Sevoflurane inhibits unstimulated and agonist-induced platelet antigen expression and platelet function in whole blood in vitro.

BACKGROUND: Previous studies have reported conflicting results about the effect of sevoflurane on platelet aggregation. To clarify this point, we investigated the effects of sevoflurane on platelet antigen expression and function in vitro. METHODS: Human whole blood was incubated for 1 h with 0.5 and 1 minimum alveolar concentration sevoflurane, 21% O(2), and 5% CO(2). A control sample was kept at the same conditions without sevoflurane. After stimulation with adenosine diphosphate or thrombin receptor agonist peptide 6, samples were stained with fluorochrome conjugated antibodies, and the expression of platelet glycoproteins GPIIb/IIIa, GPIb, and P-selectin, as well as activated GPIIb/IIIa, were measured with two-color flow cytometry. In addition, platelet function was assessed by means of thromboelastography and using the platelet function analyzer 100. RESULTS: Already in subanesthetic concentrations, sevoflurane inhibits unstimulated and agonist-induced GPIIb/IIIa surface expression and activated GPIIb/IIIa expression on platelets in whole blood. The agonist-induced redistribution of GPIb into the open canalicular system was also impaired by sevoflurane, whereas no effect on P-selectin expression in activated platelets could be found. Sevoflurane significantly reduced the maximum thromboelastographic amplitude. Furthermore, platelet function analyzer 100 closure times were significantly prolonged. CONCLUSION: The results show that sevoflurane significantly impairs platelet antigen expression in vitro. It is especially the inhibition of GPIIb/IIIa expression and activation that impairs bleeding time as reflected in thromboelastographic measurements and platelet function analyzer 100 closure times. The exact inhibitory mechanism remains unclear.     

Xenon does not affect human platelet function in vitro

We sought to determine whether xenon affects platelet glycoprotein expression and platelet-related hemostasis in vitro at a clinically relevant concentration. Human whole blood was stimulated with either adenosine diphosphate or the thrombin receptor agonist peptide (TRAP)-6 after incubation with 65% xenon. Halothane at 2 minimum alveolar anesthetic concentration was used as a positive control. Platelet function and activation were evaluated with two-color flow cytometry. The expression of the platelet glycoproteins GPIIb/IIIa, GPIb, and P selectin were detected with fluorochrome-conjugated monoclonal antibodies. In vitro measurement of platelet-related hemostasis under conditions of high shear stress was performed in citrated whole blood with a platelet function analyzer (PFA-100((R))) by using collagen/epinephrine and collagen/adenosine diphosphate cartridges. Xenon did not affect basal or agonist-induced expression of platelet membrane glycoproteins, activation-dependent conformational changes of the GPIIb/IIIa receptor, expression of P selectin, or PFA closure times. In contrast, halothane reduced TRAP-6-induced activation of the GPIIb/IIIa complex. Furthermore, collagen/epinephrine-induced PFA closure time was significantly prolonged. These results demonstrate that xenon does not affect the unstimulated or agonist-induced platelet glycoprotein expression, activation of GPIIb/IIIa, or platelet-related hemostasis.     

Platelet P-selectin and GPIIb/IIIa expression after liver transplantation and resection

Platelet dysfunction contributes to haemostatic defects, possibly leading to bleeding complications. We hypothesised that liver transplantation and liver resection, together with portal clamping time, might be a potential stimulus for platelet activation. Therefore, we determined the expression of platelet GPIIb/IIIa and P-selectin, representing important platelet activation markers, and the thrombopoietin (TPO) serum level after transplantation and resection. Twenty patients [ten that had undergone orthotopic liver transplantation (OLT), ten with liver resection (LRX)] were included in the study. From sequential venous blood samples, surface expression of GPIIb/IIIa and P-selectin was quantified by flow cytometry, and TPO serum levels were determined by ELISA. Baseline GPIIb/IIIa receptor expression on circulating platelets was significantly reduced in the OLT group compared to the LRX group and healthy volunteers. GPIIb/IIIa expression after activation with TRAP-6 increased significantly (P<0.001) in the LRX group but not in the OLT group. P-selectin expression after TRAP-6 stimulation increased significantly (P<0.001) in the LRX group, being comparable to that in healthy volunteers, whereas only a very low increase in the OLT group was found. In the OLT group, TPO serum levels were in the lower normal range and rose above the upper limit of normal values 24 h after reperfusion. These data indicate that neither liver transplantation nor liver resection influences GPIIb/IIIa and P-selectin expression on circulating platelets. There was a lack of expression in cirrhotic patients and unimpaired baseline expression and functional reserve in non-cirrhotic liver-resection patients. After liver transplantation, increasing serum TPO levels, which indicated a recovering graft function, resulted in rising peripheral platelet counts.     

Comparison of the effects of desflurane and sevoflurane on the expression of platelet surface glycoproteins in unstimulated and adenosine diphosphate-induced platelets in vitro

STUDY OBJECTIVE: To compare the effects of one minimum alveolar concentration (MAC) desflurane and sevoflurane on the expression of CD42b (glycoprotein [GP] Ib), CD41 (GPIIb), CD61 (GPIIIa), CD62P (P-selectin), and CD63 in both unstimulated and adenosine diphosphate (ADP)-stimulated platelets in vitro. SETTING: University laboratory. SUBJECTS: 15 healthy volunteers. INTERVENTIONS: Platelet-rich plasma was obtained and divided into three groups: platelet-rich plasma exposed to air (group 1); air plus one MAC desflurane (6% vol; group 2), and air plus one MAC sevoflurane (2% vol; group 3), for 40 minutes. Percentage of antigen-positive cells (%(+)) mean channel fluorescence (MCF(Sigma)), and index of platelet activation for positive platelets (IPA(+)) as expression markers for GPIb, GPIIb, GPIIIa, P-selectin, and CD63, were measured. MEASUREMENTS AND MAIN RESULTS: In unstimulated platelets, expression markers for GPIIb and GPIIIa were significantly lower in groups 2 and 3 than group 1 (P < 0.001). P-selectin expression markers were significantly higher in group 2 than in group 1 or group 3 (P < 0.016). CD63 expression markers were significantly lower in group 3 than group 1 (P < 0.016). In ADP-stimulated platelets, expression markers for all glycoproteins were significantly higher in all groups. CONCLUSION: Neither one MAC desflurane nor sevoflurane showed any significant change in ADP-stimulated platelets compared with the control group.     

Platelet GPIIb/IIIa is activated and platelet-leukocyte coaggregates formed in vivo during hemodialysis

BACKGROUND/AIM: During hemodialysis, platelets and leukocytes are activated and form platelet-leukocyte coaggregates in which GPIIb/IIIa (CD41/CD61) and CD62P (P-selectin) are involved. However, it is still controversial whether platelet activation and platelet-leukocyte coaggregate formation are dependent on the dialyzer membrane material. METHOD: We examined the appearance of activation-dependent antibody on platelets as an index of platelet activation, and the appearance of platelet-specific antigen on leukocytes as an index of platelet-leukocyte coaggregation, during hemodialysis in 7 patients treated using regenerated cellulose (RC) membrane and next using polysulfone (PS) membrane. In order to reduce the influence of factors other than dialyzer membrane material, this study was conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate. Moreover, flow cytometric techniques with whole blood were employed, which reduce artificial cell activation during the cell or plasma separation procedure. The platelet-specific monoclonal antibodies used in this study were anti-CD61, PAC-1 (which recognizes only the conformationally activated GPIIb/IIIa) and anti-CD62P. RESULTS: Changes in the percentage of PAC-1-positive platelets were significantly greater during hemodialysis with RC than with PS. However, changes in the percentage of CD62P-positive platelets were not significantly different between hemodialysis with RC and PS. Changes in the percentage of CD61- or CD62P-positive leukocytes were significantly greater during hemodialysis with RC than with PS. Although changes in percentage of PAC-1-positive platelets did not parallel those of CD62P-positive platelets during hemodialysis, there was a significant positive correlation between the percentage of CD61-positive leukocytes and the percentage of CD62P-positive leukocytes. CONCLUSION: This study, conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate in order to reduce the influence of factors other than the dialyzer membrane material, demonstrated that both the degrees of GPIIb/IIIa activation and platelet-leukocyte coaggregation were greater during hemodialysis with RC than PS.     

Reduced P-selectin expression on circulating platelets after prolonged cold preservation in renal transplantation

The uremic state in patients with terminal renal insufficiency is accompanied by a bleeding tendency connected with platelet dysfunction. Prolonged cold ischemia and inflammatory interactions between leukocytes, platelets and endothelial cells contribute to ischemia-/reperfusion (I/R) injury and may impair long-term graft survival. We evaluated the influence of the duration of cold preservation time on the expression of platelet GPIIb/IIIa and P-selectin and on the formation of leukocyte-platelet complexes after kidney transplantation. Fourteen patients undergoing kidney transplantation were divided into group I with long preservation time (26.6 +/- 1.9 h) and group II with short preservation time (8 +/- 6.1 h). Five venous blood samples (3 ml) were taken before induction of anesthesia, 12 h, 2, 7 and 14 d after transplantation. Surface expression of the GPIIb/IIIa, P-selectin and the percentage of platelet-granulocyte complexes were quantified by flow cytometry. Additionally blood from seven healthy volunteers was analyzed. GPIIb/IIIa and P-selectin expression on circulating platelets were significantly decreased in the long and the short-term graft preservation group compared with healthy volunteers. A significantly reduced P-selectin expression was found in the long-term preservation group compared with the short-term group. The percentage of platelet-granulocyte complexes also decreased in both preservation groups in the first 2 d after reperfusion and remained in this state in the long-term preservation group. Reduced expression of P-selectin on circulating platelets may be an indicator of I/R injury after prolonged kidney graft preservation.     

Paradoxical Effect of Nonphysiological Shear Stress on Platelets and von Willebrand Factor

Blood can become hypercoagulable by shear‐induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood‐shearing device. Platelet activation (P‐selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear‐induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM‐vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices.     

The effects of aprotonin on platelets in vitro using whole blood flow cytometry

We sought to evaluate the effects of aprotinin on the number and function of the platelet glycoprotein (GP) IIb-IIIa receptor and on the expression of P-selectin in vitro in order to gain insight into the potential mechanisms involved in the platelet-protective action of aprotinin during cardiopulmonary bypass. Aprotinin at 50 to 200 kallikrein inhibiting units/mL decreased the expression of activated GP IIb-IIIa complex in response to adenosine diphosphate or thrombin receptor activator peptide 6 in a dose-dependent manner in both citrated and heparinized whole blood experiments. Aprotinin inhibited adenosine diphosphate-induced platelet aggregation, but it exhibited no effect on the expression of GP IIIa and P-selectin. These results indicate that aprotinin interferes with the platelet fibrinogen receptor function during pharmacological activation. Reduced aggregability and platelet adhesion to fibrinogen adsorbed to synthetic surfaces in the presence of aprotinin may prevent platelet consumption during clinical cardiopulmonary bypass. This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated GP IIb-IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces. IMPLICATIONS: This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated glycoprotein IIb-IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces.     

The Effects of Heparinase 1 and Protamine on Platelet Reactivity

Background: Protamine is currently the most widely used drug for the reversal of heparin anticoagulation. Heparinase 1 (heparinase) is being evaluated as a possible alternative to protamine for the reversal of heparin anticoagulation. The authors evaluated the effects of equivalent doses of heparinase and protamine on platelet reactivity by measuring agonist-induced P-selectin expression.

Methods: After Institutional Review Board (IRB) approval, informed consent was obtained from 12 healthy volunteers and 8 patients undergoing surgery requiring cardiopulmonary bypass (CPB). Twenty-four ml of blood was obtained from each volunteer; 10 ml of blood was obtained from each patient before the CPB, and another 10 ml was obtained after CPB. Heparin was neutralized using heparinase or protamine. Platelet reactivity was assessed by measuring the expression of P-selectin after stimulation of platelets with increasing concentrations of a thrombin receptor agonist peptide (TRAP). Data were analyzed using analysis of variance. P < 0.05 was considered significant.

Results: For the healthy volunteers, the activated coagulation times (ACTs) of the heparinized samples returned to baseline values with heparinase (12.5 U/ml) or protamine (32.5 micro gram/ml). For the 8 patients, the ACTs returned to baseline with heparinase (20 U/ml) or protamine (50 micro gram/ml). The authors found no difference in the expression of P-selectin in samples neutralized with heparinase, but samples neutralized with protamine showed a significant decrease in the expression of P-selectin when compared with heparinized samples.     

The effect of epoprostenol on platelet activation and consumption during experimental extracorporeal perfusion

Hemorrhages are major complications experienced in 10-35% of neonates treated with extracorporeal life support (ECLS). The increased bleeding tendency is partly due to an ECLS induced thrombocytopenia and impaired platelet function. In the present study, we evaluated the effect of epoprostenol on the ECLS induced platelet consumption and activation. In terms of the methods, identical in vitro ECLS circuits were primed with fresh heparinized human blood and circulated for 24 h. Epoprostenol (2.4 microg/L blood/h) was added to one of the circuits in each pair. In total, 6 paired experiments were performed. The platelet count, neutrophil count, plasma concentration of hemoglobin, and platelet membrane expression of glycoproteins (GP) Ib and IIb/IIIa were assayed before the start and at 0.5, 1, 3, 12 and 24 h of perfusion. Higher platelet counts were observed in the experimental circuits. However, no difference in the platelet membrane expression of GPIb and GPIIb/IIIa could be observed between the circuits. In conclusion, epoprostenol reduces platelet consumption during ECLS without affecting the membrane expression of GPIb and GPIIb/IIIa.     

Sevoflurane anesthesia attenuates adenosine diphosphate-induced P-selectin expression and platelet-leukocyte conjugate formation

The expression of P-selectin on the surface of platelets and platelet-leukocyte conjugate formation are considered to be an indicator of platelet activation and are important in thrombotic and inflammatory disease. Previous studies have reported the inhibitory effects of sevoflurane on platelet aggregation. We investigated whether sevoflurane alters the expression of P-selectin on platelets and the formation of platelet-leukocyte conjugates. Twenty-five patients undergoing minor extremity surgery received sevoflurane-based general anesthesia, with mask induction and laryngeal mask airway anesthesia maintenance. Whole blood was obtained before and 40 min after sevoflurane anesthesia. Unstimulated and adenosine diphosphate-stimulated samples of whole blood and platelet rich plasma were stained with fluorochrome-conjugated antibodies. The expression of P-selectin on platelets and the formation of platelet-leukocyte conjugates were measured using flow cytometry. Sevoflurane inhibited platelet P-selectin expression. It also reduced the formation of platelet-leukocyte conjugates, both in unstimulated and adenosine diphosphate-stimulated blood samples at 3%-4% end-expiratory sevoflurane concentrations used to maintain anesthesia.     

体外循环对血小板功能影响的研究

选择心脏瓣膜替换术病人１０例，用流式细胞术（ＦＣＭ）方法在围术期定量检测血小板质膜蛋白Ｉｂ、ＩＩｂ／ＩＩａ复合物、血小板α颗粒膜蛋白、溶酶体完整膜蛋白－ＣＤ６３及血小板计数等项血小板膜糖蛋白指标进行统计学处理。结果表明体外循环中血小板计数、膜糖蛋白Ｉｂ均明显下降；α－颗粒膜蛋白１４０、溶酶体完整膜蛋白及膜糖蛋白Ｉｂ／ＩＩａ均显著增高。证实体外循环（ＣＰＢ）对血小板膜糖蛋白的影响，更深入了解ＣＰＢ导致血小板功能获得性损害的分子机制。     

Device‐induced platelet dysfunction in mechanically assisted circulation increases the risks of thrombosis and bleeding

Thrombotic and bleeding complications are the major obstacles for expanding mechanical circulatory support (MCS) beyond the current use. While providing the needed hemodynamic support, those devices can induce damage to blood, particularly to platelets. In this study, we investigated device‐induced alteration of three major platelet surface receptors, von Willebrand factor (VWF) and associated hemostatic functions relevant to thrombosis and bleeding. Fresh human whole blood was circulated in an extracorporeal circuit with a clinical rotary blood pump (CentriMag, Abbott, Chicago, IL, USA) under the clinically relevant operating condition for 4 hours. Blood samples were examined every hour for glycoprotein (GP) IIb/IIIa activation and receptor loss of GPVI and GPIbα on the platelet surface with flow cytometry. Soluble P‐selectin in hourly collected blood samples was measured by enzyme linked immunosorbent assay to characterize platelet activation. Adhesion of device‐injured platelets to fibrinogen, collagen, and VWF was quantified with fluorescent microscopy. Device‐induced damage to VWF was characterized with western blotting. The CentriMag blood pump induced progressive platelet activation with blood circulating time. Particularly, GPIIb/IIIa activation increased from 1.1% (Base) to 11% (4 hours) and soluble P‐selectin concentration increased from 14.1 ng/mL (Base) to 26.5 ng/mL (4 hours). Those device‐activated platelets exhibited increased adhesion capacity to fibrinogen. Concurrently, the CentriMag blood pump caused progressive platelet receptor loss (GPVI and GPIbα) with blood circulating time. Specifically, MFI of the GPVI and GPIbα receptors decreased by 17.2% and 16.1% for the 4‐hours sample compared to the baseline samples, respectively. The device‐injured platelets exhibited reduced adhesion capacities to collagen and VWF. The high molecular weight multimers (HMWM) of VWF in the blood disappeared within the first hour of the circulation. Thereafter the multimeric patterns of VWF were stable. The change in the VWF multimeric pattern was different from the progressive structural and functional changes of platelets with the circulation time. This study suggested that the CentriMag blood pump could induce two opposite effects on platelets and associated hemostatic functions under a clinically relevant operating condition. The device‐altered hemostatic function may contribute to thrombosis and bleeding simultaneously as occurring in patients supported by a rotary blood pump. Device‐induced damage of platelets may be an important cause for bleeding in patients supported with rotary blood pump MCS systems relative to device‐induced loss of HMWM‐VWF.     

The GP IIb/IIIa inhibitor abciximab (ReoPro) decreases activation and interaction of platelets and leukocytes during in vitro cardiopulmonary bypass simulation.

OBJECTIVE: Cardiopulmonary bypass (CPB) induces a systemic inflammatory response and increases expression of the platelet activation marker P-selectin which mediates binding of platelets to leukocytes. Inhibition of the platelet GP IIb/IIIa receptor during CPB has been shown to protect platelets without increasing bleeding complications and is assumed to reduce the inflammatory response. The aim of this study was to investigate the effect of the GP IIb/IIIa inhibitor abciximab (ReoPro) on the function and interaction of platelets and leukocytes during experimental CPB. METHODS: Heparinized (3 U/ml) fresh whole blood of healthy volunteers was treated before continuous in vitro circulation in a well established CPB model with 3.2 microg/ml abciximab (n=6) or left untreated as control (n=6). Measurements were made before (baseline) and after 30 and 60 min of circulation and comprised: percentage of platelets expressing P-selectin and percentage of platelet-bound leukocytes (flow cytometry), release of the leukocyte activation marker PMN-elastase (ELISA), and platelet and leukocyte counts. RESULTS: Abciximab almost completely prevented a CPB-induced increase of platelet P-selectin and platelet-leukocyte binding after 30 and 60 min of circulation, and significantly inhibited release of PMN-elastase after 30 min of circulation. Furthermore, abciximab significantly inhibited a CPB-induced decrease of platelet and leukocyte counts. CONCLUSIONS: Abciximab inhibits CPB-induced activation, interaction and consumption of platelets and leukocytes in vitro. GP IIb/IIIa inhibition should be considered as a promising approach not only to conserve platelet function but also to inhibit pro-inflammatory events during CPB in vivo.     

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.     

The impact of shear stress on device-induced platelet hemostatic dysfunction relevant to thrombosis and bleeding in mechanically assisted circulation

The aim of this study was to examine the impact of the nonphysiological shear stress (NPSS) on platelet hemostatic function relevant to thrombosis and bleeding in mechanically assisted circulation. Fresh human blood was circulated for four hours in in vitro circulatory flow loops with a CentriMag blood pump operated under a flow rate of 4.5 L/min against three pressure heads (70 mm Hg, 150 mm Hg, and 350 mm Hg) at 2100, 2800, and 4000 rpm, respectively. Hourly blood samples from the CentriMag pump-assisted circulation loops were collected and analyzed for glycoprotein (GP) IIb/IIIa activation and receptor shedding of GPVI and GPIbα on the platelet surface with flow cytometry. Adhesion of platelets to fibrinogen, collagen, and von Willebrand factor (VWF) of the collected blood samples was quantified with fluorescent microscopy. In parallel, mechanical shear stress fields within the CentriMag pump operated under the three conditions were assessed by computational fluid dynamics (CFD) analysis. The experimental results showed that levels of platelet GPIIb/IIIa activation and platelet receptor shedding (GPVI and GPIbα) in the blood increased with increasing the circulation time. The levels of platelet activation and loss of platelet receptors GPVI and GPIbα were consistently higher with higher pressure heads at each increasing hour in the CentriMag pump-assisted circulation. The platelet adhesion on fibrinogen increased with increasing the circulation time for all three CentriMag operating conditions and was correlated well with the level of platelet activation. In contrast, the platelet adhesion on collagen and VWF decreased with increasing the circulation time under all the three conditions and was correlated well with the loss of the receptors GPVI and GPIbα on the platelet surface, respectively. The CFD results showed that levels of shear stresses inside the CentriMag pump under all three operating conditions exceeded the maximum level of shear stress in the normal physiological circulation and were strongly dependent on the pump operating condition. The level of platelet activation and loss of key platelet adhesion receptors (GPVI and GPIbα) were correlated with the level of NPSS generated by the CentriMag pump, respectively. In summary, the level of NPSS associated with pump operating condition is a critical determinant of platelet dysfunction in mechanically assisted circulation.     

Platelet activation markers in patients with nephrotic syndrome. A comparative study of different platelet function tests

BACKGROUND/AIM: Enhanced platelet reactivity may play a significant role in the genesis of the hypercoagulable state of nephrotic syndrome. However, the role of platelet function testing in nephrosis is controversial, partly because the methods used to assess platelet function (platelet aggregation and immunoassays of plasma beta-thromboglobulin and platelet factor 4) have such marked methodological problems. In the present study, we evaluated several tests assessing platelet function in 18 adult patients with idiopathic nephrotic syndrome and normal renal function. METHODS: Platelet function was assessed by measurement of plasma beta-thromboglobulin (enzyme-linked immunosorbent assay, ELISA), plasma P-selectin (ELISA), circulating platelets exposing the activation-dependent antigens P-selectin (CD62P) and lysosomal GP53 (CD63) (flow cytometry), and by aggregation response to agonists such as ADP and collagen. Results were compared to those obtained in a group of 16 age- and gender-matched healthy subjects. RESULTS: Levels of plasma beta-thromboglobulin (p = 0.001), plasma P-selectin (p < 0.001), and CD62P/CD63-positive platelets (p < 0.001 for both) were increased in nephrotic patients as compared to healthy controls. Platelet hyperaggregability in vitro was found in 13/18 patients. The reproducibility of platelet activation markers, as assessed by blood sample collection a week later from all patients, was found to be higher for plasma P-selectin (Spearman correlation coefficient, R = 0.99) and circulating activated platelets (CD62P: R = 0.97; CD63: R = 0.96) than for plasma beta-thromboglobulin (R = 0.78). CONCLUSIONS: Pronounced platelet activation takes place in nephrotic syndrome and may contribute to the hypercoagulability of nephrosis. Whole blood flow cytometry assay of platelet activation and plasma P-selectin assay may represent useful tests to assess the hypercoagulable state in nephrotic patients.     

Platelet and leukocyte activation during aortoiliac angiography and angioplasty.

OBJECTIVE: to evaluate platelet and leukocyte activation during aortoiliac angiography and percutaneous transluminal angioplasty (PTA). METHODS: an observational study of 14 patients with aortoiliac atherosclerotic disease, nine of whom underwent PTA. The proportion of fibrinogen-, and P-selectin positive platelets, P-selectin expression on platelets, intraplatelet cGMP and cAMP, CD18 positive granulocytes, CD18 expression on granulocytes, plasma (p)-neopterin, p-TNF alpha and p- interleukin-6 were repeatedly measured in arterial blood during angiography and in venous blood before and after. RESULTS: compared to a previous venous sample, arterial intraplatelet cAMP was increased proximal to the atherosclerotic lesion before contrast infusion and PTA (median 18 [range: 14-22] vs 16 [15-21] pmol/10(9) platelets p<0.05), and intraplatelet cGMP was increased proximal to the lesion after contrast infusion and PTA (1.2 [0.8-3.9] vs 0.9 [0.6-2.5] pmol/10(9) platelets p<0.05). Four hours after angiography, both the proportion of P-selectin positive platelets (28[11-55]%) and platelet P-selectin expression (9[6-40]) had decreased (p<0.05), from arterial values distal to the lesion before contrast infusion and PTA (57 [24-78]% and 26 [10-83]). Granulocyte CD18 expression was lower during angiography than in a previous venous sample. CONCLUSIONS: the results are compatible with platelet but not leukocyte activation during peripheral angiography.     

Impaired expression of glycoproteins on resting and stimulated platelets in uraemic patients.

BACKGROUND: The increased bleeding tendency of chronic renal failure (CRF) patients has been attributed to platelet dysfunction. However, reports on various platelet functions in uraemic patients have been conflicting. The present study sought to analyse platelet function by examining their surface glycoproteins in well-identified populations of CRF patients. METHODS: Three groups were studied: 22 chronic haemodialysis (HD) patients, 25 conservatively treated patients with CRF and 30 controls. Bleeding tendency was assessed by measuring bleeding time and by recording current haemorrhagic symptoms. We measured the fibrinogen receptor GPIIbIIIa, the von Willebrand receptor GPIb, and P-selectin levels on the platelet surface using flow cytometry. RESULTS: Forty percent of CRF and 45% of HD patients had bleeding. The bleeding time was similar in the HD and CRF groups, but was longer in both groups than in controls. In resting platelets, GPIb expression was lower in CRF patients than in controls. In stimulated platelets (i) GPIb expression was higher in HD patients than in both controls and CRF patients; and (ii) GPIIbIIIa and P-selectin expression were lower in CRF and HD patients than in controls. CONCLUSIONS: These findings indicate that uraemic platelets are hyporesponsive to stimulation.     

Platelet function and anesthetics in cardiac surgery: an in vitro and ex vivo study.

We studied the effects of the anesthetics commonly used in cardiac surgery on platelet function. Fentanyl, droperidol, succinylcholine, pancuronium, thiopental, and diazepam at therapeutic concentrations were tested for their in vitro effects on the expression of platelet membrane glycoproteins Ib and IIbIIIa (GpIb, GpIIb-IIIa) and of P-selectin in anticoagulated whole blood by flow cytometry. The expression of P-selectin was determined under basal conditions, after the incubation of blood with adenosine diphosphate (ADP) 10 micromol/L, and the stable prostaglandin endoperoxide analog U46619 1 micromol/L. No drug affected the expression of P-selectin in unstimulated and ADP- or U46619-stimulated platelets, with the exception of thiopental, which markedly decreased the U46619-induced expression of P-selectin. Thiopental concentration-dependently inhibited U46619-induced and ADP-induced platelet aggregation, with effects on U46619-induced aggregation at therapeutic concentrations. To assess ex vivo effects, the same platelet markers were also assessed in blood obtained from 10 patients undergoing elective coronary surgery. Compared with basal values, platelet response to U46619 was significantly reduced just after the administration of anesthetic drugs, and the effect persisted for 48 h after surgery. Our study suggests that, at therapeutic concentrations, thiopental inhibits U46619-induced platelet activation both in vitro and ex vivo. The mechanisms responsible of this effect, together with its clinical significance, require further investigation. IMPLICATIONS: Thiopental inhibited prostaglandin-induced platelet activation at therapeutic concentrations both in vitro and ex vivo in cardiac surgical patients whereas adenosine diphosphate-induced activation was affected only at supratherapeutic drug concentrations. Thus, administration of sodium thiopental may contribute to the in vivo impairment of platelet function in patients undergoing elective cardiac surgery.