M. Pino

University of Barcelona, Barcino, Catalonia, Spain

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Publications (25)107.05 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Endothelial dysfunction seems to be a key factor in the development of several complications observed early after hematopoietic stem cell transplantation (HSCT). The conditioning regimen and many other factors associated with the procedure are responsible for this endothelial damage. The effects of immunosuppressive agents on endothelial function have not been explored in detail. We evaluated the effects of three of the drugs commonly used in HSCT: two calcineurin inhibitors, cyclosporine A (CSA) and tacrolimus (TAC), and an inhibitor of mTOR, sirolimus (SIR). We also evaluated the effect of the combination of TAC and SIR (TAC+SIR), which is used increasingly in clinical practice. Microvascular endothelial cells (HMEC-1) were exposed to these drugs to evaluate changes in: i) ICAM-1 expression on the cell surface, assessed by immunofluorescence labeling and expressed as the mean gray value (MGV); ii) reactivity of the extracellular matrix (ECM) toward platelets, upon exposure of the ECM to circulating blood; and iii) whole-blood clot formation, assessed by thromboelastometry. Studies were conducted in the absence and presence of defibrotide (DF) to assess its possible protective effect. The exposure of HMEC-1 to CSA and TAC+SIR significantly increased the expression of ICAM-1 (157.5±11.6 and 153.4±9.5 MGV, respectively, vs. 105.7±6.5 MGV in controls (both P<0.05)). TAC applied alone increased ICAM-1 slightly (120.3±8.2 MGV), and SIR had no effect (108.9±7.4 MGV). ECM reactivity increased significantly only in response to CSA (surface covered by platelets of 41.2%±5.4% vs. 30.1%±2.0%, P<0.05). DF attenuated all of these changes. No significant changes in the viscoelastic properties of clot formation were observed in any condition with blood samples incubated in vitro. In conclusion, CSA and TAC+SIR had a proinflammatory effect, but only CSA exhibited an additional prothrombotic effect. Interestingly, DF exerted clear protective anti-inflammatory and antithrombotic effects on the endothelium.
    Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 07/2013; · 3.15 Impact Factor
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    ABSTRACT: While procoagulant activities of Tissue Factor (TF) have been widely investigated, its possible pro-adhesive properties towards platelets have not been studied in detail. We explored the interaction of platelets with human Tissue Factor (hTF) firmly adsorbed on a synthetic surface of polyvinilidene difluoride (PVDF) using different shear rates. For studies at 250 and 600 s(-1), TF firmly adsorbed was exposed to flowing anticoagulated blood in flat perfusion devices. Deposition of platelets and fibrin were evaluated by morphometric, immunocytochemical and ultrastructural methods. Prothrombin fragment 1 + 2 (F1 + 2) levels were also measured. Experiments at 5000 s(-1), were performed on the Platelet Function Analyzer (PFA-100) with experimental cartridges with collagen (COL) or collagen-hTF (COL + TF). Haemostatic effect of recombinant activated FVIIa (rFVIIa) was assessed in the same experimental settings. Platelet deposition on hTF reached 19.8 +/- 1.3% and 26.1 +/- 3.4% of the total surface, at 250 and 600 s(-1), respectively. Fibrin formation was significantly higher at 250 s(-1) than at 600 s(-1) (P < 0.05). The addition of rFVIIa did not influence platelet deposition but raised fibrin formation and thrombin generation at both shear rates (P < 0.05). At 5000 s(-1), closure times (CT) in the PFA-100 were significantly shortened in the presence of hTF (154.09 +/- 14.69 s vs. 191.45 +/- 16.09 s COL alone; P < 0.05). Addition of rFVIIa did not cause a further reduction of CT. Our studies demonstrate that hTF is an adhesive substrate for platelets and suggest that the von Willebrand factor could mediate these interactions. At low and intermediate shear rates, rFVIIa enhanced the procoagulant action of hTF, but this effect was not observed at very high shear rates.
    European Journal of Clinical Investigation 01/2008; 38(1):34-42. · 3.37 Impact Factor
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    ABSTRACT: We have applied an in vitro perfusion model to explore the potential thrombogenicity of polyester annulolasty fabric used in valve repair and to investigate the possible thromboresistance characteristics conferred by a special heparin coating (Duraflotrade mark treatment). Samples of human blood from i) untreated or ii) heparin-coated extracorporeal circuits were recirculated through annular perfusion chambers containing a) untreated or b) treated annuloplasty cloth material. Perfusion experiments were performed at a shear rate of 600 s(-1) for 20 min. Platelet interaction with the material was morphometrically evaluated. In experiments performed with blood from untreated circuits and cloth material, the average cross-sectional area of platelet mass was 615 +/- 135 microm2. Treatment of cloth material with Duraflotrade mark statistically decreased the area of interacting platelets to 319 +/- 101 microm2 (*p < 0.05, n = 10). Blood samples from heparin-coated extracorporeal circuits showed a decrease of total area of platelets (308 +/- 58 microm2 vs 138 +/- 30 microm2, *p < 0.05, n = 9). The combined treatment of Duraflotrade mark in extracorporeal circuits and cloth material caused a more consistent reduction (p < 0.05). The in vitro perfusion experimental model was sensitive to evaluate the thrombogenic potential of Duraflotrade mark treatment. Our results indicate that the heparin coating of cloth material and extracorporeal circuits improves the biocompatibility of the original material and reduces the thrombogenic profile.
    Journal of Biomedical Materials Research Part A 10/2005; 75(1):192-8. · 2.83 Impact Factor
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    ABSTRACT: The ability of nitrous compounds to donate nitric oxide (NO), an agent with vasodilating and inhibitory effects on platelet function, has been considered a useful pharmacologic strategy for cardiovascular complications. The purpose of this study was to investigate the effects of a new NO donor, LA419, on platelet interaction in an ex vivo model with human blood circulating through collagen-rich surfaces. Platelet adhesive and cohesive function were analyzed by morphometric procedures after perfusion techniques. Treated blood was exposed to thrombogenic surfaces and platelet interactions were morphometrically evaluated. All the concentrations studied of LA419 (10 microM, 20 microM and 100 microM) reduced overall platelet interaction with a collagen surface (27.19 +/- 4.72; 25.52 +/- 3.52; and 23.44 +/- 3.01, P < 0.05, respectively, vs. 32.31 +/- 1.61% in the control). The antithrombotic effect was confirmed by results in cross-sectional studies performed in arterial vessels exposed to circulating blood. Values of thrombus and covered surface at 20 microM LA419 were, respectively, 13.67 +/- 4.97% and 19.01 +/- 5.89%; respect to controls 34.80 +/- 5.29% and 37.93 +/- 5.34% (P < 0.05). Moreover, LA419 reduced significantly thrombus area (88.45 +/- 21.97 microm(2); P < 0.05) with respect to controls (168.45 +/- 21.97 microm(2)) and thrombus height, from an average of 10.27 +/- 1.05 microm in nontreated blood to 7.16 +/- 0.6 microm in treated samples (P < 0.05). From the present data we can conclude that LA419 possesses a strong antiplatelet action, as demonstrated by its ability to significantly inhibit the interaction of platelet with highly thrombogenic collagen surfaces.
    European Journal of Clinical Investigation 06/2005; 35(5):337-42. · 3.37 Impact Factor
  • Blood 01/2005; 106(11):617A-617A. · 9.78 Impact Factor
  • Blood 01/2005; 106(11):617A-617A. · 9.78 Impact Factor
  • Blood 01/2005; 106(11):89B-89B. · 9.78 Impact Factor
  • Transfusion 01/2005; 44(12):1790-1. · 3.53 Impact Factor
  • Blood 01/2005; 106(11):740A-740A. · 9.78 Impact Factor
  • Blood 01/2005; 106(11):740A-740A. · 9.78 Impact Factor
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    ABSTRACT: Although it is suspected that some patients with acute coronary syndromes (ACS) could have a sub-optimal response to aspirin (SASAR), currently a fixed dose of ASA is long-term used in all individuals. This study was designed to determine SASAR and whether a SASAR is a predictor for recurrence of ischemic events in patients on low-dose ASA with a previous ACS. One hundred patients taking ASA 100 mg/day were assessed at 1 and 6 months after a first ACS. SASAR was initially defined as a failure of the ASA treatment to significantly prolong the closure time in the Platelet Function Analyzer (PFA-100). SASAR in these samples was reconfirmed by conventional aggregometry. TXB2 levels were determined in plasma. At one month 49 patients showed SASAR in the PFA-100; only 25 of them showed SASAR by conventional aggregometry. At six months, 39 of 81 patients showed SASAR by PFA-100, but conventional aggregometry detected SASAR in only 12 of the 39 patients. TXB2 levels were significantly higher in patients with SASAR. Five patients with SASAR, by both tests, died during follow-up (p = 0.013). The PFA-100 detected a high rate of SASAR in patients with ACS. This instrument could be used to screen for suboptimal response to the antiplatelet action of ASA. Whether persistence of SASAR could relate to a higher risk of recurrence and how adjusting the dose of ASA could reduce the rate of SASAR are issues that deserve further investigations.
    Platelets 12/2004; 15(7):439-46. · 2.24 Impact Factor
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    ABSTRACT: Cyclooxygenase (COX)-2-selective non-steroidal anti-inflammatory drugs have been used for anti-inflammatory therapy. However, it has also been described that they may increase risk of cardiovascular events. To study the effects of COX2 inhibitor rofecoxib on platelet function using in vitro tests. Results were compared with those obtained in a parallel experiment with acetyl salicylic acid (ASA). Studies of platelet aggregation, using different agonists, were performed by a turbidimetric method. Adhesive and cohesive function of platelets were analyzed by perfusion techniques, treated blood was exposed to thrombogenic surfaces and platelet interaction was morphometrically evaluated. Twenty-five micro M of rofecoxib induced a prolonged lag time and a reduction in the percentage of aggregation when arachidonic acid, ADP or collagen were used as agonists. In perfusion studies with parallel chamber rofecoxib 50 microM and ASA 500 microM reduced overall platelet interaction with the collagen surface (17.4 +/- 3.7, P < 0.05; vs. 32.1 +/- 2.6%P < 0.05 and 17.9 +/- 2.4, vs. 31.9 +/- 3.24, P < 0.05, respectively). In studies performed on annular chambers, 25 micro M of rofecoxib reduced platelet interaction; values of the thrombus and covered surface were 17.4 +/- 4.5%; P < 0.05 and 21.1 +/- 4.1%; P < 0.05, respectively, vs. 30.4 +/- 7.5% and 33.5 +/- 6.5 in the control. ASA did also impair thrombus formation but differences did not reach the levels of statistical significance. Moreover, rofecoxib but not ASA reduced significantly thrombus height and thrombus area (7.4 +/- 0.5 microM; P < 0.005 and 96.0 +/- 21.2 microM(2); P < 0.05 vs. control 11.2 +/- 0.9 microM and 220.0 +/- 47.7 microM(2), respectively). We conclude that under our experimental conditions, rofecoxib diminished platelet aggregation induced by different agonists and inhibited platelet-mediated thrombogenesis in an in vitro model of thrombosis.
    European Journal of Clinical Investigation 05/2004; 34(4):297-302. · 3.37 Impact Factor
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    ABSTRACT: Although it is suspected that some patients with acute coronary syndromes (ACS) could have a sub-optimal response to aspirin (SASAR), currently a fixed dose of ASA is long-term used in all individuals. This study was designed to determine SASAR and whether a SASAR is a predictor for recurrence of ischemic events in patients on low-dose ASA with a previous ACS. One hundred patients taking ASA 100 mg/day were assessed at 1 and 6 months after a first ACS. SASAR was initially defined as a failure of the ASA treatment to significantly prolong the closure time in the Platelet Function Analyzer (PFA-100®). SASAR in these samples was reconfirmed by conventional aggregometry. TXB2 levels were determined in plasma. At one month 49 patients showed SASAR in the PFA-100®; only 25 of them showed SASAR by conventional aggregometry. At six months, 39 of 81 patients showed SASAR by PFA-100®, but conventional aggregometry detected SASAR in only 12 of the 39 patients. TXB2 levels were significantly higher in patients with SASAR. Five patients with SASAR, by both tests, died during follow-up (p=0.013). The PFA-100® detected a high rate of SASAR in patients with ACS. This instrument could be used to screen for suboptimal response to the antiplatelet action of ASA. Whether persistence of SASAR could relate to a higher risk of recurrence and how adjusting the dose of ASA could reduce the rate of SASAR are issues that deserve further investigations.
    Platelets 01/2004; 15(7):439-446. · 2.24 Impact Factor
  • Annals of the Rheumatic Diseases. 01/2003; 62:263-263.
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    ABSTRACT: We explored the effect on haemostasis of different factor IX (FIX) concentrates under thrombocytopenic conditions using an in vitro perfusion technique. A moderate experimental thrombocytopenia (25 000-30 000 platelets/microl) was induced by means of a filtration procedure in blood anticoagulated with low-molecular-weight heparin. The effects of three different FIX concentrates - a prothrombin complex concentrate (PCC), an intermediate-purity concentrate (FIX/X), and a high-purity concentrate (HPFIX) - on platelet deposition and fibrin formation on subendothelium were assessed at two different shear rates (600/second and 1200/second). Activation of the coagulation system was monitored through assessment of prothrombin activation fragment 1 + 2 (F1 + 2). Fibrin deposition increased after addition of FIX concentrates, but only showed a significant increase in experiments performed after incubation of PCC at the lower shear rate (600/second) (64.25 +/- 9.61% vs. control 31.22 +/- 8.02%; P < 0.05). Addition of FIX concentrates caused a small increase in the percentage of platelet deposition and area of those aggregates. These differences reached levels of statistical significance in the presence of FIX/X and HPFIX in experiments performed at a shear rate of 600/second. F1 + 2 baseline values in anticoagulated thrombocytopenic blood were 1.15 +/- 0.13 nm and reached levels of 2.49 +/- 0.24 and 3.60 +/- 0.33 nm at shear rates of 600 and 1200/second, respectively. Increments in F1 + 2 observed after addition of different FIX concentrates always remained in the previous ranges. Data from the present study provide experimental support favouring the concept that FIX concentrates containing other activated factors could improve haemostasis under conditions of moderate thrombocytopenia.
    Vox Sanguinis 04/2002; 82(3):113-8. · 2.85 Impact Factor
  • European Heart Journal. 01/2001; 22:668-668.
  • Hepatology 01/2001; 34(4):531A-531A. · 12.00 Impact Factor
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    ABSTRACT: The potential hemostatic effect of infusible platelet membranes (IPM; Cyplex, Cypress Bioscience) prepared from outdated human platelets is investigated. Increasing concentrations of IPM were added to blood samples anticoagulated with low-molecular-weight heparin, in which platelets and WBC counts had been experimentally reduced by a filtration procedure. Thrombocytopenic blood with IPM was circulated in a perfusion chamber at various shear rates (300, 600, and 1200/sec(-1)), and platelet and fibrin deposition on the surface of a damaged vessel was measured. Prothrombin fragments 1 and 2 (F1+2) levels were also monitored. Under conditions of severe thrombocytopenia (<6000 platelets/microL) IPM did not increase platelet deposition. However, a dose-dependent increase in fibrin deposition was observed with concentrations of IPM ranging from 0.5 to 2 mg per kg in perfusions at 300 and 600 per sec(-1) (p<0.05 vs. thrombocytopenic blood). Experimental studies performed under conditions of moderate thrombocytopenia and higher shear rates (25, 000-30,000 platelets/microL; at 600 and 1200/sec(-1)) showed that IPM concentrations equivalent to 0.5 or 1 mg per kg improved fibrin deposition (33.5 +/- 9.5% and 37.7 +/- 12.8%, respectively, vs. 22.7 +/- 5.2% in controls) and also promoted a moderate increase in platelet deposition, with a concomitant significant increase in the size of platelet aggregates (p<0.05). Exposure of thrombocytopenic blood to a damaged vessel resulted in an increase of F1+2 levels from 0.8 +/- 0.15 to 1.7 +/- 0.22 nM at 300 per sec(-1) and 1.94 +/- 0.46 nM at 600 per sec(-1). Postperfusion levels of F1+2 after the addition of IPM were always similar to levels in untreated controls. IPM promotes local procoagulant activity at sites of vascular damage under conditions of severe and moderate thrombocytopenia. IPM also appears to facilitate platelet cohesive functions under conditions of moderate thrombocytopenia.
    Transfusion 09/2000; 40(9):1074-80. · 3.53 Impact Factor
  • Haemostasis. 01/2000; 30(Suppl. 1):88-.
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    ABSTRACT: Patients with end-stage renal disease or advanced cirrhosis develop bleeding disorders characterized by defective interaction of platelets with damaged subendothelium. The anemia associated with both clinical entities has a negative influence on hemostasis. We evaluated alterations of platelet function in patients suffering from end-stage renal disease (n=21) or hepatic cirrhosis (n=20) using standard aggregometric techniques and the recently developed platelet function analyzer (PFA-100 ). The impact of low hematocrit was also analyzed. The hemostatic capacity of platelets was tested in the PFA-100 using citrated blood and standard cartridges containing collagen-ADP (COL-ADP) or collagen-epinephrine (COL-Epi). The hemodynamic influence of hematocrit was also evaluated in blood aliquots in which hematocrit was experimentally increased by adding red blood cells from the same patient. Aggregation studies demonstrated abnormal responses to several agonists in both group of patients. Closure times obtained by the PFA-100 for control blood samples were 87+/-3 sec for COL-ADP and 113+/-5 sec with COL-EPi cartridges. Closure times in uremic and cirrhotic patients with average hematocrits of 0.26 and 0.27 respectively were significantly prolonged (139+/-12 and 125+/-14 sec, respectively with COL-ADP and 194+/-29 and 151+/-15 sec with COL-Epi cartridges). A 5% increase in the hematocrit caused a reduction in the closure time to 111+/-7 sec (COL-ADP) and 143+/-14 sec (COL-Epi) in the uremic group and to 86+/-4 sec (COL-ADP) and 115+/-16 sec (COL-Epi) in the cirrhotic group. Our studies confirm the platelet dysfunction in uremic and cirrhotic patients. The PFA-100 device proved to be useful for testing alterations of primary hemostasis in these acquired disorders and was sensitive enough to detect modifications in hemostasis caused by elevations in hematocrit. Conventional aggregometric tests were able to identify the intrinsic platelet abnormality in uremic and cirrhotic conditions, while the PFA-100 seemed more sensitive in detecting the negative influence of the hematocrit reduction.
    Haematologica 08/1999; 84(7):614-9. · 5.94 Impact Factor