Accelerated Fibrinolysis and Its Propagation on Vascular Endothelial Cells by Secreted and Retained tPA

We successfully visualized the secretory dynamics of tissue-type plasminogen activator (tPA) tagged by green fluorescent protein (tPA-GFP) from cultured vascular endothelial cells (VECs) using total internal reflection fluorescence (TIRF) microscopy and demonstrated that tPA-GFP secreted from VECs was retained on cell surfaces in a heavy-chain-dependent manner. Progressive binding of Alexa568-labeled Glu-plasminogen was also observed on the surface of active tPA-GFP expressing cells via lysine binding sites (LBS), which was not observed on inactive mutant tPA-GFP expressing cells. These results suggest that retained tPA on VECs effectively activated plasminogen to plasmin, which then facilitated the binding of additional plasminogen on the cell surface by proteolytically cleaving surface-associated proteins and exposing their C-terminal lysine residues. Thus prolonged retention of tPA appeared to play an important role in initiating and amplifying plasmin generation on VECs. LBS-dependent binding of plasminogen was also observed as a narrow band at the lytic front of the fibrin mesh formed on active tPA-GFP expressing cells, which expanded outward as the lytic area increased. This binding was not observed on inactive mutant tPA-GFP expressing cells or in the presence of aprotinin. The binding of plasminogen to partially digested fibrin appears to be indispensable for spontaneous fibrinolysis

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (>5min.) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high molecular weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential

Fibrinolysis in hypertensive patients with glomerulonephritis

Wstęp Rola układu fibrynolizy w chorobach nerek nie jest do końca poznana, zaś w nadciśnieniu tętniczym samoistnym stwierdza się zmniejszoną aktywność fibrynolityczną. Celem pracy była ocena układu fibrynolizy w populacji chorych z kłębuszkowym zapaleniem nerek oraz nadciśnieniem tętniczym w zależności od stosowanej przez nich terapii hipotensyjnej. Materiał i metody Objęto badaniem 34 chorych w wieku 40-57 lat, u których zdiagnozowano kłębuszkowe zapalenie nerek oraz nadciśnienie tętnicze. Przez 3 miesiące 16 chorych otrzymywało enalapril (20 mg/d.), zaś 18 nitrendipinę (20 mg/d.). Aktywność tkankowego aktywatora plazminogenu (tPA) i jego inhibitora (PAI) badano metodą amidolityczną za pomocą gotowych zestawów firmy Bioopol, Umea, Szwecja. Antygeny tPA i PAI oznaczano metodą immunoenzymatyczną na zestawach firmy Bioopol, Umea, Szwecja. Kompleksy tPA/PAI oceniano przy użyciu zestawów firmy Technoclone, Austria. Czas lizy euglobulin (ECLT) oceniano metodą Kowarzyka i Buluka. Wyniki Stwierdzono obniżoną aktywność tPA oraz wydłużenie ECLT u pacjentów z nadciśnieniem tętniczym oraz kłębuszkowym zapaleniem nerek w stosunku do grupy kontrolnej. Stężenie i aktywność PAI były istotnie wyższe u pacjentów z nadciśnieniem tętniczym oraz kłębuszkowym zapaleniem nerek w stosunku do grupy kontrolnej. Po 3 miesiącach leczenia enalaprilem stwierdzono istotny spadek stężenia i aktywności PAI, zaś w grupie leczonej nitrendipiną istotny wzrost stężenia tPA. Wnioski Upośledzenie fibrynolizy u pacjentów z nadciśnieniem oraz kłębuszkowym zapaleniem nerek może przyczyniać się do zwiększonego ryzyka powikłań zakrzepowych oraz przyspieszonego rozwoju miażdżycy z jej konsekwencjami. Stosowane leki hipotensyjne mogą wywierać korzystny wpływ na układ fibrynolizy w tej populacji chorych.Background The role of fibrinolysis and serotonin in glomerulonephritis is still the matter of controversy. In essential hypertension fibrinolytic activity is diminished. The aim of the study was to assess some fibrinolytic parameters, in patients with glomerulonephritis in relation to healthy volunteers. Material and methods Hypertensive patients with biopsyproven glomerulonephritis and normal renal function were treated for three months with nitrendipine (40 mg/day) (n = 18) or enalapril (20 mg/day) (n = 16). Healthy volunteers served as a control group. Activities of tPA and PAI-1 (amidolytic method), concentrations of tPA, PAI and tPA/PAI complexes (EIA) were studied by means of commercially available kits (Spectrolyse, Biopool, Sweden, Technoclone, Austria, respectively). Euglobulin clot lysis time (ECLT) was measured according to Kowarzyk and Buluk. Results Diminished activity of tPA and a significant prolongation of ECLT was observed in hypertensive patients with glomerulonephritis when compared to healthy volunteers (p < 0.05). Concentration and activity of PAI were found to be higher in hypertensive patients when compared to the control group (p < 0.01). After 3 months of treatment with enalapril PAI concentration and activity decreased significantly, whereas in nitrendipine group a rise in tPA concentration was seen. Conclusions Impairment in fibrinolysis in glomerulonephritis in hypertensive patients, may contribute to the increased risk of thromboembolic complications and accelerated atherosclerosis observed in these patients. Hypertensive treatment may favourably affect fibrinolysis in these patients

An update on the global use of risk assessment models and thromboprophylaxis in hospitalized patients with medical illnesses from the World Thrombosis Day steering committee: Systematic review and meta-analysis

INTRODUCTION Venous thromboembolism (VTE) is a leading cause of cardiovascular morbidity and mortality. The majority of VTE events are hospital-associated. In 2008, the Epidemiologic International Day for the Evaluation of Patients at Risk for Venous Thromboembolism in the Acute Hospital Care Setting (ENDORSE) multinational cross-sectional study reported that only approximately 40% of medical patients at risk of VTE received adequate thromboprophylaxis. METHODS In our systematic review and meta-analysis, we aimed at providing updated figures concerning the use of thromboprophylaxis globally. We focused on: (a) the frequency of patients with an indication to thromboprophylaxis according with individual models; (b) the use of adequate thromboprophylaxis; and (c) reported contraindications to thromboprophylaxis. Observational nonrandomized studies or surveys focusing on medically ill patients were considered eligible. RESULTS After screening, we included 27 studies from 20 countries for a total of 137 288 patients. Overall, 50.5% (95% confidence interval [CI]: 41.9-59.1, I$^{2}$ 99%) of patients had an indication to thromboprophylaxis: of these, 54.5% (95% CI: 46.2-62.6, I$^{2}$ 99%) received adequate thromboprophylaxis. The use of adequate thromboprophylaxis was 66.8% in Europe (95% CI: 50.7-81.1, I$^{2}$ 98%), 44.9% in Africa (95% CI: 31.8-58.4, I$^{2}$ 96%), 37.6% in Asia (95% CI: 25.7-50.3, I$^{2}$ 97%), 58.3% in South America (95% CI: 31.1-83.1, I$^{2}$ 99%), and 68.6% in North America (95% CI: 64.9-72.6, I$^{2}$ 96%). No major differences in adequate thromboprophylaxis use were found across risk assessment models. Bleeding, thrombocytopenia, and renal/hepatic failure were the most frequently reported contraindications to thromboprophylaxis. CONCLUSIONS The use of anticoagulants for VTE prevention has been proven effective and safe, but thromboprophylaxis prescriptions are still unsatisfactory among hospitalized medically ill patients around the globe with marked geographical differences

Defining trauma-induced coagulopathy with respect to future implications for patient management : Communication from the SSC of the ISTH

Peer reviewedPostprin