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C1 inhibitor is a multipotent serpin capable of inhibiting the classical and the lectin pathways of complement, the fibrinolytic system, and contact/kinin system of coagulation. Deficiency of C1 inhibitor manifest as hereditary angioedema (HAE), an autosomal dominant hereditary disease. Measuring the C1 inhibitor level is of vital importance for the diagnosis of HAE and also for monitoring patients receiving C1 inhibitor for therapy. Determination of the antigenic C1 inhibitor level by the radial immunodiffusion (RID) technique is described in detail in this chapter. The presented purification method of plasma C1 inhibitor is primarily based on its high carbohydrate content and its affinity to the lectin jacalin.

The protocol consists of running a native gel with in-gel digestion by proteases, subsequent mass spectrometrical determination of protein sequence and modifications, followed by electro-elution and conformational analysis using melting point and circular dichroism. Finally, the eluted protein is tested for preserved function. Herein, C1 esterase inhibitor is applied on a native gel; in-gel digestion by proteases is carried out and peptides are identified by nano-LC-ESI-CID/ETD-MS/MS using an ion trap for generation of peptide sequences and protein modifications. Protein from replicate bands from the same gel is electro-eluted and used for determination of the melting point and used for circular dichroism analysis. Additional bands from the native gel are either in-gel digested with asparaginase to generate deamidation or PNGase F for deglycosylation, followed by mass spectrometry, conformational and functional studies. Preserved conformation and function of the C1 esterase inhibitor was shown. This protocol can be completed in 1 week.

MASP-1 is a protease of the lectin pathway of complement. It is homologous with MASP-2, previously thought both necessary and sufficient for lectin pathway activation. Recently MASP-1 has taken centre stage with the observation that it is crucial to the activation of MASP-2 and thus central to complement activation. Numerous additional functions have been suggested for MASP-1 and its importance is obvious. Yet, thorough analyses of proteolytic activities and physiological roles in the human scenario have been hampered by difficulties in purifying or producing full-length human MASP-1. We present the successful expression of full-length recombinant human MASP-1 entirely in the zymogen form in a mammalian expression system. We found that the catalytic activity of MASP-1 suppresses its expression through rapid auto-activation and auto-degradation. This auto-degradation was not inhibited by the addition of inhibitors to the culture medium, and it was subsequently found to occur intracellularly. Numerous mutations aimed at attenuating auto-activation or preventing auto-degradation failed to rescue expression, as did also attempts at stabilizing the protease by co-expression with MBL or ficolins or expression in hepatocyte cell lines, representing the natural site of synthesis. The active protease was finally produced through co-expression with the serine protease inhibitor C1 inhibitor. We demonstrate that the expressed protease is capable of binding MBL and auto-activating, and is catalytically active. We have generalized the concept to the expression also of MASP-2 entirely in its zymogen form and with improved yields. We suggest a general advantage of expressing aggressive, autocatalytic proteases with their cognate inhibitors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5' bovine αS(1) casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1 U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (M(r)) of 67,200. Differences in M(r) on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10 ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest(®)) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Intravenous nanofiltered human C1 inhibitor (C1-INH NF) concentrate (Cinryze®) is used as a direct replacement of deficient levels of plasma C1 inhibitor in patients with hereditary angioedema (HAE). In the EU, C1-INH NF concentrate 1000 U is indicated in the treatment, pre-procedural prevention, and routine prevention of angioedema attacks in adults and adolescents with HAE. Intravenous C1-INH NF concentrate 1000 U effectively relieved angioedema attacks in patients with HAE. In a randomized, double-blind trial in pediatric and adult patients, the median time to onset of unequivocal relief from an attack was significantly shorter with C1-INH NF concentrate than with placebo. In an open-label trial, both unequivocal relief and clinical relief were shown in the majority of attacks within 1 and 4 hours of infusion of C1-INH NF concentrate, regardless of the site (i.e. gastrointestinal, cutaneous, laryngeal, or genitourinary) of the defining symptom. When administered prior to a procedure, open-label intravenous C1-INH NF concentrate 1000 U reduced the incidence of angioedema attacks during and after a variety of dental, surgical, or interventional diagnostic procedures in pediatric and adult patients with HAE. Routine preventative treatment with intravenous C1-INH NF concentrate 1000 U every 3 or 4 days reduced the number of angioedema attacks. In a randomized, double-blind, crossover trial in pediatric and adult patients with HAE, the mean normalized number of attacks per 12-week period was significantly lower during routine prevention with C1-INH NF concentrate than with placebo. Routine prevention with C1-INH NF concentrate reduced the median monthly attack rate from baseline in an open-label trial. Intravenous C1-INH NF concentrate was well tolerated in clinical trials in patients with HAE. No cases of viral transmission were reported.

We studied the efficacy of virus reduction by three process steps (polyethylene glycol 4000 (PEG) precipitation, pasteurization, and 15nm virus filtration) in the manufacturing of C1-inhibitor NF. The potential prion removing capacity in this process was estimated based on data from the literature. Virus studies were performed using hepatitis A virus (HAV) and human immunodeficiency virus (HIV) as relevant viruses and bovine viral diarrhea virus (BVDV), canine parvovirus (CPV) and pseudorabies virus (PRV) as model viruses, respectively. In the PEG precipitation step, an average reduction in infectious titer of 4.5log(10) was obtained for all five viruses tested. Pasteurization resulted in reduction of infectious virus of >6log(10) for BVDV, HIV, and PRV; for HAV the reduction factor was limited to 2.8log(10) and for CPV it was zero. Virus filtration (15nm) reduced the infectious titer of all viruses by more than 4.5log(10). The overall virus reducing capacity was >16log(10) for the LE viruses. For the NLE viruses CPV and HAV, the overall virus reducing capacities were >8.7 and >10.5log(10), respectively. Based on literature and theoretical assumptions, the prion reducing capacity of the C1-inhibitor NF process was estimated to be >9log(10).

Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with a growing number of clinical applications, but the current natural supplies are not likely to meet the clinical demands. Therefore, recombinant approaches are of interest, whereby specific attention has to be paid to the generated glycosylation patterns. Here, the N,O-glycoprotein was expressed in the mammary gland of transgenic rabbits and subjected to glycan analysis. After release of the N-glycans of recombinant-rabbit human C1 inhibitor (rhC1INH) by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the O-glycoprotein by centrifugal filtration, then fractionated by a combination of anion-exchange, normal-phase, and high-pH anion-exchange liquid chromatography. The O-glycans, released from the O-glycoprotein by alkaline borohydride treatment, were fractionated by anion-exchange high-performance liquid chromatography (HPLC). The structures of individual components were analysed by 500 MHz 1H NMR spectroscopy, in most cases combined with MALDI-TOF MS. In contrast to the structural data reported for native serum hC1INH, rhC1INH contained a broad array of different N-glycans, made up of oligomannose-, hybrid-, and complex-type structures. In the case of complex-type N-glycans (partially) (alpha2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the lower or upper antenna (Lewis x). The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicates that besides the usual N-glycan processing route, also the alternative endo-mannosidase pathway is followed. The small core 1-type O-glycans showed the usual (alpha2-3)- and (alpha2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.

C1-inhibitor is an important inhibitor of plasma kallikrein and C1, but also has inhibitory activity against numerous other plasma proteinases such as plasmin. The relevance of plasmin inhibition by the C1-inhibitor has been debated, with some evidence showing that plasmin causes significant proteolysis of C1-inhibitor. In the present study, we show that C1-inhibitor in its native state will inhibit plasmin without being significantly degraded, in a manner typical of all serpin reactions. However, if C1-inhibitor is in a denatured polymeric state (as can easily occur during storage, or as produced by heating of the native protein), it will be extensively degraded by plasmin. In addition, we show that hydrophobic interaction chromatography is an effective method to remove trace contaminants of inactive C1-inhibitor polymers.

C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH., (Copyright 2001 Academic Press.)

Human C1 inhibitor is a highly glycosylated serine protease inhibitor of the serpin family. The protein contains two disulfide bonds. In this study, an N-terminally truncated form of recombinant C1 inhibitor was overexpressed in Escherichia coli strains BL21(DE3) and AD494(DE3), the latter enabling the formation of disulfide bonds within the cytoplasm. With both strains, a major fraction of the recombinant protein produced appeared to be insoluble. However, the soluble fraction of lysates from strain AD494(DE3) inhibited the C1s target protease in functional assays. Recombinant C1 inhibitor produced in this strain also displayed the ability to complex with C1s in vitro. In contrast, lysates from strain BL21(DE3) displayed no C1 inhibitor activity. These data support the notion that glycosylation is not important, whereas disulfide bond formation appears to be essential for the production of an active recombinant C1 inhibitor. Thus, bacterial strains that permit the formation of disulfide bonds may represent a reliable system for the production of recombinant C1 inhibitor. However, a major obstacle to large-scale production will be to produce the protein in a soluble form. Attempts to increase the yield of soluble protein by coexpression of the GroEL/ES chaperonins resulted in an increase in solubility., (Copyright 2001 Academic Press.)

In this study, the protein which stimulates proplatelet formation (PPF) of megakaryocytes was purified from normal human plasma using 7 steps procedures. Two different protease inhibitors were identified based on their amino acid sequences, i.e. antithrombin III (AT III) and C1 inhibitor. They were included in high density lipoprotein (HDL). HDL was necessary for AT III to be active in PPF in vitro. The biological effects of the AT III/HDL or thrombin-AT III (TAT)/HDL were studied in vitro. PPF of murine megakaryocytes was stimulated by negative control (BSA) (1.8 +/- 0.3%), AT III (2.0 +/- 0.4%), HDL (1.2 +/- 0.9%), AT III/HDL (14.8 +/- 2.1%) or TAT/HDL (23.3 +/- 3.5%), respectively. TAT/HDL also had a synergistic effect with the mpl ligand, judging by the acetylcholinesterase (AchE) expression of murine megakaryocytes (2.7 fold increase). In vivo subcutaneous administration of AT III alone or TAT for 3 days significantly stimulated thrombocytosis (136% and 144%, respectively, p<0.05) and AT III/HDL showed rapid and further stimulation (150%, p <0.01). These results and the previous studies indicate that megakaryocytopoiesis is regulated by the mpl ligand, while a protease/protease inhibitor complex such as TAT, which is involved in the coagulation cascade associated with platelet consumption, might be one of the regulators in platelet production.

C1-inhibitor is a proteinase inhibitor in the serpin family. It is an important inhibitor of complement C1, plasma kallikrein, and factor XIIa, and as such is involved in regulating inflammatory pathways. Studies on the plasma-derived protein are hampered by the relative ease with which the protein converts to an inactive state on storage, under mild denaturing conditions, or by incubating in some unfavorable buffers. This inactivation is caused by formation of soluble polymers which can be visualized on native electrophoresis. In order to facilitate studies on both the plasma-derived protein and recombinant variants planned for the future, it was necessary to devise a method for the rapid reactivation of the polymers in high yield. It was found that nonionic detergents did not dissociate the polymers, but they were readily dissociated in 0.1% SDS. Treatment with 0.1% SDS followed by rapid removal of the SDS and refolding on an FPLC Superose 6 column allowed for recovery of about 15% of the protein in the active monomeric form. Eighty-five percent eluted as a range of higher order polymers. Using 8 M urea as the denaturant a 25% yield of active monomer was recovered. However, with 6 M guanidine hydrochloride as the denaturant, the yield of active monomer was almost 50%. The remaining material was not present as a range of polymeric species but was probably a dimer. Therefore this method is a useful technique to facilitate studies on C1-inhibitor. Moreover, the ability to produce monomer, dimer, and polymer forms of C1-inhibitor is useful for studies investigating the conformational changes which have occurred in the different forms.

Clearance rates in the guinea pig were determined for intact guinea pig and human C1 inhibitor, the complexes of both inhibitors with human Cls, beta factor XIIa and kallikrein, and for each inhibitor cleaved at its reactive centre with trypsin. Intact human and guinea pig C1 inhibitor were cleared from the circulation more slowly (t1/2s of 9-7 h and 12.1 h and fractional catabolic rates (FCRs) of 0.09 and 0.117) than any of their cleaved or complexed forms. The reactive centre-cleaved inhibitors were cleared with half-lives of 6.75 h for humans and 10.1 h for the guinea pig. The complexes with target proteases were catabolized much more rapidly, with half-lives ranging from 3-08 h to 4.3 h. The complexes with kallikrein were cleared more slowly than those with Cls and beta factor XIIa. Complexes prepared with the guinea pig and human inhibitors were cleared at equivalent rates. The free inactivated proteases were cleared at rates similar to the equivalent complexes, except for kallikrein, which was cleared more rapidly than its complex. The fact that the complexes with different target proteases differed in their catabolism and that protease and complex catabolism were similar suggests that protease may play a direct role in clearance.

Factor J (FJ) is a new inhibitor of the complement system. This work supports the fact that FJ is a cationic molecule (pI > or = 9.6 in native conditions, or pI = 8.1 in denaturing conditions) with a high sugar content (40%) that is able to interact with different lectins, suggesting a complex glycosylation. SDS impaired FJ migration in polyacrylamide gel electrophoresis. In Triton-acid-urea-polyacrylamide gel electrophoresis FJ migrated as a complex, dispersed molecule. In contrast, FJ after Smith degradation (dFJ) gave a single, smeared band of M(r) = 23.4 kDa in reducing SDS-PAGE. dFJ retained only 60% of the initial inhibitory activity of intact FJ. When digestions with different proteinases were performed, no modification of activity was observed. After beta-glucuronidase digestion, FJ lost 80% of its initial activity. Consequently, glycosylation plays an important role in the inhibitory activity of FJ.

A two-step chromatographic procedure has been developed to purify human C1-inhibitor from cryoprecipitate-poor plasma after removal of vitamin K-dependent proteins and antithrombin III. The procedure, which is fully compatible with modern plasma fractionation schemes, includes anion-exchange chromatography on DMAE-Fractogel EMD, viral inactivation by solvent-detergent treatment, adsorption on SO3-Fractogel EMD and viral removal by nanofiltration on 35- and 15-nm pore size membranes. Overall yields were about 45% and 58% for antigen and activity, respectively, providing 60-70 mg of highly purified inhibitor per litre of plasma. The purified inhibitor had a specific activity of 6.5 +/- 0.5 units/mg protein, representing a more than 400-fold increase in purity compared with plasma. C1-inhibitor purity with respect to total protein was greater than 80%. The main contaminant was complement component C3 which accounted for 4-10% of the total protein. Minor contaminants included low amounts of IgM, IgG, IgA, fibrinogen and albumin. Complement component C4 was undetectable. The purified inhibitor was stable throughout the purification process and for more than 24 h at room temperature after reconstitution of the freeze-dried material. Animal tests in rats and mice demonstrated that the C1-inhibitor concentrate was well tolerated at relatively high doses.

Factor J (FJ) is a protein present in human serum, with inhibitory activity against C1. Here we describe the quantitation of FJ in human serum by means of an ELISA inhibition assay. We have purified FJ from the urine of a normal donor following a previously published method with slight modifications. Polyclonal anti-FJ antibodies have been raised in rabbits immunized with a single dose of purified antigen injected in multiple sites. IgG from polyclonal FJ antiserum, coupled to a solid matrix (Affi-Prep gel) was able to adsorb purified FJ antigenically and functionally. Furthermore, anti-FJ specifically retained serum components antigenically related with urine FJ. Taking into account this reactivity, we have developed an inhibition enzyme-linked immunosorbent assay (ELISA) useful for measuring FJ levels in normal human serum. This immunoassay involves preincubating polyclonal anti-FJ with different dilutions of normal human serum to quantitatively reduce the antibody available to bind to purified FJ-coated microtiter plates. Binding of remaining antibody to the microtiter plate is measured spectrophotometrically using peroxidase-conjugated secondary antibody. Quantitation is accomplished by comparison with a known quantity of purified FJ. Conditions for optimization of this quantitative assay have been assessed, including trials with different blocking agents, of which nonfat milk gave the best results. Preliminary experiments showed the existence of paradoxical effects, that is, high nonspecific binding at high serum dilutions. We have eliminated these effects by including high ionic strength (0.4 M NaCl) in the sample incubation solution. Sensitivity and reproducibility parameters have also been established. FJ levels have been measured for the first time in sera from 86 healthy donors.(ABSTRACT TRUNCATED AT 250 WORDS)

The serum C1q inhibitor (C1q INH) is a chondroitin 4-sulfate proteoglycan which is composed of several polyanionic components ranging in size from 21-750 kDa. Although the activity of C1q INH has been described in terms of its ability to precipitate C1q and inhibit its hemolytic activity, not much is known about either the mechanisms of its action or its role in health and disease. This report provides evidence that a 30 kDa core protein component of the proteoglycan macromolecule contains most of the C1q inhibitory activity. This inhibitory activity occurs as a result of C1q INH binding to the C1q "heads" (gC1q) as well as to the collagen "tail" (cC1q). What may be more significant in terms of perpetuation of inflammatory processes is the ability of C1q INH to moderately activate the classical pathway leading to C2 and C4 consumption. The binding of C1q INH to C1q is enhanced at low ionic strength, but significant binding does occur under physiologic conditions which makes it likely for the inhibitor to participate in inflammatory processes especially in microenvironments of high inhibitor concentration. Such elevated concentration does occur in patients with active rheumatoid arthritis and systemic lupus erythematosus either as a result of unregulated proteoglycan synthesis or disturbances in connective tissue metabolism. Another important function of serum C1q INH is its ability to prolong the clotting time of plasma and fibrinogen solutions containing or lacking CaCl2. This potent anticoagulant activity is again displayed by the 30 kDa putative protein core which specifically binds to both the E and D domains of fibrinogen. However, the epitope(s) on the 30 kDa which binds to C1q appears to be distinct from that which binds to fibrinogen. The known presence of proteoglycans on the basement membranes and other sites may explain at least in part the presence of fibrinogen in atheromatous lesions. Furthermore, by binding to fibrinogen, soluble C1q INH-and C1q-C1q INH complexes may limit fibrin gelation in inflammatory and tissue repair microenvironments.

Incubation of highly purified human C1 inhibitor with equally pure human leukocyte proteinase 3, resulted in a dose- and time-dependent inactivation of C1 inhibitor hemolytic activity. Furthermore, this inactivation was accompanied by proteinase 3-dependent cleavage of the C1 inhibitor into an 83,000 molecular weight fragment. The formation of the 83,000 molecular weight fragment followed a time course which was similar to that observed for the inactivation of hemolytic activity. Within 120 minutes more than 90% of the hemolytic activity was lost. This inactivation of C1 inhibitor appeared to be selective as purified human C1q was not degraded in a similar time period. Moreover, when 100 micrograms IgG, isolated from each of 21 Wegener's granulomatosis patients with cytoplasmic anti-nuclear antibodies immunofluorescent titers to proteinase 3 greater then 1:64, was incubated with 3 milliunits of proteinase 3, little to no cleavage of C1 inhibitor was observed. In contrast, 100 micrograms of IgG isolated from 14 normal donors was ineffective in affording protection to C1 inhibitor upon incubation with proteinase 3. Our results suggest that neutrophil infiltration and activation could lead to local complement consumption at the tissue sites.

C1-inhibitor(Mo), a dysfunctional C1-inhibitor molecule produced in two kindred with type II hereditary angioedema, has a mutation at the P10 position (Ala436 to Thr). Like most serpins with hinge region mutations (P14, P12, P10), C1-inhibitor(Mo) loses its inhibitory activity. However, unlike the other hinge region mutations, this mutant is not converted to a substrate. As shown by nondenaturing gel electrophoresis, gel filtration, sucrose density gradient ultracentrifugation, and electron microscopy, C1-inhibitor(Mo) exists in both monomeric and multimeric forms. Polymerization probably results from reactive center loop insertion into the A sheet of an adjacent molecule. Native C1-inhibitor(Mo) was shown to have a thermal stability profile intermediate to those of intact and of cleaved normal C1-inhibitor. Native C1-inhibitor(Mo) did not bind to monoclonal antibody KII, which binds only to reactive center-cleaved normal C1-inhibitor. It did, however, react with monoclonal antibody KOK12, which recognizes complexed or cleaved C1-inhibitor but not intact normal C1-inhibitor. Native C1-inhibitor(Mo), therefore, exists in a conformation similar to the complexed form of normal C1-inhibitor.

To ascertain the mechanism for decreased synthesis of C1 inhibitor (C1 INH) in certain patients with the autosomal dominant disorder hereditary angioneurotic edema, we studied expression of C1 INH in fibroblasts in which the mutant and wild type mRNA and protein could be distinguished because of deletion of exon 7 (delta Ex7). In the HANE delta Ex7 cells, the amount of wild type mRNA (2.1 kb) was expressed at 52 +/- 2% (n = 5) of normal, whereas the mutant mRNA was 17 +/- 1% (n = 5) of normal. Rates of synthesis of both wild type and mutant proteins (11 +/- 3 and 3 +/- 1% of normal, respectively) were lower than predicted from the mRNA levels. There was no evidence of increased C1 INH protein catabolism. These data indicate that there are multiple levels of control of C1 INH synthesis in type I hereditary angioneurotic edema. Pretranslational regulation results in < 50% of the mutant truncated 1.9-kb mRNA. In addition, translational regulation results in decreased synthesis of both wild type and mutatn C1 INH proteins. These data suggest a transinhibition of wild type C1 INH translation by mutant mRNA and/or protein.

Human C1- inhibitor can be rapidly purified by the affinity chromatography procedure described by Pilatte and his associates (1989), but the inhibitor so purified breaks down during storage or is in a cleaved form when initially purified. By adding an ion-exchange chromatography procedure after the affinity chromatography, a stable, single species of C1- inhibitor molecules is obtained. It is likely that serine proteinases in trace amounts, which may be complexed with some of the C1- inhibitor, are removed during the ion-exchange procedure. This procedure provides a highly purified and useful preparation of C1- inhibitor.

This report is an analysis of the parameters involved in the preparation of a C1-esterase inhibitor concentrate. We investigated ways to improve the purification by finding the optimal pH, ionic strength, and quantity of chromatographic media to use at each step. The optimisation of the process gave a greater than two-fold increase in both yield and purity over published methods. The product retained more than 50% activity upon dry heat-treatment at 80 degrees C for 72 h, and the yield of the heat-treated product was 180 I.U./kg of plasma.

We report here the results of studies showing that inhibition of C is a property of several invertebrate paramyosins. Paramyosins from Taenia solium, Schistosoma mansoni, and the mussel Mytilus edulis bind polymeric collagen and can be isolated from crude extracts of tissues by collagen affinity. These paramyosins inhibit C1 function whether the C1 is isolated or present in C2-deficient serum. Because T. solium paramyosin was the best inhibitor, we concentrated further studies on this molecule. T. solium paramyosin binds purified C1q in solution with a dose/response similar to C1r2S2. Further studies of the C1-paramyosin interaction indicate that: 1) C4 is not activated, 2) C4b2a decay is not affected, and 3) there is no effect on the efficiency of C3-9, as provided in EDTA-chelated guinea pig serum, in lysing SRBC. Thus, paramyosin inhibition is directed at the initiation of the classical pathway. The results suggest that paramyosins of helminthic parasites may have a role as modulators of the host immune response through C inhibition at C1.

The antigen levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were assayed in the plasma and in the euglobulin fraction, and their contributions to the euglobulin clot lysis time (ECLT) and t-PA activity were analyzed. Total and free PAI-1 levels in both fractions showed significant positive correlation with ECLT (p less than 0.001), whereas t-PA antigen level did not have a high correlation coefficient with ECLT. t-PA activity showed significant negative correlation with ECLT (p less than 0.001) and positive correlation with free t-PA level (p less than 0.001), which was calculated by the ratio of the concentrations of t-PA-PAI-1 complex and the free PAI-1. Thus free t-PA seems to dissolve the euglobulin clot and its concentration seems to be controlled by the concentration of free PAI-1. These findings were confirmed by the analyses of the effects of C1-inactivator and antibody against t-PA to regular ECLT and kaolin activated ECLT, the latter of which was only inhibited by the addition of C1-inactivator whereas the former was inhibited by anti-t-PA antibody.

A case of angioedema due to acquired deficiency of the regulatory protein C1-esterase-inhibitor (C1-INH) is reported. The edematous attack occurred 3 1/2 weeks after initiation of successful therapy for autoimmune-hemolytic anemia in the course of long-standing non-Hodgkin's lymphoma. At the time of acute edema the complement profile was typical: virtual absence of C1-INH function was associated with diminished concentrations of the components of the classical pathway of complement (C1q, C1r, C1s, C2, C4) and reduced complement hemolytic activity (CH50). Anti-C1-INH-autoantibodies were not detected. The angioedema lasted for about one week, and no further attacks occurred during the five-months follow-up period. Although there was only a minor adjustment to the therapy, the C1q, C2, C4 and CH50 values gradually increased to levels close to the lower limit of the normal range, while C1r and C1s showed normal values. In contrast to most other reports, this case was characterized by angioedema which was precipitated only after initiation of appropriate treatment for the underlying disease rather than before therapy or even diagnosis of the underlying disease.

Novel procedures for structural analysis of the 'reactive-centre' residues, particularly the P1 residue, of the dysfunctional C1-inhibitor proteins found in the plasmas of type II hereditary angio-oedema (HAE) patients are described. C1-inhibitor is adsorbed directly from plasma on to Sepharose-anti-(C1 inhibitor) beads. The P1 residue of C1 inhibitor is arginine and hence a potential cleavage site for trypsin. Thus trypsin digestion of the immobilized protein, followed by SDS/PAGE of the released fragments, identifies P1 residue mutations. Pseudomonas aeruginosa elastase digestion of the immobilized protein, followed by purification of the released C-terminal peptide (by h.p.l.c.) and N-terminal sequence analysis defines the new P1 residue (or other mutations in the reactive-centre region). The techniques are both rapid and highly sensitive, requiring only 400 microliters of plasma. In addition, they permit accurate assessment of the level of normal (functional) inhibitor in a subclass of type II HAE plasmas, those containing P1-residue mutant proteins.

Hereditary angioneurotic edema is inherited as an autosomal dominant disorder and is characterized by potentially life-threatening episodic angioedema. In type II hereditary angioneurotic edema, a dysfunctional C1 inhibitor molecule is present together with low levels of normal C1 inhibitor. About 70% of these dysfunctional proteins result from reactive center (Arg-444) mutations. We describe the deletion of nucleotides encoding Lys-251 (AAG) in C1 inhibitor Ta, the dysfunctional C1 inhibitor from a family with type II hereditary angioneurotic edema. DNA sequence analysis was derived from clones obtained through polymerase chain reaction amplification of blood monocyte C1 inhibitor mRNA. As expected, clones with both normal and abnormal sequence were isolated. The deletion was verified by protein sequence analysis. These data, together with biochemical analysis of the protein and cell-free translation studies, suggest that this deletion, by altering the normal amino acid sequence from Asn-Lys-Ile-Ser to Asn-Ile-Ser, creates a new glycosylation site. The additional carbohydrate accounts for the larger size on SDS/PAGE and very likely interferes with protein function.

Purification of nine plasma proteinase inhibitors and one zymogen from a single batch of human plasma, using affinity chromatography has been accomplished. Those isolated were plasminogen (lysine-Sepharose), alpha-2-antiplasmin (plasminogen-Sepharose), high and low molecular weight kininogens (CM-papain-Sepharose), alpha-2-macroglobulin (Zn++ chelate-Sepharose), alpha-1-proteinase inhibitor, alpha-1-antichymotrypsin, Cl-inhibitor, inter-alpha-trypsin inhibitor (Blue-Sepharose) and antithrombin III (heparin-Sepharose). Alpha-2-macroglobulin and alpha-1-proteinase inhibitor required gel filtration as additional purification steps. Each protein was recovered in both high yield and purity.

An improved procedure for the isolation of the C1 inhibitor (C1-INH) component of human complement is reported. Following preliminary steps to remove plasminogen, fibrinogen, and aggregated material, three conventional chromatographic steps are used to isolate C1-INH in high (70%) overall yield. An extinction coefficient (E 1%, 1 cm 280nm) of 3.60 has been determined. The isolated protein exhibits a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a mobility corresponding to an apparent molecular weight (Mr) of 105 000. After removal of carbohydrate, the protein shows an increased mobility, corresponding to an apparent Mr of 78 000. A total carbohydrate content of 33% has been calculated, and from this and the size of the deglycosylated polypeptide, a true molecular weight of 116 000 was estimated. Further analysis of the carbohydrate has indicated a galactose:mannose ratio of 2:1 and approximately equimolar amounts of N-acetylglucosamine and N-acetylgalactosamine. This composition is unusual for a plasma protein and suggests that much of the carbohydrate is contained in linkages other than the typical N-glycosidic structures. Values found for the amino acid composition are compared with those reported previously. The amino-terminal sequence (40 residues) of C1-INH is also reported. Asparagine lies at the amino terminus. Neither high-performance liquid chromatography of the released phenylthiohydantoin derivative nor back-hydrolysis of the thiazolinone permitted identification of the residue contained at position 3. The sequence around this position is compatible, however, with an N-glycosidic linkage to residue 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequencing of trypsin fragments of C1 inhibitor gave regions of low codon degeneracy that were used for oligonucleotide probes. Human liver cDNA libraries gave clones containing most of the protein sequence, showing that the inhibitory domain belongs to the 'serpin' class of protein inhibitors. Fragments of these cDNA clones were used to probe human genomic cosmid libraries. The genomic sequence was found to be about 17 X 10(3) base pairs, with a coding sequence of approximately 1800 base pairs containing introns at amino acid positions--6, 162, 207, 275, 321, 395, and one in the 5' non-coding region. There is very little similarity of intron position amongst the serpin genes. All but one of the intron positions in the C1 inhibitor structural gene correspond to surface residues if C1 inhibitor is considered to have a structure similar to the cleaved form of alpha 1-antiproteinase. The serine and threonine residues in the N-terminal 100 amino acids of the sequence thought to carry complex carbohydrates are found in a single exon.

We have previously shown that the human monocyte-like cell line U937 synthesizes C1-INA and expresses cell surface C1-INA. In this report we provide evidence that this surface-expressed C1-INA is functionally active. Intact U937 cells demonstrated functional C1-INA activity in a hemolytic assay. This activity was blocked when the cells were incubated with monospecific antibody to C1-INA, and was not detectable in cell-free supernatants of U937 cells. SDS-PAGE analysis of radiolabeled U937 cell surface proteins purified by anti-C1-INA affinity chromatography revealed two distinct bands. One protein had a Mr of 105 kDa identical to plasma C1-INA, and the second had a Mr of 200 kDa. We were unable to determine the identity of the 200 kDa protein by Western blotting with anti-C1-INA. However, the possibility exists that this 200 kDa molecule may represent a C1-INA receptor, a dimeric form of C1-INA, or an unrelated cell surface protein with affinity for C1-INA. Furthermore, we show that treatment of U937 cells with phorbol ester resulted in an increase in the percentage of cells expressing surface C1-INA. These results suggest that U937 cells express functional cell surface C1-INA, which could function in vivo to protect these human tumor cells from lysis by host complement.

C1 inhibitor (C1-INH) acts to inhibit active enzymes of both the classical complement and Hageman factor-dependent pathways. Previously reported C1-INH purification procedures were multistep and most have been associated with significant loss in specific functional activity. We have developed a simple chromatographic procedure which yields a pure C1-INH protein from normal human plasma with a specific activity equal to or greater than the starting sample. Briefly, protease inhibitor-treated, pooled human citrated plasma was fractionated with polyethylene glycol (PEG 4000); the supernatant fraction that remained soluble at 16% was obtained. The inhibitor was precipitated with 45% PEG. The resulting precipitate was solubilized and chromatographed on DEAE Sephacel using a linear salt gradient. The eluted fractions containing the C1-INH and other contaminants were pooled and dialyzed against the starting buffer of the next chromatographic step. A unique separation procedure using zinc ion chelate-coupled agarose was employed as the second chromatographic step. The eluted C1-INH, after zinc ion chromatography, displayed a significant enhancement in purity and maintained a specific functional activity twice that of plasma. The final procedure utilized immunoadsorption chromatography using an anti-contaminant column. Under reducing conditions on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified C1-INH migrated as a single band with an apparent molecular weight of 90,000-105,000, but under non-reducing conditions, a doublet with apparent molecular weights of 94,000-100,000 and 85,000-93,000 was seen. C1-INH antigenic concentrations were measured and shown to be correlated in serum, citrate plasma, and EDTA plasma from 16 normal subjects.

New methods have been developed for the isolation, quantitative detection, and functional measurement of human complement C-1-inhibitor (C-1-INH). The two-step purification procedure for C-1-INH from human plasma or serum employs affinity chromatography with a monoclonal anti-C-1-INH antibody coupled to CNBr-activated Sepharose 4B followed by fractionation on a FPLC Mono Q HR 5/5 column. It yields functionally active, homogeneous C-1-INH with about 40% recovery. For quantitative estimation of C-1-INH an ELISA was performed. ELISA plates were coated with a polyclonal anti-C-1-INH antibody, serum or plasma was added and bound C-1-INH was detected with the monoclonal anti-C-1-INH antibody. The method has a sensitivity of 0.4 ng C-1-INH per assay corresponding to 20 ng/ml. For the detection of functionally active C-1-INH an ELISA was developed using C1-s-coated microtiter plates. After incubation with serum or plasma, C1-s-bound C-1-INH was monitored with the monoclonal anti-C-1-INH antibody. With this method it is possible to measure as little as 0.3 ng of functionally active C-1-INH in 20 microliter of a biological sample. All methods described in the present paper are easy to perform, rapid, sensitive, and highly reproducible.

The sera of three different patients from Togo, Africa were investigated with respect to their complement profile. The three patients were suffering from parasitic (Onchocerca volvulus) and bacteriological (Treponema pertenue) diseases. The total hemolytic activity (50% hemolytic complement) was markedly depressed. The analysis of the individual complement components revealed the the titers of C1, C2, C3, and C4 were lowered up to 90%, indicating an activation of the classical pathway of complement. Addition of the patients' sera to normal human serum induced a temperature-dependent consumption of C4 and C2, whereas C3 was not affected. This activity in the patients' sera eluted from a Sephadex-G-200 column with the 19 S peak and could be identified as the activated form of the first component of the complement system. The reason for the presence of activated C1, C1 in the patients' sera resides in the absence of functionally active C1 inactivator.

This report describes the purification of a novel proteinase inhibitor from bovine serum. This protein was purified to apparent homogeneity employing affinity binding to sulfated dextran and precipitation by ammonium sulfate, followed by sequential chromatography on DEAE-cellulose, heparin-Sepharose and Sephacryl S-200. Quantitative enzyme-linked immunosorbent assays revealed that the concentration of this inhibitor is approximately 3 microM in bovine serum. The inhibitor is a single polypeptide chain with an estimated Mr of 83,000 as determined by SDS-polyacrylamide gel electrophoresis. An aspartic acid was found at the amino terminus of the protein; N-terminal amino acid sequence data indicated that there was no significant homology with other reported amino acid sequences. This bovine inhibitor covalently complexed the human proteinases C1-r, C1-s, factor XIIa and plasma kallikrein, which are also complexed and inactivated by human C1-inhibitor. In addition, the bovine inhibitor complexed and inactivated bovine chymotrypsin, a feature which functionally distinguishes it from human C1-inhibitor. Although the bovine inhibitor appears functionally very similar to C1-inhibitor, we found no evidence for structural homology with the human counterpart.

Protease nexin I is a proteinase inhibitor that is secreted by human fibroblasts and forms stable complexes with certain serine proteinases; the complexes then bind to the fibroblasts and are rapidly internalized and degraded. In this report, we show that this inhibitor, which is present in very low concentrations in plasma, has functional and structural similarities to C1 inhibitor, an abundant proteinase inhibitor in plasma. Both inhibitors complex and inactivate certain proteinases that previously were known to rapidly react only with C1 inhibitor. Kinetic inhibition studies show that protease nexin I inhibits Factor XIIa and plasma kallikrein with second-order rate constants of 2.3 x 10(3) and 2.5 x 10(5) M-1 s-1, respectively, which are similar to the rate constants for inhibition of these proteinases by C1 inhibitor. Protease nexin I inhibits C1s about one-tenth as rapidly as does C1 inhibitor. Alignment of the amino acid sequences of protease nexin I and C1 inhibitor shows that these proteins have similarity at their reactive centers (from sites P7 to P1). The remaining regions of the two proteins share much less similarity. In contrast to protease nexin I, C1 inhibitor is not secreted by human fibroblasts. Although 125I-C1s-protease nexin I complexes readily bind to human fibroblasts, binding of 125I-C1s-C1 inhibitor complexes or other 125I-proteinase-C1-inhibitor complexes to these cells is not detectable. Thus, protease nexin I and C1 inhibitor may control some common regulatory proteinases in the extravascular and vascular compartments, respectively.

A clone coding for the precursor form of C1 inhibitor has been isolated from a human liver cDNA library constructed in lambda gt11. This clone contains a cDNA insert of 1777 nucleotides, which includes 63 nucleotides coding for a signal sequence of 21 amino acids, 1434 nucleotides coding for the mature protein of 478 amino acids, a stop codon of TGA, and 265 nucleotides of the 3'-noncoding sequence followed by a poly(A) tail of 12 nucleotides. The predicted amino acid sequence of mature C1 inhibitor contains a unique region (positions 43 to 97) of 14 tandemly repeated copies of the tetrapeptide Gln-Pro-Thr-Thr and variants thereof. The carboxy-terminal sequence (positions 98 to 478) comprises the typical structural elements of a serine proteinase inhibitor (serpin). These findings indicate that C1 inhibitor is unique among the known members of the serpin superfamily due to the presence of an extra-domain of highly repetitive structure located at the amino-terminus of the inhibitor.

A new purification method for C1-esterase inhibitor is described, which is essentially a three-step procedure: precipitation with poly(ethylene glycol), chromatography on DEAE-cellulose and hydrophobic interaction chromatography on hexyl-Sepharose. The final product is a single-chain glycoprotein with a molecular weight of about 100 000 and NH2-terminal asparagine. The molecule is fully active as judged by complex formation with C1s. Two of its three disulphide bridges can be easily reduced and S-carboxymethylated under non-denaturing conditions without loss of activity. However, at high dithioerythritol concentration the third disulphide bridge is also cleaved and accompanied by loss of the activity, indicating that this disulphide bridge is involved in maintaining the conformation around the reactive site in the inhibitor.

Proteins of the C1 complex, C1q, C1r, and C1s, of the classical pathway of complement activation are known to be synthesized in human skin fibroblasts. Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-PAGE, we demonstrate that human skin fibroblasts synthesize and secrete C1 inhibitor with an apparent molecular mass of 78 kDa in the cell lysate and 102 kDa in the extracellular medium. This C1 inhibitor had the capacity to bind activated C1s. Fibroblasts synthesized 30- to 50-fold more C1 inhibitor than was synthesized in monocytes. As previously reported, fibroblasts also synthesized C1r and C1s. IFN-gamma, IFN-beta 1, and TNF had significant, but distinct, effects on synthesis of C1 inhibitor, C1r, and C1s. Incubation of the cells with IFN-gamma, 1000 U/ml, for 24 h induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 4.2-, 1.9- and 1.6-fold, respectively. IFN-beta 1 had effects similar to IFN-gamma, although smaller in magnitude. TNF, 12.5 ng/ml, induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 1.5-, 1.4- and 2.6-fold. IL-1, IFN-beta 2 (IL-6), and LPS did not affect synthesis of C1 inhibitor, C1r, or C1s. Fibroblasts are present in large amounts in most tissues. Synthesis of C1 inhibitor, C1r, and C1s by these cells could provide a source of these important proteins in body tissues. In addition, fibroblasts should be a good model for the in vitro study of genetic diseases involving the synthesis of these proteins.

The primary structure of human C1 inhibitor was determined by peptide and DNA sequencing. The single-chain polypeptide moiety of the intact inhibitor is 478 residues (52,869 Da), accounting for only 51% of the apparent molecular mass of the circulating protein (104,000 Da). The positions of six glucosamine-based and five galactosamine-based oligosaccharides were determined. Another nine threonine residues are probably also glycosylated. Most of the carbohydrate prosthetic groups (probably 17) are located at the amino-terminal end (residues 1-120) of the protein and are particularly concentrated in a region where the tetrapeptide sequence Glx-Pro-Thr-Thr, and variants thereof, is repeated 7 times. No phosphate was detected in C1 inhibitor. Two disulfide bridges connect cysteine-101 to cysteine-406 and cysteine-108 to cysteine-183. Comparison of the amino acid and cDNA sequences indicates that secretion is mediated by a 22-residue signal peptide and that further proteolytic processing does not occur. C1 inhibitor is a member of the large serine protease inhibitor (serpin) gene family. The homology concerns residues 120 through the C-terminus. The sequence was compared with those of nine other serpins, and conserved and nonconserved regions correlated with elements in the tertiary structure of alpha 1-antitrypsin. The C1 inhibitor gene maps to chromosome 11, p11.2-q13. C1 inhibitor genes of patients from four hereditary angioneurotic edema kindreds do not have obvious deletions or rearrangements in the C1 inhibitor locus. A HgiAI DNA polymorphism, identified following the observation of sequence variants, will be useful as a linkage marker in studies of mutant C1 inhibitor genes.

We describe a mechanism of action of autoantibodies reactive with the C1 esterase inhibitor (C1EI) molecule found in three patients with acquired angioedema without associated diseases. All of these patients have a circulating C1EI of lower molecular weight than that of normal control subjects. When native C1EI was added to patient, but not control, plasmas, it was cleaved to a lower molecular weight fragment under conditions that allowed contact system activation. In a partially purified system, patient immunoglobulin preparations impaired stable complex formation between plasmin and C1EI and exaggerated the cleavage of the latter to a lower molecular weight fragment. We propose that the autoantibody does not interfere with the cleavage of the bait sequence of the inhibitor but reduces the availability of the reactive center of the molecule in a way that interferes with the irreversible inhibition of target enzymes. In this way unregulated activation of the kinin and complement pathways occurs, leading to disease manifestations via as yet uncharacterized mediators.

The second component of human complement (C2) in pseudoglobulin prepared from normal plasma eluted as a single peak at high conductivity (30 mS) and pH 4.5 from the cationic exchangers S-Sepharose or Mono S in the Fast Protein Liquid Chromatography (FPLC) System. The C2 was stable at pH 4.5 and 0 degrees C if enzyme inhibitors were used and the pH was raised to 6.0 after elution from the columns. After rechromatography on Mono S in the FPLC System at the median isoelectric point of 5.5 or pH 6.0, the C2 eluted as two distinct hemolytic forms: the first peaked at 16 mS, the second at 30 mS. The two forms of C2 did not correlate with the allotypic variant of C2 in individual, normal human plasmas. After elution at pH 4.5 from S-Sepharose and rechromatography at pH 5.5 or 6.0 on Mono S, the hemolytic activities of the two forms in individual plasmas eluted in 3 patterns: 1) high activity at 16 mS, low activity at 30 mS; 2) low activity at 16 mS, high activity at 30 mS; 3) high activity at 16 mS, high activity at 30 mS. The specific activities of both forms were approximately the same; both eluted the same after gel filtration at pH 5.5, and both had the same pattern on SDS-PAGE and immunoblots. The pattern of elution was characteristic for each individual plasma, and the first hemolytic form appeared to elute independent of the second form. At pH 4.5, C2 was completely separated from Factor B, a functionally and structurally similar protein of the alternative complement pathway, whereas at pH 5.5 or 6.0, the two proteins eluted together. From these results, the two forms of hemolytic C2 can be purified for structural and functional analyses.

A minority of patients with hereditary angioedema (HAE) have normal concentrations of a dysfunctional C1 inhibitor protein (C1INH) in their plasmas. We purified C1INH from the plasmas of one such patient before and during treatment with the anabolic steroid stanozolol. Both the pretreatment plasma and plasma obtained during stanozolol treatment contained varying amounts of two extremely similar C1INH proteins that were functionally distinct. The pretreatment plasma contained primarily (94%) dysfunctional C1INH that did not inactivate or complex with either purified C1s, activated Hageman factor, or kallikrein and small amounts (6%) of functionally normal C1INH. Stanozolol treatment increased the plasma concentrations of both of these proteins as well as the proportion (23%) of functional C1INH in the plasma. The purified dysfunctional and functional C1INHs had identical or nearly identical molecular sizes, charges, amino acid compositions, and amino sugar contents, and could not be distinguished physicochemically from each other or from normal C1INH. From these studies of purified C1INH proteins we concluded that HAE associated with dysfunctional C1INH is due to a defect at the structural locus for one C1INH gene and that both the dysfunctional C1INH gene and the normal C1INH gene products are present in the plasma of the affected subject. Treatment with stanozolol comparably increased the synthesis of both C1INH proteins. The disproportionate rise in the level of the normal C1INH protein is consistent with the view that it is more rapidly catabolized as a consequence of its interaction with the proteases it inactivates.

The effect of Bacteroides gingivalis W83 on various key components of the human plasma proteinase cascade systems was studied. When purified C1-inhibitor was incubated with the bacterium, the inhibitor was rapidly inactivated by limited proteolytic cleavage. In citrated whole plasma, C1-inhibitor, antithrombin, plasminogen, prekallikrein, prothrombinase complex, the clotting factor X, and most of the alpha 2-antiplasmin were functionally eliminated after 30 min of incubation with the bacterium. Fibrinogen disappeared from the plasma almost immediately upon mixing with the bacterial suspension. In contrast, there was no appreciable decrease in the bulk of other plasma proteins, such as various transport proteins (albumin, prealbumin, transferrin) and immunoglobulins, during 4 h of incubation with the bacterium. Most of the observed effects can be assigned to the proteolytic activity of the bacterium itself, since there was little evidence for generation of intrinsic plasma proteinase activity, despite the loss of proteinase inhibitory activities. B. gingivalis W83 thus seems to be equipped with proteolytic enzyme systems which selectively recognize and rapidly inactivate the most important proteinase inhibitors and proenzymes present in human plasma. This bacterium therefore seems to be able to efficiently paralyze the host's various defenses against invading microorganisms.

Plasma kallikrein and factor XIa circulate bound to high molecular weight kininogen, and such binding has been reported to protect these enzymes from inactivation by their respective inhibitors. However, this observation is controversial, and the effect of high molecular weight kininogen upon the interaction between kallikrein and C1 inhibitor (C1-INH) has been questioned. We have re-evaluated this reaction and studied the rate of inhibition of kallikrein by C1-INH in the presence and absence of high molecular weight kininogen. The second-order rate constant of inhibition of kallikrein by C1-INH was unaffected by saturating concentrations of high molecular weight kininogen. Our results suggest that although high molecular weight kininogen clearly augments the rate of formation of kallikrein and other enzymes of the contact activation pathway, it has no effect on the rate of enzyme inhibition by C1-INH.