The influence of a serine protease inhibitor, nafamostat mesilate, on plasma coagulation, and platelet activation during experimental Extracorporeal Life Support (ECLS).

INTRODUCTION: During extracorporeal circulation the contact between blood and the artificial surface of the circuit induces several changes in the hemostatic system. The objective of the present study was to assess the effect of a serine protease inhibitor--Nafamostat mesilate (FUT-175)--on coagulation and on platelets during experimental extracorporeal circulation. METHODS: Two identical Extra Corporeal Life Support (ECLS) circuits were primed with fresh, heparinized human blood and circulated for 24 h. FUT-175 was added to one of the paired circuits and the other was used as a control. The following FUT-175 concentrations were employed: (1) 7.1 mg/l/h, (2) 14.2 mg/l/h, (3) 14.2 mg/l/h + 85.5 mg given as an initial bolus, (4) 28.5 mg/l/h + 171 mg given as an initial bolus. Blood samples were collected from the circuits before the start of the perfusion and at 0.5, 1, 3, 12, and 24 h of perfusion, and analysed for platelet count, plasma betathromboglobulin (beta-TG), platelet membrane glycoprotein (GP) Ib and GPIIb/IIIa expression, thrombin/antithrombin III complex (TAT), prothrombin fragment 1+2 (F1+2), fibrinogen, D-dimer, and plasminogen activator inhibitor 1 activity (PAI-1). RESULTS: Significantly higher platelet membrane GPIb expression and lower plasma beta-thromboglobulin levels were observed in the circuits holding FUT-175, suggesting a lower degree of platelet activation. Also, a reduced activation of the coagulation system was observed in the "FUT-circuits", as reflected by the levels of F1+2 and TAT, and the PAI-1 activity that was rapidly inactivated. CONCLUSION: FUT-175 reduces the activation of platelets and plasma coagulation in an in vitro ECLS model.

Pharmacological inhibition of plasma coagulation and platelet activation during experimental long-term perfusion.

OBJECTIVE: During extracorporeal circulation, initial contact between blood and the artificial surface of the circuit induces an overall activation of the hemostatic system. The objective of this study was to investigate the combined effect of epoprostenol (PGI (2) ), nitric oxide (NO) and nafamostat mesilate (FUT-175, a serine protease inhibitor), on plasma coagulation and platelet activation during experimental long-term perfusion. DESIGN: Two identical extracorporeal life support (ECLS) circuits were primed with fresh, heparinized human blood, and circulated for 24 h. FUT was given with a bolus dose of 85 mg/l blood at the initiation of the experiment and thereafter as a continuous infusion of 14 mg/l/h. PGI (2), at a rate of 2.4 microg/l/h, was also administered to the experimental circuit, and 120 ppm NO gas was added to the oxygenator sweep gas. The other circuit was used as a control. RESULTS: Higher platelet count and platelet membrane expression of GPIb were found in the experimental circuits as compared with control circuits. The levels of thrombin/antithrombin III complex (TAT) and prothrombin fragment 1 + 2 (F1 + 2) increased significantly over time in the control circuits but remained low in the experimental circuits. Plasma levels of plasminogen activator inhibitor 1 (PAI-1) decreased rapidly in both circuits but were higher in the control circuits at each time point studied. CONCLUSION: The activation of platelets and of the coagulation system encountered during extracorporeal perfusion is consistently inhibited by a combination of PGI (2), NO and FUT-175. The combination of these drugs appears to be more effective than each drug separately.

The effect of epoprostenol on platelet activation and consumption during experimental extracorporeal perfusion.

Hemorrhages are major complications experienced in 10-35% of neonates treated with extracorporeal life support (ECLS). The increased bleeding tendency is partly due to an ECLS induced thrombocytopenia and impaired platelet function. In the present study, we evaluated the effect of epoprostenol on the ECLS induced platelet consumption and activation. In terms of the methods, identical in vitro ECLS circuits were primed with fresh heparinized human blood and circulated for 24 h. Epoprostenol (2.4 microg/L blood/h) was added to one of the circuits in each pair. In total, 6 paired experiments were performed. The platelet count, neutrophil count, plasma concentration of hemoglobin, and platelet membrane expression of glycoproteins (GP) Ib and IIb/IIIa were assayed before the start and at 0.5, 1, 3, 12 and 24 h of perfusion. Higher platelet counts were observed in the experimental circuits. However, no difference in the platelet membrane expression of GPIb and GPIIb/IIIa could be observed between the circuits. In conclusion, epoprostenol reduces platelet consumption during ECLS without affecting the membrane expression of GPIb and GPIIb/IIIa.

The effect of albumin priming solution on platelet activation during experimental long-term perfusion.

The objective of this study was to evaluate the effect of albumin priming on platelet consumption and activation during long-term perfusion. Two identical in vitro extracorporeal membrane oxygenation circuits were used; one was primed with Ringer's solution containing human serum albumin, the other with Ringer's solution only. Fresh heparinized human blood was pooled, divided between the two systems and circulated for 24 h at 37 degrees C. Platelet count, plasma concentration of betathromboglobulin (BTG), platelet membrane density of glycoprotein (GP) Ib and of GPIIb/IIIa were assayed before the start and at 0.5, 1, 3, 12 and 24 h of perfusion. In total, seven experiments were performed. We found that during the first hour of perfusion, slightly higher platelet counts (p = 0.058) and lower BTG values (p = 0.0005) were observed in the circuits primed with albumin, compared to the control circuits. No statistically significant differences were observed for the platelet membrane expression of GPIb and GPIIb/IIIa. We conclude that albumin priming appears to transiently prevent platelet consumption and activation during long-term perfusion.

Induced cell trauma during in vitro perfusion: a comparison between two different perfusion systems.

The purpose of this study was to compare blood cell activation during in vitro long-term perfusion using 2 parallel in vitro extracorporeal membrane oxygenation (ECMO) systems. We compared two substantially different perfusion systems, an assistance respiratoire extra corporelle (AREC) system on one hand, containing an AREC pump, silicon tubing, and a hollow-fiber oxygenator, and a centrifugal pump system, on the other hand, containing a Biomedicus centrifugal pump, PVC tubing, and a membrane oxygenator. We measured the platelet count using an automated blood cell counter. Platelet activation was evaluated using a flow cytometric technique for the platelet membrane expression of glycoproteins and ELISA for the plasma concentration of beta-thromboglobulin (beta-TG), a platelet specific protein released into the blood upon platelet activation. The neutrophil count was assayed using an automated blood cell counter and the plasma concentration of cytokines using an ELISA kit. A significant difference between the two systems was observed in terms of the platelet membrane expression of glycoprotein (GP)Ib (p=0.0001) and GPIIb/IIIa (p=0.0037), indicating a lower degree of platelet activation in the AREC system. The concentration of neutrophils was significantly lower in the centrifugal system (p=0.002) compared to the AREC system. The neutrophil membrane expression of CD11b was significantly lower (p=0.0067) in the AREC system, indicating a lower degree of neutrophil activation compared to the centrifugal pump system. A significantly lower degree of hemolysis, as expressed by plasma hemoglobin, was observed in the AREC pump system (p=0.0491). In conclusion, lower degrees of the platelet membrane expression of GPIb and GPIIb/IIIa and of the neutrophil membrane expression of CD11b were observed in the AREC system, indicating a lower degree of platelet and neutrophil activation in this system. No significant difference between the two systems as to the plasma concentration of interleukin (IL)-1beta, IL-6, or IL-8 could be recorded. Further studies are warranted to specify the role of each individual component of the two systems.

Cytokine release during long-term extracorporeal circulation in an experimental model.

The objective of this study was to determine the degree of leukocyte activation, as measured by cytokine release, in circulating blood during experimental extracorporeal circulation. Complete in vitro extracorporeal membrane oxygenation (ECMO) circuits were used, and 9 experiments were performed. Whole blood stored at 37 degrees C was used as the control. Blood samples were withdrawn before the start of perfusion and at 24 h of perfusion. Statistically significant releases of interleukin (IL)-1beta, IL-8, and IL-1 receptor antagonist were observed in the perfusion circuits compared to both the control blood and baseline values. Also, increases in plasma tumor necrosis factor (TNF)alpha and IL-6 were seen after 24 h of perfusion although these changes did not reach statistical significance. These results indicate that extracorporeal circulation induced leukocyte activation and cytokine release. These reactions might, as an additional trauma, deteriorate the situation in an already severely ill patient. A search for methods to counteract this untoward activation seems warranted.

Platelet activation and degradation in an experimental extracorporeal system. A comparison between a silicone membrane and a hollow-fibre oxygenator.

Blood platelets are rapidly activated in contact with biomaterials and, therefore, can be used as markers of the biocompatibility of various components in an extracorporeal system. In the present work two different oxygenators, one membrane oxygenator (Avecor) and one hollow-fibre oxygenator ("Lilliput', Dideco) were compared. Complete in vitro extracorporeal membrane oxygenation circuits were perfused with fresh, heparinized human blood for 24 h. Eight experiments were performed with the hollow-fibre oxygenator and five experiments with the membrane oxygenator. Blood gases, electrolytes, glucose and haematocrit were kept within physiological limits. Platelet count, plasma concentration of beta-thromboglobulin, platelet serotonin content, platelet membrane glycoprotein lb and its degradation product glycocalicin, as well as plasma haemoglobin concentration were assayed. As regards most of these variables, significant differences in favour of the hollow-fibre oxygenator were observed.

Influence of hemofiltration on plasma cytokine levels and platelet activation during extra corporeal membrane oxygenation.

OBJECTIVE: Extra corporeal circulation of human blood is used daily in lifesaving procedures such as open-heart surgery and extracorporeal membrane oxygenation (ECMO). But extracorporeal circulation also induces activation of various cascade reactions in the blood. The objective of this study was to evaluate the effects of hemofiltration on cytokine release and removal as well as on platelet activation and consumption. MATERIAL AND METHODS: Two complete ECMO systems, each of them holding a hollow fiber oxygenator, a bladder box, PVC tubing and a roller pump were perfused for 24 h with fresh, heparinized human blood. A hemofilter was added to one of the paired systems. Blood samples were collected from both circuits before start, and at 0.5, 1, 3, 12 and 24 h of perfusion. A total of 8 paired experiments was performed. RESULTS: The plasma concentration of interleukin (IL)1beta, IL-6 and IL-8, as well as of IL-1 receptor antagonist (IL-1ra) increased over time in both systems, but consistently lower levels were observed in the filter circuits compared to the controls. Only minor parts of these cytokines could be assayed in the ultrafiltrate. No significant difference in platelet count and platelet membrane expression of glycoprotein Ib was observed between the circuits. CONCLUSIONS: By adding a hemofilter to the ECMO circuit, it is possible to reduce the plasma concentration of interleukins without significantly affecting platelet activation and consumption.