is 60% for all recombinants. The chimeric molecule with both epidermal growth factor-like domains from factor X has about 4% normal activity in the activated partial thromboplastin time assay. In contrast, the construct containing the first epidermal growth factor-like domain of factor X shows essentially normal clotting activity. Thus, it is unlikely that this domain is involved in a unique interaction with factor VIII.

Purification and characterization of plasma protein C inhibitor

Plasma protein C inhibitor (PCI) was purified to homogeneity (>95%) with good recovery (>25%) and reproducibility, and the inhibition of a number of blood clotting and fibrinolytic enzymes by purified PCI was studied. PCI inhibited activated protein C (APC), two-chain urokinase (2c-uPA), two-chain tissue plasminogen activator (2c-tPA), thrombin, factor Xa, plasma kallikrein and factor XIa, and this inhibition was accelerated by heparin. The inhibition of each enzyme was accompanied by formation of enzyme inhibitor complexes and by degradation of the inhibitor to lower molecular weight derivatives. Plasma kallikrein and factor XIa cleaved PCI of native Mr-57,000 into two products with Mr-54,000 and 52,000 whereas the other enzymes converted the PCI to a product with Mr=54,000. PCI did not detectably inhibit α-factor XIIa or plasmin. Kinetic studies using PCI yielded the following second-order rate constants for inhibition of human APC, 2c-uPA, 2c-tPA, thrombin, factor Xa, kallikrein and factor XIa respectively: 0.65 × 10 4, 0.22 × 10 4, 0.08 × 10 4, 0.61 × 10 4, 2.01 × 10 4, 6.50 × 10 4 and 9.03 × 10 4 M −1s −1 in the absence of heparin and 1.58 × 10 6, 0.43 × 10 6, 0.03 × 10 6, 0.52 × 10 6, 0.09 × 10 6, 0.18 × 10 6 and 0.74 × 10 6 M −1s −1 in the presence of optimal concentrations of heparin. The rate constants for the inhibition of factor XIa and 2c-uPA by PCI suggest a possible role of PCI in the physiologic regulation of these enzymes. The second order rate constants for inhibition of bovine APC and Gla-domainless bovine APC by human PCI were 0.61 × 10 4 and 0.26 × 10 4 M −1s −1 in the absence of heparin and 0.54 × 10 6 and 0.71 × 10 6 M −1s −1 in the presence of heparin, respectively. Calcium ions (0.05 to 4 mM) did not affect these rate constants. The results obtained with normal and Gla-domainless APC indicate that the Gla domain of APC is not required for inactivation by PCI and is not essential for the heparin stimulation of this reaction.

Factors influencing the acceleration of human factor XIa inactivation by antithrombin III

Controversy exists in the literature concerning the potentiating effect of heparin on the inactivation rate of factor XIa by antithrombin III (AT III) in both purified systems and in plasma. We have analyzed the factors that could influence this reaction and found that ionic strength of the medium, as well as the type and concentration of the heparin preparations accounted for the major discrepancies in the literature. At I = 0.43 N, a preparation of bovine lung heparin at 1 U/mL did not augment the inactivation rate of factor XIa by inhibitors in plasma or by purified AT III. However, when ionic strength was decreased, a progressive increase in the potentiating effect was observed, reaching 6.5-fold at I = 0.15 N. At saturating concentrations of heparin, which results in the formation of 100% AT III-heparin complex, (greater than ten-fold molar excess over AT III) in purified systems, all heparin preparations (porcine, bovine, low molecular weight [LMW], and high affinity) yielded an approximately 30-fold augmentation of the factor XIa inactivation rate. However, when heparin was less than saturating, we observed that various heparin preparations affected the AT III-induced inactivation of factor XIa to different degrees even though they exhibited the same inhibitory activity (1 U/mL) against thrombin. This variation resulted from differences in the number of AT III binding sites in each heparin preparation, despite a similar Kd for each. Addition of high molecular weight kininogen (HK) to AT III-heparin complexes did not enhance their ability to inhibit factor XIa, and high concentrations of HK decreased the inactivation rate. A high therapeutic dose of heparin only permits the formation of 2.5% to 16.5% of the AT III-heparin complexes that can be achieved at saturation. We observed that 1 U/mL heparin (bovine lung heparin) (high therapeutic concentration) in virtually undiluted plasma only accelerated the inactivation rate of factor XIa (in the absence of other active enzymes) less than two-fold. These new observations further support our previous conclusion that therapeutic levels of heparin have little to no influence on the inactivation rate of factor XIa in plasma.

Factors Influencing the Acceleration of Human Factor XIa Inactivation by Antithrombin III

Controversy exists in the literature concerning the potentiating effect of heparin on the inactivation rate of factor XIa by antithrombin III (AT III) in both purified systems and in plasma. We have analyzed the factors that could influence this reaction and found that ionic strength of the medium, as well as the type and concentration of the heparin preparations accounted for the major discrepancies in the literature. At I = 0.43 N, a preparation of bovine lung heparin at 1 U/mL did not augment the inactivation rate of factor XIa by inhibitors in plasma or by purified AT III. However, when ionic strength was decreased, a progressive increase in the potentiating effect was observed, reaching 6.5-fold at I = 0.15 N. At saturating concentrations of heparin, which results in the formation of 100% AT III-heparin complex, (greater than ten-fold molar excess over AT III) in purified systems, all heparin preparations (porcine, bovine, low molecular weight [LMW], and high affinity) yielded an approximately 30-fold augmentation of the factor XIa inactivation rate. However, when heparin was less than saturating, we observed that various heparin preparations affected the AT III-induced inactivation of factor XIa to different degrees even though they exhibited the same inhibitory activity (1 U/mL) against thrombin. This variation resulted from differences in the number of AT III binding sites in each heparin preparation, despite a similar Kd for each. Addition of high molecular weight kininogen (HK) to AT III-heparin complexes did not enhance their ability to inhibit factor XIa, and high concentrations of HK decreased the inactivation rate. A high therapeutic dose of heparin only permits the formation of 2.5% to 16.5% of the AT III-heparin complexes that can be achieved at saturation. We observed that 1 U/mL heparin (bovine lung heparin) (high therapeutic concentration) in virtually undiluted plasma only accelerated the inactivation rate of factor XIa (in the absence of other active enzymes) less than two-fold. These new observations further support our previous conclusion that therapeutic levels of heparin have little to no influence on the inactivation rate of factor XIa 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.

The heparin-catalysed inhibition of human factor XIa by antithrombin III is dependent on the heparin type.

The effect of various well-characterized heparin preparations on the inactivation of human Factor XIa by human antithrombin III was studied. The heparin preparations used were unfractionated heparin and four heparin fractions obtained after anion-exchange chromatography. Inactivation of Factor XIa was monitored with S2366 as chromogenic substrate and followed pseudo-first-order reaction kinetics under all reaction conditions tested. Enhancement of the rate of inhibition of Factor XIa in the presence of unfractionated heparin correlated to the binding of antithrombin III to heparin. From the kinetic data a binding constant of 0.1 microM was inferred. The maximum rate enhancement, achieved at saturating heparin concentrations, was 30-fold. The rate enhancement achieved in the presence of each of the heparin fractions could also be correlated to the binding of antithrombin III to the heparin. The binding constant inferred from the kinetic data varied from 0.10 to 0.28 microM and the number of binding sites for antithrombin III varied from 0.06 to 0.74 site per heparin molecule. The maximum rate enhancements, achieved at saturating heparin concentrations, were strongly dependent on the type of heparin used and varied from 7-fold for fraction A to 41-fold for fraction D. Therefore, although the stimulation of Factor XIa inactivation by antithrombin III could be quantitatively correlated to the binding of antithrombin III to heparin, the heparin-catalysed inhibition of Factor XIa is dependent not only upon the degree of binding of antithrombin III to heparin but also upon the type of heparin to which antithrombin III is bound.

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)

Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants.

Trypsin, porcine pancreatic kallikrein, and several blood coagulation enzymes, including bovine thrombin, bovine factor Xa, human factor Xa, human plasma factor XIa, human plasma factor XIIa, and human plasma kallikrein, were inactivated by a number of substituted isocoumarins containing basic functional groups (aminoalkoxy, guanidino, and isothiureidoalkoxy). 3-Alkoxy-4-chloro-7-guanidinoisocoumarins were found to be the most potent inhibitors for the coagulation enzymes tested with kobsd/[I] values in the range of 10(3)-10(5) M-1 s-1. 4-Chloro-3-isothiureidoalkoxyisocoumarins show high inhibitory potency toward porcine pancreatic kallikrein, human plasma kallikrein, human factor XIa, human factor XIIa, and trypsin with kobsd/[I] values of the order of 10(4)-10(5) M-1 s-1. The inhibition of these serine proteases by the substituted isocoumarins are time dependent, and the inactivation of trypsin by 3-alkoxy-4-chloro-7-guanidinoisocoumarins and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin occured concurrently with the loss of the isocoumarin absorbance. The complex formed from inactivation of trypsin by these two types of inhibitors was very stable and regained less than 4% activity in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5) after 1 day at 25 degrees C and regained 8-45% activity upon addition of buffered 0.29 M hydroxylamine. Trypsin inactivated by other inhibitors regained full activity upon standing or addition of hydroxylamine. Thrombin inactivated by 3-alkoxy-4-chloro-7-guanidinoisocoumarins was also quite stable and only regained 9-15% activity under similar conditions. These results are consistent with a proposed mechanism, where serine proteases inactivated by aminoalkoxyisocoumarins or isothiureidoalkoxyisocoumarins form acyl enzymes that will deacylate upon standing or addition of hydroxylamine. However, the acyl enzymes formed from 3-alkoxy-4-chloro-7-guanidinoisocoumarins or 7-amino-4-chloro-3-(3-isothiureidopropoxy)-isocoumarin will decompose further, probably through a quinone imine methide, to give an irreversibly inactivated enzyme by reaction with an active-site nucleophile such as His-57. The quinone imine methide intermediate may also react with a solvent nucleophile to give an acyl enzyme that can be reactivated by hydroxylamine. The inhibitors 4-chloro-7-guanidino-3-methoxyisocoumarin and 4-chloro-3-ethoxy-7-guanidinoisocoumarin have been tested as anticoagulants in human plasma and were effective at prolonging the prothrombin time. However, they are unstable in plasma (t1/2 = 4-8 min), and their in vivo utility may be limited.

Inhibition of human blood coagulation factor XIa by C-1 inhibitor.

The inactivation of activated factor XI (factor XIa) and of its isolated light chain by C-1 inhibitor was studied. Irreversible inhibition was observed in a reaction in which no reversible enzyme-inhibitor complex was formed. The second-order rate constants for the inactivation of factor XIa or its light chain by C-1 inhibitor were 2.3 X 10(3) and 2.7 X 10(3) M-1 s-1, respectively. High molecular weight kininogen did not affect the rate of inactivation. The nature of the complexes formed between factor XIa or its light chain and C-1 inhibitor was studied by using sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis. Under nonreducing conditions, two factor XIa-C-1 inhibitor complexes were observed with apparent molecular weights of 230,000 and 300,000. Reduction of these complexes resulted in the formation of a single band with a molecular weight of 130,000. This band is also formed in the reaction of the isolated light chain of factor XIa with C-1 inhibitor. These results demonstrate that two C-1 inhibitor molecules can become bound to the light chains of a factor XIa molecule. In addition, the mechanism of interaction of factor XIa or its isolated light chain with C-1 inhibitor appears identical, and the rate of inactivation of the enzyme by C-1 inhibitor is very similar. Neither the heavy chain of factor XIa nor high molecular weight kininogen is significantly involved in the inactivation of factor XIa by C-1 inhibitor.