Hageman factor substrates. Human plasma prekallikrein: mechanism of activation by Hageman factor and participation in hageman factor-dependent fibrinolysis.

Two molecular forms of prekallikrein can be isolated from pooled normal human plasma. Their approximate molecular weights by sodium dodecyl sulfate-gel electrophoresis are 88,000 and 85,000. The two bands observed are shown to represent prekallikrein by functional, immunochemical, and structural criteria. Both forms are cleaved by activated Hageman factor, they appear to share antigenic determinants, they are not interconvertible upon incubation with activated Hageman factor or kallikrein, and the ratio of kinin-generating, and plasminogen-activating activities of the preparations are independent of the relative proportion of each band. Activated Factor XII converts prekallikrein to kallikrein by limited proteolysis and two disulfide-linked chains designated kallikrein heavy chain (Mr = 52,000) and kallikrein light chains (Mr = 36,000 or 33,000) are formed. The active site is associated with the light chains as assessed by incorporation of [3H]diisopropyl fluorophosphate. No dissociable fragments were observed in the absence of reducing agents. However, kallikrein could digest prekallikrein to diminish its molecular weight by 10,000. In addition, two factors capable of activating plasminogen to plasmin have been isolated; one is identified as kallikrein. The second principle fractionates with Factor XI and is demonstrable in normal and prekallikrein-deficient plasma.     

Naloxone attenuates the hypotension induced by Hageman factor

The hypotension induced in the pentobarbital anesthesized rat by the i.v. administration of an active Hageman factor fragment (Hff) is significantly attenuated by naloxone. This effect is specific because the opiate antagonist does not modify the hypotension elicited by rat urinary kallikrein, bradykinin or nitroglycerin. These results suggest that the contact activation of endogenous Hageman factor could result in the generation of vasoactive opioid peptides derived from circulating large molecular weight precursors.     

Activation of hageman factor and prekallikrein and generation of kinin by various microbial proteinases

Activation of the Hageman factor-kallikrein-kinin system by serratial 56-kDa proteinase was previously demonstrated (Matsumoto, K., Yamamoto, T., Kamata, T., and Maeda, H. (1984) J. Biochem. (Tokyo) 96, 739-749; Kamata, R., Yamamoto, T., Matsumoto, K., and Maeda, H. (1985) Infect. Immun. 48, 747-753). To investigate whether the activation of the system is specific for 56-kDa proteinase or is found similarly with other microbial proteinases, 11 proteinases of microbial origins were studied; the 56-kDa proteinase was the control. For in vitro studies, activation of guinea pig Hageman factor and prekallikrein was examined in purified systems as well as in plasma as a zymogen source. Specific antibodies and inhibitors confirmed the activation steps of the cascade. In the in vivo study the enhancement of vascular permeability in guinea pig skin and its sensitivity to inhibitors of activated Hageman factor, plasma kallikrein, or a kininase were examined. The results from the in vivo experiments were consistent with those in vitro. Taking all the data together, we classified the 11 microbial proteinases into three groups as follows: 1) Serratia marcescens 56-, 60-, and 73-kDa proteinases, Pseudomonas aeruginosa alkaline proteinase and elastase, and Aspergillus melleus proteinase (this group activated Hageman factor but not prekallikrein); 2) Vibrio vulnificus proteinase, subtilisin from Bacillus subtilis, and thermolysin from Bacillus stearothermophilus (this group activated both Hageman factor and prekallikrein); 3) Streptomyces caespitosus proteinase and V8 proteinase from Staphylococcus aureus (this group activated neither Hageman factor nor prekallikrein, but generated kinin from high molecular weight kininogen directly).     

Interaction of Hageman factor,prekallikrein activator and plasmin

When plasmin was incubated with active Hageman factor prepared from acetone-activated human plasma by adsorption and elution from pre-treated supercel, no change in prekallikrein activator (PKA) activity was found, even though by polyacrylamide gel electrophoresis and by supercel adsorption it was shown that the active Hageman factor had been converted to the 30, 000 molecular weight fragment. These data are in agreement with the concept that PKA is derived from Hageman factor, but do not support the concept that the conversion of plasminogen to plasmin is necessary for maximal generation of PKA activity in human plasma. Also reported is a radiochemical method for the measurement of PKA (active Hageman factor) activity which is 300 times more sensitive than the guinea pig ileum bioassay and 10 times more sensitive than the clotting tests.     

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.     

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

A rapid purification with high recovery of factor XII (Hageman factor) on immunoaffinity column: application to an abnormal clotting factor XII (factor XIITORONTO)

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.     

Activation of the contact system of coagulation by a monoclonal antibody directed against a neodeterminant in the heavy chain region of human coagulation factor XII (Hageman factor)

We studied the characteristics of two monoclonal antibodies (mAbs), F1 and F3, against human coagulation factor XII (Hageman factor). Experiments with trypsin-digested 125I-factor XII revealed that the epitope for mAb F1 is located in the NH2-terminal Mr 40,100 portion of factor XII, whereas that for mAb F3 resides in the COOH-terminal Mr 30,000 portion of this protein. Factor XII in fresh plasma (single-chain factor XII) bound approximately 190 times less to mAb F1 than factor XII in dextran sulfate-activated plasma (cleaved factor XII). However, no difference in accessibility of the epitope for mAb F1 was observed between cleaved and single-chain factor XII when bound to glass. mAb F3 appeared to bind to both single-chain and cleaved factor XII in plasma as well as when bound to glass. Neither mAb F1, nor F3 affected the amidolytic activity of factor XIIa, whereas both mAb F1 and F3 inhibited factor XII-coagulant activity to about 15 and 70%, respectively, at a molar ratio of mAb to factor XII of 20 to 1. mAb F1, as well as F(ab')2 and F(ab') fragments of this antibody induced activation of the contact system in plasma, as reflected by the generation of factor XIIa. C1 inhibitor and kallikrein. C1 inhibitor complexes. Activation was induced neither upon incubation with mAb F3, nor with that of control mAbs. mAb F1-induced contact activation required the presence of factor XII, prekallikrein, and high molecular weight kininogen and, in contrast to activation by negatively charged surfaces, was not inhibited by the presence of Polybrene. Based on these results we propose that a conformational change in factor XII is a key event in the activation process of this molecule. This conformational change can be induced by binding of factor XII to a surface as well as by proteolytic cleavage. As mAb F1 can also induce this conformational change, this antibody may provide a unique tool in studies of the activation of factor XII.     

Pumpkin seed inhibitor of human factor XIIa (activated Hageman factor) and bovine trypsin

A strong inhibitor of human Hageman factor fragment (HFf, beta-factor XIIa) and bovine trypsin was isolated from pumpkin (Cucurbita maxima) seed extracts by acetone fractionation, by chromatography on columns of diethyl-aminoethylcellulose and carboxylmethyl-Sephadex C-25, and by Sephadex G-50 gel filtration. Pumpkin seed Hageman factor inhibitor (PHFI) is unusual in its lack of inhibition of several other serine proteinases tested--human plasma, human urinary, and porcine pancreatic kallikreins, human alpha-thrombin, and bovine alpha-chymotrypsin. Human plasmin and bovine factor Xa are only weakly inhibited. PHFI also inhibits the HFf-dependent activation of plasma prekallikrein and clotting of plasma. Other properties of PHFI are a pI of 8.3, 29 amino acid residues, amino-terminal arginine, carboxyl-terminal glycine, 3 cystine residues, undetectable sulfhydryl groups and carbohydrate, and arginine at the reactive site. The minimum molecular weight of PHFI is 3268 by amino acid analysis. PHFI may be the smallest protein inhibitor of trypsin known.     

Studies of C1 inactivator-plasma kallikrein complexes in purified systems and in plasma

An enzyme-linked immunosorbent assay (ELISA) has been developed for the quantification of C1 inactivator-kallikrein (C1In-K) complexes. The formation of complexes assayed by this method parallelled the inhibition of plasma kallikrein esterase activity by C1 inactivator in purified systems. C1In-K complexes were detected when a final concentration of 5.7 nM plasma kallikrein was added to plasma, equivalent to the activation of 1% of the plasma prekallikrein. Exogenous Hageman factor fragment added to plasma induced the rapid formation of C1In-K complexes, whereas there was an appreciable delay when the plasma contact system was activated by the addition of kaolin. In both systems, the rate of formation and final amount of complex generated were directly related to the concentration of Hageman factor fragment or of kaolin added, indicating that this proteolytic pathway is tightly regulated. C1In-K complexes were not generated by kaolin in plasma congenitally deficient in Hageman factor or prekallikrein or by kallikrein in hereditary angioedema plasma deficient in C1 inactivator, thus confirming the specificity of the assay. Sucrose gradient ultracentrifugation studies showed plasma C1In-K complexes to have a molecular weight consistent with a 1:1 molar complex. In contrast, the complex displayed an anomalously high molecular weight on gel filtration chromatography. These data demonstrate that a sensitive and specific probe has been developed for documenting plasma kallikrein activation.     

Isolation and characterization of bovine plasma prekallikrein (Fletcher factor)

Prekallikrein (Fletcher factor) has been purified from bovine plasma approximately 25 000-fold with an overall yield of 14%. Purification steps included ammonium sulfate fractionation and column chromatography on heparin-agarose, DEAE-Sephadex, CM-Sephadex, benzamidine-agarose, and arginine methyl ester-agarose. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. Bovine plasma prekallikrein is a glycoprotein with a molecular weight of 82 000 as determined by sedimentation equilibrium centrifugation. It contains 12.9% carbohydrate, including 6.2% hexose, 4.5% N-acetylglucosamine, and 2.2% N-acetylneuraminic acid. Prekallikrein is a single polypeptide chain with an amino-terminal sequence of Gly-Cys-Leu-Thr-Gln-Leu-Tyr-His-Asn-Ile-Phe-Phe-Arg-Gly-Gly. This sequence is homologous to the amino-terminal sequence of human factor XI (plasma thromboplastin antecedent). Both prekallikrein and kallikrein require kaolin to correct Fletcher factor deficient plasma. Kallikrein, however, has a specific activity 3.5 times greater than prekallikrein. Prekallikrein does not correct plasma deficient in factor XII (Hageman factor), factor XI, or high molecular weight kininogen (Fitzgerald factor).     

Conformation of high molecular weight kininogen: effects of kallikrein and factor XIa cleavage

The effect of kallikrein and factor XIa proteolysis of high molecular weight kininogen (HK) was investigated. Circular dichroism (CD) spectroscopy showed that cleavage of HK by plasma kallikrein or urinary kallikrein, both of which result in an active cofactor (HKa), results in conformational change that is characterized by increase in CD ellipticity at 222 nm. This suggests an increase in organized secondary structures. By contrast, cleavage of HK by factor XIa which results in an inactive cofactor (HKi) is characterized by a dramatic decrease in CD ellipticity at 222 nm suggesting an entirely different type of conformational change. The intrinsic fluorescence of HK is enhanced after cleavage by all three proteases. These conformational changes may play a role in determining the structure and function of HKa and HKi.     

Functional properties of factor Va subunits after proteolytic alterations by activated protein C

The two-subunit structure of the factor Va molecule is essential to its function in the prothrombinase complex. In the presence of phospholipids, the cleavage of the light chain of bovine factor Va by activated protein C proceeded at the same rate as the cleavage of the heavy chain. The limited proteolysis of factor Va is accompanied by a parallel loss of factor Va activity. Evidence that loss of activity was solely the result of the cleavage of the heavy chain, was obtained from reconstitution experiments utilizing cleaved and intact chains. The pseudo first-order rate constant of factor Va inactivation by activated protein C was found to be dependent on the amount of phospholipid-bound activated protein C and not on the amount of phospholipid-bound factor Va. However, phospholipids enhance the rate of proteolysis of the phospholipid-binding subunit, i.e. the light chain, and not the cleavage of the heavy chain. Cleavage of the heavy chain and as a consequence the inactivation of factor Va by activated protein C is mediated by phospholipid-bound light chain. After cleavage of the light chain, the 'two-subunit' structure, as well as the phospholipid-binding properties of factor Va were found to be conserved.     

Regulation of C4 photosynthesis: characterization of a protein factor mediating the activation and inactivation of NADP-malate dehydrogenase.

The leaf NADP-malate dehydrogenase of Zea mays is rapidly activated when leaves are illuminated and inactivated in the dark. The present studies show that inactive enzyme isolated from darkened leaves was activated by dithiothreitol and that the active enzyme was rapidly inactivated by oxygen in dithiothreitol-free solutions. Following the fractionation of leaf extracts, both the activation and inactivation of NADP-malate dehydrogenase in vitro were partially or totally dependent upon a separate small molecular weight protein factor. Activation and inactivation were largely or solely dependent upon this factor at pH 8.0 or less, but apparently only partially factor dependent at pH 9.0. The factor was heat stable, inactivated by incubation with trypsin, and had a molecular weight of about 10,000. It was mostly associated with the chloroplasts of mesophyll cells.     

Human factor VIII: purification from commercial factor VIII concentrate, characterization, identification and radiolabeling

Human factor VIII was purified from commercial factor VIII concentrate with a 12% yield. The specific coagulant activity of purified factor VIII was 8,000 units/mg. In the presence of SDS the purified factor VIII consisted of a variety of polypeptides on polyacrylamide gels, ranging between Mr 80,000 and Mr 208,000. In the absence of SDS the purified factor VIII showed an apparent molecular weight of 270,000 upon Sephadex G200 gel-filtration. The purified factor VIII could be activated by thrombin, which resulted in the disappearance of Mr 108,000-208,000 polypeptides in favor of an Mr 92,000 polypeptide. Treatment with factor Xa also activated factor VIII, whereas treatment with activated protein C resulted in the inactivation of coagulant activity. Coagulant-active 125I-factor VIII was prepared using a lactoperoxidase radioiodination procedure. This 125I-factor had the same characteristics as unlabeled factor VIII. All polypeptides could be precipitated with monoclonal antibodies directed against factor VIII. With 125I-factor VIII a pIapp of 5.7 was found in the presence of urea.     

Detailed mechanisms of the inactivation of factor VIIIa by activated protein C in the presence of its cofactors, protein S and factor V

Factor VIIIa is inactivated by a combination of two mechanisms. Activation of factor VIII by thrombin results in a heterotrimeric factor VIIIa that spontaneously inactivates due to dissociation of the A2 subunit. Additionally, factor VIIIa is cleaved by the anticoagulant serine protease, activated protein C, at two cleavage sites, Arg(336) in the A1 subunit and Arg(562) in the A2 subunit. We previously characterized an engineered variant of factor VIII which contains a disulfide bond between the A2 and the A3 subunits that prevents the spontaneous dissociation of the A2 subunit following thrombin activation. Thus, in the absence of activated protein C, this variant has stable activity following activation by thrombin. To isolate the effects of the individual activated protein C cleavage sites on factor VIIIa, we engineered mutations of the activated protein C cleavage sites into the disulfide bond-cross-linked factor VIII variant. Arg(336) cleavage is 6-fold faster than Arg(562) cleavage, and the Arg(336) cleavage does not fully inactivate factor VIIIa when A2 subunit dissociation is blocked. Protein S enhances both cleavage rates but enhances Arg(562) cleavage more than Arg(336) cleavage. Factor V also enhances both cleavage rates when protein S is present. Factor V enhances Arg(562) cleavage more than Arg(336) cleavage as well. As a result, in the presence of both activated protein C cofactors, Arg(336) cleavage is only twice as fast as Arg(562) cleavage. Therefore, both cleavages contribute significantly to factor VIIIa inactivation.     

Activation of plasma Hageman factor and kallikrein in ongoing allergic reactions in the skin

Ten atopic subjects, sensitive to intradermal injection of less than or equal to 10 protein nitrogen units of ragweed or grass pollen antigen, underwent paired antigen and buffer skin chamber incubation over the base of denuded skin blisters. The chamber fluids were sampled over a 6-hr period for histamine and activated Hageman factor and plasma kallikrein which were complexed to C1 inhibitor. In 9 of 10 subjects significantly (p less than 0.01) increased histamine levels (74 +/- 11 ng/ml vs 1.5 +/- 0.55 ng/ml) and kallikrein-C1 inhibitor complexes (2.15 +/- 0.78 ng/ml/hr vs 0.51 +/- 0.09 ng/ml/hr, p less than 0.25) were detected at antigen sites compared with buffer sites, respectively. Increased levels of activated Hageman factor (ng/ml/hr) were detected at antigen sites (1.35 +/- 0.60) compared with buffer sites (0.11 +/- 0.05), (p less than 0.01), in 8 of 10 subjects. Whereas peak levels of histamine were obtained after 1 hr of challenge, both Hageman factor and kallikrein activation, as assessed by complex formation, tended to peak later from the 2nd to the 5th hr. This represents the first demonstration that cutaneous IgE-mediated allergic responses are associated with local activation of the intrinsic plasma coagulation-kinin pathways.     

Beta-transforming growth factor is stored in human blood platelets as a latent high molecular weight complex

Human blood platelets, the richest known source of beta-transforming Growth Factor extractable under acid conditions, release in neutral extracts (pH 7.2) a latent form of this growth factor with an apparent molecular weight of 400 Kd. This latent form, poorly active on rat NRK-49F indicator cells in soft agar assays can be activated by exposure to acid pH or 8 molar urea. The acid activated beta-Transforming Growth Factor from neutral extracts elutes on Biogel P60, in 1 molar acetic acid, as a broad peak of apparent molecular weight 15-30 Kd, like when this factor is extracted from platelets by the usual acid-ethanol procedure. Moreover, beta-Transforming Growth Factor from both acid activated neutral extracts and from acid-ethanol extracts elutes on reverse phase at 30% acetonitrile. We suggest that beta-Transforming Growth Factor is stored in human blood platelets as a poorly active high molecular weight complex which may be dissociated and activated in appropriate in vivo microenvironments.     

Relationship of eukaryotic initiation factor 3 to poliovirus-induced p220 cleavage activity.

The cleavage of the p220 subunit of eukaryotic initiation factor 4F (eIF-4F) that is induced by the poliovirus protease 2A has been shown previously to require another translation initiation factor, eIF-3. The role of eIF-3 in this cleavage reaction, however, is not known. An antiserum was raised against human eIF-3 and used to analyze the eIF-3 subunit composition in poliovirus-infected and uninfected HeLa cells and after incubation of eIF-3 in vitro with viral 2A protease. No evidence for 2Apro-dependent cleavage of any eIF-3 subunit was detected. Infected cells contain an activity that catalyzes the cleavage of p220 to a specific set of cleavage products. This activity is thought to be an activated form of a latent cellular protease. The p220-specific cleavage activity was partially purified. It was resolved from eIF-3 by both gel filtration and anion-exchange chromatography. Neither intact eIF-3 nor any detectable subunits of eIF-3 were found to copurify with the p220-specific cleavage activity. The latter activity behaves as a protein of 55,000 to 60,000 molecular weight and is inhibited by alkylating agents and metals, which indicates the presence of essential thiol groups. When this activity was incubated with partially purified p220, cleavage occurred only in the presence of eIF-3. Thus, eIF-3 appears to play a role in the p220 cleavage cascade which is subsequent to the 2Apro-induced activation of the p220-specific protease.     

Molecular changes associated with proteolysis of bovine factor V by thrombin.

Native factor V contains two major polypeptide chains, h and 1. The molecular weights determined by gel electrophoresis in the presence of sodium dodecylsulfate and dithiothreitol (125 000 and 73 000) are in reasonable agreement with those obtained by gel filtration in 5 M guanidine-HC1 (125000 and 64000). Exposure of factor V to thrombin results in cleavage of the heavier chain to an altered form with a molecular weight of 87000. The other fragment of this proteolytic reaction appears to be a carbohydrate-rich piece, which migrates abnormally slowly on gel electrophoresis conducted under denaturing and reducing conditions. Both proteolytic cleavage products remain associated with the light chain during the purification of factor V. The 87000-Mr fragment is present in samples of factor V which are isolated by immunoprecipitation of blood obtained from a single animal by venous catheter. This finding suggests that some proteolysis may occur in vivo. In contrast, the molecular weight of the light chain is unaltered after thrombin proteolysis of either purified factor V or thrombin-treated plasma.