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.     

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.     

Effect of platelet glycoprotein IIb/IIIa receptor antagonist on survival of epigastric island flaps in rats with total venous occlusions.

We evaluated the effect of tirofiban hydrochloride on the survival of epigastric island flaps in rats that had had all the veins occluded. Male Wistar Albino rats were randomly assigned to control (treated with sterile saline) and experimental (treated with tirofiban hydrochloride 1 mg/kg intravenously) groups. An epigastric island skin flap 3x6 cm was raised in each rat. All veins that drained the flap were ligated to give total venous occlusion. Blood flow was recorded by laser Doppler preoperatively (baseline), immediately after the flap had been sutured back to its original position (acute) and on postoperative days 1 and 3. The degree of necrosis was evaluated on day 3. Mean percentage necrosis and minimum laser Doppler values were compared in the two groups. Total necrosis was evident on day 1 in the control group and on day 3 in the experimental group. Macroscopic evidence was confirmed by histopathological examination. There were appreciable differences in blood flow and in the necrotic area of the flap in the experimental group compared with the control group on both days 1 and 3. Tirofiban hydrochloride might be effective in this flap model.     

The glycoprotein IIb/IIIa antagonist c7E3 inhibits platelet aggregation in the presence of heparin-associated antibodies

Purpose: Heparin-associated antibodies (HAAb), in the presence of heparin, can cause platelet activation and aggregation. The purpose of this study was to assess whether a platelet glycoprotein (GP) IIb/IIIa receptor antagonist, c7E3, would inhibit platelet aggregation in the presence of HAAb. If aggregation is inhibited by c7E3, enzyme-linked immunosorbent assays (ELISA) would be done to determine whether c7E3 interfered with the binding of heparin and the HAAb.Methods: HAAb-positive plasmas from 21 patients (determined by platelet aggregation assays) were studied. Normal donor platelet-rich plasmas (PRP) were incubated (1 minute) with either saline solution or 3 μg/ml of c7E3. Platelet-poor plasma from patients with HAAb and one of three sources of heparin (25 μl, 10 U/ml; porcine heparin, bovine heparin, and low molecular weight heparin [enoxaparin]) were added to the PRP mixture. Aggregation was determined using a platelet aggregometer by measuring time to aggregation, the slope of the aggregation curve, and the percent change in optical density.Results: Platelet aggregation occured in 100%, 100%, and 95% of the saline solution incubations exposed to porcine heparin, bovine heparin, and enoxaparin, respectively. Incubation with c7E3 caused 100% inhibition of platelet aggregation in plasma exposed to porcine heparin, bovine heparin, and enoxaparin. The optical density curves obtained from the ELISA, which were dependent on the binding of HAAb to heparin, were not significantly different when c7E3 was compared to buffer alone.Conclusions: The GP IIb/IIIa receptor antagonist, c7E3, inhibits HAAb-induced platelet aggregation via a mechanism that does not appear to interfere with the binding between heparin and HAAb. Clinical trials are warranted to assess whether GP IIb/IIIa antagonists may allow patients with HAAb to safely receive heparin. (J Vasc Surg 1997;25:124-30.)     

Prohemorrhagic and bleeding time activities of recombinant tissue plasminogen activator, heparin, aspirin, and a glycoprotein IIb/IIIa antagonist

Intracerebral hemorrhage (ICH) is the most serious side effect of antithrombotic agents, especially in cases of cerebrovascular disease. In the present study, we compared the exacerbation of ICH and prolongation of bleeding time (BT) in guinea pigs with recombinant tissue plasminogen activator (rt-PA), heparin, aspirin, and FK419, a novel nonpeptide platelet glycoprotein (GP) IIb/IIIa receptor antagonist. ICH was induced by injection of bacterial collagenase into the caudate nucleus; BT was measured with a Simplate R device. Neither heparin nor aspirin prolonged BT. In contrast, rt-PA at the highest dose used in the study did prolong BT, and FK419 caused a dose-dependent prolongation of BT. Moreover, rt-PA and heparin increased the degree of ICH in a dose-dependent manner, leading to death in more than half of the animals treated with higher doses of these drugs. These findings show that the prohemorrhagic mechanisms underlying the prolongation of BT differ from those in collagenase-induced ICH, and that the risk of an agent with antithrombotic effects potentiating hemorrhage in the collagenase-induced model of ICH more closely parallels that in stroke patients than does the effect of the agent on BT. The findings also suggest that antiplatelet agents, including FK419, may be safer than thrombolytic or anticoagulant agents for use in patients at risk for ICH, such as those with stroke or cerebral aneurysm.     

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.     

体外循环期间心腔血血小板膜糖蛋白Ib含量的变化

目的：探讨心肺转流（CPB）体外循环期间心腔血血小板膜糖蛋白Ib的变化及与凝血的关系。方法：用流式细胞术对40例先天性心脏病房或室间隔缺损患者在CPB下行心内修补术时的心腔血血小板膜糖蛋白Ib免疫荧光定量测定,同时与本组体外循环全血比较。结果：全血血小板膜糖 蛋白Ib在选定的三个时点分别下降10％、28％和25％,而心腔血则都下降50％以上。结论：CPB期间心腔血血小板表面膜糖蛋白Ib明显减少,已无凝血功能。     

Anticoagulation during cardiopulmonary bypass in patients with heparin-induced thrombocytopenia type II and renal impairment using heparin and the platelet glycoprotein IIb-IIIa antagonist tirofiban

BACKGROUND: Patients with heparin-induced thrombocytopenia type II require an alternative to standard heparin anticoagulation. However, in patients with renal impairment, anticoagulation during cardiopulmonary bypass with agents such as danaparoid sodium or r-hirudin are associated with hemorrhage. Anticoagulation with unfractionated heparins combined with prostacyclin, a potent platelet aggregation inhibitor, is associated with severe hypotension. The authors investigated a new concept using unfractionated heparins after platelet inhibition with the short-acting platelet glycoprotein IIb-IIIa antagonist tirofiban. METHODS: Ten patients with heparin-induced thrombocytopenia type II and renal impairment were enrolled in the investigation. All had heparin-induced thrombocytopenia type II antibodies present as proved by the heparin-induced platelet aggregation assay, the heparin-platelet factor 4 enzyme-linked immunosorbent assay, or both. In all patients, preoperative anticoagulation to an activated partial thromboplastin time of 40-60 s was performed with r-hirudin. Anticoagulation during cardiopulmonary bypass was achieved with a bolus of 400 IU/kg unfractionated heparins after a bolus of tirofiban 10 microg/kg followed by an infusion of tirofiban at a rate of 0.15 microg x kg(-1) x min(-1) until 1 h before conclusion of cardiopulmonary bypass. Additional unfractionated heparins were only administered if activated clotting time decreased below 480 s. Coagulation was monitored by a abciximab-modified TEG and the adenosine diphosphate-stimulated (20 microm) platelet aggregometry. D-dimer concentrations, as a marker of venous thromboembolism, were measured before and 12, 24, and 48 h after surgery. Postoperative antithrombotic therapy was started immediately with r-hirudin to anticoagulation to an activated partial thromboplastin time of 40-60 s. RESULTS: The postoperative blood loss ranged from 110 to 520 ml. No patient needed reexploration. In no patient was there clinical evidence of thrombosis or embolism in the postoperative period or of a critical increase of the D-dimer concentrations, suggesting venous thromboembolism. Transfusion of platelets was necessary in only two patients. CONCLUSIONS: The protocol is easy to perform and no increased postoperative bleeding and no thromboembolic complications occurred. The combination of unfractionated heparins and tirofiban may be an alternative to other anticoagulation strategies in patients with heparin-induced thrombocytopenia.     

Modulation of platelet surface adhesion receptors during cardiopulmonary bypass.

Alterations in platelet receptors critical to adhesion may play a role in the pathogenesis of the qualitative platelet defect associated with cardiopulmonary bypass. Using flow cytometry, we measured changes in the following platelet surface adhesive proteins: the von Willebrand factor receptor, glycoprotein Ib; the fibrinogen receptor, glycoprotein IIb/IIIa; the thrombospondin receptor, glycoprotein IV; the adhesive glycoprotein granule membrane protein 140, whose expression also reflects platelet activation and alpha-granule release; and, as a control, the nonreceptor protein HLA, A,B,C. Glycoprotein Ib decreased during cardiopulmonary bypass (P less than 0.05) and reached a nadir at 72% (P less than 0.05) of its baseline value at 2-4 h after bypass. This decrease correlated (r = 0.76) with the magnitude of platelet activation (alpha-granule release) in any given patient, but even platelets that were not activated demonstrated a decrease in glycoprotein Ib expression. Glycoprotein IIb/IIIa also decreased in both the activated (47% of baseline, P less than 0.01) and unactivated (63% of baseline, P less than 0.01) subsets of platelets at the end of cardiopulmonary bypass. Glycoprotein IV and HLA A,B,C did not decrease, but instead increased 2-4 h after cardiopulmonary bypass (P less than 0.05). We conclude that cardiopulmonary bypass produces selective decreases in surface glycoproteins Ib and IIb/IIIa as well as in platelet activation; that these two alterations are temporally but not necessarily mechanistically linked; and that these changes have the potential to adversely affect platelet function.     

Clinical significance of PlA polymorphism of platelet GP IIb/IIIa receptors during long-term VAD support

BACKGROUND: Although bleeding and thromboembolism remain major complications after implantation of ventricular assist devices (VADs), no standard anticoagulation protocols are available. Genetic polymorphism of platelet glycoprotein IIb/IIIa may contribute to the development of complications. The present study demonstrates a relationship between the PlA genotype and postoperative complications in patients implanted with pulsatile and axial flow VADs. METHODS: The PlA genotype was determined in 41 consecutive patients treated with a VAD who received anticoagulation with phenprocoumon and aspirin. Pulsatile Novacor (Novacor Corp, Oakland, CA) and Berlin Heart VADs (Berlin Heart, Berlin, Germany) were implanted in 28 patients and the axial flow MicroMed DeBakey VAD (MicroMed Technology, Inc, Houston, TX) in 13. The relationship between the PlA genotype, the anticoagulation regime, and bleeding and thromboembolic events was analyzed. RESULTS: There were no differences between patients with the A1A1 and A1A2 genotype regarding demographic characteristics, weight, or infection episodes. The international normalized ratio (INR), platelet activation tests, and doses of aspirin and dipyridamol before events were similar in both groups. Patients with the A1A1 genotype developed more bleeding complications (39% vs 0%, p = 0.021), while patients with the A1A2 genotype showed a tendency toward more thromboembolic events (13% vs 30%, p = 0.33). With regard to different types of VAD, patients with the axial flow DeBakey VAD and the A1A1 genotype developed significantly more bleeding complications (70% vs 0%, p = 0.033). CONCLUSIONS: In patients with a long-term VAD determination of PlA polymorphism and subsequent adjustment of the anticoagulation regime may lead to a reduction of bleeding and thromboembolic complications.     

Anesthetic induction with ketamine inhibits platelet activation before, during, and after cardiopulmonary bypass in baboons

The objective of this study was to investigate the effects of antifactor D monoclonal antibody (Mab) 166-32 on platelet activation during and after hypothermic cardiopulmonary bypass (CPB) in baboons. Fourteen baboons (mean weight, 15 kg) underwent hypothermic CPB. Seven of them were treated with a single injection of antifactor D Mab 166-32 (5 mg/kg) and the other seven animals were given saline as control. Each baboon was sedated with an intramuscular injection of 10 mg/kg of ketamine hydrochloride. A 20-gauge angiocatheter was placed in the cephalic vein, and 5 mg of diazepam was administered intravenously. Anesthesia was maintained with 0.80% to 2.25% isoflurane, 100% O2, and an inspiratory tidal volume of 13 mL/kg at a rate of 13 breaths per minute throughout the surgical procedure except during CPB. Pancuronium bromide, 0.1 mg/kg, was administered to achieve adequate muscle paralysis. Blood samples were collected before CPB, during CPB, and 1, 2, 3, and 6 h after CPB. Assays were performed to measure platelet activation [CD62P (P-selectin)] using immunofluorocytometric methods. There were no significant differences on CD62P expression of platelets between control and antibody groups before CPB (105 +/- 12% vs. 99 +/- 8%, P=NS), during normothermic CPB (62 +/- 6% vs. 63 +/- 19%, P=NS), during hypothermic CPB (55 +/- 8% vs. 54 +/- 13%, P=NS), and 1, 3, or 6 h after CPB (74 +/- 20% vs. 81 +/- 11%, P=NS). Anesthetic induction with ketamine caused significant reduction in the platelet activation in both groups. Ketamine did not affect complement, neutrophil, and monocyte activation or cytokine production. Further studies on the mechanisms of platelet inhibition by ketamine are warranted.     

Complement activation before, during and after cardiopulmonary bypass

Plasma levels of the complement parent molecules C3, C4, and factor B and their split products, C3d, C4d, and Ba were measured in 12 patients undergoing cardiopulmonary bypass for coronary artery surgery. Alternative and common complement pathway activation, demonstrated by statistically significant rising levels of Ba (P less than 0.05), and C3d (P less than 0.05) and by elevated Ba:B (P less than 0.05) and C3d:C3 (P less than 0.05) ratios were found before the institution of cardiopulmonary bypass but following heparin administration suggesting that heparin may itself initiate alternative pathway activation. In addition, significant depletion of parent complement components and elevation of split product concentrations was seen during bypass suggesting classical and alternate pathway activation (P less than 0.01). This study clarifies the pathways of complement activation during bypass and presents evidence that heparin administration may initially activate the complement cascade.     

肝素涂层管道对体外循环中血小板的保护作用

目的：探讨常规剂量肝素体外循环（CPB）下使用肝素涂层CPB管道（HCC）对血小板的影响。方法：机械瓣膜置换术患者随机分为HCC组（n=8）和对照组（n=15）。分别在CPB前、CPB60min、肝素中和后30、60min及手术后12h测定血小板计数（PLT），血浆α颗粒膜蛋白－140（GMP－140）的浓度及手术后12h出血量。利用电子显微镜观察两组转流后微栓过滤网上沉积物的附着情况。结果；对照组PLC在转流中、后显著低于HCC组，差异有显著性（P＜0．05），且血浆GMP－140浓度在转流60min、转流中和后30、60min显著高于HCC组，差异有显著性（P＜0．05），但手术后12h差异无显著性（P＞0．05）；HCC组手术后12h出血量较对照组少，差异有显著性（P＜0．05）；HCC组动脉过滤网表面光洁，网眼边缘清晰，偶见白细胞和血小板附着；对照组动脉过滤网表面可见纤维蛋白沉积，网眼边缘模糊，有大量白细胞及少数活化血小板粘附。结论：HCC在CPB中对血小板具有保护作用，较好地改善CPB装置的血液相容性。     

体外循环中止血芳酸对血小板的保护作用

目的：探讨止血芳酸在体外循环中对血小板的保护作用及其临床意义。方法：测定止血芳酸组和对照组血小板数量、出血量和输血量,及体外循环前后血浆α－颗粒膜蛋白（ＧＭＰ－１４０）,血栓烷Ｂ２（ＴＸＢ２）和６－酮－前列腺素Ｆ１a(6-k-PGF1a）的浓度变化。结果,体外循环术后血小板计数组明显高于对照组,出血量明显少于对照组。ＧＭＰ－１４０、ＴＸＢ２上升幅度小于对照组,二者相比差异显著（Ｐ＜０．０５）。结论：止血芳酸在体外循环术中可以起到保护血小板,减少术后出血的作用。