Plasma kallikrein-mediated activation of the renin-angiotensin system does not require prior acidification of prorenin

Activation of prorenin in the neutral phase after pH 3.3 dialysis of human plasma depends on clotting factor XII-initiated prekallikrein to kallikrein conversion. Acid dialysis may be necessary for destroying kallikrein inhibitors or rendering prorenin susceptible to attack by kallikrein. If the latter possibility proves true, it is difficult to see how the factor XII-kallikrein pathway could activate prorenin in vivo. Plasma prorenin was therefore separated from active renin and from the protease inhibitors alpha 2-macroglobulin, C1-inactivator, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, and antithrombin III by gel filtration on Sephadex G-100 and affinity chromatography on Blue Sepharose CL-6B at neutral pH. The resulting prorenin preparation could be activated at pH 7.5 by highly purified human plasma kallikrein, which was prepared from prekallikrein by activation with active factor XII fragment beta-factor XII a. Activation proceeded at 4 and 37 C at a kallikrein concentration of 2 micrograms/ml, which is approximately 5% of the prekallikrein concentration in normal plasma. It appears that an acid-induced conformational change of the prorenin molecule is not required for its activation by plasma kallikrein.     

Role of high-molecular-weight kininogen in surface-binding and activation of coagulation Factor XI and prekallikrein.

In the contact phase of activation of the kinin-forming, intrinsic clotting, and fibrinolytic systems, high-molecular-weight kininogen acts as a cofactor for the activation of Factor XI, prekallikrein, and Hageman factor. One mechanism by which high-molecular-weight kininogen acts as a cofactor has been studied by using 125I-labeled Factor XI and prekallikrein in kaolin-activated normal human plasma and plasmas deficient in high-molecular-weight kininogen and Hageman factor. High-molecular-weight kininogen was found to be essential for normal binding and cleavage of both Factor XI and prekallikrein on the kaolin surface. Hageman factor was essential for cleavage but not for binding of Factor XI and prekallikrein to kaolin. In normal plasma 80% of the activated Factor XI remained surface-bound, whereas 80% of the kallikrein was not surface-bound. These findings are consistent with the hypothesis that, in the initial phase of contact activation, high-molecular-weight kininogen links both Factor XI and prekallikrein to the exposed surface where they are activated by surface-bound activated Hageman factor. Once activated, the Factor XI molecules remain localized at the site of activation, in contrast to the kallikrein molecules which are found largely in the surrounding plasma.     

In vitro activation of the contact (Hageman factor) system of plasma by heparin and chondroitin sulfate E

A large number of negatively charged macromolecules, including DNA, glycosaminoglycans, and proteoglycans, were tested as possible activators of the contact (Hageman factor) system in vitro. Activation was assessed by conversion of prekallikrein to kallikrein, as determined by amidolytic assay and by cleavage of 125I-Hageman factor into 52,000- and 28,000-dalton fragments. Of particular interest to these studies, heparin proteoglycan and glycosaminoglycan from rat peritoneal mast cells, and squid chondroitin sulfate E, which is representative of the glycosaminoglycan from cultured mouse bone marrow derived mast cells, induced the reciprocal activation between Hageman factor and prekallikrein. In addition, naturally occurring heparin glycosaminoglycans from pig mucosa, bovine lung, and rat mast cells also induced activation. In contrast, native connective tissue matrix glycosaminoglycans and proteoglycans from several sources were inactive, although when one such chondroitin sulfate was further sulfated in vitro, it gained activity. When the negative charge of the activating agents was blocked by the addition of hexadimethrine bromide, the cleavage of 125I-Hageman factor in the presence of prekallikrein was prevented. The active negatively charged macromolecules induced cleavage of 125I-high molecular weight kininogen in normal plasma but not in Hageman factor-deficient or prekallikrein- deficient plasmas. Reconstitution of prekallikrein-deficient plasma with purified prekallikrein restored the kininogen cleavage upon addition of the active proteoglycans. These results suggest that both heparin from connective tissue mast cells and highly sulfated chondroitin sulfate E from cultured mouse bone marrow derived mast cells (which are considered synonomous with mucosal mast cells) could activate the contact system of plasma subsequent to an activation secretion response.     

Hageman-factor-dependent fibrinolysis: generation of fibrinolytic activity by the interaction of human activated factor XI and plasminogen.

Human coagulation factor XI has been purified, and upon activation with Hageman factor fragments, was found to convert the fibrinolytic proenzyme plasminogen to plasmin. This proactivator activity was shown to be functionally and antigenically distinct from prekallikrein. When the gamma-globulin fractions of plasma deficient in Hageman factor, prekallikrein and factor XI were isolated, factor-XI-deficient plasma possessed two-thirds of the plasminogen proactivator activity of the Hageman-factor-deficient plasma, while prekallikrein deficient plasma had only one-third of the plasminogen proactivator activity. Thus, the Hageman-factor-dependent plasminogen proactivator previously reported to be present in the gamma-globulin fraction of normal human plasma is a function of prekallikrein and factor XI, while the activity observed in prekallikrein-deficient plasma is attributable to factor XI. When compared utilizing digestion of iodinated fibrin, prekallikrein and factor XIa had similar potency per active site; they were, however, far less active than urokinase.     

Mechanisms for the involvement of high molecular weight kininogen in surface-dependent reactions of Hageman factor.

The mechanisms by which human high molecular weight kininogen (HMKrK) contributes to the surface-dependent activation of the Hageman factor systems have been studied. The ability of various mixtures of purified human Hageman factor (coagulation factor XII), HMrK, prekallikrein, and kaolin to activate coagulation factor XI was determined with factor XIa (activated factor XI) clotting assays. Hageman factor, HMrK and prekallikrein were required for maximal rates of activation of factor XI. A certain optimal mixture of purified Hageman factor, HMrK, prekallikrein, and kaolin gave the same rapid initial rate of activation of purified factor XI as an equivalent aliquot of factor XI-deficient plasma. This suggests that potent, surface-mediated activation of factor XI in plasma is explicable in terms of Hageman factor, HMrK, and prekallikrein. By studying separately some of the surface-dependent reactions involving Hageman factor, it was found that HMrK accelerated by at least an order of magnitude the following reactions: (i) the activation of factor XI by activated Hageman factor; (ii) the activation of prekallikrein by activated Hageman factor; and (iii) the activation of Hageman factor by kallikrein. Stoichiometric rather than catalytic amounts of HMrK gave optimal activation of factor XI. These results are consistent with the hypothesis that HMrK and Hageman factor form a complex on kaolin which renders Hageman factor more susceptible to proteolytic activation by kallikrein and which facilitates the action of activated Hageman factor on its substrate proteins, factor XI and prekallikrein.     

Interactions among Hageman factor, plasma prekallikrein, high molecular weight kininogen, and plasma thromboplastin antecedent.

To investigate the earliest steps of the intrinsic clotting pathway, Hageman factor (Factor XII) was exposed to Sephadex gels to which ellagic acid had been adsorbed; Hageman factor was then separated from the gels and studied in the fluid phase. Sephadex-ellagic acid-exposed Hageman factor, whether purified or in plasma, activated plasma thromboplastin antecedent, but only when high molecular weight kininogen was presnet. In the absence of plasma prekallikrein, maximal activation of plasma thromboplastin antecedent was slightly delayed in plasma, a delay not observed with similarly treated purified Hageman factor. Thus, high molecular weight kininogen was needed for expression of Hageman factor's clot-promoting properties and plasma prekallikrein played a minor role in the interaction of ellagic acid-treated Hageman factor and plasma thromboplastin antecedent.     

Role of surface in surface-dependent activation of Hageman factor (blood coagulation Factor XII)

The mechanism by which negatively charged substances such as celite, kaolin, or ellagic acid contribute to the surface-dependent activation of Hageman factor (Factor XII) was studied. Kinetic studies of the proteolytic activation of (125)I-labeled human Hageman factor by human plasma kallikrein, plasma, activated Factor XI, and trypsin were performed in the presence and absence of high molecular weight kininogen and surface materials such as celite, kaolin, or ellagic acid. The results showed that surface-bound Hageman factor was 500 times more susceptible than soluble Hageman factor to proteolytic activation by kallikrein in the presence of high molecular weight kininogen. Surface binding of Hageman factor enhanced its cleavage by plasmin, activated Factor XI, and trypsin by 100-fold, 30-fold, and 5-fold, respectively. On a molar basis, trypsin was twice as potent as kallikrein in the cleavage of the surface-bound Hageman factor, while plasmin and activated Factor XI were an order of magnitude less potent than kallikrein. Kallikrein even at concentrations as low as 0.5 nM (i.e., 1/1000th of the concentration of prekallikrein in plasma) was very potent in the limited proteolysis of the surface-bound Hageman factor. These results suggest that substances classically known as "activating surfaces" promote the activation of Hageman factor indirectly by altering its structure such that it is much more susceptible to proteolytic activation by other plasma or cellular proteases.     

Role of Intrinsic Inhibitors in Acid-Activation of Inactive Plasma Renin (Prorenin)

Inactive plasma renin is converted into an active form of renin during dialysis of plasma against a pH 3.3–buffer. This form of renin is inactivated at neutral pH. When plasma, after acid-dialysis, is kept at neutral pH a different form of active renin is generated, which remains active. The irreversible activation of prorenin depends on factor XII-initiated kallikrein formation. C1-inhibitor (C1-INH) and α₂–macroglobulin (α₂M) can be selectively denaturated by treatment of plasma at low pH values to which kallikrein and renin are resistant. After denaturation of C1-INH at pH 4–5, factor XII becomes capable of activating prekallikrein. The kallikrein that is formed after restoration of pH is complexed with α₂M. This complex has little activity towards natural protein substrates including prorenin. α₂M is denaturated at pH 3–4 and, as a consequence, kallikrein that is formed after restoration of pH is not complexed with α₂M. This kallikrein is able to attack both high-molecular weight kininogen and prorenin as manifested by the generation of bradykinin-forming and angiotensin-forming activities. These observations show a crucial role for C1-INH and α₂M in prorenin activation and explain, at least in part, why the pH of the acid-treatment step has to be less than 4 before irreversible activation of prorenin at neutral pH can occur.     

The Reversible Activation of Inactive Renin in Human Plasma: Role of Acid and of Plasma Kallikreing and Plasmin

Inactive renin in normal plasma, Factor XII-deficient plasma and prekallikrein-deficient plasma was fully activated by dialysis to pH 3.0 for 24 hours at 4°C. This activation was reversed after neutralization and incubation of the plasma at 37°C. The reversible activation-inactivation was not affected by the presence of soya bean trypsin inhibitor. If acidified normal plasma was neutralized and stood at 4°C, plasma kallikrein, but not plasmin, was generated. This rendered the initial acid-activation irreversible. Since no kallikrein was generated in the deficient plasmas, the acid-activation was reversible in these plasmas even after neutralization and standing at 4°C. Thus the apparent activation of inactive renin by kallikrein in acidified, neutralized plasma is not a direct action by the serine protease on inactive renin but a two-stage process in which the inactive renin is first fully activated by acid treatment and the reverse reaction is prevented by plasma kallikrein.     

The contact activation mechanism in human plasma: activation induced by dextran sulfate

Incubation of normal human plasma with dextran sulfate for 7 min at 4 degrees C generates kallikrein amidolytic activity. No kallikrein activity is generated in factor XII or prekallikrein-deficient plasma and only small amounts (8%) in high molecular weight (HMW) kininogen- deficient plasma. Addition of specific antisera directed against prekallikrein or HMW kininogen to normal plasma blocked the generation of kallikrein activity by dextran sulfate. Thus, factor XII, prekallikrein, and HMW kininogen are essential components for optimal activation of prekallikrein. The role of limited proteolysis in the activation of prekallikrein induced by dextran sulfate was studied by adding 125I-prekallikrein to plasma. The generation of kallikrein activity paralleled the proteolytic cleavage of prekallikrein as judged on SDS gels in the presence of reducing agents. The same cleavage fragments were observed as obtained by activation of purified prekallikrein by beta-factor-XIIa. Addition of 131I-HMW kininogen and 125I-factor XII or 131I-HMW kininogen and 125I-prekallikrein to normal plasma followed by activation with dextran sulfate and analysis on SDS gels indicated that the observed cleavage of prekallikrein and HMW kininogen is fast compared to the observed cleavage of factor XII, which is much slower and less extensive. During the first minutes of incubation of normal plasma with dextran sulfate, mainly alpha-factor- XIIa is formed. During prolonged incubation, beta-factor-XIIa is also formed.     

A unique precipitating autoantibody against plasma thromboplastin antecedent associated with multiple apparent plasma clotting factor deficiencies in a patient with systemic lupus erythematosus

A 42-yr-old woman with systemic lupus erythematosus without bleeding diathesis developed a prolonged activated partial thromboplastin time that was not corrected by normal plasma. An inhibitor that acted rapidly and inactivated 0.5 U/ml plasma thromboplastin antecedent (PTA, factor XI) at a 1:200 plasma dilution was demonstrated. In addition to a low titer of PTA (less than 0.01 U/ml), plasma assayed at 20-fold dilution also showed low titers of Hageman (factor XII, 0.02 U/ml), Fletcher (plasma prekallikrein, 0.02 U/ml), and Fitzgerald (high molecular weight kininogen, less than 0.01 U/ml) factors. The titer of these factors, except PTA, returned to normal upon further plasma dilution or upon removal of the inhibitor by protein A adsorption. Thus, the inhibitor appeared to interfere with these clotting factor assays, possibly by inactivating PTA in the substrate plasmas in the test system. Its specificity was further confirmed. The inhibitor did not interfere with surface-induced proteolytic cleavage of Hageman factor. Surface-induced generation of plasma kallikrein activity (amidolysis of H-D-pro-phe-arg-pNa and cold-promoted factor VII activity enhancement) requires only Hageman, Fletcher, and Fitzgerald factors and was normal. Reactions requiring all 4 contact phase factors, including PTA, such as surface-induced generation of plasmin activity (amidolysis of H-D-val-leu-lys-pNa) and activated Christmas factor (factor IXa) activity, were defective. Furthermore, the inhibitor bound to agarose-protein A inactivated and removed PTA selectively from normal plasma. The inhibitor was an IgG-lambda autoantibody that precipitated PTA. The inactivated activated PTA (factor XIa) without the requirement for an additional cofactor. Furthermore, it inhibited surface-induced activation of PTA by interfering with its proteolytic cleavage upon glass surface exposure and with its binding onto the reactive surfaces.     

Molecular assembly in the contact phase of the Hageman factor system.

Data obtained in the past few years have defined the molecular mechanisms of contact activation of the Hageman factor pathways of plasma, i.e., the kinin-forming, intrinsic clotting and fibrinolytic systems. Involved are four molecules: Hageman factor, high molecular weight (MW) kininogen, prekallikrein and factor XI. High MW kininogen serves as a surface cofactor to assemble prekallikrein or factor XI in proximity to surface-bound Hageman factor. Reciprocal proteolytic activation of Hageman factor and prekallikrein represents an essential step in the rapid activation of the contact phase. Although Hageman factor does undergo cleavage and activation in the absence of prekallikrein or high MW kininogen, the rate is approximately 50 and 100 times slower than when these molecules are present. Once Hageman factor is activated on the surface, it cleaves and activates clotting factor XI. Activated Hageman factor (HFa) exhibits two molecular forms. One of these, alpha HFa, activates prekallikrein and factor XI, and the intrinsic clotting system on the surface. alpha HFa and clotting factor XI remain surface bound. The other form of activated Hageman factor, beta HFa, leaves the surface, going into solution where it readily activates additional prekallikrein but not factor XI. Of perhaps even greater importance, kallikrein rapidly dissociates from the surface. Thus the formation of bradykinin and fibrinolysis is disseminated whereas clotting via the intrinsic system remains localized. Reviewed here is the molecular mechanism of contact activation of the Hageman factor pathways and discussed in the interaction of Hageman factor with the negatively charged surface, prekallikrein, factor XI and high MW kininogen. The multiple forms of activated Hageman factor and their potential biologic significance are also discussed.     

Urinary excretion of Hageman factor (factor XII) and the presence of nonfunctional Hageman factor in the nephrotic syndrome

Acquired deficiencies of functional Hageman factor (factor XII) and prekallikrein, proteins involved in the plasma kinin-generating system, have been previously reported in the nephrotic syndrome. The basis for these changes, however, is not fully understood. We have examined the levels of Hageman factor and prekallikrein by functional and radioimmunoassays in plasmas and urines of 11 patients with the nephrotic syndrome. All 11 patients had decreased titers of plasma Hageman factor activity (mean ± standard deviation (SD), 0.29 ± 0.15 U/ml), but essentially normal titers of immunoreactive Hageman factor (0.88 ± 0.23 U/ml). The ratio of immunoreactive Hageman factor to functional Hageman factor (2.63 ± 0.86) was significantly higher than that in nine control patients (1.08 ± 0.17). Since no circulating anticoagulants against Hageman factor were detected, these data suggest the presence of nonfunctional (altered) Hageman factor in plasmas of patients with the nephrotic syndrome. Urinary excretion of Hageman factor was present in six patients but did not appear to account for the reduced plasma Hageman factor activity. Urinary Hageman factor in one patient had the same size as plasma Hageman factor as assessed by gel filtration and sucrose density gradient centrifugation. The titers of plasma prekallikrein were within the normal range. These studies indicate urinary excretion of Hageman factor and alterations in the functional sites of plasma Hageman factor molecules in the nephrotic syndrome. Whether these changes are related to the pathogenesis of the nephrotic syndrome remains to be determined.     

Identification of prekallikrein and high-molecular-weight kininogen as a complex in human plasma.

Prekallikrein and high-molecular-weight kininogen were found associated in normal human plasma at a molecular weight of 285,000 as assessed by gel filtration on Sephadex G-200. The molecular weight of prekallikrein in plasma that is deficient in high-molecular-weight kininogen was 115,000. This prekallikrein could be isolated at a molecular weight of 285,000 after plasma deficient in high-molecular-weight kininogen was combined with plasma that is congenitally deficient in prekallikrein. Addition of purified 125I-labeled prekallikrein and high-molecular-weight kininogen to the respective deficient plasma yielded a shift in the molecular weight of prekallikrein, and complex formation could be demonstrated by incubating prekallikrein with high-molecular weight kininogen. This study demonstrates that prekallikrein and high-molecular-weight kininogen are physically associated in plasma as a noncovalently linked complex and may therefore be adsorbed together during surface activation of Hageman factor. The complex is disrupted when these proteins are isolated by ion exchange chromatography.     

Soluble mediators of injury of the microvasculature; Hageman factor and the kinin forming, intrinsic clotting and the fibrinolytic systems

Studies on Hageman factor have revealed that this protein of approximately 80,000 MW is activated in both solid and fluid phase. In solid phase, the molecule interacts with negatively charged particles without undergoing cleavage. Enzymatic activity is acquired, presumably following a conformational change in the structure of Hageman factor. In fluid phase, the enzymes kallikrein, plasmin, and plasma thromboplastin antecedent (clotting Factor XI) all activated Hageman factor, and in human plasma, the Hageman factor is readily cleaved during this activation. Evidence is presented indicating that kallikrein is the most important fluid phase activator and that the activation with kallikrein is essential for the normal function of the intrinsic clotting, fibrinolytic and kinin forming systems. Information on the role of these systems in immunopathology awaits careful analyses of the function of individual components and means of their accurate detection and quantitation.     

Human plasma inactive renin: purification and activation by proteases

A new affinity chromatographic procedure was devised to purify inactive renin by using a selective hydrophobic interaction of inactive renin to octyl-Sepharose. Additional extensive purification was accomplished by immunoaffinity chromatography on antihuman renin immunoglobulin G-Sepharose. A trace amount of active renin was removed by chromatography on pepstatin-Sepharose. Human plasma inactive renin purified by this method was free from protease inhibitors and permitted the investigation of protease-mediated activation without the acid treatment which was used previously to remove inhibitors. Human plasma kallikrein, human plasmin, cathepsin B1, and arginine esteropeptidases associated with mouse epidermis growth factor and nerve growth factor were effective activators. Human urinary kallikrein, hog pancreatic kallikrein, and rat urinary esterase A were inefficient activators of low potency. Thrombin, factor Xa, factor XIIa, and urokinase did not activate inactive renin. The in vitro activation of 56,000-dalton inactive renin by these proteases was not accompanied by a recognizable reduction in molecular weight. Activation required plasma albumin, presumably as a protecting substance. These results suggest that human inactive renin can be activated by a minimum change in its molecular size.     

Inhibitory spectrum of alpha 2-plasmin inhibitor.

alpha 2-Plasmin inhibitor (alpha 2PI) has been recently characterized as a fast-reacting inhibitor of plasmin in human plasma and appears to play an important role in the regulation of fibrinolysis in vivo. We have studied the effect of purified alpha 2PI upon various proteases participating in human blood coagulation and kinin generation. At physiological concentration (50 microgram/ml), alpha 2PI inhibited the clot-promoting and prekallikrein-activating activity of Hageman factor fragments, the amidolytic, kininogenase, and clot-promoting activities of plasma kallikrein, and the clot-promoting properties of activated plasma thromboplastin antecedent (PTA, Factor XIa) and thrombin. alpha 2PI had minimal inhibitory effect on surface-bound activated PTA and activated Stuart factor (Factor Xa). alpha 2PI did not inhibit the activity of activated Christmas factor (Factor IXa) or urinary kallikrein. Heparin (1.5-2.0 units/ml) did not enhance the inhibitory function of alpha 2PI. These results suggest that, like other plasma protease inhibitors, alpha 2PI possesses a broad in vitro spectrum of inhibitory properties.     

The contact phase of blood coagulation and renin activation in essential hypertension before and after captopril

The contact phase of blood coagulation in a group of patientssuffering from essential hypertension was studied before andafter captopril administration. The baseline levels of factorXII, factor XI and plasminogen were significantly higher thanin normals and correlated with baseline diastolic blood pressurelevels. On the contrary, plasma prekallikrein was not significantlydifferent from normal. These results suggest the presence ofa hypercoagulable state in essential hypertension. After captopriladministration, factor XII, factor XI and prekallikrein rapidlydecreased, perhaps as a consequence of the drug's effect onthe vascular endothelial surface. There was no correlation betweenthe changes of active and inactive renin and the changes ofprekallikrein and plasminogen levels. Our data do not supportthe view that factor XII-plasma kallikrein or plasmin dependentpathways are involved in the activation of inactive renin invivo. Captopril, by provoking rapid pressure changes, appearsto be able to affect the clotting system.     

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).     

Assay of prekallikrein in human plasma: comparison of amidolytic, esterolytic, coagulation, and immunochemical assays

Using the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCl, an amidolytic assay was designed to measure prekallikrein in plasma. At a substrate concentration of 1 mM (Km = 0.2 mM), the amidolysis of purified kallikrein at 1 coagulant unit/ml was observed to be 2.47 mumole/min/ml. Conditions for plasma prekallikrein activation were optimized to approach complete activation when compared to the amidolytic activity of the purified plasma kallikrein. Plasma treated with chloroform to destroy inhibitors of kallikrein was activated with dilute kaolin (final concentration 1 mg/ml) for 1 min at 25 degrees C. Activated plasma prekallikrein had 78% (1.92 mumole/min/ml) of activity of purified kallikrein at plasma concentration. Comparison of this amidolytic assay with immunochemical, esterolytic, and coagulant assays of three subject populations (normals, women on birth control pills, and patients with hepatocellular disease) showed good correlation both in normals and in the patient groups between the amidolytic and esterolytic assays (r = 0.89). Each enzymatic assay correlated with the immunochemical assay (r = 0.72, r = 0.68, respectively). However, comparison of each of these assays with the coagulant assay showed no significant correlation due to the large inherent error of the latter assay. This standardized plasma prekallikrein amidolytic assay should facilitate studies of plasma prekallikrein concentration in physiologic and pathologic conditions and help identify activation of the contact phase of coagulation in disease states.