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J Thorac Cardiovasc Surg 2005;129:782-790
© 2005 The American Association for Thoracic Surgery
Cardiopulmonary Support and Physiology |
a Departments of Pharmacology and Cardiothoracic Surgery, University of Alberta, Edmonton, Alberta, Canada
b Department of Cardiothoracic Surgery, German Heart Center Munich, Technical University of Munich, Munich, Germany
c Department of Integrative Biology and Pharmacology and Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas, Houston, Tex
Received for publication December 8, 2003; revisions received June 2, 2004; accepted for publication June 22, 2004. * Address for reprints: Marek W. Radomski, MD, PhD, DSc, Center for Vascular Biology, Institute of Molecular Medicine, University of Texas-Houston, 6770 Bertner Ave, Suite C950J, Houston, TX 77030 (E-mail: Marek.Radomski{at}uth.tmc.edu).
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Abstract |
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METHODS: Blood samples from 41 patients randomized to the control, nitric oxide (20 ppm), iloprost (2 ng · kg–1 · 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.
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Methods |
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Patient selection and experimental design
The Health Research Ethics Board of the University of Alberta, Edmonton, Canada, approved this study. Informed consent was obtained from all patients before the study. Forty-one patients were randomly assigned to one of the 4 groups: (1) routine CPB (control); (2) routine CPB plus NO (20 ppm) administered into the membrane oxygenator; (3) routine CPB plus iloprost (2 ng · kg–1 · min–1) administered into the blood phase of the oxygenator; and (4) routine CPB plus NO plus iloprost.
All patients included were elective patients scheduled for coronary artery bypass grafting. Excluded were patients undergoing combined procedures and reoperation. Further exclusion criteria were evidence for impaired platelet function and coagulation disorders, chronic inflammatory diseases, and renal failure. It was ensured that platelet inhibitor drugs, such as aspirin or clopidogrel, were discontinued at least 7 days before the operation.
NO (PulmNOx Medical Inc, Tofield, Alberta, Canada) was administered into the membrane oxygenator through a Y connector during the period of extracorporeal circulation. Iloprost (Cayman Chemical Company, Ann Arbor, Mich) was infused into the oxygenator starting from the initiation of CPB and was maintained throughout the duration of CPB. The doses of NO and iloprost were selected on the basis of preliminary studies showing significant effects of these drugs on platelet function without alterations of hemodynamics (mean arterial blood pressure) or increase in vasopressor requirement.
For safety reasons, the staff in the operating room, including surgeons, perfusionists, and anesthesiologists, was not blinded to the study. In addition, the exhaust, including the outflow from the extracorporeal circuit, was removed from the operating room through central air conditioning. However, the basic scientists involved in the sample collection, processing, and data analysis were blinded to the treatment and control groups.
Anesthesia was identical in each group, according to a standard protocol of the institution, except that aprotinin and all nitrovasodilators were excluded.
The surgical procedure was performed through a midline sternotomy, with systemic heparinization and an activating clotting time well above 400 seconds. All extracorporeal circuits used were noncoated, were identical in each patient, and consisted of a membrane oxygenator, arterial filter, and colloidal priming (1400 mL). No patient received aprotinin or other proteinase inhibitors during the study period. Cardioplegic arrest was performed with blood cardioplegia.5 The temperature during bypass grafting was between 32°C and 34°C, and rewarming was initiated after completion of the last peripheral anastomosis. Reperfusion was performed with whole blood by means of aortic declamping. The demographic characteristics, clinical parameters of the patients, number of grafts, reperfusion times, and aortic crossclamp times are given in Table 1.
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The 2 time points selected for analysis of platelet aggregation and flow cytometry were chosen to study the maximal effects of CPB on platelet function. We hypothesized that this protocol would enable us to detect the maximal difference between the control and treatment groups. In addition, technically it was impossible to perform any additional time point analysis on fresh blood samples because processing the samples would have exceeded the duration of bypass. All data affected by hemodilution were corrected for it.
Platelet aggregation
Platelet aggregation was measured by means of whole blood aggregometry with a whole blood ionized calcium lumi-aggregometer (Chronolog). Changes in impedance (ohm) were recorded as an index of aggregation. Because blood aggregation depends on many factors, including red blood cell counts, white blood cell counts, and fibrinogen and plasminogen concentrations in the plasma, the results were expressed as a percentage of the maximal aggregative response of each patient.
Flow cytometry
To determine the relative abundance of GPIb, total-active GPIIb, and P-selectin, samples were incubated for 20 minutes with saturating concentrations of FITC- or PE-conjugated antibodies, such as CD42-PE, CD41-FITC, PAC-1-FITC, or CD62P-PE. The platelet population was gated by using forward- and side-scatter parameters. Fluorescence intensity was analyzed with logarithmic scale and CELLQUEST software. The quantification of GPIb, total-active GPIIb, and P-selectin levels was expressed as the mean fluorescence intensity (MFI) from 10,000 individual events. For microparticle (MP) analysis, samples were incubated with CD42-PE MoAbs, and the MP population was distinguished by the forward-scatter cutoff that was set to the immediate left of the platelet population. MPs were reported as the percentage of PE-positive cells. Mac-1 abundance was analyzed by means of dual-labeling leukocytes with CD45-FITC and CD11b-PE MoAbs, and the MFI was determined from the FITC-positive cells. The PLA formation was expressed as 109/L and was assayed by double-labeling samples with CD45-FITC and CD62P-PE MoAbs, quantifying the percentage of leukocytes exhibiting CD62P fluorescence, and multiplying the percentage of PLA by the number of leukocytes in the samples.
Statistical analysis
All results were given as means ± SEM derived from n determinations and were analyzed by using analysis of variance with the Bonferroni or Dunnett tests, where appropriate, with Prism software (GraphPad Software, Inc, San Diego, Calif).
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Results |
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The panel of CPB-induced markers of platelet activation, such as GPIb and GPIIb downregulation and MP formation, were significantly attenuated by NO (Figure 2). In addition, there was decreased leukocyte activation and formation of PLA (Figure 3), as demonstrated by reduced Mac-1, P-selectin, and PLA levels.
Platelet aggregation showed that although the aggregative responses to collagen were preserved by the NO treatment (EC50 postpump value of 1.2 ± 0.1 vs prepump value of 1.3 ± 0.2 mg/L, P > .05), TRAP-induced aggregation remained decreased (EC50 postpump value of 14.2 ± 2.8 vs prepump value of 5.6 ± 0.2 µmol/L, P < .05).
Effects of iloprost on CPB-induced platelet-leukocyte activation
With the use of iloprost, platelet numbers were reduced by 14% ± 2%, which was less than those in control subjects. Iloprost exerted no significant effects on leukocytosis (increase by 92% ± 25%, Figure 1).
However, iloprost attenuated the effects of CPB on GPIb, GPIIb, activated GPIIb, MP, and P-selectin. In contrast, the treatment enhanced the effects of CPB on Mac-1 and PLA (Figures 2 and 3). Aggregation to collagen was preserved (EC50 postpump value of 1.2 ± 0.2 vs prepump value of 1.4 ± 0.1 mg/L, P > .05), whereas that to TRAP remained decreased (EC50 postpump value of 13.8 ± 1.1 vs prepump value of 6.3 ± 0.3 µmol/L, P > .05).
Effects of NO plus iloprost on CPB-induced platelet-leukocyte activation
The combination therapy effectively prevented thrombocytopenia but did not prevent leukocytosis (Figure 1). The drugs attenuated the effects of CPB on GPIb, total GPIIb, MP, P-selectin, Mac-1, and PLA (Figures 2 and 3).
Interestingly, aggregation to collagen and TRAP were well preserved (Figure 4, B).
Effects of NO and iloprost on chest tube loss
Figure 5 shows the effects of treatments with NO and iloprost on chest tube blood loss after cardiac surgery. Both NO and NO plus iloprost, but not iloprost alone, significantly attenuated this blood loss.
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Discussion |
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The major novel findings of our investigation showed that the combined treatment with NO and iloprost (1) abolished the CPB-induced thrombocytopenia, (2) prevented the loss of platelet aggregation to collagen and TRAP, and (3) reduced postoperative bleeding.
CPB and platelet-leukocyte dysfunction
The increased tendency for hemorrhage in patients undergoing cardiac surgery with CPB is likely to be multifactorial; it involves thrombocytopenia, increased coagulation, fibrinolysis, complement activation, and inflammation. In addition, it is known that pump and clamp times, as well as flow rates, are important determinants of platelet and leukocyte activation and function. In this regard a recent study by Greilich and colleagues7 showed that prolongation of CPB is related to increasing degrees of platelet dysfunction and that reductions in platelet contractile force are related to decreases in platelet adhesion receptors and early postoperative blood loss. This pathologic scenario is often associated with the postoperative need for transfusion of blood products and the requirement for inotropic support, both of which are risk factors for morbidity and mortality.2
Previous studies and the current investigation show that thrombocytopenia is a major contributing factor to CPB-induced bleeding.2,8,9 Early hemodilution caused by the crystalloid fluid for priming of the extracorporeal circuit might partially explain this phenomenon. However, Holloway and coworkers9 suggested that the mechanical disruption of platelets by shear forces (oxygenator) and increased platelet adhesion to the extracorporeal membrane and sequestration in organs both contribute to the true decrease in circulating platelet numbers. Our study provides a compelling indication for in-circuit activation of platelets. This is evidenced by a large increase in MP formation that derives from the membrane of activated platelets,10 downregulation of GPIb that results from its internalization,11 and the translocation of platelet 
-granule P-selectin to the surface of activated platelets.12 Downregulation of total and active GPIIb provides a strong rationale for the proposal that circuit-activated platelets are exhausted and no longer capable of responding with aggregation to hemostatic regulators in vivo.1 This hemostatic defect can be exposed by ex vivo adhesion and aggregation studies that demonstrated impaired platelet reactivity to collagen, thrombin,13–15 and, as shown in the current study, TRAP. Therefore the major mechanisms of CPB-related postoperative bleeding are both functional (platelet reactivity) and quantitative (platelet count) defects.13,16
We and others have shown that thrombocytopenia is accompanied by leukocytosis that is associated with the inflammatory response.17 The surfaces of CPB systems (ie, oxygenator and tubing) are recognized as foreign by the immune system, which subsequently activates a cytokine cascade.18 Cytokines initiate systemic inflammatory reactions, leading to leukocyte recruitment and activation and generation of oxidants and radicals, such as superoxide. Clinically, these reactions result in systemic vasodilation, increased vascular permeability, and organ dysfunction.
The role of platelets in CPB-induced inflammation is not clear; however, PLA might play a critical role in this process. Indeed, activated platelets translocate P-selectin to their surface, whereas Mac-1 (CD11b/CD18) is upregulated by activated leukocytes. The interactions of these adhesion receptors with their ligands19–21 facilitate PLA formation, endothelial adhesion, and inflammatory leukocyte extravasation. Increased Mac-1 and P-selectin levels in conjunction with enhanced PLA during CPB are well evidenced by our study. Therefore, simultaneous activation of both platelets and leukocytes with platelet adhesion, aggregation, and PLA can all contribute to circuit-induced thrombocytopenia, inflammatory responses, and increased postoperative blood loss. It is important to note that in the present study no differences between the control and treatment groups with regard to pump and clamp times, as well as flow rates, were observed (Table 1).
Effects of NO or iloprost on CPB
We also demonstrated the protective effects of NO and iloprost on platelet function. Both drugs protected platelets from activation, as evidenced by attenuation of thrombocytopenia and inhibition of changes in platelet receptors. Because NO and PGI2 and its analog iloprost are well-known regulators of platelet activation and inhibit platelet adhesion and aggregation at various levels of the activating cascade,4 the effectiveness of these drugs in limiting platelet damage is not surprising.
Indeed, the beneficial effects of NO and PGI2 have also been demonstrated in other studies. Aren and associates22 showed that PGI2 infusion into the bypass circuit reduced plasma ß-thromboglobulin levels, reflecting inhibition of platelet activation, although the dosage used (50 ng · kg–1 · min–1) was much higher than in our study (2 ng · kg–1 · min–1). Mellgren and colleagues23 demonstrated higher platelet counts in experimental perfusion circuits treated with NO (15–75 ppm); however, platelet function was not measured in this study. Inhaled NO downregulated P-selectin, platelet aggregation, and fibrinogen binding in patients with severe adult respiratory distress syndrome,24 but the concentration of NO (100-884 ppm) was again much higher than in our study (20 ppm). Furthermore, extracorporeal circuits coated with NO release polymers reduced platelet consumption in experimental animals.25 The protective effects of NO and PGI2 in CPB, however, are not without considerable debate. PGI2 infusion might cause hypotension,26 suggesting that the use of high amounts of these potent platelet inhibitors is likely to affect the hemodynamic balance by increasing vasodilatation and vascular permeability. Therefore the use of small (nonvasodilator) amounts of NO and iloprost (that proved to be efficacious in our study) applied directly to the oxygenator might be preferable in the quest to limit the CPB-induced platelet activation and damage.
Interestingly, the iloprost treatment increased Mac-1 levels in white blood cells. In contrast to platelets, where stimulation with antiaggregative prostaglandins, such as prostacyclin and prostaglandin D2, invariably leads to downregulation of integrin function, the effects of prostaglandins on white blood cells appear to be more complex. Indeed, prostaglandin D2 stimulates chemotaxis and Mac-1 expression27 in eosinophils through activation of a novel DP2 receptor. Thus, the stimulator effects of iloprost on Mac-1 could reflect activation of the DP2 receptor in white blood cells.
Effects of NO plus iloprost on CPB
The major interest of our study has been focused on the combined use of NO and iloprost during CPB. The in vitro studies clearly showed that NO and PGI2 synergize with each other as inhibitors of platelet aggregation and inducers of deaggregation. The current study has shown that the most striking advantage of using this combination of low doses of NO and iloprost is total prevention of thrombocytopenia and normalization of circuit-induced aggregative dysfunction. NO plus iloprost also reduced postoperative blood loss; however, in this action NO was as efficacious as the combination. The protective effects afforded by NO plus iloprost correlated well with reduction of MP and P-selectin levels. However, no additional protection over that seen with single-drug treatment could be detected with GPIb and GPIIb measurements. The reason or reasons for this discrepancy are not known.
Mechanisms of platelet-protective effects of NO and iloprost
NO and iloprost inhibit platelet activation by stimulating the intraplatelet levels of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), respectively.4 Indeed, increase of intracellular cAMP and cGMP levels is the most potent endogenous mechanism of platelet inhibition. Cyclic nucleotides control a number of platelet functions, such as inhibition of intracellular Ca2+ increase, cytoskeletal reorganization, integrin receptor activation, granule secretion, surface molecule expression, and mitogen-activated protein kinase function.28 All these effects result in inhibition of platelet activation. There is also evidence that the cAMP and cGMP pathways cross-talk to inhibit platelet activation. Cyclic GMP–mediated inhibition of cAMP hydrolysis through reduction of phosphodiesterase 3 activity might be one of the mechanisms that account for the synergistic interactions between NO and iloprost on platelets.28,29
As with single-compound treatment, the combined therapy exerted no significant effects on leukocytosis and only weakly affected Mac-1 and PLA levels. Thus other pharmacologic strategies should be used to decrease further circuit-induced inflammatory reactions.
Clinical implications of the present study
The most important clinical aspect to be derived from the present findings is the potential for significant reduction in postoperative bleeding by means of preservation of platelet function. This might be even more pronounced in the setting of prolonged pump time or in patients receiving long-term extracorporeal support, including extracorporeal membrane oxygenation and assist devices. Indeed, Jacobson30 has recently pointed out that NO is a potent platelet protectant against a significant reduction in platelet number and function, as observed in patients requiring long-term mechanical support.
Another important aspect of the use of the combination of NO and iloprost is cost-effectiveness. In our center the average cost for such therapy per one patient is $25 for NO and $15 for iloprost, assuming a pump time of 60 minutes. Given the potential saving effect on blood products with this treatment strategy, the overall cost might be reduced.
Study limitations
We acknowledge that there are limitations of this pilot clinical study. The first is the relatively small number of patients in the treatment groups. However, our study was of sufficient statistical power to delineate differences in platelet function and platelet-leukocyte interactions between the control and treatment groups. Furthermore, the conclusions of our study are based not on the analysis of single variable but on the use of a panel of molecular (GP receptors, MPs, P-selectin, and Mac-1 levels), functional (platelet aggregation and platelet-leukocyte aggregation), and clinical (platelet and leukocyte numbers and postoperative blood loss) variables. This analysis clearly shows the protective effects on platelets and the resultant clinical benefits (reduction of thrombocytopenia and postoperative blood loss) of nonvasodilator amounts of NO and iloprost.
Second, in this study we focused only on the acute effects of NO and iloprost on blood cells. It is possible that longer treatment regimens would have additional beneficial effects on postoperative bleeding or other thrombotic and inflammatory complications of CPB. In addition, in the present study no differences were observed between groups with regard to the administration of blood products. One reason might be that the patients included in our study were elective patients, excluding those scheduled for repeat operations and those expected to have long pump times. Thus the present study was performed in a selected group of patients not at high risk for major bleeding disorders. However, one can speculate that the finding of reduced chest tube loss and the platelet-sparing effect with NO and iloprost, as shown in the present study, might be even more pronounced in larger trials with higher-risk patients and prolonged CPB procedures, resulting in reduction of transfusion requirements.
In summary, the present study demonstrates, for the first time, that combined treatment with low amounts of NO (20 ppm) or iloprost (2 ng · kg–1 · min–1), when delivered into the oxygenator, reduced thrombocytopenia, normalized platelet aggregation, and attenuated postoperative blood loss, providing a safe method to decrease bleeding in patients subjected to CPB.
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Footnotes |
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* Drs Chung and Wildhirt contributed equally to the article. 
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