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The plasma contact activation (CAS) and kallikrein/kinin (KKS) systems consist of 4 proteins: factor XII, prekallikrein, high molecular weight kininogen, and the bradykinin B2 receptor. Murine genetic deletion of factor XII (F12(-/-)), prekallikrein (Klkb1(-/-)), high molecular weight kininogen (Kgn1(-/-)) and the bradykinin B2 receptor (Bdkrb2(-/-)) yield animals protected from thrombosis. With possible exception of F12(-/-) and Kgn1(-/-) mice, the mechanism(s) for thrombosis protection is not reduced contact activation. Bdkrb2(-/-) mice are best characterized and they are protected from thrombosis through over expression of components of the renin angiotensin system (RAS) leading to elevated prostacyclin with vascular and platelet inhibition. Alternatively, prolylcarboxypeptidase, a PK activator and degrader of angiotensin II, when deficient in the mouse leads to a prothrombotic state. Its mechanism for increased thrombosis also is mediated in part by components of the RAS. These observations suggest that thrombosis in mice of the CAS and KKS are mediated in part through the RAS and independent of reduced contact activation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The plasma contact system is a proinflammatory and procoagulant protease system. The biochemistry of the system is well established, however, its biological functions are just beginning to emerge. Here, we provide a condensed overview on mechanisms involved in activation and regulation of the contact system. These recent findings will help us to better understand the role of the enigmatic system for health and disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sulfation of curdlan, a natural, linear beta-1,3-glucan results in potent anticoagulant and antithrombotic agents. The different activity characteristics in the classical coagulation assays were shown to depend on various structural parameters. To obtain more detailed information about their structure-dependent mechanisms of action, one representative of these beta-1,3-glucan sulfates (CurS), was further investigated using several coagulation assays and amidolytic tests with chromogenic substrates. The mode of action of CurS differs from that of heparin. CurS reduces the thrombin formation by principally inhibiting the intrinsic FXa generation. As shown by amidolytic assays, it eliminates already generated thrombin mainly by accelerating the HCII-mediated thrombin inactivation, whereas its AT-mediated anti-thrombin activity is considerably lower than that of heparin. Further, it prevents the thrombin-mediated fibrin polymerization by directly interfering with the thrombin action on fibrinogen as well as by binding to fibrinogen. Finally, CurS is capable to activate the contact system with the consequence of a potential fibrinolytic effect. In conclusion, beta-1,3-glucan sulfates do not inhibit the blood coagulation nonspecifically due to their anionic character, but in dependence on their individual structure, they interfere specifically with the coagulation process at several sites.

The expression of high-molecular-weight and low-molecular-weight kininogen mRNAs in the whale liver was examined by reverse transcription-polymerase chain reaction. The nucleotide sequences of the high-molecular-weight and low-molecular-weight kininogen cDNAs were analyzed and deduced to the amino acid sequences. The high-molecular-weight kininogen composed of 609 amino acid residues with 18 signal peptides possessed the consensus sequences of the cysteine protease inhibitor domains I and II, the bradykinin domain, the histidine-rich region, and the prekallikrein-binding region. Except for the histidine-rich region, the overall homologies with bovine, human, and rat high-molecular-weight kininogens were 81%, 76%, and 62%, respectively. The low-molecular-weight kininogen is composed of 408 amino acid residues. The nucleotide sequence down to C(1200) as well as the amino acid sequence till Ile(382) is identical to that of the high-molecular-weight kininogen. The remaining low-molecular-weight kininogen-specific carboxy-terminal portion possessed an amino acid sequence similar to that of the land mammals. The overall homologies with bovine, human, and rat low-molecular-weight kininogens were 82%, 79%, and 64%, respectively. The amino acid sequences of both whale high-molecular-weight and low-molecular-weight kininogens are most similar to those of the bovine among the land mammals analyzed so far. An incubation of dolphin/whale plasma with human plasma kallikrein, or with bovine trypsin, in the presence of carboxypeptidase inhibitors generated bradykinin antigen as well as the spasmogenic activity to the estrous rat uterus. The amount of bradykinin released by the latter enzyme was almost double of the former, indicating that the dolphin/whale plasma contained similar concentrations of low-molecular-weight and high-molecular-weight kininogens.

The incubation of normal human plasma in the presence of sulphatide vesicles results in the generation of amidolytic activity due to factor XIIa (FXIIa) and to kallikrein (KRN). The progress of the generation of the enzymes distinguished a high initial rate of enzyme generation, a decline of this rate to maximum amidolytic activity ([FXIIa]m and [KRN]m) and a negative pseudo-first-order rate attributed to enzyme inactivation by plasma C1-inhibitor (C1INH). [FXIIa]m and [KRN]m were determined after the treatment of various dilutions of plasma in the presence of 4, 15, or 40 microM sulphatide vesicles. At all levels of sulphatides, [FXIIa]m and [KRN]m initially increased with the concentration of plasma, to reach a plateau at higher concentration of plasma. The plateau activities of the generated enzymes and the optimal concentration of plasma both increased with the level of sulphatide vesicles. The pseudo-first-order inactivation rate for KRN increased progressively with the concentration of plasma but the respective rate for FXIIa was independent of the plasma concentration. The data suggest that contiguous binding of plasma FXIIa, factor XII (FXII), and the complexes of high molecular weight kininogen (HK) with prekallikrein (HK-PKRN) and factor XI (HK-FXI) to an electronegative surface induces a rapid generation of FXIIa and KRN. The concentration of the electronegative surface controls the levels of generated FXIIa and KRN and their release to the bulk phase. The released FXIIa and KRN are both inactivated by C1INH.

Factor XI (FXI) deficiency is associated with an abnormal bleeding state. The extent of bleeding does not correlate well with the plasma concentration of FXI, and it has been suggested that also unknown factors interfere with the bleeding tendency. In a recent paper (Thromb. Res. 74, 477-485, 1994) we found that FXIa activated in human plasma was present in association with part of factor XIIa (FXIIa) and part of kallikrein, influencing their functional activities. Should the activity level of FXIa also be altered by the other contact factors this might provide one approach to the problem of the failure of assays of FXIa to correlate with bleeding tendency. In the present study we have developed an assay procedure for FXIa based on its amidolytic (S-2366) activity, and allowing at the same time a quantification of the amount of FXIa associated to kallikrein. The total amidase activity obtained was separated into two main fractions by use of soybean trypsin inhibitor (STI), corn inhibitor (CI) and lima bean trypsin inhibitor (LTI). One fraction contained free FXIa which could be specifically blocked by LTI. An inhibitor resistant fraction was found to contain FXIa inactive in association with kallikrein. The content of FXIa could be assessed in experiments with mixtures of normal plasma and plasma deficient in prekallikrein, and was taken into account in the calculations. This fraction increased during storage of plasma at -70 degrees C. To obtain stable and comparable assay conditions the method was based on plasma stored for at least four weeks. The specificity of the method was verified by parallel radial immunodiffusion tests. The results imply that the activity level of FXIa is dependent on kallikrein present. If the experimental results has relevance to the situation under physiological conditions, they indicate one possible cause of the failure of assays of FXI to correlate with bleeding tendency.

The plasma levels of contact activation factors were measured in women using a low estrogen dose oral contraceptive (OC). Basic values for factor XII (FXII), factor XI (FXI), prekallikrein (PK), and high molecular weight kininogen (HK) were obtained in immunoassays by comparing with control plasma. The plasma levels of FXII and PK were significantly increased in OC plasma, to 147% and 146% respectively, whereas no significant increase could be registered for FXI (106%) or for HK (107%). Functional assays carried out with different peptide substrates (S-2222 for FXIIa, and S-2222, S-2302 and Bz-Pro-Phe-Arg-pNA for kallikrein) showed increases in OC plasma to about 150% for both proteases, in accordance with results obtained in radial immunodiffusion (RID). However, when FXIIa was measured with the high molecular weight substrate PK, no significant increase could be registered. Further experiments suggested this result to be due to the low level of FXI present in OC plasma, as compared to the levels of FXII and PK. Assays were carried out in mixtures of test plasma (OC or control plasma) and plasma deficient in FXI or FXII. The results obtained suggested that FXIa was present in some kind of association with part of FXIIa and part of kallikrein present. At low concentrations of FXI the functional activity of FXIIa was reduced, and the assay data indicated that an appropriate level of FXI was required to obtain maximum rate of hydrolysis of prekallikrein by FXIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Native and reduced SDS polyacrylamide gel electrophoresis on the automated PhastSystem (Pharmacia) were used to demonstrate protein-protein binding interactions and structural changes during proteolytic activations of the proteins involved in contact activation. The "mobility shift" assay in native gels has been used to visualize the kinetics of activation of factor XII by dextran sulfate as well as the formation of kallikrein-cleaved high molecular weight kininogen. It shows the formation of prekallikrein-high molecular weight kininogen complexes and factor XII-dextran sulfate complex for the first time in gels. The use of automation makes this procedure fast and reproducible using nanogram amounts of protein in relatively short time.

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 surface dependent contact activation of human plasma was studied as a function of the molecular weight of several synthetic macromolecular and watersoluble derivatives: polystyrene sulphonates, dextran methyl-benzylamide sulphonate/carboxylates as well as dextran sulphates as reference materials. Extent of the contact activation was determined by the amidolytic activities generated from cold activation of plasma in the presence of dilutions of the polymers for varying incubation times. The activating properties of the materials have been rationalized by means of a parameter calculated from the shapes of the amidolytic rates vs. incubation time variations. It was found that all the studied polymers behave similarly: contact stimulating properties begin to increase sharply passed a molecular weight of 25,000 and thereafter gradually reach a limiting plateau for Mw = 100,000. Activating capacities estimated for these limiting values have been expressed in term of specific capacities unrelated to Mw effects. These parameters were also compared to that of standard heparin estimated in the same conditions.

Cholesterol sulfate was found to display a strong ability to trigger the activation of Factor XII and prekallikrein in the presence of HMW kininogen. Other sulfate ester derivatives of testosterone, estrone, pregnenolone and dehydroepiandrosterone and cholesterol tested did not show any effect on the activation of Factor XII and prekallikrein. The activity of cholesterol acetate and sulfodeoxycholic acid was very weak. Cholesterol sulfate markedly shortened the partial thromboplastin time of normal human plasma, but not plasmas deficient in Factor XII, Factor XI and HMW kininogen. Upon prolonged incubation, the partial thromboplastin time of prekallikrein-deficient plasma was also shortened. Moreover, as well as kaolin and sulfatide, cholesterol sulfate shortened the partial thromboplastin time of plasmas from monkey, dog, rat, guinea pig, sheep, cow, hog and horse, but not from duck and chicken. Since cholesterol sulfate is distributed in erythrocytes, various organs and body fluids, it may play an important role in the activation of the intrinsic blood coagulation system.