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The effect of anabolic-androgenic steroid (AAS) abuse on the contact activation system (CAS) is not known in detail. We hypothesized that current AAS abuse reduces the kallikrein-generating capacity of CAS significantly and investigated the impact of AAS on the proteins and capacity of CAS in current and former AAS abusers and healthy age-matched controls. Men 18 to 50 years of age were included as current AAS abusers, former AAS abusers, or controls. Blood samples were collected after overnight fasting. Kallikrein generation (lag time, peak height, and endogenous kallikrein potential [EKP]), coagulation factor XII (FXII), prekallikrein, high-molecular-weight kininogen (HK), and Complement C1 esterase inhibitor (C1inh) were assessed. Groups were compared by analysis of variance or Kruskal-Wallis test and probabilities were corrected for multiple comparisons. Associations were evaluated by linear regression models. The EKP was significantly reduced in current ( n  = 37) AAS abusers (984 ± 328 nmol/L × min) compared with former ( n  = 33) abusers (1,543 ± 481 nmol/L × min) and controls ( n  = 30) (1,521 ± 339 nmol/L × min), p  < 0.001. Current abusers had higher levels of FXII and C1inh and lower levels of prekallikrein and HK than controls, p ≤ 0.025. Stepwise regression analysis showed that EKP was associated with C1inh and prekallikrein in current AAS abusers, R 2  = 0.70, p  < 0.001. We conclude that current AAS abuse reduces the kallikrein-generating capacity of CAS by increasing the concentration of C1inh and reducing the concentration of prekallikrein. These changes may contribute to the anti-inflammatory effect of testosterone., Competing Interests: None declared., (Thieme. All rights reserved.)

Cardiovascular disease, including stroke, myocardial infarction, and venous thromboembolism, is one of the leading causes of morbidity and mortality worldwide. Excessive coagulation may cause vascular occlusion in arteries and veins eventually leading to thrombotic diseases. Studies in recent years suggest that coagulation factors are involved in these pathological mechanisms. Factors XIa (FXIa), XIIa (FXIIa), and plasma kallikrein (PKa) of the contact system of coagulation appear to contribute to thrombosis while playing a limited role in hemostasis. Contact activation is initiated upon autoactivation of FXII on negatively charged surfaces. FXIIa activates plasma prekallikrein (PK) to PKa, which in turn activates FXII and initiates the kallikrein-kinin pathway. FXI is also activated by FXIIa, leading to activation of FIX and finally to thrombin formation, which in turn activates FXI in an amplification loop. Animal studies have shown that arterial and venous thrombosis can be reduced by the inhibition of FXI(a) or PKa. Furthermore, data from human studies suggest that these enzymes may be valuable targets to reduce thrombosis risk. In this review, we discuss the structure and function of FXI(a) and PK(a), their involvement in the development of venous and arterial thrombosis in animal models and human studies, and current therapeutic strategies., Competing Interests: S.H. is an employee of Bayer AG, Germany. M.V., H.T.C. and H.M.H.S. have nothing to disclose. H.T.C. was a fellow of the Gutenberg Research Foundation and is adjunct professor at the Center for Thrombosis and Hemostasis, Gutenberg University, Mainz, Germany. H.M.H.S. and H.T.C. receive support from the Netherlands Heart Foundation: CVON2014-09, reappraisal of atrial fibrillation: interaction between hypercoagulability, electrical remodeling, and vascular destabilization in the progression of atrial fibrillation (RACE V)., (Georg Thieme Verlag KG Stuttgart · New York.)

The kallikrein/kinin system, an intravascular biochemical pathway that includes several proteins involved in the contact activation system of coagulation, renin-angiotensin activation and inflammation, may or may not play a role in venous thromboembolism (VTE) occurrence. Within a large prospective population-based study in the United States, we conducted a nested case-cohort study to test the hypothesis that higher plasma levels of high molecular weight kininogen (HK) or prekallikrein are associated with greater VTE incidence. We related baseline enzyme-linked immunosorbent assay measures of HK and prekallikrein in 1993 to 1995 to incidence VTE of the lower extremity ( n  = 612) through 2015 (mean follow-up = 18 years). We found no evidence that plasma HK or prekallikrein was associated positively with incident VTE. HK, in fact, was associated inversely and significantly with VTE in most proportional hazards regression models. For example, the hazard ratio of VTE per standard deviation higher HK concentration was 0.88 (95% confidence interval = 0.81, 0.97), after adjustment for several VTE risk factors. Our findings suggest that plasma levels of these factors do not determine the risk of VTE in the general population., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

The plasma zymogens factor XI (fXI) and prekallikrein (PK) are activated by factor XIIa (fXIIa) during contact activation. Polyanions such as DNA and RNA may contribute to thrombosis and inflammation partly by enhancing PK and fXI activation. We examined PK and fXI activation in the presence of nucleic acids, and determine the effects of the cofactor high molecular weight kininogen (HK) on the reactions. In the absence of HK, DNA and RNA induced fXI autoactivation. Proteases known to activate fXI (fXIIa and thrombin) did not enhance this process appreciably. Nucleic acids had little effect on PK activation by fXIIa in the absence of HK. HK had significant but opposite effects on PK and fXI activation. HK enhanced fXIIa activation of PK in the presence of nucleic acids, but blocked fXI autoactivation. Thrombin and fXIIa could overcome the HK inhibitory effect on autoactivation, indicating these proteases are necessary for nucleic acid-induced fXI activation in an HK-rich environment such as plasma. In contrast to PK, which requires HK for optimal activation, fXI activation in the presence of nucleic acids depends on anion binding sites on the fXI molecule. The corresponding sites on PK are not necessary for PK activation. Our results indicate that HK functions as a cofactor for PK activation in the presence of nucleic acids in a manner consistent with classic models of contact activation. However, HK has, on balance, an inhibitory effect on nucleic acid-supported fXI activation and may function as a negative regulator of fXI activation.

When triggered by factor (F) XII and nucleic acids, we showed that thrombosis in HRG-deficient mice is accelerated compared with that in wild-type mice. In this study, we set out to identify the mechanisms by which nucleic acids promote contact activation, and to determine whether HRG attenuates their effects. DNA or RNA addition to human plasma enhances thrombin generation via the intrinsic pathway and shortens the clotting time. Their effect on the clotting time is seven- to 14-fold greater in HRG-deficient plasma than in control plasma. Investigations into the mechanisms of activation reveal that nucleic acids a) promote FXII activation in the presence of prekallikrein- and high molecular weight kininogen (HK), and b) enhance thrombin-mediated FXI activation by 10- to 12-fold. Surface plasmon resonance studies show that DNA and RNA bind FXII, FXIIa, HK, FXI, FXIa and thrombin with high affinity. HRG attenuates DNA- and RNA-mediated FXII activation, and FXI activation by FXIIa or by thrombin, suggesting that HRG down regulates the capacity of DNA and RNA to activate the intrinsic pathway. Therefore, HRG attenuates the procoagulant activity of nucleic acids at multiple levels.

Plasma kallikrein (PK) is a serine protease generated from plasma prekallikrein, an abundant circulating zymogen expressed by the Klkb1 gene. The physiological actions of PK have been primarily attributed to its production of bradykinin and activation of coagulation factor XII, which promotes inflammation and the intrinsic coagulation pathway. Recent genetic, molecular, and pharmacological studies of PK have provided further insight into its role in physiology and disease. Genetic analyses have revealed common Klkb1 variants that are association with blood metabolite levels, hypertension, and coagulation. Characterisation of animal models with Klkb1 deficiency and PK inhibition have demonstrated effects on inflammation, vascular function, blood pressure regulation, thrombosis, haemostasis, and metabolism. These reports have also identified a host of PK substrates and interactions, which suggest an expanded physiological role for this protease beyond the bradykinin system and coagulation. The review summarises the mechanisms that contribute to PK activation and its emerging role in diabetes and metabolism.

Plasma kallikrein is a multifunctional serine protease involved in contact activation of coagulation. Deficiency in humans is characterised by prolonged activated partial thromboplastin time (aPTT); however, the balance between thrombosis and haemostasis is not fully understood. A study of plasma kallikrein-deficient mice revealed increased aPTT, without prolonged bleeding time. Prekallikrein antisense oligonucleotide (ASO) treatment in mice suggested potential for a positive therapeutic index. The current goal was to further define the role of plasma kallikrein in coagulation. Blood pressure and heart rate were normal in plasma kallikrein-deficient mice, and mice were completely protected from occlusion (100 ± 1.3% control flow) in 3.5% FeCl3 -induced arterial thrombosis versus heterozygotes (20 ± 11.4%) and wild-type littermates (8 ± 0%). Vessels occluded in 8/8 wild-type, 7/8 heterozygotes, and 0/8 knockouts. Anti-thrombotic protection was less pronounced in 5% FeCl3-induced arterial injury. Integrated blood flow was 8 ± 0% control in wild-type and heterozygotes, and significantly (p<0.01) improved to 43 ± 14.2% in knockouts. The number of vessels occluded was similar in all genotypes. Thrombus weight was significantly reduced in knockouts (-47%) and heterozygotes (-23%) versus wild-type in oxidative venous thrombosis. Average tail bleeding time increased modestly in knockout mice compared to wild-type. Average renal bleeding times were similar in all genotypes. These studies confirm and extend studies with prekallikrein ASO, and demonstrate that plasma kallikrein deletion prevents occlusive thrombus formation in mice with a minimal role in provoked bleeding. Additional support for the significance of the intrinsic pathway in the coagulation cascade is provided, as well as for a potential new anti-thrombotic approach.

The way by which contact of blood with foreign surface accelerates clotting has been elucidated from the discovery of four rare disorders of blood coagulation; Hageman trait, plasma thromboplastin antecedent (PTA) deficiency, Fletcher trait, and Fitzgerald trait. Interestingly, it was unexpectedly found that Fletcher factor is plasma prekallikrein and Fitzgerald factor is high-molecular-weight kininogen; components of the kinin-generating system, thus disclosing intimate relationships among clotting, fibrinolysis and kinin generation which may be viewed as body's defense reactions against injury. This review mainly reflects our research on Fletcher trait and Fitzgerald trait during the 1970s in Cleveland.

A genetically-transmissible factor (F) XII-inactivated allele has been produced in mice by targeted replacement of exons 3-8 of the FXII gene with the neomycin resistance gene. Interbreeding of these mice provided offspring homozygous for two inactivated FXII alleles (FXII(-/-)). Male and female FXII-deficient mice bred normally in all genotypic combinations of the heterozygous and homozygous states, and the offspring survived to adulthood, suggesting that a total FXII deficiency does not affect embryonic development and survival. Neither FXII transcripts nor FXII antigen was found in various tissues of adult FXII(-/-) mice. No obvious unchallenged coagulopathies were present in FXII(-/-) adult mice, despite greatly prolonged activated partial thromboplastin times in this mouse cohort. FXII(-/-) mice were then used to assess the in vivo importance of the plasma FXII/prekallikrein/kininogen pathway in provision of resting plasma bradykinin (BK) levels and in generation of plasma BK stimulated by contact with an artificial surface, using a new and greatly improved plasma BK assay developed during these studies. It was found that approximately 50% of resting BK, and all of the contact-stimulated plasma BK, was provided by this FXII-dependent pathway, without a requirement for FXI. These results provide clear evidence that surface-stimulated BK production, in mice, is dependent on the activation of FXII.

Measurement of activities of urinary (u-PA) and tissue plasminogen activator (t-PA) and analysis of euglobulin lysis times (ELTs) and prekallikrein were performed simultaneously using the plasma of normal pregnant women and nonpregnant women. The activities of plasminogen activators were determined by the method of electrophoretic zymography. The activity of low-molecular-weight (LMW) u-PA was significantly decreased at the onset of labor (p = 0.05) compared with that during the latter half of pregnancy. The same tendency was observed in the analysis of prekallikrein. On the contrary, no significance was shown in activities of high-molecular-weight (HMW) u-PA or t-PA. It is suggested that the action of kallikrein on low-molecular-weight u-PA causes it to change into plasmin at the onset of labor.

Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires > or = 50 microM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 x 10(7) or 2.4 x 10(7) sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.

Although proteins of the kinin-forming pathway are bound along the surface of endothelial cells, the mechanism of activation of this proteolytic cascade is unclear. Endothelial cell surface proteins, gC1qR and cytokeratin 1, are capable of binding Factor XII and high molecular weight kininogen (HK) in a zinc-dependent reaction thus we considered the possibility that these proteins might catalyze initiation of the cascade. Incubation of Factor XII, prekallikrein, and HK with gC1qR or cytokeratin 1 leads to a zinc-dependent and Factor XII-dependent conversion of prekallikrein to kallikrein. We also demonstrate that normal plasma is capable of activating upon interaction with the cells whereas plasma deficient in Factor XII, prekallikrein and HK do not activate. Normal plasma activation was inhibitable by antibody to gC1qR and cytokeratin 1. Thus, gC1qR and cytokeratin 1, represent potential initiating surfaces for activation of the plasma kinin-forming cascade and may do so as a result of their expression along cell surfaces.

The contribution of the various components of the contact system in the generation of factor XIIa (FXIIa) and of kallikrein (KRN) on an electronegative surface and the release of the generated enzymes to the bulk phase was examined in mixtures of normal human plasma and plasmas congenitally deficient in these components. The incubation of normal human plasma in the presence of sulphatide vesicles (40 microM) resulted in a fast generation of amidolytic activities due to FXIIa and to KRN followed by slower first-order inactivation rates of FXIIa (k'FXIIa) and of KRN (k'KRN) due to the presence of esterase inhibitors. Variation of the levels of factor XII (FXII), over a wide range, showed little effect on levels of FXIIa and of KRN but no activities were detected in 100% FXII-deficient plasma. The variation of prekallikrein (PKRN) concentration showed little effect on the generation of FXIIa but the generation of KRN declined linearly with the decrease in the level of PKRN. No activities were detected on treatment of PKRN-deficient plasma. The variation in the concentration of high molecular weight kininogen (HK) showed effects on FXIIa and KRN that were qualitatively similar to those seen on variation of PKRN but 100% HK-deficient plasma generated considerable activities of both FXIIa and KRN. The variation in the concentration of factor XI (FXI) showed no effect on the generation of FXIIa, whereas KRN levels increased linearly with the contribution of FXI-deficient in normal plasma. The present results suggest that the contiguous binding of FXIIa, FXII, PKRN-HK and FXI-HK onto the electronegative surface induces a rapid generation of FXIIa and KRN. The bound PKRN-HK complex prevents the release of generated FXIIa and therefore further binding and activation of FXII from the bulk phase. Consequently, the turnover of FXII is independent of its levels in the bulk phase and is rather related to the concentration of contact surface. The generated KRN is also protected by HK. However, since the enzyme responsible for the activation of PKRN-HK is FXIIa, the levels of generated KRN are positively related to the concentration of substrate.

It is well known that on artificial surfaces, binding and autoactivation of factor XII (FXII) is the initiating event of plasma prekallikrein (PK) activation. We performed investigations to examine whether this mechanism was true for FXII activation on endothelial cells (HUVEC). Activation of PK on HUVEC required an optimal substrate and Zn2+ concentration, the latter of which varied with the buffer's carrier protein. Maximal PK activation required the addition of 250 microM or 10 microM Zn2+ to buffers containing bovine serum albumin (BSA) or gelatin, respectively. However, the actual free Zn2+ concentration in these buffers was the same at 8 microM. In both BSA- and gelatin-containing buffers and using two different chromogenic substrates for FXII, no autoactivation of FXII on HUVEC was seen when incubated for up to 60 min. Rather, initiation of FXII enzymatic activity required the presence of PK. FXII activation after PK activation contributed to the extent of measured enzymatic activity, but its role was secondary because treatment with corn trypsin inhibitor or a neutralizing antibody to FXIIa did not abolish the measured enzymatic activity. They also reduced the activity to the level seen with PK activation alone. Alternatively, soybean trypsin inhibitor abolished the proteolytic activity associated with PK and FXII activation on HUVEC. Further, only normal human and FXII-deficient plasmas, not PK-deficient plasma, had the ability to generate proteolytic activity when incubated over endothelial cells. In a purified system, maximal PK activation was measured after a 10-15 min incubation depending upon the concentration of reactants. When FXII was added with the PK, maximal activation occurred within 7.5-10 min. In normal human or FXII-deficient plasmas, but not in PK-deficient plasma, maximal activation was seen in 4 min. These data indicate that on HUVEC, unlike artificial surfaces, PK activation when bound to HK is the initiating activation event in this system. FXII activation is secondary to PK activation and contributes to the extent of measured enzymatic activity. These data challenge the accepted dogmas of "contact activation" and suggest that on biologic membranes a new notion as to how this system is activated needs to be considered.

Factor XII has long been implicated in the intrinsic pathway of fibrinolysis, but the mechanism by which it triggers plasminogen activation and targets fibrinolysis has not been established. In the present study, the assembly and function of activated Factor XII (F.XIIa), prourokinase (pro-u-PA), high molecular weight kininogen (H-kininogen), and prekallikrein on human umbilical vein endothelial cells (HUVEC) was investigated. 125I-prekallikrein was shown to bind to HUVEC via receptor-bound H-kininogen in the presence of 50 microM ZnCl2. After the addition of F.XIIa, 78% of the 125I-prekallikrein initially bound to HUVEC was converted to 125I-kallikrein. However, only 6% of the HUVEC-bound 125I-pro-u-PA was thereby activated. This discrepancy was shown to be related to rapid dissociation (> 50% within 15 min) of prekallikrein/kallikrein, but not pro-u-PA, from HUVEC. Increasing the level of cell-bound kallikrein increased the portion of cell-bound pro-u-PA activated, indicating that their co-localization was important for this pathway. Finally, F.XIIa was shown to trigger plasminogen activation on HUVEC via this pathway. This assembly of reactants on the endothelium suggests a mechanism whereby local fibrinolysis may be triggered by blood coagulation.

The plasma of a 68-year-old man with cross reacting material (CRM)-positive prekallikrein (PK) deficiency was studied. PK clotting activity was < 0.01 U/ml, and PK antigen was 0.1 U/ml. No circulating anticoagulant against PK was detectable. The abnormal PK molecule, denoted as prekallikrein Zürich, was partially characterized by immunological and functional studies on the propositus' plasma. Immunoblotting analysis showed the abnormal PK being a single chain molecule of the same M(r) (80 kDa) as normal PK. Dextran sulfate activation of the propositus' plasma did not lead to proteolytic cleavage of the variant PK molecule, in contrast to dextran sulfate activation of a mixture of 1 volume normal plasma and 9 volumes CRM-negative PK deficient plasma. Agarose gel electrophoresis followed by immunoblotting demonstrated that PK Zürich was complexed with high molecular weight kininogen similarly to PK in normal plasma. Incubation of the propositus' plasma with purified beta-FXIIa resulted in impaired cleavage of PK Zürich when compared with PK hydrolysis in a mixture of 10% normal plasma and 90% CRM-negative PK deficient plasma. Moreover, proteolytically cleaved PK Zürich showed no enzymatic activity against factor XII and high molecular weight kininogen. These studies show that the functional defect of prekallikrein Zürich is due to an impaired cleavage by activated factor XII and probably the lack of enzymatic activity of the cleaved variant molecule.

To determine if rhesus monkeys (Macaca mulatta) could serve as a model for studying the role of the contact system in the pathophysiology of human infections, we compared structural, kinetic, and functional characteristics of plasma prekallikrein and its activation products in rhesus and humans. Three prekallikrein variants (85-, 89- and 93-kDa) were revealed in rhesus plasma as compared with the two variants (85- and 88-kDa) in human plasma by immunoblotting with the monoclonal antibody MAb 13G11. The prekallikrein concentration in rhesus plasma was 1.5-fold that in human plasma as determined by computerized immunoblot analyses (CIBA) and amidolytic activity. The electrophoretic mobility of prekallikrein from plasma of both species increased after deglycosylation. Inhibition of prekallikrein activation by MAb 13G11 was 55% (rhesus plasma) and 76% (human plasma), with similar inhibition curves. Immunoblots of activated rhesus plasma showed prekallikrein, complexes of kallikrein with C1 inhibitor, alpha 2-macroglobulin and approximately 60-kDa inhibitor(s) (viz. antithrombin III), and 45-kDa fragments, like those in activated human plasma. Concentrations and molecular masses of factor XII and high molecular weight kininogen were similar in rhesus and human plasma. The activated partial thromboplastin time (APTT) and prothrombin time were 20.1 +/- 1.6 and 9.7 +/- 0.3 s for rhesus and 32.0 +/- 5.6 and 12 +/- 0.5 s for human plasma. Human and rhesus APTTs were similar when prekallikrein concentrations in human and rhesus plasma became alike by adding human purified prekallikrein.(ABSTRACT TRUNCATED AT 250 WORDS)

We developed a very sensitive assay for functional factor XII. This assay is based on the property of glass-bound factor XII to activate prekallikrein (PK) into kallikrein in factor XII-deficient plasma, which is assessed by measuring the formation of kallikrein-C1-inhibitor complexes in this plasma by radioimmunoassay. Incubation of varying amounts of factor XII in glass tubes led to a dose-dependent increase in kallikrein-C1-inhibitor complexes in factor XII-deficient plasma with a lower limit of detection of +/- 20 pg of factor XII. The specificity of the assay for factor XII was demonstrated by experiments with plasmas deficient for factor XII, PK or high molecular weight kininogen (HK) and by incubation of factor XII with Polybrene or with a monoclonal antibody that inhibits the amidolytic and procoagulant activity of factor XII. The high sensitivity of the assay appeared to be due to the inability of C1-inhibitor to inhibit factor XII bound to glass, which resulted in a molar ratio of generated kallikrein to glass-bound factor XII of at least 100:1. This assay for factor XII may be a feasible tool in studies on structural and functional aspects of (recombinant) factor XII species synthesized by cultured cells.

The analysis of normal human plasma by fibrin autography revealed four species of plasminogen activator (PA) activity related to tissue-type PA, factor XII, prekallikrein and urokinase-type PA (u-PA). The u-PA activity increased significantly by incubating plasma with dextran sulfate. This increase was coincident with both the cleavage of factor XII and the complex formation of activated factor XII with its plasma inhibitors, which were determined by immunoblotting procedure. The dextran sulfate-dependent activation of u-PA required both factor XII and prekallikrein, but did not require either plasminogen or factor XI. High molecular weight kininogen was required only at a low concentration of dextran sulfate. Thus the results indicate that the factor XII and prekallikrein-mediated activation of single chain u-PA (scu-PA) operates as a major pathway of scu-PA activation in whole plasma in contact with dextran sulfate.

Patients treated with high doses of interleukin-2 (IL-2) because of cancer, develop hemodynamic and vasopermeability changes, that resemble those observed in sepsis. These patients thus provide a unique opportunity to study the early events in the development of septic shock. We analysed the changes that occurred in the contact system of coagulation in plasma from 4 patients, who together received seven 12-day cycles of high doses of IL-2. Levels of factor XII and prekallikrein during the cycles progressively fell to 50 and 30% of their initial levels, respectively, whereas significant increases in plasma factor XIIa- and kallikrein-C1-inhibitor complexes were not observed (in 3 out of 211 samples slightly increased levels of both complexes were found). The reductions in factor XII and prekallikrein were only in part due to protein leakage, since levels were still significantly lower, i.e., 80 and 50%, respectively, when corrected for albumin decreases. Levels of high molecular weight kininogen (HMWK) also decreased during IL-2 therapy, however, this decrease paralleled that of albumin. SDS-PAGE analysis of plasma HMWK did not reveal increased cleavage of this protein. The reduction of factor XII and prekallikrein, corrected for protein leakage, significantly correlated with albumin levels and inversely with daily cumulative weight gain in the patients. Thus, we demonstrate that factor XII and prekallikrein decrease during IL-2 therapy. As these decreases, already observed after 1 day treatment, were disproportional to that of albumin, a negative acute phase reactant, and correlated with signs of the vascular leak syndrome, we favor the explanation that they reflected activation rather than a decreased synthesis of the contact system proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Apart from tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), a third PA appears to occur in human plasma. Its activity is initiated when appropriate triggers of the contact system are added, and the activation depends on the presence of factor XII and prekallikrein in plasma. The activity of this, so-called, contact-system dependent PA accounts for 30% of the PA activity in the dextran sulphate euglobulin fraction of plasma and was shown not to be an intrinsic property of one of the contact-system components, nor could it be inhibited by inhibitory antibodies against t-PA or u-PA. We have succeeded in identifying this third PA in dextran sulphate euglobulin fractions of human plasma. Its smallest unit (SDS-PAGE) is an inactive 110 kDa single-chain polypeptide which upon activation of the contact system is converted to a cleaved, disulphide-bridged molecule with PA activity. The native form, presumably, is an oligomer, since the apparent Mr on gel-chromatography is 600,000. The IEP is 4.8, much lower than that of t-PA and u-PA. Although the active 110 kDa polypeptide cannot be inhibited by anti-u-PA, it yet comprises a 37 kDa piece with some u-PA related antigenic determinants. However, these determinants are in a latent or cryptic form, only detectable after denaturation by SDS. The 110 kDa polypeptide is evidently not a dimer of 55 kDa u-PA or a complex of u-PA with an inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of negatively-charged polymers, used in some artificial devices, on plasma clotting and kinin systems was studied in vitro using polyelectrolyte complexes. Contact activation was observed as an immediate, transient and surface-dependent phenomenon. After incubation of the plasma with the polymer a small decrease of factor XII activity was noticed, which corresponded to a greater reduction of prekallikrein activity and to a marked kinin release. No significant decrease of factor XII, prekallikrein, HMW kininogen could be detected immunologically. Only the initial contact of the plasma with the polyelectrolyte lead to activation, subsequently the surface became inert. Beside contact activation, factor V activity also decreased in the plasma. The decrease was surface and time-dependent. It was independent of contact factor activation, and appeared to be related to the sulfonated groups of the polymer. If purified factor V was used instead of plasma factor V, inactivation was immediate and not time-dependent suggesting a direct adsorption on the surface. A second incubation of the plasma-contacted polymer with fresh plasma resulted in a further loss of Factor V activity.

Components of the fibrinolytic system were studied in samples of plasma from 15 normal, young women and from 11 women taking oral contraceptives containing 30 micrograms ethinyl oestradiol and 150 micrograms levo-norgestrel. Fibrinolytic activity of euglobulins precipitated at pH 5.9 was higher than normal in the hormone group, with significant fluctuations related to the cycle. Normal women showed only minor fluctuations. The concentration of C1-inactivator was lower in euglobulins of the hormone group. However, the difference in fibrinolytic activity was retained, when C1-inactivator was inactivated with sodium flufenamate. Fluctuations of the extrinsic (tissue-type) plasminogen activator (t-PA) activity parallelled those of the euglobulin activity. The intrinsic plasminogen activator activity (dextran sulphate precipitated euglobulin) was significantly increased in the hormone group and the cyclic pattern differed from that of the normal group. The increased activity was factor XII-dependent. Plasma prekallikrein did not differ. The factor XII level was increased in the hormone group but this could not explain the increased intrinsic fibrinolytic activity, suggesting an increase in the quantity of an additional factor XII-dependent proactivator.

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

Fibrinolytic studies in euglobulin fractions of Fletcher trait plasma (deficient in prekallikrein) revealed reduced activities as compared to normal plasma. A quantitative assay for total plasminogen activator plus proactivator in plasma showed that the amount in Fletcher trait patients is about half of normal (normal = +/- 100 blood activator units [BAU]/ml). Plasma kallikrein partially purified in a high and low molecular weight form exerted plasminogen activator activity amounting to 10-15 BAU/ml plasma. So, the absence of kallikrein in the deficient plasma cannot fully account for the reduction in activator activity. Additions of kallikrein preparations or normal plasma fractions resulted in additional activator activity in Fletcher trait plasma which was assessed at 30-40 BAU/ml. This activity was assumed to originate from a previously undescribed plasminogen proactivator whose activation is kallikrein- and factor XII-dependent. Fractionation experiments demonstrated the presence of two major activities and a minor activity caused by kallikrein in normal plasma. It is concluded that plasma kallikrein has two functions in the generation of factor XII-dependent fibrinolytic activity: one as a direct plasminogen activator and another as a factor in the activation of a major factor XII-dependent plasminogen proactivator.