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At least 19 recordsLinked to original sources
PubMed1983

A comparison of the abilities of plasma kallikrein, beta-Factor XIIa, Factor XIa and urokinase to activate plasminogen.

In order to compare the relative potencies of plasma kallikrein, beta-Factor XIIa, Factor XIa and urokinase as plasminogen activators, plasminogen activation by these proteins was studied using a radiolabeled fibrin plate assay. Urokinase was approximately 20,000 times more active than kallikrein or Factor XIa and 300,000 times more active than beta-Factor XIIa. Kallikrein and Factor XIa were approximately equal in plasminogen activator activity and were 20 times more potent than beta-Factor XIIa.

Endopeptidases↗
PubMed1987

Studies on the effect of serine protease inhibitors on activated contact factors. Application in amidolytic assays for factor XIIa, plasma kallikrein and factor XIa.

Amidolytic assays have been developed to determine factor XIIa, factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic p-nitroanilide substrates Pro-Phe-Arg-NH-Np (S2302 or chromozym PK), Glp-Pro-Arg-NH-Np (S2366), Ile-Glu-(piperidyl)-Gly-Arg-NH-Np (S2337), and Ile-Glu-Gly-Arg-NH-Np (S2222) were tested for their suitability as substrates in these assays. The kinetic parameters for the conversion of S2302, S2222, S2337 and S2366 by beta factor XIIa, factor XIa and plasma kallikrein indicate that each active enzyme exhibits considerable activity towards a number of these substrates. This precludes direct quantification of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors have been tested for their ability to inhibit those contact factors selectively that may interfere with the factor tested for. Soybean trypsin inhibitor very efficiently inhibited kallikrein, inhibited factor XIa at moderate concentrations, but did not affect the amidolytic activity of factor XIIa. Therefore, this inhibitor can be used to abolish a kallikrein and factor XIa contribution in a factor XIIa assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethyl ketones. D-Phe-Pro-Arg-CH2Cl was moderately active against contact factors (k = 2.2 X 10(3) M-1 s-1 at pH 8.3) but showed no differences in specifity. D-Phe-Phe-Arg-CH2Cl was a very efficient inhibitor of plasma kallikrein (k = 1.2 X 10(5) M-1 s-1 at pH 8.3) whereas it slowly inhibited factor XIIa (k = 1.4 X 10(3) M-1 s-1) and factor XIa (k = 0.11 X 10(3) M-1 s-1). Also Dns-Glu-Gly-Arg-CH2Cl was more reactive towards kallikrein (k = 1.6 X 10(4) M-1 s-1) than towards factor XIIa (k = 4.6 X 10(2) M-1 s-1) and factor XIa (k = 0.6 X 10(2) M-1 s-1). Since Phe-Phe-Arg-CH2Cl is highly specific for plasma kallikrein it can be used in a factor XIa assay selectively to inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for factor XIIa, factor XIa and kallikrein in mixtures of contact activation factors.

Amino Acid Chloromethyl Ketones↗
PubMed1985

Amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor).

The amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor) was determined by automated Edman degradation using the peptides produced by chemical and enzymatic cleavages of intact factor XII and alpha-factor XIIa. Combining this sequence with the previously determined sequence of beta-factor XIIa (Fujikawa, K., and McMullen, B. A. (1983) J. Biol. Chem. 258, 10924-10933), the complete amino acid sequence of human factor XII has been established. The heavy chain of alpha-factor XIIa is composed of 353 amino acid residues containing one Asn-linked and six probable O-linked carbohydrate chains. The heavy chain of alpha-factor XIIa appears to contain four different domains including a "kringle," a "growth factor" domain, and the "type I" and "type II" domains of fibronectin. The domain organization of factor XII is analogous to those of several fibrinolytic proteins, including tissue plasminogen activator and urokinase, suggesting that factor XII belongs to the same protease subfamily as these two proteins.

Amino Acid Sequence↗
PubMed1977

Characterization of bovine factor XIIa (activated Hageman factor).

Factor XIIa (activated Hageman factor) was isolated from bovine plasma by ammonium fractionation followed by heparin-agarose, carboxymethylcellulose, and arginine-agarose column chromatography. It was separated from factor XII in the final step by chromatography on benzamidine-agarose. Factor XIIa has a molecular weight of approximately 74 000 and is composed of a heavy and light chain held together by a disulfide bond(s). The amino-terminal sequence of the heavy chain is Thr-Pro-Pro-Trp--Lys-Gly-Pro-Lys-Lys-His-Lys-Leu- which is the same as the precursor protein. The carobyl-terminal residue in this polypeptide chain is arginine. The amino-terminal sequence of the light chain is Val-Val-Gly-Gly-Leu-Val-Ala-Leu-Pro-Gly-Ala-?-Pro-Tyr-Ile-. This latter sequence is homologous with the amino-terminal sequence of a number of plasma serine proteases when compared with the chain containing the active-site serine residue. These data suggest that factor XII is converted to factor XIIa by the cleavage of a specific internal arginyl-valine peptide bond. Factor XIIa, in contrast to factor XII, has hydrolase activity toward arginine-containing substrates and is readily inhibited by antithrombinIII and diisopropyl phosphorofluoridate. The inhibitors, in each case, are bound to the light chain of factor XIIa which contains the active-site serine residue.

Amino Acid Sequence↗
PubMed1977

Activation of bovine factor VII (proconvertin) by factor XIIa (activated Hageman factor).

Bovine factor VII (proconvertin) is a plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. It has a molecular weight of 45 500 and is composed of a single polypeptide chain with an amino-terminal alanine residue. Factor VII is readily converted to factor VIIa by factor XIIa (activated Hageman factor) employing an enzyme to substrate weight ratio of 1:50. Factor VIIa is composed of a light and a heavy chain held together by a disulfide bond(s). The heavy chain, which is formed from the carboxyl-terminal region of the precursor, contains an amino-terminal sequence of Ile-Val-Gly-Gly-. The heavy chain also contains the active-site sequence of -Phe-Cys-Ala-Gly-Tyr-Thr-Asp-Gly-Thr-Lys-Asp-Ala-Cys-Lys-Gly-Asp-Ser-Gly-Gly-Pro-His-. This sequence is homologous with the active-site region of a number of plasma serine proteases. These data indicate that factor VII is a typical precursor of a serine protease which is converted to an enzyme by factor XIIa by the cleavage of a single, internal peptide bond.

Amino Acid Sequence↗
PubMed1995

Activation of hepatocyte growth factor by two homologous proteases, blood-coagulation factor XIIa and hepatocyte growth factor activator.

Hepatocyte growth factor (HGF) is secreted as an inactive single-chain precursor from the producing cells, and normally remains in this form associated with the extracellular matrix. In response to tissue injury, the single-chain precursor is converted to a biologically active heterodimer by a serine protease, the activity of which is induced in the injured tissue. We have previously identified HGF activator, a serum serine protease that activates single-chain HGF. The sequence of HGF activator cDNA revealed that the HGF activator is homologous to blood-coagulation factor XIIa. In this study, we found that coagulation factor XIIa has an ability to activate single-chain HGF. Factor XIIa exhibited a significant level of HGF-converting activity in the presence of dextran sulfate, although the specific activity of factor XIIa was slightly lower than that of the HGF activator. Since factor XIIa is activated during the initiation of contact activation induced by tissue injury, factor XIIa may function as an HGF-converting enzyme together with HGF activator in the injured tissue. C1-inhibitor, antithrombin III and alpha 2-antiplasmin, that regulate the blood-clotting activity of factor XIIa, were also effective against the HGF-converting activity of factor XIIa. Furthermore, factor XIIa was not active in the HGF-converting activity in serum. Thus, the HGF-converting activity of factor XIIa may be regulated by these serum inhibitors.

Factor XIIa↗
PubMed1983

Amino acid sequence of human beta-factor XIIa.

Human factor XII was activated by limited proteolysis with trypsin, and the resulting beta-factor XIIa (Mr = 30,000) was isolated by DEAE-Sephacel column chromatography. The complete amino acid sequence of beta-factor XIIa was then determined on peptides produced by enzymatic digestion with either trypsin, chymotrypsin, or Staphylococcus aureus V8 protease and by chemical cleavage at methionyl and tryptophyl bonds. beta-Factor XIIa is a glycoprotein composed of a heavy chain (243 amino acid residues) and a light chain (9 amino acid residues), and these two chains are held together by a disulfide bond. The carbohydrate is attached to asparagine residue 61 in the heavy chain. The amino acid sequence of the heavy chain shows a high degree of homology to the corresponding regions of other plasma serine proteases, such as plasmin, thrombin, factor IXa and factor Xa, as well as the pancreatic digestive enzymes. These results demonstrate that factor XII is the precursor of a typical serine protease that participates in the coagulation cascade.

Amino Acid Sequence↗
PubMed1977

Coagulation factor XIIa (activated Hageman factor) inhibitor from adult Schistosoma mansoni.

An inhibitory activity for the contact phase of the intrinsic coagulation pathway was demonstrated in an extract of adult Schistosoma mansoni. Inhibition is apparently specific for the enzymatic activation of Factor XI (pre-PTA) by Factor XIIa (activated Hageman factor). This phenomenon offers an explanation for the schistosomal evasive mechanism of the host contact hemostatic defense system.

Animals↗
PubMed1982

Pumpkin seed inhibitor of human factor XIIa (activated Hageman factor) and bovine trypsin.

A strong inhibitor of human Hageman factor fragment (HFf, beta-factor XIIa) and bovine trypsin was isolated from pumpkin (Cucurbita maxima) seed extracts by acetone fractionation, by chromatography on columns of diethyl-aminoethylcellulose and carboxylmethyl-Sephadex C-25, and by Sephadex G-50 gel filtration. Pumpkin seed Hageman factor inhibitor (PHFI) is unusual in its lack of inhibition of several other serine proteinases tested--human plasma, human urinary, and porcine pancreatic kallikreins, human alpha-thrombin, and bovine alpha-chymotrypsin. Human plasmin and bovine factor Xa are only weakly inhibited. PHFI also inhibits the HFf-dependent activation of plasma prekallikrein and clotting of plasma. Other properties of PHFI are a pI of 8.3, 29 amino acid residues, amino-terminal arginine, carboxyl-terminal glycine, 3 cystine residues, undetectable sulfhydryl groups and carbohydrate, and arginine at the reactive site. The minimum molecular weight of PHFI is 3268 by amino acid analysis. PHFI may be the smallest protein inhibitor of trypsin known.

Amino Acids↗
PubMed1987

Effect of negatively charged activating compounds on inactivation of factor XIIa by Cl inhibitor.

Human factor XII, upon exposure to negatively charged surfaces such as kaolin, sulfatides, and heparin, is converted to enzymatic forms, factor XIIa and factor XIIf. Cl inhibitor has been quantitatively demonstrated to be the primary plasma inhibitor of both factor XIIa and factor XIIf. Studies were performed to determine whether the presence of artificial, negatively charged surfaces influenced the ability of Cl inhibitor to inhibit factors XIIa and XIIf. Kaolin and sulfatides slowed the rate of inhibition of factor XIIa by Cl inhibitor 4.8- and 2-fold, respectively, whereas they had no effect on the inhibition of factor XIIf by Cl inhibitor. Heparin in a concentration of 65 U/ml decreased the inhibition rate of factor XIIa by Cl inhibitor, but, at the same concentration, had less of an effect on the ability of Cl inhibitor to inhibit factor XIIf. These studies indicate that negatively charged surfaces protect factor XIIa but not factor XIIf from inhibition from Cl inhibitor. Since the difference between factors XIIa and XIIf consists of the presence of a surface binding region in factor XIIa, the basis of this protection must reside in the surface binding residues of factor XII. These in vitro events suggest that surface-bound factor XIIa may hydrolyze its physiologic substrates, factor XI and prekallikrein, in an environment partially protected from inhibition by Cl inhibitor.

Complement C1 Inactivator Proteins↗
PubMed1993

Identification and characterization of a binding site for factor XIIa in the Apple 4 domain of coagulation factor XI.

Previously we have characterized a binding site for high M(r) kininogen in the first of four tandem-repeat (Apple) domains within the heavy chain region of factor XI (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154; Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 267, 4247-4252), whereas a substrate binding site for factor IX was localized to the second Apple (A2) domain (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 266, 24190-24197). To define the factor XI domain that binds factor XIIa, we have screened a panel of synthetic peptides for their capacity to inhibit factor XI activation by factor XIIa. Peptide Gly326-Lys357 (located in the A4 domain) is a noncompetitive inhibitor of factor XI activation by factor XIIa (Ki = 3.75 microM), whereas structurally similar peptides from the A1, A2, and A3 domains were required at > 1000-fold higher concentrations for similar effects. The same peptide (Gly326-Lys357) is a competitive inhibitor of factor XIIa amidolytic activity (Ki = 3.8 microM) suggesting that it binds near the active site of factor XIIa. Computer modeling was used to predict the secondary and tertiary structure of the A4 domain of factor XI that interacts with factor XIIa. Rationally designed, conformationally constrained peptides were synthesized comprising residues Ala317-Gly326, Lys331-Lys340, and Gly344-Gly350, which act in concert to inhibit factor XI-activation by factor XIIa. Finally, a conformationally constrained peptide spanning residues Ala317-Gly350 inhibits factor XIIa-catalyzed factor XI activation 50% at a concentration of 5 x 10(-7) M. These results, interpreted in the context of the model, suggest that the sequence of amino acids from Ala317 through Gly350 of the heavy chain of the A4 domain of factor XI contains three peptide structures, possibly consisting of three antiparallel beta-strands that together comprise a contact surface for interacting with factor XIIa.

Amino Acid Sequence↗
PubMed1985

The regulation of human factor XIIa by plasma proteinase inhibitors.

Studies of the inactivation of factor XIIa by plasma protease inhibitors in purified systems and in plasma were initiated to determine the relative importance of these inhibitors to the neutralization of factor XIIa. Factor XIIa was measured by the amidolysis of H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride or by coagulant activity. C1 inhibitor (C1INH), alpha 2-antiplasmin (alpha 2AP), alpha 2-macroglobulin (alpha 2M), and antithrombin III (ATIII) inhibited factor XIIa with second-order rate constants of 2.2 X 10(5), 1.1 X 10(4), 5.0 X 10(3), and 1.3 X 10(3) M-1 min-1. Factor XIIa activity was not affected by alpha 1-proteinase inhibitor. Incubation of 125I-radiolabeled factor XIIa resulted in 1:1 stoichiometric complexes with C1INH (Mr 190,000), ATIII (Mr 125,000), and alpha 2AP (Mr 150,000 and 125,000) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of 125I-Factor XIIa with alpha 2M resulted in a component of Mr 85,000 on a reduced sodium dodecyl sulfate-polyacrylamide gel, indicating that a subunit of factor XIIa was covalently bound to a proteolyzed portion of alpha 2M. The relative effectiveness of each inhibitor at plasma concentrations was 61:2:3:1 for C1INH, alpha 2AP, alpha 2M, and ATIII, respectively. Kinetic studies of the inactivation of purified factor XIIa added to various plasmas containing different concentrations of C1INH verified the predictions from the purified systems. Gel filtration of radiolabeled factor XIIa incubated with plasma confirmed that factor XIIa-C1INH was the major complex. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the complexes in plasma had the same molecular size as those with purified inhibitors. C1INH functions as the predominant inhibitor of factor XIIa in plasma.

Antithrombin III↗
PubMed1986

Mechanism of activation of coagulation factor XI by factor XIIa studied with monoclonal antibodies.

The interaction of Factor XIIa with Factor XI was investigated using two monoclonal antibodies, one (3Cl) directed against the heavy chain of Factor XIa and the other (5F4) against its light chain. 3C1 either as intact IgG or as Fab' fragment, enhanced the rate of Factor XIa generation in the fluid phase but inhibited it in the presence of kaolin and high molecular weight (HMW) kininogen. In contrast, the Fab' fragments of 5F4 inhibited only the fluid phase activation and had no effect on the surface-mediated activation. 3C1 was found to block the binding of Factor XI to HMW kininogen, whereas 5F4 did not. We conclude: a domain on the heavy chain region of Factor XI is essential for binding to HMW kininogen and for optimal surface-mediated activation by Factor XIIa; and binding of 3C1 to Factor XI changes its conformation rendering it a more favorable substrate for Factor XIIa in the fluid phase.

Antibodies, Monoclonal↗
PubMed1995

The Apple 1 and Apple 4 domains of factor XI act synergistically to promote the surface-mediated activation of factor XI by factor XIIa.

Binding sites for high molecular weight kininogen (HK) and for factor XIIa are present in the Apple 1 (A1) and the A4 domains of factor XI, respectively. To define the roles of these two sites in surface-mediated factor-XI activation we prepared conformationally constrained synthetic peptides and recombinant A1 domain (rA1) and determined their effects on the activation of factor XI by factor XIIa in the presence of HK and either kaolin or dextran sulfate. Surface-mediated factor-XI activation by factor XIIa was inhibited by a conformationally constrained A4 peptide (Ala317-Gly350), by an A1 peptide (Phe56-Ser86), and by rA1 (Glu1-Ser90). When used in combination at equimolar concentrations, rA1 and A4 peptide were 10-fold more effective than either one alone in inhibiting surface-mediated activation of factor XI by factor XIIa. The A4 peptide was a competitive inhibitor of factor XIIa amidolytic activity and a noncompetitive inhibitor of factor-XI activation by factor XIIa, whereas rA1 and the A1 peptide did not inhibit factor XIIa. The rA1 domain inhibited factor XI binding to HK, whereas the A4 peptide did not. We conclude that specific sequences exposed on the surfaces of the A1 (Val59-Lys83) and A4 (Ala317-Gly350) domains of factor XI act synergistically to promote surface-mediated factor-XI activation by factor XIIa in the presence of HK by binding factor XI to surface-bound HK (A1 domain) and by binding factor XIIa near the cleavage site (Arg369-Ile370) of factor XI (A4 domain).

Dextran Sulfate↗
PubMed1991

Mitogenic effects of coagulation factor XII and factor XIIa on HepG2 cells.

The structure of coagulation factor XII (Hageman factor), inferred from its DNA sequence, includes two epidermal growth factor (EGF)-homologous domains in its amino-terminal region. This suggests that factor XII may exhibit EGF-like activities. Reciprocal antigenic cross-reactivity between factor XII and EGF was shown by exposing purified human factor XII or mouse EGF to anti-mouse EGF or anti-human factor XII. Western blot analysis showed that anti-mouse EGF recognized intact factor XII at 80 kDa. Together, these results suggest that the EGF-homologous domains are accessible for anti-EGF binding in native factor XII. To determine whether factor XII has mitogenic activity, HepG2 or L cells (10(4) cells per well) were grown in serum-free medium in the presence or absence of factor XII or kaolin-activated factor XII (factor XIIa). Both factors XII and XIIa (6.0 micrograms/ml) enhanced cell proliferation by approximately 2-fold (P less than 0.001 and P less than 0.005, respectively). In contrast, L cells, which are not EGF target cells, were not affected by either factor XII or factor XIIa. Various doses of factor XII enhanced cell proliferation, [3H]thymidine incorporation, and [3H]leucine incorporation in HepG2 cells cultured under the same conditions. These data indicate that factor XII, like EGF, is a mitogen for HepG2 cells and suggest a possible autocrine role in the liver.

Antibodies↗
PubMed1988

Interaction of bovine factor XIIa with an inhibitor from bovine plasma.

An inhibitor of factor XIIa has been purified from bovine plasma and characterized (Thornton, R.D. and Kirby, E.P. (1987) J. Biol. Chem. 262, 12714-12721). This inhibitor interacts with XIIa to form a very stable complex with a 1:1 stoichiometry. The active site of XIIa, located on the light chain, is directly involved in the interaction, and complex formation between factor XIIa inhibitor and XIIa can be blocked by diisopropyl fluorophosphate, corn trypsin inhibitor, or the chromogenic substrate S2302. Incubation of the complex with excess XIIa does not result in cleavage of the complex. The complex does not spontaneously dissociate and is stable to boiling, SDS, thiocyanate, acid, and hydroxylamine or Tris at pH 7-10. In addition to complex formation, a cleaved form of factor XIIa inhibitor can be observed. We suggest that the inhibitor is acting as a mechanism-based inactivator, using the criteria of time-dependent inactivation under pseudo-first-order conditions, 1:1 stoichiometry, active site involvement, kinetic protection by substrate or by an active site inhibitor, and partitioning between cleavage of factor XIIa inhibitor and inactivation by complex formation.

Animals↗
PubMed1986

Purified plasma factor XIIa aggregates human neutrophils and causes degranulation.

Plasma kallikrein has been shown to aggregate human neutrophils and release human neutrophil elastase. However, neutrophils resuspended in factor XII-deficient plasma released only 30% of the elastase compared with normal plasma. Isolated human neutrophils were aggregated in a concentration-dependent fashion by 0.06 to 0.6 U/mL factor XIIa (0.022 to 0.22 mumol/L). Factor XIIa (0.1 to 1.0 U/mL) also induced neutrophil degranulation as evidenced by a concentration-dependent release of the specific granule protein, lactoferrin, and azurophilic granule protease, elastase. The release of neutrophil elastase was biphasic, reaching 40% of maximum at 15 seconds with maximal release by 90 minutes. The active site of factor XIIa was required, since the synthetic inhibitor, D-Pro-Phe-Arg-CH2Cl, which reacts with an essential histidine, and the natural plasma inhibitor, Cl-inhibitor, which interacts with the critical serine, both inhibit by more than 90% the release of elastase. The heavy chain is also required, since factor XII fragments failed to aggregate neutrophils or stimulate degranulation. Factor XIIa (0.6 U/mL) can completely correct the defect in elastase release evident in factor XII-deficient plasma. These studies demonstrate that factor XIIa, at concentrations potentially obtainable in plasma in disease states, can activate neutrophils, and thus may participate in the inflammatory response.

Cell Aggregation↗