Plasminogen Activity Research Articles

Critical Roles of Rho Kinase and Mek/Erk Pathways for Angiotensin Ii-Induced Plasminogen Activator Inhibitor-1 Gene Expression

P157 Plasminogen activator inhibitor-1 (PAI-1) plays an integral role not only in the regulation of plasminogen activity and fibrinolytic system but also in the pathogenesis of atherosclerosis and hypertension. Because angiotensin II (Ang II) is also involved in these processes, we investigated its role in the intracellular signaling cascade leading to PAI-1 gene expression in vascular smooth muscle cells (VSMC). Ang II increased the PAI-1 mRNA and protein levels through Ang II type 1 receptor. Although PAI-1 mRNA stability was not increased by Ang II, PAI-1 gene promoter activity, which was measured by luciferase assay, was significantly increased by Ang II. This process did not require de novo protein synthesis. BAPTA-AM, genistein and AG1478 completely inhibited the Ang II-induced PAI-1 mRNA upregulation, suggesting that intracellular calcium, tyrosine kinase and epidermal growth factor (EGF) receptor transactivation were involved in this process. However, inhibition of protein kinase C (PKC) by calphostin C, GF109203, or prolonged exposure to PMA failed to abolish the Ang II-induced PAI-1 upregulation, suggesting PKC pathway was not involved. PD98059 suppressed Ang II-induced PAI-1 upregulation, whereas SB203580 did not, suggesting that MEK/ERK1/2 pathway rather than p38 MAP kinase pathway was crucial in this process. Furthermore, adenovirus-mediated expression of dominant negative form of Rho kinase or Rho kinase inhibitor Y27632 also completely suppressed PAI-1 induction by Ang II without affecting Ang II-induced ERK1/2 activation. These data suggest that activation of both MEK/ERK1/2 and Rho kinase pathways will be necessary for the upregulation of PAI-1 gene expression and these two pathways may act synergically to promote PAI-1 gene transcription at least at the downstream of ERK1/2 in VSMC. These findings are important biological and therapeutical implications for the evolution of arterial wall thrombus and the pathogenesis of atherosclerosis by Ang II.

Critical Roles of Rho Kinase and Mek/Erk Pathways for Angiotensin Ii-Induced Plasminogen Activator Inhibitor-1 Gene Expression

P157 Plasminogen activator inhibitor-1 (PAI-1) plays an integral role not only in the regulation of plasminogen activity and fibrinolytic system but also in the pathogenesis of atherosclerosis and hypertension. Because angiotensin II (Ang II) is also involved in these processes, we investigated its role in the intracellular signaling cascade leading to PAI-1 gene expression in vascular smooth muscle cells (VSMC). Ang II increased the PAI-1 mRNA and protein levels through Ang II type 1 receptor. Although PAI-1 mRNA stability was not increased by Ang II, PAI-1 gene promoter activity, which was measured by luciferase assay, was significantly increased by Ang II. This process did not require de novo protein synthesis. BAPTA-AM, genistein and AG1478 completely inhibited the Ang II-induced PAI-1 mRNA upregulation, suggesting that intracellular calcium, tyrosine kinase and epidermal growth factor (EGF) receptor transactivation were involved in this process. However, inhibition of protein kinase C (PKC) by calphostin C, GF109203, or prolonged exposure to PMA failed to abolish the Ang II-induced PAI-1 upregulation, suggesting PKC pathway was not involved. PD98059 suppressed Ang II-induced PAI-1 upregulation, whereas SB203580 did not, suggesting that MEK/ERK1/2 pathway rather than p38 MAP kinase pathway was crucial in this process. Furthermore, adenovirus-mediated expression of dominant negative form of Rho kinase or Rho kinase inhibitor Y27632 also completely suppressed PAI-1 induction by Ang II without affecting Ang II-induced ERK1/2 activation. These data suggest that activation of both MEK/ERK1/2 and Rho kinase pathways will be necessary for the upregulation of PAI-1 gene expression and these two pathways may act synergically to promote PAI-1 gene transcription at least at the downstream of ERK1/2 in VSMC. These findings are important biological and therapeutical implications for the evolution of arterial wall thrombus and the pathogenesis of atherosclerosis by Ang II.

Post-exercise Alcohol Ingestion Perturbs Blood Haemostasis During Recovery

It is known that exercise induces modification in blood haemostasis. It is, however, not known whether alcohol consumption post-exercise influences these modifications during recovery. Eleven moderately active young men were studied immediately after a standardised cycle ergometer test and during the 24-hour period of recovery. Alcohol (0.7 g/kg body mass) was given 1 hour after exercise on one test occasion, while an equal volume of alcohol-free solution was administered on the other. Exercise induced a significant increase in factor VIII activity with a significant shortening of activated partial thromboplastin time. Parallel increases in tissue plasminogen activity and antigen with a concomitant decrease in tissue plasminogen activator inhibitor-1 activity were also observed after exercise. During recovery, while the increase in factor VIII activity post-exercise persisted in both trials, fibrinolytic activity demonstrated a sharp fall. The elevated factor VIII activity was significantly higher at 5 and 22 hours during the alcohol trial compared with the control. Although no demonstrable effect of alcohol on tissue plasminogen activator activity was present from 1 hour after ingestion onward, tissue plasminogen activator antigen and tissue plasminogen activator inhibitor-1 antigen increased significantly 22 hours following alcohol ingestion. Further comparison between trials revealed a higher plasminogen activator inhibitor-1 activity 5 hours after alcohol ingestion. In conclusion, exercise-induced changes to blood haemostasis are balanced during exercise but not during recovery. Alcohol consumption after physical exercise further perturbs blood haemostasis and could constitute a thrombotic risk.

Efeitos do contraceptivo oral contendo 20 µg de etinilestradiol e 150 µg de desogestrel sobre os sistemas de coagulação e fibrinólise

O uso de contraceptivos orais está associado a um risco aumentado de doenças tromboembólicas, o que pode ser explicado pelos seus efeitos sobre o sistema hemostático. Tem sido descrito que o uso de contraceptivos orais promovem alterações pró-coagulantes, e que essas alterações são acompanhadas dos aumentos da atividade fibrinolítica e dos inibidores naturais da coagulação, o que causaria um restabelecimento do equilíbrio hemostático. O objetivo deste estudo foi avaliar os efeitos do contraceptivo oral contendo 20 µg de etinilestradiol e 150 µg de desogestrel sobre os sistemas de coagulação e fibrinólise. Participaram do estudo 11 voluntárias que foram avaliadas antes e após seis meses de uso do contraceptivo oral. Os parâmetros analisados foram: atividades dos fatores VII, VIII, IX, X e XII (plasmas deficientes em fatores com detecção foto-óptica do coágulo), atividades da antitrombina, plasminogênio e a2-antiplasmina (ensaios cromogênicos), quantificação dos antígenos t-PA, produtos de degradação da fibrina e proteínas C e S (ELISA), TP, TTPA e concentração plasmática de fibrinogênio (detecção foto-óptica do coágulo). Observamos as seguintes alterações estatisticamente significantes (nível de significância de p<0,05): aumento das atividades dos fatores VIII, IX, X e XII, redução do TTPA, aumento da atividade do plasminogênio, aumento de proteína C e diminuição de proteína S. Com esses resultados sugerimos que o uso do contraceptivo oral testado promove um estado pró-coagulante. Entretanto, os mecanismos de restabelecimento do equilíbrio hemostático não podem ser garantidos, já que não observamos alterações suficientes que indiquem isso. Não observamos aumento nos níveis dos produtos de degradação da fibrina, o que indica que não ocorreram alterações na gênese e degradação de fibrina. Portanto, sugerimos que o uso do contraceptivo oral pode aumentar o risco de doenças tromboembólicas, principalmente em associação com outros fatores de riscos genéticos e/ou adquiridos.

Aging, physical conditioning, and exercise-induced changes in hemostatic factors and reaction products.

The influence of age on training-induced changes in resting and stimulated hemostatic potential was studied in three age categories (Cat I-III; 20-30 yr, 35-45 yr, and 50-60 yr, respectively) of sedentary men before and after 12 wk of training. Coagulation, fibrinolytic activity, and activation markers (reflecting fibrin formation and degradation) were determined. Physical conditioning resulted in a more pronounced increase in von Willebrand factor (vWF) and factor VIII clotting activity (FVIII:c) in Cat I and II and a more pronounced shortening of the activated partial thromboplastin time in all categories at maximal exertion and during recovery. Enhanced increases in tissue-type plasminogen activator (t-PA) antigen and activity and single-chain (sc) urokinase-type plasminogen activator (u-PA) at maximal exercise and 5 min of recovery were observed in all age groups after training. The effects on FVIII:c, vWF, and scu-PA were most pronounced in the youngest age group (Cat I). Increases in the marker of thrombin generation were highest in Cat III; no effect was seen on thrombin-antithrombin complex, plasmin-antiplasmin complex, and D-dimer in any of the age groups. We concluded that training enhances both coagulation and fibrinolytic potential during strenuous exercise. The effect on FVIII/vWF and t-PA/u-PA is most pronounced in younger individuals, whereas thrombin formation is most pronounced in older individuals.

Kinetic and structural characterization of a two-domain streptokinase: dissection of domain functionality.

The mammalian protease plasminogen can be activated by bacterial activators, the three-domain (alpha, beta, gamma) streptokinases and the one-domain (alpha) staphylokinases. These activators act as plasmin(ogen) cofactors, and the resulting complexes initiate proteolytic activity of host plasminogen which facilitates bacterial colonization of the host organism. We have investigated the kinetic mechanism of the plasminogen activation mediated by a novel two-domain (alpha, beta) streptokinase isolated from Streptococcus uberis (Sk(U)) with specificity toward bovine plasminogen. The interaction between Sk(U) and plasminogen occurred in two steps: (1) rapid association of the proteins and (2) slow transition to the active complex Sk(U)-PgA. The complex Sk(U)-PgA converted plasminogen to plasmin with the following parameters: K(m) < or = 1.5 microM and k(cat) = 0.55 s(-)(1). The ability of proteolytic fragments of Sk(U) to activate plasminogen was investigated. Only two C-terminal segments (97-261 and 123-261), which both contain the beta-domain (126-261), were shown to be active. They initiated plasminogen activation in complex with plasmin, but not with plasminogen, and thereby exhibited functional similarity to the staphylokinase. The fusion protein His(6)-Sk(U) (i.e., Sk(U) with a small N-terminal tag) acted exclusively in complex with plasmin as well. These observations demonstrate that (1) the N-terminal alpha-domain, including a native N-terminus, was necessary for "virgin" activation of the associated plasminogen in the Sk(U)-PgA complex and (2) the C-terminal beta-domain of Sk(U) is important for recognition of the substrate in the Sk(U)-PgA complex.

Fibrinolytic function in diuretic-induced volume depletion

Accumulating evidence suggests that the renin-angiotensin system (RAS) may participate in the regulation of fibrinolytic function. In clinical studies, however, angiotensin-converting enzyme (ACE) inhibitors and the angiotensin II receptor antagonist losartan have failed to consistently affect endogenous fibrinolysis. Because such an effect may depend on the degree of prestimulation of the RAS, we have studied parameters of fibrinolytic function in 15 healthy volunteer subjects during baseline (day 1) and after 10 days of treatment with 25 mg of hydrochlorothiazide (HCT)/day (day 11). On the last day of the study (day 12), a single oral dose of 50 mg of losartan was given to the volunteers in addition to HCT and fibrinolytic function was assessed at the peak effect of losartan (5 h later). Plasma renin activity (PRA) was significantly stimulated during diuretic treatment (1.35 ± 0.21 v 0.34 ± 0.06 ng mL −1 · h −1 [ P < .001]) and further increased after losartan (6.39 ± 1.16 ng mL −1 · h −1 [ P < .001]). No effects of either the diuretic or losartan could be observed on tissue-type plasminogen activator (t-PA) antigen concentration and activity. However, 10 days of treatment with HCT significantly increased plasminogen activator inhibitor-1 (PAI-1) antigen (26.8 ± 5.8 v 21.1 ± 3.4 ng/mL [p = .037]). In addition, PAI-1 activity was also tentatively raised by HCT treatment (5.48 ± 1.82 v 3.88 ± 0.79 IU/mL [ P = .067]). In spite of the marked further rise in PRA after losartan, the stimulation of PAI-1 antigen and activity was blunted by losartan (24.4 ± 3.6 ng/mL and 4.55 ± 0.99 IU/mL, respectively). Our results demonstrate that volume depletion induced by HCT treatment is associated with a rise in PAI-1. Acute administration of losartan is capable of blunting this effect, suggesting that the angiotensin II type 1 receptor may participate in this effect of angiotensin II.

Regulation of plasminogen activation by TGF-β in cultured human retinal endothelial cells

BACKGROUND/AIMSRegulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor β (TGF-β) affected...

Isolation and characterization of cell lines with reduced urokinase binding.

Six cell lines have been generated from the human fibrosarcoma HT-1080 by mutagenesis. They were selected on the basis of reduced urokinase (uPA) binding on replicate polyester filters. Single cell clones were then isolated by limited dilution cloning. All cloned cells showed less uPA binding on filters, and as cell monolayers. These cell lines were able to bind only 10 to 65% as much uPA as the wild-type HT-1080 cells. Surface-bound uPA proteolytic activity and surface activation of plasminogen from these cells were also reduced relative to the wild-type. uPA could activate MAP kinases in the wild-type and two of the cell lines with the least uPA-binding, but the amount of the activated forms of the signalling molecules were reduced. Immunoblotting using two different anti-uPA receptor antibodies showed two cross-reacting protein species of approximately 53 kDa and approximately 38 kDa. The proportion of the lower Mr band to the higher Mr band was found to be reduced in all the cell lines relative to the wild-type. Chemical cross-linking with single-chain urokinase (scuPA) showed only one high-molecular-weight adduct, with Mr approximately 90 kDa, in all the cell lines tested. Similarly, cross-linking with the amino terminal fragment of uPA yielded a single approximately 70 kDa adduct. These would indicate that only the approximately 53 kDa band was responsible for cross-linking reactions. Equilibrium binding experiments showed that only one set of high-affinity binding sites for the wild-type cells. However, the binding of scuPA to two of these cell lines was best fitted to a two-site model, one of which was similar to the high-affinity binding sites of the wild-type, although the number of sites was reduced, while the other was of much lower affinity but was large in number. These results are discussed in relation to changes in the structure of ligand binding machinery in these cells, which affect other cellular functions.

Immunoassays for the Quantitation of Porcine PAI-1 Antigen and Activity in Biological Fluid Samples

Two monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) for the quantitation of porcine plasminogen activator inhibitor-1 (PAI-1) antigen and activity in plasma were constructed and validated. The intra-assay, interassay, and interdilution coefficients of variation were 4.3, 13, and 8%, respectively, for the antigen ELISA and 5, 16, and 11% for the activity assay. Assay recoveries, in the antigen ELISA. of either latent or active recombinant porcine PAI-1 (10 and 50 ng/ml) added to plasma were 86 +/- 9% and 92 +/- 22%, respectively, for the latent form and 89 +/- 99% and 87 +/- 7% for the active form (mean +/- SD, n = 3 to 4). In the immunofunctional assay, recoveries for the same concentrations of active PAI-1 were 108 +/- 16% and 92 +/- 21%, respectively. In male porcine plasma the level of PAI-1 antigen was 31 +/- 11 ng/ ml and the activity, 34 +/- 16 ng/ml (mean +/- SD, n = 10). In female plasma PAI-1 antigen levels were 20 +/- 5.2 ng/ml and the PAI-1 activity 42 +/- 17 ng/ml (n = 13). A linear correlation was found between PAI-1 antigen and activity levels in male (r = 0.60) and female (r = 0.70) plasma. Immunodepletion resulted in a decrease of >95% of the original PAI-1 antigen or activity levels. Incubation of plasma samples at 37 degrees C for 16 h resulted in a significant decrease (70 to 85%) of PAI-1 activity. Under these conditions (37 degrees C, 16 h) PAI-1 antigen levels remained unchanged in males whereas the response of the female samples in the PAI-1 antigen assay increased two-fold. In lysed platelet-rich plasma males had 990 +/- 470 ng/ml antigen and 160 +/- 80 ng/ml activity and females, 920 +/- 500 ng/ml antigen and 150 +/- 98 ng/ml activity corresponding to 2.1 +/- 0.77 fg PAI-1 antigen per platelet. Only 16% of PAI-1 released from platelets was found to be active. Linear correlations between PAI-1 antigen and activity were found for both males (r = 0.61) and females (r = 0.67). The assays are both sensitive and specific and may, therefore, aid the elucidation of the pathophysiological role of PAI-1 in swine experimental models of atherosclerosis and other thrombotic disorders.

State-of-the-Art-Review : Combined Heterozygous Plasminogen Deficiency and Factor V Leiden Defect in the Same Kindred

Combined plasminogen deficiency and resistance to activated protein C defect (factor V Leiden) have been described in a few families and associated with a variable occurrence of thrombotic events. Here we describe a new family with thrombophilia and the presence of hypoplasminogenemia and factor V Leiden mutation. In addition, a brief review of the literature is presented. Nine patients belonging to this kindred underwent coagulation study for hereditary thrombophilia, which included plasminogen antigen and activity assays, an activated protein C resistance test, and genetic analysis for factor V Leiden mutation and for prothrombin variant 20210A. The proposita, a 40-year-old asymptomatic female with a family history of thrombotic diathesis, was affected by heterozygous plasminogen deficiency. Hypoplasminogenemia was found also in her two sisters, in one instance associated with factor V Leiden mutation. The mother was the putative carrier of hypoplasminogenemia, but she refused to be studied. The symptomatic father was heterozygous for factor V Leiden mutation, but presented with normal plasminogen levels. Among the available siblings investigated from the paternal side, resistance to activated protein C due to factor V Leiden mutation was found in three patients, one of whom experienced venous thromboembolism. Another uncle with a history of thrombotic disease showed no coagulation abnormalities. These findings together with the data from literature confirm the role of factor V Leiden as an independent risk factor for venous thromboembolism, whereas isolated hypoplasminogenemia does not seem to increase the risk for thrombosis. There is no clear evidence that the coinheritance of these two defects may be associated with an additional risk for thrombosis compared with the presence of factor V Leiden mutation alone.

Tetranectin Binds to the Kringle 1-4 Form of Angiostatin and Modifies Its Functional Activity.

Tetranectin is a plasminogen kringle 4 domain-binding protein present in plasma and various tissue locations. Decreased plasma tetranectin or increased tetranectin in stroma of cancers correlates with cancer progression and adverse prognosis. A possible mechanism through which tetranectin could influence cancer progression is by altering activities of plasminogen or the plasminogen fragment, angiostatin. Tetranectin was found to bind to the kringle 1-4 form of angiostatin (ASTK1-4). In addition, tetranectin inhibited binding of plasminogen or ASTK1-4 to extracellular matrix (ECM) deposited by endothelial cells. Finally, tetranectin partially counteracted the ability of ASTK1-4 to inhibit proliferation of endothelial cells. This latter effect of tetranectin was specific for ASTK1-4 since it did not counteract the antiproliferative activities of the kringle 1-3 form of angiostatin (ASTK1-3) or endostatin. These findings suggest that tetranectin may modulate angiogenesis through interactions with AST.

Cloning, expression, sequence analysis, and characterization of streptokinases secreted by porcine and equine isolates of Streptococcus equisimilis.

Streptokinases secreted by nonhuman isolates of group C streptococci (Streptococcus equi, S. equisimilis, and S. zooepidemicus) have been shown to bind to different mammalian plasminogens but exhibit preferential plasminogen activity. The streptokinase genes from S. equisimilis strains which activated either equine or porcine plasminogen were cloned, sequenced, and expressed in Escherichia coli. The streptokinase secreted by the equine isolate had little similarity to any known streptokinases secreted by either human or porcine isolates. The streptokinase secreted by the porcine isolate had limited structural and functional similarities to streptokinases secreted by human isolates. Plasminogen activation studies with immobilized (His)(6)-tagged recombinant streptokinases indicated that these recombinant streptokinases interacted with plasminogen in a manner similar to that observed when streptokinase and plasminogen interact in the fluid phase. Analysis of the cleavage products of the streptokinase-plasminogen interaction indicated that human, equine, and porcine plasminogens were all cleaved at the same highly conserved site. The site at which streptokinase was cleaved to form altered streptokinase (Sk*) was also determined. This study confirmed not only the presence of streptokinases in nonhuman S. equisimilis isolates but also that these proteins belong to a family of plasminogen activators more diverse than previously thought.

Effects of angiotensin converting enzyme and angiotensin II receptor inhibition on impaired fibrinolysis in systemic hypertension

Abnormalities in fibrinolysis have been reported in hypertension. Angiotensin converting enzyme (ACE) inhibitors have been shown to improve altered fibrinolytic balance in hypertensive patients. It has not been documented, however, whether this is due to a decrease in angiotensin II (Ang-II) generation or is a consequence of elevated local levels of bradykinin. Accordingly, the aim of this study was to determine the effects of an ACE inhibitor (perindopril) and an Ang-II receptor antagonist (losartan) on fibrinolytic kinetics. We have examined the serum levels of the plasminogen activator inhibitor type-1 (PAI-1) antigen and activity, tissue plasminogen activator (t-PA) antigen and activity, soluble thrombomodulin (sTM), and tissue factor pathway inhibitor (TFPI) before and after reaching the target blood pressure (< 140/90 mm Hg) in 13 hypertensive patients receiving perindopril (mean age 40 ± 11 years, 6 women, 7 men) and in 12 patients receiving losartan (mean age 38 ± 9 years, 6 women, 6 men). We also compared the baseline fibrinolytic activity of hypertensive patients with that of 12 normotensive control persons (mean age 40 ± 9 years, 6 women, 6 men). The mean basal plasma levels of PAI-1 antigen, PAI-1 activity, and sTM were significantly higher in the hypertensive patients than in normal controls ( P < .005). The values of other analytes were similar in both groups. Increased plasma levels of PAI-1 antigen, PAI-1 activity, and sTM were reduced in patients after they were given perindopril and losartan ( P < .005); the reductions in losartan-receiving group were more pronounced ( P < .05). There were no significant effects on the plasma levels of t-PA antigen, t-PA activity, and TFPI in patients receiving the two therapeutic regimens ( P > .05). In conclusion, chronic hypertension is associated with hypofibrinolysis. The beneficial effect of ACE inhibitors on fibrinolysis seems to be related to the blockade of Ang-II, and increased kinin activity does not appear to play a major role.

Response of dairy ewes in late lactation to recombinant bovine somatotropin

This study was conducted to evaluate the effect of recombinant bovine somatotropin (rbST) administration to dairy ewes in late lactation on milk yield, composition, clotting parameters, and plasmin/plasminogen system. Eighty multiparous lactating Comisana ewes (186±18 days in lactation) were randomly assigned to one of two treatments: control and rbST. Slow-release rbST (120 mg/ewe) was administered for two injection cycles of 21 days each. Recombinant bovine somatotropin treatment increased milk (589.7 vs. 483.7 g/d; p < 0.01), protein (35.5 vs. 31.4 g/d; p < 0.05) and lactose (27.3 vs. 23.1 g/d; p < 0.05) yields. The percentage of milk fat (8.2% vs. 8.9%; p < 0.01) and protein (6.0% vs. 6.2%; p < 0.05) decreased, while somatic cells count and pH were unaffected by rbST treatment. Administration of rbST decreased milk coagulation time ( r) (12 vs. 13.1 min; p < 0.05), while firming rate ( k 20) and curd firmness ( a 30) were unaffected. Plasminogen activity of milk was lower in rbST-treated ewes (18.11 vs. 21.89 U/ml; p < 0.01).

α and β-adrenergic influences on the tissue and urokinase plasminogen activator systems

Objective: To study the influence of a- and β-adrenoceptor stimulation in vivo on fibrinolysis in healthy male volunteers. Design: Infusions were given in a single blind fashion and in a randomized order if placebo infusions were included. Setting: University Department (Karolinska Hospital). Subjects: Thirty-four healthy, non-smoking male volunteers aged 21–38 years. Interventions: Study 1 - The volunteers participated in two or three separate experiments; 30 min infusions of saline placebo, phentolamine (500 μg/min) or propranolol (bolus dose of 140 μg/kg + 0.5 μg/kg/min) followed by the addition of a 30 min adrenaline infusion (0.4 nmol/kg/min), n=10 in each group. Study 2 - Infusion of a low and an intermediate-high dose of noradrenaline (0.15 and 0.75 nmol/kg/min; 20 min at each dose level), n=11. Study 3— Infusion of a low and high dose of isoprenaline (5 and 20 ng/kg/min), each during 20 min, n=10. Main outcome measures: Venous plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) antigen and activity levels, and plasma levels of urokinase (u-PA) antigen. Results: t-PA activity and antigen levels were markedly increased during adrenaline infusion (by 309±35 and 81±16% respectively, both P<0.01) with a concomitant decrease in PAI-1 activity. U-PA antigen levels increased to a lesser extent. Propranolol, but not phentolamine, attenuated or blunted the effects of adrenaline. Non-selective β-adrenoceptor stimulation by isoprenaline caused smaller, but similar and dose-dependent alterations in fibrinolytic parameters as observed during adrenaline infusions, except for u-PA antigen levels that remained unaltered. A small increase in t-PA activity (by 55±13%, P<0.001) was observed during noradrenaline infusion, whereas t-PA antigen levels only tended to increase, PAI-1 activity decreased and u-PA antigen levels were not affected. Conclusion: Catecholamine-induced increases in plasma levels of t-PA, and to some extent u-PA, are mainly β-adrenoceptor mediated, whereas α-adrenoceptors are of minor importance.

Relation between long-term steroid treatment after heart transplantation, hypofibrinolysis and myocardial microthrombi generation

Background Thrombotic complications and transplant coronary artery disease are among the main causes of morbidity and mortality after heart transplantation. A thrombophilic state has been described in transplant recipients, and correlated to immunosuppressive therapy with Cyclosporine A or Azathioprine, whereas the prothrombotic effects of steroids, even though always given, have never been duly considered. A reduced fibrinolytic capacity due to high levels of PAI-1, the most important inhibitor of plasminogen activators, was suggested to play a role in the development of cardiovascular diseases and transplant coronary artery disease. A severe hypofibrinolytic state secondary to PAI-1 increase has been found in patients with Cushing’s disease, and in hypercorticism secondary to long-term steroid treatment after renal transplantation. Methods We evaluated plasma clotting and fibrinolytic behaviors in 2 groups of heart transplant patients treated with (26 cases) or without (23 cases) steroids together with Cyclosporine A and Azathioprine. Twenty-five healthy subjects were studied as normal controls. The following tests were assayed at least 1 year after transplantation: fibrinogen, factor VIII coagulant activity, von Willebrand factor antigen, Euglobulin Lysis Time, tissue plasminogen activator antigen and activity, PAI-1 antigen and activity. In addition, the presence of cardiac microthrombi was evaluated on 2 endomyocardial biopsy specimens obtained in each patient both on Day 7 after heart transplantation (first control) and usually 1 year or more later (last control). Results Plasma levels of fibrinogen, factor VIII and von Willebrand factor were significantly higher in both groups of patients than in normal controls. Fibrinolytic activity was significantly reduced in transplant patients treated with steroids, compared with steroid-free patients and normal controls. In steroid-treated heart transplant recipients, the hypofibrinolytic state was due to a significant and pathological increase in PAI-1 antigen and activity levels. The fibrinolytic impairment was more evident in patients transplanted for ischemic heart disease and treated with steroids than in patients with previous dilated cardiomyopathy and treated either with or without steroids. Myocardial microthrombi were found in 2/49 cases at the first biopsy control, and in 12/49 cases at the last biopsy control after transplantation. This different prevalence was statistically significant (χ 2 = 8.33, p = .003). Plasma PAI-1 activity was significantly higher and, as a consequence, Euglobulin Lysis Time was more prolonged in microthrombi-positive patients than in microthrombi-negative ones. Among the 12 transplant recipients who developed cardiac microthrombi, 7 patients were treated with steroids and showed higher PAI-1 levels and more reduced fibrinolytic activity than the 5 steroid-free patients. Conclusions Our data confirm the prothrombotic state induced by long-term steroid treatment, characterized by an increase in PAI-1 levels and secondary impairment of fibrinolytic capacity. In heart transplant patients, steroid-related hypofibrinolysis might constitute a further risk factor for transplant coronary artery disease.

Increased tissue-type plasminogen activator antigen release is not accompanied by increased systemic fibrinolytic activity in severe neonatal respiratory distress syndrome.

Intravascular and intraalveolar fibrin depositions in preterm infants with severe respiratory distress syndrome (RDS) have been attributed to activation of clotting. We questioned whether in the face of activated clotting, fibrinolysis is sufficient in these infants. We found, in infants with severe RDS within 6 to 12 h of birth, increased median thrombin-antithrombin III complex formation (11.1 versus 1.3 ng/mL in the group with mild-to-moderate RDS, p < 0.001), indicating activation of clotting. Simultaneously, we found increased tissue-type plasminogen activator antigen (t-PA) release in plasma of these infants represented by increased median t-PA plasma concentrations (8.3 versus 2.5 ng/mL in the group with mild-to-moderate RDS, p < 0.01). This increased t-PA release was not accompanied with more plasminogen and antiplasmin consumption and with more fibrin and fibrinogen degradation than in the infants with mild-to-moderate RDS because plasma plasminogen and antiplasmin activity and total fibrin and fibrinogen degradation product concentrations were similar in both groups. We have found that activated clotting and t-PA plasma concentrations are positively correlated with arterial-to-alveolar oxygen tension ratio and ventilator efficiency index values. Plasminogen and antiplasmin activity, and total fibrin and fibrinogen degradation product concentrations were not correlated with these continuous measures of RDS severity. In neonatal RDS, clotting activity contributes to disease severity. Insufficient fibrinolysis likely facilitates the deleterious effects of activated clotting.

Functions of the fibrinolytic system in human Ito cells and its control by basic fibroblast and platelet-derived growth factor.

During liver fibrogenesis, hepatic stellate cells (HSC) proliferate and migrate under the influence of growth factors, including platelet-derived growth factor (PDGF) and basic-fibroblast growth factor (b-FGF). The plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We evaluated the expression and biological functions of the plasminogen activation system in human HSC and its interaction with PDGF and b-FGF. Urokinase-plasminogen activator receptors (u-PAR) were measured by radioligand binding, cell cross-linking, immunoassay, and RNAse protection assay. u-PA and plasminogen activator inhibitors (PAIs) expression and activities were analyzed by zymography, immunoassay, and RNase protection assay. Cell migration and proliferation, studied in Boyden chambers and by microscopic counting, were evaluated after the addition of PDGF, b-FGF, and blockade with anti-u-PA, anti-u-PAR antibodies, and antisense oligodeoxynucleotides (aODN) against u-PAR mRNA. We have shown that HSC produce u-PAR, u-PA, and PAI-1. PDGF and b-FGF up-regulate u-PA and u-PAR, but not PAI-1, and exogenous addition of u-PA stimulates HSC proliferation, chemotaxis, and chemoinvasion. Inhibition of u-PA/u-PAR with antibodies against u-PA or u-PAR and with u-PAR aODN inhibit the proliferative, chemotactic, and chemoinvasive activity of PDGF and b-FGF. These findings indicate that u-PA and u-PAR are required for the mitogenic and chemoinvasive activity of PDGF and b-FGF on HSC.

Expression of plasminogen-related gene B varies among normal tissues and increases in cancer tissues

We previously found that the promoter activity of plasminogen (PLG)-related gene B (PRGB) was 5-fold that of PLG. We have since examined the transcript levels of PRGB among various normal human tissues, and compared these findings with those of PLG. Reverse transcription-PCR analysis revealed that the PRGB expression varied widely among different tissues, while PLG was expressed only in the liver and kidney. RNA samples obtained from cultured cell lines also demonstrated differing PRGB expression. Furthermore, increased PRGB expression was observed in several fresh samples of cancer tissue obtained from cancer patients when compared with surrounding normal tissues.