Accelerating effect of zinc ions on the surface-mediated activation of factor XII and prekallikrein

The effect of zinc ions on the surface-mediated activation of factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine and human plasmas. The sulfatide-mediated activation of factor XII and prekallikrein in the presence of high-molecular-weight (HMW) kininogen was remarkably accelerated by 10(-5) M zinc ions. This accelerating effect was observed only in the presence of HMW kininogen. The kinetic analysis of the accelerating effect of zinc ions demonstrated that zinc ions reduce the Km values and increase the Vmax values on the activation of factor XII by kallikrein and on the activation of prekallikrein by factor XIIa. The value of Vmax/Km increased 26.4-fold in the former reaction and 2.8-fold in the latter reaction, indicating that zinc ions accelerate mainly the activation of factor XII by kallikrein. In the presence of 5 x 10(-4) M zinc ions, typical difference spectra due to a red shift of tryptophan and/or tyrosine residues were observed for HMW kininogen and its derivatives but not low-molecular-weight (LMW) kininogen. Since the concentration of zinc ions required to induce the difference spectra is comparable with that to enhance the activation of factor XII and prekallikrein, it appears that there is some correlation between the conformational change of HMW kininogen and the enhancement of the activation.     

Kinetic studies on surface-mediated activation of bovine factor XII and prekallikrein. Effects of kaolin and high-Mr kininogen on the activation reactions

The kaolin-mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high-Mr kininogen and kaolin surface on the kinetics of these activation reactions were studied. High-Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high-Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 X 10(7) s-1 X M-1, respectively. The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high-Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 microM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 X 10(4) to 1.1 X 10(6) s-1 X M-1). These results suggest that high-Mr kininogen accelerates the surface-mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.     

Isolation and characterization of bovine plasma prekallikrein (Fletcher factor)

Prekallikrein (Fletcher factor) has been purified from bovine plasma approximately 25 000-fold with an overall yield of 14%. Purification steps included ammonium sulfate fractionation and column chromatography on heparin-agarose, DEAE-Sephadex, CM-Sephadex, benzamidine-agarose, and arginine methyl ester-agarose. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. Bovine plasma prekallikrein is a glycoprotein with a molecular weight of 82 000 as determined by sedimentation equilibrium centrifugation. It contains 12.9% carbohydrate, including 6.2% hexose, 4.5% N-acetylglucosamine, and 2.2% N-acetylneuraminic acid. Prekallikrein is a single polypeptide chain with an amino-terminal sequence of Gly-Cys-Leu-Thr-Gln-Leu-Tyr-His-Asn-Ile-Phe-Phe-Arg-Gly-Gly. This sequence is homologous to the amino-terminal sequence of human factor XI (plasma thromboplastin antecedent). Both prekallikrein and kallikrein require kaolin to correct Fletcher factor deficient plasma. Kallikrein, however, has a specific activity 3.5 times greater than prekallikrein. Prekallikrein does not correct plasma deficient in factor XII (Hageman factor), factor XI, or high molecular weight kininogen (Fitzgerald factor).     

The role of bovine high-molecular-weight (HMW) kininogen in contact-mediated activation of bovine factor XII: interaction of HMW kininogen with kaolin and plasma prekallikrein

Previous studies from our laboratories (Sugo et al. (1980) Biochemistry 19, 3215-3220) have shown that bovine high-molecular-weight (HMW) kininogen remarkably accelerates the kaolin-mediated activation of Factor XII in the presence of prekallikrein, and that both fragment 1.2 and the light chain regions located in the COOH terminal half of the kininogen molecule are essential for the activation. In the present study, we demonstrate that the accelerating effect of HMW kininogen is mediated through its adsorption on the kaolin surface through the fragment 1.2 region and its complex formation with prekallikrein through the light chain region. The evidence is as follows: 1. HMW kininogen radio-labeled with 125I was adsorbed on kaolin and the adsorption was inhibited by the prior treatment of kaolin with fragment 1.2, fragment 1.2-light chain, kinin-free protein or HMW kininogen, but not with kinin- and fragment 1.2-free protein, light chain or low molecular-weight (LMW) kininogen. 2. The complex formation of HMW kininogen with prekallikrein in bovine plasma or in the purified system was examined by gel-filtration on a column of Sephacryl S-200 In bovine plasma, prekallikrein was eluted in the same fraction as HMW kininogen, showing an apparent molecular weight of 250,000, whereas purified prekallikrein was eluted in the fraction corresponding to an apparent molecular weight of 100,000. When purified prekallikrein was mixed with purified HMW kininogen in a mol ratio of 1 to 2, all prekallikrein was found to be associated with HMW kininogen. Furthermore, purified prekallikrein mixed with kininogen derivatives, such as kinin- and fragment 1.2-free protein, fragment 1.2-light chain or light chain, was eluted in the higher molecular weight fraction. HMW kininogen did not form a complex with prekallikrein. Using the same technique, it was shown that kinin- and fragment 1.2-free protein forms a complex not only with prekallikrein but also with kallikrein.     

Activation of factor XII and prekallikrein with polysaccharide sulfates and sulfatides: comparison with kaolin-mediated activation

The activation of Factor XII and prekallikrein by polysaccharide sulfates and sulfatides in the presence of high-molecular-weight (HMW) kininogen was studied, and compared with the kaolin-mediated activation reaction. Among a variety of artificially-sulfated polysaccharides and native polysaccharide sulfates, amylose sulfate (M.W.= 380,000 and sulfur content, 19.1%) and sulfatide were found to have the most efficient ability to trigger the activation of prekallikrein by Factor XII. The effects of these two kinds of negatively-charged surfaces on the following three activation reactions were compared; the activation of prekallikrein by Factor XII (reaction 1), the activation of Factor XII by kallikrein (reaction 2) and the activation of prekallikrein by Factor XIIa (reaction 3). All three reactions mediated by the selected surfaces were strongly accelerated by HMW kininogen and its derivatives, kinin-free protein and fragment 1.2-linked light chain, like the kaolin-mediated activation. However, this accelerating effect of HMW kininogen on the amylose sulfate- and sulfatide-mediated activations (reaction 1) was diminished after treatment with fluorescein iso-thiocyanate, whereas the effect on the kaolin-mediated activation was not influenced by fluorescein-labeling. In addition, reaction 2 mediated by amylose sulfate and sulfatide was extremely slow even in the presence of HMW kininogen, and the results also differed from those with kaolin. The sulfatide-mediated activation of reaction 1 was not inhibited by fragment 1.2 (His-rich fragment), which is released from HMW kininogen by the action of kallikrein, and is known to be a potent inhibitor of the kaolin-dependent activation. These results indicate that the mechanisms responsible for surface activation triggered by soluble amylose sulfate, sulfatide micelles and kaolin differ from each other as regards the molecular interaction with the contact factors.     

Inositolphospholipid-accelerated activation of prekallikrein by activated factor XII and its inhibition by beta 2-glycoprotein I

Inositolphospholipid-accelerated activation of prekallikrein by alpha-factor XIIa was determined by measuring the appearance of kallikrein amidolytic activity towards the chromogenic substrate, D-prolyl-phenylalanyl-arginyl p-nitroanilide (S-2302). The activation reaction did not exhibit normal Michaelis-Menten kinetics. The Hill coefficient was found to be 1.6 indicating that the activation followed an allosteric reaction mechanism. The temperature dependence of the reaction showed a thermal transition at 30 degrees C, which in addition to the allosteric reaction mechanism is indicative of a conformational change of prekallikrein following binding to the inositolphospholipid. The reaction exhibited pH optimum at pH 7.2 and ionic strength optimum at 50 mM NaCl. At optimal conditions the apparent KA value and the kcat/KA value for factor XIIa on prekallikrein were calculated to be 73 nM and 9.3 x 10(6) s-1 M-1, respectively. Kinetic constants could not be calculated at salt concentrations higher than the optimal concentrations, as Lineweaver-Burk plots were curvilinear in agreement with the Hill coefficient greater than unity. The activation was inhibited competitively by beta 2-glycoprotein I with a Ki value of 77 nM as determined by the Dixon plot.     

Binding and activation properties of human factor XII, prekallikrein, and derived peptides with acidic lipid vesicles

The binding of human factor XII and prekallikrein to vesicles of various compositions and the relationship to activation of factor XII were studied. Factor XII, factor XIIa, and the 40-kilodalton binding fragment of factor XII bound tightly to all of the negatively charged lipids investigated, including sulfatide, phosphatidylserine, and phosphatidylethanolamine, but not to the neutral lipid phosphatidylcholine. Binding could be reversed by high salt, and the dissociation constant for binding to sulfatide vesicles was in the nanomolar range at an ionic strength of 0.15 M. Prekallikrein did not bind significantly to either sulfatide or phosphatidylethanolamine vesicles under the conditions used. Stopped-flow studies showed that the association rate for the factor XII-sulfatide interaction was biphasic and very rapid; the faster rate corresponded to about 30% collisional efficiency. The kinetics of activation of factor XII was investigated and was in agreement with previous studies; sulfatide promoted activation but phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine did not. Autoactivation rates correlated closely with the packing density of factor XII and factor XIIa on the vesicle surface. In contrast, kallikrein activation of factor XII correlated with the amount of sulfatide-bound factor XII and was relatively insensitive to the density of factor XII on the vesicle surface. When the concentration of factor XII was reduced to only several molecules per vesicle, the autoactivation rate dropped very low whereas kallikrein activation held relatively constant. These results indicated that the autoactivation and the kallikrein activation of factor XII were dependent on different properties of the surface component.     

Isolation and amino acid sequence of bovine platelet factor 4

Bovine platelet factor 4 was isolated by affinity chromatography using dextran sulfate Sepharose and purified by subsequent gel filtration. The complete amino acid sequence of this 88-residue, 9505-Da protein was determined by isolation and analysis of the overlapping peptides from tryptic and Staphylococcus aureus hydrolysates of reduced, carboxymethylated, and reductive methylated protein. Primary structure comparison was made between bovine platelet factor 4, human platelet factor 4, and human beta-thromboglobulin. The bovine platelet factor 4 amino-terminal region, which contains two unique phenylalanine residues, is extended by 15 residues relative to human platelet factor 4. The bovine carboxy-terminal region is extended by three residues relative to human platelet factor 4 and differed from beta-thromboglobulin in the absence of two additional terminal residues. Bovine platelet factor 4 shares sequence similarities proportionately with both human platelet factor 4 and beta-thromboglobulin. The sequences of the lysine-rich carboxy-terminal putative heparin binding domains are essentially identical for all three proteins. The heparin neutralizing potencies of bovine and human platelet factor 4 are similar: 40 USP units of heparin neutralized per milligram protein, as measured by a modified chromogenic substrate assay. Heparin neutralization was lost by reduction of the disulfide bonds, but only attenuated by tryptic digestion of the intact protein.     

The inositol-phospholipid-accelerated activation of prekallikrein by activated factor XII at physiological ionic strength requires zinc ions and high-Mr kininogen

In a system consisting of purified proteins inositol-phospholipid-accelerated activation of prekallikrein by alpha-factor XIIa was determined by measuring the appearance of kallikrein amidolytic activity towards the chromogenic substrate, H-D-Pro-Phe-Arg-NH-PhNO2 (PhNO2, 4-nitrophenyl). The activation reaction was ionic-strength dependent. In the absence of high-Mr kininogen optimal activity was recorded at I = 50 mM. Searching for conditions, which could change this optimum towards physiological values, high-Mr kininogen was added. This resulted in an inhibition of the activity, with no change in ionic strength optimum. If, however, Zn2+ were added concomitant with high-Mr kininogen, the inhibition was abolished and optimal activity recorded at physiological ionic strength. The optimal Zn2+ concentration was found to be 0.1 mM. Kinetic analysis of the reaction demonstrated that the kcat/Km was 1.2 x 10(5) M-1 s-1 in the absence and 1.1 x 10(6) M-1 s-1 in the presence of Zn2+. Zn2+ were also required for inositol-phospholipid-accelerated initiation of the contact activation in whole plasma.     

Determination of factor XII, plasma prekallikrein and factor V in patients with thrombophilic states

In patients with thrombophilic states the plasma level of factor XII was lower by about 15 per cent, while the factor V content was higher by about 20 per cent than in healthy subjects. Plasma prekallikrein was unchanged. It is not clear whether these changes are the cause or the consequence of thrombotic events.     

Kinetics of activation and autoactivation of human factor XII

The kinetics of the enzymic reactions that participate in the contact activation system of human plasma were examined. These reactions are potentiated by dextran sulfate, a negatively charged solute that mimics many of the effects of glass or kaolin on this system. The reactions of reciprocal activation, consisting of activation of factor XII by kallikrein and of prekallikrein by activated factor XII, follow Michaelis-Menten kinetics; values of kcat and Km for each of these reactions were determined in the presence of dextran sulfate and in its absence. In the presence of dextran sulfate, the catalytic efficiency for factor XII activation was increased 11 000-fold, and that for prekallikrein was increased 70-fold. Autoactivation of factor XII in the presence of dextran sulfate also follows Michaelis-Menten kinetics with kcat = 0.033 s-1 and Km = 7.5 microM. This finding supports the concept that autoactivation is an enzymic process, initiated by traces of activated factor XII which are invariably present in factor XII preparations. At prekallikrein and factor XII levels equal to those in plasma, reciprocal activation is approximately 2000-fold more rapid than autoactivation. Thus, reciprocal activation is the predominant mode of factor XII activation in normal plasma.     

Isolation and characterization of the bovine hypothalamic corticotropin-releasing factor

A 41 amino acid peptide with high intrinsic corticotropin-releasing activity was isolated from 1000 bovine hypothalami by means of immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The primary structure of the amino terminal 39 amino acids was characterized by gas phase sequence analysis. The sequence of the amidated carboxyl terminal dipeptide was established by digestion of the intact natural product with Staphylococcus aureus V8 protease, dansylation of the digest and comparative reverse phase liquid chromatography studies with the synthetic dansylated dipeptides Ile-Ala-NH2, Ile-Ala-OH, Ala-Ile-NH2 and Ala-Ile-OH. The complete structure of the bovine corticotropin-releasing factor was established as: Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val- Leu- Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Asn-Asn-Arg-Lys-Leu- Leu- Asp-Ile-Ala-NH2 using approximately 650 pmol of material.     

Interaction of factor XII, high-molecular-weight (HMW) kininogen and prekallikrein with sulfatide: analysis by fluorescence polarization

The interaction of high-molecular-weight (HMW) kininogen, Factor XII and prekallikrein with sulfatide was studied by fluorescence polarization. Fluorescein-conjugated derivatives of HMW kininogen, Factor XII and prekallikrein were prepared by reacting the purified bovine factors with fluorescein isothiocyanate (FITC). The apparent dissociation constant (Kd) for the binding of FITC-labeled HMW kininogen (F-HMW kininogen) with sulfatide was calculated to be 3.2 (+/- 0.3) X 10(-8) M. This binding was partially inhibited by three kininogen derivatives, fragment 1 X 2, kinin-free protein and fragment 1 X 2-light chain, but not by kinin and fragment 1 X 2-free protein. In the presence of Factor XII, the binding of F-HMW kininogen with sulfatide was strongly inhibited, suggesting that the zymogen and the protein cofactor compete for the same or a closely related binding site on the sulfatide surface. In contrast, the binding of FITC-labeled Factor XII (F-Factor XII) with sulfatide was weakly inhibited by HMW kininogen but not by prekallikrein. The Kd value for binding of F-Factor XII with sulfatide was calculated to be 2.0 (+/- 0.3) X 10(-8) M. F-Prekallikrein did not interact with sulfatide. Moreover, the fluorescence polarization value of F-HMW kininogen decreased in the presence of prekallikrein, leveling off at a one-to-one molar ratio of prekallikrein to F-HMW kininogen. The Kd value for binding of F-HMW kininogen-light chain (F-light chain) with prekallikrein was calculated to be 3.8 (+/- 0.6) X 10(-8) M and the stoichiometry was estimated as 1 to 1.2 on a molar basis from the Scatchard plot.     

Isolation and characterization of bovine factor VII.

Factor VII (proconvertin) has been purified approximately 5 x 10(5)-fold from bovine plasma with an overall yield of 30%. The isolation procedure involves barium sulfate adsorption and elution, DEAE-Sephadex batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis. The final product was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A minimal molecular weight of 45,500 was determined by sedimentation equilibrium. The molecular weight estimated by sodium dodecyl sulfate gel electrophoresis was 54,000. Factor VII is composed of a single polypeptide chain possessing an amino-terminal sequence of Ala-Asn-Gly-Phe-Leu-. The amino acid and carbohydrate compositions of factor VII are also reported.     

Human factor XII binding to the glycoprotein Ib-IX-V complex inhibits thrombin-induced platelet aggregation

Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ibalpha (glycocalicin) without activation, we now report that factor XIIa (0. 37 microm), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nm) binding to gel-filtered platelets and, at concentrations of 50 nm, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ibalpha. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by co-localizing with HK, to control the extent of platelet aggregation in vivo.