The role of inter-alpha-trypsin inhibitor and other proteinase inhibitors in the plasma clearance of neutrophil elastase and plasmin

The plasma clearance of neutrophil elastase, plasmin, and their complexes with human inter-alpha-trypsin inhibitor (I alpha I) was examined in mice, and the distribution of the proteinases among the plasma proteinase inhibitors was quantified in mixtures of purified inhibitors, in human or murine plasma, and in murine plasma following injection of purified proteins. The results demonstrate that I alpha I acts as a shuttle by transferring proteinases to other plasma proteinase inhibitors for clearance, and that I alpha I modulates the distribution of proteinase among inhibitors. The clearance of I alpha I-elastase involved transfer of proteinase to alpha 2-macroglobulin and alpha 1-proteinase inhibitor. The partition of elastase between these inhibitors was altered by I alpha I to favor formation of alpha 2-macroglobulin-elastase complexes. The clearance of I alpha I-plasmin involved transfer of plasmin to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Results of distribution studies suggest that plasmin binds to endothelium in vivo and reacts with I alpha I before transfer to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Evidence for this sequence of events includes observations that plasmin in complex with I alpha I cleared faster than free plasmin, that plasma obtained after injection of plasmin contained a complex identified as I alpha I-plasmin, and that a murine I alpha I-plasmin complex remained intact following injection into mice. Plasmin initially in complex with I alpha I more readily associated with alpha 2-plasmin inhibitor than did free plasmin.     

Hepatocyte uptake of alpha 1-proteinase inhibitor-trypsin complexes in vitro: evidence for a shared uptake mechanism for proteinase complexes of alpha 1-proteinase inhibitor and antithrombin III

In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.     

Plasma proteinase inhibitors in Morris hepatoma-bearing rats: changes in the blood level and synthesis in tissue slices

The antiproteinase activities against trypsin, chymotrypsin, elastase, papain and rat leucocyte proteinases were determined in plasma from control and Morris hepatoma-bearing rats. Bovine trypsin and chymotrypsin were similarly inhibited by the two types of plasma whereas porcine pancreatic elastase, papain and rat leucocyte neutral proteinases were more efficiently inhibited by plasma from tumour-bearing rats. The increased plasma concentrations of some proteinase inhibitors, as determined by rocket immunoelectrophoresis, are suggested to be responsible for the observed differences in inhibition. The highest increases in plasma of tumour-bearing rats were observed for alpha 2-macroglobulin and alpha 1-acute-phase globulin. The synthesis and secretion of six proteinase inhibitors: antithrombin III, alpha 1-proteinase inhibitor, alpha 1-macroglobulin, alpha 2-macroglobulin, alpha 1-acute-phase globulin and haptoglobin, as well as albumin, were measured in tissue slices from rat liver and Morris hepatoma after incubation with [14C]leucine. Local inflammation inflicted upon the tumour-bearing rats increased formation of acute-phase proteins in liver slices but not in hepatoma slices.     

Isolation of nine human plasma proteinase inhibitors by sequential affinity chromatography.

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.     

alpha 1-Proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors alpha1-proteinase inhibitor, alpha1-antichymotrypsin, and alpha2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.     

Inhibition of inflammatory proteinase, medullasin, by alpha 2-macroglobulin and ovomacroglobulin

The in vitro activity of inflammatory proteinase, medullasin, was stoichiometrically inhibited by a serum proteinase inhibitor, alpha 2-macroglobulin, and its homolog, chicken ovomacroglobulin. The two inhibitors were cleaved by medullasin only in the bait region. The effectiveness of alpha 2-macroglobulin to inhibit medullasin in competition with alpha -1-proteinase inhibitor was measured under a simulated in vivo condition and an estimation was made that about 60-70% medullasin is inhibited by alpha-1-inhibitor and 30-40% by alpha 2-macroglobulin.     

Immunolocalisation of BPTI-like serine proteinase inhibitory proteins in mast cells, chondrocytes and intervertebral disc fibrochondrocytes of ovine and bovine connective tissues. An immunohistochemical and biochemical study

A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against alpha1-proteinase inhibitor (human and sheep), inter-alpha-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, alpha2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus     

Repression of alpha 2-macroglobulin and stimulation of alpha 1-proteinase inhibitor synthesis in human mononuclear phagocytes by endotoxin

Mononuclear phagocytes are a bone-marrow-derived subgroup of white blood cells which circulate as monocytes and, after differentiation into macrophages, become resident in many tissues. By synthesizing the important proteinase inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor mononuclear phagocytes contribute to the control of proteolysis both in blood and tissues. Applying a culture system which enables human blood monocytes to differentiate into macrophages in vitro, synthesis of alpha 2-macroglobulin and alpha 1-proteinase inhibitor was studied. The normal course of monocyte-macrophage maturation is accompanied by a strong increase of specific alpha 2-macroglobulin synthesis and a concomitant slight decrease of alpha 1-proteinase inhibitor. alpha 2-Macroglobulin can be designated as a marker protein of the monocyte/macrophage differentiation. Endotoxin (Salmonella typhi) in a concentration as low as 100 ng/ml strongly represses alpha 2-macroglobulin synthesis both in monocytes and macrophages. Furthermore, endotoxin completely abolishes the induction of alpha 2-macroglobulin synthesis during the course of normal monocyte in vitro cultivation, indicating that endotoxin is a strong inhibitor of the monocyte-macrophage maturation. In contrast to alpha 2-macroglobulin, alpha 1-proteinase inhibitor synthesis is strongly stimulated by endotoxin in monocytes as well as in macrophages.     

Alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, or alpha(2)-macroglobulin is required for vascular smooth muscle cell spreading in three-dimensional fibrin gel

It is assumed that vitronectin and other adhesion molecules induce cell spreading. We found that vascular smooth muscle cells require unidentified plasma components besides adhesion molecules to spread in fibrin gel, a likely provisional matrix at wound sites. By purification, the plasma components were found to be alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, and alpha(2)-macroglobulin. The chemically inactivated alpha(1)-proteinase inhibitor and alpha(2)-macroglobulin lose the spreading activity, indicating that these proteins function as proteinase inhibitors but not as adhesion molecules. Not only anti-integrin (alpha(v)beta(3) and alpha(5)beta(1)) antibodies but also anti-fibronectin antibodies inhibit the cell spreading. The spreading occurs without the addition of fibronectin and integrins, suggesting that cells produce these molecules. In the absence of the proteinase inhibitors, Western blot analysis shows that the fibronectin is degraded in fibrin gel, while it is intact in the presence of the inhibitors. Thus, the proteinase inhibitors prevent adhesion molecules such as fibronectin from being degraded by a cell-derived proteinase(s) and thus play a role in cell spreading.     

Interactions in vitro and in vivo between anionic and cationic porcine trypsin and porcine plasma proteinase inhibitors

A method for purifying porcine anionic and cationic trypsin is presented. Reaction mixtures with increasing amounts of the two porcine trypsins and porcine serum were studied in vitro to evaluate the relative importance of alpha 1-macroglobulin and alpha 2-macroglobulin as well as alpha 1-proteinase inhibitor in the rapid binding of porcine anionic and cationic trypsin. Porcine cationic trypsin was preferentially bound to alpha 1-macroglobulin, while anionic trypsin exhibited equal binding to both alpha-macroglobulins. Both trypsins were also bound by the alpha 1-proteinase inhibitor but not until alpha 1-macroglobulin approached saturation. Trypsin-alpha-macroglobulin complexes were cleared from plasma with a half-life of 6 min. For trypsin-alpha 1-proteinase inhibitor-complexes the half-life was 120 min. These findings are in accordance with results for other mammalian species, including man.     

Interactions of the complex between human urinary trypsin inhibitor and human leukocyte elastase with alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The urinary trypsin inhibitor was recently shown to inhibit human leukocyte elastase. Complexes of human urinary trypsin inhibitor with human leukocyte elastase or human trypsin were produced and subjected to gel filtration. The complexes were found to be sufficiently stable up to 24 h incubation (at least 70% recovery). When human serum was added, elastase and trypsin dissociated from the urinary trypsin inhibitor and associated with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The addition of alpha 1-proteinase inhibitor to a complex of urinary trypsin inhibitor and leukocyte elastase caused a rapid dissociation of the complex (kdiss = 3.2 X 10(-2) s-1).     

Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.     

The major plasma kallikrein inhibitor of guinea pig plasma.

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.     

Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis.

A procedure is presented for purifying a novel proteinase inhibitor in human plasma whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clots induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, and immunoelectrophoresis. Its molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium is 67,000 and 63,000, respectively. The inhibitor is a glycoprotein consisting polypeptide chain containing 11.7% carbohyrate. It migrates in the alpha2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma. Inhibition of plasmin by the inhibitor is almost instantaneous even at 0 degrees, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. The inhibitor also inhibits trypsin. Consequently, it is suggested that this newly identified inhibitor is named alpha2-plasmin inhibitor or alpha2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor was specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitory in the serum of a healthy man is in or near the range of 5 to 7 mg/100 ml, which is the lowest concentration among the concentration of the proteinase inhibitors in plasma. The inhibitor and plasmin, trypsin, or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis and is associated with the cleavage of a unique peptide bone, which is most clearly demonstrated in the interaction of the inhibitor and beta-trypsin. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin. It is suggested that, in a fashion analogous to complex formation between alpha1-antitrypsin and trypsin, the cross-links are formed between the active site serine of the enzyme and the newly formed COOH-terminal residue of the inhibitor, with cleavage of a peptide bond.     

In vivo catabolism of alpha 1-antichymotrypsin is mediated by the Serpin receptor which binds alpha 1-proteinase inhibitor, antithrombin III and heparin cofactor II

The in vivo catabolism of 125I-labeled alpha 1-antichymotrypsin was studied in our previously described mouse model. Native alpha 1-antichymotrypsin cleared with an apparent t1/2 of 85 min, but alpha 1-antichymotrypsin in complex with chymotrypsin or cathepsin G cleared with a t1/2 of 12 min. Clearance of the complex was blocked by a large molar excess of unlabeled complexes of proteinases with either alpha 1-antichymotrypsin or alpha 1-proteinase inhibitor. These studies indicate that the clearance of alpha 1-antichymotrypsin-proteinase complexes utilizes the same pathway as complexes with the homologous inhibitor alpha 1-proteinase inhibitor. Previous studies have demonstrated that this pathway is also responsible for the catabolism of two other serine proteinase inhibitors, antithrombin III and heparin cofactor II. This pathway is thus responsible for removing several proteinases involved in coagulation and inflammation from the circulation, thereby decreasing the likelihood of adventitious proteolysis.     

Components of human split ejaculates. II. Enzymes and proteinase inhibitors.

Lysozyme, alpha-amylase, neutral proteinase and plasminogen activator were most concentrated in the initial portion of the ejaculate that consists mostly of Cowper's gland and prostate gland fluids as well as spermatozoa. The concentration of the high molecular weight proteinase inhibitors, alpha1-antitrypsin and alpha1X-antichymotrypsin, was essentially unaltered throughout the ejaculate fractions, although their absolute amounts showed an increase towards the final fraction. By contrast, the total inhibitory activity towards pancreatic trypsin was highest both in concentration and amount in the last fraction, thus indicating that the seminal vesicles are its primary source. Plasminogen, prothrombin, Factor XIII, and the proteinase inhibitors antithrombin III, alpha2-macroglobulin, inter-alpha-trypsin inhibitor and C1S-inactivator could not be detected immunochemically in whole ejaculates, and indicates the dissimilarity between the coagulation/liquefaction processes of semen and blood.     

Kinetics of association of human proteinases with human alpha 2-macroglobulin

Association rates have been determined for the interaction of human alpha 2-macroglobulin with human neutrophil elastase, cathepsin G, and human plasma kallikrein. Both of the neutrophil enzymes are rapidly inactivated by this inhibitor; however, the inactivation of plasma kallikrein is much slower. Comparison of the rates of inactivation with those already established for other inhibitors clearly indicate that alpha 1-proteinase inhibitor is the controlling inhibitor for neutrophil elastase and alpha 1-antichymotrypsin for cathepsin G, alpha 2-macroglobulin acting only as a secondary inhibitor. The control of plasma kallikrein would appear to be rather poor since neither alpha 2-macroglobulin nor C1-inhibitor appears to react very rapidly with this proteinase. Thus, a primary role for alpha 2-macroglobulin in directly inactivating proteinases in blood, under normal physiological conditions, remains to be established.     

Characterization of the inactive fragment resulting from limited proteolysis of human alpha1-proteinase inhibitor by Crotalus adamanteus proteinase II.

The inactive 50,000-dalton fragment of human plasma alpha1-proteinase inhibitor resulting from limited proteolysis of the inhibitor by Crotalus adamanteus proteinase II has been isolated and partially characterized. The amino acid composition of the inactivated inhibitor indicates the loss of a peptide fragment from the intact inhibitor. Both intact and inactivated inhibitor contain COOH-terminal lysine. However, the NH2 terminus of the intact inhibitor is Glx, whereas that of inactivated inhibitor is methionine. NH2-terminal analysis of the inactive inhibitor fragment revealed the following sequence: -Met-Phe-Leu-Glu-Ala-Ile-Pro-Met-Ser-Ile-Pro-Pro-Gln-Val-Lys-Phe-Asn. The data show that the venom proteinase has inactivated alpha1- proteinase inhibitor by cleavage of a single bond which differs from that reported for trypsin or papain.     

Primary structure of a proteinase inhibitor released from goat serum inter-alpha-trypsin inhibitor

An acid-resistant trypsin inhibitor was released from goat serum inter-alpha-trypsin inhibitor and isolated by affinity chromatography. The primary structure of the inhibitor was established and the inhibitory properties were estimated. The inhibitor designed gIK-14 was characterized as a serine proteinase inhibitor from the family of the double-headed Kunitz-type inhibitors as suggested.     

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.