scholarly journals Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion

1997 ◽  
Vol 110 (9) ◽  
pp. 1083-1089 ◽  
Author(s):  
S. Bonavaud ◽  
C. Charriere-Bertrand ◽  
C. Rey ◽  
M.P. Leibovitch ◽  
N. Pedersen ◽  
...  
Keyword(s):  
Cell Migration ◽  
Plasminogen Activator ◽  
Cell Fusion ◽  
Muscle Cell ◽  
Myogenic Cell ◽  
Mrna Levels ◽  
Amino Terminal ◽  
Pai 1

Urokinase can form a tripartite complex binding urokinase receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1), a component of the extracellular matrix (ECM). The components of the tripartite complex are modulated throughout the in vitro myogenic differentiation process. A series of experiments aimed at elucidating the role of the urokinase tripartite complex in the fusion of human myogenic cells were performed in vitro. Myogenic cell fusion was associated with increased cell-associated urokinase-type plasminogen activator (uPA) activity, cell-associated uPAR, and uPAR occupancy. Incubation of cultures with either uPA anticatalytic antibodies, or the amino-terminal fragment of uPA (ATF), which inhibits competitively uPA binding to its receptor, or anti-PAI-1 antibodies, which inhibit uPA binding to PAI-1, resulted in a 30 to 47% decrease in fusion. Incubation of cultures with the plasmin inhibitor aprotinin did not affect fusion. Decreased fusion rates induced by interfering with uPAR/uPA/PAI-1 interactions were not associated with significant changes in mRNA levels of both the myogenic regulatory factor myogenin and its inhibitor of DNA binding, Id. Incubation of cultures with purified uPA resulted in a decrease in fusion, likely due to a competitive inhibition of PAI-1 binding of endogenous uPA. We conclude that muscle cell fusion largely depends on interactions between the members of the urokinase complex (uPAR/uPA/PAI-1), but does not require proteolytic activation of plasmin. Since the intrinsic muscle cell differentiation program appears poorly affected by the state of integrity of the urokinase complex, and since cell migration is a prerequisite for muscle cell fusion in vitro, it is likely that the urokinase system is instrumental in fusion through its connection with the cell migration process. Our results suggest that the urokinase tripartite complex may be involved in cell migration in a non conventional way, playing the role of an adhesion system bridging cell membrane to ECM.

Effect of PAI-1 Specific RNA Aptamers On Cell Adhesion and Motility.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2135-2135
Author(s):  
Yolanda Fortenberry ◽  
Charlene M Blake ◽  
Bruce A Sullenger
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Binding Site ◽  
Plasminogen Activator ◽  
Umbilical Vein ◽  
Rna Aptamers ◽  
Type Plasminogen Activator ◽  
Pai 1

Abstract Abstract 2135 Poster Board II-112 Introduction: The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1) binds to and inhibits the plasminogen activators tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). This results in both a decrease in plasmin production, as well as a decrease in the dissolution of fibrin clots. PAI-1 is also associated with the pathophysiology of several diseases, including cancer and cardiovascular disease. Both experimental and clinical studies have shown that increasing the plasma and vessel wall PAI-1 levels positively correlates with an increased risk of cardiovascular-related events. Consequently, the pharmacological suppression of PAI-1 might prevent or treat vascular disease. Unfortunately, since PAI-1 is a multifunctional protein, complete inhibition of PAI-1 might hinder its ability to regulate fibrinolysis, which can provoke bleeding. However, eliminating the pathological functions of PAI-1 without hindering its physiological functions might be beneficial in treating a variety of diseases. Extracellular matrix vitronectin (VN) increases at sites of vessel injury and is also present in fibrin clots. In response to injury, vitronectin facilitates cell adhesion, thereby increasing vascular cell migration by binding to integrins and to surface-bound uPA. PAI-1 competes with integrins and the urokinase-type plasminogen activator receptor (uPAR) for VN binding, resulting in the detachment of cells from the extracellular matrix. The binding of PAI-1 to VN prevents integrins from binding to VN, and inhibits cell adhesion and migration. Objective: The goal of this study was to develop RNA aptamers to interfere with a single PAI-1 function, without obstructing its other functions. The present study concentrated on developing PAI-1 aptamers to the vitronectin binding site of PAI-1. Aptamers are single-stranded nucleic acids, either DNA or RNA, that bind to their target protein with high affinity and specificity. Methods: Our aptamers were generated by the systematic evolution of ligands by exponential enrichment (SELEX). Adopting the SELEX in vitro selection technique ensures creation of nuclease-resistant RNA molecules that will bind to target proteins. We used in vitro assays to determine the effect of the aptamers on the adhesion and migration of smooth muscle (SM) and human umbilical vein endothelial cells (EC). Results: Recently, we published a paper that showed the generation of PAI-1 specific RNA aptamers that bind to the heparin/vitronectin binding site of PAI-1 (Blake et al., 2009). We showed that PAI-1 specific aptamers prevented the detachment of cancer cells from vitronectin in the presence of PAI-1, resulting in an increase in cell adhesion. We have expanded these studies to include smooth muscle (SMC) and human umbilical vein endothelial cells (EC). We demonstrated that the PAI-1 specific aptamers (SM-20 and WT-15) dose dependently increase SMC and EC attachment in the presence of vitronectin (compared to the control aptamer). Interestingly, SM-20 (the aptamer to stable PAI-1) was more effective than WT-15 (aptamer to wild-type PAI-1). Whereas PAI-1 significantly inhibited cell migration (in the presence of vitronectin), the PAI-1 specific aptamers were able to restore migration of both SMC and EC cells. Additionally, the PAI-1 aptamers were unable to bind to the PAI-1 vitronectin binding mutant, further suggesting that these aptamers bind to the PAI-1's vitronectin binding site. Importantly, these aptamers did not affect the antiprotease activity of PAI-1. Conclusions: We have shown that we are able to inhibit one of PAI-1's functions without hindering its other functions. By promoting smooth muscle and endothelial cell migration, these aptamers can potentially eliminate the adverse effects of elevated PAI-1 levels in the pathogenesis of vascular disease. Disclosures: Sullenger: Regado Biosciences Inc.: Equity Ownership, Scientific Founder.

scholarly journals Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3631-3636 ◽  
Author(s):  
C Krishnamurti ◽  
C Bolan ◽  
CA Colleton ◽  
TM Reilly ◽  
BM Alving
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Fibrin Deposition ◽  
Plasminogen Activator Inhibitor 1 ◽  
Elevated Activity ◽  
Activator Inhibitor ◽  
Pai 1

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P “ .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.

scholarly journals Influence of fibrinolitic potential reduction in ischemic brain disease development

2008 ◽  
Vol 61 (5-6) ◽  
pp. 247-251 ◽  
Author(s):  
Biljana Vuckovic ◽  
Mirjana Djeric ◽  
Marija Zarkov
Keyword(s):  
Plasminogen Activator ◽  
Brain Disease ◽  
Fibrinolytic System ◽  
Brain Diseases ◽  
Primary Role ◽  
Plasminogen Activator Inhibitor 1 ◽  
Ischemic Brain ◽  
Pai 1

Introduction Vascular brain diseases are the third leading cause of morbidity and mortality today. Among them ischemic brain disease caused mostly by cerebral atherosclerosis makes almost 80%. The main risk factors for this disease are arterial hypertension, dislipoproteinemia and diabetes but, recently hemostatic system disorders have also been underlined, especially fibrinolytic disorders. Physiology of fibrinolysis The primary role of fibrinolytic system is to make blood vessels passable by prevention of thromb forming or by removing the existing ones. This role is enabled by constant dynamic balance between activators and inhibitors of fibrinolysis. The leading activators are tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) and the most important inhibitors are 2-antiplasmin, plasminogen activator inhibitor 1 (PAI-1) and thrombin activated fibrinolytic inhibitor (TAFI). Fibrinolysis and thrombosis In patients with atherosclerosis there is clear insufficiency of fibrinolysis caused mainly by the increased activity of fibrinolytic inhibitors. There is evidence that the level of PAI-1 connected to fibrin directly affects thromb resistance to fibrinolysis and that velocity of thromb lysis in vitro predominantly depends on TAFI concentration. Factor XIII also has influence on thromb structure and stability and Lp(a) lipoprotein is one more factor that is responsible for resistency of thromb to fibrinolysis. Fibrinolysis in ischemic brain disease Reduced fibrinolytic capacity is noticed in patients with ischemic brain disease resulting from the increased PAI-1 activity. Higher risk for ischemic brain disease is verified among people with 4G/4G PAI-1 genotype. Raised level of t-PA antigene can also induce reduction of endogenous fibrinolysis. Conclusion In conclusion we want to underline that a possible pathophysiological significance of fibrinolytic system is assumed in a high percentage of patients with ischemic brain disease. Because of that we need further investigations to establish precise role of fibrinolytic mechanisms in genesis of this disease.

T cell lymphokines modulate bFGF-induced smooth muscle cell fibrinolysis and migration

1997 ◽  
Vol 272 (2) ◽  
pp. C392-C398 ◽  
Author(s):  
W. Wang ◽  
H. J. Chen ◽  
A. Schwartz ◽  
P. J. Cannon ◽  
L. E. Rabbani
Keyword(s):  
Smooth Muscle ◽  
T Cell ◽  
Smooth Muscle Cell ◽  
Plasminogen Activator ◽  
Muscle Cell ◽  
Ifn Gamma ◽  
Type Plasminogen Activator ◽  
And Migration ◽  
Pai 1

Smooth muscle cell (SMC) fibrinolysis is necessary for SMC migration. To determine whether the T cell lymphokines interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) modulate SMC fibrinolysis and migration induced by basic fibroblast growth factor (bFGF), we examined the effects of IL-4 and IFN-gamma on human SMC tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor 1 (PAI-1) antigen production, determined by enzyme-linked immunosorbent assays. Although IL-4 had no effects on SMC tPA, UPA, and PAI-1 production, it potentiated bFGF-induced tPA, UPA, and PAI-1 antigens. IL-4 plus bFGF resulted in a net increase in SMC fibrinolytic activity. IFN-gamma did not significantly affect bFGF induction of SMC tPA and PAI-1 antigens. However, IFN-gamma significantly decreased bFGF-mediated induction of SMC UPA antigen. IFN-gamma decreased the IL-4 plus bFGF induction of both tPA and UPA antigens. IL-4 increased and IFN-gamma abrogated bFGF induction of in vitro SMC migration through a modified micro-Boyden chamber. Therefore, IL-4 and IFN-gamma modulate bFGF-mediated induction of in vitro vascular SMC fibrinolysis and migration.

scholarly journals Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3631-3636 ◽  
Author(s):  
C Krishnamurti ◽  
C Bolan ◽  
CA Colleton ◽  
TM Reilly ◽  
BM Alving
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Fibrin Deposition ◽  
Plasminogen Activator Inhibitor 1 ◽  
Elevated Activity ◽  
Activator Inhibitor ◽  
Pai 1

Abstract The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P “ .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.

scholarly journals Platelets inhibit fibrinolysis in vitro by both plasminogen activator inhibitor-1-dependent and -independent mechanisms

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 351-356 ◽  
Author(s):  
WP Fay ◽  
DT Eitzman ◽  
AD Shapiro ◽  
EL Madison ◽  
D Ginsburg
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Clot Lysis ◽  
Homozygous Mutation ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Platelet-rich thrombi are resistant to lysis by tissue-type plasminogen activator (t-PA). Although platelet alpha-granules contain plasminogen activator inhibitor-1 (PAI-1), a fast-acting inhibitor of t-PA, the contribution of PAI-1 to the antifibrinolytic effect of platelets has remained a subject of controversy. We recently reported a patient with a homozygous mutation within the PAI-1 gene that results in complete loss of PAI-1 expression. Platelets from this individual constitute a unique reagent with which to probe the role of platelet PAI-1 in the regulation of fibrinolysis. The effects of PAI-1-deficient platelets were compared with those of normal platelets in an in vitro clot lysis assay. Although the incorporation of PAI-1-deficient platelets into clots resulted in a moderate inhibition of t-PA-mediated fibrinolysis, normal platelets markedly inhibited clot lysis under the same conditions. However, no difference between PAI-1-deficient platelets and platelets with normal PAI-1 content was observed when streptokinase or a PAI-1-resistant t-PA mutant were used to initiate fibrinolysis. In addition, PAI-1-resistant t-PA was significantly more efficient in lysing clots containing normal platelets than wild-type t-PA. We conclude that platelets inhibit t-PA-mediated fibrinolysis by both PAI- 1-dependent and PAI-1-independent mechanisms. These results have important implications for the role of PAI-1 in the resistance of platelet-rich thrombi to lysis in vivo.

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