Nitric oxide gas infusion to the oxygenator enhances the biocompatibility of heparin coated extracorporeal bypass circuits

Heparin coated bypass circuits have been reported to improve the biocompatibility of extracorporeal circulation, although it is still insufficient and improvable. Nitric oxide (NO) is known to inhibit platelet activation and inflammatory reactions. In this study, the authors evaluated exogenous NO infusion in enhancing the effect of a heparin coated bypass circuit on the biocompatibility of an extracorporeal circuit, especially in view of the attenuation of the inflammatory response. A miniature closed bypass circuit, including an oxygenator (BioActive surface; Carmeda, Stockholm, Sweden) was primed with fresh human heparinized blood and perfused with a centrifugal pump. Either pure N2 gas (control group: n = 7) or NO gas (NO group [100 ppm in N2]: n = 7) was infused to the oxygenator. NO metabolites (nitrite and nitrate), platelet count, thrombin-antithrombin III complex (TAT), alpha2-plasmin-plasminogen inhibitor complex (PIC), beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), serotonin, complement 3 activation products (C3a), granulocyte elastase, and bradykinin were measured at 0, 30, 60, 120, and 180 min after starting perfusion. At every sampling point, platelet counts were significantly higher, and TAT, beta-TG, and bradykinin were lower in the NO group than in the control group. PF4, C3a, and granulocyte elastase were significantly lower in the NO group at 60, 120, and 180 min. These results suggest that NO gas infusion to the oxygenator enhances the biocompatibility of heparin coated extracorporeal circuits.     

Platelet and neutrophil distributions in pump oxygenator circuits. III. Influence of nitric oxide gas infusion

The authors used quantitative gamma scintigraphy to evaluate the influence of nitric oxide gas on platelet and neutrophil deposition in Cobe Duo microporous oxygenators during cardiopulmonary bypass (CPB). The effects of nitric oxide gas on circulating platelet and neutrophil counts and platelet function also were assessed. Animals were prepared by standard methods. Cells were harvested, labeled (111 In platelet and 99mTc neutrophil), infused, and recirculated. Nitric oxide gas, a guanylate cyclase pathway promoter, was infused int he Duo gas port at 500 ppm (t = 0-60 min), increased to 1,000 (t = 60-80 min), and stopped (final, 10 min). Images were taken at 10-15 min intervals during CPB. Standard isotope image corrections were made. No differences between nitric oxide gas and control experiments were observed for flow, pressure, hematocrit, or replacement volume. Nitric oxide gas infusion significantly (p < 0.05) reduced both platelet adherence to the oxygenator and in vitro platelet aggregation. Neutrophil adhesion tended to be lower, and circulating platelet and neutrophil counts tended to be higher with nitric oxide gas infusion. Results of in vitro aggregometry studies using rabbit platelets indicate that the class V phosphodiesterase inhibitor zaprinast can strongly enhance the inhibitory effects of nitric oxide. The authors conclude nitric oxide gas is a promising platelet sparing agent in the setting of CPB.     

CD8+ T cells are the effectors of the contact dermatitis induced by urushiol in mice and are regulated by CD4+ T cells

Nitric oxide (NO) reduces platelet aggregation in vitro. However, repeated measurements of platelet aggregation in infants and small children are impossible due to the large blood samples required. Instead, the expression of different platelet receptors mediating platelet adhesion (CD 36 and CD 42b), activation (CD 42b and CD 61) and aggregation (CD 41a) was measured repeatedly by flow cytometry. First, the expression of platelet receptors was quantified in platelet suspensions of 20 healthy volunteers after incubation with different concentrations of NO (0, 25, 100 and 640 ppm) and compared to changes in platelet aggregation and intrathrombocytic cGMP levels. It was then studied in 21 infants and children before, during and up to 3 days after cardiopulmonary bypass surgery. Seven of these patients required NO inhalation postoperatively. The in vitro experiments showed a reduced expression of the CD 41a, CD 42b and CD 61 receptors with increasing doses of NO, predominantly affecting the CD 41a receptor (-11% at 100 ppm and -20% at 640 ppm). This significant effect is in keeping with the observed NO-induced inhibition of platelet aggregation (-44% at 100 ppm) and the rise in platelet cGMP levels (+69% at 100 ppm). In patients without inhaled NO, the expression of CD 41a was slightly attenuated during cardiopulmonary bypass surgery (-15%) but increased significantly afterwards (2 h: +31%, 1st day: +129%, 2nd day: +120%, 3rd day: +111%). Comparable results were obtained regarding the other adhesion molecules CD 36, CD 42b and CD 61. In patients with inhaled NO the same pattern was observed and analysis of variance did not reveal any significant difference between both groups of patients. CONCLUSIONS: NO (> or = 100 ppm) decreases the expression of different platelet adhesion molecules and platelet aggregation, presumably via an increase in intracellular cGMP. However, due to the low dose range used in the clinical setting (1-40 ppm) this is clinically not relevant. Immediately after cardiopulmonary bypass surgery the expression of these adhesion molecules is reduced, but recovers on the 1st postoperative day.     

Development of a silicone hollow fiber membrane oxygenator for ECMO application

A new silicone hollow fiber membrane oxygenator for extracorporeal membrane oxygenation (ECMO) was developed using an ultrathin silicone hollow fiber, with a 300 microm outer diameter and a wall thickness of 50 microm. The hollow fibers were mechanically cross-wound on the flow distributor to achieve equal distribution of blood flow without changing the fiber shape. The housing, made of silicone coated acryl, was 236 mm long with an inner diameter of 60 mm. The surface area was 1.0 m2 for prototype 211, and 1.1 m2 for prototype 209. The silicone fiber length was 150 mm, and the silicone membrane packing density was 43% for prototype 211 and 36% for prototype 209. Prototype 211 has a priming volume of 208 ml, and prototype 209 has a priming volume of 228 ml. The prototype 211 oxygenator demonstrates a gas transfer rate of 120 +/- 5 ml/min (mean +/- SD) for O2 and 67 +/- 12 ml/min for CO2 under 2 L of blood flow and 4 L of O2 gas flow. Prototype 209 produced the same values. The blood side pressure drop was low compared with the silicone sheet oxygenator (Avecor, 1500ECMO). These results showed that this new oxygenator for ECMO had efficiency similar to the silicone sheet oxygenator that has a 50% larger surface area. These results suggest that the new generation oxygenator using an ultrathin silicone hollow fiber possesses sufficient gas transfer performance for long-term extracorporeal lung support.     

Development of a membrane oxygenator for ECMO using a novel fine silicone hollow fiber

One of the limitations of conventional silicone hollow fiber oxygenators compared with microporous membrane oxygenators is poor gas permeability. However, the silicone hollow fiber is free from plasma leakage, which is the major life limiting factor of the microporous membrane oxygenator. It has been difficult to fabricate a fine, thin hollow fiber for reduction of resistance to gas permeability because of the poor mechanical strength of conventional silicone materials. The authors developed a novel silicone material with sufficient mechanical strength, and a fine silicone hollow fiber with a diameter of 30 microns and wall thickness of 50 microns, which is approximately half that of a conventional silicone hollow fiber. Using this newly developed silicone hollow fiber, the authors developed a compact extracapillary flow membrane oxygenator. The oxygenator consists of fine silicone hollow fibers inserted in a housing made of polycarbonate. The housing is a cylindrical case, 20 cm long and 55 mm in inside diameter. The hollow fibers are cross-wound. The surface area of the membrane is 2.0 m2, and priming volume is 230 ml. Gas transfer performance of the newly developed oxygenator was evaluated by in vitro experiments. Oxygen and carbon dioxide transfer rates were 195 ml/min and 165 ml/min, at a blood flow rate 3 L/min. The novel silicone membrane oxygenator developed in this study can be used for extended duration in such applications as extracorporeal membrane oxygenation.     

Emboli from an extraluminal blood flow hollow fiber oxygenator with and without an arterial filter during cardiopulmonary bypass in a pig model

The effect of an arterial filter on visceral emboli was quantified with autologous indium-111 labeled platelets (INPLT) during cardiopulmonary bypass (CPB) in Yorkshire pigs. Biodistribution of INPLT was determined in 12 control pigs (30-35 kg, unoperated control [n = 6] and sham operated control [n = 6]). CPB was carried out with (n = 6) and without (n = 6) an arterial filter in 12 pigs at a flow rate of 2.5-3.5 L/min. Platelets labeled with In-111 tropolone (650-780 microCi) were injected intravenously 24 hr before CPB. All pigs were systemically heparinized (activated coagulation time > 400 sec); CPB was instituted with a roller pump, an extraluminal blood flow oxygenator (Bentley Univox, 1.8 m2), and an arterial filter (0.25 m2) and continued for 3 hr. Platelet kinetics, pooling, and counts were monitored by a Geiger probe and a Coulter counter. The thrombi in the oxygenator and arterial filter and emboli in viscera and brain were imaged with a gamma camera and measured with an ion chamber and gamma counter. Percentage of INPLT (mean +/- SD) in organs, tissues, and components of the circuit in four groups of pigs was calculated. Flow cytometry with antibodies to CD61 (GPIIIa) and CD62P (GMP-140: control) of porcine platelets was carried out with blood samples taken before, during, and after CPB for estimation of circulating platelet aggregates and platelet microparticles. Pulmonary, renal, cardiac, and cerebral emboli in pigs undergoing CPB with and without a filter were similar (p < 0.1). The amount of filter adherent thrombi was small (0.04 +/- 0.01%); oxygenator adherent thrombus in both groups was similar (p < 0.1). Emboli were found in the cerebral medulla, hippocampus, and posterior cerebral cortex in both groups. During CPB, the arterial filter functioned minimally as a trap for platelet thrombi detached from the oxygenator and circulating emboli. Flow cytometry of blood demonstrated the shift of equilibria from single platelets to platelet aggregates and microparticles during CPB and their gradual reversal to single platelets after CPB; the loosely adherent emboli disaggregated and further shifted these equilibria to single platelets and smaller aggregates, probably through the action of endogenous nitric oxide and prostacyclin. The emboli were trapped in organs and tissues and microparticles were sequestered by the reticuloendothelial system.     

Antioxidant status and alpha1-antiproteinase activity in subarachnoid hemorrhage patients

Grafting of polyethylene glycol chains onto cellulosic membrane can be expected to reduce the interaction between blood (plasma protein and cells) and the membrane surface. Alkylether carboxylic acid (PEG acid) grafted high flux cellulosic membranes for hemodialysis, in which the polyethylene glycol chain bears an alkyl group at one side and a carboxyl group at the other side, have been developed and evaluated. PEG acid-grafted high flux cellulosic membranes with various grafting amounts have been compared with respect to platelet adhesion, the contact phase of blood coagulation, and complement activation in vitro. A new method of quantitating platelet adhesion on hollow-fiber membrane surfaces has been developed, which is based on the determination of lactate dehydrogenase (LDH) activity after lysis of the adhered platelets. PEG acid-grafted high flux cellulosic membranes showed reduced platelet adhesion and complement activation effects in grafting amounts of 200 ppm or higher without detecting adverse effects up to grafting amounts of 850 ppm. The platelet adhesion of a PEG acid-grafted cellulosic membrane depends on both the flux and grafting amounts of the membrane. It is concluded that the grafting of PEG acid onto a cellulosic membrane improves its biocompatibility as evaluated in terms of platelet adhesion, complement activation, and thrombogenicity.     

Estrogen receptor alpha and beta expression in upper gastrointestinal tract with regulation of trefoil factor family 2 mRNA levels in ovariectomized rats

STUDY OBJECTIVE: To investigate the effect of short-term inhalation of nitric oxide (NO) on transpulmonary angiotensin II formation in patients with severe ARDS. DESIGN: Prospective, clinical study. SETTING: Anesthesiology ICU of a university hospital. PATIENTS: Ten ARDS patients who responded to inhalation of 100 ppm NO by decreasing their pulmonary vascular resistance (PVR) by at least 20 dyne x s x cm(-5) were included in the study. INTERVENTIONS AND MEASUREMENTS: In addition to standard treatment, the patients inhaled 0, 1, and 100 ppm NO in 20-min intervals. Fraction of inspired oxygen was 1.0. Hemodynamics were measured and recorded online. Mixed venous (pulmonary arterial catheter) and arterial (arterial catheter) blood samples were taken simultaneously for hormonal analyses at the end of each inhalation period. RESULTS: Pulmonary arterial pressure decreased from 33+/-2 mm Hg (0 ppm NO, mean+/-SEM) to 29+/-2 mm Hg (1 ppm NO, p<0.05), and to 27+/-2 mm Hg (100 ppm NO, p<0.05, vs 0 ppm). PVR decreased from 298+/-56 (0 ppm NO) to 243+/-45 dyne x s x cm(-5) (1 ppm NO, not significant [NS]), and to 197+/-34 dyne x s x cm(-5) (100 ppm NO, p<0.05, vs 0 ppm). Arterial oxygen pressure increased from 174+/-23 mm Hg (0 ppm NO) to 205+/-26 mm Hg (1 ppm NO, NS), and to 245+/-25 mm Hg (100 ppm NO, p <0.05, vs 0 ppm). Mean plasma angiotensin II concentrations were 85+/-20 (arterial) and 57+/-13 pg/mL (mixed venous) during 0 ppm NO and did not change during inhalation of 1 and 100 ppm NO. Mean transpulmonary plasma angiotensin II concentration gradient (=difference between arterial and mixed venous blood values) was 28+/-8 pg/mL (range, 0 to 69) during 0 ppm NO and did not change during inhalation of 1 and 100 ppm NO. Mean transpulmonary angiotensin II formation (transpulmonary angiotensin II gradient multiplied with the cardiac index) was 117+/-39 ng/min/m2 (range, 0 to 414) during 0 ppm NO and did not change during inhalation of 1 and 100 ppm NO. Mean arterial plasma cyclic guanosine monophosphate concentration was 11+/-2 pmol/mL (0 ppm NO), did not change during 1 ppm NO, and increased to 58+/-8 pmol/mL (100 ppm NO, p<0.05). Arterial plasma concentrations of aldosterone (142+/-47 pg/mL), atrial natriuretic peptide (114+/-34 pg/mL), angiotensin-converting enzyme (30+/-5 U/L), and plasma renin activity (94+/-26 ng/mL/h of angiotensin I) did not change. CONCLUSION: The decrease of PVR by short-term NO inhalation in ARDS patients was not accompanied by changes in transpulmonary angiotensin II formation. Our results do not support any relationship between transpulmonary angiotensin II formation and the decrease in PVR induced by inhaled NO.     

Inhaled nitric oxide inhibits human platelet aggregation, P-selectin expression, and fibrinogen binding in vitro and in vivo

BACKGROUND: Recent data suggest that inhaled NO can inhibit platelet aggregation. This study investigates whether inhaled NO affects the expression level and avidity of platelet membrane receptors that mediate platelet adhesion and aggregation. METHODS AND RESULTS: In 30 healthy volunteers, platelet-rich plasma was incubated with an air/5% CO2 mixture containing 0, 100, 450, and 884 ppm inhaled NO. ADP- and collagen-induced platelet aggregation, the membrane expression of P-selectin, and the binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor were determined before (t0) and during the 240 minutes of incubation. In addition, eight patients suffering from severe adult respiratory distress syndrome (ARDS) were investigated before and 120 minutes after the beginning of administration of 10 ppm inhaled NO. In vitro, NO led to a dose-dependent inhibition of both ADP-induced (3+/-3% at 884 ppm versus 70+/-6% at 0 ppm after 240 minutes; P<.001) and collagen-induced (13+/-5% versus 62+/-5%; P<.01) platelet aggregation. Furthermore, P-selectin expression (36+/-7% of t0 value; P<.01) and fibrinogen binding (33+/-11%; P<.01) were inhibited. In patients with ARDS, after two who did not respond to NO inhalation with an improvement in oxygenation had been excluded, an increase in plasma cGMP, prolongation of in vitro bleeding time, and inhibition of platelet aggregation and P-selectin expression were observed, and fibrinogen binding was also inhibited (19+/-7% versus 30+/-8%; P<.05). CONCLUSIONS: NO-dependent inhibition of platelet aggregation may be caused by a decrease in fibrinogen binding to the platelet GP IIb/IIIa receptor.     

Gas transfer and in vitro and in vivo blood compatibility of a fluorinated polyimide membrane with an ultrathin skin layer

The authors have synthesized fluorinated polyimides to develop a novel membrane oxygenator combining excellent gas transfer and blood compatibility. Gas exchange membranes of fluorinated polyimides prepared by a dry/wet process showed an asymmetric structure and consisted of an ultrathin and defect-free skin layer supported by a porous substructure. The asymmetric polyimide membranes never incurred plasma leakage because of the defect-free skin layer of the membrane surface. The calculated, apparent defect-free skin layer thickness of the asymmetric membrane was approximately 20 nm. Carbon dioxide and oxygen transfer rates through the membranes were dramatically enhanced because of the ultrathin skin layer and were 96 and 64 times larger than those determined in currently available oxygenator polymer membranes, such as polydimethylsiloxane (PDMS). For the evaluation of in vitro blood compatibility, platelet adhesion and plasma protein adsorption on the polyimide membranes were measured by using scanning electron microscopic examination and an amino acid analyzer. Deformation and aggregation of platelets adherent to the membranes were not observed, and the number of platelets was 1.6 micrograms/cm2, which was one-sixth less than the value measured in PDMS. For in vivo evaluation, the polymer tubes were implanted in the femoral vein of a mongrel dog for 7 days. Thrombus formation and fibrin were found on the surface of PDMS. However, thrombus formation was not observed on the polyimide. These results indicate that the fluorinated polyimides show excellent blood compatibility and are a promising membrane material for an oxygenator.     

Effects of nitric oxide inhalation on pulmonary serial vascular resistances in ARDS

The pulmonary vasculature site of action of nitric oxide (NO) in patients with acute respiratory distress syndrome (ARDS) is still unknown. Seven patients were studied during the early stage of ARDS. The bedside pulmonary artery single-occlusion technique, which allows estimation of the pulmonary capillary pressure (Pcap) and segmental pulmonary vascular resistance, was used without NO or with increasing inhaled NO concentrations (15 and 25 parts per million [ppm]). Systemic circulatory parameters remained unaltered during 15 ppm NO inhalation, whereas 25 ppm NO inhalation slightly decreased mean systemic arterial pressure from 76.7 +/- 5.1 (mean +/- SEM) to 69 +/- 5.2 mm Hg (p < 0.01). Mean pulmonary arterial pressure (Ppam) and mean pulmonary capillary pressure (Pcapm) fell during 25 ppm NO inhalation from 27.4 +/- 3.5 to 21 +/- 2.2 mm Hg (p < 0.001) and from 14.8 +/- 1.5 to 10.7 +/- 1.4 mm Hg (p < 0.001) respectively, the total pulmonary resistance decreased by 28% (p < 0.01). The resistance of the capillary-venous compartment fell during 25 ppm NO inhalation from 100 +/- 16 to 47 +/- 16 dyn x s x m(2) x cm(-5) (p < 0.01), whereas the pulmonary arterial resistance was unchanged. In these patients NO inhalation during the early stage of ARDS reduces selectively Ppam and Pcapm by decreasing the pulmonary capillary-venous resistance. This latter effect may reduce the filtration through the capillary bed and hence alveolar edema during ARDS.     

A new blood compatible and permselective hollow fiber membrane for hemodialysis

The authors have prepared a blood compatible and highly permselective hemodialysis membrane composed of polyether segmented nylon. This block copolymer was synthesized by polycondensation of bis-3-aminopropyl-poly(tetramethylene oxide) (PTMO) and poly(imino-1,3-bismethyl-cyclohexyl-iminoisophtharoyl) (NyBl) prepolymer obtained by polycondensation of 1,3-bis(aminomethyl)cyclohexane (B) and isophthalic acid (I). The molecular weight (MW) calculated from the number of end-groups was 16,000-21,000. In vitro blood compatibility was evaluated in terms of platelet adhesion onto the surface. PTMO-NyBl surfaces showed excellent platelet adhesion preventing properties. The PTMO-NyBl hollow fiber membrane was obtained by a dry-wet spinning process. The membranes had higher permeability coefficients for macromolecules ranging from MW 10,000 to 20,000 than polysulfone hollow fiber membrane (PS membrane), and had acceptably low albumin permeability for use as a dialysis membrane. The ex vivo blood compatibilities of PTMO-NyBl membrane and PS membrane were investigated by extracorporeal circulation in a pig model. The PTMO-NyBl membrane gave excellent results when assessing hemodialysis leukopenia, oxidative burst, and free platelet count decrease.     

Acute effects of inhaled nitric oxide in adult respiratory distress syndrome

This study evaluated the dose-response effect of inhaled nitric oxide (NO) on gas exchange, haemodynamics, and respiratory mechanics in patients with adult respiratory distress syndrome (ARDS). Of 19 consecutive ARDS patients on mechanical ventilation, eight (42%) responded to a test of 10 parts per million (ppm) NO inhalation with a 25% increase in arterial oxygen tension (Pa,O2,) over the baseline value. The eight NO-responders were extensively studied during administration of seven inhaled NO doses: 0.5, 1, 5, 10, 20, 50 and 100 ppm. Pulmonary pressure and pulmonary vascular resistance exhibited a dose-dependent decrease at NO doses of 0.5-5 ppm, with a plateau at higher doses. At all doses, inhaled NO improved O2 exchange via a reduction in venous admixture. On average, the increase in Pa,O2, was maximal at 5 ppm NO. Some patients, however, exhibited maximal improvement in Pa,O2 at 100 ppm NO. In all patients, the increase in arterial O2 content was maximal at 5 ppm NO. The lack of further increase in arterial O2 content above 5 ppm partly depended on an NO-induced increase in methaemoglobin. Respiratory mechanics were not affected by NO inhalation. In conclusion, NO doses < or =5 ppm are effective for optimal treatment both of hypoxaemia and of pulmonary hypertension in adult respiratory distress syndrome. Although NO doses as high as 100 ppm may further increase arterial oxygen tension, this effect may not lead to an improvement in arterial O2 content, due to the NO-induced increase in methaemoglobin. It is important to consider the effect of NO not only on arterial oxygen tension, but also on arterial O2 content for correct management of inhaled nitric oxide therapy.     

A baboon model for hematologic studies of cardiopulmonary bypass

Objective investigation of new inhibitors of blood protein or cellular systems that are activated during cardiopulmonary bypass (CPB) is impeded by the absence of a satisfactory animal model. Because most baboon hematologic proteins immunologically cross-react with those used for human assays, we developed a robust, reusable baboon model of CPB. Blood samples were obtained from adult baboons at six time intervals before, during, and after 60 minutes of partial CPB at 37 degrees C with peripheral cannulas. Both membrane (n = 7) and bubble oxygenators (n = 7) were investigated. We measured platelet and white blood cell counts; platelet response to adenosine diphosphate and release of beta-thromboglobulin; fibrinopeptide A, prothrombin fragment F1.2, thrombin-antithrombin complex, D-dimer, and plasmin-antiplasmin complex; activated complement (C3b/c and C4b/c); elastase-alpha1 proteinase inhibitor complex; and bleeding times. Adherent glycoprotein IIIa antigen in Triton X-100 washes of the perfusion circuit was also measured. Markers of baboon platelet, complement, and neutrophil activation and thrombosis significantly increased during CPB with bubble oxygenator systems but did not change appreciably in membrane oxygenator circuits. Markers of fibrinolysis, D-dimer, and plasmin-antiplasmin complex did not change with either oxygenator. The baboon model of CPB, when a bubble oxygenator is used, is a robust, reusable animal model for evaluating inhibitors of platelet, complement, and neutrophil activation and thrombosis during and after CPB.     

Optimal antagonism of GPIIb/IIIa favors platelet adhesion by inhibiting thrombus growth. An ex vivo capillary perfusion chamber study in the guinea pig

To evaluate the involvement of the glycoprotein (GP) IIb/IIIa-dependent process in platelet deposition and thrombus growth on capillaries coated with human type III collagen, the effects of incremental doses of Lamifiban, a potent specific synthetic GPIIb/IIIa antagonist, were studied in ex vivo capillary perfusion chambers using guinea pig blood. In this model, nonanticoagulated blood was perfused for 4.5 minutes at three shear rates: 100, 650, and 1600 s-1. Platelet deposition was quantified by computer-assisted morphometry and expressed as platelet adhesion (percentage of capillary surface covered with spread and contact platelets and platelets implicated in thrombus), mean thrombus height, and total thrombus cross-sectional area. In control untreated guinea pigs, platelet adhesion and thrombus height were 63% and 2.5 microns at 100 s-1, 60.5% and 13.8 microns at 650 s-1, and 45% and 28.1 microns at 1600 s-1, respectively. At 100 s-1, Lamifiban had no effect on platelet deposition at any of the three doses administered to the guinea pigs (0.3, 1, and 3 mg/kg). At 0.3 mg/kg and shear rates of 650 and 1600 s-1, Lamifiban had no effect on platelet adhesion or thrombus size, but at 1 and 3 mg/kg and shear rates of 650 and 1600 s-1, it significantly reduced thrombus size. At 1600 s-1, 1 mg/kg Lamifiban significantly increased platelet adhesion from 45% to 62.5%, whereas at 3 mg/kg it induced a significant overall decrease from 45% to 25% and qualitatively increased the ratio of contact to spread platelets. These data suggest that at high shear rates, GPIIb/IIIa participates in platelet spreading and that there is a balance between platelet involvement in adhesion to the thrombogenic surface and the growth of the already formed thrombus. This indicates that important clinical implications of an optimal therapeutic degree of GPIIb/IIIa antagonism could be expected.     

Experimental antitumour activity of S 16020-2 in a panel of human tumours

OBJECTIVE: To compare the effects of inhaled nitric oxide (NO) and extracorporeal membrane oxygenation (ECMO) on oxygenation, hemodynamics, and lymphatic drainage in an oleic acid lung injury model in sheep. DESIGN: Prospective, randomized study. SETTING: Animal research laboratory. ANIMALS: Thirty female sheep, weighing 35 to 40 kg. INTERVENTIONS: Acute lung injury was induced by central venous injection of oleic acid (0.5 mL/kg body weight). A chronic lymph fistula had been prepared through a right thoracotomy 3 days before the experiment. Animals were assigned randomly to the NO group (n = 14) or the ECMO group (n = 16). When a lung injury score of > 2.5 was achieved, the animals were given NO in dosage increments of 2, 5, 10, 20, and 40 parts per million (ppm), or placed on ECMO with an FIO2 of 0.21 (ECMO-21) and then 1.0 (ECMO-100) at the oxygenator. Mechanical ventilator parameters were kept constant to isolate the effects of NO and ECMO on systemic and pulmonary hemodynamics, cardiac output, oxygenation parameters, lymph/plasma protein ratio, and lymph flow. Measurements and calculations were performed after 1 hr at each individual step of NO concentration or FIO2. MEASUREMENTS AND MAIN RESULTS: In the ECMO group, PVRI and MPAP did not change and were significantly different from the NO group. In the NO group, there was a dose-dependent decrease in venous admixture, maximal at 10 ppm NO and decreasing from 40 +/- 6% to 23 +/- 10% (p < .05). This decrease was significantly different from the ECMO group, where there was no change. There was a significant increase in PaO2/FIO2 in the NO group, maximal at 10 ppm NO (84 +/- 11 to 210 +/- 90, p < .05), but a greater increase in PaO2/FIO2 on ECMO-21 (81 +/- 14 to 265 +/- 63) and a further increase on ECMO-100 (398 +/- 100) (p < .05). The lymph/plasma protein ratio remained unchanged in both groups after induction of lung injury by oleic acid. However, lymph flow decreased by 11 +/- 6% in the NO group, whereas it increased by 14 +/- 17% in the ECMO group (p < .05). CONCLUSIONS: In an oleic acid-induced sheep model of acute lung injury, there were significant differences between the effects of NO and ECMO on acute pulmonary hypertension, hypoxemia, hypercarbia, and lymph flow. NO significantly decreases pulmonary hypertension, whereas pulmonary hemodynamics were not substantially affected by ECMO. Both interventions reversed hypoxemia, but ECMO did so to a greater degree, and only ECMO improved hypercarbia. Only NO decreased lymph flow, possibly as an effect of decreased microvascular filtration pressure. This study did not attempt to evaluate the impact of these interventions on ventilatory requirements, barotrauma, or outcome. However, this model suggests that NO therapy may moderate pulmonary hypertension and improve lymph flow in acute lung injury. Clinical studies are needed to assess whether NO therapy might be beneficial in treatment of severe acute lung injury in older children and adults.     

Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model)

Carbohydrate moieties on leukocytes adhere to activated platelets via P-selectin under static binding condition studies. We characterize polymorphonuclear cell (PMN) surface interactions with surface adherent platelets and the PMNs response, under physiologic flow conditions corresponding to a shear of 100 s-1, in an in vitro flow chamber. Fluorescent labeled PMNs with red blood cells were drawn through a transparent flow channel and visually quantitated over 30 minutes, interacting with a confluent monolayer of activated, shear-spread platelets expressing P-selectin. PMN adhesion was saturable (2,250 +/- 350/mm2), and time and cation (Ca2+, Mg2+) dependent, and PMNs did not bind to the experimental surface in the absence of a platelet monolayer. P-selectin antibodies completely abolished PMN adhesion in a concentration-dependent manner with half inhibition at 70 micrograms/mL. Antibodies to a putative P-selectin receptor CD15 (80H5 and MMA) maximally inhibited PMN adhesion by 73% and 10%, respectively. Adherent PMNs appeared morphologically activated and flow cytometric analysis of adherent PMNs confirmed activation because CD11b and CD18 surface expression was upregulated (100% and 27%, respectively), whereas L-selectin was downregulated (55%) compared with control nonadherent PMNs. In the presence of the metabolic inhibitor sodium azide (0.02% and 0.1%) there was a 23% +/- 9% and 51% +/- 3% decrease, respectively, in PMN adhesion at 100 s-1. Thus, P-selectin is required for PMN adhesion to a pathophysiologic surface of activated adherent platelets at physiologic shear rates. Furthermore, a secondary step involving PMN activation after platelet binding appears necessary for complete (irreversible) adhesion to occur. This unique flow cell provides a model to explore, under controlled conditions, biologic mechanisms and ligands involved in leukocyte-platelet binding that play important roles in PMN localization at sites of thrombosis and vascular injury.     

Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen

Sera of 34 patients with heparin-associated thrombocytopenia (HAT), giving a positive result in the serotonin release assay (SRA), were assessed in a platelet factor 4 (PF4)/heparin ELISA. Three sera revealing indeterminate results in the SRA and 10 control sera were also investigated. Both tests correlated closely (Kappa 0.742; p = 2.67 x 10(-7)), but one positive serum in the SRA was negative in the pF4/heparin ELISA. We have isolated the HAT antibodies by absorbtion and elution of HAT sera using endothelial cells (HUVEC). Eluates gave similar results as the sera in the PF4/heparin ELISA (Kappa 0.837, p = 9.26 x 10(-9)), and they also correlated very closely with the SRA (Kappa 0.888; p = 8.89 x 10(-10)). This demonstrates that HAT antibodies bind to the same epitope on platelets and on endothelial cells. High heparin concentrations released PF4 in a dose dependent manner from microtiter plates if PF4/heparin, but not if PF4 alone, was covalently linked. Concomitant to the release of PF4, binding of HAT antibodies to PF4/heparin decreased, as indicated by the median optical density (OD) values of OD 0.88 in the presence of buffer compared to OD 0.181 in the presence of 100 IU/ml heparin. The latter values were similar to those obtained when plates were coated with PF4 alone (median OD 0.203). Binding of three eluates was not inhibited by high heparin concentrations and they reacted also with PF4 alone. We conclude that multimolecular PF4/heparin complexes represent the major antigen in HAT. These multimolecular complexes might present several epitopes and form immune complexes after HAT antibody binding which activate platelets via the FcRII. In a few cases, PF4 alone can be recognized by the antibody. However, there is also evidence that other molecules might be involved in some patients.     

Initial interactions of platelets and plasma proteins in flowing non-anticoagulated human blood with the artificial surfaces Dacron and PTFE

The purpose of the present study was to investigate and to compare the interactions of platelets and proteins in flowing non-anticoagulated human blood with the biomaterials polyethylene-terephthalate (Dacron) and polytetrafluoroethylene (PTFE, Teflon). The respective biomaterials were positioned in a parallel-plate perfusion chamber, and exposed to flowing blood for 5 min at wall shear rates characteristic for veins (100/s), medium sized (650/s) and moderately stenosed arteries (2,600/s). Blood-material interactions were morphologically quantified as platelet-surface adhesion, thrombus volume and fibrin deposition. Platelet adhesion to Dacron was highest at the lowest shear rate (13%) and decreased with increasing shear (4% at 2600/s). In contrast, platelet adhesion to PTFE was shear rate independent (17-19%), and significantly higher than the adhesion to Dacron at 2600/s (P < 0.05). A hallmark of the platelets adherent to PTFE and Dacron was the large percentage of platelets not spread out on the surface. This indicates that both materials were poor platelet activators, even though immunostaining demonstrated the adsorption of the platelet adhesive proteins von Willebrand factor and fibronectin. Adsorption of fibrinogen was also prevailing on both materials. Virtually no thrombi formed on Dacron, while a few small platelet thrombi were observed on PTFE. Less than 1% of the Dacron and PTFE surfaces were covered by fibrin, irrespective of the shear rate. Thus, Dacron and PTFE interact differently with flowing non-anticoagulated human blood, and Dacron is apparently the least thrombogenic material.     

Treatment of septic shock in rats with nitric oxide synthase inhibitors and inhaled nitric oxide

OBJECTIVE: To evaluate the effect of treatment with a combination of nitric oxide synthase inhibitors and inhaled nitric oxide on systemic hypotension during sepsis. DESIGN: Prospective, randomized, controlled study on anesthetized animals. SETTING: A cardiopulmonary research laboratory. SUBJECTS: Forty-seven male adult Sprague-Dawley rats. INTERVENTIONS: Animals were anesthetized, mechanically ventilated with room air, and randomized into six groups: a) the control group (C, n=6) received normal saline infusion; b) the endotoxin-treated group received 100 mg/kg i.v. of Escherichia coli lipopolysaccharide (LPS, n=9); c) the third group received LPS, and 1 hr later the animals were treated with 100 mg/kg i.v. Nw-nitro-L-arginine (LNA, n=9); d) the fourth group received LPS, and after 1 hr, the animals were treated with 100 mg/kg i.v. aminoguanidine (AG, n=9); e) the fifth group received LPS and 1 hr later was treated with LNA plus 1 ppm inhaled nitric oxide (LNA+NO, n=7); f) the sixth group received LPS and 1 hr later was treated with aminoguanidine plus inhaled NO (AG+NO, n=7). Inhaled NO was administered continuously until the end of the experiment. MEASUREMENTS AND MAIN RESULTS: Systemic mean blood pressure (MAP) was monitored through a catheter in the carotid artery. Mean exhaled NO (ENO) was measured before LPS (T0) and every 30 mins thereafter for 5 hrs. Arterial blood gases and pH were measured every 30 mins for the first 2 hrs and then every hour. No attempt was made to regulate the animal body temperature. All the rats became equally hypothermic (28.9+/-1.2 degrees C [SEM]) at the end of the experiment. In the control group, blood pressure and pH remained stable for the duration of the experiment, however, ENO increased gradually from 1.3+/-0.7 to 17.6+/-3.1 ppb after 5 hrs (p< .05). In the LPS treated rats, MAP decreased in the first 30 mins and then remained stable for 5 hrs. The decrease in MAP was associated with a gradual increase in ENO, which was significant after 180 mins (58.9+/-16.6 ppb) and reached 95.3+/-27.5 ppb after 5 hrs (p< .05). LNA and AG prevented the increase in ENO after LPS to the level in the control group. AG caused a partial reversal of systemic hypotension, which lasted for the duration of the experiment. LNA reversed systemic hypotension almost completely but only transiently for 1 hr, and caused severe metabolic acidosis in all animals. The co-administration of NO with AG had no added benefits on MAP and pH. In contrast, NO inhalation increased the duration of the reversal in MAP after LNA, alleviated the degree of acidosis, and decreased the mortality rate (from 55% to 29%). CONCLUSIONS: In this animal model, LPS-induced hypotension was alleviated slightly and durably after AG, but only transiently after LNA. Furthermore, co-administration of NO with AG had no added benefits but alleviated the severity of metabolic acidosis and mortality after LNA. We conclude that nitric oxide synthase (NOS) inhibitors, given as a single large bolus in the early phase of sepsis, can exhibit some beneficial effects. Administration of inhaled NO with NOS inhibitors provided more benefits in some conditions and therefore may be a useful therapeutic combination in sepsis. NO production in sepsis does not seem to be a primary cause of systemic hypotension. Other factors are likely to have a major role.