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.     

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.     

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.     

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.     

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 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.     

The effect of C1 inhibitor upon Hageman factor autoactivation

Using components purified from human plasma, we have examined the effects of C1 inhibitor (C1 INH), the primary inhibitor of activated Hageman Factor (HFa) and Hageman factor fragment (HFf), on Hageman Factor (HF) autoactivation. When Hageman factor was exposed to a negatively charged surface, provided by either a glass cuvette or dextran sulfate, the addition of C1 INH gave a dose-dependent inhibition of the activity observed. The ability of C1 INH to decrease the maximal enzymatic activity generated was markedly temperature dependent with inhibition increasing as the temperature was raised from 4 degrees C to 37 degrees C. Although the rates of both autoactivation and inhibition were decreased at lower temperatures (4 degrees C), the latter rate was more sensitive to temperature modulation. When HF (final concentration 1 mumol/L) was incubated with C1 INH (0.54, 1.07, and 2.14 mumol/L) in the absence of an initiating surface, no increases in enzymatic activity were observed for up to 48 hours regardless of the C1 INH concentration. However, SDS polyacrylamide gel electrophoresis of the incubation mixture revealed that HF autodigestion had occurred by 48 hours despite the presence of C1 INH. In addition, the appearance of a new band suggested that a complex had been formed between the inhibitor and activated HF. Our findings indicate that C1 INH does not prevent HF autoactivation but rather inactivates the products of HF autodigestion.     

Interactions of C1(-)-inhibitors from normal persons and patients with type II hereditary angioneurotic edema with purified activated Hageman factor (factor XIIa)

Activated high molecular weight Hageman factor (75 Kd) and Hageman factor carboxy-terminal fragments both formed complexes with purified C1(-)-inhibitor, but the Hageman factor fragments appeared to have a higher affinity for the C1(-)-inhibitor than activated Hageman factor. Therefore, the clot-promoting activity of activated Hageman factor might be relatively unimpaired if Hageman factor fragments are also present. Normal C1(-)-inhibitor was cleaved by Hageman factor fragments. Clot-promoting activity was not generated in Hageman factor by exposure to Hageman factor fragments, nor was Hageman factor cleaved by Hageman factor fragments. When Hageman factor was cleaved by streptokinase-activated plasminogen, a 40 Kd fragment was released. In contrast to their interactions with other proteinases, which are blocked by normal C1(-)-inhibitor, Type II C1(-)-inhibitors from plasmas of affected members of eight different kindred with this form of hereditary angioneurotic edema all inhibited the specific coagulant activity of activated Hageman factor to some degree. They did not all form complexes with activated Hageman factor that were stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Production and characterization of a murine monoclonal antibody against a heavy chain of Hageman factor (factor XII)

A murine hybridoma cell line that produces a monoclonal antibody to human Hageman factor (HF, factor XII) is described. The antibody (P 5-2- 1) consists of mouse IgG2b heavy chains and lambda light chains, selectively neutralizes HF procoagulant activity, and prevents the proteolytic cleavage of HF during contact activation in plasma. When HF is exposed to P 5-2-1 before the absorption of HF to kaolin, HF procoagulant activity is markedly inhibited. In contrast, P 5-2-1 does not interfere with HF activity after the adsorption of HF to kaolin. P 5-2-1 does not inactivate the prekallikrein-activating activity of 28,000-mol wt HF fragments (HFf). P 5-2-1 binds exclusively to the 40,000-mol wt portion of a heavy chain of HF and inhibits the adsorption of HF to negatively charged surfaces. P 5-2-1 immobilized on Sepharose can be used to deplete HF from normal human plasma. This immunoaffinity-depleted plasma is indistinguishable from congenital HF- deficient plasma and can be used as the substrate for HF procoagulant activity assay.     

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.     

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.     

Studies on the inhibition of ellagic acid-activated Hageman factor (factor XII) and Hageman factor fragments

Hageman factor (HF, factor XII) that has been exposed to Sephadex- ellagic acid gels is a single-chain species (HFea) with amidolytic properties for the synthetic substrate H-D-phenylalanyl-L-pipecolyl-L- arginine p-nitroanilide. Earlier we reported that amidolysis was suppressed by incubation of HFea with specific antiserum. The present study provides additional evidence that the amidolytic properties of preparations of HFea are ascribable to this substance through an examination of a number of protease inhibitors. HFea's amidolytic properties were inhibited by alpha 2-plasmin inhibitor, antithrombin III in the presence of heparin, and Cl esterase inhibitor (Cl-INH). Additionally, it was inhibited by popcorn inhibitor, leupeptin, hexadimethrine bromide, protamine sulfate, dansyl-arginine N-(3-ethyl- 1,5-pentanediyl) amide (DAPA), diisopropylphosphofluoridate (DFP), aprotinin, and at excessively high concentrations, soybean and lima bean trypsin inhibitors. The spectrum of action of agents that did or did not inhibit HFea supports the view that amidolysis by preparations of HFea is attributable to this enzyme. In general, the enzymatically active carboxy-terminal fragment of HF (HFf) was inhibited by the same agents that inhibited HFea, but aprotinin, protamine sulfate and hexadimethrine bromide were more effective against HFf than HFea, while the reverse was true of lima bean trypsin inhibitor.     

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.     

Inhibition of the activation of hageman factor (factor XII) by eosinophils and eosinophilic constituents

Several syndromes characterized by striking eosinophilia may be complicated by thrombosis. The experiments described indicate that, paradoxically, eosinophils and certain of their constituents inhibit the activation of Hageman factor (HF, factor XII). In earlier studies, suspensions of mixed types of granulocytes, other nucleated peripheral blood cells, and platelets inhibited activation of Hageman factor by ellagic acid, glass, and sulfatides. After these cells were sedimented by centrifugation, the supernatant fluids were also inhibitory. No attempt had been made earlier to distinguish among different granulocytic species. In the present study, suspensions of eosinophils and the supernatant fluid after eosinophils had been separated by centrifugation inhibited activation of Hageman factor by ellagic acid. The protein concentration of that amount of supernatant fluid that inhibited activation by about half was 16 μg/ml, approximately the same as had been described for suspensions of peripheral blood mononuclear cells. Activation of Hageman factor by ellagic acid was also inhibited by certain constituents of eosinophils, including eosinophil peroxidase, eosinophil major basic protein and eosinophil cationic protein. Inhibition was not specific for ellagic acid-induced activation of Hageman factor, as inhibition was also observed with sulfatide-induced activation. Inhibition was presumably related to neutralization of the negative charge of activators of Hageman factor. Thus, bismuth subgallate, a particulate activator of Hageman factor, was no longer effective after it had been exposed to eosinophil cationic protein. The observations reported here raise the question of whether in vivo eosinophils modulate certain of the defense reactions ascribed to Hageman factor. © 1993 Wiley-Liss, Inc.     

Hageman Factor Activation in the Generalized Shwartzman Reaction Induced by Endotoxin

S ummary . The role of Hageman factor (factor XII) in triggering intravascular coagulation was studied in rabbits injected with Thorotrast and endotoxin. Besides changes of coagulation, parameters characteristic of consumption coagulopathy (e.g. decreases in platelet counts and fibrinogen levels), a drop in Hageman factor activity was observed. However, this decrease in Hageman factor activity does not appear responsible for triggering intravascular coagulation, becuase it was found that: (1) A decrease in Hageman factor activity occurred only in those animals which developed a generalized Shwartzman reaction with a drop in fibrinogen levels. A small single dose of endotoxin did not lower Hageman factor activity. (2) Coumarin pretreatment prevented the drop in Hageman factor activity after Thorotrast and endotoxin injections, although this would not be expected if Hageman factor was the first coagulation factor to be activated. (3) Inhibition of Hageman factor activation by lysozyme infusion did not prevent the generalized Shwartzman reaction elicited by Thorotrast and endotoxin.
We conclude that endotoxin triggers intravascular coagulation by a mechanism different from that of the intrinsic pathway of coagulation.     

The influence of estrogen and prolactin on Hageman factor (factor XII) titer in ovariectomized and hypophysectomized rats

The synthesis of prothrombin in hepatic microsomes is augmented in intact estrogen-treated rats and in hypophysectomized rats treated with purified prolactin. We investigated the influence of these gonadal and pituitary hormones on the titer of Hageman factor (factor XII), reportedly elevated in women using oral contraceptives. Rats were ovariectomized to minimize the influence of endogenous estrogen and progesterone on the Hageman factor titer. The administration of progesterone did not alter the plasma concentration of Hageman factor. In contrast, the infusion of 17 beta-estradiol induced a marked elevation of the plasma Hageman factor titer, as measured functionally and immunologically. The titer of Hageman factor was directly related to both plasma estradiol and prolactin concentrations, indicating that prolactin may play a role in the regulation of plasma Hageman factor titers. In agreement with this, hypophysectomy induced a marked decrease in the Hageman factor level. In hypophysectomized ovariectomized animals, the administration of estradiol restored the Hageman factor titer to normal levels, whereas the infusion of prolactin induced a dramatic rise in the Hageman factor titer to the degree observed in nonhypophysectomized estrogen-treated rats. No further increase in the Hageman factor titer was observed in rats treated with both estradiol and prolactin. These data indicate that estrogens increase the plasma Hageman factor titer both directly and through its release of prolactin and that prolactin may also increase the titer of Hageman factor through estrogen-independent mechanisms.     

F XII

Summary The plasma protein F XII (Hageman factor) has been shown to be linked with the plasma defence systems of coagulation, fibrinolysis, kallikrein-kinin and complement. It can be activated by surface contact activation and in solution. Surface contact activation is a complex phenomenon involving negatively charged surfaces, F XII, high molecular weight kininogen and plasma kallikrein. Fluid-phase activation can be effected by a variety of serine proteases. In both types of activation the F XII zymogen is converted to active enzymes. F XII levels in plasma are low or undetectable in both inherited deficiencies and in a variety of clinical conditions. F XII levels can also be elevated in some clinical conditions. Although discovered as a clotting protein F XII appears to play an important role in the kallikrein-kinin and fibrinolytic systems and also has effects on cells. Recent studies suggest that therapeutic blockade of activation of F XII can be of benefit in certain clinical conditions.     

Initiation of plasma prorenin activation by Hageman factor-dependent conversion of plasma prekallikrein to kallikrein.

Plasma prorenin is an inactive form of renin (EC 3.4.99.19) that can be converted to active renin in acid-treated plasma by an endogenous serine protease that is active at alkaline pH (alkaline phase activation). To identify this enzyme we first tested the ability of Hageman factor fragments, plasma kallikrein (EC 3.4.21.8), and plasmin (EC 3.4.21.7) to activate prorenin in acid-treated plasma. All three enzymes initiated prorenin activation; 50% activation was achieved with Hageman factor fragments at 1 microgram/ml, plasma kallikrein at 2-4 microgram/ml, or plasmin at 5-10 microgram/ml. We then showed that the alkaline phase of acid activation occurred normally in plasminogen-free plasma but was almost completely absent in plasmas deficient in either Hageman factor or prekallikrein; alkaline phase activation was restored to these latter plasmas when equal parts were mixed together. Therefore, both Hageman factor and prekallikrein were required for alkaline phase activation to occur. We then found that, although plasma kallikrein could activate prorenin in plasma deficient in either Hageman factor or prekallikrein, Hageman factor fragments were unable to activate prorenin in prekallikrein-deficient plasma. These studies demonstrate that alkaline phase prorenin activation is initiated by Hageman factor-dependent conversion of prekallikrein to kallikrein which, in turn, leads to activation of prorenin. In this fashion, we have revealed a possible link between the coagulation-kinin pathway and the renin-angiotensin system.     

Enzymatic activities of activated and zymogen forms of human Hageman factor (factor XII)

Pro-Phe-Arg chloromethylketone (PPACMK) at 5.26 microM inactivated the amidolytic activity of native human Hageman factor with an apparent first-order rate constant of 0.75 min-1. The activated forms of Hageman factor, Hfa and HFf, were also inactivated by PPACMK with rate constants 0.82 and 0.72 min-1. These numbers indicate that the activity detectable in native Hageman factor is due to contamination with activated species. Uncleaved Hageman factor reacts slowly with 40 mM diisopropyl fluorophosphate with concomitant loss of its procoagulant activity. Incubation of native Hageman factor with PPACMK does not destroy its procoagulant activity, even in the presence of the activator dextran sulphate, but PPACMK inhibits autoactivation of Hageman factor, suggesting that no active site is formed in uncleaved, surface-bound Hageman factor. The activation of prekallikrein by Hageman factor under initial-rate conditions occurs after a lag and is prevented by an inhibitor of Hageman factor from corn. The kinetics of prekallikrein activation and the effects of inhibitors provide evidence that the amidolytic and proteolytic activities of human Hageman factor reside in the activated forms derived by limited proteolysis of the native molecule.     

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.