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Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human βFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.

Human blood coagulation factor XII (FXII; 80 kDa) contains a C-terminal serine protease zymogen domain, which becomes activated upon contacting a negative surface. Activated FXII (alphaFXIIa) brings about reciprocal activation of FXII and kallikrein that by further hydrolysis produces the free catalytic domain (betaFXIIa; 28 kDa). Increased levels of alphaFXIIa are associated with coronary heart disease, sepsis, and diabetes. Biophysical investigation of the structural basis of activation, substrate specificity, and regulation of FXII requires an efficient bacterial system for producing the wild-type and mutant recombinant proteins. Here, the cDNA of the zymogen domain of FXII (betaFXII) was cloned into the pET-28a(+) vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) and overexpressed. The multi-disulfide, recombinant protein, His(6)-betaFXII (rbetaFXII), expressed as an inclusion body, was purified by means of a Ni(2+)-charged resin. The matrix-bound rbetaFXII was subjected to refolding with the glutathione redox system and activated by the in vivo activator, kallikrein. The active form, rbetaFXIIa, obtained in milligram quantities, exhibited similar structural and comparable functional properties relative to human betaFXIIa, as indicated by circular dichroism spectroscopy and kinetics of substrate hydrolysis. Thermodynamics of enzyme:inhibitor complex formation, including the expected 1:1 stoichiometry, was determined for rbetaFXIIa by isothermal calorimetric titration with a specific recombinant protein inhibitor, Cucurbita maxima trypsin inhibitor-V (rCMTI-V; 7kDa).

A procedure of isolation of human blood plasma prekallikrein, coagulation factor XII and active fragment beta-XIIa has been developed. This procedure includes the traditional chromatography steps and FPLC. Disc-electrophoresis revealed that the preparations of factor XII and beta-XIIa were homogeneous. Their specific activity was 70.6 U and 2.5 U, respectively. The procedure described is less time consuming and it allows to isolate these factors in the preparative quantities.

In the present study it is shown that a preparation of highly purified plasma kallikrein (specific activity 81 S-2302 U/mg) still contained small amounts of an IgG fraction. Amidase assays of the fresh enzyme with four peptide substrates (S-2302, Bz-Pro-Phe-Arg-pNA, S-2366, S-2222) did not reveal any inhomogeneity, and immunoblot experiments with antibodies against prekallikrein yielded only an 85 kD double band. After a storage period (2 years at -70 degrees C), S-2366 and S-2222 amidase activities not reflecting the initial kallikrein appeared. Immunoblot studies with Fc-specific antibodies against IgG showed a 170 kD band, and immunoblots with a monoclonal antibody against prekallikrein demonstrated that, in addition to the 85 kD band, a band with a mol weight of about 152 kD could also be detected. Immunoblots showed that only 85 kD kallikrein was recovered in the eluate from Protein G columns, and amidase assays based on S-2302 and Bz-Pro-Phe-Arg-pNA showed a recovery of about 70%. The other part of the kallikrein (30%) was removed along with the additional S-2366 and S-2222 activities and the IgG3 fraction present. The theory is advanced that the additional activity reflects a kallikrein fraction with a mol weight of about 152 kD, and is present in the fresh enzyme preparation in inactive complex with IgG. Storage of the kallikrein preparation led to some weakening of this complex and the appearance of functional activities of the kallikrein fraction involved.

When porcine plasma was subjected to four steps of ion-exchange column chromatographies, factor XII (F. XII) was separated into two fractions, F. XII-1 and F.XII-2, and about 8.5 mg of F. XII-1 and 2.3 mg of F. XII-2 were obtained from 675 ml of the plasma. The purified proteins were found to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular masses of F. XII-1 and XII-2 were estimated to be about 84 kDa by SDS-PAGE, and consisted of a single polypeptide chain. However, F. XII-1 converted to active form after removing Polybrene, but F. XII-2 was not activated. Activation of F. XII-2 took place on incubation with porcine plasma kallikrein, and the activation rate was increased only in the presence of negatively charged surfaces, such as sulfatide. When the preparation was incubated with sulfatide, spontaneous activation was not observed on the condition that we used. Activation of porcine F. XII-2 by porcine plasma kallikrein was found to involve the cleavage of the peptide bond on the disulfide-bridged polypeptide chain, and further fragmentation of activated form was observed during prolonged incubation time.

The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable.

We partially purified the glycoproteins prokallikrein and kallikrein from rat plasma. The purification of rat plasma kallikrein may result in two forms: an intact form (alpha, M(r) 84-87 kDa) and a partially degraded form (beta, M(r) 46-51 kDa). The alpha-form is composed of a heavy chain (M(r) 50 kDa) and a light chain (M(r) 34-37 kDa) linked by a disulfide bond. The catalytic site is found on the light chain. The beta-form has a partially degraded heavy chain (M(r) 28 kDa). Using a preparation of exsanguinated and perfused rat liver, we verified that rat plasma prokallikrein is not activated by the liver and that neither the proenzyme nor the light chain is removed by the organ. Both forms (alpha and beta) of the active enzyme are similarly removed from the perfusate. We also observed that the clearance of plasma kallikrein is temperature-dependent, and not affected by substances that inhibit binding to galactosyl-, mannosyl-, fucosyl- or phosphomannosyl-specific lectins, but inhibited by beta-galactosides. We suggest that: (a) the binding site to hepatocytes is latent on prokallikrein and is located on its heavy chain, more specifically on the 28-kDa fragment still present in the beta form of the active enzyme and (b) plasma kallikrein is recognized by an S-type lectin.

The Hageman factor fragment (HFf) concentrate was prepared from acetone and dextran sulphate activated human plasma by chromatography on a CM-Sephadex column. The preparation was free of the main kallikrein inhibitors--Cl-inactivator, alpha 2-macroglobulin, antithrombin III and alpha 1-antitrypsin. The HFf concentrate can serve as an activator of prekallikrein in patient plasma.

Our observations indicates that the plasma contact system is activated in ascites from patients with gastrointestinal cancer: Factor XII is activated, plasma kallikrein is present in complex with the protease inhibitor alpha 2-macroglobulin, and the plasma kallikrein substrate high molecular weight kininogen, is highly degraded. Contact activation seems to take place in spite of a high level of inhibition. Activation of the contact system generates mediators, which may play a role in the accumulation of ascites.

We examined in purified systems and in human plasma whether heparin serves as a contact system activating compound. Purified human factor XII zymogen was not activated by heparin through an autoactivation mechanism, but was activated in the presence of purified prekallikrein. Zn2+ (12 microM) did not support autoactivation by heparin. The activation of factor XII and the contact system by heparin in plasma anticoagulated with citrate or with hirudin (not chelating ions) was examined by the cleavage of 125I-labeled factor XII and high molecular weight kininogen (HK). Heparin at 1.6 and 16 USP U/ml was not able to produce activation, in contrast to dextran sulfate (20 micrograms/ml) which supported activation of both factor XII and HK. This study indicates that heparinized plasma does not support activation of the contact system mediated through activation of factor XII. It is not expected that heparin anticoagulant therapy will contribute to activation of the contact system.

The specific clot promoting activity of factor XII (F XII) in plasma samples from 50 healthy adults was between 30 and 48 U/mg, whereas the specific activity of purified F XII ranged from 55 to 66 U/mg. This difference was neither due to partial proteolytic activation during purification of F XII nor to the influence of plasma protease inhibitors. Purified F XII showed normal size and charge, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing, respectively. The increase of the specific F XII activity during the purification process mainly occurred after anion exchange chromatography on DEAE-Sephadex and after the final gel filtration step. Upon dextran sulfate activation, proteolytic cleavage of F XII and generation of kallikrein-like amidolytic activity was faster in F XII deficient plasma containing purified F XII than in F XII deficient plasma containing a corresponding amount of pooled normal plasma (NHP). The binding to kaolin was similar for both, purified F XII and plasma F XII. In conclusion, purification alters the properties of F XII in an unknown way, resulting in an increased specific clot promoting activity.

The plasma levels of FXII, prekallikrein (PK), and high- and low molecular weight kininogens (HK and LK) were studied in pregnant women in the last trimester and in non-pregnant controls. FXIIa and plasma kallikrein were assayed in acetone-treated citrated plasma (CPLa) with the tetrapeptide S-2222 as substrate, using soybean trypsin inhibitor and corn inhibitor to exclude kallikrein and FXIIa respectively. No difference in PK-level could be registered for the two kinds of plasma, but the level of FXII had increased to about 150% in the pregnancy plasma. No difference in HK-level was observed, whereas the LK-level was significantly higher in pregnancy plasma, about 250% and 160% in rocket immunoassay and bioassay respectively. In fractions from gel filtration of plasma acetone-activated in the presence of benzamidine (BPLa), kallikrein was assayed as S-2302 amidase, HK and LK were measured in rocket immunoassay, and HK and FXII were studied in PAGE immunoblot experiments. In contrast to previous results obtained upon gel filtration of CPLa, not only kallikrein and HK, but in addition also FXII now appeared together in the same fractions and as two separate peaks. One peak eluting in early fractions (gel mol. wt. 300-400 KD), and one late eluting peak of proteins adsorbed to the gel material. The first peak was notably marked in pregnancy plasma. The results provide support for the assumption of an association in plasma between the three contact activation factors studied.

The separation of the Hageman factor fragment (HFf) activity from human serum albumin by chromatography on Blue Sepharose CL-6B is described. The complete separation cannot be achieved in a single chromatography step due to complex formation between HFf and albumin.

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.

A procedure of isolation of the Hageman factor from human blood plasma, including ion-exchange chromatography on QAE-Sephadex A-50 and preparative disc-electrophoresis is proposed. The active fragment of the Hageman factor was obtained by contact activation of the latter followed by repeated disc-electrophoresis. The peculiarities of the Hageman factor activation by trypsin were studied. The conditions for maximal activation of prekallikrein by the Hageman factor and its active fragment were selected. It was found that the kallikrein formed therupon possesses a higher thermal lability than the trypsin-activated prekallikrein.

To define the factors responsible for the inactivation of the active fragment derived from Factor XII (Factor XIIf ) in plasma, we studied the inactivation kinetics of Factor XIIf in various purified and plasma mixtures. We also analyzed the formation of 125I-Factor XIIf -inhibitor complexes by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In purified systems, the bimolecular rate constants for the reactions of Factor XIIf with C-1-inhibitor, alpha 2-antiplasmin, and antithrombin III were 18.5, 0.91, and 0.32 X 10(4) M-1 min-1, respectively. Furthermore, SDS-PAGE analysis revealed that 1:1 stoichiometric complexes were formed between 125I-Factor XIIf and each of these three inhibitors. In contrast, kinetic and SDS-PAGE studies indicated that Factor XIIf did not react with alpha 1-antitrypsin or alpha 2-macroglobulin. The inactivation rate constant of Factor XIIf by prekallikrein-deficient plasma was 14.4 X 10(-2) min-1, a value that was essentially identical to the value predicted from the studies in purified systems (15.5 X 10(-2) min-1). This constant was reduced to 1.8 X 10(-2) min-1 when Factor XIIf was inactivated by prekallikrein-deficient plasma that had been immunodepleted (less than 5%) of C-1-inhibitor. In addition, after inactivation in normal plasma, 74% of the active 125I-Factor XIIf was found to form a complex with C-1-inhibitor, whereas 26% of the enzyme formed complexes with alpha 2-antiplasmin and antithrombin III. Furthermore, 42% of the labeled enzyme was still complexed with C-1-inhibitor when 125I-Factor XII was inactivated in hereditary angioedema plasma that contained 32% of functional C-1-inhibitor. This study quantitatively demonstrates the dominant role of C-1-inhibitor in the inactivation of Factor XIIf in the plasma milieu.

The contact phase of intrinsic clotting involves Factor XI, Factor XII, Fletcher factor, and a fourth activity that we call contact activation cofactor (CAC). All four of these activities are reduced or absent in Dicalite-adsorbed plasma. A modified activated partial thromboplastin time assay for CAC has been defined by using a substrate of Dicalite-adsorbed plasma combined with partially purified sources of Factors XI and XII, and Fletcher factor. The following properties of CAC in plasma have been determined by using the assay: it is stable up to 60 min at 56 degrees C; gradually loses activity at 80 degrees C; is stable between pH 6 and 9; is precipitated by ammonium sulfate between 40% and 50% saturation; is slightly adsorbed by A1(OH)3; and is eluted from DEAE-cellulose after the major protein peaks. A purification procedure has been devised that separates CAC from other known clotting factors. Isolated CAC was less stable than CAC in plasma, but in the presence of dilute human serum albumin it retained full activity for 80 min at 56 degrees C. On gel filtration CAC had an apparent mol wt of 220,000 daltons. These properties are consistent with those described for Fitzgerald factor, which further supports the conclusion that CAC and Fitzgerald factor represent the same activity. Isolated CAC promoted the generation of activated Factor XI (XIa) in a mixture containing purified Factor XI, Factor XII, and kaolin. The amount of Factor XIa generated was proportional to the amount of added CAC. No time-consuming reaction between Factor XI or Factor XII and CAC could be demonstrated.

The effect of divalent metal ions on the rate of dextran sulfate dependent autocatalytic activation of human blood coagulation factor XII was studied at pH 7.4 and 25 degrees C. Zn2+ and Cu2+, but not Co2+, increased the rate of factor XII activation induced by dextran sulfate with optimum effects at approximately 5 and 1 microM, respectively, while Ca2+ acceleration required much higher concentrations (millimolar). Further investigation of the effect of Zn2+ on factor XII activation demonstrated a complete dependence on the presence of dextran sulfate, lack of inhibition by soybean trypsin inhibitor, the appearance of alpha-XIIa as the primary reaction product, and reaction kinetics characteristic of an autocatalytic process. These results were consistent with Zn2+ affecting only the rate of surface-mediated factor XII autoactivation. The initial turnover velocity of dextran sulfate induced factor XII autoactivation increased linearly with factor XII concentration in the absence of Zn2+ up to 0.9 microM factor XII but showed saturation behavior over this same concentration range in the presence of 5 microM Zn2+, indicating that Zn2+ increased the reaction rate primarily by lowering the apparent Km. Comparison of the kinetics of autoactivation at mu = 0.15 and 0.24 revealed that the enhancement in the apparent kcat/Km brought about by Zn2+ increased from 19-fold to 520-fold, respectively, due to a differential dependence of the Zn2+-stimulated and unstimulated reactions on ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

beta-Factor XIIa (beta-XIIa, Mr approximately 30,000) was isolated from human plasma by a procedure which utilized, as an initial step, the adsorption of Factor XII to celite. Activation of Factor XII and subsequent release of beta-XIIa was brought about by the proteolytic action of co-adsorbed kallikrein. Two successive chromatographic procedures were then used to achieve a final purification of 4,420-fold over plasma and an overall field of 2.3 mg of beta-XIIa per liter.

We have compared the cleavage of purified human Hageman factor (HF) by an activated form of human Hageman factor (HFa) (autodigestion) and by kallikrein. In each case, an initial cleavage is seen which produces HFa with Mr = 80,000 consisting of a heavy chain of Mr = 52,000 disulfide-linked to a light chain of Mr = 28,000. As autodigestion proceeds, HFa is shown to be further digested to yield a major active product at a molecular weight of 40,000 as well as Hageman factor fragment (HFf), which appear as two closely related molecular species of Mr = 28,000 and 30,000. A minor active product of Mr = 70,000 is also seen. Upon reduction of each of the active forms, a chain with Mr = 28,000 is released which contains the active site. HF digestion by kallikrein results in rapid formation of HFa, followed by HFa digestion to HFf and degradation of the heavy chain region to an inactive fragment at 40,000 daltons, which is then degraded to an end product of Mr = 36,000. Production of the active species with Mr = 40,000 and 70,000 is greatly diminished when kallikrein is the HF activator, and these active forms are shown to be formed primarily by autodigestion. The time course of HFa and HFf formation indicates that the rate of activation of Hageman factor by kallikrein is much faster than the rate of autoactivation; the addition of high molecular weight kininogen increases the rate of HFa and HFf formation as well as the extent of HG digestion. These data indicate that HFa is the active intermediate from which other active species are derived. The patterns of HF and HFa digestion by HFa and kallikrein are distinct; a model for HF digestion is presented.

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.

Purified bovine factor XII was radiolabeled with iodine-125 and its binding to kaolin studied. Binding was rapid and was not readily reversible upon adding unlabeled factor XII. The optimum pH for binding was in the region of pH 5-7. The isoelectric point of factor XII was pH 5.7. High concentrations of urea or increasing the ionic strength of the medium did not inhibit binding. Polyvalent macromolecules, such as Polybrene and polylysine, were effective inhibitors of factor XII binding to kaolin. Polylysine caused the release of factor XII that had bound to the kaolin surface.

The structure of Hageman factor, isolated from human plasma, was analyzed before and after enzymatic activation. The purified molecule is a single polypeptide chain of 80,000 molecular weight (mol wt) sedimenting at 4.5S. An amino acid analysis has been performed. The concentration of Hageman factor in normal human plasma was found to be 29 mug/ml with variation between individuals ranging from 15 to 47 mug/ml. Treatment of the molecule with kallikrein, plasmin, or trypsin resulted in cleavage at two primary sites, yielding fragments of 52,000, 40,000, and 28,000 mol wt. No further changes occurred in the fragments with subsequent reduction. Prekallikrein-activating ability was associated exclusively with the 28,000 moiety.

Human factor XII (Hageman Factor) was isolated from human plasma to apparent homogeneity using a four step procedure with yields up to 30%. The method, which is more rapid than the current conventional procedures, consists of a 25-50% ammonium sulfate fractionation, two affinity chromatography steps using Zinc Chelate Sepharose, followed by gel filtration. The isolated zymogen factor XII was a single protein component when examined by SDS PAGE with a Mr of 80,000. Activation of zymogen factor XII to its active enzymatic forms by kallikrein resulted in factor XIIa and factor XIIf as observed with factor XII purified by other procedures.

A rapid purification procedure with high recovery of blood coagulation factor XII (Hageman factor, HF) was established. Homogeneous HF was isolated in 6 days on a monoclonal antibody-immunoaffinity column chromatography followed by gel filtration. Approximately 4,300-fold purification of HF was attained with 31% yield on average (n = 4). Using this method, an abnormal HF was purified from plasma of a patient with cross-reacting material (CRM)-positive Hageman trait (factor XIITORONTO). The abnormal HF was found to be a single chain polypeptide with the same molecular weight (80,000) as the normal HF. Both abnormal and normal HF had similar amino acid compositions.

Proteolysis of 125I-high molecular weight (Mr) kininogen occurred in kaolin-activated plasma which was deficient in prekallikrein, but not in plasma which lacked both prekallikrein and Hageman factor (HF) activity. The implication of this observation is that HF itself might be capable of releasing kinin from high Mr kininogen. This concept was further supported by the following studies. In a purified protein system rabbit (Mr = 80,000) two-chain activated HF (alpha-HFa) incubated with human 125I-high Mr kininogen caused rapid proteolysis of the kininogen in the presence, but not in the absence, of kaolin. This was in contrast to the effect of kallikrein on proteolysis of high Mr kininogen which was inhibited 10-fold by the presence of kaolin. The possibilities that the alpha-HFa preparation was contaminated by rabbit kallikrein or that the human high Mr kininogen preparation was contaminated by human prekallikrein were excluded by using specific antibodies (IgG) against these proteins. The proteolytic fragments of 125I-high Mr kininogen generated by both alpha-HFa and kallikrein were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis. Bioassayable kinin was released from high Mr kininogen following incubation with alpha-HFa in the presence of kaolin. The amount of kinin released in proportion to the extent of proteolysis was the same for both kallikrein and alpha-HFa. The data show that activated Hageman factor may cause release of kinin by proteolytic cleavage of high Mr kininogen. This phenomenon occurs not only in a purified system of proteins but also in kaolin-activated plasma.

An inhibitor of factor XIIa has been purified from bovine plasma and characterized (Thornton, R.D. and Kirby, E.P. (1987) J. Biol. Chem. 262, 12714-12721). This inhibitor interacts with XIIa to form a very stable complex with a 1:1 stoichiometry. The active site of XIIa, located on the light chain, is directly involved in the interaction, and complex formation between factor XIIa inhibitor and XIIa can be blocked by diisopropyl fluorophosphate, corn trypsin inhibitor, or the chromogenic substrate S2302. Incubation of the complex with excess XIIa does not result in cleavage of the complex. The complex does not spontaneously dissociate and is stable to boiling, SDS, thiocyanate, acid, and hydroxylamine or Tris at pH 7-10. In addition to complex formation, a cleaved form of factor XIIa inhibitor can be observed. We suggest that the inhibitor is acting as a mechanism-based inactivator, using the criteria of time-dependent inactivation under pseudo-first-order conditions, 1:1 stoichiometry, active site involvement, kinetic protection by substrate or by an active site inhibitor, and partitioning between cleavage of factor XIIa inhibitor and inactivation by complex formation.

The ability of human Hageman factor (coagulation factor XII) to bind to a glass surface and its susceptibility to limited proteolytic cleavage during the contact activation of plasma have been studied using normal human plasma and plasmas genetically deficient in factor XI, prekallikrein, or high molecular weight kininogen (HMWK). When diluted normal plasma containing (125)I-Hageman factor was exposed to a glass surface for varying times, the Hageman factor was found to bind to the surface, and within 5 min became maximally cleaved from its native 80,000 mol wt to yield fragments of 52,000 and 28,000 mol wt. Hageman factor in factor XI-deficient plasma behaved similarly. In prekallikrein-deficient plasma, the binding of Hageman factor to the glass surface occurred at the same rate as in normal plasma but the cleavage was significantly slower, and did not reach maximum until 60 min of incubation. Cleavage of Hageman factor in HMWK-deficient plasma occurred at an even slower rate, with greater than 110 min of incubation required for maximal cleavage, although the rate of binding to the glass was again the same as in normal plasma. Normal rates of cleavage of Hageman factor were observed for the deficient plasmas after reconstitution with purified human prekallikrein or HMWK, respectively. These observations suggest that normal contact activation in plasma is associated with proteolytic activation of surfacebound Hageman factor. The cleavage of the surface-bound Hageman factor molecule responsible for the formation of the 52,000-and 28,000-mol wt fragments occurred at two closely situated sites, one of which was within a disulfide loop. Cleavage at the site external to the disulfide bond resulted in the release from the surface of the 28,000-mol wt fragment. Cleavage at the site within the disulfide loop resulted in the formation of a 28,000-mol wt fragment which remained surface bound, presumably by virtue of the disulfide linkage to the larger fragment.

In this article we have reviewed the current knowledge regarding the involvement of Factor XII in contact activation. Clearly in the past decade an overwhelming amount of data and hypotheses have been published regarding the central role of this zymogen in the initiation and further propagation of contact activation reactions. Therefore we feel that it will be helpful to conclude this article with a figure that summarizes those interactions and reactions that are generally believed to reflect the major molecular events occurring during surface-dependent contact activation. The contact factors are capable of very efficient interation with each other, provided a suitable negatively charged surface is present. Such surfaces are thought to stimulate the interactions between the contact factors through binding of the proteins and thus bringing the proteins together. Factor XII readily binds to the negatively charged surface, but for the binding of prekallikrein and Factor XI, the cofactor HMW kininogen is likely to be necessary. Bound at the surface, the zymogens Factor XII and prekallikrein are thought to be involved in a so-called reciprocal activation mechanism in which Factor XIIa activates prekallikrein to kallikrein, which in turn converts Factor XII to Factor XIIa. The formation of Factor XIIa is further promoted by the fact that surface-bound Factor XII is likely more susceptible to proteolytic cleavage and by the fact that the activated Factor XIIa is capable of auto-activating its own zymogen Factor XII. However, the latter effect, although undoubtedly contributing to the formation of Factor XIIa at the surface, seems to be of less importance than the reciprocal activation mechanism. This is underscored by the fact that Factor XII activation is rather slow in prekallikrein-deficient plasma. Surface-bound Factor XIIa is then responsible for the activation of Factor XI to Factor XIa, thereby propagating the initial trigger. Presumably, Factor XIa must leave the surface in order to be able to become involved in the activation of blood coagulation Factor IX.

The kallikrein substrate H-D-Pro-Phe-Arg-p-nitroanilide was used in a direct spectrophotometric assay for activated Hageman factor (HF). An 80,000-dalton two-chain, disulfide-linked enzyme, termed HFa, and a 28,000-dalton Hageman factor cleavage product, HFf, were not distinguished in this assay and had a Km of 190 microM and kcat of 15/s. Treatment of HF with 10(-2) M diisopropylfluorophosphate yielded preparations containing 0.2 to 0.9% activated HF as assessed in plastic cuvettes. In quartz cuvettes, concave upward progress curves were obtained. Secondary plots of absorbance/time against time were also nonlinear and consistent with an increased rate of formation of activated enzyme. The curves varied with total protein content and synthetic substrate concentration in a manner consistent with autoactivation; increasing synthetic substrate competed with native HF for interaction with activated HF. Accelerated cleavage of surface-bound radiolabeled HF was demonstrated after incubation with HFa but not HFf, indicating that HFa is the form of active enzyme responsible for autocleavage. The autoactivation described herein may provide a sufficient initial concentration of activated HF to initiate the intrinsic coagulation, fibrinolytic, and kinin-forming cascade.

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

Human factor XII, upon exposure to negatively charged surfaces such as kaolin, sulfatides, and heparin, is converted to enzymatic forms, factor XIIa and factor XIIf. Cl inhibitor has been quantitatively demonstrated to be the primary plasma inhibitor of both factor XIIa and factor XIIf. Studies were performed to determine whether the presence of artificial, negatively charged surfaces influenced the ability of Cl inhibitor to inhibit factors XIIa and XIIf. Kaolin and sulfatides slowed the rate of inhibition of factor XIIa by Cl inhibitor 4.8- and 2-fold, respectively, whereas they had no effect on the inhibition of factor XIIf by Cl inhibitor. Heparin in a concentration of 65 U/ml decreased the inhibition rate of factor XIIa by Cl inhibitor, but, at the same concentration, had less of an effect on the ability of Cl inhibitor to inhibit factor XIIf. These studies indicate that negatively charged surfaces protect factor XIIa but not factor XIIf from inhibition from Cl inhibitor. Since the difference between factors XIIa and XIIf consists of the presence of a surface binding region in factor XIIa, the basis of this protection must reside in the surface binding residues of factor XII. These in vitro events suggest that surface-bound factor XIIa may hydrolyze its physiologic substrates, factor XI and prekallikrein, in an environment partially protected from inhibition by Cl inhibitor.

The activation of Factor XII occurs via fragmentation of this zymogen into a diverse spectrum of enzymatically potent molecular species. To study the interaction of antithrombin-heparin cofactor and heparin with activated Factor XII, we have employed two forms of this enzyme with widely differing physical characteristics and biologic potencies. Antithrombin-heparin cofactor was found to be a progressive, time-dependent inhibitor of both forms. The addition of heparin dramatically accelerated the rates of these interactions. Furthermore, sodium dodecyl sulfate gel electrophoresis of reduced proteins has indicated that antithrombin-heparin cofactor functions by forming an undissociable complex with either species of the enzyme. This complex represents a 1:1 stoichiometric combination of activated Factor XII and inhibitor. In the presence of heparin, both species undergo virtually instantaneous complex formation with antithrombin-heparin cofactor without exhibiting alterations in dissociability or stoichiometry.

Proteolytic cleavage and activation of isolated, single chain, zymogen Hageman factor was observed in the presence of kaolin alone. The rate of cleavage of kaolin-bound Hageman factor was enhanced 50-fold by the presence of prekallikrein and high molecular weight kininogen. The two-chain 82,000 dalton form of activated Hageman factor (alpha-HF(a)) also cleaved kaolin- bound single-chain Hageman factor in a dose-dependent manner, yielding fragments of 28,000 and, 50,000 dahons under reducing conditions. Cleavage of kaolin-bound single-chain Hageman factor was not inhibited by preincubation with diisopropylfluorophosphate (12 mM) for 10 min, but long-term incubation of Hageman factor with diisopropylfluorophosphate (up to 48 h) resulted in inhibition of cleavage of kaolin-bound Hageman factor to an extent proportional to the inhibition of procoagulant Hageman factor activity. Hageman factor cleavage was maximal when the kaolin concentration was {approximately} 10-fold greater than the Hageman factor concentration (wt:wt), and was partially inhibited by high molecular weight kininogen. Kaolin-bound Hageman factor cleaved clotting factor XI in an amount which correlated with the extent of cleavage of the Hageman factor. These findings are compatible with the concept that single-chain Hageman factor and alpha- HF(a), are both capable of cleaving and activating kaolin-bound Hageman factor and that a close molecular association of kaolin-bound Hageman factor molecules is required for this reaction.