Platelet glycoprotein IIb/IIIa receptor antagonist (abciximab) inhibited platelet activation and promoted skin flap survival after ischemia/reperfusion injury

BACKGROUND: Evidence has shown that platelets play an important role in the pathogenesis of flap failure. Employing a rat inferior epigastric artery skin flap as a flap reperfusion injury model, we investigated whether platelet activation was involved in the skin flap failure and whether administration of abciximab (ReoPro, chimeric 7E3 Fab) could decrease platelet activation/aggregation and promote flap survival. METHODS: Normal saline and abciximab (0.06 mg/kg; 0.2 mg/kg; 1 mg/kg) were injected intravenously into skin flaps 30 min before reperfusion and 1 h after reperfusion (each subgroup n = 6). Platelet activation as demonstrated by P-selectin (CD62P) was analyzed by flow cytometry. P-selectin expression on flap vessels was detected by immunohistochemical staining. Platelet aggregation was induced with adenosine diphosphate (ADP). Laser Doppler flowmetry monitored tissue perfusion. The surviving area was evaluated 7 days postoperatively. RESULTS: CD62P progressively increased after reperfusion. The peak CD62P occurred after reperfusion for 12 h. Immunohistochemical staining showed CD62P significantly deposited on the endothelium after reperfusion. Administration of abciximab (1 mg/kg) effectively improved flap survival rate (P = 0.003), significantly decreased ADP-induced platelet aggregation (P < 0.001), and suppressed CD62P expression on blood platelets (P = 0.002) and its deposition on the flap vessels. CONCLUSION: Abciximab promotion of skin flap survival is due to blocked platelet activation/aggregation and decreased activated-platelet deposition on the vascular endothelium. Thus, administration of a platelet glycoprotein IIb/IIIa receptor antagonist such as abciximab may save the skin flap from reperfusion injury after a long period of ischemia.     

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.     

Emergency coronary artery bypass surgery in the era of glycoprotein IIb/IIIa receptor antagonist use

The use of intravenous glycoprotein (GP) IIb/IIIa platelet receptor antagonists in the management of patients with acute coronary syndrome or those undergoing percutaneous coronary intervention (PCI) has become increasingly common in recent years. There are three GP IIb/IIIa receptor antagonists currently available for clinical use. Patients on GP IIb/IIIa receptor antagonists who require emergency surgical revascularization may be at increased risk for excessive peri- and postoperative bleeding. The duration of action of eptifibatide and tirofiban are short because they bind reversibly to the GP IIb/IIIa receptor and have a short half-life. Therefore, within a relatively short time after discontinuation of these agents, surgery can be performed with little or no increased risk of bleeding and without the need for additional hemostatic measures. Abciximab has a short plasma half-life but a long duration of action due to its high-affinity binding of GP IIb/IIIa receptors. Early retrospective studies demonstrated a higher incidence of major bleeding and requirement for blood transfusion, especially in those undergoing surgery within 12 hours of the discontinuation of abciximab. However, platelet transfusion has been shown to successfully reduce the incidence of these complications. The current evidence therefore indicates that, with appropriate measures, urgent surgical revascularization can be safely performed in patients who have received a GP IIb/IIIa receptor antagonist with little added risk. The benefits of these agents in the treatment of patients with an acute coronary syndrome or undergoing PCI are not obviated by the need for emergency bypass surgery.     

Emergency Coronary Artery Bypass Surgery in the Era of Glycoprotein IIb/IIIa Receptor Antagonist Use

Abstract The use of intravenous glycoprotein (GP) IIb/IIIa platelet receptor antagonists in the management of patients with acute coronary syndrome or those undergoing percutaneous coronary intervention (PCI) has become increasingly common in recent years. There are three GP IIb/IIIa receptor antagonists currently available for clinical use. Patients on GP IIb/IIIa receptor antagonists who require emergency surgical revascularization may be at increased risk for excessive peri‐and postoperative bleeding. The duration of action of eptifibatide and tirofiban are short because they bind reversibly to the GP IIb/IIIa receptor and have a short half‐life. Therefore, within a relatively short time after discontinuation of these agents, surgery can be performed with little or no increased risk of bleeding and without the need for additional hemostatic measures. Abciximab has a short plasma half‐life but a long duration of action due to its high‐affinity binding of GP IIb/IIIa receptors. Early retrospective studies demonstrated a higher incidence of major bleeding and requirement for blood transfusion, especially in those undergoing surgery within 12 hours of the discontinuation of abciximab. However, platelet transfusion has been shown to successfully reduce the incidence of these complications. The current evidence therefore indicates that, with appropriate measures, urgent surgical revascularization can be safely performed in patients who have received a GP IIb/IIIa receptor antagonist with little added risk. The benefits of these agents in the treatment of patients with an acute coronary syndrome or undergoing PCI are not obviated by the need for emergency bypass surgery.     

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。     

Differential platelet receptor expression following hydroxyethyl starch infusion in thrombocytopaenic orthotopic liver transplantation recipients

BACKGROUND AND OBJECTIVE: Platelet function abnormalities influence the haemostatic defect in patients with liver failure. Patients after orthotopic liver transplantation present thrombocytopaenia associated with bleeding problems, which may be aggravated by the interaction of hydroxyethyl starches with platelets. METHODS: From 12 patients after liver transplantation venous blood samples (3 mL) were taken before, 20 and 120 min after infusion of hydroxyethyl starch of medium molecular weight (200 kDa/0.5) 6% 10 mL kg(-1) over a period of 30 min. Surface expression of glycoprotein IIb/IIIa and P-selectin were quantified by flow cytometry as well as the percentage of platelet-leucocyte complexes. RESULTS: A significant decrease of P-selectin expression following administration of hydroxyethyl starch after 120 min (89.1 +/- 4.2%, P = 0.029) and a corresponding significant reduction in the formation of platelet-monocyte complexes (81.1 +/- 7.8%, P = 0.001) were observed. There was no alteration in the glycoprotein IIb/IIIa expression after hydroxyethyl starch infusion. CONCLUSIONS: Infusion of hydroxyethyl starch 200 kDa/0.5 in clinically relevant doses does not alter glycoprotein IIb/IIIa expression in thrombocytopaenic patients with pre-existing platelet dysfunction after orthotopic liver transplantation. Accordingly, infusion of hydroxyethyl starch may have a beneficial effect on microvascular graft perfusion through the resulting haemodilution and reduced P-selectin expression with subsequent reduced leucocyte-platelet complexes and endothelial adhesion.     

Effects of ε-Aminocaproic Acid and Aprotinin on Leukocyte-Platelet Adhesion in Patients Undergoing Cardiac Surgery

Background: The administration of aprotinin during cardiopulmonary bypass (CPB) is hypothesized to decrease activation of leukocytes and platelets and possibly reduce their adhesion. Although [epsilon]-aminocaproic acid (EACA) shares the ability of aprotinin to inhibit excessive plasmin activity after CPB, its effect on leukocyte and platelet activation and leukocyte-platelet (heterotypic) adhesion is largely unknown. This study was performed to determine the relative effectiveness of the antifibrinolytics, aprotinin and EACA, at reducing leukocyte and platelet activation and leukocyte-platelet conjugate formation in patients undergoing CPB.

Methods: Thirty-six patients scheduled to undergo cardiac surgery with CPB were randomized in a double-blind fashion to receive EACA, aprotinin, or saline (placebo). Markers of plasmin activity (D-dimer concentrations), platelet activation (CD62P), leukocyte activation (CD11b), and leukocyte-platelet adhesion (monocyte- and neutrophil-platelet conjugates) were measured before, during, and after CPB.

Results: Platelet CD62P (P-selectin), monocyte CD11b, and monocyte-platelet conjugates were all significantly increased (compared with baseline) in the saline group during and after CPB. Despite equivalent reductions in D-dimer formation in patients receiving EACA (P < 0.0001) and aprotinin (P < 0.0001), decreases in platelet CD62P and monocyte CD11b expression were incomplete (not significantly different from saline control). In contrast, peak monocyte-platelet conjugate formation was significantly reduced by both EACA (P = 0.026) and aprotinin (P = 0.039) immediately after CPB.     

Effects of epsilon-aminocaproic acid and aprotinin on leukocyte-platelet adhesion in patients undergoing cardiac surgery

BACKGROUND: The administration of aprotinin during cardiopulmonary bypass (CPB) is hypothesized to decrease activation of leukocytes and platelets and possibly reduce their adhesion. Although epsilon-aminocaproic acid (EACA) shares the ability of aprotinin to inhibit excessive plasmin activity after CPB, its effect on leukocyte and platelet activation and leukocyte-platelet (heterotypic) adhesion is largely unknown. This study was performed to determine the relative effectiveness of the antifibrinolytics, aprotinin and EACA, at reducing leukocyte and platelet activation and leukocyte-platelet conjugate formation in patients undergoing CPB. METHODS: Thirty-six patients scheduled to undergo cardiac surgery with CPB were randomized in a double-blind fashion to receive EACA, aprotinin, or saline (placebo). Markers of plasmin activity (D-dimer concentrations), platelet activation (CD62P), leukocyte activation (CD11b), and leukocyte-platelet adhesion (monocyte- and neutrophil-platelet conjugates) were measured before, during, and after CPB. RESULTS: Platelet CD62P (P-selectin), monocyte CD11b, and monocyte-platelet conjugates were all significantly increased (compared with baseline) in the saline group during and after CPB. Despite equivalent reductions in D-dimer formation in patients receiving EACA (P < 0.0001) and aprotinin (P < 0.0001), decreases in platelet CD62P and monocyte CD11b expression were incomplete (not significantly different from saline control). In contrast, peak monocyte-platelet conjugate formation was significantly reduced by both EACA (P = 0.026) and aprotinin (P = 0.039) immediately after CPB. CONCLUSIONS: EACA seems to be as effective as aprotinin at reducing peak monocyte-platelet adhesion after CPB. Furthermore, inhibition of excessive plasmin activity seems to influence monocyte-platelet adhesion. The findings suggest that monocyte-platelet conjugate formation may be a useful marker of monocyte and platelet activation in this clinical setting.     

Effect of laser welding with human serum albumin on the expression of P-selectin on platelets

BACKGROUND AND OBJECTIVE: Artery repair by means of laser energy induces activation of platelets with a risk of thrombosis and local inflammatory reactions. The aim of this study was to investigate the effect of human serum albumin, the most common solder in laser surgery, on platelet activation. STUDY DESIGN/MATERIALS AND METHODS: Platelet activation was evaluated in canine blood by using two-color flow cytometry with a phycoerythrin-labeled antibody to a common platelet marker, glycoprotein IIb/IIIa and fluorescein isothiocyanate-labeled antibody to a platelet activation molecule, P-selectin. Human serum albumin was applied in vitro and in vivo, as a solder during laser reconstruction of canine arteries. RESULTS: In vitro, albumin significantly (P < 0.01) reduces the expression of P-selectin on platelets. This is most likely related to the blockage of P-selectin by albumin, which binds to the platelet surface, as confirmed by flow cytometry with fluorescein isothiocyanate-labeled albumin. In vivo, application of albumin solder tended to result in a lower percentage of P-selectin-expressing platelets in laser-repaired arteries compared to suture-repaired arteries. CONCLUSION: Albumin decreases the percentage of P-selectin-expressing platelets in vitro. Further research may allow the platelet activation inhibiting properties of albumin to be further optimized in vivo.     

Combined administration of nitric oxide gas and iloprost during cardiopulmonary bypass reduces platelet dysfunction: a pilot clinical study

BACKGROUND: Thrombocytopenia and platelet dysfunction are major mechanisms of cardiopulmonary bypass-induced postoperative hemorrhage. This study evaluated the effects of low amounts of nitric oxide, iloprost (prostacyclin analog), and their combination administered directly into the oxygenator on platelet function, platelet-leukocyte interactions, and postoperative blood loss in patients undergoing coronary artery bypass grafting. METHODS: Blood samples from 41 patients randomized to the control, nitric oxide (20 ppm), iloprost (2 ng x kg -1 x 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.     

The effect of s-nitroso-glutathione on platelet and leukocyte function during experimental extracorporeal circulation

Treatment with extracorporeal membrane oxygenation ECMO) is associated with side effects, e.g., blood cell consumption and activation. Our group has earlier shown that nitric oxide administered as a gas reduces platelet consumption and activation. In the present work we have studied the effect of the NO-donor S-nitroso-glutathione GSNO) on platelets and leukocytes in an in vitro extracorporeal circuit. Two complete ECMO circuits were perfused with fresh heparinized human blood for 24 hours. GSNO was administered as a continuous infusion to one circuit at a rate of 0.7 mg/hour in four paired experiments and at a rate of 3.5 mg/hour in another four paired experiments. The other circuit was used as a control. Blood samples were withdrawn from both circuits before the start of the experiments and at 0.5, 1, 3, 12, and 24 hours of perfusion. The samples were analyzed for red blood cell count, leukocyte count, platelet count, platelet membrane expression of glycoproteins GP) Ib and GPIIb/IIIa, leukocyte membrane expression of cluster of differentiation CD) 11b/CD18, as well as plasma concentration of tumor necrosis factor TNF)-alpha, interleukin IL)-1beta, and IL-8. No difference in these parameters between the GSNO and the control circuit at any time point was assayed. In this study, no significant effect of GSNO on circulating platelets or leukocytes during experimental extracorporeal circulation could be shown.     

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.     

Efficacy of FK633, an ultra-short acting glycoprotein IIb/IIIa antagonist on platelet preservation during and after cardiopulmonary bypass.

OBJECTIVE: Temporary pharmacologic inhibition of platelet function during and after cardiopulmonary bypass (CPB) (platelet anesthesia) is an attractive strategy for preserving platelets during CPB. We examined the efficacy of FK633, an ultra-short acting glycoprotein IIb/IIIa antagonist. METHODS: The study was carried out in six mongrel dogs that received an intravenous bolus of 0.1 mg/kg of FK633 at the time of administration of heparin (group F), and six control dogs (group C). All animals underwent 60 min of normothermic CPB followed by a 2-h observation period. Blood samples for platelet count, platelet aggregation to adenosine diphosphate and parameters concerning the coagulation system were obtained at eight time points. Hemodynamics, bleeding time, and postoperative blood loss were assessed serially. Scanning electron micrograph of the oxygenator's membrane was investigated. RESULTS: FK633 significantly protected platelet number (group F, 59+/-10% versus group C, 38+/-15% of the pre-CPB value; P < 0.01), and inhibited platelet aggregation to adenosine diphosphate (group F, 13+/-12% versus group C, 35+/-9% of the pre-CPB value; P < 0.01) during CPB. Postoperative blood loss did not significantly differ between the two groups, but there was a tendency of less bleeding in group F (group F, 73+/-23 ml versus group C, 111+/-44 ml; P = 0.09). In group F, scanning electron micrograph of the oxygenator's membrane showed that its surface was free from platelets. There were no significant differences between the groups in hemodynamics. CONCLUSIONS: An ultra-short acting glycoprotein IIb/IIIa antagonist, FK633, is effective in preventing both platelet aggregation and thrombocytopenia during CPB, and may be effective for minimizing postoperative bleeding.     

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.     

Inhibition of platelet aggregation by the GPIIb/IIIa antagonist Reopro does not significantly prolong xenograft survival in an ex vivo model

Effects on hyperacute rejection were studied in a discordant model with the platelet GPIIb/IIIa antagonist Reopro. Pig kidneys perfused with human blood survived median 118 min in the Reopro group and 103 min in the controls (P = 0.22). Platelet and leukocyte counts decreased, whereas plasma thrombospondin and soluble as well as platelet membrane P-selectin increased significantly in both groups without significant intergroup differences. beta-Thromboglobulin and myeloperoxidase increased significantly more in the control group than in the Reopro group (P = 0.009 and P = 0.02, respectively). The classical complement pathway was substantially and similarly activated in both groups. Light and electron microscopy revealed arterial thrombi and numerous glomerular platelet aggregates in the control group in contrast to the Reopro group. In conclusion, Reopro reduced platelet aggregation, and platelet and leukocyte activation to some extent, but had no effect on complement activation and did not significantly prolong xenograft survival, even though better preservation of morphology was shown.     

Epinephrine enhances platelet-neutrophil adhesion in whole blood in vitro

Previous studies showed that alpha- or beta-adrenoceptor stimulation by catecholamines influenced neutrophil function, cytokine liberation, and platelet aggregability. We investigated whether adrenergic stimulation with epinephrine also alters platelet-neutrophil adhesion. This might be of specific interest in the critically ill, because the increased association of platelets and neutrophils has been shown to be of key importance in inflammation and thrombosis. For this purpose, whole blood was incubated with increasing concentrations of epinephrine (10 nM, 100 nM, and 1 microM). To distinguish receptor-specific effects, a subset of samples was incubated with propranolol (10 microM) or phentolamine (10 microM) before exposure to epinephrine. After incubation, another subset of samples was also stimulated with 100 nM of N-formyl-methionyl-leucyl-phenylalanine. All samples were stained, and platelet-neutrophil adhesion and CD45, L-selectin, CD11b, P-selectin glycoprotein ligand-1, glycoprotein IIb/IIIa, and P-selectin expression were measured by two-color flow cytometry. Epinephrine significantly enhanced platelet-neutrophil adhesion and P-selectin and glycoprotein IIb/IIIa expression on platelets. CD11b and L-selectin expression on unstimulated neutrophils remained unchanged, whereas N-formyl-methionyl-leucyl-phenylalanine-induced upregulation of CD11b and downregulation of L-selectin were suppressed by epinephrine. beta-Adrenergic blockade before incubation with epinephrine increased platelet-neutrophil aggregates and adhesion molecule expression (CD11b, P-selectin, and glycoprotein IIb/IIIa) even further. These results demonstrate that epinephrine enhances platelet-neutrophil adhesion. The alpha-adrenergic receptor-mediated increase in P-selectin and glycoprotein IIb/IIIa expression on platelets may contribute substantially to this effect. Our study shows that inotropic support enhances the platelet-neutrophil interaction, which might be crucial for critically ill patients.     

Local glycoprotein IIb/IIIa receptor inhibitor delivery from the pump surface attenuates platelet adhesion in continuous flow ventricular assist devices

Shear-induced platelet activation (SIPA) has been identified to induce platelet adhesion and thrombus formation in continuous flow left ventricular assist devices (LVAD). Platelet glycoprotein (GP) IIb/IIIa receptor inhibitors are effective to prevent SIPA. However, systemic GP IIb/IIIa receptor inhibitor application is associated with severe bleeding complications. The aim of the study was to evaluate (i) the feasibility of absorption and elution of the GP IIb/IIIa receptor blocker TAK-029 from the Ti6Al4V surface of the pump; and (ii) the effect of local GP IIb/IIIa receptor blocker delivery regarding platelet adhesion on the surface of a continuous flow VAD model. Saturating concentrations of TAK-029 were adsorbed on the surface of a centrifugal pump. Whole human blood was perfused in circulatory mock loops using untreated (control), albumin-coated, or TAK-029-coated pumps. Peripheral resistance of the circulatory systems were adjusted accordingly to generate 5 L flow per min with impeller rotational speeds of 3500 (high-shear group) and 1500 rpm (low-shear group), respectively. Platelet adhesions on the respective impellers were quantified by ELISA and scanning electron microscopy (SEM). TAK-029 elution and half-life time were determined by ELISA. Compared with control, albumin-coated pumps showed 64 and 20% less platelet adhesions in the high- and low-shear group, respectively. TAK-029 coated pumps reduced platelet adhesion by additional 33 and 65%, respectively, compared with the albumin group. Elution of TAK 029 was initially very rapid and continued slowly. The results show that it is possible to adsorb and elute a small molecular weight GP IIb/IIIa receptor blocker from the pump surface. This drug elution reduced platelet adhesion on the pump significantly. Further studies are necessary to find a suitable drug bonding that will prolong the antiplatelet effect and preclude any bleeding complication caused by this procedure.     

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.     

Management of heparin resistance during cardiopulmonary bypass: the effect of five different anticoagulation strategies on hemostatic activation

OBJECTIVE: Attenuation of hemostatic activation is a central goal during CPB. However, this poses a problem in patients insensitive to heparin. The present investigation was performed to assess different strategies of managing patients with heparin resistance during CPB. DESIGN: A randomized, prospective clinical investigation. SETTING: A major European heart center. PARTICIPANTS: Five groups with 20 patients each were investigated. INTERVENTIONS: The groups were handled as follows: (1). maintenance of a target ACT, (2). maintenance of the target unfractionated heparin (UFH) level and supplementation of a UFH level-based strategy with (3). AT III, (4). the direct thrombin inhibitor r-hirudin, or (5). the short-acting platelet glycoprotein (GP) IIb/IIIa antagonist tirofiban. Platelet count and generation of contact factor XIIa, thrombin, and soluble fibrin were assessed. Samples were obtained before CPB and after CPB before protamine infusion. MEASUREMENTS AND MAIN RESULTS: There were no differences observed in the generation of factor XIIa. The UFH-based strategy and supplementation with AT III, r-hirudin, and tirofiban resulted in significantly reduced (p < 0.05) thrombin generation compared with ACT management. A significant reduction of fibrin formation was seen only in patients who received AT III, r-hirudin, or tirofiban supplementation to the UFH. The administration of tirofiban resulted in a significant preservation of the platelet count compared with the other groups. There were no significant differences in the postoperative blood loss. CONCLUSIONS: Activation of hemostasis during CPB in heparin-resistant patients most likely has to be attributed to stimulation of the tissue factor pathway. Even the sole use of high concentrations of UFH does not effectively inhibit this activation. Therefore, in these patients anticoagulation during CPB with UFH should be supplemented with either AT III, a short-acting direct thrombin inhibitor, or a short-acting platelet glycoprotein IIb/IIIa antagonist.     

Glycoprotein IIb/IIIa receptor inhibitor attenuates platelet aggregation induced by thromboxane A2 during in vitro nonpulsatile ventricular assist circulation

A recent development in antithrombotic research allows the inhibition of platelet aggregation via protection of the glycoprotein IIb/IIIa receptor on the platelet membrane. We hypothesized that a GP IIb/IIIa receptor inhibitor would inhibit thromboxane-induced platelet aggregation during circulation in our in vitro ventricular assist device (VAD) circuit and preserve long-term platelet function. Twenty-one in vitro nonpulsatile centrifugal VAD circuits were simulated for 4 days using 450 ml of fresh human whole blood with or without glycoprotein IIb/IIIa receptor inhibitor (tirofiban). Platelet aggregation and degranulation were measured in whole blood induced by ristocetin, collagen, ADP, and thromboxane A2 (TXA2). The tirofiban-treated group preserved the platelet count and tended to exert these beneficial effects by inhibiting pathologic platelet aggregation induced by TXA2, collagen, and ADP as well as degranulation. Tirofiban may be useful in preserving platelet number and function during clinical VAD use.