Human alpha- to zeta-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained.

Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Human leukocyte cathepsin G and elastase specifically suppress thrombin-induced prostacyclin production in human endothelial cells.

Polymorphonuclear leukocytes (PMN) when activated release products that can potentially injure endothelial cells or alter endothelial function. Exposure of cultured human umbilical vein endothelial cells to cathepsin G and elastase isolated from human PMN at concentrations reached in vivo (100 ng/mL to 10 micrograms/mL) selectively inhibited thrombin-induced prostacyclin production and the thrombin-induced rise in cytosolic free calcium ([Ca++]i) concentration. These proteases also blocked thrombin-induced release of arachidonic acid from prelabeled endothelial cells (EC). In contrast, induction of prostacyclin (PGI2) production by arachidonate, histamine, or the calcium ionophore A23187 was not altered by treatment of EC with these proteases. The effects of the proteases were concentration-dependent, were blocked by serum or serum protease inhibitors, and were reversed when the endothelial cells were further cultured for 24 hours in the absence of the proteases. Elastase, but not cathepsin G, also produced detachment of endothelial cells. Thus, the major leukocyte proteases selectively suppress thrombin-induced prostacyclin production by human vascular endothelial cells and may alter the hemostatic balance at sites of PMN activation.

Human neutrophil elastase alters human alpha-thrombin function: limited proteolysis near the gamma-cleavage site results in decreased fibrinogen clotting and platelet-stimulatory activity.

During blood coagulation, polymorphonuclear leukocytes release elastase in amounts that can exceed 100 nmol/L. We therefore studied the interaction between human leukocyte elastase and human alpha-thrombin. Elastase cleaved the thrombin B chain (Ala 150-Asn 151) near the gamma-cleavage site, resulting in two fragments held together by noncovalent interactions. The NH2-terminal fragment (FI), mol wt approximately 18,000, was disulfide-linked to the thrombin A chain. The COOH-terminal fragment (FII), mol wt approximately 13,000, contained the active-site serine and formed a covalent bond with antithrombin III. Heparin accelerated proteolysis of alpha-thrombin by elastase. Proteolyzed alpha-thrombin (T theta) retained full amidolytic activity; however, the concentration of T theta causing 50% maximal platelet aggregation and adenosine triphosphate (ATP) release was 7.9 nmol/L (1.1 nmol/L for alpha-thrombin and 220 nmol/L for gamma-thrombin). Fibrinogen clotting activity of T theta and gamma-thrombin was 32% and 1% that of alpha-thrombin, respectively. Elastase released during the coagulation process may modulate thrombin activity. In addition, elastase-modified thrombin may be a useful probe of the structure and function of the gamma-cleavage region.

Immunodetection with streptavidin-acid phosphatase complex on Western blots.

A technique for the detection of nanogram amounts of protein blotted onto nitrocellulose membranes has been developed using nonradioactive probes. Protein transferred to nitrocellulose membranes is detected by a specific antibody followed by incubation with biotinylated anti-antibody. After addition of streptavidin-acid phosphatase complex, incubation with fast violet B salt produces sharp magenta bands. This method allows detection of bands containing less than 20 ng of protein. The procedure does not use radioactive or carcinogenic materials.

Human neutrophil elastase modulates platelet function by limited proteolysis of membrane glycoproteins.

During blood coagulation human polymorphonuclear leukocytes release elastase in amounts that can exceed 100 nmol/liter. We therefore studied the effect of elastase on platelet structure and function. Physiologic concentrations of elastase specifically inhibited thrombin-induced platelet aggregation and ristocetin-induced agglutination of washed platelets in a time- and dose-dependent manner. This was associated with a decrease in the number of high affinity thrombin binding sites on the platelet surface (analysis by "Ligand" program) from 31 per platelet to 12 per platelet (P less than 0.05). As analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, treatment of 3H-labeled platelets with elastase resulted in a decrease in the percent glycoprotein at 130,000-150,000 Mr = and an increase in the percent protein at Mr = 102,000. The supernatant from elastase-treated platelets contained a Mr = 88,000 glycoprotein not found in the supernatant from untreated platelets. Immunoprecipitation studies with monoclonal antiglycoprotein Ib demonstrated that treatment of whole platelets with physiologic concentrations of elastase resulted in proteolytic cleavage of glycoprotein Ib. Elastase treatment of glycoprotein immunoisolated with monoclonal antiglycoprotein Ib antibody resulted in formation of a glycopeptide with the same electrophoretic mobility as the Mr = 102,000 membrane-related glycopeptide. In contrast, analysis by Western blot technique using antiglycoprotein IIb and IIIa antibodies demonstrated that elastase did not degrade glycoproteins IIb or IIIa. We conclude that elastase inhibition of thrombin-induced platelet stimulation is accompanied by (a) a reduction in the number of thrombin binding sites per platelet and (b) proteolysis of glycoprotein Ib.

Treatment of advanced ovarian carcinoma with hexamethylmelamine, doxorubicin, and cis-platinum (HAC): results in both untreated and previously treated patients.

Twenty-two patients with advanced (FIGO stages III and IV) adenocarcinoma of the ovary were treated with 28-day cycles of hexamethylmelamine, doxorubicin, and cis-dichlorodiamine-platinum (II) (HAC). After 45 months, there were 21 evaluable patients. The median survival was 16 months. Response was achieved in 82% (9/11) who had received no prior chemotherapy, and in 50% (5/10) previously treated. HAC therapy was readily administered on an outpatient basis, with comparatively low major toxicities, primarily hematologic, neurologic, and gastrointestinal. These results indicate that HAC therapy is an effective regimen for patients with advanced ovarian carcinoma, regardless of their prior treatment status.

Alpha-1-antitrypsin-human leukocyte elastase complexes in blood: quantification by an enzyme-linked differential antibody immunosorbent assay and comparison with alpha-2-plasmin inhibitor-plasmin complexes.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the quantification of alpha 1-antitrypsin-human leukocyte elastase (alpha 1AT-E) complexes. In the ELISA, the alpha 1AT-E complex is bound to a surface by rabbit antileukocyte elastase antibody, and the inhibitor-proteinase complex is quantified by a second antibody, rabbit anti-alpha 1-antitrypsin F(ab')2, labeled with alkaline phosphatase. alpha 1AT-E complexes were detected when a final concentration of 2.2 nmol/liter of leukocyte elastase was added to plasma. The concentration of these complexes increased with additional elastase. In clotting blood, alpha 1AT-E complexes were generated in parallel with the conversion of 125I-fibrinogen to fibrin, whereas alpha 2-plasmin inhibitor-plasmin (alpha 2PI-P) complexes were not formed. The concentration of alpha 1AT-E complexes in 19 of 21 controls was less than 2.2 nmol/liter. Patients with laboratory evidence for disseminated intravascular coagulation (DIC) demonstrated elevated alpha 2PI-P complexes with either increased or normal concentrations of alpha 1AT-E complexes. Patients without evidence for DIC, but who demonstrated prolonged reptilase clotting times, were studied. This group had increased alpha 1AT-E but normal alpha 2PI-P complex levels, raising the possibility that elastase release in vivo may be accompanied by limited degradation of fibrinogen. These assays thus serve as useful probes for the study of leukocyte activation and of the interactions between cellular and plasma proteolytic enzyme systems.

Proteolytic cleavage and inactivation of alpha 2-plasmin inhibitor and C1 inactivator by human polymorphonuclear leukocyte elastase.

This study has examined the interaction between human leukocyte elastase and alpha 2-plasmin inhibitor, or C1 inactivator, inhibitors of proteases of the complement, kinin, coagulation, and fibrinolytic enzyme systems. Leukocyte elastase, in catalytic concentrations, progressively inactivates the plasmin inhibitory activity of both inhibitors. The C1s binding function of C1 inactivator is also destroyed by leukocyte elastase. The nature of the molecular events underlying the inactivation of these protease inhibitors was examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Loss of functional activity was accompanied by limited proteolytic cleavage of both inhibitors with the production of several characteristic derivative peptide chains. Leukocyte elastase cleaved alpha 2-plasmin inhibitor at two separate sites and generated lower molecular weight fragments similar to those produced by bovine beta-trypsin. C1 inactivator was hydrolyzed at three different regions on the molecule whereas beta-trypsin cleaved two regions in common with leukocyte elastase. These findings suggest that inactivation of alpha 2-plasmin inhibitor and C1 inactivator by leukocyte elastase released in the inflammatory reaction may potentiate pathological proteolysis. The limited digestion of these inhibitory proteins by leukocyte elastase may prove useful in studies of their primary structure.