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.     

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.     

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.     

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.     

Isolation of bovine platelet cationic proteins which inhibit the surface-mediated activation of factor XII and prekallikrein

A possible role of bovine platelets in the surface-mediated activation of Factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine plasma. The washed platelets before and after aggregation by ADP, thrombin or collagen did not show any ability to trigger or accelerate the activation of Factor XII and prekallikrein. On the contrary, these aggregates showed a potent inhibitory activity on the activation of those zymogens triggered by kaolin, amylose sulfate and sulfatide. The inhibitory substances from the supernatant of the thrombin-induced aggregates were separated into two major fractions, a low affinity fraction and a high affinity fraction, on a heparin-Sepharose column. The high affinity protein was identified as platelet factor 4, based on the amino acid composition. From the low affinity fraction, a beta-thromboglobulin (beta-TG)-like substance and three kinds of unknown proteins, named LA1, LA2, and LA3, were isolated by gel-filtration on a column of Sephadex G-100 or Sephadex G-75 followed by chromatography on a column of Mono S. The molecular weights of LA1, LA2, and LA3 were estimated to be 35,000, 26,000, and 11,000, respectively, on SDS-PAGE. LA2 was identified as a carbohydrate-less LA1, as judged from the amino acid composition and carbohydrate content. The inhibitory activities of these five cationic proteins on the activation of Factor XII and prekallikrein mediated with amylose sulfate, sulfatide and kaolin were different from each other. In the case of kaolin-mediated activation, LA3 was the most potent inhibitor, while platelet factor 4 and beta-TG-like substance did not show any significant inhibitory activity. Moreover, the inhibitory activities of all the cationic proteins were not correlated with their anti-heparin activities. Since these proteins were rapidly liberated from platelets by the action of the stimulants, the present results demonstrate a negative role of platelets in the surface-mediated activation of Factor XII and prekallikrein.     

Rat plasma high-molecular-weight kininogen. A simple method for purification and its characterization

High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.     

Human high molecular weight kininogen. Studies of structure-function relationships and of proteolysis of the molecule occurring during contact activation of plasma.

Human high Mr kininogen was purified from normal plasma in 35% yield. The purified high Mr kininogen appeared homogeneous on polyacrylamide gels in the presence of sodium dodecyl sulfate and mercaptoethanol and gave a single protein band with an apparent Mr = 110,000. Using sedimentation equilibrium techniques, the observed Mr was 108,000 +/- 2,000. Human plasma kallikrein cleaves high Mr kininogen to liberate kinin and give a kinin-free, two-chain, disulfide-linked molecule containing a heavy chain of apparent Mr = 65,000 and a light chain of apparent Mr = 44,000. The light chain is histidine-rich and exhibits a high affinity for negatively charged materials. The isolated alkylated light chain quantitatively retains the procoagulant activity of the single-chain parent molecule. 125I-Human high Mr kininogen undergoes cleavage in plasma during contact activation initiated by addition of kaolin. This cleavage, which liberates kinin and gives a two-chain, disulfide-linked molecule, is dependent upon the presence of prekallikrein and Factor XII (Hageman factor) in plasma. Addition of purified plasma kallikrein to normal plasma or to plasmas deficient in prekallikrein or Factor XII in the presence or absence of kaolin results in cleavage of high Mr kininogen and kinin formation.     

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.     

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.     

Human plasma kallikrein: purification, enzyme characterization and quantitative determination in plasma.

Human plasma kallikrein was isolated from a plasma fraction related to Cohn fraction IV4 by affinity- and Sephadex G-150 chromotography yielding a material with 17.3 TAME-U/A280 unit. The preparation was characterized by immunological and enzymatic methods. Complex formation with alpha2-macroglobulin, C1-inactivator and aprotinin was demonstrated by immunoelectrophoresis. The bradykinin release from high-molecular weight kininogen and its inhibition by antibodies to kallikrein, AT III and AT III-heparin complex were measured using a biological test system (rat uterus). Time dependent inactivation of kallikrein by AT III, and AT III-heparin complex was shown by means of a synthetic kallikrein substrate: Bz-Pro-Phe-Arg-pNan. The same substrate was used to measure the activation of prekallikrein in plasma by kaolin and F XII a. Antibodies raised against kallikrein were shown to inhibit the reaction specifically. A quantitative determination of plasma prekallikrein by electroimmunodiffusion according to Laurell was developed: the plasma concentration in normal individuals was found to be 1.8 - 2.2 TAME-U/ml related to kallikrein activity; this corresponds approximately to 9 - 11 mg antigen/100 ml plasma.     

The inhibition of activated factor XII (Hageman factor) by antithrombin III: the effect of other plasma proteinase inhibitors.

Human factor XII was activated by adsorption onto kaolin in the presence of high molecular weight kininogen. The washed kaolin-containing precipitates activate prekallikrein to kallikrein. When antithrombin III was added to the reaction mixture, the conversion of prekallikrein to kallikrein was inhibited, the degree of inhibition depending on the concentration of antithrombin and the time of incubation. Heparin had a slight enhancing effect with low concentrations of antithrombin and short incubation times. However, the inhibition of the generated kallikrein by antithrombin III was markedly enhanced by heparin. Antithrombin III inhibited also the effect of activated factor XII on the partial thromboplastin time, using factor XII-deficient plasma. Of other plasma proteinase inhibitors used (α₁-antitrypsin, α₂-macroglobulin, C$\text{l}$-inactivator) only C$\text{l}$-inactivator inhibited activated factor XII.     

Localization of distinct functional domains on prekallikrein for interaction with both high molecular weight kininogen and activated factor XII in a 28-kDa fragment (amino acids 141-371).

The predominant autolytic form of human kallikrein, beta-kallikrein, was used to localize the high molecular weight kininogen (HK) binding site on kallikrein as well as the substrate recognition site for activated factor XII on prekallikrein. beta-Kallikrein is formed by autolysis of the kallikrein heavy chain to give two fragments of approximately 18 and 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A ligand binding technique established that the HK binding site on kallikrein residues on the 28-kDa fragment of the heavy chain. Limited NH2-terminal sequencing of this portion of beta-kallikrein showed that this fragment of the heavy chain consists of the COOH-terminal 231 amino acids of the heavy chain. A panel of five murine monoclonal antibodies to human prekallikrein (PK) were found to have epitopes on this same fragment of the heavy chain. None of the monoclonal antibodies were able to block binding of HK to PK. Three of the monoclonal antibodies (13G11, 13H11, and 6A6) were able to inhibit the activation of PK to kallikrein in both a plasma system and a purified system. The 28-kDa fragment of the PK heavy chain was purified and was able to compete with HK for binding to PK. The HK binding site and the site of recognition of factor XII are separate and distinct on PK, and both are contained in the COOH-terminal 231 amino acids of the PK heavy chain.     

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 fragments of bovine high molecular weight kininogen promote osteoblast proliferation in vitro

High molecular weight (HMW) kininogen is known to be a large plasma protein and cleaved by plasma proteinase kallikrein, then it generates four fragments in the blood coagulation cascade: heavy chain, bradykinin, fragment 1.2, and light chain. The fragment 1.2 has also been found in the basic protein fraction of bovine milk as a bioactive protein which promotes osteoblast proliferation. The milk basic protein has been shown to be a multi functional edible protein which promotes bone formation and inhibits bone resorption. In the present study, we purified the fragment 1.2 from bovine plasma and assessed it could promote osteoblast proliferation and posses the activity after pepsin digestion. Purified plasma HMW kininogen did not promote the proliferation, however, the kallikrein-cleaved HMW kininogen promoted the proliferation. The fragment 1.2, purified from the proteolysate, also promoted the proliferation. The pepsin digestion was performed according to the method of the assessment of allergenesity of genetically modified crops. After pepsin digestion, the fragment 1.2 generated resistant fragments and showed the promoting activity of osteoblast proliferation. These results suggest that the enzymatically-digested fragments of bovine HMW kininogen are able to be a naturally occurred active protein that promotes the bone formation by oral administration.     

Bovine milk kininogen fragment 1.2 promotes the proliferation of osteoblastic MC3T3-E1 cells

The active component on the proliferation of osteoblastic MC3T3-E1 cells was purified and identified from bovine milk. The growth-promoting activity was measured by [(3)H]thymidine incorporation on the cell. The purified protein showed a molecular size of 17 kDa on SDS-PAGE. Its amino-terminal amino acid sequence was very similar to the internal sequence of bovine high molecular weight (HMW) kininogen, which comprises fragment 1.2. The promotion of proliferation was specific for osteoblastic MC3T3-E1 cells, not for fibroblast BALB/3T3 cells. In blood coagulation, HMW kininogen is considered to be cleaved by a specific enzyme kallikrein. HMW kininogen then releases two peptides, a biologically active peptide bradykinin and fragment 1.2, but the fate of fragment 1.2 is unknown. This milk-derived protein that comprises to fragment 1.2 showed a growth-promoting activity of osteoblasts. We propose the possibility that milk plays an important role in bone formation by supplying the active agent for osteoblasts as well as supplying calcium.     

Studies on human high molecular weight (HMW) kininogen. II. Structural change of HMW kininogen by the action of human plasma kallikrein

We have investigated in detail the cleavage of human high molecular weight (HMW) kininogen by human plasma kallikrein and revealed the formation of a nicked kininogen and a novel kinin-free protein (KFP) as intermediate cleavage products. The cleavage of a single chain HMW kininogen (Mr=120,000) by plasma kallikrein was a three-step reaction. The first cleavage yielded a nicked kininogen composed of two disulfide-linked 62,000 and 56,000 daltons chains. The second cleavage yielded kinin and an intermediate kinin-free protein, KFP-I, which was apparently of equal size to the nicked kininogen. The third cleavage yielded a stable kinin-free protein, KFP-II, composed of two disulfide-linked 62,000 and 45,000 daltons chains. The liberation of an 8,000 daltons fragment was identified when the 56,000 daltons chain isolated by SP-Sephadex C-50 chromatography of reduced and alkylated KFP-I was cleaved by plasma kallikrein into the 45,000 daltons chain. Although the antiserum against HMW kininogen cross-reacted with low molecular weight (LMW) kininogen, the antiserum against the 45,000 daltons chain was specific for HMW kininogen. These results suggest that the antigenic determinant groups common to HMW and LMW kininogens are located in the 62,000 daltons heavy chain, while those specific for HMW kininogen are located in the 45,000 daltons light chain, which is known to retain blood coagulation activity.     

Surface-dependent activation of human factor XII (Hageman factor) by kallikrein and its light chain

In this paper we report the effect of sulfatides on the rate constants of factor XII activation by kallikrein and its isolated light chain (the domain of kallikrein that contains the active site of the enzyme). In the absence of sulfatides, kallikrein and the light chain were equally effective in factor XII activation (k1 = 1.57 X 10(3) M-1 s-1 at pH 7.0). The pH optima were the same (pH 7.0) and the reaction was not affected by variation of the ionic strength. Sulfatides strongly increased the rate constants of factor XIIa formation. In the presence of sulfatides kallikrein was, however, much more active than its light chain. At 330 microM sulfatides, pH 7.0 and 100 mM NaCl the rate constants of factor XII activation were 5.34 X 10(6) M-1 s-1 and 4.17 X 10(4) M-1 s-1 for kallikrein and its light chain, respectively. The pH optimum of factor XII activation by kallikrein in the presence of sulfatides was shifted to pH 6.3, and the reaction became highly ionic-strength-dependent. The rate constant increased considerably at decreasing NaCl concentrations. The optimum pH for light-chain-dependent factor XII activation in the presence of sulfatides remained unaltered and the reaction was not affected by the ionic strength. Binding studies revealed that both kallikrein and factor XII bind to the sulfatide surface, whereas no binding of the light chain of kallikrein was detectable. The isolated heavy chain of kallikrein had the same binding properties as kallikrein, which indicates that the heavy-chain domain contains the functional information for kallikrein binding to sulfatides. Since the effects of pH and ionic strength on the rate constants of kallikrein-dependent factor XII activation in the presence of sulfatides correlated with effects on the binding of kallikrein, it is concluded that under these conditions surface-bound factor XII is activated by surface-bound kallikrein. Our data suggest that sulfatides stimulate kallikrein-dependent factor XII activation by two distinct mechanisms: by making factor XII more susceptible to peptide bond cleavage by kallikrein and by promoting the formation of the enzyme-substrate complex through surface binding of kallikrein and factor XII.     

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.     

Hageman factor-dependent pathways: mechanism of initiation and bradykinin formation

The concentration of bradykinin in human plasma depends on its relative rates of formation and destruction. Bradykinin is destroyed by two enzymes: a plasma carboxypeptidase (anaphylatoxin inactivator) removes the COOH-terminal arginine to yield an inactive octapeptide, and a dipeptidase (identical to the angiotensin-converting enzyme) removes the COOH-terminal Phe-Arg to yield a fragment of seven amino acids that is further fragmented to an end product of five amino acids. Formation of bradykinin is initiated on binding of Hageman factor (HF) to certain negatively charged surfaces on which it autoactivates by an autodigestion mechanism. Initiation appears to depend on a trace of intrinsic activity present in HF that is at most 1/4000 that of activated HF (HFa); alternatively traces of circulating HFa could subserve the same function. HFa then converts coagulation factor XI to activated factor XI (XIa) and prekallikrein to kallikrein. Kallikrein then digests high-molecular-weight kininogen (HMW-kininogen) to form bradykinin. Prekallikrein and factor XI circulate bound to HMW-kininogen and surface binding of these complexes is mediated via this kininogen. In the absence of HMW-kininogen, activation of prekallikrein and factor XI is much diminished; thus HMW-kininogen has a cofactor function in kinin formation and coagulation. Once a trace of kallikrein is generated, a positive feedback reaction occurs in which kallikrein rapidly activates HF. This is much faster than the HF autoactivation rate; thus most HFa is formed by a kallikrein-dependent mechanism. HMW-kininogen is also therefore a cofactor for HF activation, but its effect on HF activation is indirect because it occurs via kallikrein formation. HFa can be further digested by kallikrein to form an active fragment (HFf), which is not surface bound and acts in the fluid phase. The activity of HFf on factor XI is minimal, but it is a potent prekallikrein activator and can therefore perpetuate fluid phase bradykinin formation until it is inactivated by the C1 inhibitor. In the absence of C1 inhibitor (hereditary angioedema) HFf may also interact with C1 and activate it enzymatically. The resultant augmented bradykinin formation and complement activation may account for the pathogenesis of the swelling characteristic of hereditary angioedema and the serologic changes observed during acute attacks.     

Autoactivation of human plasma prekallikrein

Incubation of purified human plasma prekallikrein with sulfatides or dextran sulfate resulted in spontaneous activation of prekallikrein as judged by the appearance of amidolytic activity toward the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide. The time course of generation of amidolytic activity was sigmoidal with an apparent lag phase that was followed by a relatively rapid activation until finally a plateau was reached. Soybean trypsin inhibitor completely blocked prekallikrein activation whereas corn, lima bean, and ovomucoid trypsin inhibitors did not. The Ki of the reversible inhibitor benzamidine for autoactivation (240 microM) was identical to the Ki of benzamidine for kallikrein. Thus, spontaneous prekallikrein activation and kallikrein showed the same specificity for a number of serine protease inhibitors. This indicates that prekallikrein is activated by its own enzymatically active form, kallikrein. Immunoblotting analysis of the time course of activation showed that, concomitant with the appearance of amidolytic activity, prekallikrein was cleaved. However, prekallikrein was not quantitatively converted into two-chain kallikrein since other polypeptide products were visible on the gels. This accounts for the observation that in amidolytic assays not all prekallikrein present in the reaction mixture was measured as active kallikrein. Kinetic analysis showed that prekallikrein activation can be described by a second-order reaction mechanism in which prekallikrein is activated by kallikrein. The apparent second-order rate constant was 2.7 X 10(4) M-1 s-1 (pH 7.2, 50 microM sulfatides, ionic strength I = 0.06, at 37 degrees C). Autocatalytic prekallikrein activation was strongly dependent on the ionic strength, since there was a considerable decrease in the second-order rate constant of the reaction at high salt concentrations. In support of the autoactivation mechanism it was found that increasing the amount of kallikrein initially present in the reaction mixture resulted in a significant reduction of the lag period and a rapid completion of the reaction while the second-order rate constant was not influenced. Our data support a prekallikrein autoactivation mechanism in which surface-bound kallikrein activates surface-bound prekallikrein.