Hageman factor fragment inhibitor in corn seeds: purification and characterization

A specific, potent inhibitor of Hageman factor fragment (HF_f) was isolated from sweet corn kernels. Three variants with pI's of 5.1, 6.3, and 7.7, and M_r 11,000–14,000 were found. The corn HF_f inhibitor (CHFI) forms a 1:1 molar complex with HF_f or trypsin, and prolongs the activated partial thromboplastin time of human plasma. The K_i of CHFI 6.3 with HF_f was 2.4 × 10^-8 M. CHFI does not inhibit human plasma or urinary kallikreins, plasmin, α-thrombin, hog pancreatic kallikrein, bovine Factor X_a or α-chymotrypsin. CHFI appears to be identical with the corn trypsin inhibitor (CTI) previously reported (Swartz et al., $\text{J. Biol. Chem. 252}$, 8105, 1977). The corn inhibitor is unique in its strong inhibition of HFf and very narrow spectrum of inhibition of other serine proteinases.     

Preparation of β-XIIa (Hageman factor fragment) from human plasma

β-Factor XIIa (β-XIIa, $\text{M}$_r ～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 β-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 yield of 2.3 mg of β-XIIa per liter.     

Falls in plasma levels of prekallikrein, high molecular weight kininogen, and kallikrein inhibitors during lethal endotoxin shock in dogs

Plasma levels of prekallikrein, kallikrein, high molecular weight kininogen, and inhibitors of plasma kallikrein were determined in samples from dogs infused with a lethal dose of E.coli endotoxin. Considerably reduced levels of prekallikrein and high molecular weight kininogen were found in the samples taken at collapse. Free kallikrein and kallikrein bound to α₂-macroglobulin were also detected. “Fast” and “slow” inhibitors of plasma kallikrein were studied, using purified human plasma kallikrein and a chromogenic peptide substrate for this enzyme. Significantly reduced values for both inhibitory activities were observed 9 hours after commencement of the infusion. Plasma samples taken pre-infusion and at collapse were fractionated by Sephadex G-200 gel filtration. Subsequent analysis for kallikrein inhibition revealed two major inhibitory components, one corresponding to α₂-macroglobulin and the other eluting from the column in the same volume as α₁-antitrypsin. At collapse, levels of both of these inhibitors were significantly lower than preinfusion values.     

Mouse plasma trypsin inhibitors: inhibitory spectrum of contrapsin and alpha-1-antitrypsin

We have studied the effects of murine alpha-1-antitrypsin and contrapsin, a new trypsin inhibitor (Takahara, H. and Sinohara, H. (1982) J. Biol. Chem. 257, in press), on several serine proteases participating in blood clotting, fibrinolysis, kinin generation, and complement activation. Bovine plasmin and human plasma kallikrein were inactivated by contrapsin but not by alpha-1-antitrypsin, whereas bovine alpha-thrombin and porcine pancreas kallikrein were inhibited by alpha-1-antitrypsin but not by contrapsin. Heparin protected thrombin from inactivation by alpha-1-antitrypsin. Both inhibitors had virtually no effects on canine C1 esterase.     

The mechanism by which the light chain of cleaved hmw-kininogen augments the activation of prekallikrein,factor XI and hageman factor

HMW-kininogen binds prekallikrein and factor XI to form two bimolecular complexes in plasma; upon addition of certain negatively charged surfaces, it functions as a coagulation cofactor and facilitates the activation of Hageman factor, prekallikrein and factor XI. We have investigated the mechanism by which HMW-kininogen functions in each of these steps. HMW-kininogen was found to augment the binding of prekallikrein and factor XI to kaolin in a plasma system sùggesting that the prekallikrein-HMW kininogen complex and the factor XI-HMW kininogen complex are attached to surfaces via HMW-kininogen. The attachment was shown to be mediated by the light chain derived from cleaved HMW-kininogen and is consistent with previous data demonstrating that the light chain is the coagulant part of the molecule. Prekallikrein can be readily activated when bound to the surface by this HMW-kininogen linkage; however, when an equal quantity of prekallikrein was bound directly to the surface, it was not activatable even after the addition of HMW-kininogen. Thus, binding of prekallikrein (and factor XI) to the surface via HMW-kininogen appears to place them in a conformationally favorable position for activation by activated Hageman factor. This appears to be the major function of HMW-kininogen as a coagulation cofactor. In addition, HMW-kininogen attachment of prekallikrein (kallikrein) to the surface is a reversible interaction which is not the case when direct binding to the surface occurs. In this fashion, a portion of the kallikrein formed can dissociate and interact with other Hageman factor molecules. These data suggest that the effect of HMW-kininogen upon Hageman factor activation is indirect and acts to increase the effective concentration of kallikrein available for Hageman factor cleavage.     

Kininogen in factor VIII-deficient plasma (haemophilia A)

The HM_r and LM_r molecular forms of kininogen were found to occur in normal distribution in plasma from a patient with severe haemophilia A. Present findings indicate that lack of Factor VIII does not influence, the normal content and distribution of immunoreactive kininogens determined by single radial immunodiffusion and radioimmunoassay. The partially purified HM_r and LM_r kininogens were antigenically identical with the kininogens in normal human plasma determined using the monospecific antisera raised in rabbits against purified kininogen antigens. Low kallikrein activity on H-D-Pro-Phe-Arg-pNA in the haemophilic plasma was apparently due to preparative procedures. This concept is supported by the normal binding ratio of trypsin to pure α₂-macroglobulin isolated from the haemophilic plasma.     

Interaction of trypsin, beta-factor XIIa, and plasma kallikrein with a trypsin inhibitor isolated from barley seeds: a comparison with the corn inhibitor of activated Hageman factor

A trypsin inhibitor was purified from barley seeds by a modification of published procedures. We determined the dissociation constant, Ki, for the complexes of the barley inhibitor with trypsin, beta-Factor XIIa, and plasma kallikrein. We compared these constants for those of the same enzymes with the corn Hageman Factor inhibitor, which is a homolog of the barley inhibitor. The strength of interaction of the barley inhibitor with the three enzymes was: trypsin greater than beta-Factor XIIa greater than plasma kallikrein. In contrast, the corn inhibitor inhibits beta-Factor XIIa most strongly and does not inhibit plasma kallikrein at all. A possible structural basis for the difference in inhibition specificity is discussed.     

Similarity and dissimilarity in the stereogeometry of the active sites of thrombin, trypsin, plasmin and glandular kallikrein

The relationship between chemical modifications of arginine derivatives and inhibitory activity to trypsin, plasmin and glandular kallikrein was investigated comparing with that of thrombin and concluded as follows: The hydrophobic binding pocket, which has been reported previously to be stereogeometrically very similar in trypsin and thrombin, corresponded to the length of ethylpiperidine. Concerning the site (termed the P site) next to the hydrophobic binding pocket, there were large differences in stereogeometry between trypsin and thrombin; the binding site of trypsin extended further to allow propyl and phenyl group attached to piperidine, while that of thrombin would be much narrower and unable to allow them. The P sites of plasmin and glandular kallikrein resembled that of trypsin in being able to allow phenyl group. To substantialize the hydrophobic binding pocket and the P site, a (2R, 4R)-MQPA-trypsin complex model was generated using the results of X-ray crystallography of (2R, 4R)-MQPA and BPTI-trypsin complex by calculation to minimize van der Waals contacts, and it was of great use for understanding the geometry of the active sites of trypsin, thrombin, plasmin and glandular kallikrein.     

l-[H]carnosine binding in the olfactory bulb. II. Biochemical and biological studies

During the first 10 days after peripheral deafferentation of the mouse olfactory bulb stereoselective binding ofl-[³H]carnosine declines markedly. The initial phase of this decline is due to a decrease in binding site stereoselectivity, which is then followed by a loss of assayable binding sites. The specificity of inhibition ofl-[³H]carnosine binding by various peptides is also altered after denervation. Competitive inhibitors of carnosine binding become less potent after denervation, while analogues which are not competitive inhibitors remain equipotent before and after denervation. Several carnosine analogues that are normally poor inhibitors become more potent after denervation. Treatment of bulb membranes with trypsin, RNase and hyaluronidase, but not DNase or collagenase, resulted in significant alterations in carnosine binding.l-, but notd-carnosine, protected the binding site from trypsin digestion, and induced additional binding in bulb membranes in a dose-and temperature-dependent fashion. Preincubation of membranes withl-carnosine also led to the induction of additional carnosine binding in membranes from cerebral cortex, cerebellum and deafferentated bulbs but not from muscle. Bulbs from newborn mice contain about one-half of the adult levels of binding and no significant sex differences in carnosine binding were detected in bulbs from adult rats.l-[³H]carnosine binding was two-fold higher in the anterior compared to the posterior portion of the bulb, but there were no significant differences in binding of opiate, GABA, α-adrenergic, muscarinic cholinergic, benzodiazepine or glutamic acid receptor ligands.     

Interaction between factor XII (Hageman factor), high molecular weight kininogen and prekallikrein

Adsorption of factor XII and plasma kallikrein onto kaolin induces activation of factor XII. Trace amounts of high molecular weight (HMW)-kininogen markedly enhances the rate and the amount of activation of factor XII. Activation of factor XII was measured by its capacity to convert prekallikrein to kallikrein. This potentiating effect is dependent on the amount of HMW-kininogen. Low molecular weight kininogen has no enhancing effect. When the reactants are adsorbed to kaolin and the latter separated by centrifugation the prekallikrein-converting activity is detectable only in the kaolin-containing precipitates. Less activation of factor XII occurs when the HMW-kininogen is added not initially to the kaolin-factor XII mixture, but to the prekallikrein, indicating that the HMW-kininogen probably acts when factor XII is being activated, rather than at a later stage. Treatment of HMW-kininogen with excess trypsin, plasmin or kallikrein abolishes its potentiating effect. When HMW-kininogen is treated with increasing concentrations of kallikrein there is an inverse relationship between the generated kinin and the ability of the kininogen to enhance the activation of factor XII.     

The effect of surfactants on the interaction of factor XII with surfaces

Factor XII (Hageman factor) binds irreversibly to plastic as well as glass surfaces. In dilute solution this leads to significant losses of protein onto the walls of tubes, etc. This could be prevented most efficiently by the presence of surfactants in solution. Rinsing the surfaces after contact with the protein, or precoating the surfaces were less effective. Triton X-100 was found to be non-denaturing and effective at concentrations as low as 0.001%. In its presence higher specific activities were obtained in spectrophotometric assay. The autoactivation of factor XII exposed to glass was inhibited but not prevented by Triton X-100. Similar effects on the binding and assay of (Pre) kallikrein were also found.     

Functional correlation between kallikrein and factor XII activated in human plasma

Plasma kallikrein and FXIIa were assayed in acetone-treated human citrated plasma (CPLa) with the chromogenic peptide Bz-Ile-Glu-Gly-Arg-pNA (S-2222) as substrate. In end point assays with short incubation periods (1–10 min.) nearly all kallikrein present could be blocked by a low concentration of soybean trypsin inhibitor (STI). In 30 min. assays the main part of the kallikrein was recovered in a functional state not inhibited by STI, and at the same time the level of FXIIa (as amidase activity blocked by corn inhibitor, C.I.) was reduced to about 2/3 of the initial value. The formation of an association between FXIIa and kallikrein is suggested. In fractions from gel filtration of CPLa kallikrein was assayed as S-2302 amidase, high molecular weight kininogen (HK) was measured in rocket immunoassays, and HK and FXII were studied in PAGE immunoblot experiments. Kallikrein appeared as one peak together with HK (gel mol. wt. 300 KD), about 40% of HK was free (220 KD), and no FXII was observed in the kallikrein or HK peaks, but in two areas corresponding to 78–79 KD and 39–42 KD. When experiments, however, were carried out with plasma acetone-activated and gel filtered in the presence of benzamidine (5 mM), part of the amidase activity present in kallikrein peak fractions was blocked by C.I. This observation supports the above suggestion of an association between FXIIa and kallikrein.     

The activation of factor IX by tissue factor-factor VII in a bovine plasma system lacking factor X

The activation of Factor IX by tissue factor-Factor VII has been studied in a bovine plasma system under conditions that minimize the activation of Factor VII. The plasma was defibrinated, then passed twice through a column of anti-Factor X coupled to Sepharose in order to lower the Factor X level below its limit of assay ($\text{ca.}$ 5 ng/ml), and once through an anti-Factor IX column to remove Factor IX. Varying levels of tritium-labelled Factor IX were then added back to the plasma, permitting measurement of its activation upon the addition of tissue factor and Ca²⁺. Despite the absence of significant levels of Factor X in the system, the course of Factor IX activation was initially characterized by some upward curvature, which suggested activation of the plasma Factor VII during the incubation. In order to obtain linear activation of Factor IX three proteolytic inhibitors were added to the system: 1) a Factor X_a inhibitor, 1,2-bis-(5-amidinobenzimidazole)-ethane, 2) aprotinin, and 3) heparin. Under these conditions the apparent K_m of non-activated Factor VII (+ tissue factor) on Factor IX was 17.3 +/- 2.5 nM (SE), and the maximum velocity was 0.12 nM/min. In parallel experiments the plasma Factor VII was activated by first treating the plasma with Factor X_a for 30 seconds before the addition of inhibitors and the final addition of substrate. Under these conditions the maximum velocity rose to 4.2 nM/min, and the K_m increased to 53.3 +/- 6.0 nM (SE). This change in the K_m is highly significant (P < 0.002), and indicates that the activation of Factor IX by nonactivated plasma Factor VII cannot be due only to traces of Factor VII_a in the plasma. At least in part, activation of Factor IX in the presence of tissue factor is suggested to be a result of the action of Factor VII itself.     

Identification of T-kininogen in high and low molecular weight kininogens deficient rat (brown Norway Katholiek strain)

Kinin release in Brown Norway Katholiek (B/N-Ka) rat plasma was compared with those of Brown Norway Kitasato and Sprague-Dawley rats by treating with rat plasma kallikrein, rat urinary kallikrein, snake venom kininogenase and trypsin. B/N-Ka rat plasma yielded no detectable amount of kinin by either plasma kallikrein, urinary kallikrein or snake venom kininogenase, but yielded variable amount of kinin by trypsin. The released kinin was proved to be isoleucylseryl-bradykinin by high performance liquid chromatography and bioassay profiles. B/N-Ka rat plasma formed a precipitation line against antiserum to T-kininogen, but no line against antiserum to HMW kininogen-light chain.     

Demonstration of the third kininogen in high and low molecular weight kininogens-deficient brown norway katholiek rat

A gel filtration profile of the plasma of Brown Norway Katholiek (B/N-Ka) rat was compared with those of B/N-Kitasato (B/N-Ki) and Sprague-Dawley (SD) rats. In the chromatograms of B/N-Ki and SD rat plasmas, high-molecular weight (HMW) kininogen was eluted together with pre-kallikrein. In lower molecular weight fractions, there were two kininogens, one of which released kinin by urinary kallikrein, snake venom kininogenase (SVK) and trypsin, and the other released kinin only by trypsin. In the chromatogram of B/N-Ka rat plasma, there was no fraction which released kinin by plasma kallikrein, urinary kallikrein or SVK. However, the kinin-release only by trypsin was found in the lower molecular weight fraction, which corresponds to the third peak of kininogen in the chromatograms of B/N-Ki and SD rat plasmas. These results indicate that B/N-Ka rat plasma is deficicent in HMW kininogen, and also deficient in the LMW kininogen susceptible to urinary kallikrein and SVK, but it contains the third kininogen responsive only to trypsin.     

L-[3H]Carnosine binding in the olfactory bulb. II. Biochemical and biological studies.

During the first 10 days after peripheral deafferentation of the mouse olfactory bulb stereoselective binding of L-[3H]carnosine declines markedly. The initial phase of this decline is due to a decrease in binding site stereoselectivity, which is then followed by a loss of assayable binding sites. The specificity of inhibition of L-[3H]carnosine binding by various peptides is also altered after denervation. Competitive inhibitors of carnosine binding become less potent after denervation, while analogues which are not competitive inhibitors remain equipotent before and after denervation. Several carnosine analogues that are normally poor inhibitors become more potent after denervation. Treatment of bulb membranes with trypsin, RNase and hyaluronidase, but not DNase or collagenase, resulted in significant alterations in carnosine binding. L-, but not D-carnosine, protected the binding site from trypsin digestion, and induced additional binding in bulb membranes in a dose-and temperature-dependent fashion. Preincubation of membranes with L-carnosine also led to the induction of additional carnosine binding in membranes from cerebral cortex, cerebellum and deafferentated bulbs but not from muscle. Bulbs from newborn mice contain about one-half of the adult levels of binding and no significant sex differences in carnosine binding were detected in bulbs from adult rats. L-[3H]carnosine binding was two-fold higher in the anterior compared to the posterior portion of the bulb, but there were no significant differences in binding of opiate, GABA, alpha-adrenergic, muscarinic cholinergic, benzodiazepine of glutamic acid receptor ligands.     

Serine protease specificity for peptide chromogenic substrates.

Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160 (N-Bz-Phe-Val-Arg-pNA, HCl), S-2238 (H-D-Phe-Pip-Arg-pNA, 2HCl), S-2222 (N-Bz-Ile-Glu-Gly-Arg-pNA, HCl), and S-2251 (H-D-Val-Leu-Lys-pNA, 2HCl) from AB Kabi Peptide Research and Chromozym TH (Z-Gly-Pro-Arg-pNA, HCl) from Pentapharm Limited were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha thrombin, and bovine factor Xa. S-2160, S-2238, and Chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All 3 substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin. In contrast, S-2222 is sensitive to Xa and insensitive to thrombin. S-2251 is relatively plasmin-specific, being resistant to the clotting enzymes thrombin and Xa. S-2251 exhibits even greater sensitivity to the SK-plasmin complex than to plasmin. In addition, the substrate Chromozym PK (N-Bz-Pro-Phe-Arg-pNA, HCl) was evaluated and found to be relatively specific for plasma kallikrein. Assays for antithrombin III and heparin using S-2222 as the substrate and factor Xa as the enzyme, plasma plasminogen and plasmin inhibitors using S-2251 as the substrate, and plasma prekallikrein and kallikrein inhibitors using Chromozym PK as the substrate have been developed. Synthetic peptides mimicking amino acid sequences adjacent to proteolytic activation cleavage of plasma serine protease precursors appear to be sensitive and relatively specific tools applicable to kinetical and clinical studies of these enzymes and their inhibitors.     

Discovery of glycyrrhetinic acid as an orally active,direct inhibitor of blood coagulation factor xa

Introduction

Factor Xa (FXa) plays an important role in blood coagulation. This study investigated glycyrrhetinic acid, a small molecule derived from Chinese herbs, and whether it has a direct inhibitory effect on FXa to display its anticoagulant activity.

Materials and Methods

Enzyme activities of FXa, plasmin, trypsin and thrombin, inhibition of FXa enzyme kinetics and plasma clotting time by glycyrrhentinic acid were performed in vitro. A rat tail-bleeding model and a rat venous stasis model were also used to evaluate in vivo tail-bleeding time and thrombus formation, respectively.

Results

Glycyrrhetinic acid in vitro directly inhibited FXa uncompetitivly with IC₅₀ of 32.6 ± 1.24 μmol/L, and displayed 2-, 14- and 20-fold selectivity for FXa when compared to plasmin, thrombin and trypsin, respectively. The plasma clotting time was increased in a dose-dependent manner. The prothrombin time doubled (PT₂), when the concentration of glycyrrhetinic acid reached 2.02 mmol/L. During in vivo experiments intragastric administration of glycyrrhetinic acid caused a dose-dependent reduction in thrombus weight on the rat venous stasis model (all P < 0.05). 50 mg/kg glycyrrhetinic acid resulted in 34.8% of venous thrombus weight lost, compared to the control. In addition, 200, 300 and 400 mg/kg doses of glycyrrhetinic acid caused a moderate hemorrhagic effect in the rat tail-bleeding model by prolonging bleeding time 1.1-, 1.5- and 1.9-fold compared to the control, respectively.

Conclusions

Glycyrrhetinic acid is a direct inhibitor of FXa that is effective by oral administration, and with further research could be used to treat blood coagulation disorders.     

Anticoagulant effect of cardiotoxins

Variation of the potent thrombin inhibitors derived from Nα-arylsulfonyl-4-amidinophenylalanine was carried out by interposition of an ω-aminoalkylcarboxylic acid between the Nα-arylsulfonyl residue and the 4-amidinophenylalanine part. The use of glycine as spacer renders the compounds tight binding inhibitors of thrombin. The K_i of the most potent inhibitor reaches the nmol/l range. The inhibitory effect is specifically directed against thrombin, the K_i values for inhibition of trypsin, plasmin and factor Xa are some orders of magnitude higher than those for thrombin inhibition.     

The relationship between plasma and placental tissue factor, and tissue factor pathway inhibitors in severe pre-eclampsia patients

Objective

To investigate the mechanism underlying the hypercoagulable state in severe pre-eclampsia.

Methods

Plasma tissue factor (TF) and tissue factor pathway inhibitor (TFPI) expression from pre-eclampsia patients and healthy pregnant controls were determined by ELISA. Placental TF and TFPI gene and protein expression were detected by quantitative RT-PCR, immunohistochemistry, and Western analysis.

Results

The plasma TF level in the pre-eclampsia group was significantly higher than the control group (p < 0.01), and surprisingly, the plasma TFPI-1 and TFPI-2 in the pre-eclampsia group were significantly lower (p < 0.01). Placental TF gene and protein expression levels in the pre-eclampsia group was significantly higher than the control group, while TFPI-1 and TFPI-2 levels were significantly lower (p < 0.05). Lastly, a significant correlation was found between plasma and placental TF protein levels in the pre-eclampsia group (p < 0.01).

Conclusion

Higher expression and/or release of TF from the placenta may contribute towards a pathological hypercoagulable state in pre-eclampsia patients.