http://hdl.handle.net/10171/3494 2013-05-26T06:28:32Z http://hdl.handle.net/10171/22872 Title: Development and clinical application of a new ELISA assay to determine plasmin-alpha2-antiplasmin complexes in plasma Author(s) : Montes, R. (Ramón); Paramo, J.A. (José Antonio); Angles-Cano, E. (Eduardo); Rocha, E. (Eduardo) Abstract: Plasmin-alpha2-antiplasmin complexes (PAP) are considered good markers of fibrinolytic activation in vivo. The presence of neoantigens in these complexes offers the possibility to develop specific immunoassays to determine PAP levels. We have developed a sensitive PAP purification method in vitro by adding urokinase to fresh plasma followed by affinity chromatography to lysine-sepharose and elution with epsilon-aminocaproic acid. This material, characterized by SDS-PAGE and Western blotting, was used to raise monoclonal antibodies (MoAbs). We describe a new enzyme linked immunosorbent assay (ELISA) to quantify PAP complexes in plasma. The assay follows the sandwich principle and is based on two MoAbs, CPL12 and CPL15, that bind to the modified alpha2-antiplasmin moiety and the plasmin moiety of the complex respectively. The calibration curve was constructed with definite concentrations of purified PAP. The lower limit of the assay is 75 ng/ml and the variation coefficients are 3.5% (intra-assay) and 10-6% (interassay). A mean value of 573.5+/-131.4 ng/ml was obtained from PAP concentration in a healthy population (n = 30). Significantly higher PAP levels were observed under diverse clinical conditions in which fibrinolysis is activated: clinical sepsis, acute myocardial infarction (AMI), malignancy, diabetes, pregnancy, elderly people and thrombolytic therapy. From our results we conclude that this ELISA is suitable to measure in vivo plasma PAP levels. 1995-12-31T23:00:00Z http://hdl.handle.net/10171/22811 Title: Papel del PAI-1 en los procesos trombóticos Author(s) : Orbe, J. (Josune); Montes, R. (Ramón); Paramo, J.A. (José Antonio) 1998-12-31T23:00:00Z http://hdl.handle.net/10171/22337 Title: Hemostatic markers in surgery: a different fibrinolytic activity may be of pathophysiological significance in orthopedic versus abdominal surgery Author(s) : Lopez, Y. (Y.); Paramo, J.A. (José Antonio); Valenti, J.R. (J.R.); Pardo, F. (Fernando); Montes, R. (Ramón); Rocha, E. (Eduardo) Abstract: Without prophylaxis, patients subjected to major abdominal surgery have a risk of deep vein thrombosis of approximately 30%, while the rate varies between 40% and 60% in orthopedic surgery. The reasons for this discrepancy are not completely understood. The present study was designed to compare the pre- and postoperative behavior of different coagulation and fibrinolysis parameters in patients undergoing both types of surgery, receiving low molecular weight heparin prophylaxis. Samples were taken before operation and on postoperative days 1, 3, and 7. The following parameters were assessed: prothrombin fragment 1 + 2, thrombin-antithrombin III complexes, fibrinopeptide A, tissue plasminogen activator, plasminogen activator inhibitor, plasmin-alpha 2-antiplasmin complexes, and fibrin degradation products. We found a significant increase in the clotting markers postoperatively compared with preoperative values (P < 0.05), both in abdominal and orthopedic surgery, indicating a marked hemostatic activation which remained until postoperative day 7. A significant increase in plasminogen activator inhibitor (P < 0.01) and a decrease in tissue plasminogen activator and plasmin-alpha 2-antiplasmin complexes was also observed early after operation. The plasminogen activator inhibitor activity decreased, while tissue plasminogen activator and plasmin-alpha 2-antiplasmin levels increased significantly on days 3 and 7 (P < 0.05). Fibrin degradation products significantly increased throughout the postoperative period (P < 0.01). Preoperatively, we found higher plasminogen activator inhibitor activity and lower tissue plasminogen activator and plasmin-alpha 2-antiplasmin complexes (P < 0.05) in patients undergoing hip replacement compared with abdominal surgery. Fibrin degradation products were also significantly lower on postoperative day 3 in patients undergoing hip replacement (P < 0.01). We suggest that the lower preoperative fibrinolytic activation observed in patients undergoing orthopedic surgery compared with abdominal surgery might have pathophysiological consequences. Our results also indicate that the hemostatic activation persists beyond the 7th postoperative day despite prophylaxis. 1996-12-31T23:00:00Z http://hdl.handle.net/10171/22334 Title: Purification and characterization of a variant of human prothrombin: prothrombin Segovia Author(s) : Collados, M.T. (María T.); Fernandez, J. (Javier); Paramo, J.A. (José Antonio); Montes, R. (Ramón); Borbolla, J.R. (José R.); Montaño, L.F. (Luis F.); Rocha, E. (Eduardo) Abstract: A dysprothrombin designated prothrombin Segovia was isolated from the plasma of an individual with normal prothrombin antigen and prothrombin activity lesser than 25% of the control prothrombin activity. Activation by prothrombinase complex showed a lower amidolytic than clotting activity, which suggests a lesser generation of active intermediates than normal prothrombin. When prothrombin Segovia was activated by prothrombinase complex in the absence of factor Va, no thrombin formation was found by functional activities. SDS-PAGE analysis of the molecules derived by activation with prothrombinase complex, Taipan snake venom and Echis carinatus venom showed an accumulation of molecules not cleaved at bond Arg320-Ile321. This was more evident with Echis carinatus venom, which only acts on this bond. Our data suggest that the alteration of prothrombin Segovia impairs the scission of bond Arg320-Ile321. 1996-12-31T23:00:00Z http://hdl.handle.net/10171/22333 Title: The increase of plasminogen activator inhibitor activity is associated with graft occlusion in patients undergoing aorto-coronary bypass surgery Author(s) : Rifon, J. (José); Paramo, J.A. (José Antonio); Panizo, C. (Carlos); Montes, R. (Ramón); Rocha, E. (Eduardo) Abstract: Early graft occlusion is a common complication in patients undergoing aorto-coronary bypass surgery. Both mechanical and haemostatic factors play a role in the pathogenesis of thrombotic occlusion. Several studies have demonstrated a relationship between fibrinolytic activity and venous or arterial thrombosis. We undertook this study to evaluate the possible contribution of the fibrinolytic system to postoperative occlusion in patients undergoing aorto-coronary bypass graft (CABG). A venous occlusion (VO) test was performed preoperatively in 82 patients undergoing revascularization procedures. Before and after VO the euglobulin fibrinolytic activity and tissue type plasminogen activator (t-PA) activity and antigen were measured. Plasminogen activator inhibitor (PAI) activity and antigen and fibrinogen were also assessed in the preocclusion sample. An angiography performed 10d postoperatively showed graft occlusion in 23% of patients. Patients with graft occlusion had significantly higher preoperative PAI activity than patients without occlusion (P < 0.001). Reduced fibrinolytic response and t-PA capacity was also observed in the group of patients with graft occlusion (P < 0.03 and P < 0.02 respectively). We found a reduced preoperative fibrinolytic response, mainly related to high plasma PAI activity in patients with postoperative graft occlusion. These results suggest that increased PAI activity might have a predictive value for early thrombosis in patients undergoing CABG. 1996-12-31T23:00:00Z http://hdl.handle.net/10171/22332 Title: Long-term cardiac rehabilitation program favorably influences fibrinolysis and lipid concentrations in acute myocardial infarction Author(s) : Paramo, J.A. (José Antonio); Olavide, I. (Isidro); Barba, J. (Joaquín); Montes, R. (Ramón); Panizo, C. (Carlos); Muñoz, M.C. (María Carmen); Rocha, E. (Eduardo) Abstract: BACKGROUND AND OBJECTIVE: The control of well-known atherosclerotic risk factors represents the optimal strategy in the prevention of acute coronary syndromes. It was the aim of this work to analyze the effects of a long-term cardiac rehabilitation program on the changes of fibrinolysis parameters and plasma lipid profile in coronary patients. DESIGN AND METHODS: The study was carried out in 30 (M/F:22/8, mean age 47 years) survivors of a first acute myocardial infarction (AMI) and in 30 healthy controls who underwent a cardiac rehabilitation program (9 months duration). Samples were taken before, at 3 and 9 months after the beginning of the program to measure: tissue-type plasminogen activator (t-PA) antigen and plasminogen activator inhibitor (PAI-1) activity and antigen. A lipid profile including cholesterol (both HDL and LDL) and lipoprotein(a) was also assessed. The Wilcoxon and Mann-Whitney tests were used for statistical comparisons. RESULTS: There was a marked decrease of functional PAI-1 after 3 and 9 months as compared with baseline in AMI patients (p < 0.01). Results showed a significant increase of HDL-cholesterol (p < 0.01) and decrease of lipoprotein(a) levels after the exercise program (p < 0.01). INTERPRETATION AND CONCLUSIONS: The cardiac rehabilitation program improved fibrinolysis, by reducing the functional levels of PAI-1, and ameliorated the lipid profile by decreasing lipoprotein(a) and increasing HDL-cholesterol in patients with AMI. A long-term cardiac rehabilitation has positive effects on some risk factors for coronary disease. 1997-12-31T23:00:00Z http://hdl.handle.net/10171/22273 Title: Autoantibodies against EPCR are found in antiphospholipid syndrome and are a risk factor for fetal death Author(s) : Hurtado, V. (Verónica); Montes, R. (Ramón); Gris, J.C. (Jean-Christophe); Bertolaccini, M.L. (María L.); Alonso, A. (Alvaro); Martinez-Gonzalez, M.A. (Miguel Angel); Khamashta, M.A. (Munther A.); Fukudome, K. (Kenji); Lane, D.A. (David A.); Hermida, J. (José) Abstract: The antiphospholipid syndrome (APS) is associated with thrombosis and fetal death but the pathologic mechanisms are poorly understood. Since endothelial protein C receptor (EPCR) plays a role in the anticoagulant system and in placental development, we hypothesized that anti-EPCR autoantibodies may be involved in clinical manifestations of APS and in fetal loss. The levels of immunoglobulin M (IgM) and IgG anti-EPCR autoantibodies were analyzed by enzyme-linked immunosorbent assay (ELISA) in 43 patients with APS and 43 controls. Anti-EPCR levels were higher in APS patients than in controls. Interestingly, one of the IgM anti-EPCR autoantibodies inhibited the generation of activated protein C on endothelium. Since markedly high anti-EPCR levels were found in women with fetal death, 87 patients with a first episode of unexplained fetal death were subsequently analyzed and their anti-EPCR levels were compared with 87 matched controls. We found that anti-EPCR autoantibodies constitute an independent risk factor for a first fetal death episode: the adjusted odds ratios (ORs) for anti-EPCR autoantibodies above the 95th percentile were 23.0 (95% confidence interval [CI], 2.0-266.3) for IgM and 6.8 (95% CI, 1.2-38.4) for IgG. Anti-EPCR autoantibodies can be detected in APS patients and are independent risk factors for fetal death. 2003-12-31T23:00:00Z http://hdl.handle.net/10171/22268 Title: Acute generalized, widespread bleeding. Diagnosis and management Author(s) : Rocha, E. (Eduardo); Paramo, J.A. (José Antonio); Montes, R. (Ramón); Panizo, C. (Carlos) Abstract: BACKGROUND AND OBJECTIVE: Acute generalized, widespread bleeding is often related to disseminated intravascular coagulation (DIC), a pathologic process which complicates the clinical course of many diseases and is characterized by huge amounts of thrombin and plasmin within the circulation. The final result is the consumption of platelets, coagulation factors and inhibitors, as well as secondary hyperfibrinolysis, all leading to diffuse hemorrhage and microthromboses. This review article examines the present attitudes to the diagnosis and treatment of overt DIC in clinical practice, emphasizing the importance of an accurate differential diagnosis from some other processes characterized by acute generalized, widespread bleeding. INFORMATION SOURCES: The authors have been working in this field, both at experimental and clinical levels, contributing original papers for many years. In addition, material examined in this review includes articles published in journals covered by MedLine, recent reviews in journals with high impact factor and in relevant books on hemostasis and thrombosis. STATE OF ART AND PERSPECTIVES: DIC is an intermediary mechanism of disease which complicates the clinical course of many well-known disorders. Although the systemic hemorrhagic syndrome is the predominant clinical manifestation, massive intravascular thrombosis frequently occurs contributing to ischemia and associated organ damage, making the mortality rate of this condition high. Current concepts on the pathophysiology, laboratory diagnosis and management of DIC are presented. Complex pathophysiological interrelations make the diagnosis of the etiology of the DIC difficult in clinical practice, although simple tests are useful for identification of patients with the process. Laboratory diagnosis of DIC is mainly based on screening assays, which allow a rapid diagnosis, whereas some other highly sensitive but more complex assays are not always available to routine clinical laboratories. The management of DIC is based on the treatment of the underlying disease, supportive and replacement therapies and the control of the coagulation mechanisms. Although some advances have been achieved, management decisions are still controversial, so that therapy should be highly individualized depending on the nature of the DIC and severity of clinical symptoms. Many syndromes sharing common findings with DIC, such as primary hyperfibrinolysis or thrombotic thrombocytopenic purpura, should be excluded. Finally, new therapeutic approaches to the management of this potentially catastrophic syndrome are required 1997-12-31T23:00:00Z http://hdl.handle.net/10171/22267 Title: Changes in the fibrinolytic components of cultured human umbilical vein endothelial cells induced by endotoxin, tumor necrosis factor-alpha and interleukin-1alpha Author(s) : Orbe, J. (Josune); Chorda, C. (Carlos); Montes, R. (Ramón); Paramo, J.A. (José Antonio) Abstract: BACKGROUND AND OBJECTIVE: Vascular fibrinolysis, a major natural defense mechanism against thrombosis, is a highly regulated process. The aim of this study was to evaluate the effect of endotoxin, tumor necrosis factor-alpha (TNFalpha) and interleukin-1alpha (IL-1alpha), on the fibrinolytic potential of cultured human umbilical vein endothelial cells (HUVEC). DESIGN AND METHODS: Samples of stimulated conditioned media were collected over a period of 24 hours to determine: plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activity, PAI-1 mRNA, tissue-type plasminogen activator (t-PA) antigen and urokinase-type plasminogen activator (u-PA) antigen. RESULTS: Similar changes were observed after endotoxin and cytokine stimulation: there was a significant increase of PAI activity (p<0.01), starting at 6 hours, which remained 24 hours after stimulation. PAI-1 mRNA also showed an important rise with these agents, although cytokines induced an earlier and more intense inhibitor response (up to 6-fold increase). PA activity increased significantly at 6 hours (p<0.01) to drop at 24 hours and was mainly related to the presence of u-PA. INTERPRETATION AND CONCLUSIONS: We conclude that endotoxin,+TNFalpha and IL-1alpha induce profound alterations in the fibrinolytic potential of HUVEC, characterized by an initial rise of activators (u-PA) followed by a strong increase of PAI-1. These changes may be of pathophysiologic significance for thrombosis and inflammatory reactions. 1998-12-31T23:00:00Z http://hdl.handle.net/10171/22262 Title: Evidence that heparin but not hirudin reduces PAI-1 expression in cultured human endothelial cells Author(s) : Orbe, J. (Josune); Montes, R. (Ramón); Zabalegui, N. (Natalia); Perez-Ruiz, A. (Ana); Paramo, J.A. (José Antonio) Abstract: Heparin and other antithrombotic drugs besides their anticoagulant action could have a profibrinolytic effect. We have analyzed the effect of unfractionated heparin (UFH) and hirudin on PAI-1 gene expression in human umbilical vein endothelial cells (HUVEC). Cells were stimulated with UFH (1 and 10 IU/ml) and hirudin (20 and 100 TIU/ml). Samples were obtained before and 2, 6, and 24 hours after stimulation. mRNA analysis was conducted by reverse transcription followed by polymerase chain reaction, and PAI-1 antigen was determined by ELISA. Addition of UFH (10 IU/ml) to HUVEC resulted in a decrease of PAI-1 mRNA at 6 hours (40% reduction) and 24 hours (60% reduction) and PAI-1 antigen. Hirudin, however, did not modify significantly the PAI-1 mRNA nor the inhibitor secretion. The addition of UFH (10 or 100 IU/ml) to endotoxin-stimulated HUVEC also reduced the increased PAI-1 mRNA and antigen secretion (45%), whereas no effect could be observed with hirudin. Our results suggest that UFH, but not hirudin, by reducing the endothelial expression of PAI-1 might have a profibrinolytic effect. 1998-12-31T23:00:00Z http://hdl.handle.net/10171/22241 Title: Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation Author(s) : Dominguez, M. (Miguel); Montes, R. (Ramón); Paramo, J.A. (José Antonio); Angles-Cano, E. (Eduardo) Abstract: Binding of plasminogen to fibrin and cell surfaces is essential for fibrinolysis and pericellular proteolysis. We used surface plasmon resonance and enzyme kinetic analyses to study the effect of two mAbs (A10.2, CPL15) on plasminogen binding and activation at fibrin surfaces. A10.2 is directed against the lysine-binding site (LBS) of kringle 4, whereas CPL15 recognises a region in kringle 1 outside the LBS. In the presence of CPL15 and A10.2 mAbs, binding of plasminogen (K(d)=1.16+/-0.22 micromol/l) to fibrin was characterised by a mAb concentration-dependent bell-shaped isotherm. A progressive increase in the concentration of mAbs at the surface was also detected, and reached a plateau corresponding to the maximum of plasminogen bound. These data indicated that at low mAb concentration, bivalent plasminogen-mAb-plasminogen ternary complexes are formed, whereas at high mAb concentration, a progressive shift to monovalent plasminogen-mAb binary complexes is observed. Plasmin formation in the presence of mAbs followed a similar bell-shaped profile. Monovalent Fab fragments of mAb A10.2 showed no effect on the binding of plasminogen, confirming the notion that a bivalent mAb interaction is essential to increase plasminogen binding and activation at the surface of fibrin. 2001-12-31T23:00:00Z http://hdl.handle.net/10171/22234 Title: Indications for bone marrow transplantation Author(s) : Prosper, F. (Felipe); Rifon, J. (José); Cuesta, B. (Braulia); Hermida, J. (José); Panizo, C. (Carlos); Hernandez, M. (M.); Rocha, E. (Eduardo) 1993-12-31T23:00:00Z http://hdl.handle.net/10171/22228 Title: Differential effects of 2C9*3 and 2C9*2 variants of cytochrome P-450 CYP2C9 on sensitivity to acenocoumarol Author(s) : Hermida, J. (José); Zarza, J. (José); Alberca, I. (Ignacio); Montes, R. (Ramón); Lopez, M.L. (María Luz); Molina, E. (Eva); Rocha, E. (Eduardo) Abstract: The 2C9*3 and 2C9*2 polymorphisms of cytochrome P-450 CYP2C9 are associated with hypersensitivity to warfarin and bleeding. The effect of these polymorphisms on sensitivity to acenocoumarol is unknown. Three groups of patients, with low, medium, or high acenocoumarol-dose requirements, were studied. Age influenced the acenocoumarol sensitivity. Bearing the 2C9*3 allele was associated with the need for a lower acenocoumarol dose (odds ratio [OR], 6.02; 95% confidence interval [CI], 1.50-24.18); 80% of carriers of the 2C9*3 allele required a low dose. The 2C9*2 allele was associated with a lower acenocoumarol-dose requirement (OR, 2.70; 95% CI, 1.11-6.58) because of a reduced risk of the need for a high acenocoumarol dose (4.8% of the patients in the high-dose group carried the 2C9*2 allele versus 34.1% and 30.2%, respectively, in the medium-dose and low-dose groups). Therefore, carriers of 2C9*3 may need a low initial loading dose of acenocoumarol. Because acenocoumarol sensitivity with the 2C9*2 variant does not seem to be clinically relevant, the drug could be an alternative to warfarin in 2C9*2 carriers 2001-12-31T23:00:00Z http://hdl.handle.net/10171/22226 Title: Identification and characterization of a natural R96C EPCR variant Author(s) : Hermida, J. (José); Hurtado, V. (V.); Villegas-Mendez, A. (A.); Catto, A.J. (A.J.); Philippou, H. (H.) 2002-12-31T23:00:00Z http://hdl.handle.net/10171/22122 Title: Effect of the administration of recombinant hirudin and/or tissue-plasminogen activator (t-PA) on endotoxin-induced disseminated intravascular coagulation model in rabbits Author(s) : Muñoz, M.C. (M.C.); Montes, R. (Ramón); Hermida, J. (José); Orbe, J. (Josune); Paramo, J.A. (José Antonio); Rocha, E. (Eduardo) Abstract: We evaluated the effect of r-hirudin and/or tissue-plasminogen activator (t-PA) in a model of DIC in rabbits induced by i.v. infusion of 100 micrograms/kg/h/6 h endotoxin. Rabbits were treated with saline (endotoxin control group), r-hirudin at 0.3 mg/kg/h/6 h, t-PA at 0.3 mg/kg for 90 min and r-hirudin plus t-PA at the doses described above. The best results were achieved when r-hirudin and t-PA were infused together. This treatment reduced the consumption of platelets and protein C and attenuated the increase of PAI-1 more efficiently than r-hirudin or t-PA alone. r-Hirudin plus t-PA also resulted in the lowest formation of fibrin deposits in the kidneys. Finally, mortality at 24 h dropped from 70% in the endotoxin control group to 40%, 10% and 0% in the t-PA, r-hirudin and r-hirudin plus t-PA groups respectively. None of the t-PA-infused rabbits which had died by 24 h showed macroscopic signs of haemorrhage. r-Hirudin alone was better than t-PA alone, as was shown by fibrin deposits and mortality. We conclude that r-hirudin and t-PA given simultaneously were more efficient than either given alone in this model of DIC. Effective thrombin inhibition, which could influence other pathophysiological mechanisms apart from coagulation, together with the improvement in fibrinolysis, would explain these results. 1998-12-31T23:00:00Z