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PubMed1984

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.

Animals↗
PubMed1994

Mechanism-based isocoumarin inhibitors for blood coagulation serine proteases. Effect of the 7-substituent in 7-amino-4-chloro-3-(isothioureidoalkoxy)isocoumarins on inhibitory and anticoagulant potency.

A series of 7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-CiTPrOIC) derivatives with various substituents at the 7- and 3-positions have been synthesized as inhibitors of several blood coagulation enzymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irreversible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoethoxy group at the 3-position and a large hydrophobic group at the 7-position are better inhibitors for thrombin, factor VIIa, factor Xa, factor XIa, factor IIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-CiTEtOIC (14), (S)-Ph(CH3)CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHCONH-CiTEtOIC (26) inhibit thrombin quite potently and have kobs/[I] values of (1-4) x 10(4) M-1 s-1. Modeled structures of several isocoumarins noncovalently complexed with human alpha-thrombin suggest that H-bonding between the 7-substituent and the Lys-60F NH3+ relates to the inhibitory potency. Thrombin inhibited by 14, 25, or 26 is quite stable, and only 4-16% of enzymatic activity is regained after incubation for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 65% of enzyme activity, respectively, is regained with the addition of 0.38 M hydroxylamine. With normal citrated pig or human plasma, these isocoumarin derivatives prolong the prothrombin time ca. 1.3-3.1-fold and also prolong the activated partial thromboplastin time more than 3-7-fold at 32 microM. Thus, these compounds are effective anticoagulants in vitro and may be useful in vivo.

Amino Acid Sequence↗
PubMed2005

Activation of hepatocyte growth factor by urokinase-type plasminogen activator is ionic strength-dependent.

The hepatocyte growth factor (HGF) is a multifunctional cytokine that is produced as latent scHGF (single chain HGF). Various proteases reportedly cleave scHGF to generate the active two-chain form (HGF), including u-PA (urokinase-type plasminogen activator), t-PA (tissue-type plasminogen activator), kallikrein, Factor XIa, Factor XIIa, HGF activator and matriptase. Considerable evidence indicates that, in vivo, u-PA activates scHGF in the liver; however, the in vivo results have not been uniformly supported by in vitro experiments. We now report that cleavage of scHGF by high-molecular-mass u-PA (abbreviated u-PA throughout) is sensitive to ionic strength. scHGF cleavage by u-PA was accelerated as the ionic strength was decreased. This result was equivalent irrespective of whether the predominant anion was chloride or acetate. Lmw-u-PA (low-molecular-mass u-PA) was ineffective at cleaving scHGF, regardless of ionic strength. Although scHGF shares homology with plasminogen, EACA (-amino-caproic acid) did not regulate u-PA-mediated scHGF cleavage. Soluble HGF receptor (MET) and soluble u-PAR (u-PA receptor) inhibited the scHGF cleavage. These results support a model in which the ability of u-PA to activate scHGF in vivo may be highly dependent on local conditions within the extracellular space.

Hepatocyte Growth Factor↗
PubMed1994

Inhibition of serine proteases of the blood coagulation system by squash family protease inhibitors.

Squash family inhibitors are the smallest protein serine protease inhibitors, being composed of approximately 30 amino acid residues. We isolated 8 squash family inhibitors from the seeds of bitter gourd, squash, gourd and luffa and examined their effect on serine proteases of the blood coagulation system. Five of them prolonged the activated partial thromboplastin time of human plasma to various extents, but three did not. Only Momordica charantia (bitter gourd) trypsin inhibitor-II prolonged the prothrombin time of human plasma. All inhibitors inhibited the amidolytic activities of factor XIIa, plasma kallikrein, factor Xa, but did not inhibit significantly those of factor XIa, factor IXa, factor VIIa, and thrombin. Ki values for factor XIIa, plasma kallikrein, and factor Xa were in the order of 10(-6)-10(-9), 10(-4)-10(-5), and 10(-4)-10(-6)M, respectively. The prolongation of the activated partial thromboplastin time by inhibitors appeared to correspond to their inhibitory potencies for factor XIIa. Momordica charantia trypsin inhibitor-II, which has the strongest inhibitory potency toward the amidolytic activity of factor Xa, with a Ki value 10-100 times smaller than those of other inhibitors, inhibited the activation of factor X by factor VIIa-tissue factor complex or factor IXa, while others did not.

Amino Acid Sequence↗
PubMed1975

[Activation of the blood coagulation system during gram-negative infections and endotoxemias].

Blood coagulation may be activated by the extrinsic or intrinsic pathways. The extrinsic clotting system is put into action by tissue thromboplastin, originating from injured tissue cells, but also from damaged leukocytes and erythrocytes. Tissue thromboplastin is a phospholipoprotein with an enzymatic component, capable of converting the clotting factor VII to its activated form, factor VIIa, which in turn activates factor X. The factor Xa-complex (containing also factor Va, phospholipid, and calcium) is the prothrombinconverting principle. The intrinsic clotting system is based on factors which are contained in the circulating blood. Its activation requires the availability of phospholipid and of activated factor XII (factor XIIa), or factor XIa. Factor XII is activated by collagen, i.e., whenever the vascular endothelium is injured, and to a lesser extent also by "activated" blood platelets. Platelets in turn are activated primarily by thrombin, collagen, and, in a self-perpetuating process, since all these materials are released from activated platelets, also by adenosine-5-diphosphate, adrenaline, and serotonin. The activation of platelets leads to a variety of morphological and biochemical alterations, culminating in their aggregation and in the selective release from storage organelles of different substances, among them those mentioned above. Of particular importance is the fact that in the course of platelet alterations, procoagulant phospholipid also becomes available on the platelet surface. The significance of the activation of the intrinsic system is seen in the possibility of the initiation of a self-sustained process which, after a primary event, e.g. vascular or cellular injury, will continue to convert prothrombin into thrombin. The effects of endotoxin on the blood clotting system show striking species differences. In the rabbit, endotoxin, with the involvement of factors of the complement system, will directly act upon blood platelets and thus initiate intravascular, intrinsic coagulation. In man, endotoxin remains without a direct effect on platelets and alternative possibilities of initiating thrombin formation must be considered. One possibility is extrinsic activation via tissue thromboplastin from injured leukocytes. Another pathway, which is supported by several experimental findings, starts out with endotoxin-mediated endothelial damage. Endothelial cells are in fact severely affected by endotoxin and may even be removed from the vascular wall, thus making accessible the subendothelial activator of factor XII. Thrombin in turn affects the vascular endothelium: therefore, one initiated, the process of intravascular activation of coagulation will perpetuate, this the more as platelets in turn will be stimulated into activity. The possible intervention of other vasoactive factors must also be considered...

Animals↗
PubMed1989

Amidolytic detection of prothrombin activation products after SDS-gel electrophoresis.

In this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation. The detection limit of the so-called "amidoblot" is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting. It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

Blood Coagulation Tests↗
PubMed1982

Role of charged groups in factor XI/XIa activity.

To elucidate the role of charged groups in expression of factor XI coagulant activity, the charged groups of purified human blood coagulation factor XI/XIa containing 125I-XI/XIa were derivatized: free amino groups by succinylation, guanido groups of arginine by reaction with phenylglyoxal hydrate, and free carboxyl groups by reaction with ethylenediamine. The modified proteins were tested for: 1) ability to adsorb to glass, 2) ability to be cleaved by trypsin or factor XII-high molecular weight kininogen, 3) coagulant activity. The amino group-modified factor XI had a significantly decreased ability to bind to glass; modification of arginine or carboxyl groups did not affect adsorption. Trypsin cleaved factor XI with modified free amino, guanido, or carboxyl groups. Factor XII-high molecular weight kininogen could cleave only the arginine-modified factor XI. Amino group-modified factor XI and carboxyl group-modified factor XI lost all their factor XI assay activity, whereas arginine-modified factor XI retained 50% of the original activity. Amino group-modified factor XI could not be activated by trypsin, but arginine-modified and carboxyl group-modified factor XI could be activated by trypsin to 50% of the original activity. Succinylation of the amino groups of factor XIa destroyed all its factor XIa activity. Arginine-modified and carboxyl group-modified factor XIa retained 50% of their factor XIa activity. We conclude that epsilon-amino groups are essential for adsorption; activation by factor XII-high molecular weight kininogen requires free amino and carboxyl but not guanido groups; free amino, carboxyl, and guanido groups in factor XIa all appear to be critical for interaction of factor XIa with factor IX.

Blood Coagulation↗
PubMed1994

Characterization of a factor IX variant with a glycine207 to glutamic acid mutation.

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.

Benzamidines↗
PubMed1994

In vitro mutagenesis study of two critical glutamic acids in the calcium binding loop of the factor IX heavy chain.

We investigated the structural and functional significance of calcium binding in the factor IXa heavy chain by introducing point mutations into the probable calcium binding site (residues 235 and 245). According to factor IXa computer modelling based on trypsin x-ray structure, side chains of two glutamic acid residues, 235 and 245, together with backbone carbonyl groups of residues 237 and 240, bind a calcium ion. Factor IX clotting activity decreased approximately 25 percent on substitution of glutamic acid 235 with lysine. Activity decreased more than 90 percent on substitution of glutamic acid 245 with lysine. Activity also decreased more than 90 percent on substitution of both glutamic acids by lysines. Factor XIa cleavage of factor IXGlu235Lys and factor IXGlu245Lys appeared normal by polyacrylamide gel analysis. (Factor IXGlu235Lys: Factor IX with Lysine substituted for Glutamic acid at residue 235. Factor IXGlu245Lys: Factor IX with Lysine substituted for Glutamic acid at residue 245. Factor IXGlu235&245Lys: Factor IX with Lysine substituted for Glutamic acid at residues 235 and 245.) Activated factor IXGlu235Lys bound the fluorescent active site probe, p-aminobenzamidine, normally, while factor XIa cleaved factor IXGlu245Lys and factor IXGlu235&245Lys failed to bind p-aminobenzamidine. Plasma purified factor IX titrated with terbium showed an increase in luminescence; however, factor IXGlu235Lys and factor IXGlu245Lys had no effect on terbium luminescence. Radioimmunoassays indicate that in calcium's absence, factor IXGlu245Lys adopts a conformation similar to normal factor IX in the presence of calcium. By contrast, factor IXGlu245Lys's conformation in the presence of calcium is similar to that of plasma purified factor IX in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens↗
PubMed1989

Functional domains in the heavy-chain region of factor XI: a high molecular weight kininogen-binding site and a substrate-binding site for factor IX.

To probe the molecular interactions of factor XI we have prepared two monoclonal antibodies (MoAbs; 5F7 and 3C1), each of which binds the heavy chain of reduced and alkylated factor XIa. Competitive solid phase radioimmunoassay (RIA) binding studies revealed that 5F7 and 3C1 are directed against different epitopes within factor XI. One antibody (5F7) blocked the surface-mediated proteolytic activation of factor XI and its binding to HMW kininogen, but had no effect on factor-XIa-catalyzed factor IX activation. The other antibody (3C1) is a competitive inhibitor of factor-IX activation by factor XIa, but blocked factor-XI binding to HMW kininogen only at 1,000-fold higher concentration than 5F7. Moreover, HMW kininogen had no effect on the kinetics of factor-XIa-catalyzed factor-IX activation. Furthermore, factor XI CNBr peptide fragments that bind to the 5F7 and 3C1 antibodies were isolated. The peptides that bound to the 5F7 antibody blocked the binding of HMW kininogen to factor XI but did not inhibit factor-XIa-catalyzed factor-IX activation. However, the peptides isolated by the 3C1 antibody inhibited factor-XIa-catalyzed factor-IX activation and had no effect on factor-XI binding to HMW kininogen. Our results indicate that distinct functional domains within the heavy chain region of factor XI are important for the binding of factor XI to HMW kininogen and for activation of factor IX by factor XIa.

Antibodies, Monoclonal↗
PubMed1990

Platelet coagulation factor XIa-inhibitor, a form of Alzheimer amyloid precursor protein.

An inhibitor of coagulation factor XIa was purified from serum-free conditioned medium of HepG2 liver cells. Platelets stimulated with thrombin or calcium ionophore (A23187) secrete a protein functionally and immunologically identical to the inhibitor, implying a role for this inhibitor in hemostasis. Analysis of the amino-terminal amino acid sequence and immunologic reactivity showed the inhibitor to be a truncated form of the Alzheimer's amyloid precursor protein that contains a Kunitz-type serine protease inhibitor domain and at least a portion of the amyloid beta protein. It inhibits factor XIa and trypsin with a Ki of 450 +/- 50 pM and 20 +/- 10 pM, respectively. Heparin (1 unit/ml) did not significantly effect inhibition of trypsin, but inhibition of XIa was 15 times greater (Ki = 25 +/- 15 pM) in the presence of heparin.

Amino Acid Sequence↗
PubMed1988

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.

Animals↗
PubMed1996

Identification of a factor IX binding site on the third apple domain of activated factor XI.

Activated factor XI (factor XIa) participates in blood coagulation by activating factor IX. Previous work has demonstrated that a binding site for factor IX is present on the noncatalytic heavy chain of factor XIa (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). Recombinant factor XI proteins were expressed in which each of the four apple domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding domain from the homologous but functionally distinct protease prekallikrein (PK). To identify the site of factor IX binding, the chimeric proteins were activated with factor XIIa and tested for their capacity to activate factor IX in plasma coagulation and purified protein assays. The chimera with the substitution in the third apple domain (factor XI/PKA3) had <1% of the coagulant activity of wild type factor XIa in a plasma coagulation assay, whereas the chimeras with substitutions in A1, A2, and A4 demonstrated significant activity (68-140% of wild type activity). The Km for activation of factor IX by factor XIa/PKA3 (12. 7 microM) is more than 30-fold higher than the Km for activation by wild type factor XIa or the other factor XI/PK chimeras (0.11-0.37 microM). Two monoclonal antibodies (2A12 and 11AE) that recognize epitopes on the factor XI A3 domain were potent inhibitors of factor IX activation by factor XIa, whereas antibodies against the A2 (1A6) and A4 (3G4) domains were poor inhibitors. The data indicate that a binding site for factor IX is present on the third apple domain of factor XIa.

Antibodies, Monoclonal↗
PubMed1992

Assembly and expression of an intrinsic factor IX activator complex on the surface of cultured human endothelial cells.

Endothelial cells expose specific receptors for blood clotting factors and, upon perturbation, can initiate and propagate the reactions of the extrinsic pathway of blood coagulation leading to fibrin formation on the cell surface. The existence of an intrinsic mechanism of Factor IX activation on cultured human umbilical vein cells (HUVECs) was investigated by studies of the interaction between HUVECs and two proteins of the contact activation system, the cofactor high molecular weight kininogen (H-kininogen) and the zymogen Factor XI. In the presence of zinc ions (10-300 microM), 125I-labeled H-kininogen bound to HUVECs in a time-dependent, reversible, and saturable manner, with calcium ions exerting an inhibitory effect on the zinc-dependent binding. Analysis of the binding data by the LIGAND computer program indicated that HUVECs, in the presence of 2 mM CaCl2 and 100 microM ZnCl2 at 37 degrees C, bound 1.14 x 10(7) H-kininogen molecules per cell with an apparent dissociation constant of 55 nM. HUVEC-bound H-kininogen functions as the cell surface receptor for both 125I-labeled Factor XI and 125I-labeled Factor XIa, since HUVECs cultured in contact factor-depleted serum do not detectably bind either the zymogen or the enzyme in the absence of H-kininogen and zinc ions. In the presence of saturating concentrations of H-kininogen, 2 mM CaCl2 and 100 microM ZnCl2, the binding of 125I-labeled Factor XI and Factor XIa to HUVECs was time-dependent, reversible, and saturable, with apparent dissociation constants of 4.5 and 1.5 nM, respectively. HUVEC-bound complexes of H-kininogen and Factor XI generated Factor XIa activity only after the addition of purified Factor XIIa, and cell-bound Factor XIa in turn activated Factor IX, as documented by a 3H-labeled activation peptide release assay for 3H-Factor IX activation. The results indicate that cultured HUVECs provide a surface for the assembly and expression of an intrinsic Factor IX activator complex that may participate in the initiation of blood coagulation at sites of vascular injury.

Binding, Competitive↗
PubMed2000

Intrinsic coagulation pathway: an activation threshold.

The activation threshold for the intrinsic pathway of coagulation was experimentally determined in stirred recalcified plasma from the dependence of plasma clotting time after activation by celite. Concentration of factor XIa was used as a measure of activation. At free calcium concentrations below 0.45 mM, plasma clotting time depended nonlinearly on the factor XIa concentration: with decreasing concentration of factor XIa (or celite), the clotting time dramatically increased until no coagulation was observed at concentrations of factor XIa below the threshold. As the free calcium concentration increased, the threshold concentration of factor XIa sharply decreased, from 30 pM at 0.35 mM free calcium to less than 3 pM at 0.45 mM. In the range of free calcium concentrations from 0.45 mM to physiologic ones, plasma coagulated even in the absence of celite in plastic cuvettes. This fact and extremely low threshold concentrations of factor XI (on the order of 0.5 pM) preclude determining the factor XI threshold at physiologic free calcium. As factor XIa is localized to the activating surfaces, observing the local surface concentrations of factor XIa and the dynamics of fibrin formation in systems without stirring may solve the problem.

Adult↗
PubMed1984

Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates.

A series of 14 tripeptide 4-nitroanilide substrates of the type Z-AA-Gly-Arg-NA and Z-AA-Phe-Arg-NA where AA = Ala, Asn, Glu, Lys, Phe, Pro, or Ser were used to map the S3 subsite of several serine proteases involved in blood coagulation. The enzymes studied included bovine thrombin, factor IXa, factor Xa, factor XIa, human beta-factor XIIa (factor XIIa fragment), and activated bovine and human protein C. Kinetic constants (kcat, KM, and kcat/KM) for the enzymatic hydrolysis of the substrates by each enzyme were determined and used to compare the relative reactivities of the individual enzymes. Most of the enzymes reacted with all the substrates, although a few showed considerable specificity. Human beta-factor XIIa showed the highest reactivity of all the coagulation proteases studied and was also very substrate specific (kcat/KM ranged over 470-fold). The best substrate was Z-Lys-Phe-Arg-NA with kcat/KM = 140 000 M-1 s-1. Activated bovine protein C (best substrate = Z-Ser-Phe-Arg-NA), factor Xa (best substrate = Z-Glu-Gly-Arg-NA), and thrombin (best substrate = Z-Lys-Gly-Arg-NA) were the group of enzymes that showed next highest reactivity toward the substrates. Activated bovine protein C, thrombin, and factor Xa displayed relatively little substrate specificity. Activated human protein C (best substrate = Z-Ser-Phe-Arg-NA) and factor XIa (best substrate = Z-Glu-Gly-Arg-NA) are moderately reactive enzymes. Activated human protein C is an extremely specific enzyme since it has such a large range of kcat/KM values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

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.

Animals↗
PubMed1995

Evaluation of factor XIa-alpha 1-antitrypsin in plasma, a contact phase-activated coagulation factor-inhibitor complex, in patients with coronary artery disease.

Excess activated factor XI (FXIa) in plasma indicates increased activation during the contact phase of blood coagulation. To investigate the relationship between such elevations and coronary atherosclerosis, we examined FXIa values in patients with coronary artery disease (CAD) by an enzyme-linked immunosorbent assay method that we developed that detects FXIa in plasma samples as an FXIa-alpha 1-antitrypsin complex (FXIa-alpha 1AT). The presence and extent of CAD were documented by coronary angiography and assessed by a recently developed scoring system for semiquantitative estimation of coronary atherosclerosis. Plasma FXIa-alpha 1AT levels were significantly increased in patients with angiographically proven CAD (13.9 +/- 3.0 micrograms/L, n = 42) compared with age-matched, healthy control subjects (11.9 +/- 1.7 micrograms/L, n = 20) as well as patients with angiographically normal coronary arteries (12.0 +/- 2.3 micrograms/L, n = 25). Moreover, in the total patient population, the FXIa-alpha 1AT level was related to the number of significant coronary artery stenoses as well as to the total coronary score. FXIa-alpha 1AT showed a positive correlation with thrombin-antithrombin III complex, fibrinogen, and Lp(a) and an inverse correlation with apo A-I, as determined by multi-variate analysis. Our studies provide evidence that increased activation of the contact pathway occurs in patients with CAD and is related to the severity of the disease. Although it is unknown whether this abnormality is the cause or the result of the vascular lesion, it may be important for progression of the underlying atherosclerosis or for propagation of the atherosclerotic process itself.

Antithrombin III↗