Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma.

Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine-labelled plasminogen and the plasmin-inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7-S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one. Both components were purified with high yield by a combination of affinity chromatography on lysine-agarose and gel filtration, and investigated by dodecylsulphate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against alpha1-antitrypsin, antithrombin III, C1-esterase inhibitor, inter-alpha-trypsin inhibitor or alpha1-antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and alpha2-macroglobulin which reacted with antisera against human plasminogen and against alpha2-macroglobulin. The component of the second peak had a molecular weight (Mr) of 120000-140000 by dodecyl-sulphate-polyacrylamide gel electrophoresis and lpon reduction displayed a doublet band with an Mr of 65000-70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above-mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno-absorption resulted in disappearance of the fast-reacting antiplasmin activity whereas alpha2-macroglobulin was found to represent the slower-reacting plasmin-neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of alpha1-antitrypsin, antithrombin III or C1-esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in quimolar amounts to that of plasminogen. It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast-reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and alpha2-macroglobulin, which reacts more slowly.     

Identification and characterization of trypsin, chymotrypsin and elastase inhibitors in porcine serum.

Characterization of the trypsin-, chymotrypsin- and elastase-inhibiting properties of porcine serum was carried out by gel filtration on Ultrogel, AcA 44, and agarose gel electrophoresis with subsequent processing for protease-inhibiting activity. Moreover, by allowing the fractions obtained from gel filtration to react with antibodies to porcine serum protease inhibitors, the specific inhibiting properties of these inhibitor molecules were identified. At least six protease inhibitors were identified and partially characterized in porcine serum. Two alpha 2 -macroglobulins (alpha 2 Mf and alpha 2 Ms), homologues to human alpha 2 -macroglobulin, with slightly different electrophoretic mobilities, were both found to exhibit trypsin, chymotrypsin and elastase inhibiting activity. Alpha 1 -Protease inhibitor (Mr 51 000), a homologue to human alpha 1 -protease inhibitor (alpha 1 -antitrypsin), also showed trypsin-, chymotrypsin- and elastase-inhibiting properties. Inter-alpha-trypsin inhibitor (Mr 162 000 and 129000), a porcine serum counterpart to human inter-alpha-trypsin inhibitor, showed trypsin- and chymo-trypsin-inhibiting properties. In addition, a specific trypsin inhibitor, alpha 2 -antigrypsin (Mr 58 000), and a specific elastase inhibitor, beta-elastase inhibitor, were characterized in porcine serum, and these seem to have no counterparts in human serum.     

Immunological analysis of alpha 1-microglobulin in different mammalian and chicken serum. alpha 1-Microglobulin is 5-8 kilodaltons larger in primates

Heterologous radioimmunoassays for a semiquantitative analysis of alpha 1-microglobulin were developed, exploiting the binding between polyclonal rabbit or goat antisera against human, guinea pig, or rat alpha 1-microglobulin and 125I-labeled human, guinea pig, or rat alpha 1-microglobulin. Homologues of this protein were detected in human, guinea pig, Rhesus monkey, rat, mouse, rabbit, goat, horse, and cow serum by inhibition of a set of heterologous radioimmunoassays. Serum proteins were separated by gel chromatography, and fractions were pooled, concentrated, and radiolabeled with 125I. By immunoprecipitation of the radioiodinated serum pools with heterologous anti-alpha 1-microglobulin-sera, and separating the precipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analogues of alpha 1-microglobulin were isolated from serum of man, guinea pig, Rhesus monkey, rat, mouse, horse, and chicken. The apparent molecular weight of alpha 1-microglobulin was 31,000-32,000 in human and monkey serum and 24,000-26,000 in guinea pig, rat, mouse, horse, and chicken serum. The possibility of an addition of a 5,000-8,000-Da peptide in primate alpha 1-microglobulin is discussed.     

The in vitro interactions of rat pancreatic elastase and normal and inflammatory ray serum.

The partition of labelled rat pancreatic elastase (EC 3.4.21.11) between the different protease inhibitors of rat plasma was studied at different levels of saturation of the inhibitors of rat plasma was studied at different levels of saturation of the inhibitor capacity of plasma with the enzyme. The reaction mixtures were analysed by immunoelectrophoretic methods utilizing specific antisera against the different inhibitors and by gel filtration on Sephadex G-200. Rat serum was shown to contain four elastase binding proteins. alpha 1-antitrypsin, alpha 1-macroglobulin and alpha 2-acute phase protein and alpha 1-inhibitor 3 which exhibits immunologic cross-reaction with human inter-alpha-trypsin inhibitor and is of similar molecular weight. With minute amounts of labelled elastase the partition among the binding protein was alpha 1-macroglobulin 60%, alpha 1-antitrypsin 24% and alpha 1-I3 16%. The 60% value of alpha 1-M bound radioactivity in normal serum corresponds to the sum of alpha 1-M and alpha 2-AP labelling in inflammatory serum.     

New variants of alpha 1-antitrypsin: comparison of Pi typing techniques.

Four new rare inherited variants (Pi types) of alpha 1-antitrypsin (alpha 1-protease inhibitor) are described. Each variant has been compared with previously reported genetic variants by several techniques used for Pi typing: isoelectric focusing in polyacrylamide gel, starch gel electrophoresis, and agarose gel electrophoresis. Some variants are identical or very similar by one technique but can be clearly distinguished by another technique. Crossed immunoelectrophoresis and gel immunofixation have been used to identify the gel bands as alpha 1-antitrypsin.     

Identification and characterization of trypsin, chymotrypsin and elastase inhibitors in the hedgehog, Erinaceus europaeus, and their immunological relationships to those of other mammals (rat, pig and human)

1. Hedgehog plasma was separated by gel filtration on Sephacryl S-200, the fractions resolved by electrophoresis and the electrophoregrams characterized for trypsin, chymotrypsin and elastase inhibiting activities with both low and high molecular weight substrates. Approximate molecular weights were also determined. 2. At least ten protease inhibitors were characterized in hedgehog plasma including three macroglobulins. 3. The hedgehog protease inhibitors were identified by immunoelectrophoresis. Four protease inhibitors showed homologies with specific human, rat or swine antisera. These were alpha 2-and beta-macroglobulins, alpha 1-protease inhibitor, and alpha 2-antithrombin.     

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

Interaction between human pancreatic elastase and plasma protease inhibitors

1 ml of human serum inhibits about 0.9 mg of purified human pancreatic elastase owing to complexation with alpha 1-antitrypsin and alpha 2-macroglobulin. On addition to serum, elastase is preferentially bound by alpha 2-macroglobulin. The complexes between elastase and alpha 1-antitrypsin and alpha 2-macroglobulin, respectively, migrate as alpha 2-globulin on agarose gel electrophoresis. Elastase bound by alpha 1-antitrypsin is precipitated by antibodies against enzyme as well as inhibitor, while the alpha 2-macroglobulin-bound elastase is only precipitated by antibodies against the inhibitor. The molar combining ratio for elastase/alpha 1-antitrypsin is 1:1 and for elastase/alpha 2-macroglobulin 2:1. The elastase bound by alpha 2-macroglobulin retains its activity against low molecular weight substrates, while that bound by alpha 1-antitrypsin is enzymologically inactive.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human alpha 1-antitrypsin in double immunodiffusion. PI-1 corresponding to alpha 1-antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200-300 Kd) than that of PI-1, and did not cross-react with antisera against human alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, alpha 2-plasmin inhibitor, inter-alpha-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including alpha 1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Alterations in the mouse and human proteome caused by Huntington's disease

Huntington's disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In our effort to identify proteins involved in processes upstream or downstream of the disease-causing huntingtin, we studied the proteome of a well established mouse model by large gel two-dimensional electrophoresis. We could demonstrate for the first time at the protein level that alpha1-antitrypsin and alphaB-crystalline both decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver and testes in mice. Reduced expression of the serine protease inhibitors alpha1-antitrypsin and contraspin was found in liver, heart, and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. In three brain regions obtained post-mortem from Huntington's disease patients, alpha1-antitrypsin expression was also altered. Reduced expression of the major urinary proteins not found in the brain was seen in the liver of affected mice, demonstrating that the disease exerts its influence outside the brain of transgenic mice at the protein level. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington's disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.     

Proteomic analysis revealed a strong association of a high level of alpha1-antitrypsin in gastric juice with gastric cancer

To investigate the pathology of gastric disorders, we compared the proteomic patterns of gastric juice from patients with various gastric disorders. In healthy subjects pepsin A, pepsin B and gastric lipase were the major proteins detected by two-dimensional gel electrophoresis. These digestive enzymes were not detected in 60% of gastric cancer cases (18 out of 30 analyzed cases). Interestingly, an extraordinary amount of alpha(1)-antitrypsin was observed in these cases. In contrast to gastric cancer cases, alpha(1)-antitrypsin was detected in only 5% of patients (three out of 56) with chronic atrophic gastritis, and the detection frequency went up as the disease developed (one of four intestinal metaplasia cases, two of seven tubular adenoma cases, a single examined case of hyperplastic polyp and 60% of gastric cancer). Zymography showed that a 60 kDa protease strongly associated with alpha(1)-antitrypsin and mass spectrometric analysis revealed that the gastric alpha(1)-antitrypsin was a protease-cleaved form. Our data suggest that alpha(1)-antitrypsin and 60 kDa protease may serve as good diagnostic and prognostic markers for conditions associated with gastric cancer.     

Protease inhibitors in porcine serum and their immunological relationships to human protease inhibitors.

A close molecular relationship exists between the protease inhibitors of porcine serum and those of human serum as shown by studying their immunological cross-reactivities with gel diffusion and immunoelectrophoretic methods. On studying seven different antisera to human protease inhibitors, five were found to cross-react with porcine serum, and on this bisis it was possible to identify alpha 2 -macroglobulin f, alpha 2 -macroglobulin s, alpha 1 -protease inhibitor, inter-alpha-trypsin inhibitor, antithrombin and alpha 2 -antiplasmin in porcine serum. Antisera to four of these porcine serum inhibitors (alpha 2 -macroglobulin f, alpha 2 -macroglobulin s, alpha 1 -protease inhibitor and inter-alpha-trypsin inhibitor) were produced and were shown to react immunologically with their human serum protease inhibitor counterparts.     

Interactions in vitro and in vivo between rat serum protease inhibitors and anodal and cathodal rat trypsin and chymotrypsin.

Reaction mixtures of increasing amounts of the pancreatic homologous proteases, anodal and cathodal chymotrypsin and trypsin, respectively, and normal rat serum were analyzed by immunoelectrophoretic methods in order to determine their distribution on serum protease inhibitors. This paper concerns three proteins occurring in normal serum and capable of binding protease viz. alpha1-macroglobulin, alpha1-antitrypsin and alpha1-inhibitor 3. The distribution of the enzymes among these protease inhibitors differed significantly from one protease to another. The distribution of the proteases among the serum protease inhibitors following intravenous injection of 125I-labelled proteases corresponded to that in vitro. Complexes formed with alpha1-macroglobulin and alpha1-inhibitor 3 were quickly eliminated irrespective of the enzyme species used, whereas those formed with alpha1-antitrypsin persisted much longer in the circulation.     

Two-dimensional electrophoresis of the plasma proteins of alpacas and llamas: genetic polymorphism of alpha 1B-glycoprotein and three other proteins

Plasma samples of alpacas and llamas were analysed by a simple method of two-dimensional (2-D) agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis, followed by general protein staining of gels. Genetic polymorphism in both species is described for alpha 1B-glycoprotein (alpha 1B) and three other unidentified proteins designated prealbumin (Pr), postalbumin 1 and 2 (Pa1 and Pa2). alpha 1B was identified by cross-reactivity with antisera for human and pig alpha 1B. Altogether, two alleles of Pr, two of Pa1, five of alpha 1B and three of Pa2 are described. Most of the alleles were present in alpacas and llamas. Alpacas showed a high degree of polymorphism at all four loci. Llamas showed considerable polymorphism at only the Pa1 and Pa2 loci. The theoretical probability of exclusion (PE) of an incorrectly assigned parent was estimated to be about 80% in each species by typing for the six polymorphic plasma proteins reported so far in these species. The given method of 2-D electrophoresis revealed no fixed differences in protein mobilities that discriminate between llamas and alpacas.     

Micro-quantitative determination of alpha1-antitrypsin by unidimensional capillary counter-immunodiffusion]

The immunoprecipitation reaction for the determination of microamounts of alpha1-antitrypsin was conducted in the capillaries filled with a microvolume of a 1% agarose gel (the gel length was 7--8 mm, and the diameter--0.6 mm). Microamounts of the antigen and antiserum were applied to the surface of the opposite ends of the gel. Precision control of these manipulations was carried out by means of an ocular micrometer of a binocular lens. The amount of the formed immunoprecipitate in gel was calculated by the summated optic density after staining with amido black recorded by the method of double-wave microspectrophotometry. Sensitivity of the method was 0.3--1.0 ng of alpha1-antitrypsin. The linear plot between the amount of alpha1-antitrypsin and the amount of the precipitate formed was found within the range of 1--25 ng of protein. The error was about 10%.     

Physiologic inhibition of human activated protein C by alpha 1-antitrypsin

The plasma antithrombotic enzyme activated protein C (APC) has two major plasma inhibitors. One is heparin-dependent, has been characterized, and is known as protein C inhibitor. The second inhibitor was isolated based on its heparin-independent ability to inhibit and complex with APC. The purified inhibitor had the amino acid composition and NH2 terminus of alpha 1-antitrypsin and reacted with monoclonal antibodies to alpha 1-antitrypsin. The inhibitor was greater than 95% pure alpha 1-antitrypsin as judged by electroimmunoassay, inactivation of trypsin, and electrophoresis in two gel systems. To identify the second major plasma inhibitor of APC, immunoblot studies of enzyme-inhibitor complexes were made to compare APC addition to normal plasma and to plasma deficient in protein C inhibitor or alpha 1-antitrypsin. The results showed that alpha 1-antitrypsin is the second major plasma APC inhibitor. Given the association rate constant of alpha 1-antitrypsin for APC of 10 M-1 s-1 and its plasma concentration of approximately 40 microM, it accounts for approximately half of the heparin-independent APC inhibitory activity of plasma. Based on immunoblot analysis plasmas of 15 patients with intravascular coagulation contained APC-alpha 1-antitrypsin complexes suggesting that this inhibition reaction occurs in vivo. Thus, alpha 1-antitrypsin is a major physiologic inhibitor of APC.     

Immunodiffusion Analysis of Yaba Poxvirus Structural and Associated Antigens

Yaba poxvirus virions were extracted and purified from Rhesus monkey tumors. A saline-soluble virion fraction (Y-xp), obtained by mechanical fractionation of purified virions with an X-press, contained seven components in acrylamide gel electrophoresis; five of these components were reactive in immunodiffusion with whole virion and Y-xp antisera produced in rabbits and monkeys. The saline-insoluble residue remaining after X-press treatment was hydrolyzed with sodium dodecyl sulfate, urea, and 2-mercaptoethanol (SUM). This fraction, Y-sum, contained five components, four of which were demonstrable by immunodiffusion. There was no evidence of antigenic relationships between Y-xp and Y-sum antigens in immunodiffusion. In acrylamide gel electrophoresis, one Y-xp and one Y-sum component had similar mobilities. Y-xp but not Y-sum antisera contained viral-neutralizing antibodies. Virus-free saline extracts of Yaba tumor prepared with Genetron (YS) were essentially devoid of virion structural antigens. They failed to induce precipitating antibodies for virion antigens, were nonreactive in immunodiffusion with virion antisera, and gave low complement-fixation titers with virion antisera. Yaba virion antigens were recovered from the Genetron tumor sediment by SUM and alkaline hydrolysis. Antisera prepared to YS extracts gave a maximum of 17 precipitin lines in immunodiffusion with YS extracts; none was identified as a virion structural antigen. Saline extracts of tumor prepared without Genetron contained immunogenic amounts of 5 virion antigens and 12 to 14 associated antigens. Animals immunized with infected cell culture extracts (virus-free) formed antibodies to six to seven virion antigens. The implications of using extracts of Yaba poxvirus-infected tissues in complement-fixation tests to measure virion antibodies were discussed.     

Protein C inhibitor. Purification from human plasma and characterization

Protein C inhibitor was isolated from human plasma using conventional chromatographic technique consisting of barium citrate adsorption, polyethylene glycol fractionation, DEAE-Sepharose CL-6B treatment, ammonium sulfate fractionation, dextran sulfate-agarose chromatography, gel filtration on ACA-44, and DEAE-Sephacel chromatography. The purified protein C inhibitor is a single polypeptide chain with an apparent Mr = 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor is heterogeneous in pI: six pIs exist between pH 7.4 and 8.6. The inhibitor was shown to be different from the already known plasma protease inhibitors by chemical and immunological analyses. It migrates to the late alpha 1-globulin region on agarose gel electrophoresis. The inhibitor reduced the amidolytic activity of activated protein C noncompetitively by forming a 1:1 molar complex with the enzyme, determined by the use of a fluorogenic substrate toward activated protein C (Boc-Leu-Ser-Thr-Arg-4-methylcoumaryl-7-amide). The inhibition constant (Ki) of the inhibitor against activated protein C was 5.8 x 10(-8) M. The inhibitor also blocked the prolongation of activated partial thromboplastin time by activated protein C. The immunoglobulin which was produced by the inhibitor completely removed the inhibitory activity present in normal human plasma against activated protein C. This suggests that the inhibitor which we have isolated is the only inhibitor in plasma against activated protein C.     

Isolation, purification and properties of aortic elastase.

An elastase from pig aorta has been partially purified and characterised; it exhibits immunological cross reaction with pig pancreatic elastase. Its proteolytic (k-elastin gel and polymeric elastin substrates) and esterolytic (N-succinoyl-trialanine paranitroanilide) activities as well as its degree of inhibition by serum protease inhibitors (alpha1-antitrypsin and alpha2-macro-globulin) differ sensibly from those of pancreatic elastase [14,16].     

Horse alpha-1 protease inhibitors: relationship between the slow (S) and fast (F) isoforms

Horse alpha-1 protease isoinhibitors were isolated in a highly purified form using individually designed fractionation procedures. The isoinhibitors, running in agarose gel electrophoresis at pH 8.6 as two distinct bands, were designated S alpha-1 and F alpha-1. The molecular relationships between S alpha-1 and F alpha-1 were investigated by using classical electrophoretic and immunoelectrophoretic methods. No differences between the inhibitors were revealed with respect to their antiproteolytic activity, determined by fibrinogen agarose gel electrophoresis assays. No immunological differences between the isoforms were detected. These observations, together with others reported in this paper, suggest that the two isoinhibitors are probably the monomeric and dimeric form of the same molecule.