The contact activation and kallikrein/kinin systems: pathophysiologic and physiologic activities.

The contact activation system (CAS) and kallikrein/kinin system (KKS) are older recognized biochemical pathways that include several proteins that skirt the fringes of the blood coagulation, fibrinolytic, complement and renin-angiotensin fields. These proteins initially were proposed as part of the hemostatic pathways because their deficiencies are associated with prolonged clinical assays. However, the absence of bleeding states with deficiencies of factor XII (FXII), prekallikrein (PK) and high-molecular-weight kininogen indicates that the CAS and KKS do not contribute to hemostasis. Since the discovery of the Hageman factor 60 years ago much has been learned about the biochemistry, cell biology and animal physiology of these proteins. The CAS is a pathophysiologic surface defense mechanism against foreign proteins, organisms and artificial materials. The KKS is an inflammatory response mechanism. Targeting their activation through FXIIa or plasma kallikrein inhibition when blood interacts with the artificial surfaces of modern interventional medicine or in acute attacks of hereditary angioedema restores vascular homeostasis. FXII/FXIIa and products that arise with PK deficiency also offer novel ways to reduce arterial and venous thrombosis without an effect on hemostasis. In summary, there is revived interest in the CAS and KKS due to better understanding of their activities. The new appreciation of these systems will lead to several new therapies for a variety of medical disorders.

Antigen levels of coagulation factor XII, coagulation factor XI and prekallikrein, and the risk of myocardial infarction and ischemic stroke in young women.

BACKGROUND: High levels of activated protein­inhibitor complexes of the intrinsic coagulation proteins are associated with ischemic stroke (IS) but not with myocardial infarction (MI). This study was aimed at determining whether the antigen levels of coagulation factors(factor XII, FXII, and FXI and prekallikrein (PK)are associated with MI and IS, and whether this association is independent of levels of activated protein­inhibitor complexes. PATIENTS AND METHODS: The RATIO study included young women (< 50 years) with MI (N = 205)and IS (N = 175), and 638 healthy controls. Antigen levels of FXII, FXI and PK were measured and expressed as percentages of of those in pooled normal plasmas. Odds ratios (ORs) and corresponding 99% confidence intervals (CIs) were calculated for high levels (i.e. ≥ 90th percentile of controls) as measures of rate ratios. RESULTS: After adjustment for potential confounders, high levels of FXII antigen were not associated with MI risk or IS risk(OR(MI) 1.18, 99% CI 0.51­2.74; ORIS 1.03, 9% CI 0.41­2.55). High levels of FXI antigen were slightly associated with an increase in MI risk (OR(MI) 1.55, 9% CI 0.74­3.21), whereas there was a substantial association with IS risk (ORIS 2.65, 9% CI 1.27­5.56). PK antigen was slightly associated with MI risk but not with IS risk(ORMI 1.54, 9% CI 0.67­3.52; ORIS 0.90, 9% CI 0.35­2.33). All associations remained similar after adjustment for levels of protein­inhibitor complexes. CONCLUSION: Increased levels of FXI antigen were associated with an increase in IS risk, whereas they showed only a marginal association with MI risk. FXII antigen and PK antigen levels were not substantially associated with MI risk and IS risk.

Fine mapping of the H-kininogen binding site in plasma prekallikrein apple domain 2.

Plasma prekallikrein (PPK), the zymogen of the contact phase protease plasma kallikrein, forms a non-covalent complex with its substrate H-kininogen (HK). HK binds to cell surface proteoglycans, indirectly anchoring this bradykinin-generating protease to endothelial cells. The heavy chain of PPK consisting of four apple domains designated A1 to A4. Previous studies indicated that a major HK binding site on PPK is within the A2 domain, with additional contributions to binding provided by the N-terminal portion of Al and the central part of A4. To precisely map the relevant binding segments in A2, we employed a monoclonal anti-PPK antibody (PKH6) that binds to A2 and blocks HK-PPK complex formation with an apparent IC50 of 8 nM. Using recombinant A2 C-terminal deletion mutants, we mapped the target epitope of PKH6 to the N-terminal portion of A2, residues 92-153. C-terminal deletion of A2 to residue 145 resulted in a loss of PKH6 binding, as did proteolytic cleavage of A2 at Lys140-Arg141. A comparison of HK binding to various A2 deletion mutants revealed that the major HK binding site is localized to residues 145-153 in the central portion of A2, where it overlaps with the PKH6 epitope. This sequence is conserved in the A2 domain of the related protease factor XI, explaining the unusual strong cross-reactivity of PHK6 with factor XI, as well as the similar HK-binding characteristics of PPK and factor XI.

Autoantibodies to contact proteins in patients with recurrent pregnancy losses.

The kallikrein-kinin system or plasma contact system consists of three essential plasma proteins. These are coagulation factor XII, prekallikrein and high molecular weight kininogen. Deficiencies of these proteins are not associated with clinical bleeding despite marked prolongation of in vitro surface-activated coagulation time. Paradoxically, studies suggest that contact proteins have anticoagulant, profibrinolytic functions in a physiologic millieu, on endothelial cells. Recently, evidence has accumulated for the presence of the kallikrein-kinin system or plasma contact system in the fetoplacental unit. Kinins which are released within the placenta may play a role in regulating placental blood flow. This suggests that the plasma contact system may also have an important role in pregnancy. Several studies have reported the presence of autoantibodies to the contact proteins in patients with recurrent early pregnancy losses. Disruption of this system may be a risk factor for early gestational losses.

Certain autoantibodies to phosphatidylethanolamine (aPE) recognize factor XI and prekallikrein independently or in addition to the kininogens.

Recent evidence shows that many antiphospholipid antibodies (aPL) to negatively-charged phospholipid (PL) do not target anionic PL per se, but are specific for anionic PL-binding plasma proteins, for example, beta(2)-glycoprotein I (beta(2)-GPI) and prothrombin. We also reported that certain antiphosphatidylethanolamine antibodies (aPE) are not specific for phosphatidylethanolamine (PE) per se, but are directed to PE-binding plasma proteins, high molecular weight kininogen (HK), and low molecular weight kininogen (LK). Additional studies have shown that certain aPE failed to recognize purified kininogens but continued to produce aPE ELISA reactivity in the presence of semipurified HK preparations containing the HK binding proteins, factor XI (FXI) and prekallikrein (PK). We therefore investigated if certain of these aPE recognized FXI and/or PK. In this study we observed that aPE can recognize contact proteins FXI and PK independently or in combination with HK. Since contact proteins such as HK, PK and factor XII (FXII) have anti-coagulant and profibrinolytic functions, the pathophysiological role of aPE has yet to be elucidated. We propose that aPE of different specificities may initiate or promote characteristics pathological conditions in patients with thrombosis or recurrent pregnancy losses.

The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia.

Prostate-specific antigen (PSA, hK3) is a diagnostic marker for prostatic cancer but lacks the specificity to sufficiently distinguish between prostatic cancer and benign prostatic hyperplasia (BPH). Human glandular kallikrein 2 (hK2) has been proposed as a potential diagnostic marker for prostate cancer that could complement the current PSA test. Recently we demonstrated that proPSA is present in prostate cancer sera. This study examines the expression of prohK2 in prostate cells and its presence in human sera. Western blot analysis was used to assess prohK2 expression in the human carcinoma cell line, LNCaP. A highly specific and sensitive dual monoclonal immunoassay for prohK2 was developed and used to assess the presence of prohK2 in human sera. prohK2 was detected in the spent media of LNCaP cells. Furthermore, prohK2 was present at immunodetectable concentrations in human sera, and its concentration was increased in prostatic cancer and BPH. These results indicate for the first time that prohK2 is secreted by human prostate cells and is a major component of uncomplexed (free) hK2 in human sera. In addition, prohK2 in human sera is associated with prostate disease and thus may be a useful marker for prostatic cancer and BPH.

Mapping of the discontinuous kininogen binding site of prekallikrein. A distal binding segment is located in the heavy chain domain A4.

Prekallikrein, the precursor to the serine proteinase kallikrein, circulates in plasma in an equimolar complex with H-kininogen. The binding to H-kininogen is mediated by the kallikrein heavy chain consisting of four "apple" domains, A1-A4, which attaches to H-kininogen with high specificity and affinity (KD = 83 nM). At least two distinct portions of the kallikrein heavy chain form this H-kininogen binding site: a proximal segment located in the NH2-terminal fragment of the heavy chain encompassing A1, and distal segment(s) located in COOH-terminal fragment spanning domains A2-A4. The proximal binding segment has been located to amino acid positions 56-86 of A1. To precisely map the distal binding segment, we have identified monoclonal antibodies directed to the COOH-terminal fragment which interfere with the H-kininogen-prekallikrein complex formation. Monoclonal antibody 13G11 binds to recombinant apple domain A4 but not to domain A3 of the prekallikrein heavy chain. Deletion mutagenesis of domain A4 narrowed down the target epitope of 13G11 to the center portion of domain A4, positions 284-331. Direct binding studies of H-kininogen to various domain A4 constructs revealed that the distal H-kininogen binding portion is located on a segment of 48 residues, which overlaps the 13G11 epitope. Hence the tight interaction of H-kininogen and prekallikrein is mediated by at least two separate sequence segments located in domains A1 and A4, respectively, of the prekallikrein heavy chain. The isolated distal binding segment significantly prolongs the partial thromboplastin time of reconstituted Williams plasma thus stressing the critical role of the prekallikrein-H-kininogen complex formation in the initiation of the endogenous blood coagulation cascade.

Activation of the intrinsic pathway of coagulation in children with meningococcal septic shock.

Meningococcal septic shock (MSS) is complicated by activation of coagulation, fibrinolytic, and complement systems. We studied the contact system of the intrinsic pathway of coagulation in thirteen children with MSS. Activation was assessed upon admittance to the intensive care unit and 48 h thereafter, based on the measurement of factor XII- (FXII), prekallikrein- and factor XI (FXI) antigen levels, as well as on the detection of FXIa-FXIa inhibitor, FXIIa-C1-inhibitor, and kallikrein-C1-inhibitor complexes, respectively. Levels of FXII, prekallikrein and FXI were reduced to about 50% in all patients on admission, and were significantly higher 48 h later. FXIIa-C1-inhibitor complexes were elevated in 7 patients, and kallikrein-C1-inhibitor complexes in 2 patients. FXIa-alpha 1-antitrypsin complexes were elevated in all patients, FXIa-C1-inhibitor complexes in nine, and FXIa-antithrombin III complexes in one patient. We conclude that patients with MSS have activation of the contact system, which may contribute to activation of coagulation, and thus to morbidity and mortality.

An autoantibody to human plasma prekallikrein blocks activation of the contact system.

A patient without a history of bleeding or thromboembolism presented with an activated partial thromboplastin time (aPTT) of 55.1 s (normal 24-38 s). Incubation of the patient plasma with an equal volume of normal plasma failed to correct the aPTT, suggesting the presence of an inhibitor. The MRVVT (modified Russell Viper venom time) was normal, and the anti-cardiolipin antibody titres were not elevated, indicating that the presence of a lupus anticoagulant was unlikely. Plasma prekallikrein (PK) measured by a coagulant assay (2 U/dl) was very low, but PK was in the low normal range (approximately 65%) when measured by an enzymatic assay (amidolytic) or by an antigenic assay (ELISA). The purified patient IgG reacted with purified PK, the heavy chain, and the 28 kD fragment of the heavy chain, indicating that it contained an autoantibody to PK. The purified IgG did not directly inhibit the amidolytic activity of kallikrein, but it did inhibit the activation of PK to kallikrein by activated factor XII. Activation of the contact system by dextran sulphate, as reflected by the cleavage of HK on a Western blot, was inhibited when the patient IgG was added to pooled normal plasma. The antibody appears to be oligoclonal with IgG1 being most abundant, followed by IgG4. This report appears to be the first of a spontaneously occurring antibody to prekallikrein.

Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate.

An automated assay of plasma prekallikrein is described. Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the hydrolytic activity of kallikrein to H-D-Pro-Phe-Arg-paranitroanilide subsequently measured. The conversion was complete within 8 minutes and the amidolytic activity remained stable at least another 10 min at 37 degrees C. This method worked in plasma deficient in Hageman factor (blood coagulation factor XII). Using anti-prekallikrein antibody and plasma deficient in prekallikrein, the amidolytic activity generated in normal plasma was identified as due to kallikrein. With plasma samples, the coefficients of variation (CV) for multiple measurements within run (n = 10) and between run (n = 10) were as low as 5.0% and 6.6%, respectively, and the minimum measurable concentration of prekallikrein in plasma was 10% of the normal level.

Alpha 2-macroglobulin-kallikrein complexes detect contact system activation in hereditary angioedema and human sepsis.

Activation of the contact system has been documented in severe sepsis and hereditary angioedema, but a sensitive, specific, and quantitative assay for assessing the degree of involvement of this proteolytic enzyme cascade is not yet available. We have developed a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the alpha 2-macroglobulin-kallikrein (alpha 2M-Kal) complex using an F(ab')2 derivative of a monospecific polyclonal antibody against alpha 2 M as the capture antibody and a unique murine monoclonal antibody, 13G11, against the heavy chain of kallikrein as the detector antibody. The assay does not detect complexes in normal plasma but reacts with complexes generated by activating normal plasma with dextran sulfate at 4 degrees C in a range of 5 to 375 nmol/L. A close correlation of the ELISA with an amidolytic assay for alpha 2M-Kal was documented. Patients with sepsis syndrome but negative bacterial blood cultures did not show elevated plasma complexes, whereas a majority of those with positive blood cultures did show modest elevation and a single patient with septic shock showed a very high level of alpha 2M-Kal complex. Similarly, a patient with classic hereditary angioedema (HAE) showed increased concentration of complexes on three separate occasions during attacks but normal levels between attacks. Two other HAE patients did not show elevated levels at quiescent periods. The ELISA for alpha 2M-Kal appears to be sensitive, specific, and quantitative, and it can be used to reflect the degree of contact system activation in human sepsis and in HAE attacks.

Localization of distinct functional domains on prekallikrein for interaction with both high molecular weight kininogen and activated factor XII in a 28-kDa fragment (amino acids 141-371).

The predominant autolytic form of human kallikrein, beta-kallikrein, was used to localize the high molecular weight kininogen (HK) binding site on kallikrein as well as the substrate recognition site for activated factor XII on prekallikrein. beta-Kallikrein is formed by autolysis of the kallikrein heavy chain to give two fragments of approximately 18 and 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A ligand binding technique established that the HK binding site on kallikrein residues on the 28-kDa fragment of the heavy chain. Limited NH2-terminal sequencing of this portion of beta-kallikrein showed that this fragment of the heavy chain consists of the COOH-terminal 231 amino acids of the heavy chain. A panel of five murine monoclonal antibodies to human prekallikrein (PK) were found to have epitopes on this same fragment of the heavy chain. None of the monoclonal antibodies were able to block binding of HK to PK. Three of the monoclonal antibodies (13G11, 13H11, and 6A6) were able to inhibit the activation of PK to kallikrein in both a plasma system and a purified system. The 28-kDa fragment of the PK heavy chain was purified and was able to compete with HK for binding to PK. The HK binding site and the site of recognition of factor XII are separate and distinct on PK, and both are contained in the COOH-terminal 231 amino acids of the PK heavy chain.

Association of Graves' disease and prekallikrein congenital deficiency in a patient belonging to the first CRM+ prekallikrein-deficient Italian family.

Severe prekallikrein (Fletcher factor) deficiency was diagnosed in a 49-year-old woman and in 3 of her siblings. Functional prekallikrein (PKK) activity was found below 1% by clotting assay and 20% by amidolytic assay in all the affected subjects; PKK cross-reacting material (CRM) was present in all the patients (antigen levels from 34% to 54%). This is the first CRM+ PKK-deficient family identified in Italy. The index patient was affected from Graves' disease: such association was previously reported in another patient with PKK congenital defect.

High molecular weight kininogen-binding site of prekallikrein probed by monoclonal antibodies.

A panel of monoclonal antibodies against human prekallikrein was raised in mice and characterized with respect to the major antigenic epitopes. Of 18 antibodies, nine were directed against the light chain portion performing the proteolytic function of activated kallikrein, and nine recognized the heavy chain mediating the binding of prekallikrein to high molecular weight (H-)kininogen. Among the anti-heavy chain antibodies, one (PK6) interfered with the procoagulant activity of prekallikrein, and prolonged in a concentration-dependent manner the activated partial thromboplastin time of reconstituted prekallikrein-deficient plasma (Fletcher type). Antibody PK6 was subtyped IgG1,k and had an apparent Kass of 6.8 +/- 0.44.10(8) M-1 for prekallikrein. Functional analyses revealed that PK6 does not interfere with prekallikrein activation by activated Hageman factor (beta-F XIIa), and has no effect on the kininogenase function of activated kallikrein. Monoclonal antibody PK6 but none of the other anti-heavy chain antibodies completely prevented complex formation of prekallikrein with H-kininogen, and readily dissociated preformed complexes of prekallikrein and H-kininogen. Likewise, Fab' and F(ab')2 fragments of PK6 blocked H-kininogen binding to prekallikrein. A synthetic peptide of 31 amino acid residues encompassing the entire prekallikrein binding region of H-kininogen effectively competed with PK6 for prekallikrein binding indicating that the target epitope of PK6 is juxtaposed to, if not incorporated in the H-kininogen-binding site of prekallikrein. Extensive cross-reactivity of PK6 with another H-kininogen-binding protein of human plasma, i.e. factor XI, suggested that the structure of the target epitope of PK6 is well conserved among prekallikrein and factor XI, as would be expected for the kininogen-binding site shared by the two proteins. It is anticipated that monoclonal antibody PK6 will be an important tool for the precise mapping of the hitherto unknown kininogen-binding site of prekallikrein.

Purification and immunological properties of human urinary kallikrein and prokallikrein.

Human urinary prokallikrein and kallikrein have been purified from the same source of urine simultaneously. The anti-kallikrein and anti-prokallikrein antibodies were raised in rabbits using the purified preparations. With respect to solid phase enzyme immunoassay (EIA), immunoaffinity column chromatography, and single radial immunodiffusion, reactivity of each antibody with kallikrein was distinctly different from that with prokallikrein. Kallikrein could be determined by anti-kallikrein antibody-immobilized EIA below 20 ng per ml, whereas prokallikrein was undetectable. Prokallikrein became detectable at higher concentrations, although it was less reactive than kallikrein. The anti-prokallikrein antibody-immobilized EIA detected both kallikrein and prokallikrein with the same sensitivity. However, the binding capacity for kallikrein was about one-third less than that for prokallikrein. The results show that kallikrein in human urine may be determined directly and selectively. Similar difference in reactivity was observed with immunoaffinity column chromatography and single radial immunodiffusion. The presence of 3-4 antigenic sites per molecule was indicated by quantitative precipitin reaction, and it is suggested from analysis of amino acid sequence of kallikrein by the method of Hopp and Woods that four hydrophilic regions exist in kallikrein molecule.

A monoclonal anti-human plasma prekallikrein antibody that inhibits activation of prekallikrein by factor XIIa on a surface.

Of five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was approximately 80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein-HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited approximately 70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (light chain).

Immunoblotting studies of coagulation factor XII, plasma prekallikrein, and high molecular weight kininogen.

Immunoblotting techniques for the qualitative and quantitative analysis of FXII, PK, and HMWK in whole plasma are presented. Sensitive, specific, and quantitative immunodetection of FXII and PK can be achieved by developing the blots with polyclonal antiserum followed by radiolabeled FXII or PK, respectively. This approach is based on the assumption that bivalent antibodies bind monovalently to the NC-bound antigen and have available binding sites to bind radiolabeled antigen derived from the fluid phase. This radiolabeled antigen overlay principle may be generally useful for immunodetection of any trace protein in complex mixtures, provided that the radiolabeled purified antigen is available. Immunoblotting may also be helpful for the partial characterization of the structural or functional abnormalities of CRM-positive variant molecules. For example, earlier studies of a FXII-variant molecule that had been purified and characterized were supported by immunoblotting studies of the CRM-positive deficient plasma. Quantitative measurement of HMWK is possible using a monoclonal antibody directed against the light chain of HMWK followed by radiolabeled secondary antibody. Quantitation of cleaved and single-chain HMWK is possible using dilutions of dextran sulfate-activated NHP on unreduced SDS-PAGE and dilutions of unactivated NHP with reduced SDS-PAGE as standards. These assays allow assessment of the degree of in vivo activation of the contact system in various disease states.