Support vector machine (SVM) based multiclass prediction with basic statistical analysis of plasminogen activators.

BACKGROUND: Plasminogen (Pg), the precursor of the proteolytic and fibrinolytic enzyme of blood, is converted to the active enzyme plasmin (Pm) by different plasminogen activators (tissue plasminogen activators and urokinase), including the bacterial activators streptokinase and staphylokinase, which activate Pg to Pm and thus are used clinically for thrombolysis. The identification of Pg-activators is therefore an important step in understanding their functional mechanism and derives new therapies. METHODS: In this study, different computational methods for predicting plasminogen activator peptide sequences with high accuracy were investigated, including support vector machines (SVM) based on amino acid (AC), dipeptide composition (DC), PSSM profile and Hybrid methods used to predict different Pg-activators from both prokaryotic and eukaryotic origins. RESULTS: Overall maximum accuracy, evaluated using the five-fold cross validation technique, was 88.37%, 84.32%, 87.61%, 85.63% in 0.87, 0.83,0.86 and 0.85 MCC with amino (AC) or dipeptide composition (DC), PSSM profile and Hybrid methods respectively. Through this study, we have found that the different subfamilies of Pg-activators are quite closely correlated in terms of amino, dipeptide, PSSM and Hybrid compositions. Therefore, our prediction results show that plasminogen activators are predictable with a high accuracy from their primary sequence. Prediction performance was also cross-checked by confusion matrix and ROC (Receiver operating characteristics) analysis. A web server to facilitate the prediction of Pg-activators from primary sequence data was implemented. CONCLUSION: The results show that dipeptide, PSSM profile, and Hybrid based methods perform better than single amino acid composition (AC). Furthermore, we also have developed a web server, which predicts the Pg-activators and their classification (available online at http://mamsap.it.deakin.edu.au/plas_pred/home.html). Our experimental results show that our approaches are faster and achieve generally a good prediction performance.

Expression of plasminogen activator-related genes in the adipose tissue of lactating dairy sheep in the early post-weaning period.

There is growing evidence that plasminogen activator inhibitor type 1 (PAI-1) is expressed in adipose tissue and its expression is implicated in inflammation that accompanies obesity-associated diseases. The physiological role of other genes implicated in the plasminogen-activating cascade such as urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and plasminogen activator inhibitor type 2 (PAI-2) in ovine adipose tissue remains unknown. The objective of this study was to examine the changes in the expression of four plasminogen activator (PA)-related genes during the early post-weaning period in dairy ewes. A total of 21 subcutaneous adipose tissue samples were obtained from seven lactating dairy ewes of the Chios breed at weeks 1, 2 and 4 after weaning. Results indicated that expression of all PA-related genes was detected in most of the samples examined. Greatest expression of u-PAR corresponded to highest (week 1), while greatest expression of PAI-2 corresponded to lowest (week 4) rate of lipolysis, as indicated by the expression of hormone-sensitive lipase, in the ovine adipose tissue. There were no significant differences in the expression of the other two PA-related genes (u-PA, PAI-1) throughout the experimental period. Plasminogen activator-related genes are not expressed in a coordinated manner in the adipose tissue of lactating dairy sheep in the early post-weaning period. In conclusion, adipose tissue mobilization is correlated with highest expression of u-PAR and lowest expression of PAI-2.

Fibrin specificity of plasminogen activators, rebound generation of thrombin, and their therapeutic implications.

Optimal induction of coronary thrombolysis depends in part upon the nature of the specific plasminogen activator used. The two general classes of plasminogen activators available clinically differ in a fundamental respect delineated by the term, clot selectivity. Clot selective agents are less prone to induce plasminemia and consequent occult activation of the coagulation cascade than are non-selective agents. However, under clinical conditions, all plasminogen activators result in some activation of the cascade with consequent generation of thrombin. Accordingly, optimal therapy requires the use of conjunctive anticoagulation to preclude the deleterious effects of rebound generation of thrombin, which has been well documented biochemically. The potential value of antiplatelet agents that can attenuate the positive feedback loop between activation of platelets and markedly amplified generation of thrombin in the setting of coronary thrombolysis is under active exploration. With appropriate monitoring of the efficacy of such agents in vivo it should be possible to enhance even further the benefits that can be conferred by pharmacologically induced coronary thrombolysis.

The influence of hyperestrogenism during in vitro fertilization on the fibrinolytic mechanism.

OBJECTIVE: To evaluate levels of various fibrinolytic factor antigens in women during ovulation induction using controlled ovarian hyperstimulation. METHODS: Plasma tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and the plasminogen activator inhibitors (PAI-1 and PAI-2) were evaluated and compared with plasma 17 beta-estradiol levels, ranging from 20 pg/mL to > 5,000 pg/mL during the course of treatment. Sixteen patients undergoing IVF were compared prior to (Controls) and following treatment with leuprolide acetate down-regulation followed by menopausal gonadotropin-CG ovulation induction for 14 days. RESULTS: A significant positive correlation was found between tPA and PAI-1 during treatment, while tPA and PAI-1 were negatively correlated with estradiol levels. Mean levels of tPA and PAI-1 significantly decreased as estradiol levels increased. CONCLUSIONS: As the plasminogen activator decreased with increasing estradiol levels, this suggests a potential for thrombosis. However, the major plasminogen activator inhibitor (PAI-1) also decreased; thus, the net clinical effect in terms of increased potential for thrombosis should be minimal. Furthermore, the levels of both tPA and uPA were still within normal ranges. The overall data from this study suggest that ovarian hyperstimulation with fertility-enhancing drugs does not enhance the potential for thrombosis even though there are elevated 17 beta-estradiol levels.

Production of various forms of plasminogen activator and plasminogen activator inhibitor by cultured mammary epithelial cells.

We examined amounts and types of plasminogen activator and plasminogen activator inhibitor produced by cultured bovine mammary epithelial cells. The MAC-T and two other mammary epithelial cell lines, MACT-UV1 and MACT-UV2 derived from the parental MAC-T cells by subcloning, were used as model systems. Cells were cultured in a medium free of serum and protein. Data showed that MACT-UV2 cells produced 6.2 and 17.2% more plasminogen activator than MACT-UV1 and parental MAC-T cells, respectively. Addition of amiloride, a specific urokinase-plasminogen activator inhibitor, dramatically decreased the activity in the culture medium of parental and subclonal lines, indicating that urokinase-plasminogen activator was present. Zymography revealed the presence of urokinase-plasminogen activator with an approximate molecular mass of 50,000 kDa in the culture medium of parental MAC-T cells. The culture medium of the subclonal lines contained urokinase-plasminogen activator and tissue-plasminogen activator with approximate molecular masses of 50,000 and 75,000 kDa, respectively. Complexes of both types of plasminogen activators with plasminogen activator-inhibitor-1 were detected in the culture medium of subclonal lines.

Regulation of plasminogen activation in isolated perfused rat kidney.

To better understand the mechanism and regulation of plasminogen activation within the kidney, the release and excretion of plasminogen activator activities was studied in the isolated perfused rat kidney in the absence and presence of plasminogen substrate. In the absence of plasminogen, the kidneys released a constant amount of plasminogen activator activity into both the urine and the perfusate. On continuous infusion of purified human plasminogen into the perfusate, the release of plasminogen activator activity into the urine slightly increased, and plasmin generated could be detected in both urine and perfusate. With the use of specific antibodies against the tissue-type (t-PA) and the urokinase-type plasminogen activator (u-PA), respectively, the activity in the perfusate could be identified as t-PA, whereas the activity in the urine could be ascribed to u-PA. A bolus injection of either antibody into the plasminogen-supplemented perfusion medium completely inhibited plasminogen activator activity and generation of plasmin in the vascular or tubular compartment. Furthermore, intrarenal inhibition of t-PA activity by the specific antibody significantly increased the concentration of plasminogen in the perfusate, indicating decreased consumption. This effect was accompanied by increased excretion of u-PA into the urine, suggesting that the availability of intact plasminogen in the renal circulation directly or indirectly might participate in the regulation of u-PA excretion into the urine.

Increased release of plasminogen activator inhibitor type 2 accompanies the human mononuclear cell tissue factor response to lipopolysaccharide.

Human peripheral blood mononuclear cells (PBM) respond to lipopolysaccharide (LPS) with increased release of a plasminogen activator (PA) inhibitor. This response is dose dependent and parallels the LPS-induced expression of PBM tissue factor activity. The PA inhibitors of control and LPS-stimulated PBMs appear identical as both are identified by antibodies to PA inhibitor type 2 of human placenta, but not by antibodies to type 1 inhibitor of bovine aortic endothelial cells. The PA inhibitor is specific for urokinase type PA as determined by the 125I-fibrin plate assay, and direct cleavage of 125I-plasminogen; it does not effectively inhibit tissue-type PA. The inhibitor forms an active site-dependent complex with 125I-urokinase, which then demonstrates an increase in mol wt from 33 kd to 68 kd on reduced sodium dodecyl sulfate (SDS) polyacrylamide gels. PBMs neither secrete nor express active PA. Hence, the exposure of PBMs to LPS results in conditions highly favorable to fibrin deposition and persistence: increased procoagulant and antifibrinolytic activities, accompanied by no measurable PA. Such modulation of these effectors may be important in the pathogenesis of fibrin characteristically found in tissue lesions of endotoxin-initiated processes.

Molecular mechanism of action of newer thrombolytic agents.

Recombinant tissue-type plasminogen activator (rt-PA) and single chain urokinase-type plasminogen activator (scu-PA) are thrombolytic agents, characterized by a high but not absolute degree of fibrin specificity that is mediated through different molecular mechanisms. Both activators are still under clinical investigation but it has become apparent that their therapeutic dose in humans is high and associated with a variable degree of systemic activation of the fibrinolytic system and fibrinogen breakdown. Therefore, the quest for further improvement of agents and therapeutic schemes continues. Research is being pursued in this area along the following lines: 1) tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator in molar ratios of 4:1 to 1:4 do not act synergistically on thrombolysis in a plasma environment in vitro, but display significant synergism in animal models of thrombosis. In pilot studies in patients with coronary artery occlusion, rt-PA and scu-PA are markedly synergistic and efficient thrombolysis can be obtained with a fivefold lower combined dose than that of the separate agents. The combined dose does not seem to induce systemic fibrinogen breakdown. 2) Deletion mutants of rt-PA can be constructed with a significantly prolonged half-life in vivo, and a better thrombolytic potential after bolus intravenous injection. 3) Cleavage site-specific mutants of scu-PA that abolish the conversion to urokinase may have a higher fibrin specificity. The mutants constructed thus far, however, seem to have a lower specific thrombolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinolysis in cornea and conjunctiva: evidence of two types of activators.

The nature of corneal and conjunctival plasminogen activators (PAs) from human and rabbit eyes was examined in tissue culture. The fibrinolytic activity of culture fluid from human corneas was low, roughly one-eighth of that of rabbits. The main activity was found to be of the urokinase (UK) type, as demonstrated by crossed immunoelectrophoresis against alpha 2-antiplasmin after incubation with mixed plasma. The fibrinolytic activity of culture fluid from human conjunctival tissue was higher and a mixed secretion of tissue type (tPA) and UK activator was demonstrated. These activators were separated by affinity chromatography against Sepharose-immobilized antibodies against tPA. Fibrinolytic activity in tears was quenched by antibodies against tPA but not by the use of anti-UK antibodies. Thus, in man, tear fibrinolytic activity may depend primarily on a release of PAs from conjunctival tissue.

Cultured granulosa cells produce two plasminogen activators and an antiactivator, each regulated differently by gonadotropins.

Although treatment of cultured granulosa cells with gonadotropins increases their fibrinolytic activity, the biochemical nature of this effect is unclear. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fibrin autography techniques to characterize the fibrinolytic components secreted by granulosa cells. The fibrinolytic activity of these cells results from the production of both a tissue-type plasminogen activator (t-PA) and a urokinase-like activator (u-PA). The cells also produce an inhibitor of fibrinolysis (antiactivator). FSH and LH stimulate t-PA activity and suppress antiactivator activity, while u-PA activity is not affected by the gonadotropins. The differential regulation of these molecules by the gonadotropins may be essential for ovulation.

Novel properties of human monocyte plasminogen activator.

Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing supernatants of human lymphocytes, could be shown to produce and secrete appreciable activities of a 52 000-Mr plasminogen activator. This enzyme was suppressed in control and stimulated cultures by dexamethasone (0.1 microM). Monocyte plasminogen activator could only be assayed under conditions of low ionic strength and had no detectable activity at 0.15 M NaCl. Intracellular enzyme was present as a proenzyme, requiring activation by preincubation with plasminogen containing traces of plasmin, before its activity could be seen on sodium dodecyl sulphate/polyacrylamide gel electrophoresis by a fibrin overlay method. Secreted enzyme was in the active form. Further incubation of lysate or supernatant plasminogen activator with plasminogen did not produce any active enzyme species of Mr 36 000, unlike incubations of urokinase with plasminogen. Moreover, comparisons with other plasminogen activators of Mr 52 000 from transformed cell lines showed that the monocyte activator was unique in its resistance to monocyte minactivin, a specific inactivator of urokinase-type plasminogen activators, and in its sensitivity to human alpha 2-macroglobulin. It was therefore concluded that human monocyte plasminogen activator, although sharing an Mr of 52 000 in common with other such activators, is not identical to the high Mr form of urokinase or the plasminogen activators of transformed cells. On present evidence it is the least likely of these enzymes to be active extracellularly under normal physiological conditions.