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The regulation of plasmin generation on cell surfaces is of critical importance in the control of vascular homeostasis. Cellderived microparticles participate in the dissemination of biological activities. However, their capacity to promote plasmin generation has not been documented. In this study, we show that endothelial microparticles (EMPs) from tumor necrosis factor ␣ (TNF␣) - IntroductionMicroparticles (MPs) are vesicles resulting from the blebbing of the cellular membrane of most activated or apoptotic cells. 1 These microvesicles have been described in various cellular models and in different pathological conditions as reliable hallmarks of cell damage. 2 Because they convey various bioactive effectors originating from the parent cells, MPs may exhibit a spectrum of biological activities: they regulate endothelial or blood cell functions, participate in inflammatory responses or angiogenesis, and propagate biological responses involved in hemostatic balance. 3 We previously reported the capacity of endothelial cells to release microparticles after inflammatory stimulation and the presence of increased levels of circulating endothelial microparticles (EMPs) in patients with thrombotic disorders. 4 Since this initial report, elevated levels of EMPs have been documented in various pathological conditions including coronary syndromes, 5 renal failure, 6 diabetes, 7 antiphospholipid syndrome, 8 thrombotic thrombocytopenic purpura, 9 and sickle cell disease, 10 in which they reflect endothelial dysfunction and are associated with a poor clinical outcome.EMPs provide procoagulant phospholipid surfaces for the assembly and activation of coagulation factors, mainly through phosphatidylserine translocation to the exoplasmic leaflet as a result of membrane remodeling. Their involvement in thrombin generation also results from their capacity to harbor, deliver, or induce tissue factor activity. 11-13 However, a more complex contribution to the hemostatic balance is suggested by their expression of thrombomodulin, tissue factor pathway inhibitor, and endothelial protein C receptor, thus providing a possible antithrombotic counterbalance. 14,15 Another key regulator of the vascular homeostasis is the plasminogen activation system. Plasminogen activation is mediated by 2 serine proteases: tissue-type plasminogen activator (tPA), which is mainly implicated in fibrinolysis, and urokinase-type plasminogen activator (uPA), which is critically involved in pericellular proteolysis due to its high affinity cell-surface receptor uPAR. 16 Plasmin generation induced by uPA and subsequent activation of matrix metalloproteinases (MMPs) promote cell migration through interstitial matrix and participate in processes such as tissue remodeling, cancer invasion, and angiogenesis. [17][18][19] Importantly, we have shown that uncontrolled plasminogen activation can have deleterious consequences by inducing cell detachment and apoptosis. 20,21 The regulation of plasmin generation at the endothelial surface is therefore of critical importa...

Abstract. A single-chain 55,000-mol wt form of urokinase (UK), similar to that previously isolated from urine, was purified from a transformed kidney cell culture medium and characterized; and its fibrinolytic properties were evaluated. The preparation immunoprecipitated with UK antiserum, had a low intrinsic amidolytic activity that was 0.1% of its active derivative, and resisted diisopropyl fluorophosphate treatment and inactivation by plasma inhibitors. The single-chain UK was therefore designated pro-UK. In

Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard '251-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (< 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA.Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.