Complement (C3) binding to platelets in autoimmune thrombocytopenia

Summary. In view of conflicting reports on the occurrence of complement binding to platelets in idiopathic autoimmune thrombocytopenic purpura (AITP) we performed measurements of platelet bound C3 in patients with AITP who had elevated levels of platelet bound IgG. Using a quantitative antiglobulin consumption technique 38 out of 42 patients were found to have fixed abnormally large amounts of C3 to their platelets, and a significant positive correlation between the amounts of platelet bound IgG and C3 was shown to exist. In additional experiments antibody eluates were prepared from AITP platelets and were shown to cause the fixation of C3 to normal donor platelets in vitro. Taken together these findings strongly suggest that the C3 binding in AITP is specifically related to the disease process.     

幼猪体外循环脑灌注血流/压力动物模型的构建

目的:本研究拟通过构建幼猪体外循环(CPB)脑灌注血流/压力动物模型,探讨CPB期间动脉血压、灌注流量对脑血流自动调节的影响。方法:20只,幼猪,随机分五组:对照组、高血压高流量组、高血压低流量组、低血压高流量组、低血压低流量组。在CPB开始前10min、CPB开始后10min、升主动脉阻断后30min和升主动脉开放后10min 4个时间点检测血清S100钙结合蛋白B(S100 B)和神经元特异性烯醇化酶(NSE)浓度。免疫组织化学检测大脑海马组织缺氧诱导因子-1α(HIF-1α)、凋亡诱导因子(AIF)和Caspase-3的表达。结果:在升主动脉阻断期间,低流量灌注动物脑灌注流量远远低于高流量组(P=0.009),低流量低血压组r SO2低于其他各组r SO2值(P0.05),血清S100B水平显著高于高血压低流量组(P=0.032)。海马组织HIF-1α、AIF和Caspase-3的表达无组间差异。结论:低CPB血流可以造成全身和脑灌注不足,脑血管自我调节机制不能抵消低流量造成的脑灌注不足损伤。维持较高的动脉灌注压力,有助于在一定程度上代偿脑灌注不足引起的缺氧状况。     

Reduction of per- and postoperative blood loss with aprotinin (Trasylol) during extracorporeal circulation]

Aprotinin is a pharmacological agent which, when given in high doses during cardiopulmonary bypass (CPB), seems to reduce postoperative blood loss significantly and thereby reduces the need for blood transfusion. This study was undertaken to confirm these claims and to show that there was also decreased peroperative bleeding and a shorter operation time. The immediate postoperative clinical course was also assessed. The study was a prospective, randomised double-blind trial versus placebo in 60 coronary patients undergoing at least 2 aorto-coronary bypass grafts for the first time within a 3 month period. During surgery after stopping the CPB the blood loss recorded by aspiration was 49 +/- 61 ml in the aprotinin group and 90 +/- 84 ml in the placebo group (p less than 0.05). The quality of haemostasis in the operated area evaluated independently by the anaesthetist was judged to be excellent in 30 patients in the aprotinin group compared with only 19 in the placebo group (p less than 0.001). The time between coming off CPB and skin closure was significantly shorter in the aprotinin group (42 +/- 10 min versus 49 +/- 12 min) and the dose of protamine injected at the end of the operation was 19 +/- 38 mg in the aprotinin group compared to 43 +/- 46 mg in the placebo group (p less than 0.05). The blood loss recorded over 48 hours in the intensive care unit was nearly three times less in the aprotinin group (380 +/- 125 ml) than with placebo (852 +/- 523 ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide and inflammatory response in simulated extracorporeal circulation

BACKGROUND: Simulated extracorporeal circulation (SECC) induces inflammatory reaction. Nitric oxide (NO) has pro-and anti-inflammatory properties. NO role in SECC-related inflammatory response is unclear. The aim of this study was to clarify if NO affects the foreign-surface induced leukocyte activation during SECC. METHODS: Human blood was circulated through SECC during 3 hours. Control group C was ventilated with oxygen/air mixture and the study group with oxygen/air mixture and NO. Leukocyte activation was measured as serum levels of myeloperoxidase (MPO), human neutrophil lipocalin (HNL), lactoferrin (LF), interleukin-1-beta (IL-1 beta) and interleukin-10 (IL-10). Oxygen free radical production capacity was evaluated with chemiluminescence. NO metabolites nitrite/nitrate were estimated in serum. RESULTS: Leukocyte granule release increased over time. Addition of NO significantly increased MPO, HNL and LF release. The average difference increased with SECC duration. NO addition did not significantly affect measured interleukins concentration or oxygen free radical production capacity. NO metabolites increased significantly in the NO circuits. CONCLUSIONS: Results indicate that NO addition during SECC is pro-inflammatory and has no effect on oxygen free radical production and interleukin release.     

Changes in coagulation and fibrinolytic parameters caused by extracorporeal circulation

Summary During cardiopulmonary bypass (CPB) mechanical stress and the contact of blood with artificial surfaces lead to the activation of pro- and anticoagulant systems and the complement cascade, and to changes in cellular components. This phenomenon causes the postperfusion-syndrome, with leukocytosis, increased capillary permeability, accumulation of interstitial fluid, and organ dysfunction. In this study, we focused on the influence of the extracorporeal circulation, sternotomy, and heparin administration on the activation of coagulation and fibrinolysis. In 15 patients we investigated coagulation parameters before, during and post CPB, i.e., fibrinogen, antithrombin (AT) III, thrombin-antithrombin complex (TAT), prothrombin fragments F1 + 2 (F1 + 2), factor (F) XIIa, tissue factor (TF), and parameters of the fibrinolytic system, i.e., plasmin-antiplasmin-complex (PAP), D-dimer, tissueplasminogen-activator (tPA), urokinase-type plasminogen activator (uPA), and plasminogen-activator inhibitor type 1 (PAI 1). The results demonstrate distinct alterations in the above mentioned parameters. Despite administration of a high dose of heparin (activated clotting time [ACT] > 450s) combined with a low dose of aprotinin, activation of the coagulation and fibrinolytic pathways was observed. We found this activation was mainly caused by CPB and not by sternotomy. The activation of coagulation was due to foreign surface contact (F XII F XIIa) as well as to an effect of tissue factor release in the late phase of CPB. The enhanced fibrinolytic activity during CPB was, at least in part, caused by tPA and was followed by PAI 1 release.     

体外循环心肌损伤及心肌保护的研究进展

人工心肺体外循环(ECC)是一项比较成熟的心外科生命支持技术,ECC的发明使切开心脏和大血管进行直视手术成为可能。但ECC诱发的心脏等重要脏器的损伤是除手术效果外影响患者预后的决定性因素,严重者会对手术患者的预后造成显著影响。尽管在过去几十年里,随着ECC保护措施的进步,使ECC后心肌等重要脏器的损伤显著下降,但是ECC自身的技术特点决定了ECC导致的心功能下降仍然存在。本综述浅述了ECC诱导心肌损伤的部分病理机制以及降低心肌损伤的可行措施。     

Fibrinogen binding to the integrin alpha(IIb)beta(3) modulates store-mediated calcium entry in human platelets

Effects of the occupation of integrin alpha(IIb)beta(3) by fibrinogen on Ca(++) signaling in fura-2-loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca(++)] concentrations ([Ca(++)](i)) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca(++) but not in the absence of external Ca(++) or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca(++) entry. Fibrinogen also inhibited store-mediated Ca(++) entry (SMCE) activated after Ca(++) store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to alpha(IIb)beta(3) was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca(++) chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60(src) to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin alpha(IIb)beta(3) inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60(src). This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected.     

TPA decreases 1,25(OH)2D3 binding and calbindin D-28K in renal (MDBK) cells.

The effect of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) on vitamin D receptors (VDRs) was studied in MDBK cells, a normal bovine renal epithelial cell line. 24 h treatment of MDBK cells with TPA resulted in down-regulation of VDR number, with no change in the binding affinity for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or approximate molecular weight determined by fast protein liquid chromatography (FPLC). TPA treatment also reduced the level of calbindin D-28K, a vitamin D-dependent renal protein. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), an inactive phorbol ester, did not affect either 1,25(OH)2D3 binding or calbindin D-28K levels. TPA elicited a significant decrease in membrane-associated protein kinase C (PKC) activity which coincided with the reduction in VDR number and calbindin D-28K. These data support a link between TPA, PKC activity and vitamin D actions in kidney.     

体外循环在胸外科手术中应用的现状和进展

探讨体外循环在胸外科手术中的应用现状和进展。随着体外循环技术的不断发展,ECC不仅广泛用于心脏大血管手术,也可应用于胸外科的气管肿瘤切除、纵隔巨大肿瘤、肺癌食道癌等手术中,使过去单纯胸外科无法切除的部分肺癌、食道癌和气管肿瘤的手术切除得以实现。不但保证了麻醉和手术的安全,明显提高手术的成功率,而且使患者获得了长期生存和良好的生活质量。     

Coronary artery bypass grafting in octogenarians--outcome with and without extracorporeal circulation

Octogenarians are increasingly considered for coronary artery bypass grafting (CABG), but still represent a high-risk patient group with increased mortality and morbidity. In recent years off-pump surgery has been successfully established in CABG. The avoidance of extracorporeal circulation (ECC) seems to be of particular benefit for this patient group. We retrospectively analyzed our experience with CABG surgery with and without ECC in octogenarians to define the potential benefit of these different approaches in this high-risk group of patients. We analyzed the outcome after isolated CABG of 344 consecutive patients (219 male, 125 female, age: 82 +/- 2.4 years) who were aged 80 or older. Patients were divided into two groups according to the use of ECC. The on-pump group consisted of 237 patients (151 male, 86 female, 82 +/- 2.8 years) and the off-pump group consisted of 107 patients (68 male, 39 female, 82 +/- 1.9 years). The predicted EuroSCORE and EuroSCORE mortality risk were similar for both patient groups. The overall hospital mortality rate was 5.5 % (n = 17): 14 patients (5.9 %) in the on-pump group (n = 237, 100 %) and five patients (4.6 %) in the off-pump group (n = 107, 100 %). The average number of grafts in the on-pump group was 2.8 +/- 0.4 and it was 2.4 +/- 0.6 in the off-pump group ( P = 0.05). Morbidity was comparable in both groups. Significant variables in multivariate regression were preoperative atrial fibrillation ( P = 0.03; RR = 2.7), COPD ( P = 0.0001; RR = 6.5) and prolonged intubation ( P = 0.005; RR = 4.1). Isolated CABG in octogenarians can be performed with good clinical results, although a substantial mortality remains. The results of coronary surgery in this patient group with and without ECC are comparable with respect to mortality and morbidity.     

Fibronectin binding to thrombin-stimulated platelets: evidence for fibrin(ogen) independent and dependent pathways

Plasma fibronectin binds in a specific and saturable manner to thrombin- stimulated platelets. gamma-Thrombin stimulated 80% as much fibronectin binding to platelets as alpha-thrombin with conversion of less than or equal to 1% of platelet fibrinogen to fibrin. Afibrinogenemic and normal platelets bound similar quantities of fibronectin in the presence of calcium or magnesium-ethylene glycol tetra-acetic acid (EGTA). These observations indicate that fibronectin can interact with platelets without involvement of fibrin or fibrinogen. Nevertheless, two different effects of fibrin(ogen) on fibronectin binding were observed. First, exogenous fibrinogen inhibited fibronectin binding to thrombin-stimulated platelets. This inhibition was unidirectional, as fibronectin did not inhibit fibrinogen binding to ADP or thrombin- stimulated cells. Second, formaldehyde-fixed cells with surface- associated fibrin bound significant quantities of fibronectin. This interaction required calcium and did not occur on fixed cells with or without surface-bound fibrinogen. A portion of the ligand bound to fixed cells with surface-associated fibrin was modified to form a derivative with a molecular weight identical to that of the fibronectin subunit cross-linked to the alpha-chain of fibrin. This high mol wt derivative was also observed to a variable extent with living cells in the presence of magnesium or calcium but not in the presence of magnesium-EGTA. Thus, fibronectin binds to platelets by at least two mechanisms: (1) a fibrin(ogen)-independent pathway that requires divalent ions and is inhibited by exogenous fibrinogen; and (2) a fibrin-dependent pathway with an absolute calcium requirement. With nonaggregated, thrombin-stimulated platelets, the former pathway appears to predominate.     

Endotoxin-induced effects on platelets and monocytes in an in vivo model of inflammation

Aims Chronic inflammation is a major contributing factor to atherosclerosis and various markers of inflammation, fibrinolysis and coagulation are upregulated in patients with established atherosclerotic disease. The aim of this study was to investigate the direct and short-term effects of inflammation on platelet and monocyte activation with an in vivo model of endotoxemia in healthy volunteers. Methods and results In this study, 13 healthy male subjects with a mean age of 29.5±5.4 years received intravenous administration of lipopolysaccharide (LPS; 20 IU/kg IV). The kinetics of CD40-ligand and CD62P expression on platelets, tissue-factor binding on monocytes and platelet-monocyte aggregates were measured by whole blood flow cytometry at baseline and at 1, 2, 4, 6 and 24 hours after LPS administration. Plasma levels of soluble CD40-ligand were measured with an ELISA over the same time course. Platelet-monocyte aggregates, tissue-factor binding on monocytes and surface expression of platelet CD40L significantly increased in experimental endotoxemia in vivo, reaching peak values 1 hour after LPS administation. All values returned to baseline after 24 hours. Surface expression of CD62P on platelets and plasma levels of sCD40L did not change significantly in response to LPS. Conclusions In vivo administration of endotoxin leads to an activation of platelets and monocytes with an upregulation of proatherogenic CD40L on platelets. These findings underpin the role of inflammation in early atherogenesis through platelet and monocyte activation in an in vivo model. *authors contributed equally     

Effect of sternotomy and extracorporeal circulation on pulmonary neutrophil kinetics in pigs

Pulmonary margination of neutrophils may contribute to lung damage after extracorporeal circulation for cardiac surgery. We evaluated single–pass pulmonary neutrophil kinetics using the multiple indicator–dilution technique in control pigs (n = 10), after sternotomy alone (sterno, n = 10) or after 30 min of observation following a period of 90 min extracorporeal circulation (n = 7). Blood neutrophils increased in the control and sterno groups (p < 0.05) but remained unchanged in the extracorporeal circulation group. The transfer coefficient for neutrophil margination from the circulating to the lung–marginated pool (k_c–m) and pulmonary neutrophil clearance (Cl_c–m) were similar between the three groups. There was an inverse correlation between k_c–m and the degree of lung tissue perfusion evaluated from the tracer–accessible extravascular lung water (r = –0.54, p < 0.01). There was no arterio–venous gradient of neutrophils in any of the groups, suggesting a dynamic equilibrium of the margination/demargination processes. We conclude that extracorporeal circulation does not significantly modify single pass pulmonary neutrophil kinetics 30 min after reperfusion. The rate of neutrophil margination to the tracer–accessible lung tissue suggests that lung tissue de–recruitment is associated with increased neutrophil margination.     

体外循环术中致炎性和抗炎性细胞因子失衡的动态观察

目的研究体外循环术对致炎性细胞因子和抗炎性细胞因子异常释放的影响,并探讨其在术后炎症反应中的作用.方法ELISA测定7例风湿性心脏病瓣膜置换术不同时点血浆肿瘤坏死因子α(TNFα)、白细胞介素(IL)-6、IL-10和IL-1β浓度,同时用流式细胞术检测淋巴细胞内相应细胞因子的表达.结果4种细胞因子在体外循环术中和(或)术后均明显增高(P＜0.05),它们之间存在明显相关.手术开始后以TNFa、IL-6和IL-1β为主[峰值时间和浓度分别为体外循环术开始后30minTNFa(24.5±4.9)pg/ml,体外循环术停止后4hIL-6(361.8±75.1)pg/ml和IL-1β(18.4±2.8)pg/ml],而术后24h以IL-10为主[(957.4±206.6)pg/ml].淋巴细胞内3种细胞因子表达无明显变化(P＞0.05).结论体外循环术中致炎性细胞因子与抗炎性细胞因子平衡失调启动炎症反应并通过抑制细胞和体液免疫反应促进炎症反应.