Fitzgerald factor (high molecular weight kininogen) clotting activity in human plasma in health and disease in various animal plasmas.

Fitzgerald factor (high molecular weight kininogen) is an agent in normal human plasma that corrects the impaired in vitro surface-mediated plasma reactions of blood coagulation, fibrinolysis, and kinin generation observed in Fitzgerald trait plasma. To assess the possible pathophysiologic role of Fitzgerald factor, its titer was measured by a functional clot-promoting assay. Mean +/- SD in 42 normal adults was 0.99+/-0.25 units/ml, one unit being the activity in 1 ml of normal pooled plasma. No difference in titer was noted between normal men and women, during pregnancy, or after physical exercise. Fitzgerald factor activity was significantly reduced in the plasmas of eight patients with advanced hepatic cirrhosis (0.40+/-0.09 units/ml) and of ten patients with disseminated intravascular coagulation (0.60+/-0.30 units/ml), but was normal in plasmas of patients with other congenital clotting factor deficiencies, nephrotic syndrome, rheumatoid arthritis, systemic lupus erythematosus, or sarcoidosis, or under treatment with warfarin. The plasmas of 21 mammalian species tested appeared to contain Fitzgerald factor activity, but those of two avian, two repitilian, and one amphibian species did not correct the coagulant defect in Fitzgerald trait plasmas.     

Generation of vasoactive peptide bradykinin from human umbilical vein endothelium-bound high molecular weight kininogen by plasma kallikrein.

High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. It is an inhibitor of sulfhydryl proteases and has procoagulant properties. It is also a source of the vasoactive peptide bradykinin (BK). It has been previously shown that HK binds to human umbilical vein endothelial cells (HUVEC) in culture. We have further characterized that interaction herein. Immunohistochemical experiments have indicated that when freshly obtained umbilical vein segments were treated with HK, washed, and probed with anti-HK antibodies, HK was localized on the endothelium. We next determined whether HUVEC-bound HK can be cleaved by plasma kallikrein to release BK. Cultured HUVEC were incubated with unlabeled HK for varying times, washed, and the kinetics of BK release by plasma kallikrein were assayed by radioimmunoassay. Results indicated that kallikrein released BK from HUVEC in proportion to the initial amount of bound HK. No release of BK occurred in the absence of kallikrein. Also, there was no BK release upon kallikrein treatment of the HUVEC not treated with exogenous HK. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of HUVEC-bound 125I-HK indicated that addition of kallikrein resulted in cleavage of HK, thus corroborating the BK release experiments. Comparison of cleavage patterns has also indicated that cell-bound HK is slightly less susceptible to digestion by kallikrein than free HK. Therefore, our data suggest that human HK can bind to vascular endothelium in situ and that plasma kallikrein can recognize endothelial-bound HK as a substrate and liberate the vasoactive peptide BK.     

Immunologic studies of human coagulation factor XI and its complex with high molecular weight kininogen

Coagulation factor XI was purified from human plasma using ion-exchange chromatography and affinity chromatography on high molecular weight kininogen-Sepharose. A monospecific precipitating antiserum was prepared and used to study factor XI antigen. Factor XI did not migrate during electrophoresis at pH 8.3. High molecular weight kininogen (HMWK), an alpha globulin, reversibly associates with factor XI. Complex formation between HMWK and factor XI was observed under conditions of crossed-immunoelectrophoresis. Using Laurell rocket immunoelectrophoresis, it was shown that the isolated alkylated light chain of kinin-free HMWK formed a complex with factor XI. In contrast to previous studies of prekallikrein, titrations of factor XI with increasing amounts of HMWK did not give a simple titration curve, suggesting that factor XI dissociates from the complex during electrophoresis. Prekallikrein and factor XI were shown to compete for the same HMWK molecules under the conditions of immunoelectrophoresis, and prekallikrein appeared to have a higher affinity for binding to HMWK than factor XI. Quantitative determinations of factor XI antigen in plasma by rocket immunoelectrophoresis were made. The average amount of factor XI measured in plasma samples from 20 normal individuals was 4.5 micrograms/ml (range 3-6). No factor XI antigen was detected in plasma from a patient deficient in factor XI. Normal factor XI antigen levels were detected in 3 different HMWK-deficient plasmas only if the plasmas were reconstituted with purified HMWK (2 U/ml). Addition of HMWK to normal plasma resulted in an increase of the factor XI antigen rocket. At HMWK levels of 2 U/ml, no further increase of the factor XI antigen rocket was observed. Therefore, accurate measurement of factor XI antigen by rocket immunoelectrophoresis is possible only if an excess of HMWK is present.     

Influence of high molecular weight factor VIII on the measurement of low molecular weight factor VIII procoagulant in different assay systems

Factor VIII procoagulant activity (VIIIC) is exerted by a low molecular weight (LMW) moiety of the factor VIII molecule that can be separated from a high molecular weight (HMW) moiety by high ionic strength buffers. In this investigation the procoagulant activity of the LWM moiety of factor VIII prepared by immuno-adsorbent chromatography and its relationship to the HMW moiety of haemophilic plasma was studied by means of different VIIIC assay systems and using different substrates in regard to their content of the HMW VIII moiety. LMW VIIIC was prepared by immunoadsorbent chromatography; HMW VIII without VIIIC was prepared by chromatographing cryoprecipitate from a coagulant antigen negative severe haemophiliac on 4% agarose. The LMW VIIIC obtained by immunoadsorbent chromatography gave higher VIIIC values when tested in the one-stage partial thromboplastin time (PTT) system using von Willebrand's disease plasma as substrate than using haemophilic plasma as substrate. This finding was shown to be related to the HMW VIII measured as VIII related antigen (VIII: Ag) in the substrate plasmas. When the VIIIR: Ag was removed from the haemophilic substrate plasma by immunoadsorption, the VIIIC values obtained for the LMW VIIIC were higher. Also, adding HMW VIII purified from haemophilic plasma to the von Willebrand's disease substrate plasma resulted in lower VIIIC values for the LMW VIIIC in the PTT system.     

Kinetic analysis of the interaction of human tissue kallikrein with single-chain human high and low molecular weight kininogens.

Human low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK) have been purified to apparent homogeneity as intact, single-chain molecules. When they interacted with homologous urinary kallikrein, 0.9 mol of kinin per mol of substrate was released from LMWK and 0.7 mol of kinin per mol of substrate was released from HMWK. These functionally and structurally intact substrates have been used to obtain the kinetic constants for kinin release by purified human tissue kallikreins. With human urinary kallikrein, apparent second-order rate constants (kcat/Km) of 1.46 X 105, 8.6 X 104, and 5.08 X 104 M-1.S-1 were obtained with LMWK, HMWK, and alpha-N-p-tosyl-L-arginine methyl ester (TAMe), respectively; with human pancreatic kallikrein, values of 8.7 X 103 and 7.3 X 104 M-1.S-1 were obtained with HMWK and TAMe. These values, which are comparable to those obtained for other enzyme-protein substrate interactions, indicate that LMWK is only slightly preferred to interactions, indicate that LMWK is only slightly preferred to HMWK as the natural substrate for urinary kallikrein and that HMWK as the natural substrate for urinary kallikrein and that HMWK is a somewhat better substrate for urinary kallikrein than for pancreatic kallikrein. Although the data obtained have been shown by NaDodSO4/polyacrylamide gel electrophoresis to reflect cleavage of the substrate at two points, the linear Line-weaver-Burk plots suggest that one cleavage is rate limiting. Because the plasma concentrations of both LMWK and HMWK are approximately 1/10th the Km values obtained, substrate concentration may also play a role in determining the rate at which tissue kallikreins release kinins from kininogen substrates either in the circulation or extravascularly.     

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

Plasma levels of prekallikrein, kallikrein, high molecular weight kininogen and kallikrein inhibitors were measured in samples from dogs infused with E. coli endotoxin. Markedly reduced levels of prekallikrein and high molecular weight kininogen were seen at circulatory collapse and free kallikrein was detected. Both 'fast-reacting' and 'time-dependent' inhibitors of plasma kallikrein were significantly reduced. Fractionation of pre-infusion and collapse samples by gel filtration together with immunological procedures indicated that both alpha2-macroglobin and alpha1-antitrypsin were the major kallikrein inhibitors in dog plasma, and that they were significantly reduced in the collapse samples.     

Changes of high molecular weight kininogen in various states

Changes of high molecular weight kininogen (HMW-K) clotting activity, antigen and cleavage in the plasma in the health and various diseases were studied. In 20 healthy individuals clotting activity of HMW-K, as measured by APTT one stage method, was 99 +/- 12% (male) and 84 +/- 15% (female). Antigen as measured by Laurell method were 106 +/- 24% (male) and 91 +/- 21% (female). In 35 patients with disseminated intravascular coagulation (DIC), both activity (78 +/- 33%) and antigen (69 +/- 31%) were statistically lower than those in normal individuals (p less than 0.01). In DIC both activity and antigen of HMW-K was correlated with serum albumin level. These results suggest that the cause of the lower level of HMW-K in DIC especially with septicemia is the result of lower production rather than consumption. In vivo cleavage of HMW-K was detected in plasma of a patient with septicemia and DIC by immunoblotting. The change of HMW-K was also assessed in other pathological states including liver cirrhosis, collagen disease, cardiopulmonary bypass and pregnant women.     

Mechanisms for the involvement of high molecular weight kininogen in surface-dependent reactions of Hageman factor.

The mechanisms by which human high molecular weight kininogen (HMKrK) contributes to the surface-dependent activation of the Hageman factor systems have been studied. The ability of various mixtures of purified human Hageman factor (coagulation factor XII), HMrK, prekallikrein, and kaolin to activate coagulation factor XI was determined with factor XIa (activated factor XI) clotting assays. Hageman factor, HMrK and prekallikrein were required for maximal rates of activation of factor XI. A certain optimal mixture of purified Hageman factor, HMrK, prekallikrein, and kaolin gave the same rapid initial rate of activation of purified factor XI as an equivalent aliquot of factor XI-deficient plasma. This suggests that potent, surface-mediated activation of factor XI in plasma is explicable in terms of Hageman factor, HMrK, and prekallikrein. By studying separately some of the surface-dependent reactions involving Hageman factor, it was found that HMrK accelerated by at least an order of magnitude the following reactions: (i) the activation of factor XI by activated Hageman factor; (ii) the activation of prekallikrein by activated Hageman factor; and (iii) the activation of Hageman factor by kallikrein. Stoichiometric rather than catalytic amounts of HMrK gave optimal activation of factor XI. These results are consistent with the hypothesis that HMrK and Hageman factor form a complex on kaolin which renders Hageman factor more susceptible to proteolytic activation by kallikrein and which facilitates the action of activated Hageman factor on its substrate proteins, factor XI and prekallikrein.     

Apparent molecular weight of purified human factor VIII procoagulant protein compared with purified and plasma factor VIII procoagulant protein antigen

We have compared apparent molecular weights of purified factor VIII procoagulant protein (VIII:C) and VIII:C antigen (VIII:CAg) by two different NaDodSO4 gel electrophoretic techniques. In a discontinuous NaDodSO4-7.5% polyacrylamide system, reduced and unreduced VIII:C, purified from commercial factor VIII concentrates by a monoclonal antibody immunoadsorption technique, showed a major doublet at mol wt 0.79 and 0.8 X 10(5) and less intense bands extending up to 1.9 X 10(5). In NaDodSO4-4% polyacrylamide/0.5% agarose gels (NaDodSO4-4% PAAGE), purified VIII:C had a major band of mol wt 1.0 X 10(5), with minor bands of mol wt 0.96, 1.1, 1.4, 1.6, 1.8, 2.2, and 2.4 X 10(5). In NaDodSO4-4% PAAGE of 125I-anti-VIII:C-Fab-VIII:CAg complexes, the major and minor forms of VIII:CAg in purified VIII:C had the same molecular weight as above when calculated by subtracting the molecular weight of 125I-Fab from 125I-Fab-VIII:CAg. In both plasma and factor VIII concentrate, a band of mol wt 2.4 X 10(5) predominated, and minor VIII:CAg forms of mol wt 2.6, 1.8, 1.2 and 1.0 X 10(5) were also visible. We conclude that the molecular weight of plasma VIII:CAg forms agree with those derived from protein stains of purified VIII:C in the NaDodSO4-4% PAAGE system, but that consistently lower molecular weight values are obtained for purified VIII:C in the discontinuous system. Both native and either disaggregated or proteolyzed VIII:C species are present in the purified VIII:C preparation.     

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.     

Interaction of high molecular weight kininogen, factor XII, and fibrinogen in plasma at interfaces

Using ellipsometry, anodized tantalum interference color, and Coomassie blue staining in conjunction with immunologic identification of proteins adsorbed at interfaces, we have previously found that fibrinogen is the main constituent deposited by plasma onto many man- made surfaces. However, the fibrinogen deposited from normal plasma onto glass and similar wettable materials is rapidly modified during contact activation until it can no longer be identified antigenically. In earlier publications, we have called this modification of the fibrinogen layer "conversion," to indicate a process of unknown nature. Conversion of adsorbed fibrinogen by the plasma was not accompanied by marked change in film thickness, so that we presumed that this fibrinogen was not covered but replaced by other protein. Conversion is now showen to be markedly delayed in plasma lacking high molecular weight kininogen, slightly delayed in plasma lacking factor XII, and normal in plasma that lack factor XI or prekallikrein. We conclude that intact plasma will quickly replace the fibrinogen it has deposited on glass-like surfaces by high molecular weight kininogen and, to a smaller extent, by factor XII. Platelets adhere preferentially to fibrinogen-coated surfaces; human platelets adhere to hydrophobic nonactivating surfaces, since on these, adsorbed firbinogen is not exchanged by the plasma. The adsorbed fibrinogen will be replaced on glass-like surfaces during surface activation of clotting, and platelets failing to find fibrinogen will not adhere.     

Studies of binding of prekallikrein and Factor XI to high molecular weight kininogen and its light chain.

Prekallikrein and Factor XI have been reported to circulate as complexes with the coagulation cofactor high molecular weight (HMW)-kininogen. In this study we have shown that native HMW-kininogen possesses a strong binding site for prekallikrein and Factor XI with association constants of 3.4 x 10(7) M-1 and 4.2 x 10(8) M-1, respectively. The diminished binding of prekallikrein relative to Factor XI may, in part, account for the ability of kallikrein to leave the surface and interact with other molecules of Hageman factor and HMW-kininogen. Prekallikrein and Factor XI appear to compete for binding to HMW-kininogen, suggesting a single (or closely overlapping) binding site(s). The purified light chain derived from kinin-free HMW-kininogen is shown to compete with native MHW-kininogen for binding to Hageman factor substrates and direct binding of the isolated light chain to prekalikrein and Factor XI is demonstrated. This binding of the light chain to prekallikrein and Factor XI appears to be essential to the function of HMW-kininogen as a coagulation cofactor and further digestion of the light chain with excess kallikrein destroys its coagulant activity.     

Role of arginine residues in the coagulant activity of high molecular weight kininogen

High molecular weight (HMW) kininogen, the cofactor for activation of the contact system of plasma proteolysis, transports and optimally positions prekallikrein and factor XI on a negatively charged surface, allowing those zymogens to be activated by surface-bound factor XIIa. HMW kininogen circulates in plasma as a procofactor that, after cleavage by kallikrein or factor XIIa, gains ability to bind to the surface. The mechanism responsible for this increased affinity for the surface is unknown. We hypothesized that modification of arginine residues may prevent cleavage of HMW kininogen, since the initial kallikrein-induced cleavage sites on the HMW kininogen molecule are at the NH2 terminal and the COOH terminal of the bradykinin-containing portion of the molecule, each of which contains arginine. We found that modification with butanedione of four arginine residues in the HMW kininogen molecule prevented bradykinin release, which results from cleavage of HMW kininogen. Furthermore, HMW kininogen coagulant activity was lost, in proportion to the degree of arginine modification, until 6.6 residues had been modified. Complex formation with prekallikrein, however, was found to be uneffected by the modification of modified HMW kininogen. To account for the loss of coagulant activity, we also examined the ability of modified HMWKa (active cofactor) to bind to an activating surface. The affinity of modified HMWKa for kaolin was tenfold less than the affinity of unmodified HMWKa. These data suggest that arginine residues play a critical role in the ability of HMW kininogen to function as an activation cofactor, both by preventing the cleavages that produce HMWKa as well as by decreasing the affinity of HMWKa for the surface.     

Characterization of a tissue kallikrein in human prolactin-secreting adenomas

Immunoreactive tissue kallikrein was co-localized with prolactin in all the eleven prolactin-secreting adenomas of the human anterior pituitary gland examined in this study. The intracellular distribution of immunoreactivity in the prolactin-secreting cells suggests that tissue kallikrein is located within the Golgi complex of these cells. Both the intracellular hormone-processing action and the kininogenase activity of tissue kallikrein may be of functional importance in human prolactinomas.     

Domain 5 of high molecular weight kininogen (kininostatin) down-regulates endothelial cell proliferation and migration and inhibits angiogenesis

We have demonstrated that high molecular weight kininogen (HK) binds specifically on endothelial cells to domain 2/3 of the urokinase receptor (uPAR). Inhibition by vitronectin suggests that kallikrein-cleaved HK (HKa) is antiadhesive. Plasma kallikrein bound to HK cleaves prourokinase to urokinase, initiating cell-associated fibrinolysis. We postulated that HK cell binding domains would inhibit angiogenesis. We found that recombinant domain 5 (D5) inhibited endothelial cell migration toward vitronectin 85% at 0.27 microM with an IC(50) (concentration to yield 50% inhibition) = 0.12 microM. A D5 peptide, G486-K502, showed an IC(50) = 0.2 microM, but a 25-mer peptide from a D3 cell binding domain only inhibited migration 10% at 139 microM (IC(50) > 50 microM). D6 exhibited weaker inhibitory activity (IC(50) = 0.50 microM). D5 also potently inhibited endothelial cell proliferation with an IC(50) = 30 nM, while D3 and D6 were inactive. Using deletion mutants of D5, we localized the smallest region for full activity to H441-D474. To further map the active region, we created a molecular homology model of D5 and designed a series of peptides displaying surface loops. Peptide 440-455 was the most potent (IC(50) = 100 nM) in inhibiting proliferation but did not inhibit migration. D5 inhibited angiogenesis stimulated by fibroblast growth factor FGF2 (97%) in a chicken chorioallantoic membrane assay at 270 nM, and peptide 400-455 was also inhibitory (79%). HK D5 (for which we suggest the designation, "kininostatin") is a potent inhibitor of endothelial cell migration and proliferation in vitro and of angiogenesis in vivo. (Blood. 2000;95:543-550)     

Conversion of high molecular weight human epidermal growth factor (hEGF)/urogastrone (UG) to small molecular weight hEGF/UG by mouse EGF-associated arginine esterase.

Human epidermal growth factor (hEGF) has previously been isolated from urine and appears to be identical to beta-urogastrone (UG), an inhibitor of stimulated gastric acid secretion. A high molecular weight (HMW) form of hEGF/UG has recently been found in human urine which is fully immunoreactive but is less bioactive as measured by receptor binding activity. A specific arginine esterase, the EGF-binding protein from mouse submandibular glands, was capable of cleaving HMW-hEGF to yield a small molecular weight (SMW)-hEGF with full immunoreactivity and bioactivity, whereas trypsin produced a SMW-hEGF with much less bioactivity. SMW-hEGF produced by the arginine esterase appeared to be immunologically, biologically (both by receptor binding and mitogenic activity) and chromatographically similar to highly purified hEGF. These data suggest that HMW-hEGF may play a precursor role in the biosynthesis of hEGF/UG in man.     

Interactions among Hageman factor, plasma prekallikrein, high molecular weight kininogen, and plasma thromboplastin antecedent.

To investigate the earliest steps of the intrinsic clotting pathway, Hageman factor (Factor XII) was exposed to Sephadex gels to which ellagic acid had been adsorbed; Hageman factor was then separated from the gels and studied in the fluid phase. Sephadex-ellagic acid-exposed Hageman factor, whether purified or in plasma, activated plasma thromboplastin antecedent, but only when high molecular weight kininogen was presnet. In the absence of plasma prekallikrein, maximal activation of plasma thromboplastin antecedent was slightly delayed in plasma, a delay not observed with similarly treated purified Hageman factor. Thus, high molecular weight kininogen was needed for expression of Hageman factor's clot-promoting properties and plasma prekallikrein played a minor role in the interaction of ellagic acid-treated Hageman factor and plasma thromboplastin antecedent.     

Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies

Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti- light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.