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At least 19 recordsLinked to original sources
PubMed1986

Characterization of a variant prekallikrein, prekallikrein Long Beach, from a family with mixed cross-reacting material-positive and cross-reacting material-negative prekallikrein deficiency.

Studies of plasma prekallikrein in a family with prekallikrein deficiency were made. Three children had no clotting activity but approximately 35% antigen levels, and the mother and five children had twice as much prekallikrein antigen as clotting activity, suggesting the presence of a dysfunctional molecule. A nonfunctional variant form of prekallikrein was purified that contained no prekallikrein clotting activity. The variant and normal molecules were both 80,000 mol wt, immunologically indistinguishable and complexed similarly with high molecular weight kininogen. Isoelectric focusing studies suggested a difference of one charged amino acid residue. The variant was cleaved by beta-Factor XIIa 200 times slower than the normal molecule, and no amidolytic activity was detected for the cleaved variant. These data and other observations suggest that an amino acid was substituted in the variant near the NH2-terminal end of the kallikrein light chain resulting in slower cleavage by beta-Factor XIIa and the absence of enzymatic activity.

Adult↗
PubMed1987

A monoclonal anti-human plasma prekallikrein antibody that inhibits activation of prekallikrein by factor XIIa on a surface.

Of five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was approximately 80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein-HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited approximately 70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (light chain).

Antibodies, Monoclonal↗
PubMed1984

Plasma prekallikrein assay: reversible inhibition of C-1 inhibitor by chloroform and its use in measuring prekallikrein in different mammalian species.

The assay of plasma prekallikrein requires activation of prekallikrein to kallikrein and sufficient inactivation of the plasma protease inhibitors of kallikrein to accurately measure the generated kallikrein activity. One method of elimination of the plasma protease inhibitors to kallikrein is to chemically pretreat the plasma. Methylamine has previously been employed to selectively inactivate alpha 2-macroglobulin. Our study examines the effect of sequential preincubation of plasma with chloroform and methylamine on the plasma prekallikrein assay. Chloroform was demonstrated to be a chemical inhibitor of purified C-1 inhibitor, but alpha 2-macroglobulin was not. Chloroform inhibition of C-1 inhibitor was not caused by precipitation of the protein into the interface between the water and organic solvent phase. Greater than 95% of C-1 inhibitor antigen was recovered in the supernatant of chloroform-treated purified C-1 inhibitor, and chloroform-saturated buffer inhibited purified C-1 inhibitor. Chloroform did not dissociate a preformed complex of kallikrein and C-1 inhibitor, but its inhibition of C-1 inhibitor was reversible. The addition of methylamine to plasma pretreated with chloroform in the plasma prekallikrein assay allowed for only a slight increase in the amount of kallikrein measured at 1 minute kaolin activation times, but provided for sustained measurement of activated prekallikrein when kaolin activation times were 5 to 7 minutes. Without chemical pretreatment, prekallikrein was not measurable in rabbit plasma. Both rabbit and pig plasma prekallikrein was measurable after exposure of the plasma to chloroform and methylamine, although the peak activation times and the contribution of each animals' protease inhibitors varied with the species. Our results show that chloroform is a reversible inhibitor of C-1 inhibitor, and that the plasma prekallikrein assay in which it is used is useful for the measurement of prekallikrein in nonhuman mammalian plasma samples.

Animals↗
PubMed1980

Studies on prekallikrein of bovine plasma. II. Activation of prekallikrein with proteinases and properties of kallikrein activated by bovine Hageman factor.

Activation of bovine plasma prekallikrein was investigated with several proteinases. Highly purified bovine plasma prekallikrein was rapidly activated to kallikrein [EC 3.4.21.8] by bovine activated Hageman factor, trypsin [EC 3.4.21.4] and Pronase P (proteinases from Streptomyces griseus) and more gradually by papain [EC 3.4.22.2] and ficin [EC 3.4.22.3]. Activation of prekallikrein was also observed with bovine plasmin [EC 3.4.21.7], but not with bovine clotting factors Xa (Stuart factor) [EC 3.4.21.6] and IXa (Christmas factor) or thrombin [EC 3.4.21.5]. Urokinase [EC 3.4.99.26], Reptilase, collagenase [EC 3.4.24.3], elastase [EC 3.4.21.11], alpha-chymotrypsin [EC 3.4.21.1], Nagarse [EC 3.4.21.14], and stem bromelain [EC 3.4.22 4] did not convert prekallikrein to kallikrein. Plasma kallikrein activated to Hageman factor released kinin rapidly from bovine high molecular weight (HMW) kininogen. However, from bovine low molecular weight (LMW) kininogen, liberation of kinin was extremely slow. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI), Trasylol, diisopropylfluorophosphate (DFP), and N-alpha-tosyl-L-lysine chloromethylketone (TLCK), but not by egg-white trypsin inhibitor (EWTI), lima bean trypsin inhibitor (LBTI), heparin or hexadimethrine bromide (Polybrene). The kallikrein formed an enzyme-inhibitor complex with SBTI and Trasylol, but not with LBTI. Prekallikrein did not react with SBTI. Prekallikrein consists of a single polypeptide chain of molecular weight about 90,000, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Activation of prekallikrein by Hageman factor was found to involve cleavage of the single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. The peptide bond cleaved in prekallikrein by the activation was an Arg-X peptide bond on a disulfide-bridged polypeptide chain.

Animals↗
PubMed1977

Prekallikrein deficiency in a kindred with kininogen deficiency and Fitzgerald trait clotting defect. Evidence that high molecular weight kininogen and prekallikrein exist as a complex in normal human plasma.

Plasma from an individual with a hereditary deficiency of kininogens is deficient in kininogen antigens; heterozygous relatives are partially deficient in plasma kininogen antigens. In addition, plasma from the proband is partially deficient in functional and antigenic properties of a plasma prekallikrein, and the relatives heterozygous for kininogen deficiency are also partially deficient in the plasma prekallikrein. It is possible that the defects are both inherited and that the inheritance of a deficiency of prekallikrein is genetically linked to the inheritance of a deficiency of kininogen. Alternatively, it is possible that the deficiency of prekallikrein may be due to its hypercatabolism which could be a consequence of a deficiency of high molecular weight kininogen that may stabilize the prekallikrein in plasma. Evidence to support this possibility is presented by the fact that prekallikrein and high molecular weight kininogen apparently exist as a complex in normal plasma, because monospecific antiserum to kininogen removed both high molecular weight kininogen and prekallikrein from plasma, and vice versa. Moreover, prekallikrein was not adsorbed from kininogen-deficient plasma by antiserum to kininogen unless high molecular weight kininogen was first added to the plasma. Low molecular weight kininogen did not participate in these reactions.

Blood Coagulation↗
PubMed1971

A prealbumin activator of prekallikrein. II. Derivation of activators of prekallikrein from active Hageman factor by digestion with plasmin.

Activation of a plasma fraction containing unactivated Hageman factor and prekallikrein followed by chromatography of this fraction on DEAE-cellulose revealed four peaks having bradykinin-generating activity. Peak 1 contained kallikrein; peaks 2-3, 4, and 5 each contained prekallikrein-activating activity. Elution of peaks 2-3, 4, and 5 from disc gels after electrophoresis at pH 9.3 revealed peaks of prekallikrein-activating activity located at 5-8, 11-12, 15-16, and 20-26 mm, each of which was associated with a peak of clot-promoting activity which specifically corrected Hageman factor deficiency. Conversion of peak 2 to peaks 3, 4, and 5 was associated with a progressive decrease in size, increase in net negative charge, increased prekallikrein-activating activity, and decreased ability to correct Hageman factor deficiency. Plasminogen and plasmin were found on a DEAE-cellulose chromatogram of serum overlapping peaks 2 and 3. Incubation of active Hageman factor with streptokinase-activated plasminogen resulted in enhanced ability of the mixture to activate prekallikrein. Assessment of the products of this reaction by disc gel electrophoresis demonstrated the formation of the prealbumin prekallikrein activator corresponding to the major prekallikrein activator generated by contact activation of human plasma. The conversion of plasminogen to plasmin and the subsequent cleavage of Hageman factor by plasmin to form activators of prekallikrein represents one pathway in which coagulation, fibrinolysis, and inflammation are linked.

Bradykinin↗
PubMed1992

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase. II. Kinetic analysis and identification of scissile bond of prekallikrein in the activation.

Activation of human plasma prekallikrein by a bacterial metalloendopeptidase, Pseudomonas aeruginosa elastase, was reported (Shibuya et al. (1991) Biochim. Biophys. Acta 1097, 23-27). Details of the activation process were presently studied. The activation accompanied limited proteolysis of a peptide bond inside of a disulfide bridge of prekallikrein molecule. Amino acid sequencing analysis of the newly generated amino-terminal revealed that the cleavage site was Arg371-Ile372 bond which is the scissile bond in the activation of prekallikrein with trypsin-type proteinases. A pentapeptide substrate, 2-aminobenzoyl-Ser-Thr-Arg-Ile-Val-4- nitrobenzylamide, which contained the amino acid sequence identical to that around the scissile bond of prekallikrein was synthesized. Pseudomonal elastase, indeed, hydrolyzed the substrate at Arg-Ile bond with the kinetic parameters of Km = 118 microM, kcat = 1.56/s and kcat/Km = 1.33.10(4)/s M. These results indicated that the Arg371-Ile372 bond was sensitive not only to trypsin-type serine proteinases, but also a bacterial metalloproteinase. Kinetic analysis of the prekallikrein activation by pseudomonal elastase, however, revealed that the activation rate was slow, though the Km values was good enough to expect an occurrence of this activation in vivo (Km = 248 nM, kcat = 6.8.10(-4)/s, and kcat/Km = 2.7.10(3)/s M). The activation rate of prekallikrein by pseudomonal elastase in Hageman factor deficient plasma was remarkably improved when the plasma was reconstituted with purified Hageman factor molecule. From the results, a biological significance of the proteinase cascade in the plasma kinin generation was also indicated. The present in vitro study might support the hypothesis that the Hageman factor/kallikrein-kinin system plays an important role in bacterial infection including the pseudomonal one.

Amino Acid Sequence↗
PubMed1980

Kinetics of activation of prekallikrein by prekallikrein activator.

A 28 000 molecular weight activator of prekallikrein was isolated from human plasma, and the kinetics of its enzymic activity toward prekallikrein was investigated. The activation follows Michaelis-Menten kinetics with a kcat of approximately 3 S(-1); Km is strongly dependent upon the ionic strength. Under suitable conditions the activation obeys first-order kinetics, and the first-order rate constant may be used to quantitate prekallikrein activator activity. Thus a two-stage assay, in which the first step involves the activation of prekallikrein by the activator and the second step quantitates the kallikrein generated, was developed to allow the measurement of prekallikrein activator in biologic samples. The prekallikrein activator content of therapeutic protein solutions, as determined by means of this assay, correlated well with the hypotensive activity of these solutions as determined in an animal model.

Enzyme Activation↗
PubMed1980

Purification of guinea-pig plasma prekallikrein. Activation by prekallikrein activator derived from guinea-pig skin.

Prekallikrein was purified from guinea-pig plasma. The prekallikrein appeared homogeneous as a single-chain protein on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. The apparent molecular weight was 82 000 by SDS-polyacrylamide gel electrophoresis, 99 000 by gel filtration on a Sephadex G-150 column and 84 500 (protein part) by amino acid analysis. The isoelectric point was approx. 9.0. The purification method yielded 3.8 mg (A280 3.800) of prekallikrein from 500 ml of plasma. Kallikrein was generated from the prekallikrein by limited proteolytic action of a prekallikrein activator which was derived from guinea-pig skin. From analysis using SDS-polyacrylamide gel electrophoresis, the kallikrein has two fragments with apparent molecular weights of 52 000 and 40 000 which are linked by disulfide bond(s). The 40 000 molecular weight fragment was shown to incorporate [3H]diisopropylfluorophosphate. The kallikrein hydrolyzed the synthetic substrates containing the Phe-Arg sequence at the COOH-terminal, and it cleaved carbobenzyloxy-Phe-Arg-4-methylcoumaryl-7-amide more readily than Pro-Phe-Arg-methylcoumaryl-7-amide. The Km for the kallikrein with carbobenzyloxy-Phe-Arg-methylcoumaryl amide was 2 times 104 M. Also, the kallikrein showed negligible activities on peptide-methylcoumaryl amide-substrate for alpha-thrombin, Factor Xa or plasmin.

Amino Acids↗
PubMed1993

Mapping of the high molecular weight kininogen binding site of prekallikrein. Evidence for a discontinuous epitope formed by distinct segments of the prekallikrein heavy chain.

Prekallikrein, a glycoprotein involved in contact phase activation, circulates in plasma in the form of a binary complex with high molecular weight kininogen (H-kininogen). The binding to H-kininogen is mediated by the prekallikrein heavy chain consisting of four repetitive domains, A1-A4. To define more precisely the region(s) involved in kininogen binding, we have employed an affinity cross-linking strategy with a synthetic peptide of 31 residues which mimics the prekallikrein binding site of H-kininogen. Cross-linking of the radiolabeled peptide to (pre)kallikrein revealed a binding segment in the NH2-terminal portion of the prekallikrein heavy chain; another binding segment was located in the COOH-terminal part of the heavy chain. The latter binding segment is harbored by a previously identified fragment of the kallikrein heavy chain involved in H-kininogen binding (Page, J.D., and Colman, R.W. (1991) J. Biol. Chem. 266, 8143-8148). Chemical cleavage of the heavy chain cross-linked with the radiolabeled peptide mapped the NH2-terminal binding segment to 60 residues (positions 53-112) of A1. Synthesis of a peptide (positions 56-86) and development of specific antibodies to this peptide narrowed down the kininogen binding segment to 31 residues of the center portion of A1. This NH2-terminal segment is equivalent to a kininogen binding site previously identified in factor XI (Baglia, F.A., Jameson, B.A., and Walsh, P.N. (1992) J. Biol. Chem. 267, 4247-4252). We conclude that prekallikrein exposes at least two segments on its heavy chain portion which form a continuous surface thereby facilitating the intimate binding of the zymogen to its nonenzymatic cofactor, H-kininogen.

Amino Acid Sequence↗
PubMed1990

Association of Graves' disease and prekallikrein congenital deficiency in a patient belonging to the first CRM+ prekallikrein-deficient Italian family.

Severe prekallikrein (Fletcher factor) deficiency was diagnosed in a 49-year-old woman and in 3 of her siblings. Functional prekallikrein (PKK) activity was found below 1% by clotting assay and 20% by amidolytic assay in all the affected subjects; PKK cross-reacting material (CRM) was present in all the patients (antigen levels from 34% to 54%). This is the first CRM+ PKK-deficient family identified in Italy. The index patient was affected from Graves' disease: such association was previously reported in another patient with PKK congenital defect.

Cross Reactions↗
PubMed1978

Dextran-induced lowering of prekallikrein proactivator and prekallikrein in rat plasma.

The intravenous injection into rats of dextran (average MW 70,000) 10 mg/100 g caused marked hypotension after a delay of about 5 minutes. Blood samples collected by cardiac puncture at this time were tested for the amounts of prekallikrein activator (PKA) and kallikrein after acetone- and then kaolin activation of the plasminogen-free plasma. PKA was assayed by measuring the initial rate of release of benzoyl arginine esterase (BAEe) activity in a preparation of partially purified human prekallikrein, and kallikrein was assayed by measuring the BAEe esterase activity. Significant reductions of both parameters were registered, and the amount of high molecular weight kininogen (HMWK) present in the plasma was also reduced. Pretreatment of the rats with epsilon-aminocaproic acid intraperitoneally (200-300 mg/100 g) abolished the dextran-caused decreases in the plasma levels of the above mentioned factors, and reduced the fall in blood pressure. The addition of purified human HMWK to the plasma before the acetone activation procedure was started, increased the yield of PKA activity in the final enzyme preparation. When PKA was assayed after kaolin activation of plasma at 0 degrees using the method developed by Laake & Vennerød (1973a & b) for the determination of PKA (activated factor XII) in human plasma, no differences were registered between plasma from rats treated with dextran and plasma obtained from control rats. It is suggested that the low PKA activity of the acetone activated enzyme preparation from plasma of rats treated with dextran was due to the loss of HMWK or a fraction of HMWK.

Acetone↗
PubMed1978

Determination of prekallikrein in human plasma: optimal conditions for activating prekallikrein.

A method for the assay of human plasma prekallikrein in which a chromogenic synthetic tripeptide, PPAN, is used as a substrate for kallikrein is described. The conversion of prekallikrein to kallikrein is achieved by cold activation (0 degrees C) with water-soluble dextran sulfate. Conditions for obtaining optimal amounts of free kallikrein with respect to concentration of dextran sulfate, activation time, inhibitors (C-1-inactivator), and requirement of factor XII have been determined. The activation procedure is compared to other known procedures. The assay system was worked out for pooled normal plasma and is applicable to any plasma sample not liable to unwanted preactivation or incomplete inactivation, as revealed by control experiments. A survey in 15 apparently health individuals showed a mean activity of 476 +/- 58 (S.D.) mU/ml with a range of 385 to 586 mU/ml.

Acetone↗
PubMed1972

A prealbumin activator of prekallikrein. 3. Appearance of chemotactic activity for human neutrophils by the conversion of human prekallikrein to kallikrein.

Human plasma kallikrein has been shown to directly and selectively attract human neutrophils from a mixed leukocyte population. The capacity of plasma kallikrein to be chemotactic and to generate the nonapeptide bradykinin was maintained during progressive purification. While neither highly purified prekallikrein nor the prealbumin Hageman factor fragments were chemotactic alone, their interaction so as to convert prekallikrein to kallikrein yielded both chemotactic and kinin-generating activity. Both functions of kallikrein were inhibited by treatment with diisopropyl fluorophosphate, indicating an essential role for the active site of the enzyme in the expression of its chemotactic activity.

Binding Sites↗
PubMed1980

Function and immunochemistry of prekallikrein-high molecular weight kininogen complex in plasma.

Plasma from individuals with high molecular weight (HMW) kininogen deficiency has been reported to be deficient in prekallikrein as measured by radial immunodiffusion, prekallikrein coagulant activity, and/or kaolin-activated arginine esterase activity. The discovery that prekallikrein and HMW kininogen circulate as a complex in plasma led us to reevaluate the antigenic and functional properties of prekallikrein in HMW kininogen-deficient plasma as well as in normal plasma. The low prekallikrein antigen level in an individual with HMW kininogen deficiency was corrected to the normal range (80-95%) by the addition of 0.2 U/ml of purified HMW kininogen. A similar increase in apparent prekallikrein antigen was observed when purified prekallikrein and HMW kininogen were combined. The correction of the apparent prekallikrein defect in this HMW kininogen-deficient plasma coincided with the formation of a prekallikrein-HMW kininogen complex as demonstrated by immunoelectrophoresis. Similar findings were demonstrated with purified prekallikrein and HMW kininogen by immunoelectrophoresis as well as crossed immunoelectrophoresis. The coagulant activity in HMW kininogen-deficient plasma was increased in a dose-dependent manner by the addition of HMW kininogen, reaching 85% of normal level at a concentration of 0.2 U/ml. Kaolin-activated arginine esterase activity (kallikrein) in HMW kininogen-deficient plasma was fully corrected when HMW kininogen was added to the deficient plasma after depletion of kallikrein inhibitors. The functional and antigenic concentration of prekallikrein in plasma from four other HMW kininogen-deficient individuals was similarly corrected to normal after adding HMW kininogen. Addition of HMW kininogen increased the apparent prekallikrein activity in native normal plasma (as measured by esterase activity) but not in normal plasma in which inhibitors were inactivated. The apparent prekallikrein antigen concentration (as measured by radial immunodiffusion or electroimmunodiffusion) increased upon addition of HMW kininogen. Immunoelectrophoresis as well as gel filtration of normal plasma revealed the presence of free prekallikrein (17-38% of the total) in addition to the HMW kininogen-prekallikrein complex previously reported. This study emphasizes the influence of HMW kininogen on both functional and immunologic determinations of prekallikrein.

Antigens↗
PubMed1990

High molecular weight kininogen-binding site of prekallikrein probed by monoclonal antibodies.

A panel of monoclonal antibodies against human prekallikrein was raised in mice and characterized with respect to the major antigenic epitopes. Of 18 antibodies, nine were directed against the light chain portion performing the proteolytic function of activated kallikrein, and nine recognized the heavy chain mediating the binding of prekallikrein to high molecular weight (H-)kininogen. Among the anti-heavy chain antibodies, one (PK6) interfered with the procoagulant activity of prekallikrein, and prolonged in a concentration-dependent manner the activated partial thromboplastin time of reconstituted prekallikrein-deficient plasma (Fletcher type). Antibody PK6 was subtyped IgG1,k and had an apparent Kass of 6.8 +/- 0.44.10(8) M-1 for prekallikrein. Functional analyses revealed that PK6 does not interfere with prekallikrein activation by activated Hageman factor (beta-F XIIa), and has no effect on the kininogenase function of activated kallikrein. Monoclonal antibody PK6 but none of the other anti-heavy chain antibodies completely prevented complex formation of prekallikrein with H-kininogen, and readily dissociated preformed complexes of prekallikrein and H-kininogen. Likewise, Fab' and F(ab')2 fragments of PK6 blocked H-kininogen binding to prekallikrein. A synthetic peptide of 31 amino acid residues encompassing the entire prekallikrein binding region of H-kininogen effectively competed with PK6 for prekallikrein binding indicating that the target epitope of PK6 is juxtaposed to, if not incorporated in the H-kininogen-binding site of prekallikrein. Extensive cross-reactivity of PK6 with another H-kininogen-binding protein of human plasma, i.e. factor XI, suggested that the structure of the target epitope of PK6 is well conserved among prekallikrein and factor XI, as would be expected for the kininogen-binding site shared by the two proteins. It is anticipated that monoclonal antibody PK6 will be an important tool for the precise mapping of the hitherto unknown kininogen-binding site of prekallikrein.

Antibodies, Monoclonal↗
PubMed1987

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.

Dextran Sulfate↗