Activation Of Plasma Prekallikrein Research Articles

The Effect of Collagen Mediated Platelet Release on Plasma Prekallikrein Activation

Collagen mediated platelet aggregation caused -5.6 +/- 6.7% inhibition and +39.1 +/- 15.2% potentiation of prekallikrein activation in plasma from normal healthy volunteers between 20-40 and 50-65 years of age, respectively (n = 15, p less than 0.01). The amounts of platelets factor-four (PF4) released in the two groups were not significantly different. Collagen treatment in the presence of indomethacin caused +11.5 +/- 3.6% and +59.6 +/- 19.5% potentiation in the 20-40 and 50-65 age groups respectively (p less than 0.02). Adrenaline mediated platelet aggregation caused -55.2 +/- 7.1% and -35.2 +/- 8.3% inhibition in the 20-40 and 50-65 age groups, respectively. Collagen treatment of platelet-deficient-plasma and platelet-rich-plasma in EDTA also caused potentiation of prekallikrein activation. The results indicate that the observed degree of prekallikrein activation after platelet aggregation is a net result of the inhibitory effect of PF4 and the potentiatory effect of activated platelets. The potentiatory effect was greater after collagen treatment as compared to adrenaline treatment, and in the 50-65 age group as compared to the 20-40 age group.

A platelet derived inhibitor of plasma prekallikrein activation

Platelet products released by ADP mediated platelet aggregation or by repeated freeze-thawing of platelet-rich-plasma caused inhibition of the dextran sulphate induced activation of prekallikrein, measured amidolytically, in subsequently prepared platelet-poor-plasma. Platelet products did not inhibit the amidolytic activity of contact activated-plasma. Addition of antiserum to platelet factor-4 (PF4) neutralized the inhibitory effect of platelet products towards prekallikrein activation. When platelet released products were fractionated by heparin affinity chromatography, 93% of the PF4 applied and 86% of the inhibitory activity of the starting material was recovered in the high-affinity fraction.

Effects of cyclooxygenase inhibitors on plasma and urinary kallikrein.

In vitro studies were performed to investigate the direct effects of the cyclooxygenase inhibitors (COI), meclofenamate, imidazol, acetylsalicylic acid (ASA), indomethacin, and acetaminophen on the plasma kallikrein system and on urinary kallikrein excretion. Biological (guinea pig ileum) and colorimetric (synthetic substrate) methods were used. Results showed that all COIs except ASA affected both the activation of pre-kallikrein in plasma and the direct activity of plasma kallikrein, but none of the COIs tested was able to alter the urinary kallikrein excretion. Additionally, in Wistar rats we studied the effects of chronic administration of ASA, meclofenamate, and indomethacin on the plasma kallikrein system and urinary kallikrein excretion. In contrast to the in vitro studies, administration of these COIs reduced the amount of total 24-hour urinary kallikrein excretion. Moreover, the pre-plasma kallikrein and total plasma kallikrein levels were diminished in all experimental groups. However, only ASA and meclofenamate increased the free-plasma kallikrein levels despite the fact the three COIs used reduced the high molecular weight kininogen. Thus, in vitro data suggest an important and direct effect of meclofenamate, imidazol, indomethacin, and acetaminophen on the plasma kallikrein system, without any effect on urinary secretion. ASA was the only COI that showed no direct effect in these experiments. Chronic COI administration in Wistar rats suggests that ASA, meclofenamate, and indomethacin affect both the plasma kallikrein-kinin system and kallikrein excretion. When these drugs are used to evaluate the interactions between prostaglandins and the kallikrein-kinin system, their possible effects through both mechanisms, directly or via prostaglandin inhibition, should be considered.

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.

Assay of Prekallikrein in Human Plasma: Comparison of Amidolytic, Esterolytic, Coagulation, and Immunochemical Assays

Using the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCl, an amidolytic assay was designed to measure prekallikrein in plasma. At a substrate concentration of 1 mM (Km = 0.2 mM), the amidolysis of purified kallikrein at 1 coagulant unit/ml was observed to be 2.47 mumole/min/ml. Conditions for plasma prekallikrein activation were optimized to approach complete activation when compared to the amidolytic activity of the purified plasma kallikrein. Plasma treated with chloroform to destroy inhibitors of kallikrein was activated with dilute kaolin (final concentration 1 mg/ml) for 1 min at 25 degrees C. Activated plasma prekallikrein had 78% (1.92 mumole/min/ml) of activity of purified kallikrein at plasma concentration. Comparison of this amidolytic assay with immunochemical, esterolytic, and coagulant assays of three subject populations (normals, women on birth control pills, and patients with hepatocellular disease) showed good correlation both in normals and in the patient groups between the amidolytic and esterolytic assays (r = 0.89). Each enzymatic assay correlated with the immunochemical assay (r = 0.72, r = 0.68, respectively). However, comparison of each of these assays with the coagulant assay showed no significant correlation due to the large inherent error of the latter assay. This standardized plasma prekallikrein amidolytic assay should facilitate studies of plasma prekallikrein concentration in physiologic and pathologic conditions and help identify activation of the contact phase of coagulation in disease states.

Assay of prekallikrein in human plasma: comparison of amidolytic, esterolytic, coagulation, and immunochemical assays

Using the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCl, an amidolytic assay was designed to measure prekallikrein in plasma. At a substrate concentration of 1 mM (Km = 0.2 mM), the amidolysis of purified kallikrein at 1 coagulant unit/ml was observed to be 2.47 mumole/min/ml. Conditions for plasma prekallikrein activation were optimized to approach complete activation when compared to the amidolytic activity of the purified plasma kallikrein. Plasma treated with chloroform to destroy inhibitors of kallikrein was activated with dilute kaolin (final concentration 1 mg/ml) for 1 min at 25 degrees C. Activated plasma prekallikrein had 78% (1.92 mumole/min/ml) of activity of purified kallikrein at plasma concentration. Comparison of this amidolytic assay with immunochemical, esterolytic, and coagulant assays of three subject populations (normals, women on birth control pills, and patients with hepatocellular disease) showed good correlation both in normals and in the patient groups between the amidolytic and esterolytic assays (r = 0.89). Each enzymatic assay correlated with the immunochemical assay (r = 0.72, r = 0.68, respectively). However, comparison of each of these assays with the coagulant assay showed no significant correlation due to the large inherent error of the latter assay. This standardized plasma prekallikrein amidolytic assay should facilitate studies of plasma prekallikrein concentration in physiologic and pathologic conditions and help identify activation of the contact phase of coagulation in disease states.

Responses of active and inactive plasma renin and changes in urinary kallikrein and plasma prekallikrein to various conditions in normal subjects.

Little is known about changes in inactive plasma renin in various conditions or the in vivo activation mechanism of inactive renin. The effects of various factors known to stimulate or suppress renin release on active and inactive PRA were examined in normal subjects. Inactive PRA was determined as the difference between the total PRA after trypsin activation and active PRA. Concurrent measurements of urinary kallikrein excretion and plasma prekallikrein activity were performed to assess the possible role of renal or plasma kallikrein in in vivo activation of inactive renin. Short term stimulation with iv furosemide and ambulation, infusion of isoproterenol, and administration of captopril increased active PRA, but had little or no effect on inactive PRA. Sodium restriction and sodium loading, each for 4 days, induced parallel changes in active and inactive PRA. The administration of propranolol for 4 days decreased active PRA but did not change inactive PRA. There were no significant correlations between the changes in urinary kallikrein excretion and those in active PRA or in the proportion of active to total PRA after any short term treatments, except furosemide administration. Plasma prekallikrein activity was correlated with the proportion of active renin only during the long term sodium balance study. The present data suggest that the mechanisms ofr the control of inactive and active renin are different. Neither renal nor plasma kallikrein seems to be consistently involved in the in vivo activation of inactive renin.

Effect of captopril on inactive renin and contact phase of coagulation system.

It has been suggested that a Factor XII-plasma Prekallikrein dependent pathway might play an important role in the activation of inactive renin. Since Captopril has the potential to affect the kinin-kallikrein system, we have studied in a group of 16 patients with essential hypertension its acute effect both on the levels of active, inactive and total renin, and on the contact phase of the coagulation system. Our results show that a single dose of Captopril (25 mg) induces a rapid and persistent increase of active and total renin, while inactive renin tends to decrease. Together with blood pressure, plasma Prekallikrein(PK), Factor XII(FXII) and Factor XI(FXI) concomitantly decrease, although not significantly, and their values seem to return to basal levels soonafter. However, no correlation was found at any time between the levels of any of these coagulation factors, including PK, and those of inactive, active or the ratio inactive/total renin. In spite of that, it is still possible that an activation of PK, which is likely to occur under Captopril administration, may affect at least the conversion of vessel-bound prorenin rather than the circulating form.

Mechanism of activation of inactive renin in human plasma by puff adder venom

Venom of the puff adder ( Bitis arietans) contains a potent, basic, M r 24 000 metalloproteinase activity that can destroy all detectable trypsin and chymotrypsin inhibitory activity, when venom is incubated with human plasma. We have found that during such incubation, concomitant activation of inactive renin occurs. In an examination of the mechanism involved we now report the activation, in addition, of plasma prekallikrein and serine proteinase activity, but not plasminogen, when human plasma is incubated with venom. Furthermore, venom was not able to release active trypsin from its complex with α 1-proteinase inhibitor and human renin was not inhibited by α 1-proteinase inhibitor. The activities in venom and venom/plasma mixtures were analysed using Sephacryl S-200 gel filtration and the effect of 10 mM EDTA and 5 mM phenylmethanesulphonyl fluoride on activities in column fractions was tested. The inactive-renin-activating, plasma prekallikrein-activating and serine proteinase-activating activities could be accounted for to a large extent by a venom metalloproteinase which was estimated to have a M r of 24 000 by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. This enzyme activity appeared to complex with α 2-macroglobulin when venom was mixed with plasma. Since both EDTA and phenylmethanesulphonyl fluoride could inhibit the activation of inactive renin by this metalloproteinase, it is suggested that the enzyme activates serine proteinase(s), which then activate inactive renin. Plasma kallikrein may have a role in this process. Additional peaks of inactive-renin-activating activity eluted from Sephacryl S-200 at M r 30 000 and 80 000 (minor) and an additional, minor peak of caseinolytic activity eluted at M r 60 000. The M r 24 000 metalloproteinase in venom may have considerable utility in activating inactive renin at physiological pH owing to its ability to destroy plasma proteinase inhibitors at the same time.

Surface activation of blood coagulation, fibrinolysis and kinin formation.

The activation of plasma prekallikrein by single-chain factor XII has been studied in the presence of high molecular weight kininogen and kaolin. The data indicate that factor XII can initiate blood coagulation, fibrinolysis or kinin generation in the presence of kaolin and does so by converting prekallikrein to kallikrein. An enzyme cascade is then generated leading to the formation of fibrin, plasmin or bradykinin in three closely related physiological events.

Prekallikrein (Fletcher factor) deficiency in clinical disease states

Plasma prekallikrein activity was measured in a clotting test in 315 consecutive patients undergoing coagulation profiles during the seven month period between January 1, 1979, and August 3, 1979, using substrate from one of our patients with severe Fletcher factor deficiency. Among the 68 patients with significantly reduced Fletcher factor activity (< 0. 50 U/ml), 20 had chronic liver disease, 13 had disseminated intravascular coagulation (DIC), 11 had sepsis, 4 had chronic renal failure, 4 had Vitamin K deficiency, 3 had nonspecific blocking inhibitors, 3 had dilutional product replacement, 2 had thrombotic phenomena, 1 had Fletcher trait, and 7 were of miscellaneous etiology. Partial thromboplastin time (APTT) was prolonged in 42 of the 68. The most commonly associated coagulation abnormality in patients with prekallikrein deficiency was factor VII deficiency, observed in 39 suggesting a link of the intrinsic to the extrinsic coagulation pathway. There was no significant difference in Fletcher factor activity between males and females. Comparison of our findings with studies in the literature is undertaken, with speculation into the underlying pathogenetic mechanism involved in Fletcher factor deficiency in each disease state.

Studies on prekallikrein of bovine plasma. II. Activation of prekallikrein with proteinases and properties of kallikrein activated by bovine Hageman factor.

Activation of bovine plasma prekallikrein was investigated with several proteinases. Highly purified bovine plasma prekallikrein was rapidly activated to kallikrein [EC 3.4.21.8] by bovine activated Hageman factor, trypsin [EC 3.4.21.4] and Pronase P (proteinases from Streptomyces griseus) and more gradually by papain [EC 3.4.22.2] and ficin [EC 3.4.22.3]. Activation of prekallikrein was also observed with bovine plasmin [EC 3.4.21.7], but not with bovine clotting factors Xa (Stuart factor) [EC 3.4.21.6] and IXa (Christmas factor) or thrombin [EC 3.4.21.5]. Urokinase [EC 3.4.99.26], Reptilase, collagenase [EC 3.4.24.3], elastase [EC 3.4.21.11], alpha-chymotrypsin [EC 3.4.21.1], Nagarse [EC 3.4.21.14], and stem bromelain [EC 3.4.22 4] did not convert prekallikrein to kallikrein. Plasma kallikrein activated to Hageman factor released kinin rapidly from bovine high molecular weight (HMW) kininogen. However, from bovine low molecular weight (LMW) kininogen, liberation of kinin was extremely slow. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI), Trasylol, diisopropylfluorophosphate (DFP), and N-alpha-tosyl-L-lysine chloromethylketone (TLCK), but not by egg-white trypsin inhibitor (EWTI), lima bean trypsin inhibitor (LBTI), heparin or hexadimethrine bromide (Polybrene). The kallikrein formed an enzyme-inhibitor complex with SBTI and Trasylol, but not with LBTI. Prekallikrein did not react with SBTI. Prekallikrein consists of a single polypeptide chain of molecular weight about 90,000, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Activation of prekallikrein by Hageman factor was found to involve cleavage of the single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. The peptide bond cleaved in prekallikrein by the activation was an Arg-X peptide bond on a disulfide-bridged polypeptide chain.

Kinin-inactivating enzyme from the mushroom Tricholoma conglobatum. V. Its application to anaphylactic shock in rats.

In order to clarify the involvement of the kallikrein-kinin system in anaphylactic shock, Shimeji kininase, which is able to block kinin action in the body due to rapid kinin destruction, was tested on anaphylactic shock in rats. The anaphylactic shock in rats at 15 days after sensitization with egg albumin was hardly suppressed by Shimeji kininase alone or mepyramine alone, but the rats were markedly protected when this enzyme was used together with mepyramine. The rats at 30 days after sensitization were completely protected against shock by mepyramine alone and were also protected to some extent by Shimeji kininase alone. Thus, at the early phase after sensitization, both kinins and histamine appear to play important roles in anaphylaxis in rats, but in the late phase after sensitization, histamine is considered to play a more important role than kinins. In the rats at 15 days after sensitization, the high molecular weight kininogen level in plasma did not alter on challenge with antigen, and plasma prekallikrein activation was not detected. On the other hand, a slight but significant decrease of low molecular weight kininogen level and an increase of kinin level on challenge with antigen were observed.

Kinin-inactivating enzyme from the mushroom Tricholoma conglobatum. IV. Its effects on the kallikrein-kinin system.

A potent kinin-inactivating enzyme, Shimeji kininase, may have applications in research on the kallikrein-kinin system in the body. In order to test its suitability, the effects of this enzyme, in addition to kinin destruction, on substances related to the kallikrein-kinin system were investigated. This enzyme hardly affected the esterase activities of the glandular kallikreins. It destroyed the heat-denatured kininogens of various species, but did not attack the native rat low molecular weight kininogen molecule. The vasodilative activity of this enzyme assayed in the dog was almost negligible, and no kinin activity could be detected in incubation mixtures of this enzyme and heatdenatured kininogens of various species by the Magnus method. Therefore, this enzyme does not have kininogenase activity, and its destruction of the heat-denatured kininogens was not due to kinin liberation from the kininogens. Intravenous injection of this enzyme in the rat caused the depletion of plasma high molecular weight kininogen, presumably due to plasma prekallikrein activation, while the low molecular weight kininogen level in plasma was not significantly changed.

Activation of human plasma prekallikrein: Influence of activators, activation time and temperature and inhibitors

Plasma prekallikrein (PKK) was activated with Cephotest and dextran sulphate (DS) under various conditions and the resulting kallikrein (KK) assayed on a chromogenic peptide substrate for this enzyme. Cephotest: plasma ratios of 30:1 and DS concentrations of between 0.001 and 0.01% (equal volumes plasma and DS) gave maximal activities when activation proceeded at 0°C. However, enzyme activity was also dependent on incubation time and temperature and activation times of 3 minutes (Cephotest) and 15 minutes (DS) at 0°C gave highest activities. Incubation at 37°C gave progressively lower KK yields with Cephotest and very little KK activity was detected with DS at this temperature. These results were shown to be due to progressive inhibition of plasma KK at 37°C. With C 1-esterase inhibitor deficient plasma higher KK activities were obtained with both activators at 0°C and the loss of activity on incubation at 37°C was markedly reduced. The addition of pure C 1-esterase inhibitor to the deficient plasma prior to activation resulted in a normal activity and normal inhibition profile. Gel filtration studies on normal and C 1-esterase deficient plasmas after activation at 0°C with Cephotest revealed that both free KK and KK- α 2 macroglobulin complexes were present in the activated plasmas. In the deficient plasma both activities were higher indicating that in normal plasma some KK is immediately bound by α 2-macroglobulin and C 1-esterase inhibitor even when activation takes place at 0°C.

Increased plasma kallikrein activity and tumour growth suppression associated with intralesional bradykinin injections in hamsters.

Daily intralesional injections of bradykinin (250 microgram) into syngeneic SV40 virus-induced fibrosarcomas in inbred hamsters for 21 days produced marked inhibition of tumour growth, marked lymphoid cell infiltration of tumours, and significant elevations in plasma kallikrein and prekallikrein activity, compared with intralesional saline injections. All tumours were of comparable size and growth rates before injections were begun. Plasma kallikrein levels of animals which received the standard tumour cell inoculum subcutaneously and did not develop tumours were elevated significantly above normal levels and above levels of animals with large tumours. Kallikrein inhibitor activity of animals which did not develop tumours was depressed below normal levels. Intralesional injections of saline produced a significantly lesser response in plasma kallikrein and plasma prekallikrein activity than was encountered with subcutaneous saline injections in normal animals. Intralesional injections of bradykinin produced a response equivalent to that encountered with subcutaneous bradykinin injections in normal animals.

Human plasma kallikrein: purification, enzyme characterization and quantitative determination in plasma.

Human plasma kallikrein was isolated from a plasma fraction related to Cohn fraction IV4 by affinity- and Sephadex G-150 chromotography yielding a material with 17.3 TAME-U/A280 unit. The preparation was characterized by immunological and enzymatic methods. Complex formation with alpha2-macroglobulin, C1-inactivator and aprotinin was demonstrated by immunoelectrophoresis. The bradykinin release from high-molecular weight kininogen and its inhibition by antibodies to kallikrein, AT III and AT III-heparin complex were measured using a biological test system (rat uterus). Time dependent inactivation of kallikrein by AT III, and AT III-heparin complex was shown by means of a synthetic kallikrein substrate: Bz-Pro-Phe-Arg-pNan. The same substrate was used to measure the activation of prekallikrein in plasma by kaolin and F XII a. Antibodies raised against kallikrein were shown to inhibit the reaction specifically. A quantitative determination of plasma prekallikrein by electroimmunodiffusion according to Laurell was developed: the plasma concentration in normal individuals was found to be 1.8 - 2.2 TAME-U/ml related to kallikrein activity; this corresponds approximately to 9 - 11 mg antigen/100 ml plasma.

Subunits of human plasma kininase II generated by plasma kallikrein

Partially purified human plasma kininase II was incubated with proteolytic enzymes such as plasma kallikrein, prekallikrein activator, plasmin, thrombin and trypsin. Digestion of plasma kininase II by plasma kallikrein generated 180,000 (I) and 95,000 (II) mol. wt subunits while plasmin, thrombin and trypsin inactivated plasma kininase II. These subunits have two activities just as plasma kininase has: they hydrolyze bradykinin, and they convert angiotensin I into angiotensin II. K m values of the two subunits for bradykinin were 0.17 × 10 −6 M, indicating that this value was identical with plasma kininase II. V max values of the two subunits for bradykinin were higher than that of plasma kininase II. The bradykininase activities of the two subunits were significantly inhibited by inhibitors such as HgCl 2, ethylenediamine tetraacetic acid. p-chloromercuriphenyl sulfonic acid, SQ 20881(BPF9a), soy bean trypsin inhibitor and Trasylol. The behaviour of the two subunits towards these above inhibitors was also identical with plasma kininase II. A 180,000 mol. wt kininase was partially purified from human plasma. The kininase had bradykinin-inactivating and angiotensin I-converting activities. Its K m value and the behaviour towards the inhibitors described above were the same as the subunit I.

Activation of protein mediators of inflammation and evidence for endotoxemia in Borrelia recurrentis infection

Fifteen patients with Borrelia recurrentis infection were studied to evaluate the roles of certain plasma proteins and endotoxin in the pathophysiology of both the acute illness and the Jarisch-Herxheimer-like reaction. The causative spirochetes disappeared from the blood during the Jarisch-Herxheimer-like reaction, which occurred about 2 hours after antibiotic therapy. The mean titers of Hageman factor, plasma prekallikrein and serum hemolytic complement activity were decreased at the time of admission and 2 hours after treatment, and rose to normal values during convalescence. Serum properdin titers were decreased in 14 patients at the time of admission, in 12 patients 2 hours after treatment, and in none during convalescence. The frequency of elevated levels of fibrinogen-related antigens increased from three patients at the time of admission to 12 patients 2 hours after treatment. Results of plasma limulus tests for endotoxin-like material were positive in 11 patients at the time of admission and in 13 patients 2 hours after treatment. These findings demonstrated that Hageman factor, prekallikrein and proteins of the complement system are activated in B. recurrentis infection and that endotoxin may play a role in both the acute illness and in the development of the Jarisch-Herxheimer-like reaction after treatment.

Inactivation and binding of human plasma kallikrein by antithrombin III and heparin

Purified human antithrombin III was found to inactivate the esterase, the kininogenase, and the plasminogen activator activity of purified plasma kallikrein. The reaction rate was increased by heparin. Kinetic studies indicated second-order kinetics for the reaction between kallikrein and antithrombin III, and a catalytic effect of heparin. SDS polyacrylamide gel electrophoresis revealed that three new bands were generated during the inactivation of kallikrein by antithrombin III. Heparin did not influence the factor XII-dependent activation of prekallikrein in human plasma, whereas the inactivation of kallikrein elapsed slightly more rapidly in plasma samples where heparin was added. It is concluded that antithrombin III probably is of minor importance for the inactivation of kallikrein in plasma under physiological circumstances.