The effect of isoflurane on neutrophil selectin and beta(2)-integrin activation in vitro

Isoflurane is reported to reduce ischemia-reperfusion injury. Lower expression of CD11b may be responsible for attenuated postischemic neutrophil adhesion to vascular endothelium. However, neutrophil adhesion to vascular endothelium is a multistep process involving several selectins and beta(2)-integrins. Therefore, we assessed whether isoflurane affects the activation of the selectins P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin and the beta(2)-integrins CD11a and CD11b. Whole blood was incubated for 60 min with 0.5 or 1 minimum alveolar anesthetic concentration (MAC) isoflurane. After incubation, neutrophils were activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol-12-myristate-13-acetate (PMA). Activation of adhesion molecules was evaluated via flow cytometry, and 1 MAC isoflurane reduced the expression of CD11a in the unstimulated samples. After stimulation with FMLP and PMA, shedding of L-selectin was lower in the presence of isoflurane. Furthermore, 1 MAC isoflurane reduced FMLP-induced activation of CD11a and CD11b compared with unexposed blood samples. These results demonstrate that isoflurane affects the activation of three adhesion molecules involved in the multistep process of neutrophil recruitment. First, isoflurane inhibits the activation of L-selectin, which mediates the neutrophil tethering and rolling on the vascular endothelium. Second, isoflurane attenuates the activation of both beta(2)-integrins-CD11a and CD11b-which mediate firm adhesion and transendothelial migration. IMPLICATIONS: Adhesion of neutrophils to endothelial cells in reperfusion injury is mediated by different adhesion molecules. This study indicates that the inhibiting effect of isoflurane on neutrophil recruitment may be mediated by a decreased activation of the L-selectin and by attenuation of the activation of the beta(2)-integrins CD11a and CD11b.     

Inhibition of platelet receptors involved in neutrophil-platelet interaction in model cardiopulmonary bypass

We investigated the interactions between neutrophils, platelets, and artificial surfaces, and whether blocking of relevant receptors on platelets reduced unwanted activation responses in model cardiopulmonary bypass. Isolated neutrophils and platelets resuspended in heparin-anticoagulated plasma were recirculated with and without blocking antibodies to CD62P, CD42b, or junctional adhesion molecule C (JAM-C) in polyvinyl chloride tubing using a roller pump. Platelet adhesion to the tubing was inhibited by anti-CD42b and anti-CD62P, and adhesion of neutrophils by anti-JAM-C. Formation of platelet-neutrophil and platelet aggregates was reduced by anti-CD62P. Anti-JAM-C decreased platelet-neutrophil aggregation at low concentrations and platelet macroaggregates at high concentrations. Anti-CD62P increased neutrophil CD11b expression but not degranulation. Anti-JAM-C substantially increased neutrophil degranulation and slightly increased CD11b expression. Platelet activation increased when CD62P was blocked and decreased with anti-CD42b antibody. High-dose anti-JAM-C reduced platelet activation. In conclusion, inhibiting platelet and neutrophil-platelet interactions had useful effects but no single blocking antibody seemed capable of inducing only beneficial effects.     

Functional and phenotypic changes in polymorphonuclear neutrophils induced by catecholamines

PURPOSE: To elucidate differential functional and phenotypic changes in response to relevant catecholamines, the generation of oxidative free radicals by PMN, and changes in the expression of L-selectin and Mac-1 on the surface of PMN were examined in the presence of epinephrine, norepinephrine and dopamine in physiological and pharmacological concentrations. MATERIALS AND METHODS: Human polymorphonuclear neutrophils were obtained from healthy donors and pretreated with 0.5 nM or 500 nM epinephrine; 1.18 nM or 1 180 nM norepinephrine; or 0.26 nM or 261 nM dopamine, followed by stimulation with FMLP. Stimulated neutrophils were incubated with antibodies against CD 11 b or CD 62 l and assessed by flow cytometry. Additional probes were assessed by flow cytometry for the generation of oxidative free radicals. RESULTS: All catecholamines in high concentration inhibited the suppression of CD 62 l expression and CD 11 b upregulation following stimulation with FMLP. A high concentration of epinephrine suppresses generation of oxidative free radicals. CONCLUSIONS: The effect of catecholamines on the expression of CD 62 l explains the increased expression of L-selection on PMN observed after trauma. The suppression of CD 11 b reduces leukocyte adherence and consecutive abnormalities in microvascular flow. Epinephrine inhibits the generation of oxidative free radicals by PMN with potentially detrimental effects with respect to bacterial clearance.     

Effect of halothane and isoflurane on binding of ADP- and TRAP-6- activated platelets to leukocytes in Whole blood.

BACKGROUND: Adhesion of activated platelets to neutrophils and monocytes has an important role in the regulation of inflammatory processes. This study investigates whether halothane and isoflurane affect binding of activated platelets to leukocytes in human whole blood. METHODS: Citrated whole blood was incubated for 60 min with either 1 or 2 minimum alveolar concentration (MAC) halothane or isoflurane. After stimulation with adenosine-5-diphosphate (ADP) or the thrombin receptor agonist protein TRAP-6, platelet-leukocyte adhesion and surface expression of CD62P on platelets were evaluated by flow cytometry. RESULTS: Halothane led to an inhibition of agonist-induced adhesion of activated platelets to neutrophils and monocytes. One MAC halothane reduced the formation of TRAP-6-induced platelet-monocyte conjugates. After exposure to 2 MAC halothane, agonist-induced platelet-monocyte and platelet-neutrophil adhesion were inhibited. Surface expression of CD62P on ADP- and TRAP-6-stimulated platelets were significantly reduced after 1 and 2 MAC halothane. After 2 MAC isoflurane, the authors observed an increase of the percentage of lymphocytes with bound platelets after activation with ADP. The percentage of neutrophils with bound platelets after activation with ADP or TRAP-6 was also increased in this group. Two MAC isoflurane led to an increase of the percentage of platelets expressing CD62P in the unstimulated and TRAP-6 stimulated samples, and of the amount of CD62P epitopes on the surface of platelets in the ADP-stimulated samples. CONCLUSION: This study indicates that halothane inhibits, whereas isoflurane enhances, adhesion of agonist-activated platelets to leukocytes. Interaction of both anesthetics with the expression of CD62P on platelets contribute to theses effects.     

Differential platelet receptor expression following hydroxyethyl starch infusion in thrombocytopaenic orthotopic liver transplantation recipients

BACKGROUND AND OBJECTIVE: Platelet function abnormalities influence the haemostatic defect in patients with liver failure. Patients after orthotopic liver transplantation present thrombocytopaenia associated with bleeding problems, which may be aggravated by the interaction of hydroxyethyl starches with platelets. METHODS: From 12 patients after liver transplantation venous blood samples (3 mL) were taken before, 20 and 120 min after infusion of hydroxyethyl starch of medium molecular weight (200 kDa/0.5) 6% 10 mL kg(-1) over a period of 30 min. Surface expression of glycoprotein IIb/IIIa and P-selectin were quantified by flow cytometry as well as the percentage of platelet-leucocyte complexes. RESULTS: A significant decrease of P-selectin expression following administration of hydroxyethyl starch after 120 min (89.1 +/- 4.2%, P = 0.029) and a corresponding significant reduction in the formation of platelet-monocyte complexes (81.1 +/- 7.8%, P = 0.001) were observed. There was no alteration in the glycoprotein IIb/IIIa expression after hydroxyethyl starch infusion. CONCLUSIONS: Infusion of hydroxyethyl starch 200 kDa/0.5 in clinically relevant doses does not alter glycoprotein IIb/IIIa expression in thrombocytopaenic patients with pre-existing platelet dysfunction after orthotopic liver transplantation. Accordingly, infusion of hydroxyethyl starch may have a beneficial effect on microvascular graft perfusion through the resulting haemodilution and reduced P-selectin expression with subsequent reduced leucocyte-platelet complexes and endothelial adhesion.     

Stimulation of neutrophil integrin expression during coronary artery bypass grafting: comparison of crystalloid and blood cardioplegic solutions

OBJECTIVES: This study was designed (1) to evaluate the influence of plasma obtained from patients undergoing coronary artery bypass grafting on L-selectin, CD11b, and CD18 expression on human neutrophils and (2) to determine the influence of the use of crystalloid or blood cardioplegia during bypass grafting on plasma-mediated expression of adhesion molecules on polymorphonuclear neutrophils.Patients and methods: Patients undergoing coronary artery bypass grafting were divided into 2 groups to receive crystalloid or blood cardioplegic solutions. Peripheral vein, radial artery, and coronary sinus blood samples were drawn at aortic crossclamping, aortic crossclamp release, and 30 minutes after reperfusion. Human neutrophils were incubated with patients' plasma, and the expression of CD11b, CD18, and L-selectin was determined with flow cytometry. RESULTS: In patients receiving crystalloid cardioplegic solutions, plasma samples collected from the coronary sinus at aortic clamp release and 30 minutes thereafter induced significantly higher expression of neutrophil CD11b and CD18 than plasma samples obtained from a peripheral vein or artery at the same time points. The expression of L-selectin on polymorphonuclear neutrophils was significantly reduced with plasma obtained 30 minutes after reperfusion as compared with samples collected at aortic crossclamp release. In the group receiving blood cardioplegia, no significant differences in CD11b, CD18, or L-selectin expression were found. CONCLUSIONS: (1) Ischemia/reperfusion after coronary artery bypass grafting is associated with the release of factors capable of neutrophil activation from myocardium into the circulating blood. (2) The release of soluble stimuli for neutrophils during bypass grafting may be modified by the cardioplegic solution.     

Phosphodiesterase III inhibition affects platelet-monocyte aggregate formation depending on the axis of stimulation

OBJECTIVE: The purpose of this study was to investigate the effect of the phosphodiesterase (PDE) type 3 inhibitor milrinone on the adhesion of platelets to monocytes in vitro. DESIGN: Prospective study. SETTING: University experimental laboratory. PARTICIPANTS: Ten healthy volunteers. INTERVENTIONS: Whole blood was incubated with 1, 10, or 100 micromol/L of milrinone. After stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or adenosine-5-diphosphate (ADP), platelet-monocyte adhesion and CD11b, PSGL-1, GPIIb/IIIa, and P-selectin expression were measured by flow cytometry. MEASUREMENTS AND RESULTS: The formation of platelet-monocyte conjugates after PDE3 inhibition depended on the type of stimulation. In unstimulated and FMLP-stimulated blood platelet monocytes, aggregation was enhanced by increasing concentrations of milrinone. This augmentation was accompanied by a rise in P-selectin expression in platelets. In ADP-stimulated blood the number of platelet-monocyte aggregates decreased with increasing concentrations of milrinone. Concurrent with the reported antiinflammatory properties of PDE-inhibition, an inhibition of CD11b expression was found in monocytes after stimulation with FMLP. In contrast, in unstimulated samples lower concentrations of milrinone caused an increase in CD11b. CONCLUSIONS: These findings suggest that the effects of PDE3 inhibition on platelets and monocytes are modified by the type of stimulation and only partially suppress the inflammatory response of platelets and monocytes. The increase in platelet-monocyte conjugates in unstimulated and FMLP-stimulated blood suggested that PDE3 inhibition may also trigger proinflammatory reactions.     

Effect of isoflurane on monocyte adhesion molecule expression in human whole blood

BACKGROUND: Recruitment of monocytes to inflamed tissue is a crucial step in the acute inflammatory reaction. Adherence of monocytes to endothelial cells followed by transmigration depends on monocyte surface adhesion molecules, inflammatory cytokines and chemoattractant chemokines. In the present study, we determined the effect of isoflurane on monocyte adhesion receptor expression in vitro. METHODS: Citrated whole blood was incubated for 60 min with either 0.5 or 1 MAC isoflurane. In unstimulated blood samples and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) monocyte cell-surface expression of the selectins PSGL-1 and L-selectin, and the beta2-integrins CD11a and CD11b were evaluated by flow cytometry. RESULTS: Isoflurane reduced significantly the expression of PSGL-1 on unstimulated monocytes, whereas the remaining selectins and beta2-integrins were not affected. At both concentrations, the FMLP-induced removal of PSGL-1 from the monocyte surface was increased. Furthermore, at 1 MAC isoflurane the FMLP-induced increase in CD11a expression was significantly inhibited. The surface expression of L-selectin and CD11b was not affected following exposure to isoflurane. CONCLUSION: Isoflurane increases the removal of the selectin PSGL-1 from the monocyte surface. Since PSGL-1 is important during the initial step of monocyte adhesion to endothelial P-selectin, the decrease in monocyte surface PSGL-1 may have profound effects on monocyte-endothelial interactions. Furthermore, the effects of isoflurane on monocyte adhesion molecule expression are different from those reported for neutrophils.     

Effect of Halothane and Isoflurane on Binding of ADP- and TRAP-6- activated Platelets to Leukocytes in Whole Blood

Background: Adhesion of activated platelets to neutrophils and monocytes has an important role in the regulation of inflammatory processes. This study investigates whether halothane and isoflurane affect binding of activated platelets to leukocytes in human whole blood.

Methods: Citrated whole blood was incubated for 60 min with either 1 or 2 minimum alveolar concentration (MAC) halothane or isoflurane. After stimulation with adenosine-5-diphosphate (ADP) or the thrombin receptor agonist protein TRAP-6, platelet-leukocyte adhesion and surface expression of CD62P on platelets were evaluated by flow cytometry.

Results: Halothane led to an inhibition of agonist-induced adhesion of activated platelets to neutrophils and monocytes. One MAC halothane reduced the formation of TRAP-6-induced platelet-monocyte conjugates. After exposure to 2 MAC halothane, agonist-induced platelet-monocyte and platelet-neutrophil adhesion were inhibited. Surface expression of CD62P on ADP- and TRAP-6-stimulated platelets were significantly reduced after 1 and 2 MAC halothane. After 2 MAC isoflurane, the authors observed an increase of the percentage of lymphocytes with bound platelets after activation with ADP. The percentage of neutrophils with bound platelets after activation with ADP or TRAP-6 was also increased in this group. Two MAC isoflurane led to an increase of the percentage of platelets expressing CD62P in the unstimulated and TRAP-6 stimulated samples, and of the amount of CD62P epitopes on the surface of platelets in the ADP-stimulated samples.     

Xenon modulates neutrophil adhesion molecule expression in vitro

BACKGROUND AND OBJECTIVE: Xenon reduces the infarct size after regional ischaemia in the rabbit heart in vivo, but the underlying mechanisms are unknown. Since adhesion molecules on neutrophils are closely involved in the pathophysiology of ischaemia/reperfusion injury and modulation of neutrophil function, we investigated the effect of xenon on neutrophil adhesion molecule expression in vitro. METHODS: Freshly isolated neutrophils were incubated with 30% or 60% xenon for 60 min. In unstimulated and after stimulation with either N-formyl-methionyl-leucyl-phenylalanine or phorbol-12-myristate-13-acetate neutrophil surface expression of PSGL-1, L-selectin, CD11a and CD11b were measured by flow cytometry. RESULTS: At both concentrations, xenon reduced the surface expression of PSGL-1 by 10% (P < 0.05), and of L-selectin by 15% (P < 0.05) in the 60% xenon group. Furthermore, N-formyl-methionyl-leucyl-phenylalanine activated neutrophils showed an increased removal of L-selectin from the neutrophil surface following incubation with xenon (30% compared to controls, P < 0.05). Neutrophil beta2-integrin expression was not altered by xenon. CONCLUSIONS: Xenon increases the removal of the selectins PSGL-1 and L-selectin from the neutrophil surface in vitro. Since both selectins are involved in the initial contact between neutrophils and endothelial cells, xenon may affect neutrophil adhesion to endothelium during ischaemia/reperfusion injury. However, because the beta2-integrin expression was unaffected by xenon, further investigations are required to clarify whether xenon may modulate neutrophil transmigration through endothelial cells in vivo.     

Activated neutrophils and platelet microaggregates impede blood filterability through microchannels during simulated extracorporeal circulation

BACKGROUND: Neutrophil sequestration and platelet microaggregates in organ capillaries have been implicated in the inflammatory response associated with cardiopulmonary bypass. We examined the filterability of neutrophils and platelet microaggregates through silicon microchannels during simulated extracorporeal circulation. We hypothesize that blood contact with artificial surfaces over time decreases the ability of neutrophils, platelets, and their aggregates to pass through microchannels. METHODS: Fresh human blood from donors (n = 9) was recirculated for 120 minutes in a simulated extracorporeal circuit. Blood samples were obtained from a donor at 0, 30, 60, and 120 minutes of recirculation. The microchannel transit time and the flow behavior of blood cells were evaluated by a silicon microchannel array flow analyzer. CD11b, L-selectin, and F-actin of neutrophils were measured by flow cytometry. Neutrophil and platelet counts and platelet aggregation to adenosine diphosphate were measured. RESULTS: The microchannel transit time was prolonged during recirculation, reaching 185.9% +/- 25.6% of baseline at 120 minutes. The video microscope showed that neutrophils and platelet microaggregates plugged the microchannels. CD11b, L-selectin, and F-actin levels changed significantly by 120 minutes. Platelet counts decreased and platelet aggregability was attenuated. CONCLUSIONS: Simulated extracorporeal circulation caused a progressive loss in the ability of neutrophils, platelets, and their aggregates to pass through the microchannels independent of neutrophil adhesion molecule expression.     

Pharmacologic platelet anesthesia by glycoprotein IIb/IIIa complex antagonist and argatroban during in vitro extracorporeal circulation

OBJECTIVE: Contact between blood and the synthetic surfaces of a cardiopulmonary bypass circuit leads to platelet activation, and resultant platelet dysfunction contributes to postoperative bleeding. We compared the effects of various platelet inhibitors on preservation of platelet function during simulated cardiopulmonary bypass circulation. METHODS: Fresh human blood was recirculated in an in vitro cardiopulmonary bypass model circuit. We measured various platelet activation markers including expressions of PAC-1 and P-selectin, annexin V binding, and microparticle formations by means of whole-blood flow cytometry. RESULTS: Two types of glycoprotein IIb/IIIa complex antagonists, peptide-mimetic FK633 and abciximab and prostaglandin E(1), significantly prevented platelet loss and the increase in binding of PAC-1, an antibody specific for fibrinogen receptor on activated platelets, during extracorporeal circulation of heparinized blood. These antagonists significantly suppressed but did not abolish P-selectin expression, annexin V binding, and microparticle formation. Anti-von Willebrand factor monoclonal antibody and aurin tricarboxylic acid (an inhibitor of glycoprotein Ib) had no effect on platelet activation during simulated cardiopulmonary bypass circulation. These data suggest that inhibition of fibrinogen binding glycoprotein IIb/IIIa complex is partly effective in attenuating platelet activation in a heparinized cardiopulmonary bypass model circuit. The direct thrombin inhibitor argatroban prevented platelet loss and expression of P-selectin significantly more than did heparin. A combination of FK633 with argatroban as a substitute for heparin further prevented platelet loss and platelet secretion during simulated cardiopulmonary bypass circulation, although the inhibition of microparticle formation was less. CONCLUSION: The inhibition of both platelet adhesion and thrombin may be effective to preserve platelet number and function during cardiopulmonary bypass circulation.     

Leukocyte depletion attenuates expression of neutrophil adhesion molecules during cardiopulmonary bypass in human beings

BACKGROUND: On the basis of scanty information, the effects of a leukocyte filter during cardiac operations in human beings have been examined from the viewpoint of the expression of neutrophil adhesion molecules. This study was therefore designed to determine whether leukocyte depletion during cardiopulmonary bypass may interfere with neutrophil adhesion properties. METHODS: Twenty-four patients undergoing elective heart operations were randomly allocated to a leukocyte-depletion group or a control group. Blood samples were collected at 7 points: before sternotomy, at 10, 30, and 60 minutes of cardiopulmonary bypass, at termination of cardiopulmonary bypass, 5 minutes after protamine administration, and 2 hours after cardiopulmonary bypass. The expression of the neutrophil surface adhesion molecules L-selectin and beta2-integrins was determined by flow cytometric analysis in whole blood. RESULTS: (1) CD11a expression did not change significantly in either group. There were no significant differences between control and leukocyte-depletion groups (P =.63). (2) There was a significantly higher expression of CD11b on the neutrophils during cardiopulmonary bypass in the control group than in the leukocyte-depletion group (P =.01). (3) CD11c expression was initially up-regulated from the onset of cardiopulmonary bypass, reaching a peak at 60 minutes after bypass in the control group (P =.02). The expression of CD11c did not differ significantly between groups (P =.23). (4) L-selectin expression was significantly lower in the leukocyte-depletion group than in the control group (P =.03). CONCLUSIONS: The major findings of the present study in human subjects undergoing elective cardiac operations with cardiopulmonary bypass are as follows: (1) bypass was associated with an up-regulation of the adhesion molecules L-selectin, CD11b, and CD11c but with no significant change in CD11a expression, and (2) the clinical use of a leukocyte-depleting filter could down-regulate the expression of CD11b and L-selectin.     

Influence of polyclonal ATGs on expression of adhesion molecules: an experimental study

BACKGROUND: The aim of our study was to assess the influence of polyclonal antithymocyte globulins (ATGs) on the expression of adhesion molecules on lymphocytes, neutrophils, and thrombocytes by means of flow cytometry. ATGs are employed in various regimens for solid organ transplantation. Immunosuppression with ATGs may influence the expression of adhesion molecules on thrombocytes, lymphocytes, and neutrophils due to nonspecific antibodies directed against myeloid and nonmyeloid cells. MATERIAL AND METHODS: Depletion, activation, and expression of adhesion molecules on thrombocytes (CD41, CD42, CD62p and CD107a), neutrophils, and lymphocytes (CD11, CD18, CD62L) were studied in vitro in whole blood of healthy volunteers by means of flow cytometry after incubation with different dosages of three ATGs. RESULTS: Our data showed no ATG-mediated cytotoxic activity against platelets. ATGs were able, however, to induce activation of platelets through increased expression of P-selectin and hLAMP-1. ATGs also influenced the expression of adhesion molecules on lymphocytes and neutrophils by reducing the expression of CD62L. Furthermore, the effects of ATG on CD11/CD18 were dependent on the dosage and type of ATG. CONCLUSION: Polyclonal ATGs induced expression of adhesion molecules and activation of unstimulated thrombocytes as well as reduced the expression of adhesion molecules on lymphocytes and neutrophils. Increased adhesion of thrombocytes may be responsible for the undesirable side effects observed in clinical practice such as thrombocytopenia. However, reduction in the expression of adhesion molecules on lymphocytes and neutrophils may decrease the effects of ischemia/reperfusion injury.     

Human neutrophil cathepsin G is a potent platelet activator

Purpose: Neutrophil activation has been implicated in the pathophysiologic condition of ischemia-reperfusion injury, the formation of arterial aneurysms, the progression of myocardial ischemia, and the initiation of deep venous thrombosis. Activated neutrophils release cathepsin G, a serine protease, from their granules, which may cause platelet activation that leads to intravascular thrombosis, tissue infarction, and systemic release of the thrombogenic products of platelet granules. This study used flow cytometry to quantify the extent of cathepsin G–induced platelet activation and degranulation through changes in the expression of platelet surface glycoproteins.Methods: Increasing concentrations of human neutrophil–derived cathepsin G were incubated with washed platelets or whole blood from healthy human donors. The platelet surface expression of glycoproteins, including P-selectin, a platelet membrane glycoprotein only expressed after platelet alpha granule release, were determined by quantifying the platelet binding of a panel of fluorescently labeled monoclonal antibodies. Results were compared with the effect of a maximal dose of thrombin, the most potent known platelet activator.Results: In a washed platelet system, cathepsin G increased platelet surface expression of P-selectin (an activation-dependent neutrophil binding site), the glycoprotein IIb/IIIa complex (fibrinogen receptor), and glycoprotein IV (thrombospondin receptor), and decreased surface expression of glycoprotein Ib (von Willebrand factor receptor) to an extent comparable to maximal thrombin. However, these effects were not observed in a whole blood system. Further experiments revealed that preexposure to plasma completely inhibited cathepsin G–induced washed platelet activation and degranulation. Prostacyclin treatment of washed platelets markedly inhibited cathepsin G–induced platelet activation.Conclusions: Cathepsin G is a very potent platelet agonist and degranulator, comparable to maximal thrombin, which alters platelet surface glycoprotein expression for enhanced neutrophil binding and effective platelet aggregation. This study helps to elucidate a possible pathway through which neutrophils may directly activate platelets, leading to intravascular thrombosis, irreversible ischemia, and tissue death in cardiovascular disease states. Patients with diseased endothelium that is deficient in prostacyclin production may be particularly prone to the detrimental effects of neutrophil-derived cathepsin G platelet activation. (J VASC SURG 1994;19:306-19.)     

Modulation of platelet surface adhesion receptors during cardiopulmonary bypass.

Alterations in platelet receptors critical to adhesion may play a role in the pathogenesis of the qualitative platelet defect associated with cardiopulmonary bypass. Using flow cytometry, we measured changes in the following platelet surface adhesive proteins: the von Willebrand factor receptor, glycoprotein Ib; the fibrinogen receptor, glycoprotein IIb/IIIa; the thrombospondin receptor, glycoprotein IV; the adhesive glycoprotein granule membrane protein 140, whose expression also reflects platelet activation and alpha-granule release; and, as a control, the nonreceptor protein HLA, A,B,C. Glycoprotein Ib decreased during cardiopulmonary bypass (P less than 0.05) and reached a nadir at 72% (P less than 0.05) of its baseline value at 2-4 h after bypass. This decrease correlated (r = 0.76) with the magnitude of platelet activation (alpha-granule release) in any given patient, but even platelets that were not activated demonstrated a decrease in glycoprotein Ib expression. Glycoprotein IIb/IIIa also decreased in both the activated (47% of baseline, P less than 0.01) and unactivated (63% of baseline, P less than 0.01) subsets of platelets at the end of cardiopulmonary bypass. Glycoprotein IV and HLA A,B,C did not decrease, but instead increased 2-4 h after cardiopulmonary bypass (P less than 0.05). We conclude that cardiopulmonary bypass produces selective decreases in surface glycoproteins Ib and IIb/IIIa as well as in platelet activation; that these two alterations are temporally but not necessarily mechanistically linked; and that these changes have the potential to adversely affect platelet function.     

Differential expression of neutrophil adhesion molecules during coronary artery surgery with cardiopulmonary bypass.

BACKGROUND: Activation of neutrophil adhesion molecules and subsequent neutrophil adhesion to vascular endothelium are key events initiating inflammatory organ dysfunction after cardiopulmonary bypass and ischemic reperfusion. OBJECTIVES: We sought to characterize neutrophil integrin CD11b and L-selectin activation associated with coronary artery bypass graft surgery and to determine whether neutrophil activation contributes to their sequestration on postbypass reperfusion. METHODS: Twenty patients undergoing routine coronary artery bypass were studied. Heparinized whole blood was simultaneously sampled from a central venous line, aorta, coronary sinus, and right and left atrium before, during, and up to 20 minutes after cardiopulmonary bypass. Neutrophil counts were obtained, and neutrophil CD11b and L-selectin expression was determined by flow cytometric analysis in whole blood. RESULTS: CD11b expression on circulating neutrophils increased during cardiopulmonary bypass, peaking at 145% of baseline level after release of the aortic clamp and then declined by 20 minutes after bypass (analysis of variance, P =.003). No change in neutrophil L-selectin expression was observed during cardiopulmonary bypass. Neutrophils responded to ex vivo stimulation by C5a and leukotriene B(4) during cardiopulmonary bypass but not at 24 hours after the operation. After reperfusion, neutrophil loss, but not local activation, was demonstrated in the coronary and pulmonary circulations. CONCLUSIONS: Upregulated CD11b expression on neutrophils is likely to contribute to neutrophil sequestration in the heart and lungs after bypass, but neutrophil activation may be limited by their reduced responsiveness to agonist stimulation. CD11b represents a potential therapeutic target for diminishing inflammation after cardiac operations.     

Combined administration of nitric oxide gas and iloprost during cardiopulmonary bypass reduces platelet dysfunction: a pilot clinical study

BACKGROUND: Thrombocytopenia and platelet dysfunction are major mechanisms of cardiopulmonary bypass-induced postoperative hemorrhage. This study evaluated the effects of low amounts of nitric oxide, iloprost (prostacyclin analog), and their combination administered directly into the oxygenator on platelet function, platelet-leukocyte interactions, and postoperative blood loss in patients undergoing coronary artery bypass grafting. METHODS: Blood samples from 41 patients randomized to the control, nitric oxide (20 ppm), iloprost (2 ng x kg -1 x min -1 ), or nitric oxide plus iloprost groups were collected during cardiopulmonary bypass. Platelets and leukocytes were enumerated. Platelet membrane glycoprotein Ib and glycoprotein IIb/IIIa, P-selectin, platelet-derived microparticles, leukocyte CD11b/CD18 (Mac-1), and platelet-leukocyte aggregate were quantified by means of flow cytometry. Collagen and thrombin receptor-activating peptide-induced platelet aggregation in whole blood was analyzed by means of aggregometry. RESULTS: Both nitric oxide or iloprost attenuated cardiopulmonary bypass-induced thrombocytopenia, reduction of glycoprotein Ib and glycoprotein IIb levels, translocation of P-selectin, microparticle formation, Mac-1 upregulation, and suppression of collagen-induced aggregation. Nitric oxide plus iloprost was significantly more effective in preventing thrombocytopenia, microparticle formation, and P-selectin translocation. Moreover, this treatment preserved thrombin receptor-activating peptide-induced aggregation, which was not rescued by single treatments. Both nitric oxide and nitric oxide plus iloprost attenuated postoperative blood loss. CONCLUSIONS: Nitric oxide plus iloprost reduced the deleterious effects of cardiopulmonary bypass, such as thrombocytopenia, platelet activation, platelet-leukocyte aggregate formation, and suppression of platelet aggregative responses. The reduced postoperative bleeding observed with this treatment suggests that this is a new and clinically feasible therapeutic option for patients subjected to cardiopulmonary bypass.     

Activation of neutrophil granulocytes in an in vitro model of a cardiopulmonary bypass

Activated neutrophils play a central role in the pathogenesis of postoperative organ dysfunction after surgery with cardiopulmonary bypass. The researchers used an in vitro roller pump model to investigate the relative importance of the biomaterial, platelets, plasma proteins including activated complement, and flow mode on neutrophil activation as shown by the adhesion, degranulation, and increased the surface expression of CD11b. Neutrophil adhesion to the biomaterial increased with platelet addition, but not with plasma. Biomaterial contact activated neutrophils in a serum-free buffer, but was significantly increased by activated complement. Platelets increased neutrophil degranulation in a serum-free buffer but tended to reduce it in plasma. CD11b expression increased in both media. Complement activation was higher with neutrophils alone than with neutrophils and platelets combined. The roller pump reduced neutrophil adhesion and increased degranulation compared to passive rotation. Neutrophil interaction with platelets and complement were more important for activation than biomaterial contact and use of the roller pump. Improvement of biocompatibility is dependent on modifying complement activation and platelet interaction with neutrophils.     

The effects of sevoflurane and desflurane in vitro on platelet-leukocyte adhesion in whole blood

The interaction between platelets and leukocytes plays an important role in inflammatory and thrombotic processes. We investigated whether the volatile anaesthetics sevoflurane and desflurane alter the formation of platelet-leukocyte aggregates and the expression of P-selectin on platelets. Whole blood was incubated with 1 and 2 minimum alveolar concentration (MAC) sevoflurane or desflurane. Unstimulated and adenosine diphosphate, or thrombin receptor agonist peptide-6-stimulated samples were stained with fluorochrome-conjugated antibodies. The formation of platelet-leukocyte conjugates and the expression of P-selectin on platelets were measured using flow cytometry. Sevoflurane was found to enhance the binding of platelets to lymphocytes, neutrophils and monocytes, it also increased the expression of P-selectin on platelets especially in the stimulated samples. Desflurane decreased the percentage of lymphocyte-platelet, neutrophil-platelet and monocyte-platelet conjugates principally in unstimulated samples. The results show that these two volatile anaesthetics have differing effects on the formation of platelet-leukocyte conjugates in vitro. Sevoflurane also enhanced the expression of P-selectin on platelets.