Evaluation of a microassay for human plasma prekallikrein

Current methods for determining plasma prekallikrein, one of three zymogens of the contact phase of plasma proteolysis, are laborious and impractical for general use in a clinical laboratory. Therefore, we have developed a simple, reliable assay using commercially available reagents. By use of the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCI (S-2302), a functional assay, performed in a 96-well microplate, was designed to measure prekallikrein in plasma. Measures were taken to destroy the naturally occurring plasma protease inhibitors of kallikrein without affecting the integrity of the plasma prekallikrein, which allowed complete activation of the zymogen to virtually 100% of predicted activity when compared with that of purified kallikrein. Besides permitting full activation, the use of low pH to destroy critical plasma protease inhibitors allowed the conversion of prekallikrein to kallikrein in as many as 44 plasma samples at one time without the tedious individual timing step usually required to activate each sample. An excellent correlation was found (r = 0.92) when this functional microassay was compared with a functional spectrophotometric assay performed in three subject populations: normal individuals, women receiving oral contraceptives (who frequently exhibit high plasma prekallikrein concentrations), and patients with liver disease (who manifest low plasma prekallikrein levels). This plasma prekallikrein microassay should facilitate the increased determination of plasma prekallikrein in pathophysiologic conditions as well as the monitoring of the progression of various diseases in which contact activation occurs.     

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.     

Plasma prekallikrein: quantitative determination by direct activation with Hageman factor fragment (beta-XIIa)

This report describes a plasma prekallikrein assay which, unlike methods that employ contact activation, is not affected by the factor XII or HMW kininogen content of the plasma analyzed. In this assay beta-XIIa, a potent fluid-phase activator of prekallikrein, is added to diluted plasma in the presence of 20% acetone (to inactivate kallikrein inhibitors) at 30 degrees C and the kallikrein generated is measured with the chromogenic substrate S-2302. Prekallikrein is fully activated under these conditions and the activity remains stable for at least 30 hr. The mean prekallikrein concentration in plasma samples from 24 healthy individuals was 1.50 +/- 0.35 (S.D.) S-2302 U/ml, corresponding to 20.3 +/- 4.7 micrograms/ml prekallikrein (the specific activity of highly purified human prekallikrein was determined to be 74 S-2302 U/mg). In contrast, the mean concentration in five plasma samples from patients deficient in HMW kininogen was 0.38 +/- 0.02 S-2302 U/ml. No activity was generated in prekallikrein-deficient plasma, and essentially normal levels (1.35 +/- 0.18 S-2302 U/ml) were measured in plasmas from three patients with factor XII deficiency. Plasma prekallikrein was also quantitated by radial immunodiffusion, which gave results similar to those obtained by functional assay with beta-XIIa. The determination of plasma prekallikrein by direct activation with beta-XIIa in the presence of acetone offers several advantages over the use of contact activators such as dextran sulfate. These advantages include complete inactivation of kallikrein inhibitors and total activation of prekallikrein (even in plasmas deficient in other contact factors) without simultaneous generation of plasmin.     

Prorenin-renin conversion by the contact activation system in human plasma: role of plasma protease inhibitors

Acid-pretreated normal human plasma generates renin activity at 0 degree C and neutral pH by the activation of prorenin. The activation is caused by kallikrein generated from prekallikrein by activated factor XII. Nonacidified plasma also generates renin at 0 degree C, but at a lower rate (cold-promoted activation). In normal plasma, 14% +/- 1% of prorenin (mean +/- SEM, n = 30) was activated during incubation at 0 degree C for 7 days (range 6% to 26%). Cold-promoted activation of prorenin was within the normal range in plasma deficient in factor XI, X, IX, VIIIC, VII, V, prothrombin, or high mol wt kininogen. Cold-promoted activation of prorenin was less than or equal to 1% in plasma deficient in factor XII or prekallikrein. Reconstitution of these plasmas with highly purified factor XII or prekallikrein restored normal prorenin activation. Correction of high mol wt kininogen deficiency had no effect. Thus cold-promoted activation of prorenin depends on the presence of factor XII and prekallikrein, whereas the other clotting factors are not essential. The influence of the inhibitors C1 esterase-inhibitor, alpha 2-macroglobulin, antithrombin III, and alpha 1-antitrypsin on the activation of prorenin was studied in factor XII-deficient plasma from which one or more of these inhibitors had been selectively removed by immunoadsorption. Factor XII was subsequently added, and the generation of renin at 37 degrees C was observed after complete factor XII-high mol wt kininogen-mediated activation of prekallikrein induced by dextran sulfate. No activation of prorenin was observed at 37 degrees C after depletion of C1 esterase inhibitor, alpha 2-macroglobulin, antithrombin III, or alpha 1-antitrypsin. When prekallikrein was activated in plasma depleted of both C1 esterase-inhibitor and alpha 2-macroglobulin, 6% of prorenin was activated in 2 hours at 37 degrees C. After additional depletion of antithrombin III, the activation increased to 47%. These results indicate that the contact activation system is capable of activating prorenin in plasma at physiologic pH and temperature when the three most important kallikrein inhibitors, C1 esterase-inhibitor, alpha 2-macroglobulin, and antithrombin III, are absent.     

HUMAN PLASMA ALPHA 2-MACROGLOBULIN : AN INHIBITOR OF PLASMA KALLIKREIN

Activation of plasma kallikrein arginine esterase activity by kaolin resulted in peak activity at 1 min of incubation and a 50% reduction in activity at 5 min in normal plasma, and 30% reduction in the plasma of patients with hereditary angioneurotic edema who lacked the C1 inactivator. The peak esterolytic activity was inhibited by soybean trypsin inhibitor whereas the 5 min activity was resistant to this inhibitor. Acid treatment of normal and hereditary angioneurotic edema plasma destroyed the factor responsible for the fall in esterase activity at 5 min and the factor which rendered the esterase resistant to soybean trypsin inhibitor. Purified α₂-macroglobulin inhibited approximately 50% of the TAMe esterase activity of purified plasma kallikrein without changing its activity toward basic amino acid esters. The interaction between the α₂-macroglobulin and kallikrein resulted in alterations in the gel filtration chromatographic pattern of the TAMe esterase and biologic activity of kallikrein, indicating that kallikrein was bound to the α₂-macroglobulin. The TAMe esterase activity of this complex, isolated by column chromatography, was resistant to C1 inactivator and SBTI. Studies of incubation mixtures of kallikrein, α₂-macroglobulin and C1 inactivator suggested that these inhibitors compete for the enzyme, and that the α₂-macroglobulin partially protects the esterase activity of kallikrein from C1 inactivator. The α₂-macroglobulin isolated from kaolin-activated plasma possessed 240 times the esterolytic activity of the α₂-macroglobulin purified from plasma treated with inhibitors of kallikrein and of its activation. The α₂-macroglobulin blocked the uterine-containing activity and vascular permeability-inducing effects of plasma kallikrein. These studies suggest that the α₂-macroglobulin is a major plasma inhibitor of kallikrein and provide a new example of an interrelationship between the coagulation, fibrinolytic, and kallikrein enzyme systems.     

Anaphylactic release of a prekallikrein activator from human lung in vitro.

We have demonstrated the in vitro IgE-mediated release of a prekallikrein activator from human lung. The lung prekallikrein activator was partially purified by sequential chromatography on sulfopropyl-Sephadex, DEAE-Sephacel, and Sepharose 6B. Purified human prekallikrein was converted to its active form (kallikrein) by the lung protease. The generated kallikrein was shown to be biologically active; that is, it generates bradykinin from purified human high-molecular weight kininogen and also cleaves benzoyl-propyl-phenyl-arginyl-p-nitroanilide, a known synthetic substrate of kallikrein. The lung prekallikrein activator differs from the known physiologic activators of prekallikrein (the activated forms of Hageman factor) with respect to: (a) size (it has a mol wt of approximately 175,000); (b) synthetic substrate specificity (D-propyl/phenyl/arginyl-p-nitroanilide is a substrate for the activated forms of Hageman factor, but not the lung protease); (c) antigenic specificity (an anti-Hageman factor immunoadsorbent column did not remove significant amounts of the lung protease, while it removed most of the activity of activated Hageman factor fragments); and (d) inhibition profile (the lung proteases was not inhibited by corn trypsin inhibitor). This prekallikrein activator provides a physiologic mechanism by which prekallikrein can be directly activated during IgE-mediated reactions of the lung. While the role of this lung prekallikrein activator in immediate hypersensitivity reactions and in other inflammatory processes is not clear, it does represent a first and important interface between IgE-mediated reactions and the Hageman factor-dependent pathways of the inflammatory response.     

Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate.

An automated assay of plasma prekallikrein is described. Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the hydrolytic activity of kallikrein to H-D-Pro-Phe-Arg-paranitroanilide subsequently measured.

The conversion was complete within 8 minutes and the amidolytic activity remained stable at least another 10 min at 37 ° C. This method worked in plasma deficient in Hageman factor (blood coagulation factor XII). Using anti-prekallikrein antibody and plasma deficient in prekallikrein, the amidolytic activity generated in normal plasma was identified as due to kallikrein. With plasma samples, the coefficients of variation (CV) for multiple measurements within run (n = 10) and between run (n = 10) were as low as 5.0% and 6.6%, respectively, and the minimum measurable concentration of prekallikrein in plasma was 10% of the normal level.     

The role of prekallikrein and high-molecular-weight kininogen in the contact activation of Hageman factor (factor XII) by sulfatides and other agents

Hageman factor (HF, factor XII), adsorbed to negatively charged agents, is transformed to an activated state in which it initiates reactions of the intrinsic pathway of thrombin formation by activating plasma thromboplastin antecedent (PTA, factor XI). High-molecular-weight kininogen (HMWK, Fitzgerald factor) and plasma prekallikrein accelerate these early steps in the clotting process. Questions have been raised about the role of HMWK in the activation of Hageman factor by surfaces. In the present studies, we report that the activation of purified human HF by sulfatides, ellagic acid, kaolin, or glass occurred in the absence of HMWK. Indeed, small amounts of HMWK inhibited activation of HF by ellagic acid. Physiological concentration of HMWK had little or no influence on the activation of HF by sulfatides, kaolin, or glass, but higher concentrations (3 to 6 times more) showed the same inhibitory effect as after activation by ellagic acid. This inhibitory property of HMWK may be attributed to competitive binding of HF and HMWK on the surface of the activating agents. In fact, when HF was added to kaolin or glass that had been incubated with HMWK and then washed, the inhibitory effect persisted, indicating HMWK that was bound to the surface blocked activation of HF. Studies with 125I-HF and 125I-HMWK supported this interpretation. Plasma prekallikrein accelerated activation of the amidolytic properties of HF by sulfatides, kaolin, or glass but did not influence activation of HF by ellagic acid. In the presence of plasma kallikrein, HMWK at moderate concentrations slightly accelerated the rate of activation of HF by activating agents other than ellagic acid. Higher concentrations of HMWK counteracted both the accelerating effect of prekallikrein and the inhibitory effect observed when HF was incubated with an excess of kaolin. These experiments, then, support the view that the procoagulant function of HMWK is localized to a point subsequent to the activation of HF.     

Immunochemical Studies of Plasma Kallikrein

A monospecific antibody against human plasma kallikrein has been prepared in rabbits with kallikrein further purified to remove gamma globulins. The antisera produced contained antikallikrein and also anti-IgG, in spite of only 8% contamination of kallikrein preparation with IgG. The latter antibody was removed by adsorption of antisera with either Fletcher factor-deficient plasma or with purified IgG. Both kallikrein and prekallikrein (in plasma) cross-react with the antibody with no apparent difference between the precipitation arcs developed during immunoelectrophoresis and no significant difference in reactivity when quantified by radial immunodiffusion.Kallikrein antibody partially inhibits the esterolytic and fully inhibits the proteolytic activity of kallikrein. In addition, the antibody inhibits the activation of prekallikrein, as measured by esterase or kinin release. The magnitude of the inhibition is related to the molecular weight of the activator used. Thus, for the four activators tested, the greatest inhibition is observed with kaolin and factor XII(A), while large activator and the low molecular weight prekallikrein activators are less inhibited.With the kallikrein antibody, the incubation of kallikrein with either plasma or partially purified C1 esterase inactivator results in a new precipitin arc, as detected by immunoelectrophoresis. This finding provides physical evidence for the interaction of the enzyme and inhibitor. No new arc could be demonstrated between kallikrein and alpha(2)-macroglobulin, or alpha(1)-antitrypsin, although the concentration of free kallikrein antigen decreases after interaction with the former inhibitor.By radial immunodiffusion, plasma from healthy individuals contained 103+/-13 mug/ml prekallikrein antigen. Although in mild liver disease, functional and immunologic kallikrein are proportionally depressed, the levels of prekallikrein antigen in plasma samples from patients with severe liver disease remains 40% of normal, while the functional kallikrein activity was about 8%. These observations suggest that the livers of these patients have synthesized a proenzyme that cannot be converted to active kallikrein.     

Human plasma prekallikrein (Fletcher factor) clotting activity and antigen in health and disease

Human plasma prekallikrein (Fletcher factor) clotting activity and antigen levels have been examined in various clinical conditions. Prekallikrein antigen was measured by a newly developed, specific, and sensitive radioimmunoassay. The assay had no demonstrable cross-reactivity with human urinary kallikrein nor, in the species tested, animal plasma prekallikrein. This assay was able to measure plasma kallikrein after its biological functions had been inactivated by plasma inhibitors. Normal human pooled plasma contained approximately 50 microgram/ml prekallikrein. Quantitative measurement of plasma prekallikrein was possible for concentrations as low as 0.3% of that of normal pooled plasma. A good correlation (correlation coefficient = 0.71) existed between titers of plasma prekallikrein measured by Fletcher factor clotting assays and radioimmunoassays among 40 normal subjects. Both prekallikrein clotting activity and antigen were significantly reduced in plasmas of patients with advanced hepatic cirrhosis or DIC. Prekallikrein activity and antigen were mildly decreased in plasmas or serums of patients with chronic renal failure and nephrotic syndrome but were normal in those of patients under treatment with warfarin or suffering from SLE, rheumatoid arthritis, sarcoidosis, or HANE. Human cord serum contained a lower titer of prekallikrein antigen than adult serum. Strenuous physical exercise did not significantly change plasma prekallikrein levels.     

Plasma Kallikrein Activation and Inhibition during Typhoid Fever

As an ancillary part of a typhoid fever vaccine study, 10 healthy adult male volunteers (nonimmunized controls) were serially bled 6 days before to 30 days after ingesting 10(5)Salmonella typhi organisms. Five persons developed typhoid fever 6-10 days after challenge, while five remained well. During the febrile illness, significant changes (P < 0.05) in the following hematological parameters were measured: a rise in alpha(1)-antitrypsin antigen concentration and high molecular weight kininogen clotting activity; a progressive decrease of platelet count (to 60% of the predisease state), functional prekallikrein (55%) and kallikrein inhibitor (47%) with a nadir reached on day 5 of the fever and a subsequent overshoot during convalescence. Despite the drop in functional prekallikrein and kallikrein inhibitor, there was no change in factor XII clotting activity or antigenic concentrations of prekallikrein and the kallikrein inhibitors, C1 esterase inhibitor (C1-INH) and alpha(2)-macroglobulin. Plasma from febrile patients subjected to immunoelectrophoresis and crossed immunoelectrophoresis contained a new complex displaying antigenic characteristics of both prekallikrein and C1-INH; the alpha(2)-macroglobulin, antithrombin III, and alpha(1)-antitrypsin immunoprecipitates were unchanged. Plasma drawn from infected-well subjects showed no significant change in these components of the kinin generating system. The finding of a reduction in functional prekallikrein and kallikrein inhibitor (C1-INH) and the formation of a kallikrein C1-INH complex is consistent with prekallikrein activation in typhoid fever. The correlation of these changes with the drop in platelet count suggests that a common mechanism may be responsible.     

Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate

An automated assay of plasma prekallikrein is described. Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the hydrolytic activity of kallikrein to H-D-Pro-Phe-Arg-paranitroanilide subsequently measured.The conversion was complete within 8 minutes and the amidolytic activity remained stable at least another 10 min at 37 ° C. This method worked in plasma deficient in Hageman factor (blood coagulation factor XII). Using anti-prekallikrein antibody and plasma deficient in prekallikrein, the amidolytic activity generated in normal plasma was identified as due to kallikrein. With plasma samples, the coefficients of variation (CV) for multiple measurements within run (n = 10) and between run (n = 10) were as low as 5.0% and 6.6%, respectively, and the minimum measurable concentration of prekallikrein in plasma was 10% of the normal level.     

Fletcher factor deficiency. A diminished rate of Hageman factor activation caused by absence of prekallikrein with abnormalities of coagulation, fibrinolysis, chemotactic activity, and kinin generation

Fletcher factor-deficient plasma is deficient in prekallikrein and therefore generates no bradykinin upon activation with kaolin. It also possesses a diminished rate of kaolin-activable coagulation and fibrinolysis and possesses a defect in kaolin-activable chemotactic activity. These abnormalities are also corrected by reconstitution with purified prekallikrein. Addition of intact activated Hageman factor corrected the coagulation, fibrinolytic, and chemotactic defects and addition of Hageman factor fragments corrected the fibrinolytic defect and partially corrected the chemotactic defect; neither of these corrected the kinin-generating defect. Although the Hageman factor-dependent pathways appear to be initiated by contact activation of Hageman factor, the kallikrein generated activates more Hageman factor; this feedback is necessary for the Hageman factor-dependent pathways to proceed at a normal rate. It is the absence of this feedback in Fletcher factor-deficient plasma that accounts for the diminished rate of activation of Hageman factor and therefore a diminished rate of activation of the coagulation and fibrinolytic pathways. The ability of prekallikrein to correct the coagulation, fibrinolytic, kinin-generating, and chemotactic defects of Fletcher factor-deficient plasma is consistent with the identity of the Fletcher factor and prekallikrein.     

Alteration of factor VII activity by activated Fletcher factor (a plasma kallikrein): a potential link between the intrinsic and extrinsic blood-clotting systems.

Although surface contact is known to accelerate the one-stage prothrombin time of human plasma through the participation of Hageman factor (factor XII) and factor VII, it has not been clear whether Hageman factor interacts with factor VII directly or indirectly. Recently, Gj?nnaess reported experiments suggesting that plasma kallikrein was an intermediate between Hageman factor and factor VII. The present study was undertaken to elucidate the interaction of plasma kallikrein and factor VII. Incubation of Fletcher-trait plasma (deficient in a plasma prekallikrein) with kaolin at 0 degrees C. did not induce shortening of the Thrombotest time or enhancement of factor VII activity, in contrast to studies of normal plasma. Monospecific rabbit antiserum against plasma kallikrein blocked the shortening of the Thrombotest time of normal plasma brought about by kaolin. Purified Hageman factor fragments (prekallikrein activator) induced an increase in factor VII activity in normal or Hageman-trait plasma, but not in Fletcher-trait plasma. A purified plasma kallikrein preparation enhanced factor VII activity in all plasmas, including that of Fletcher-trait plasma. The effect of the kallikrein preparation was blocked by soybean trypsin inhibitor, Trasylol, or rabbit antiserum against kallikrein, but not by lima bean trypsin inhibitor or antiserum against Hageman factor. The activity of partially purified factor VII was enhanced by purified kallikrein in the presence, but not in the absence of factor VII-deficient plasma. These results further support the idea that the enhancement of factor VII activity by surface contact is via Hageman factor and plasma kallikrein, suggesting a possible link between the intrinsic and extrinsic pathway of blood clotting. The significance of this phenomenon in hemostasis in vivo remains to be elucidated.     

Studies on the prekallikrein (kallikreinogen)—kallikrein enzyme system of human plasma: II. Evidence relating the kaolin-activated arginine esterase to plasma kallikrein

Evidence is presented in this paper that the kaolin-activated arginine esterase of plasma is related to plasma kallikrein activity. Such a relationship is based on studies that (1) establish a constant ratio of esterase activity on various synthetic substrates for the kaolin-activated arginine esterase, purified kallikrein(s), and preparations obtained during the fractionation procedure; (2) exclude other known plasma and tissue arginine esterases; (3) confirm the requirement for factor XII in the activation of the enzyme precursor; and (4) show similarities in behavior between the plasma esterase and purified kallikrein(s) toward a variety of inhibitors.Based on this probable identification, evidence is provided that the concentration of active factor XII determines the rate of activation of plasma kallikreinogen, and that the activation may be blocked by polybrene. Once activated, plasma kallikrein is rapidly inactivated by the naturally occurring plasma inhibitor, but the inhibition is incomplete. Acid or chloroform treatment of plasma rapidly inactivates the plasma inhibitor without affecting the concentration of plasma kallikreinogen.Another plasma arginine esterase with properties suggestive of permeability factor is activated by factor XII in the presence of synthetic substrates, but only at low ionic strength. The data suggest that this enzyme is closely related to plasma kallikrein and that it arises from a common precursor.     

Cleavage of human high molecular weight kininogen markedly enhances its coagulant activity. Evidence that this molecule exists as a procofactor.

High molecular weight kininogen (HMW)-kininogen, the cofactor of contact-activated blood coagulation, accelerates the activation of Factor XII, prekallikrein, and Factor XI on a negatively charged surface. Although prekallikrein and Factor XI circulate as a complex with HMW-kininogen, no physical association has been demonstrated between Factor XII and HMW-kininogen, nor has the order of adsorption to surfaces of these proteins been fully clarified. In this report we explore the requirements for adsorption of HMW-kininogen to a clot-promoting surface (kaolin), in purified systems, as well as in normal plasma and plasma genetically deficient in each of the proteins of the contact system. The fraction of each coagulant protein associated with the kaolin pellet was determined by measuring the difference in coagulant activity between the initial sample and supernatants after incubation with kaolin, or by directly quantifying the amount of 125I-HMW-kininogen that was associated with the kaolin pellet. In normal plasma, the adsorption of HMW-kininogen to kaolin increased as the quantity of kaolin was increased in the incubation mixture. However, the HMW-kininogen in Factor XII-deficient plasma did not absorb appreciably to kaolin. Furthermore, the quantity of HMW-kininogen from prekallikrein-deficient plasma that adsorbed to kaolin was decreased as compared with normal plasma. These observations suggested that HMW-kininogen in plasma must be altered by a reaction involving both Factor XII and prekallikrein in order for HMW-kininogen to adsorb to kaolin, and to express its coagulant activity. Subsequently, the consequence of the inability of HMW-kininogen to associate with a negatively charged surface results in decreased surface activation. This assessment was derived from the further observation of the lack of prekallikrein adsorption and the diminished Factor XI adsorption in both Factor XII-deficient and HMW-kininogen-deficient plasmas, since these two zymogens (prekallikrein and Factor XI) are transported to a negatively charged surface in complex with HMW-kininogen. The percentage of HMW-kininogen coagulant activity that adsorbed to kaolin closely correlated (r = 0.98, slope = 0.97) with the amount of 125I-HMW-kininogen adsorbed, suggesting that adsorption of HMW-kininogen results in the expression of its coagulant activity. Since kallikrein, which is known to cleave HMW-kininogen, is generated when kaolin is added to plasma, we tested the hypothesis that proteolysis by kallikrein was responsible for the enhanced adsorption of HMW-kininogen to kaolin. When purified HMW-kininogen was incubated with purified kallikrein, its ability to absorb to kaolin increased with time of digestion until a maximum was reached. Moreover, (125)I-HMW-kininogen, after cleavage by kallikrein, had markedly increased affinity for kaolin than the uncleaved starting material. Furthermore, fibrinogen, at plasma concentration (3 mg/ml), markedly curtailed the adsorption of a mixture of cleaved and uncleaved HMW-kininogen to kaolin, but was unable to prevent fully cleaved HMW-kininogen from adsorbing to the kaolin. Addition of purified kallikrein to Factor XII-deficient plasma, which bypasses Factor XII-dependent contact-activation amplified the ability of its HMW-kininogen to adsorb to kaolin. These observations indicate that HMW-kininogen is a procofactor that is activated by kallikrein, a product of a reaction which it accelerates. This cleavage, which enhances its association with a clot-promoting surface in a plasma environment, is an event that is necessary for expression of its cofactor activity. These interactions would allow coordination of HMW-kininogen adsorption with the adsorption of Factor XII, which adsorbs independently of cleavage, to the same negatively charged surface.     

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.     

Enzymes of the contact phase of blood coagulation: kinetics with various chromogenic substrates and a two-substrate assay for the joint estimation of plasma prekallikrein and factor XI

Kinetic constants (Km and kcat) of kallikrein and factor XIa for the chromogenic substrates H-D-L-prolyl-L-phenylanyl-L-arginine-p-nitroanilide (S-2302) and L-pyroglutamyl-L-propyl-L-arginine-p-nitroanilide (S-2366) were determined. The determined constants allow the use of S-2302 and S-2366 in an assay that leads to the joint estimation of factor XI and prekallikrein in activated plasma. The assay reports approximately 3.1 micrograms/ml factor XIa and 34.5 micrograms/ml kallikrein in kaolin-activated plasma (kaolin content 2 mg/ml). The dual-substrate amidolytic assay shows good correlation with the coagulant assay of both factors (0.92 with the prekallikrein assay and 0.98 with the factor XI assay). It is capable, through the joint estimation of factor XI and prekallikrein levels, of differentiating among plasma samples deficient in components of the contact phase of blood coagulation. Kinetic constants of factor beta-XIIa (factor XII fragment) for these substrates and for N-benzol-L-isoleusyl-L-glutamyl-glycyl-L-arginine-P-nitro ani lide (S-2222) were determined, and they allowed the assessment of the contribution of this factor to this assay and its estimation in the activated phase.     

Inactivation of kallikrein in human plasma

Human plasma kallikrein is inactivated by plasma protease inhibitors. This study was designed to determine the nature of these protease inhibitors and to assess their relative importance in the inactivation of kallikrein. Therefore, the kinetics of kallikrein inactivation and the formation of kallikrein inhibitor complexes were studied in normal plasma and in plasma depleted of either alpha 2-macroglobulin (alpha 2M), C1 inhibitor, or antithrombin (AT III). Prekallikrein was activated by incubation of plasma with dextran sulfate at 4 degrees C. After maximal activation, kallikrein was inactivated at 37 degrees C. Inhibition of kallikrein amidolytic activity in AT III-deficient plasma closely paralleled the inactivation rate of kallikrein in normal plasma. The inactivation rate of kallikrein in alpha 2M-deficient plasma was slightly decreased compared with normal plasma, but in contrast to normal, C1 inhibitor-deficient, and AT III-deficient plasma, no kallikrein amidolytic activity remained after inactivation that was resistant to inhibition by soybean trypsin inhibitor. Suppression of kallikrein activity in C1 inhibitor-deficient plasma was markedly decreased, and this was even more pronounced in plasma deficient in both C1 inhibitor and alpha 2M. The pseudo first-order rate constants for kallikrein inactivation in normal, AT III-deficient, alpha 2M-deficient, C1 inhibitor-deficient plasma, and plasma deficient in both alpha 2M and C1 inhibitor, were 0.68, 0.60, 0.43, 0.07, and 0.016 min-1, respectively. Sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis showed that during inactivation of kallikrein in plasma, high-Mr complexes were formed with Mr at 400,000-1,000,000, 185,000, and 125,000-135,000, which were identified as complexes of 125I-kallikrein with alpha 2M, C1 inhibitor, and AT III, respectively. In addition, the presence of an unidentified kallikrein-inhibitor complex was observed in AT III-deficient plasma. 52% of the 125I-kallikrein was associated with C1-inhibitor, 35% with alpha 2M, and 13% with AT III and another protease inhibitor. A similar distribution of 125I-kallikrein was observed when the 125I-kallikrein inhibitor complexes were removed from plasma by immunoadsorption with insolubilized anti-C1 inhibitor, anti-alpha 2M, or anti-AT III antibodies. These results suggest that only covalent complexes are formed between kallikrein and its inhibitors in plasma. As a function of time, 125I-kallikrein formed complexes with C1 inhibitor at a higher rate than with alpha 2M. No difference was observed between the inactivation rate of kallikrein in high-Mr kininogen-deficient plasma and that in high-Mr kininogen-deficient plasma reconstituted with high-Mr kininogen; this suggests that high-Mr kininogen does not protect kallikrein from inactivation in the plasma milieu. These results have quantitatively demonstrated the major roles of C1 inhibitor and alpha 2M in the inactivation of kallikrein in plasma.     

Plasma kallikrein and prorenin in patients with hereditary angioedema

Recent evidence indicates that plasma kallikrein is activated during acute attacks of hereditary angioedema. Plasma kallikrein is known to convert inactive renin, or prorenin, into an active proteolytic enzyme in plasma exposed to acid or low temperatures as well as in purified systems. To establish whether plasma kallikrein could activate prorenin under physiologic or pathologic conditions, prorenin to renin conversion was assessed at neutral pH in plasma deficient in C1 inhibitor (hereditary angioedema). In these plasma samples lacking the two major inhibitors of kallikrein and possessing less than 10% of the inhibitory activity of normal plasma, prorenin was not converted to an active enzyme despite conditions under which prekallikrein was completely activated to plasma kallikrein and despite normal prorenin concentrations and activability.