Plasma kallikrein/kinin system: a revised hypothesis for its activation and its physiologic contributions

Recent studies indicate that assembly of high molecular weight kininogen on its multiprotein receptor allows for prekallikrein activation. On endothelial cells, factor XII activation is secondary to prekallikrein activation and amplifies it. The immediate consequence of plasma prekallikrein activation is the cleavage of high molecular weight kininogen (HK) with liberation of bradykinin. Cleaved high molecular weight kininogen is antiangiogenic. Bradykinin stimulates tPA liberation and nitric oxide formation. In addition, formed plasma kallikrein promotes single-chain urokinase activation and subsequent plasminogen activation. Kininogens and their breakdown products also are antithrombins. The angiotensin converting enzyme breakdown product of bradykinin prevents canine coronary thrombosis. The author presents a new hypothesis for physiologic assembly and activation of the plasma kallikrein/kinin system and discusses its influence on vascular biology.     

Potential role of platelet FcgammaRIIA in collagen-mediated platelet activation associated with atherothrombosis

Increased levels of hemostatic factors may play a role in the pathogenesis of myocardial infarction by triggering thrombin formation. We measured factor XII (FXII), factor XI (FXI), plasma prekallikrein (PK) and high-molecular-weight kininogen (HK) in 200 patients having survived myocardial infarction for at least 2 months, and in 100 healthy controls. We found significantly elevated levels of FXI clotting activity (FXI:C), HK:C and of the amidolytic activity of PK (PK:Am) among the patients as compared to the controls. Plasma levels of FXI:C, HK:C and PK:Am in the highest quartile were associated with an odds ratio of 1.9 (95% CI: 1.0-3.8), 2.0 (95% CI: 1.0-4.0) and 5.4 (95% CI: 2.6-11.2), respectively, compared to the respective plasma levels in the lowest quartile. After correction for established clinical and laboratory risk factors, the association between PK:Am plasma levels and myocardial infarction remained significant (P=0.0007). Combination of high PK:Am plasma levels and smoking or arterial hypertension, respectively, resulted in a more than additive relative risk for myocardial infarction.     

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.     

Physiopathology of kinin forming system in reproduction

We studied contact factors and kinin-kallikrein in normal non-pregnant and pregnant women, FXII deficient toxemia and DIC. The results obtained are as follows: 1. The levels of plasma prekallikrein, high molecular weight kininogen, kallikrein inhibitor, and C-1 INA were gradually decreased at delivery, and the levels of kallikrein like activity and bradykinin were increased during pregnancy and at the time of parturition. These facts indicate that kinin kallikrein systems played important role in uterine contraction. 2. The levels of contact factors (FXII and FXI) were lower at delivery than those of term. 3. In rat uterus, specific binding of bradykinin was observed by the method of radio receptor assay in the pelet of 10,000 X g fetal membranes, and its activity was 38%. 4. A synthetic kallikrein inhibitor (OS-291, MS) and bradykinin antagonist inhibited completely spontaneous uterine contraction of Wistar rats during delivery. 5. In the case of FXII deficiency, the levels of plasma prekallikrein, high molecular weight kininogen were normal, but at delivery, these levels were lower than those of term. The levels of kallikrein like activity which was half of normal parturition level was increased at parturition. 6. In cases of DIC (17) and severe toxemia (22), plasma prekallikrein levels were lower than the normal controls. The decrease was due to consumption of plasma prekallikrein to kallikrein activation.     

Activation of the kallikrein-kinin system after endotoxin administration to normal human volunteers

The objective of this study was to determine the role of the kallikrein- kinin system in healthy humans after intravenous administration of either Escherichia coli endotoxin or saline. We studied a total of 18 healthy nonsmoking volunteers, 23 to 38 years old, in an open-label study at the Critical Care Medicine Department, Clinical Center, National Institutes of Health (Bethesda, MD) in which some of the patients served as their own controls. After baseline data collection, the subjects received intravenously either E coli endotoxin (n = 15, 4 ng/kg of body weight) or saline (n = 8, controls). Signs, symptoms, systemic blood pressure, factor XII, plasma prekallikrein (PK), factor XI (FXI), antithrombin III (AT-III), high molecular weight kininogen (HK), and alpha 2-macroglobulin-kallikrein complexes were measured at baseline and 1, 2, 3, 5, and 24 hours after injection of either saline or endotoxin. After infusion of endotoxin, we found the functional plasma levels of FXI decreased at 2 hours (P < .05) and at 5 hours (P < .05). Functional PK was significantly depressed by 2 hours (P < .05), at 5 hours (P < .05), and at 24 hours (P < .01), whereas the PK antigen was only low at 5 hours (P < .05). These changes were accompanied by a significant increase in circulating alpha 2-macroglobulin-kallikrein complexes at 3 hours (P < .05) and 5 hours (P < .01). No significant changes occurred in the plasma levels of factor XII or HK. We concluded that clinical response to intravenous endotoxin in healthy human volunteers is associated with activation of the kallikrein-kinin systems. Further investigation is needed with specific inhibitors of the kallikrein-kinin system to define its primary or secondary role in the endotoxin-mediated reactions.     

Kininogen-cytokeratin 1 interactions in endothelial cell biology

This review brings available data together to put in perspective the binding properties of high molecular weight kininogen (HK) to cytokeratin 1 (CK1) to direct future investigations. We summarize our knowledge of the structure and function of HK and discuss the influence of HK on the biologic activities of the plasma kallikrein/kinin system. To better understand the role of HK in regulating the activation of the kallikrein/kinin system, we discuss the interaction of HK with endothelial cell CK1 and its influence on prekallikrein activation. Last, we address the question on how a cytoskeletal protein could be a membrane-binding site and its possible role in disease states.     

Monoclonal antibody to human high-molecular-weight kininogen recognizes its prekallikrein binding site and inhibits its coagulant activity

We developed a mouse monoclonal antibody (MoAb 115-21) to human high- molecular-weight kininogen (HK) that recognizes its prekallikrein binding site (residues 565 through 595 of HK). The corresponding synthesized 31-amino acid peptide (peptide IV) was recently shown to retain native HK's prekallikrein binding property. The same peptide bound factor XI also, although less avidly. Our MoAb recognizes purified HK, peptide IV, and the light chain moiety of HK (where the peptide IV resides), as shown by enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments. The apparent dissociation constant for the HK and MoAb 115-21 interaction was 2.2 nmol/L. It does not recognize low-molecular-weight kininogen (LK) with which HK shares its heavy chain moiety or any antigens in human plasma congenitally deficient in kininogens. The binding of MoAb 115-21 to purified light chain of HK was competitively inhibited by peptide IV. In addition, the antibody inhibits HK-dependent clotting activity of normal human plasma and dextran sulfate-mediated activation of prekallikrein in plasma and retards cleavage of HK in normal plasma after contact activation with dextran sulfate. Also, purified Fab fragments of MoAb 115-21 inhibited the HK-dependent coagulant activity and dextran sulfate-mediated prekallikrein activation in normal plasma. Since the kd for HK-MoAb 115- 21 interaction is ten times lower than that of HK-prekallikrein, our data suggest that binding of MoAb 115-21 to HK's peptide IV site increases the free prekallikrein concentration in plasma and thus results in the decreased efficiency of factor XIIa-mediated activation of prekallikrein. Decreased levels of kallikrein thus formed may be responsible for the inhibition of HK-dependent clotting activity and the decrease in rate and extent of HK cleavage in normal plasma on contact activation with dextran sulfate. MoAb 115-21 may thus prove very useful, especially with its high affinity for HK, in further delineation of the role of HK and prekallikrein in contact activation and kinin-related human pathology.     

Activation of factor XII by dextran sulfate: the basis for an assay of factor XII

A system was developed for studying the activation of factor XII (Hageman factor) in the presence of dextran sulfate (DS). Salient features of the system included low ionic strength (0.08), low concentration of factor XII (approximately 1/10,000 that in normal plasma), and an excess of exogenous prekallikrein (PK). In this system, factor XII was rapidly converted to the 80,000 molecular weight (mol wt) form of factor XIIa (alpha-factor-XIIa). Once formed, the factor XIIa converted PK to kallikrein at a rate that was proportional to the amount of factor XII originally present in the incubation mixture. This system was used to construct a simple sensitive assay for factor XII in plasma and other biologic samples. The kallikrein produced was measured spectrophotometrically with the chromogenic substrate (H-D-Pro-Phe-Arg- p-nitroanilide (S-2302). This assay was shown to be independent of the high molecular weight kininogen and the PK content of the sample being analyzed. The measurements obtained were consistent with fundamental enzymologic principles and, if desired, could be processed with a simple calculator program to achieve linear standard curves. When applied to the quantitation of factor XII in plasma, the assay yielded values in close agreement with those determined by coagulant assay or by radial immunodiffusion.     

Naturally occurring human antibodies against two distinct functional domains in the heavy chain of FXI/FXIa

We have isolated and probed the mechanism of action of two naturally occurring antibodies (Baltimore and Winston-Salem) against factor XI (FXI), that developed in patients congenitally deficient in FXI after replacement therapy. Purification on immobilized protein A and neutralization with monospecific antibodies against IgG heavy and light chain subtypes indicated that both antibodies were of restricted heterogeneity. Both Winston-Salem (IgG3 kappa) and Baltimore (IgG1 kappa) completely inhibited FXI coagulant activity at titers of 200 and 8 Bethesda units, respectively. Immunoaffinity columns prepared from each antibody were able to bind the heavy but not the light chain of reduced and alkylated activated FXI (FXIa). The activation of purified FXI by activated bovine factor XII (FXIIa), a reaction independent of high molecular weight kininogen (HK), was not inhibited by either antibody. The active site on the FXIa light chain was unaffected by either patient's IgG, as measured by its amidolytic activity. In contrast, one antibody (Baltimore) or its Fab' blocked the surface-mediated proteolytic activation of FXI by human FXIIa in a concentration-dependent fashion by preventing its binding to HK, but had no effect on the rate of activation of FIX by FXIa. In contrast, the other antibody (Winston-Salem) or its Fab' inhibited the activation of FIX by FXIa in a concentration-dependent fashion but did not inhibit binding of FXI to HK. We conclude that each of these two naturally occurring antibodies is directed against a specific, separate, and distinct epitope located in the heavy chain of FXIa, one near or at the domain essential for the activation of FIX by FXIa and the other close to the domain required for binding to HK.     

Heat shock protein 90 catalyzes activation of the prekallikrein-kininogen complex in the absence of factor XII

Bradykinin is a major mediator of swelling in C1 inhibitor deficiency as well as the angioedema seen with ACE inhibitors and may contribute to bronchial hyperreactivity in asthma. Formation of bradykinin occurs in the fluid phase and along cell surfaces requiring interaction of factor XII, prekallikrein, and high M(r) kininogen (HK). Recent data suggest that activation of the kinin-forming cascade can occur on the surface of endothelial cells, even in the absence of factor XII. We sought to further define this factor XII-independent mechanism of kinin formation. Both cytosolic and membrane fractions from endothelial cells possessed the ability to catalyze prekallikrein conversion to kallikrein, and activation depended on the presence of HK and zinc ion. We fractionated the cytosol by ion exchange chromatography and affinity chromatography by using corn trypsin inhibitor as ligand. The fractions with peak activity were subjected to SDS gel electrophoresis and ligand blot with biotinylated corn trypsin inhibitor, and positive bands were sequenced. Heat shock protein 90 (Hsp90) was identified as the protein responsible for zinc-dependent prekallikrein activation in the presence of HK. Zinc-dependent activation of the prekallikrein-HK complex also depended on addition of either alpha and beta isoforms of Hsp90 and the activation on endothelial cells was inhibited on addition of polyclonal Ab to Hsp90 in a dose-dependent manner. Although the mechanism by which Hsp90 activates the kinin-forming cascade is not understood, this protein represents the cellular contribution to the reaction and may become the dominant mechanism in pathologic circumstances in which Hsp90 is highly expressed or secreted.     

Assay of factors of the kinin system in plasma from patients with specific exogenous allergies.

Factors of significance for the release of kinin were estimated in plasma from patients with specific, exogenous allergies and in that from healthy individuals. There was no evidence that the rapid initial kinin release in plasma from allergic patients caused by submaximum concentrations of hog pancreas kalikrein or by acetone-activated human plasma (2) was due to an increased level of prekallikrein activator (activated factor XII), to prekallikrein itself or to a factor possibly positioned between active factor XII and prekallikrein. In addition, the rapid halt in kinin release observed for the patient plasma could not be ascribed to an increased level of alpha2-macroglobulin, CI-inactivator or alpha1-antitrypsin. It is suggested that the differences in kinin release registered between the patient plasma and the normal plasma might reflect differences in the relative amounts of kininogen fractions present.     

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.     

Changes in high molecular weight kininogen levels during and after cardiopulmonary bypass surgery measured using a chromogenic peptide substrate assay.

High molecular weight kininogen (HK) is a co-factor in the blood-contact activation system. A chromogenic peptide substrate assay for HK (HKcs) has been developed in which test plasmas are mixed with diluted HK-deficient plasma and incubated with a soluble contact system activator that activates prekallikrein and factor XII. Calcium chloride, a synthetic thrombin inhibitor and a chromogenic peptide substrate for activated factor X (FXa) are then added. The FXa generated cleaves the FXa substrate releasing p-nitroanaline, which is measured photometrically. Test plasma HK values were calculated from a standard curve generated using a pooled normal plasma. Acceptable intra-assay and inter-assay precision values were obtained and levels of HK up to 200% were measurable. The assay measured HK in plasmas deficient in factor XII, prekallikrein and factor XI, was not affected by antiphospholipid antibodies and gave an acceptable correlation (r = 0.95) when normal plasmas and mixtures of HK-deficient and normal pooled plasma, calculated to give HK levels of 25 and 50%, were compared using HKcs and a HK one-stage clotting assay. The HKcs was used to measure HK levels in seven patients undergoing cardiopulmonary bypass (CPB). HK levels fell significantly during CPB (P = 0.0014) and were significantly higher (P = 0.016) 6 days after CPB, suggesting that HK may be a positive acute-phase reacting protein.     

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).     

Monoclonal antibody F1 binds to the kringle domain of factor XII and induces enhanced susceptibility for cleavage by kallikrein

In a previous study we have shown that monoclonal antibody F1 (MoAb F1), directed against an epitope on the heavy chain of factor XII distinct from the binding site for anionic surfaces, is able to activate factor XII in plasma (Nuijens JH, et al: J Biol Chem 264; 12941, 1989). Here, we studied in detail the mechanism underlying the activation of factor XII by MoAb F1 using purified proteins. Formation of factor XIIa was assessed by measuring its amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302) in the presence of soybean trypsin inhibitor and by assessing cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Upon incubation with MoAb F1 alone, factor XII was auto-activated in a time-dependent fashion, activation being maximal after 30 hours. Factor XII incubated in the absence of MoAb F1 was hardly activated by kallikrein, whereas in the presence of MoAb F1, but not in that of a control MoAb, the rate of factor XII activation by kallikrein was promoted at least 60-fold. Maximal activation of factor XII with kallikrein in the presence of MoAb F1 was reached within 1 hour. This effect of kallikrein on the cleavage of factor XII bound to MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator could not substitute for kallikrein. Also, trypsin could easily activate factor XII, but in contrast to kallikrein, this activation was independent of MoAb F1. SDS-PAGE analysis showed that the appearance of amidolytic activity correlated well with cleavage of factor XII. MoAb F1-induced activation of factor XII in this purified system was not dependent on the presence of high- molecular-weight kininogen (HK), in contrast to the activation of the contact system in plasma by MoAb F1. Experiments with deletion mutants revealed that the epitopic region for MoAb F1 on factor XII is located on the kringle domain. Thus, this study shows that binding of ligands to the kringle domain, which does not contribute to the proposed binding site for negatively charged surfaces, may induce activation of factor XII. Therefore, these findings point to the existence of multiple mechanisms of activation of factor XII.     

Plasma Kallikrein and Diabetic Macular Edema

Recent proteomic studies have identified components of the kallikrein kinin system, including plasma kallikrein, factor XII, and kininogen, in vitreous obtained from individuals with advanced diabetic retinopathy. In rodent models, activation of plasma kallikrein in vitreous increases retinal vascular permeability; whereas inhibition of the kallikrein kinin system reduces retinal leakage induced by diabetes and hypertension. These findings suggest that intraocular activation of the plasma kallikrein pathway may contribute to excessive retinal vascular permeability that can lead to diabetic macular edema. The kallikrein kinin system contains two separate and independently regulated serine proteases that generate bradykinin peptides: plasma kallikrein and tissue kallikrein. Tissue kallikrein is expressed in the retina and ciliary body, where it has been implicated in exerting autocrine or paracrine effects via bradykinin receptors that are colocalized in these tissues. Emerging evidence suggests that plasma kallikrein inhibitors may provide a new therapeutic opportunity to reduce retinal vascular permeability.     

A different cleavage site for high molecular weight kininogen in vivo following intravenous injection of dextran sulfate in the rabbit

Purified radiolabeled rabbit Hageman factor, prekallikrein, and high molecular weight kininogen were used to examine Hageman factor system molecular dynamics after the intravenous injection of heparin-like dextran sulfate polymer in the rabbit. Hageman factor system proteins rapidly disappeared from the circulation following dextran sulfate injection, as measured by radial immunodiffusion, by kaolin-releasable kinin formation, and by measuring circulating levels of radiolabeled Hageman factor, prekallikrein, and high molecular weight kininogen. 125I-Hageman factor was distributed mainly to lung, liver, and spleen following dextran sulfate injection. Proteolysis of circulating 125I-Hageman factor occurred at a site within a disulfide loop into fragments of 50,000 and 30,000 molecular weight. Proteolysis of 125I-prekallikrein also occurred with visualization of a 50,000 molecular weight fragment. Although extensive proteolysis of 131I-high molecular weight kininogen was observed, the cleavage fragments were not the same as those generated during contact activation in vitro. The major fragment of high molecular weight kininogen observed in vivo was at 80,000 molecular weight, in contrast to the 65,000 molecular weight fragment generated by kallikrein in vitro. These results indicate that high molecular weight kininogen can undergo proteolysis in vivo into fragments not known to be associated with kinin release.     

An autoantibody to human plasma prekallikrein blocks activation of the contact system

. A patient without a history of bleeding or thromboembolism presented with an activated partial thromboplastin time (aPTT) of 55.1 s (normal 24-38 s). Incubation of the patient plasma with an equal volume of normal plasma failed to correct the aPTT. suggesting the presence of an inhibitor. The MRVVT (modified Russell Viper venom time) was normal, and the anti-cardiolipin antibody titres were not elevated, indicating that the presence of a lupus anticoagulant was unlikely. Plasma prekallikrein (PK) measured by a coagulant assay (2 U/dl) was very low, but PK was in the low normal range (65%) when measured by an enzymatic assay (amidolytic) or by an antigenic assay (ELISA). The purified patient IgG reacted with purified PK. the heavy chain, and the 28 kD fragment of the heavy chain, indicating that it contained an autoantibody to PK. The purified IgG did not directly inhibit the amidolytic activity of kallikrein, but it did inhibit the activation of PK to kallikrein by activated factor XII. Activation of the contact system by dextran sulphate, as reflected by the cleavage of HK on a Western blot, was inhibited when the patient IgG was added to pooled normal plasma. The antibody appears to be oligoclonal with IgG1 being most abundant. followed by IgG4. This report appears to be the first of a spontaneously occurring antibody to prekallikrein.     

Effect of surfaces on fluid-phase prekallikrein activation

The activation of prekallikrein by factor XII fragments (XIIf), during incubation in plastic tubes was previously noted to be increased by high molecular weight (HMW) kininogen as well as other plasma proteins. In this report, we investigated the mechanism responsible for this increase. Although we confirmed that HMW kininogen, bovine serum albumin, fibrinogen, cold insoluble globulin, and mixed phospholipids apparently increased prekallikrein activation, we found that the product of prekallikrein activation (kallikrein) lost substantial activity in less than 0.5 min after exposure to a variety of fresh surfaces. This loss was partially prevented by the presence of various proteins and phospholipids. Similar protection against inactivation of XIIf, the enzyme in this reaction, was also found. In contrast, no loss of the substrate, prekallikrein, was observed during incubation. The loss of kallikrein activity was found to be proportional to the surface area of the incubation vessel as well as the concentration of kallikrein. Further loss of kallikrein activity could also be prevented by pretreating the vessel with kallikrein. We therefore conclude that various substances apparently affect prekallikrein activation in a purified system by preventing the enzyme and product in the reaction mixture from losing activity due to adsorption to a surface.     

Properties of sulfatides in factor-XII-dependent contact activation

Incubation of normal human plasma with low amounts of sulfatides resulted in the initiation of intrinsic coagulation and the appearance of kallikrein activity. The optimal initiation of procoagulant and kallikrein amidolytic activity was dependent on the presence of factor XII, high molecular weight kininogen, and prekallikrein. Since the activated partial thromboplastin clotting times in prekallikrein- deficient plasma approach normal values upon prolonged incubation with kaolin, this phenomenon of autocorrection was studied and found to be even more pronounced in the presence of sulfatides. Autocorrection was essentially completed in 5 min in the presence of sulfatides, whereas a preincubation of 15-20 min was required in the presence of kaolin. The limited proteolysis of 125I-factor XII in plasma during incubation with activating material or during clotting was determined. Cleavage of factor XII was more rapid and more extensive in the presence of sulfatides than in the presence of kaolin. In prekallikrein-deficient plasma, factor XII cleavage was completed within 5 min in the presence of sulfatides and within 15 min in the presence of kaolin. Thus, the appearance of factor-XII-dependent coagulant activity correlates with the limited proteolysis of factor XII when normal or prekallikrein- deficient plasma is activated by sulfatides or kaolin.