A sensitive assay for tissue plasminogen activator

Rånby, Mats; Norrman, Bo; Wallén, Per

doi:10.1016/0049-3848(82)90012-3

Cited by 191 publications

(71 citation statements)

References 5 publications

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“…1 A , left ). Because PAI-1 can circulate in both active and latent forms (24), plasma PAI-1 activity was measured using a microtiter system which monitors PAI-1-mediated inhibition of plasminogen-activator activity (16,17). These experiments demonstrated a time-dependent increase in plasma PAI-1 activity (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Plasminogen activator inhibitor activity was determined by a functional rate assay described by Ranby et al (16), and its adaptation to plasma samples, as described by Wiman et al (17). In brief, samples from normoxic or hypoxic conditions were added to reaction mixture containing a known quantity of tPA, soluble fibrin (Desafib; American Diagnostica), and a plasmin substrate (Spectrozyme PL; American Diagnostica).…”

Section: Methodsmentioning

confidence: 99%

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Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.

Pinsky¹,

Liao²,

Lawson³

et al. 1998

J. Clin. Invest.

182

109

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Oxygen deprivation, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. To investigate the effects of low ambient oxygen tension on the fibrinolytic system, mice were placed in a hypoxic environment with pO 2 Ͻ 40 Torr. Plasma levels of plasminogen activator inhibitor-1 (PAI-1) antigen, detected by ELISA, increased in a time-dependent fashion after hypoxic exposure (increased as early as 4 h, P Ͻ 0.05 vs. normoxic controls), and were accompanied by an increase in plasma PAI-1 activity by 4 h ( P Ͻ 0.05 vs. normoxic controls). Northern analysis of hypoxic murine lung demonstrated an increase in PAI-1 mRNA compared with normoxic controls; in contrast, transcripts for both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) decreased under hypoxic conditions. Immunocolocalization studies identified macrophages as the predominant source of increased PAI-1 within hypoxic lung. Using a transformed murine macrophage line, striking induction of PAI-1 transcripts occurred under hypoxic conditions, due to both increased de novo transcription as well as increased mRNA stability. Consistent with an important role of the fibrinolytic system in hypoxia-induced fibrin accumulation, PAI-1 ϩ / ϩ mice exposed to hypoxia exhibited increased pulmonary fibrin deposition based upon a fibrin immunoblot, intravascular fibrin identified by immunostaining, and increased accumulation of 125 I-fibrinogen/fibrin in hypoxic tissue. In contrast, mice deficient for the PAI-1 gene (PAI-1 Ϫ / Ϫ ) similarly exposed to hypoxic conditions did not display increased fibrin accumulation compared with normoxic PAI-1 ϩ / ϩ controls. Furthermore, homozygous null uPA (uPA Ϫ / Ϫ ) and tPA (tPA Ϫ / Ϫ ) mice subjected to oxygen deprivation showed increased fibrin deposition compared with wildtype controls. These studies identify enhanced expression of PAI-1 as an important mechanism suppressing fibrinolysis under conditions of low oxygen tension, a response which may be further amplified by decreased expression of plasminogen activators. Taken together, these data provide insight into an important potential role of macrophages and the fibrinolytic system in ischemia-induced thrombosis.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.

Pinsky¹,

Liao²,

Lawson³

et al. 1998

J. Clin. Invest.

182

109

View full text Add to dashboard Cite

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“…These tissues were placed in buffer (0.05 M Tris, 0.15 M NaCl, 500 units/ml heparin, final pH 7.6) on ice and homogenized. Plasmin digestion was performed by a modification of the methods of Francis (36), as described previously (34). Human plasmin (0.32 units/ml; Sigma) was added to the tissue homogenate, followed by agitation at 37°C for 6 h. More plasmin (0.32 units/ml) was then added, and samples were agitated for an additional 2 h, and then the mixture was centrifuged at 2300 ϫ g for 15 min, and the supernatant was aspirated.…”

Section: Methodsmentioning

confidence: 99%

“…PAI and tPA Activity Assay-PAI/tPA activity was determined by a functional rate assay described by Ranby et al (34) and its adaptation to plasma samples, as described by Wiman et al (35). Blood samples (F, n ϭ 9; IL-10 (ϩ/ϩ), n ϭ 9; IL-10 (Ϫ/Ϫ), n ϭ 9; and IL-10 (Ϫ/Ϫ) plus rmIL-10, n ϭ 9) were drawn at the end of survival experiments and acidified by acetate buffer immediately.…”

Section: Methodsmentioning

confidence: 99%

“…PAI and tPA Activity Assay-Because PAI can circulate in both active and latent forms (40), plasma PAI activity was measured using a microtiter system that monitors PAI-mediated inhibition of plasminogen activator activity (34,35) as well as tPA activity. IL-10 (Ϫ/Ϫ) mice showed significantly higher PAI activity compared with that of IL-10 (ϩ/ϩ) mice.…”

Section: Pai-1 Mrna Expression In Il-10 (ϩ/ϩ) (ϫ/ϫ) and (ϫ/ϫ) Plus mentioning

confidence: 99%

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Potentiation of Endogenous Fibrinolysis and Rescue from Lung Ischemia/Reperfusion Injury in Interleukin (IL)-10-reconstituted IL-10 Null Mice

Okada

Fujita

Minamoto

et al. 2000

Journal of Biological Chemistry

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Little is known about interactions between endogenous anti-inflammatory paradigms and microvascular thrombosis in lung ischemia/reperfusion (I/R) injury. Interleukin (IL)-10 suppresses macrophage activation and down-regulates proinflammatory cytokine production, but there are no available data to suggest a link between IL-10, thrombosis, and fibrinolysis in the setting of I/R. We hypothesized that hypoxia/ischemia triggers IL-10 production, to dampen proinflammatory cytokine and adhesion receptor cascades and to restore vascular patency by fibrinolytic potentiation. Studies were performed in a mouse lung I/R model. IL-10 mRNA levels in lung were increased 43-fold over base line by 1 h of ischemia/2 h of reperfusion, with a corresponding increase in plasma IL-10. Expression was prominently localized in bronchial epithelial cells and mononuclear phagocytes. To study the link between IL-10 and fibrinolysis in vivo, the induction of plasminogen activator inhibitor-1 (PAI-1) was evaluated. Northern analysis demonstrated exaggerated pulmonary PAI-1 expression in IL-10 (؊/؊) mice after I/R, with a corresponding increase in plasma PAI/tissue-type plasminogen activator activity. In vivo, IL-10 (؊/؊) mice showed poor postischemic lung function and survival after I/R compared with IL-10 (؉/؉) mice. Despite a decrease in infiltration of mononuclear phagocytes in I/R lungs of IL-10 (؊/؊) mice, an increased intravascular pulmonary fibrin deposition was observed by immunohistochemistry and Western blotting, along with increased IL-1 expression. Recombinant IL-10 given to IL-10 (؊/؊) mice normalized the PAI/tissue-type plasminogen activator ratio, reduced pulmonary vascular fibrin deposition, and rescued mice from lung injury. Since recombinant hirudin (direct thrombin inhibitor) also sufficed to rescue IL-10 (؊/؊) mice, these data suggest a preeminent role for microvascular thrombosis in I/R lung injury. Ischemiadriven IL-10 expression confers postischemic pulmonary protection by augmenting endogenous fibrinolytic mechanisms. Ischemia/reperfusion (I/R)1 lung injury plays a significant role in clinical situations such as lung transplantation (1-3). Lung failure associated with I/R is characterized by increased microvascular permeability, pulmonary vascular resistance with subsequent edema formation and impairment of gas exchange, and microembolism. The lungs are particularly susceptible to ischemia/reperfusion injury, presumably due to the rich vascularity of the lungs and the relatively large surface area over which blood-borne components can interact with endothelium. The proximate mechanisms of ischemic lung injury are diverse and include leukocyte activation and recruitment (1), complement activation (4), abnormalities in pulmonary vascular tone, and increased procoagulant activity, resulting in microcirculatory failure, cellular dysfunction, edema, and cell death. The local production of proinflammatory cytokines, such as IL-1␣ and tumor necrosis factor-␣, is considerably increased in I/R injury (5, 6), which can also feed...

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Anabolic‐Androgenic steroid abuse in weight lifters: Evidence for activation of the hemostatic system

et al. 1995

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Anabolic-androgenic steroid abuse has recently been linked with acute vascular events in athletes. To date, the relationship between steroid abuse and the potential for cardiovascular disease has been considered almost exclusively in terms of lipid metabolism. However, recent reports of thrombosis in androgen abusing athletes with no evidence of atherosclerosis suggests the hypothesis that thrombosis risk in such athletes could be mediated through androgen induced abnormalities of coagulation. To determine if anabolic-androgenic steroid abuse in weight lifters is associated with an activation of the hemostatic system we studied forty-nine weight lifters recruited through advertisements. History of androgen use or abstinence was confirmed via urine assays. Plasma was assayed for clotting and fibrinolytic activity by measuring thrombin/antithrombin complexes (TAT), prothrombin fragment 1 + 1 (F1 + 2), and D-dimers (D-di); markers of the endothelial based fibrinolytic components were assayed by measuring tissue plasminogen activator antigen (t-PA Ag) and its inhibitor (PAI-1); finally, the activity of antithrombin III, protein C, and protein S were measured. Abnormally high concentrations of TAT complexes were noted in 16% of our confirmed steroid using weight lifters compared to 6% of our confirmed nonusers (P = .01). Steroid users also demonstrated abnormally high concentrations of F1 + 2 and D-dimers when compared to nonusers (44 vs. 24%, P < .001, and 9 vs. 0%, respectively). Non-steroid users were more likely to have elevated levels of t-PA Ag and PAI-1 than our steroid using weight lifters (both P < .001). The activities of antithrombin III and protein S were more likely to be higher in users compared to nonusers (22 vs. 6%, P = .005; 19 vs. 0%, respectively). Some anabolic-androgenic steroid using weight lifters have an accelerated activation of their hemostatic system as evidence by increased generation of both thrombin and plasmin. These changes could reflect a thrombotic diatheses that may contribute to vascular occlusion reported in young athletes using these drugs. The predictive value of these coagulation abnormalities in terms of risk of thrombosis to individual steroid using weight lifters or the population as a whole remains to be studied.

show abstract

A sensitive assay for tissue plasminogen activator

Cited by 191 publications

References 5 publications

Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.

Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.

Potentiation of Endogenous Fibrinolysis and Rescue from Lung Ischemia/Reperfusion Injury in Interleukin (IL)-10-reconstituted IL-10 Null Mice

Anabolic‐Androgenic steroid abuse in weight lifters: Evidence for activation of the hemostatic system

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