Modification of bovine somatotropin (growth hormone) with plasmin

Although much work has been reported on modification of human somatotropin with plasmin and has revealed important information about structure-function relationships, plasmin modification of nonprimate somatotropins (which differ markedly in structure and biological actions from the human hormone) has been little studied. Therefore we investigated plasmin digestion of bovine somatotropin. Digestion was less rapid but more extensive than that of human somatotropin. Structural characterization of digestion products resolved by gel filtration suggested that a major product after 24 h was a disulfide-linked fragment comprising residues 1–133 plus 140–177. Further cleavage products were found in aggregated material and minor fractions. In radioimmunoassay for bovine somatotropin, activity was retained only by the unfractionated digest and aggregated material. In radioreceptor assay for somatotropin using receptors from livers of late-pregnant rabbit all preparations retained some activity. The results suggest that receptor- and antibody-binding sites in bovine somatotropin are not identical and that greater activity may result from specific association of fragments that are less active or inactive once separated.     

Follicular plasminogen and plasminogen activator and the effect of plasmin on ovarian follicle wall.

Plasminogen, plasminogen activator, protease inhibitors, and a proteolytic activity are shown to be present in bovine follicular fluid. Much of the proteolytic activity appears to be due to plasmin. In addition, plasminogen activator activity can be demonstrated in follicle wall homogenates. Evidence that plasmin decreases the tensile strength of follicle wall preparations is also reported. The potential for the involvement of these substances in ovulation is discussed.     

The effect of heparin on the proteolytic and fibrinolytic activity on plasmin(ogen) and fibrin clot lysis

The effect of heparin on the proteolytic and fibrinolytic activities of plasmin and plasminogen was studied. Heparin at a concentration of 6.3.10(-6) M did not change the caseinolytic activity of plasmin and plasminogen stimulated by streptokinase but suppressed their fibrinolytic activity. At concentrations from 2.10(-8) to 0.5.10(-6) M heparin increased, whereas at 1.10(-6)-4.10(-6) M reduced the time of desAAfibrin clot half-lysis by plasmin. Within the concentration range of 2.10(-8) to 4.10(-6) M heparin did not change the time of the clot half-lysis by glu-plasminogen and slightly decreased the time of fibrin clot half-lysis by lys-plasminogen in the presence of the tissue activator. It was supposed that heparin inhibits the fibrinolytic effect of plasmin by way of formation of complexes with plasmin and reduction of plasmin specificity to the solid phase substrate, i. e., polymeric fibrin.     

3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex.

The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.     

Inactivation of factor Va by plasmin

The inactivation of Factor Va by plasmin was studied in the presence and absence of phospholipid vesicles and calcium ions. The cleavage patterns of bovine Factor Va and its isolated subunits were analyzed using polyacrylamide gel electrophoresis, and the progress of inactivation was monitored by clotting assays and measurements of prothrombin activation using 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5-penta nediyl)amide. In addition, the ability of prothrombin and Factor Xa to protect Factor Va from inactivation by human plasmin was examined. The data presented indicate that the cofactor Factor Va is inactivated rapidly upon its interaction with human plasmin. The rate of inactivation is significantly enhanced in the presence of phospholipid vesicles, suggesting that the inactivation process is a membrane-bound phenomenon. The isolated D component (heavy chain of factor Va) was found to be slowly degraded by human plasmin, giving rise to cleavage products different from those obtained with activated protein C and Factor Xa. However, the 48- and 30-kDa fragments obtained from human plasmin degradation of component E (light chain of Factor Va) appear to be similar to those obtained following the proteolysis of the same subunit by activated protein C and Factor Xa.     

Study on the localization of proteases of mitochondrial origin

A marked proteolytic activity against casein can be demonstrated in rat liver mitochondria. The proteases degrading casein appear distributed between a sedimentable fraction (Po) and a soluble extract (So). Part of the soluble fraction activity, which may be recovered in the mitochondrial intermembrane space, results from a contamination by lysosomal proteases and can be eliminated by previously washing the mitochondria with digitonin. The pre-exposure to digitonin causes an enhancement of the caseinolytic activity associated with the membrane fragments, proving that this activity is not due to lysosomal enzymes. When rats have been injected in vivo with the compound 48/80 which, by degranulating the mast cells prevents contamination of the mitochondrial preparations by mast cell proteases, the membrane fraction (Po) retains a caseinolytic activity of the order of 80 per cent of the control preparations. A similar value of activity is observed in the membranes of brain mitochondria, isolated by a method which removes the rare mast cells they may contain. This shows that the greater part of the caseinolytic activity associated with the rat liver membranes does not originate from mast cell granules. Liver mitochondria pre-exposed to digitonin to eliminate lysosomal contaminants, have been subfractionated into matrix, intermembrane space, inner and outer membrane. Each of the fractions exhibits a caseinolytic activity, but the largest part is localized in the inner compartments of mitochondria: the matrix and the inner membrane. The optimal pH and the sensitivity to inhibitors of the proteases in the different compartments indicate that we are dealing with distinct enzymes.     

Human thrombins. Production, evaluation, and properties of alpha-thrombin.

Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.     

Inactivation of factor X by plasmin]

Plasmin does not activate factor X into the enzyme--factor Xa. On the contrary, the enzyme inactivates factor X, rendering it incapable of conversion into factor Xa during incubation in 25% sodium citrate. After proteolysis by plasmin the prothrombin preparations contaminated with factor X lose their ability to generate thrombin. This ability is partially restored by an addition of factor X.     

Murine lymphocyte cell surface proteolytic activity is strain related

A/J and C57 Br/cdj mice bear the H-2 haplotypes which are generally associated with high responses to bovine gamma globulin and type 3 pneumococcal polysaccharide. A/J has been reported, however, to produce higher levels of antibody than C57 Br/cdj mice against the antigens. The two strains of mice were used as model systems in this study to determine whether the level of lymphoid cell surface proteolytic activity is also genetically controlled. The results of this study illustrate that the level of lymphoid cell surface proteolytic activity is strain related. Since A/J lymphocytes were found to have a significantly higher rate of proteolysis than C57 Br/cdj lymphocytes, a correlation between lymphoid cell surface caseinolytic activity and immune responsiveness is suggested.     

Studies on the proteolytic activity of γ-globulin preparations

1. The proteolytic activities of several gamma-globulin preparations were tested. These included sulphate-precipitated human and bovine preparations and human and bovine Cohn fraction II preparations as well as purified gamma-globulin preparations. Up to 14mg. of diffusible peptides and glycopeptides/g. of gamma-globulin was liberated after dialysis and up to 10mg. of peptides/g. after incubation and trichloroacetic acid precipitation, as products of the degradation process in incubated gamma-globulin. 2. in-Aminohexanoic acid and p-chloromercuribenzoic acid, as well as heating at 60 degrees for 40min., were shown to inhibit strongly these proteolytic activities. Streptokinase was shown to activate strongly the proteolytic activity of all the human preparations (sulphate-precipitated, Cohn fraction II, and purified gamma-globulin). 3. Two distinct pH optima were shown for human and bovine gamma-globulin preparations: one at pH8, the other at pH3.8 (the latter activity could be demonstrated only in the presence of cysteine). 4. Both (131)I-labelled human Cohn fraction II and bovine fibrinogen were attacked by a sulphate-precipitated preparation of gamma-globulin. Of the synthetic substrates tested toluene-p-sulphonyl-l-arginine methyl ester was hydrolysed by both the sulphate-precipitated and Cohn fraction II preparations, as was benzoyl-l-arginine amide at pH5, but only in the presence of cysteine. 5. These data are interpreted to indicate that at least two enzymes are present in gamma-globulin preparations, one being similar to the plasmin system, the other similar to cathepsin B.     

Activation of plasminogen by the early bovine embryo

Activation of the plasma zymogen plasminogen to the enzyme plasmin by the early bovine embryo was evaluated. Sixteen-cell embryos to early morulae were collected at death from handmated synchronized and superovulated crossbred beef cows. Embryos were cultured in Ham's F-12 medium supplemented with 15 mg/ml bovine serum albumin containing 0, 15, 30, 60 or 120 micrograms/ml plasminogen in a humidified atmosphere of 5% CO2 in air at 37 degrees C. Cultures were observed every day, and stage of development was recorded. Medium was collected at 24-h intervals, starting at initiation and continuing through 288 h of culture. Plasminogen activator and plasmin levels in the culture media were determined, using a caseinolytic assay. The percentages of embryos developing to the initiating hatching blastocyst, hatched blastocyst, attached blastocyst, and attached blastocyst with trophoblastic outgrowth stages were not significantly different between the five levels of plasminogen. Initiation and completion of hatching, however, accelerated as plasminogen concentration increased in the culture media. Plasminogen activator production, expressed as milliunits X ml-1 X h-1 X viable embryo-1, was low for the first 48 h of culture, increased between 48-120 h, and tended to plateau thereafter. Plasminogen activation, measured indirectly as the plasmin concentration in a microdrop of medium and expressed as microgram plasmin X ml-1 X h-1 X viable embryo-1, followed plasminogen activator production, and was consistently low for the first 48-72 h of culture. Embryonic activation of plasminogen increased sharply thereafter, and also plateaued after 120 h.     

Identification of the human plasma protein which inhibits fibrinolysis associated with malignant cells

Mixed cultures of mouse fibroblasts and mouse fibroblasts transformed with Kirsten murine sarcoma virus were grown in petri dishes and overlayed with casein. The appearance of focal lysis zones required the presence of transformed cells in the culture and plasminogen in the overlay, indicating that caseinolysis was due to plasminogen activator released by the malignant cells. Caseinolysis was inhibited by addition of human plasma or bovine pancreatic trypsin inhibitor to the overlay, 1 ml of plasma being equivalent to 67 ± 18 (mean ± S.E.) kallikrein inhibitor (KI) units of trypsin inhibitor.The culture fluid of a human melanoma line induced lysis of a fibrin clot, 1 ml of culture fluid being equivalent to 250 CTA units of urokinase (EC 3.4.99.26). Fibrinolysis was inhibited by addition of human plasma or trypsin inhibitor, 1 ml of plasma being equivalent to 94 ± 34 KI units of trypsin inhibitor.Specific removal of antiplasmin, the fast-reacting plasmin inhibitor (Collen, D. (1976) Eur. J. Biochem. 69, 209), from plasma by immunoabsorption completely abolished its inhibitory activity, both in the caseinolytic and fibrinolytic assays. It is therefore concluded that antiplasmin is the only protein in human plasma capable of inhibiting the fibrinolytic activity associated with oncogenic transformation or neoplasia. Whether this effect is exclusively due to inhibition of formed plasmin or also to interference with plasminogen activvtion remains unsettled.     

Design of a novel plasminogen activator based on the structure of hirudin

Using a phage library, seven peptide sequences with high affinity to human microplasminogen were obtained. Caseinolytic assay indicated that only the synthesized peptide P07 had slight fibrinolytic activity. To enhance its plasminogen activation ability, peptide P07 was fused into loop 32-35 of hirudin. In vitro assay demonstrated that this hirudin-like fusion protein can activate human plasminogen and retain the function of thrombin inhibition. Fusing the sequence ＂SPDASRL＂ into hirudin generated a plasminogen activation activity 100 times higher than peptide P07 in chromogenic and radial caseinolytic assay. This significant functional improvement might originate from a more specific active structure due to the hirudin scaffold.     

Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity

Cultured bovine capillary endothelial (BCE) cells synthesize heparan sulfate proteoglycans (HSPG), which are both secreted into the culture medium and deposited in the cell layer. The nonsoluble HSPGs can be isolated as two predominant species: a larger 800-kD HSPG, which is recovered from preparations of extracellular matrix, and a 250-kD HSPG, which is solubilized by nonionic detergent extraction of the cells. Both HSPG species bind bFGF. 125I-bFGF bound to BCE cell cultures is readily released by either heparinase or plasmin. When released by plasmin, the growth factor is recovered from the incubation medium as a complex with the partly degraded high molecular mass HSPG. Endogenous bFGF activity is released by a proteolytic treatment of cultured BCE cells. The bFGF-binding HSPGs are also released when cultures are incubated with the inactive proenzyme plasminogen. Under such experimental conditions, the release of the extracellular proteoglycans can be enhanced by treating the cells either with bFGF, which increases the plasminogen activating activity expressed by the cells, or decreased by treating the cells with transforming growth factor beta, which decreases the plasminogen activating activity of the cells. Specific immune antibodies raised against bovine urokinase also block the release of HSPG from BCE cell cultures. We propose that this plasminogen activator-mediated proteolysis provides a mechanism for the release of biologically active bFGF-HSPG complexes from the extracellular matrix and that bFGF release can be regulated by the balance between factors affecting the pericellular proteolytic activity.     

The role of the membrane in the inactivation of factor va by plasmin. Amino acid region 307-348 of factor V plays a critical role in factor Va cofactor function

The mechanism of inactivation of bovine factor Va by plasmin was studied in the presence and absence of phospholipid vesicles (PCPS vesicles). Following 60-min incubation with plasmin (4 nm) membrane-bound factor Va (400 nm) is completely inactive, whereas in the absence of phospholipid vesicles following a 1-h incubation period, the cofactor retains 90% of its initial cofactor activity. Amino acid sequencing of the fragments deriving from cleavage of factor Va by plasmin demonstrated that while both chains of factor Va are cleaved by plasmin, only cleavage of the heavy chain correlates with inactivation of the cofactor. In the presence of a membrane surface the heavy chain of the bovine cofactor is first cleaved at Arg(348) to generate a fragment of M(r) 47,000 containing the NH(2)-terminal part of the cofactor (amino acid residues 1-348) and a M(r) 42,000 fragment (amino acid residues 349-713). This cleavage is associated with minimal loss in cofactor activity. Complete loss of activity of the membrane-bound cofactor coincides with three cleavages at the COOH-terminal portion of the M(r) 47,000 fragment: Lys(309), Lys(310), and Arg(313). These cleavages result in the release of the COOH terminus of the molecule and the production of a M(r) 40,000 fragment containing the NH(2)-terminal portion of the factor Va molecule. Factor Va was treated with plasmin in the absence of phospholipid vesicles followed by the addition of PCPS vesicles and activated protein C (APC). A rapid inactivation of the cofactor was observed as a result of cleavage of the M(r) 47,000 fragment at Arg(306) by APC and appearance of a M(r) 39,000 fragment. These data suggest a critical role of the amino acid sequence 307-348 of factor Va. A 42-amino acid peptide encompassing the region 307-348 of human factor Va (N42R) was found to be a good inhibitor of factor Va clotting activity with an IC(50) of approximately 1.3 microm. These data suggest that plasmin is a potent inactivator of factor Va and that region 307-348 of the cofactor plays a critical role in cofactor function and may be responsible for the interaction of the cofactor with factor Xa and/or prothrombin.     

The role of a contaminant in thrombin in the human plasmin assay system

Many of the anomalous results obtained in the fibrinolytic assay of human plasmin systems were shown to be simply explained if bovine plasminogen had been introduced into the assay system on the addition of thrombin. Experimental investigation of the proteolytic and fibrinolytic activity of systems containing plasmin and thrombin showed that enzyme activity was influenced by the presence and quantity of thrombin. The quantity of bovine plasminogen present as a contaminant in bovine fibrinogen was shown to be responsible for only 1/25th of the observed enhanced activity. Thrombin in the amounts commonly used for clotting contained sufficient proenzyme to account for all this activity. A highly purified thrombin preparation obtained from another laboratory, and thrombin purified in this laboratory by starch electrophoresis brought about no enhancement of activity. The material separated from thrombin by starch electrophoresis was shown to be enzymatically identical with bovine plasminogen and, on labelling with radioactive iodine, was shown to behave physically like bovine plasminogen. Several experiments reported in the literature were reinterpreted in the light of this observation.     

Ability of bacteria living in the sea for the biosynthesis of fibrinolytic enzymes

Biosynthesis of fibrinolytic enzymes was studied in bacteria isolated from sea water (the Black Sea, the Balearic Sea, the Caribbean Sea) and from different habitats (water, detritus, fish intestines). Strains with the highest fibrinolytic activity belonged to the Bacillus genus and were isolated from mineral detritus and ruff intestines in the Black Sea. A procedure was developed for the isolation of enzyme preparations with a high specific fibrinolytic activity and a low caseinolytic activity.     

New fluorogenic peptide substrates for plasmin

Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The Km values estimated from Line-weaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10(-4) M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, alpha-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond. These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys- of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.     

Expression, isolation, and characterization of an active site (serine 528----alanine) mutant of recombinant bovine prothrombin

An active site mutant bovine prothrombin cDNA (Ser528----Ala) has been constructed, subcloned, and expressed in Chinese hamster ovary cells. The recombinant mutant prothrombin, expressed at the level of 1.5-2.0 micrograms/ml of cell medium, was fully carboxylated (9.9 +/- 0.4 mol of gamma-carboxyglutamic acid/mol of prothrombin). The mutant prothrombin could be activated to thrombin by Taipan snake venom and activated to meizothrombin by ecarin in a manner comparable to native bovine prothrombin or recombinant wild-type bovine prothrombin. The mutant meizothrombin thus formed was stable and did not autolyze. The initial rate of cleavage of mutant prothrombin catalyzed by the full prothrombinase was only 28% of the rate of cleavage of native prothrombin, while recombinant wild-type prothrombin was cleaved at the same rate as the native molecule. The mutant thrombin, obtained from the mutant prothrombin in situ by prothrombinase or Taipan snake venom activation, showed no enzymatic activity toward either fibrinogen or a synthetic chromogenic substrate, D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride (S2238). The mutant thrombin also bound dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, a specific fluorescent inhibitor of the thrombin active site, with a weaker binding affinity (kd = 5.4 x 10(-8) M) than did native thrombin (kd = 1.7 x 10(-8) M). These results indicate that the mutant recombinant prothrombin described here is a useful tool for the study of meizothrombin or thrombin without the complications arising from the proteolytic activities of these molecules. Study of the activation of this mutant has already revealed a functional link between the site of initial cleavage by the prothrombinase and the conformation at the nascent active site of prothrombin.     

Comparison of Bovine Milk Protease with Plasmin

Comparative studies of bovine milk protease and bovine plasmin were performed. It was found that milk protease was very similar to plasmin in various properties such as optimum pH, pH-stability, heat-stability, inhibition by various inhibitors and molecular weight. The changes of casein by both enzymes as observed by polyacrylamide gel electrophoresis were also quite similar. From these results, it is suggested that milk protease may be plasmin itself transported from bovine plasma.