Biphasic pro-thrombotic and inflammatory responses after coronary artery bypass surgery
2003; Elsevier BV; Volume: 1; Issue: 3 Linguagem: Inglês
10.1046/j.1538-7836.2003.00109.x
ISSN1538-7933
AutoresNailin Li, R Astudillo, Torbjörn Ivert, Paul Hjemdahl,
Tópico(s)Venous Thromboembolism Diagnosis and Management
ResumoSummaryEarly graft failure after coronary artery bypass grafting (CABG) is related to thrombosis and inflammation in the grafted vessel(s). The time courses of, and relationships between, pro-thrombotic and inflammatory responses to CABG surgery have, however, not been well defined. Fifteen patients undergoing CABG were examined before, and 1 h, 1 day, 7 days, and 3 months after surgery. Cellular markers of platelet and leukocyte activation were monitored by whole blood flow cytometry, and plasma markers of pro-thrombotic and inflammatory responses were analyzed by immunoassays. CABG immediately increased circulating P-selectin-positive platelets, leukocyte CD11b expression, and platelet-leukocyte aggregates (PLAs). Thrombin generation (F1 + 2 levels) and cytokine release [tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and IL-10], soluble P-selectin, and soluble E-selectin also increased immediately. These alterations persisted during the first week after surgery, with re-bound increases of circulating activated platelets and PLAs, TNF-α, and F1 + 2 on day 7. Platelet and PLA responsiveness to in vitro stimulation was suppressed immediately after CABG, but markedly enhanced 1 week after surgery. After 3 months, plasma soluble P-selectin, F1 + 2, and IL-10, and monocyte CD11b expression were still slightly elevated compared with baseline. In conclusion, CABG induces marked pro-thrombotic and inflammatory responses, which persist for at least 1 week. Platelet activation, platelet reactivity, PLA formation, thrombin generation, and TNF-α release show a second peak 1 week after surgery. These findings suggest that intensified and prolonged antithrombotic/inflammatory treatment should be considered after CABG surgery. Early graft failure after coronary artery bypass grafting (CABG) is related to thrombosis and inflammation in the grafted vessel(s). The time courses of, and relationships between, pro-thrombotic and inflammatory responses to CABG surgery have, however, not been well defined. Fifteen patients undergoing CABG were examined before, and 1 h, 1 day, 7 days, and 3 months after surgery. Cellular markers of platelet and leukocyte activation were monitored by whole blood flow cytometry, and plasma markers of pro-thrombotic and inflammatory responses were analyzed by immunoassays. CABG immediately increased circulating P-selectin-positive platelets, leukocyte CD11b expression, and platelet-leukocyte aggregates (PLAs). Thrombin generation (F1 + 2 levels) and cytokine release [tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and IL-10], soluble P-selectin, and soluble E-selectin also increased immediately. These alterations persisted during the first week after surgery, with re-bound increases of circulating activated platelets and PLAs, TNF-α, and F1 + 2 on day 7. Platelet and PLA responsiveness to in vitro stimulation was suppressed immediately after CABG, but markedly enhanced 1 week after surgery. After 3 months, plasma soluble P-selectin, F1 + 2, and IL-10, and monocyte CD11b expression were still slightly elevated compared with baseline. In conclusion, CABG induces marked pro-thrombotic and inflammatory responses, which persist for at least 1 week. Platelet activation, platelet reactivity, PLA formation, thrombin generation, and TNF-α release show a second peak 1 week after surgery. These findings suggest that intensified and prolonged antithrombotic/inflammatory treatment should be considered after CABG surgery. In spite of increasing use of percutaneous coronary interventions, coronary artery bypass grafting (CABG) still accounts for approximately half of the invasive treatment of coronary heart disease. The use of arterial conduits is increasing, but autologous saphenous vein grafts are still the most common conduits used in CABG [1Mehta D. Izzat M.B. Bryan A.J. Angelini G.D. Towards the prevention of vein graft failure.Int J Cardiol. 1997; 62: S55-63Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Refinement of surgical techniques and antithrombotic therapy have improved graft patency, but early graft failure may still occur in ∼10% of vein grafts during the first month after CABG surgery [2Fitzgibbon G.M. Kafka H.P. Leach A.J. Keon W.J. Hooper G.D. Burton J.R. Coronary bypass graft fate and patient outcome angiographic follow-up of 5,065 grafts related to survival and reoperation in 1,388 patients during 25 years.J Am Coll Cardiol. 1996; 28: 616-26Crossref PubMed Google Scholar]. The failure rate may be up to 15–30% during the first year [1Mehta D. Izzat M.B. Bryan A.J. Angelini G.D. Towards the prevention of vein graft failure.Int J Cardiol. 1997; 62: S55-63Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Motwani J.G. Topol E.J. Aortocoronary saphenous vein graft disease.Pathogenesis, Predisposition, Prevention Circulation. 1998; 97: 916-31Google Scholar]. Thus, saphenous vein graft failure remains a major clinical problem. Early vein graft failure is due almost exclusively to thrombotic occlusion, whereas late failure is associated with inflammation and atherogenesis in the graft wall, as well as subsequent thrombosis. It is well established that antiplatelet therapy with aspirin reduces early graft failure [4Chesebro J.H. Clements I.P. Fuster V. Elveback L.R. Smith H.C. Bardsley W.T. Frye R.L. Holmes Jr, D.R. Vlietstra R.E. Pluth J.R. Wallace R.B. Puga F.J. Orszulak T.A. Piehler J.M. Schaff H.V. Danielson G.K. A platelet-inhibitor-drug trial in coronary-artery bypass operations: benefit of perioperative dipyridamole and aspirin therapy on early postoperative vein-graft patency.N Engl J Med. 1982; 307: 73-8Crossref PubMed Scopus (416) Google Scholar, 5Antiplatelet Trialists' Collaboration.Collaborative overview of randomised trials of antiplatelet therapy – II. Maintenance of vascular graft or arterial patency by antiplatelet therapy.BMJ. 1994; 308: 159-68Crossref PubMed Scopus (20) Google Scholar]. However, the continued high incidence of early thrombotic graft failure with established antiplatelet regimens calls for further improvement of the preventive strategy. This has stimulated investigation of new therapeutic principles, such as gene therapy and nitric oxide donor therapy [3Motwani J.G. Topol E.J. Aortocoronary saphenous vein graft disease.Pathogenesis, Predisposition, Prevention Circulation. 1998; 97: 916-31Google Scholar]. Improved understanding of thrombotic and inflammatory processes in patients undergoing CABG is important for the design of future clinical trials and the improvement of clinical management. CABG surgery, performed with cardiopulmonary bypass (CPB), may evoke activation and interaction of platelets, leukocytes, and vessel wall cells, which are all involved in the pathogenesis of graft failure [1Mehta D. Izzat M.B. Bryan A.J. Angelini G.D. Towards the prevention of vein graft failure.Int J Cardiol. 1997; 62: S55-63Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Thus, CABG/CPB surgery acutely induces platelet activation, seen as increased circulating activated platelets and elevated platelet-released substances in plasma, which is followed by platelet consumption, and subsequent depression of platelet function [6Weerasinghe A. Taylor K.M. The platelet in cardiopulmonary bypass.Ann Thorac Surg. 1998; 66: 2145-52Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar]. CABG/CPB also activates leukocytes, as indicated by increased surface expression of activation markers [7Rinder C.S. Bonan J.L. Rinder H.M. Mathew J. Hines R. Smith B.R. Cardiopulmonary bypass induces leukocyte-platelet adhesion.Blood. 1992; 79: 1201-5Crossref PubMed Google Scholar, 8Asimakopoulos G. Taylor K.M. Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules.Ann Thorac Surg. 1998; 66: 2135-44Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar] and elevated plasma elastase [8Asimakopoulos G. Taylor K.M. Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules.Ann Thorac Surg. 1998; 66: 2135-44Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar]. Bypass surgery increases platelet–leukocyte, mainly platelet–monocyte and platelet–neutrophil, aggregate formation [7Rinder C.S. Bonan J.L. Rinder H.M. Mathew J. Hines R. Smith B.R. Cardiopulmonary bypass induces leukocyte-platelet adhesion.Blood. 1992; 79: 1201-5Crossref PubMed Google Scholar]. This may facilitate leukocyte adhesion onto and migration into the vessel wall, and contribute to the rapid accumulation of leukocytes in and around the vein graft [9Angelini G.D. Bryan A.J. Williams H.M. Morgan R. Newby A.C. Distention promotes platelet and leukocyte adhesion and reduces short-term patency in pig arteriovenous bypass grafts.J Thorac Cardiovasc Surg. 1990; 99: 433-9Abstract Full Text PDF PubMed Google Scholar]. Furthermore, bypass surgery induces a systemic inflammatory response, as evidenced by markedly elevated plasma levels of inflammatory mediators, such as interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) [10Ferroni P. Speziale G. Ruvolo G. Giovannelli A. Pulcinelli F.M. Lenti L. Pignatelli P. Criniti A. Tonelli E. Marino B. Gazzaniga P.P. Platelet activation and cytokine production during hypothermic cardiopulmonary bypass – a possible correlation?.Thromb Haemost. 1998; 80: 58-64Crossref PubMed Scopus (38) Google Scholar, 11Zahler S. Massoudy P. Hartl H. Hahnel C. Meisner H. Becker B.F. Acute cardiac inflammatory responses to postischemic reperfusion during cardiopulmonary bypass.Cardiovasc Res. 1999; 41: 722-30Crossref PubMed Scopus (137) Google Scholar], which may regulate inflammatory and pro-thrombotic responses. Previous data on pro-thrombotic and inflammatory responses to CABG/CPB surgery were mainly obtained during or 1–3 days after CABG, and pro-thrombotic and inflammatory alterations thereafter have not been well documented. Furthermore, the various pro-thrombotic and inflammatory responses have not been directly compared in those studies, even though they are closely integrated pathophysiological processes in this clinical setting. Therefore, we investigated the short- and long-term dynamic changes of platelet and leukocyte activation, systemic inflammatory markers, and pro-thrombotic responses in patients undergoing CABG surgery. We also wished to clarify the relationship between pro-thrombotic and inflammatory responses after bypass surgery. Fifteen patients undergoing elective primary CABG surgery were included after informed consent. Their clinical characteristics are listed in Table 1. The study was approved by the Ethics Committee of the Karolinska Institute.Table 1Clinical characteristics of the study patientsAge (years)68 ± 2Female/male1/14Body mass index (kg m−2)27.7 ± 5.3Diagnoses Stable/unstable angina10/5 Previous acute myocardial infarction11 Hypertension4 Diabetes mellitus2 Hyperlipidemia4Pre-/post-operative medication Aspirin12/12 Clopidogrel3/3 NO donors10/0 β-blockers15/15 ACE inhibitor4/4 Calcium antagonist2/1 Hypoglycemic drug1/1 Lipid lowering drug11/11 Open table in a new tab Blood samples were obtained on five occasions: 1 day before, 1 h after the termination of CPB, and 1 day, 1 week, and 3 months after the surgery. Blood was collected from antecubital veins, using sodium citrate (final concentration 0.38%; for flow cytometry and plasma analyses) or EDTA vacutainers (5 mmol L−1; for hematological analyses). One-hour samples were obtained from a central venous catheter (first 20 mL discarded). Citrated blood for plasma preparation was kept on ice, and centrifuged within 15 min (1400 × g at 4 °C, 10 min); plasma was aliquoted and stored at − 80 °C. The patients were premedicated with morphine 0.125 mg kg−1 and scopolamine 5 mg kg−1 1 h before surgery. Anesthesia was induced with fentanyl (7–15 µg kg−1) or ketamine (1–2 mg kg−1 in unstable angina patients) and midazolam (40–70 µg kg−1), and was maintained with fentanyl (3–5 µg kg−1 min−1) and midazolam (30–50 µg kg−1 min−1). CPB was conducted at moderate hypothermia (32–34 °C) using a centrifugal pump (Bio-80; Biomedicus, Minneapolis, MN, USA) and a hollow-fiber membrane oxygenator (Affinity-NT; Medtronic, Minneapolis, MN, USA). Systemic heparinization was achieved before bypass with 300 U kg−1 heparin infusion. The activated clotting time was kept above 480 s throughout CPB. Heparinization was reversed after CPB with protamine at a weight ratio of 1 : 1. All patients received 1–2 arterial grafts in addition to saphenous vein grafts (median graft number of 3; range 2–5). The durations of CPB and aortic clamp were 71 ± 2 min and 37 ± 2 min, respectively. Within 3 min of collection, 5 µL blood was added to 45 µL of Hepes-buffered saline containing fluorescent antibodies for the detection of platelet P-selectin expression, leukocyte CD11b expression, or platelet-leukocyte conjugation. Thrombin receptor activating peptide (TRAP) or adenosine diphosphate (ADP) was added in some samples to elucidate platelet and leukocyte reactivity in vitro. Samples were incubated at room temperature in the dark for 20 min, and then diluted and mildly fixed with 0.5% (v/v) formaldehyde saline before measurement using a Beckman-Coulter EPICS XL-MCL flow cytometer (Beckman-Coulter Corp., Hialeah, FL, USA). Whole blood flow cytometric assays for platelet P-selectin expression [12Li N. Soop A. Sollevi A. Hjemdahl P. Multi-cellular activation in vivo by endotoxin in humans – limited protection by adenosine infusion.Thromb Haemost. 2000; 84: 381-7Crossref PubMed Scopus (26) Google Scholar], leukocyte CD11b expression [13Li N. Halldén G. Hjemdahl P. A whole-blood flow cytometric assay for leukocyte CD11b expression using fluorescence signal triggering.Eur J Haematol. 2000; 65: 57-65Crossref PubMed Scopus (20) Google Scholar], platelet-platelet aggregates (PPAs) [12Li N. Soop A. Sollevi A. Hjemdahl P. Multi-cellular activation in vivo by endotoxin in humans – limited protection by adenosine infusion.Thromb Haemost. 2000; 84: 381-7Crossref PubMed Scopus (26) Google Scholar], and platelet-leukocyte aggregates (PLAs) [14Li N. Goodall A.H. Hjemdahl P. Efficient flow cytometric assay for platelet-leukocyte aggregates in whole blood using fluorescence signal triggering.Cytometry. 1999; 35: 154-61Crossref PubMed Scopus (93) Google Scholar] have been described previously. Platelet P-selectin expression data are reported as percentages of P-selectin-positive cells in the platelet population or the number of circulating activated single platelets, which is calculated as the product of the single platelet count and the percentage of P-selectin-positive platelets in an unstimulated blood sample. Leukocyte CD11b expression is reported as mean fluorescence intensity (MFI) of total leukocytes and leukocyte subpopulations. Circulating PPA data are presented as numbers of PPAs (109 L−1) in blood samples, and are the product of platelet counts and percentages of aggregate-sized events in the total CD42a-positive population. Heterotypic aggregates are presented as percentages of platelet-conjugated leukocytes in the total leukocyte population (PLA), lymphocytes (P-Lym), monocytes (P-Mon), and neutrophils (P-Neu), and the absolute counts of the conjugates were calculated as the products of the corresponding percentages and leukocyte counts. Enzyme immunoassay kits were used to determine plasma soluble P-selectin (R & D Systems, Abingdon, UK), soluble E-selectin (R & D), elastase (DPC Biermann GmbH, Bad Nauheim, Germany), and prothrombin fragment 1 + 2 (F1 + 2) (Behringwerke AG, Marburg, Germany). Plasma TNF-α, IL-8, and IL-10 were determined using immunoassay kits from R & D Systems. All plasma parameters have been adjusted to the hematocrit before CABG, to eliminate the influences of hemodilution. Red blood cell counts, total and differential leukocyte counts, platelet counts, hematocrit, and hemoglobin were analyzed by a Technicon H.3 RTX cell counter (Miles Inc., Tarrytown, NY, USA). Data are presented as mean ± SEM. Individual measurements were compared with Wilcoxon's signed rank test (statview 4.5; Abacus Concepts, Berkeley, CA, USA), while agonist effects and possible differences in platelet and leukocyte reactivity over time were analyzed by two factor repeated measures anova (superanova; Abacus). P < 0.05 was considered statistically significant. There were marked decreases of erythrocyte counts, hematocrit, and hemoglobin after CABG (Table 2). Total leukocyte counts increased immediately after CABG, reached a peak on day 1, and remained elevated on day 7. The increase was mainly seen in neutrophils, and a mild increase of monocytes was found on day 1. Lymphocyte counts were reduced during the first week after CABG. Platelets decreased immediately, remained low on day 1, but showed a rebound increase on day 7. After 3 months, most variables had returned to their basal levels.Table 2Hematological analysis in CABG patients during 3 monthsTime−1 day+1 h+1 day+1 week+3 monthsHematocrit (%)42 ± 131 ± 2**P < 0.01 compared with corresponding data before CABG/CPB.32 ± 1**P < 0.01 compared with corresponding data before CABG/CPB.31 ± 1**P < 0.01 compared with corresponding data before CABG/CPB.40 ± 1Erythrocytes (×1012 L−1)4.7 ± 0.13.4 ± 0.2**P < 0.01 compared with corresponding data before CABG/CPB.3.6 ± 0.1**P < 0.01 compared with corresponding data before CABG/CPB.3.5 ± 0.1**P < 0.01 compared with corresponding data before CABG/CPB.4.6 ± 0.1Hemoglobin (g L−1)140 ± 2103 ± 5**P < 0.01 compared with corresponding data before CABG/CPB.107 ± 4**P < 0.01 compared with corresponding data before CABG/CPB.105 ± 3**P < 0.01 compared with corresponding data before CABG/CPB.134 ± 3*P < 0.05,Leukocytes (×109 L−1)7.4 ± 0.49.6 ± 0.8**P < 0.01 compared with corresponding data before CABG/CPB.11.7 ± 1.0**P < 0.01 compared with corresponding data before CABG/CPB.8.6 ± 0.6**P < 0.01 compared with corresponding data before CABG/CPB.6.8 ± 0.3Neutrophils (×109 L−1)4.6 ± 0.47.8 ± 0.7**P < 0.01 compared with corresponding data before CABG/CPB.9.7 ± 0.9**P < 0.01 compared with corresponding data before CABG/CPB.6.0 ± 0.5**P < 0.01 compared with corresponding data before CABG/CPB.4.2 ± 0.3Lymphocytes (×109 L−1)1.9 ± 0.21.2 ± 0.1**P < 0.01 compared with corresponding data before CABG/CPB.1.1 ± 0.1**P < 0.01 compared with corresponding data before CABG/CPB.1.5 ± 0.1*P < 0.05,1.6 ± 0.1*P < 0.05,Monocytes (×109 L−1)0.41 ± 0.040.36 ± 0.040.62 ± 0.07*P < 0.05,0.49 ± 0.040.43 ± 0.04Platelets (×109 L−1)263 ± 27169 ± 18**P < 0.01 compared with corresponding data before CABG/CPB.202 ± 24**P < 0.01 compared with corresponding data before CABG/CPB.370 ± 33**P < 0.01 compared with corresponding data before CABG/CPB.†P < 0.05 compared with 1 day after CABG/CPB.240 ± 20* P < 0.05,** P < 0.01 compared with corresponding data before CABG/CPB.† P < 0.05 compared with 1 day after CABG/CPB. Open table in a new tab Circulating activated (P-selectin-positive) single platelets increased immediately after CABG, and showed a re-surge on day 7 (Fig. 1a). The first surge was related to an increased percentage of P-selectin-positive single platelets (from 1.6 ± 0.2% to 3.1 ± 0.4%; P < 0.01). This percentage returned towards baseline thereafter: 1.8 ± 0.2% on day 1, 1.7 ± 0.2% on day 7, and 1.5 ± 0.2% at 3 months. The second surge was due to the marked thrombocytosis on day 7 (Table 2). The number of circulating platelet microaggregates (PPAs) decreased immediately after CABG (from 2.45 ± 0.18 to 1.48 ± 0.17 × 109 L−1; P < 0.01), remained low on day 1 (1.70 ± 0.20 × 109 L−1; P < 0.01), and returned to basal levels after 1 week (data not shown). Plasma soluble P-selectin (Table 3) increased immediately after surgery, remained elevated during the first week after CABG, and still tended to be elevated 3 months after surgery.Table 3Changes of plasma parameters in CABG patients during 3 monthsTime−1 day+1 h+1 day+1 week+3 monthsSoluble P-selectin (ng mL−1)49.2 ± 4.168.8 ± 5.6**P < 0.01, and68.7 ± 5.8**P < 0.01, and61.3 ± 4.9**P < 0.01, and54.1 ± 4.6†P = 0.06 compared with before CABG/CPB.Elastase (ng mL−1)20.4 ± 2.2215.7 ± 38.1**P < 0.01, and68.6 ± 5.3**P < 0.01, and47.8 ± 3.2**P < 0.01, and23.0 ± 2.9Soluble E-selectin (ng mL−1)44.4 ± 4.047.3 ± 3.9*P < 0.0555.9 ± 4.8**P < 0.01, and50.1 ± 4.1**P < 0.01, and45.2 ± 4.1F1 + 2 (nmol L−1)0.57 ± 0.053.82 ± 0.45**P < 0.01, and1.28 ± 0.13**P < 0.01, and1.72 ± 0.14**P < 0.01, and‡P < 0.05 compared with 1 day after CABG/CPB.0.72 ± 0.07*P < 0.05TNF-α (pg mL−1)2.5 ± 0.38.1 ± 1.2**P < 0.01, and3.7 ± 0.3**P < 0.01, and4.3 ± 0.4**P < 0.01, and‡P < 0.05 compared with 1 day after CABG/CPB.2.8 ± 0.3IL-8 (pg mL−1)8.1 ± 0.551.1 ± 8.5**P < 0.01, and16.1 ± 1.6**P < 0.01, and15.2 ± 2.0**P < 0.01, and7.6 ± 0.3IL-10 (pg mL−1)5.6 ± 1.2106.2 ± 16.2**P < 0.01, and29.5 ± 3.3**P < 0.01, and13.7 ± 1.9**P < 0.01, and8.6 ± 2.7*P < 0.05* P < 0.05** P < 0.01, and† P = 0.06 compared with before CABG/CPB.‡ P < 0.05 compared with 1 day after CABG/CPB. Open table in a new tab TRAP (Fig. 2a) and ADP (Fig. 2c) dose-dependently enhanced platelet P-selectin expression. The platelet responsiveness to TRAP was depressed immediately after CABG (P < 0.01), but markedly enhanced 1 week after CABG (P < 0.001). The platelet responsiveness to ADP tended to be lower immediately after bypass (P = 0.07), but did not differ from baseline 1 week after CABG (P = 0.36). Platelet responses to ADP or TRAP on day 1 and 3 months after surgery were similar to those before bypass (data not shown). CABG immediately increased CD11b expression of total leukocytes in unstimulated blood (Fig. 1b), which remained elevated after 24 h, and then returned to baseline levels. When leukocyte subpopulations were analyzed, marked increases of CD11b expression were found immediately after surgery in neutrophils (Fig. 3b) and monocytes (Fig. 3e), while a small but significant increase was seen in lymphocytes (data not shown). One day after CABG, CD11b expression returned to basal levels in neutrophils (Fig. 3b) and lymphocytes. Monocyte CD11b expression, however, remained elevated during the first week, and was still slightly but significantly elevated after 3 months (Fig. 3e). CABG/CPB immediately induced a marked elevation of plasma elastase, which declined gradually afterwards, but remained two- to threefold above basal levels during the first week (Fig. 3c). After 3 months, elastase levels were normalized. TRAP and ADP increased leukocyte CD11b expression at the highest concentrations tested (10 µmol L−1), from 0.66 ± 0.04 to 0.97 ± 0.10 (P < 0.01) and 1.21 ± 0.11 (P < 0.01), respectively. CABG/CPB had little influence on leukocyte responsiveness to TRAP or ADP stimulation (data not shown). CABG/CPB enhanced platelet–leukocyte interaction. PLAs increased from 3.7 ± 0.4% to 4.7 ± 0.6% (P < 0.05) immediately after operation. PLAs returned to baseline after 24 h, but showed a marked rebound (to 5.7 ± 0.8%; P < 0.01) at 1 week. When the numbers of circulating platelet-conjugated leukocytes were calculated, it was found that circulating PLAs (Fig. 1c) increased immediately after CABG, declined slightly on day 1, showed a marked re-bound on day 7, and returned to baseline after 3 months. Similar changes were seen for P-Neus (Fig. 3a) and P-Mons (Fig. 3d), whilst circulating P-Lyms decreased 1 day after CABG (from 4.1 ± 0.5 to 1.8 ± 0.3 × 109 L−1). TRAP and ADP enhanced PLA formation in vitro dose dependently (Fig. 2b,d), with more pronounced increases among neutrophils and monocytes than among lymphocytes (data not shown). Both TRAP- and ADP-induced PLA formation were depressed immediately after surgery (P < 0.05 for both), but markedly enhanced after 1 week (P < 0.001 for both; Fig. 2b,d). Plasma soluble E-selectin, a marker of endothelial cell activation, increased immediately after surgery, reached a peak on day 1, remained elevated 1 week after CABG, and returned to baseline levels at 3 months (Table 3). CABG/CPB immediately elevated plasma F1 + 2 approximately sixfold (Fig. 3f, Table 3). F1 + 2 levels declined, but remained higher than basal levels on day 1, and showed a re-surge on day 7, with higher levels than on day 1. After 3 months, F1 + 2 levels were still slightly, but significantly, higher than before surgery. TNF-α, IL-8, and IL-10 increased markedly immediately after CABG, and remained elevated during the first week (Table 3). TNF-α showed a rebound on day 7, with significantly higher levels than on day 1. After 3 months, TNF-α and IL-8 levels had returned to baseline, whilst IL-10 levels were still elevated. The present study investigated the time courses of pro-thrombotic and inflammatory responses in patients undergoing CABG/CPB. During the acute phase (i.e. 1 h after surgery), in vivo activation of platelets, leukocytes, and endothelial cells, but depressed platelet and platelet-leukocyte conjugation responsiveness to in vitro stimulation were found. CABG/CPB also triggered marked thrombin generation and cytokine release in vivo. These changes persisted at least 1 week. A novel finding is that there was a resurgence of circulating activated platelets, PLAs, cytokine release, thrombin generation, and enhanced platelet responsiveness and PLA formation in the subacute phase, i.e. 1 week after surgery, when effects of CPB had presumably waned. Interestingly, some signs of residual pro-thrombotic and inflammatory activity were found even at the 3-month follow-up. CABG/CPB has previously been shown to induce acute pro-thrombotic and inflammatory responses [7Rinder C.S. Bonan J.L. Rinder H.M. Mathew J. Hines R. Smith B.R. Cardiopulmonary bypass induces leukocyte-platelet adhesion.Blood. 1992; 79: 1201-5Crossref PubMed Google Scholar, 8Asimakopoulos G. Taylor K.M. Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules.Ann Thorac Surg. 1998; 66: 2135-44Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 10Ferroni P. Speziale G. Ruvolo G. Giovannelli A. Pulcinelli F.M. Lenti L. Pignatelli P. Criniti A. Tonelli E. Marino B. Gazzaniga P.P. Platelet activation and cytokine production during hypothermic cardiopulmonary bypass – a possible correlation?.Thromb Haemost. 1998; 80: 58-64Crossref PubMed Scopus (38) Google Scholar, 11Zahler S. Massoudy P. Hartl H. Hahnel C. Meisner H. Becker B.F. Acute cardiac inflammatory responses to postischemic reperfusion during cardiopulmonary bypass.Cardiovasc Res. 1999; 41: 722-30Crossref PubMed Scopus (137) Google Scholar]. The present investigation confirms that CABG/CPB induces platelet activation in vivo, with increased percentages and numbers of circulating activated (i.e. P-selectin positive) single platelets and elevated levels of soluble P-selectin in plasma. The latter may also, in part, be related to activated endothelial cells, as soluble E-selectin levels were elevated during the first week after surgery. Platelet responsiveness to in vitro stimulation was depressed immediately after surgery, reflecting consumption of sensitive platelets in the extracorporeal circuit. A novel finding in the present study is the marked re-surge of platelet counts, circulating activated platelets, and platelet reactivity one week after surgery. These findings indicate a marked release of young and reactive platelets after the initial period with excessive platelet activation and consumption. Circulating neutrophils and plasma elastase were elevated at least 1 week after CABG, suggesting continuous mobilization of cells from the storage pools and neutrophil activation, while monocyte CD11b expression was increased throughout the first week, and remained slightly increased even after 3 months. CABG/CPB-enhanced platelet and leukocyte activation led to enhanced platelet–leukocyte interaction, as shown by increased circulating P-Neus and P-Mons. PLA formation seems to be closely related to platelet activity, and both P-Neu and P-Mon showed delayed increases when platelet counts and the numbers of circulating activated single platelets surged 1 week after surgery. When platelet reactivity was depressed immediately after CABG/CPB, agonist-induced PLA formation was also reduced. When platelet reactivity was enhanced at 1 week, PLA formation was also enhanced. Platelet activation, monocyte activation, and P-Mon formation may also increase the risk of thrombosis via procoagulant effects. In our study, circulating activated single platelets, circulating P-Mons, and plasma F1 + 2 levels showed biphasic responses to CABG/CPB with both immediate and delayed increases. Furthermore, monocyte activation, and the elevations of soluble P-selectin and F1 + 2 persisted throughout the period of investigation. Activated monocytes express tissue factor and the negatively charged phospholipid surface of leukocyte-bound activated platelets, which incorporates factor Va, may serve as a fully functioned thrombin generation unit. The present results provide further evidence for platelet–leukocyte collaboration in thrombosis and coagulation, and support the hypothesis that P-Mon formation may facilitate thrombin generation in vivo[15Reverter J.C. Béguin S. Kessels H. Kumar R. Hemker H.C. Coller B.S. Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody.J Clin Invest. 1996; 98: 863-74Crossref PubMed Scopus (385) Google Scholar, 16Li N. Wallén N.H. Hjemdahl P. Evidence for prothrombotic effects of exercise and limited protection by aspirin.Circulation. 1999; 100: 1374-9Crossref PubMed Google Scholar]. Cytokines may regulate the intensity and duration of inflammatory and thrombotic responses by stimulating or inhibiting the activation, proliferation, differentiation, and interaction of various cells. Our data show that CABG/CPB elevates both pro-inflammatory (TNF-α and IL-8) and anti-inflammatory (IL-10) cytokines in plasma during the first week. Of note, TNF-α showed a rebound at 1 week, and IL-10 levels remained elevated even 3 months after surgery. These changes may contribute to thrombocytosis and platelet hyper-reactivity, since TNF-α and IL-8, and other cytokines not measured in the present study, are known to stimulate megakaryocytopoiesis and increase platelet reactivity [17Esmon C.T. Possible involvement of cytokines in diffuse intravascular coagulation and thrombosis.Baillières Best Pract Res Clin Haematol. 1999; 12: 343-59Crossref PubMed Scopus (92) Google Scholar]. Our findings highlight that CABG/CPB evokes a marked and immediate activation of coagulation and inflammation, but also induces a re-bound of these responses despite conventional antiplatelet treatment. Our findings indicate that actions should be taken to intensify and prolong antithrombotic and anti-inflammatory therapies, to counteract the hypercoagulant and inflammatory states after CABG/CPB. This may improve graft patency, and reduce postoperative complications, such as cerebral embolism, which may occur during the first week after cardiac surgery [18Libman R.B. Wirkowski E. Neystat M. Barr W. Gelb S. Graver M. Stroke associated with cardiac surgery. Determinants, timing, and stroke subtypes.Arch Neurol. 1997; 54: 83-7Crossref PubMed Scopus (99) Google Scholar]. Furthermore, a recent meta-analysis of 25 clinical trials on antiplatelet treatment after CABG/CPB [19Antithrombotic Trialists' Collaboration.Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients.BMJ. 2002; 324: 71-86Crossref PubMed Google Scholar] showed a risk reduction of only 4%, which stresses the continuing need for improvement of current antiplatelet therapies. In conclusion, CABG with CPB evokes immediate activation of platelets, leukocytes, and platelet–leukocyte conjugation, and induces marked activation of coagulation and inflammatory responses. These responses persisted up to 1 week, and slight increases in platelet and monocyte activity and thrombin generation were seen even after 3 months. Of special interest is that platelet reactivity, PLA formation, thrombin generation, and TNF-α showed re-bound increases at 1 week, and that pro-thrombotic and inflammatory responses seem to be closely related in this clinical setting. These findings may be of pathophysiological and therapeutic interest in the treatment of patients after CABG. The authors are grateful to Maud Daleskog and Maj-Christina Johansson for their expert technical assistance, and to the staff of the intensive care unit at the Thoracic Clinic, Karolinska Hospital, for kind assistance. The project was supported by grants from the Swedish Research Council (5930), the Swedish Heart-Lung foundation, the Stockholm County Council, and the Karolinska Institute.
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