Jayne B. Tracey

Role of C3 Cleavage in Monocyte Activation During Extracorporeal Circulation

BACKGROUND We previously demonstrated that inhibiting formation of terminal complement components (C5a and C5b-9) prevents platelet and neutrophil (PMN) but not monocyte activation during simulated extracorporeal circulation (SECC). This study examined whether earlier complement inhibition during SECC, blocking C3a formation, would additionally prevent monocyte activation. METHODS AND RESULTS SECC was established by recirculating heparinized whole blood from human volunteers on a membrane oxygenator. CAB-2, a chimeric protein constructed from genes encoding the complement regulatory proteins CD46 and CD55, inactivates the C3/C5 convertases and blocks in vitro generation of C3a, C5a, and C5b-9. CAB-2 was used in 4 experiments at a final concentration of 300 micrograms/mL and 4 experiments at 30 micrograms/mL; 4 control runs used vehicle alone. Samples were assayed for C3a and C5b-9, monocyte activation (CD11b upregulation), PMN activation (CD11b upregulation and elastase release), and platelet activation (P-selectin expression and monocyte-platelet conjugate formation). CAB-2 at both doses significantly inhibited formation of C3a and C5b-9 during SECC. High-dose CAB-2 significantly blocked monocyte and PMN CD11b upregulation and PMN elastase release. CAB-2 also inhibited formation of platelet activation-dependent monocyte-platelet conjugates. CONCLUSIONS Blockade of complement activation early in the common pathway inhibited monocyte CD11b upregulation during SECC, suggesting that early complement components contribute most to monocyte activation during SECC. As expected, PMN and platelet activation were blocked by terminal complement inhibition. This investigation further elucidates the relation between complement and blood cell activation during simulated cardiopulmonary bypass.

Reversibility of severe metabolic stress in stored platelets after in vitro plasma rescue or in vivo transfusion: restoration of secretory function and maintenance of platelet survival.

BACKGROUND:  Determining reversible aspects of the platelet storage lesion may result in improved function and survival of transfused platelets.

Lymphocyte and monocyte subset changes during cardiopulmonary bypass : effects of aging and gender

Complications of cardiopulmonary bypass (CPB) may be associated with either immune suppression or immune activation, but the specific effects of CPB on many lymphocyte and monocyte subsets are unclear. In addition, the increasing age of patients undergoing cardiac surgery raises the possibility of even greater effects on the immune system in elderly patients. We measured immunophenotypic alterations of circulating lymphocytes and monocytes after CPB in male and female cardiac surgery patients who were either younger than 60 or older than 75 years of age. The total lymphocyte counts in all patients decreased postoperatively; older patients had significantly lower counts at all time points. The absolute decline was greatest among T cells and particularly CD4+ T cells, which reached an average nadir of 251 cells/microl on postoperative day 1 in the older patients. The percentages of CD8+, CD4+CD45RA+, and CD4+CD45RO+ T cells did not change significantly, whereas the percentages of B cells and natural killer cells increased. Both T and B lymphocytes and monocytes showed evidence of activation, with increased percentages of CD3+HLADr+, CD3+IL2R+, and CD19+CD23+ lymphocytes and increased expression of CD11b on monocytes. By contrast, expression of class II major histocompatibility antigen (HLADr) monocytes decreased significantly. We conclude that CPB produces a profound alteration in the pool of circulating lymphocytes and monocytes, evidenced by decreased numbers of lymphocyte subsets including CD4+ cells and decreased expression of monocyte surface membrane proteins important for antigen presentation; CPB also activates a variety of specific circulating mononuclear cell subsets. Older patients showed patterns of lymphocyte and monocyte activation comparable to those of younger patients; however, they had consistently lower lymphocyte numbers and a trend toward decreased monocyte HLADr expression, potentially placing them at greater risk for infectious complications. Gender had no effect.

Selective blockade of membrane attack complex formation during simulated extracorporeal circulation inhibits platelet but not leukocyte activation

OBJECTIVE Complement activation is induced by cardiopulmonary bypass, and previous work found that late complement components (C5a, C5b-9) contribute to neutrophil and platelet activation during bypass. In the present study, we blocked C5b-9 formation during extracorporeal recirculation of whole blood to assess whether the membrane attack complex was responsible for both platelet and leukocyte activation. METHODS In a simulated extracorporeal model that activates complement (C3a and sC5b-9), platelets (CD62P expression, leukocyte-platelet conjugate formation), and leukocytes (increased CD11b expression and neutrophil elastase), we examined an anti-human C8 monoclonal antibody that inhibits C5b-9 generation for its effects on cellular activation. RESULTS Anti-C8 significantly inhibited sC5b-9 formation but did not block C3a generation. Anti-C8 also significantly inhibited the increase in platelet CD62P and monocyte-platelet conjugate formation seen with control circulation. Moreover, compared with control circulation, in which the number of circulating platelets fell by 45%, addition of anti-C8 completely preserved platelet counts. In contrast to blockade of both C5a and sC5b-9 during simulated extracorporeal circulation, neutrophil activation was not inhibited by anti-C8. However, circulating neutrophil and monocyte counts were preserved by addition of anti-C8 to the extracorporeal circuit. CONCLUSIONS The membrane attack complex, C5b-9, is the major complement determinant of platelet activation during extracorporeal circulation, whereas C5b-9 blockade has little effect on neutrophil activation. These data also suggest a role for platelet activation or C5b-9 (or both) in the loss of monocytes and neutrophils to the extracorporeal circuit.

Acadesine inhibits neutrophil CD11b up-regulation in vitro and during in vivo cardiopulmonary bypass ☆ ☆☆ ★ ★★ ♢

Granulocyte adhesion to ischemic tissue, mediated in large part by beta 2 integrin receptors, is important in the pathophysiology of reperfusion injury. Acadesine, a drug that modulates adenosine levels in ischemic tissue, has been shown to reduce reperfusion injury in animal models of ischemia. The purpose of this study was to measure changes in granulocyte CD11b/CD18 in an in vitro assay and in an in vivo trial of acadesine administered during cardiopulmonary bypass to determine whether this agent might modulate up-regulation of this adhesion receptor. In vitro, whole blood was incubated with acadesine or control diluent, stimulated with N-formyl-methionyl-leucyl-phenylalanine, and granulocyte CD11b measured. Acadesine significantly (p < 0.01) inhibited N-formyl-methionyl-leucyl-phenylalanine-induced granulocyte CD11b up-regulation by a mean of 61%. In similar experiments, adenosine also inhibited N-formyl-methionyl-leucyl-phenylalanine-induced granulocyte CD11b up-regulation (p < 0.01). In vivo, 34 patients at our institution participating in a multicenter trial of acadesine during cardiopulmonary bypass were randomized to placebo, low-dose, or high-dose acadesine infusion perioperatively. Combining low- and high-dose treatment groups, there was significant (p = 0.05) inhibition of granulocyte CD11b up-regulation in patients receiving acadesine; granulocyte CD11b expression in the acadesine group peaked at 2.8 times baseline versus 4.3 for placebo. By contrast, monocyte CD11b up-regulation (peaking after cardiopulmonary bypass at 3 times baseline) was not affected by acadesine. Acadesine and adenosine inhibit up-regulation of granulocyte CD11b in vitro, and acadesine is capable of a similar inhibition during in vivo cardiopulmonary bypass. This inhibition may contribute to the ability of these agents to decrease in vivo reperfusion injury.

The effects of heparin, protamine, and heparin/protamine reversal on platelet function under conditions of arterial shear stress.

Platelet dysfunction contributes to blood loss after cardiopulmonary bypass. This study examined the antiplatelet effects of heparin, protamine, and varying heparin/protamine ratios in an in vitro physiologic model and further elucidated the mechanism of the antiplatelet and anticoagulant effects of protamine. We used the Clot Signature Analyzer (CSA™), a system that analyzes coagulation in flowing whole blood, to test two aspects of platelet function, with different concentrations of heparin and protamine, under conditions simulating arterial flow: collagen-induced thrombus formation (CITF) under moderate shear and high shear platelet activation, platelet hemostasis time (PHT). In addition, platelet aggregometry, celite activated clotting time (Hepcon™ ACT), prothrombin time (PT), and partial thromboplastin time (PTT) were measured. Both PHT and the CITF were prolonged by heparin at 20 &mgr;g/mL, protamine at 20 and 40 &mgr;g/mL, and heparin/protamine ratios of 1:1 and 1:2, but not at 1:1.5. The Hepcon ACT was prolonged by heparin 20 &mgr;g/mL and protamine alone at 20 and 40 &mgr;g/mL, was normal at a ratio of 1:1, and was prolonged at 1:1.5 and 1:2. Protamine 80 &mgr;g/mL prolonged the PT and PTT. Dependency on thrombin, protein kinase C activation, and nonspecific charge effects were examined. The direct thrombin inhibitor D-phenylalanyl-l-prolyl-l-arginyl-chloromethyl ketone prolonged the PHT and ACT, but not the CITF, whereas the polycationic molecules polyarginine and polylysine prolonged the CITF, but not the PHT. The effect of protamine on the PTT, but not PT, could be shortened by the addition of excess phospholipid. Therefore, heparin inhibits both high shear collagen-independent and moderate shear collagen-dependent platelet activation; however, the latter is not mediated by its antithrombin activity. Protamine’s antithrombin effect may explain its inhibition of platelet activation at high shear stress. Protamine’s nonspecific charge effects are more important for inhibiting moderate shear collagen-induced platelet activation.

Nitroprusside Inhibition of Platelet Function Is Transient and Reversible by Catecholamine Priming

BACKGROUND The time course and reversibility of sodium nitroprussides in vivo inhibition of platelet function are unclear. METHODS Platelet aggregation and P-selectin expression as measures of platelet dense and alpha-granule release, respectively, were examined before and after administration of sodium nitroprusside (18 mg) to human volunteers and in in vitro studies. Hypotension occurring with sodium nitroprusside administration was treated with intravenous crystalloid and/or phenylephrine. RESULTS Compared with preinfusion studies, platelet aggregation to epinephrine was significantly inhibited immediately and 4 min after discontinuation of the sodium nitroprusside infusion but returned to baseline at 8 and 12 min after discontinuing sodium nitroprusside. However, both dense and alpha-granule release to adenosine diphosphate after in vivo sodium nitroprusside were never significantly inhibited even at the time when sodium nitroprusside infusion was maximal. In contrast to our in vivo findings, in vitro incubation of platelet-rich plasma with sodium nitroprusside resulted in significant inhibition of dense and alpha-granule release to adenosine diphosphate. These in vitro inhibitory effects of sodium nitroprusside were reversed by pretreatment with epinephrine but not phenylephrine. CONCLUSIONS In normal volunteers, sodium nitroprusside inhibits platelet aggregation to epinephrine but not adenosine diphosphate; inhibition was reversed within 8-12 min after discontinuing sodium nitroprusside. Sodium nitroprusside in vitro inhibition of platelet function to adenosine diphosphate was reversed by epinephrine pretreatment. Because of the rapid reversibility of its antiplatelet effect, sodium nitroprusside may be clinically useful even when there is the potential for impaired coagulation.

Antithrombin reduces monocyte and neutrophil CD11b up regulation in addition to blocking platelet activation during extracorporeal circulation

BACKGROUND:  Patients undergoing cardiac surgery requiring cardiopulmonary bypass develop a systemic inflammatory reaction. Antithrombin III (AT) has anticoagulant effects but also shows evidence of anti‐inflammatory activity. The aim of this study was to examine whether exogenous AT could reduce white blood cell activation (CD11b up regulation or elastase release), in addition to inhibiting platelet (PLT) activation and fibrin generation, during simulated cardiopulmonary bypass (sCPB), undertaken in the absence of endothelium.