Protection of platelet glycoprotein IIb/IIIa receptor complex preserves platelet function during in vitro ventricular assisted circulation

Platelet dysfunction probably contributes to bleeding associated with ventricular assist devices (VADs). Previous evidence suggests that VAD associated platelet dysfunction may be due to dysfunction of the platelet fibrinogen receptor. The purpose of this investigation was to test the hypothesis that selective protection of platelet fibrinogen receptor preserves platelet aggregating ability during in vitro ventricular assisted circulation. Eight in vitro nonpulsatile centrifugal VAD circuits were simulated for four days using 450 ml of fresh human whole blood. Temperature, activated clotting time, pH, PCO2, PO2, Ca2+, and glucose were maintained at physiologic values. Flow was maintained at a constant 2.0 L/min/m2. We examined whole blood platelet aggregation induced by ristocetin, collagen, and adenosine diphosphate (ADP). We added a highly specific reversible inhibitor (MK-383) of the glycoprotein (GP) IIb/IIIa receptor complex before start of circulation to the final four VAD experiments. ADP induced aggregation decreased within the first hour of circulation. Ristocetin and collagen induced aggregation decreased to negligible levels after 10 hours of circulation. With MK-383, ristocetin induced aggregation was preserved. Addition of MK-383 did not alter the decrease of ADP and collagen induced aggregation. These results suggest platelet aggregating ability is maintained with protection of the platelet fibrinogen receptor during in vitro ventricular assisted circulation.     

Differential effects of glycoprotein IIb/IIIa antagonists on platelet microaggregate and macroaggregate formation and effect of anticoagulant on antagonist potency. Implications for assay methodology and comparison of different antagonists

BACKGROUND: Citrated platelet-rich plasma (PRP) turbidimetry is used for assessing pharmacodynamic effects of glycoprotein (GP) IIb/IIIa antagonists in clinical trials. However, citrate can enhance the potency of at least eptifibatide (Integrilin), and turbidimetry is insensitive to microaggregate formation. We compared PRP turbidimetry, as a measure of macroaggregate formation, with single-platelet counting in both whole blood and PRP as a measure of microaggregate formation, using both citrate and hirudin anticoagulation. METHODS AND RESULTS: Three GP IIb/IIIa antagonists, eptifibatide, MK-0852, and GR144053, were compared in PRP (turbidimetry) and whole blood (platelet counting with an Ultra-Flo 100 Platelet Counter), with ADP and collagen used as agonists. Compared with hirudin, citrate enhanced the potency of eptifibatide by up to 4-fold in both PRP and whole blood (P<0.0005), modestly enhanced MK-0852 potency (P=0.001), and had no effect on GR144053. Potency measured in PRP was 2- to 3-fold greater compared with whole blood for MK-0852 and GR144053 but 3- to 4-fold greater for eptifibatide. Simultaneous turbidimetry and platelet counting performed in PRP indicated that this is because GP IIb/IIIa antagonists are more potent inhibitors of in vitro macroaggregation than microaggregation, this effect being greater for eptifibatide in hirudinized PRP compared with GR144053 (P=0.032). CONCLUSIONS: GP IIb/IIIa antagonist potency is variably enhanced by citrate. Macroaggregation is inhibited more effectively than microaggregation, most markedly in the case of eptifibatide in hirudinized blood. These observations have implications for the interpretation and comparison of pharmacodynamic assays and possibly for the risk/benefit ratio of different agents.     

Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (alphaIIbbeta3) inhibitors

The blockade of platelet integrin glycoprotein (GP) IIb/IIIa is a promising new antiplatelet strategy. The binding of ligands or of the ligand-mimetic peptide RGD causes a conformational change of GP IIb/IIIa from the nonactivated to the activated state. Because several blocking agents/inhibitors are ligand-mimetics, the current study evaluates whether these agents have the intrinsic property to activate GP IIb/IIIa. Fibrinogen binding to GP IIb/IIIa on platelets or on CHO cells expressing recombinant GP IIb/IIIa was evaluated by flow cytometry or 125I-labeled fibrinogen. Incubation with the monoclonal antibody (MoAb) fragment c7E3 (abciximab) results in fibrinogen binding to GP IIb/IIIa and in the access of ligand-induced binding sites. At low concentrations (0.01 to 0.1 microgram/mL), this intrinsic activating property of c7E3 can result in platelet aggregation. The disintegrin flavorodin and the RGD analogue fradafiban also induce fibrinogen binding, whereas the blocking MoAbs 2G12 and P2 and the activation-specific MoAb PAC-1 do not. Aspirin and indomethacin cannot block c7E3-induced fibrinogen binding to GP IIb/IIIa, but can inhibit c7E3-induced platelet aggregation. Thus, we conclude that GP IIb/IIIa inhibitors can demonstrate an intrinsic activating property, which can result in fibrinogen binding to GP IIb/IIIa and consequently in platelet aggregation. Cyclooxygenase inhibitors can inhibit platelet aggregation caused by GP IIb/IIIa inhibitors. Further studies will have to evaluate the clinical relevance of the potential intrinsic activating property of GP IIb/IIIa inhibitors and define consequences for the future drug development and evaluation of these potent antiplatelet agents.     

Pharmacodynamic profile of short-term abciximab treatment demonstrates prolonged platelet inhibition with gradual recovery from GP IIb/IIIa receptor blockade

BACKGROUND: The glycoprotein (GP) IIb/IIIa receptor antagonist abciximab is approved for use in high-risk percutaneous coronary interventions. The purpose of the present study was to establish the pharmacodynamic profile and platelet-bound life span of abciximab. METHODS AND RESULTS: The pharmacodynamics of abciximab (inhibition of ex vivo platelet aggregation and GP IIb/IIIa receptor blockade) were measured in 41 individuals who were randomized to receive a 0.25-mg/kg bolus and a 12-hour infusion of either 10 microg/min (EPIC regimen) or 0.125 microg x kg(-1) x min(-1) (EPILOG regimen) of the antiplatelet agent. At extended times, the amount and distribution of platelet-bound abciximab were monitored by flow cytometry. The EPIC and EPILOG infusion regimens exhibited equivalent blockade of both GP IIb/IIIa receptors and platelet aggregation throughout the duration of abciximab treatment. Flow cytometry revealed a single, highly fluorescent platelet population during treatment, consistent with complete saturation and homogeneous distribution of abciximab on circulating platelets. For 15 days after treatment, the fluorescence histograms remained unimodal with gradually diminishing fluorescence intensity, indicating decreasing levels of platelet-bound abciximab. At 8 and 15 days, which exceeds the normal circulating life span of platelets, median relative fluorescence intensity corresponded to 29100 (29% GP IIb/IIIa receptor blockade) and 13300 (13% GP IIb/IIIa receptor blockade) abciximab molecules bound per platelet, respectively. CONCLUSIONS: These results are consistent with continuous reequilibration of abciximab among circulating platelets and may explain the gradual recovery of platelet function and long-term prevention of ischemic complications by abciximab after coronary intervention.     

Staphylococcus aureus induces platelet aggregation via a fibrinogen-dependent mechanism which is independent of principal platelet glycoprotein IIb/IIIa fibrinogen-binding domains

Platelet aggregation by bacteria is felt to play an important role in the pathogenesis of infective endocarditis. However, the mechanisms involved in bacterium-induced platelet aggregation are not well-defined. In the present study, we examined the mechanisms by which Staphylococcus aureus causes rabbit platelet aggregation in vitro. In normal plasma, the kinetics of S. aureus-induced platelet aggregation were rapid and biphasic. The onset and magnitude of aggregation phase 1 varied with the bacterium-platelet ratio, with maximal aggregation observed at a ratio of 5:1. The onset of aggregation phase 2 was delayed in the presence of apyrase (an ADP hydrolase), suggesting that this later aggregation phase may be triggered by secreted ADP. The onset of aggregation phase 2 was delayed in the presence of prostaglandin I2-treated platelets, and this phase was absent when paraformaldehyde-fixed platelets were used, implicating platelet activation in this process. Platelet aggregation phase 2 was dependent on S. aureus viability and an intact bacterial cell wall, and it was mitigated by antibody directed against staphylococcal clumping factor (a fibrinogen-binding protein) and by the cyclooxygenase inhibitor indomethacin. Similarly, aggregation phase 2 was either delayed or absent in three distinct transposon-induced S. aureus mutants with reduced capacities to bind fibrinogen in vitro. In addition, a synthetic pentadecapeptide, corresponding to the staphylococcal binding domain in the C terminus of the fibrinogen delta-chain, blocked aggregation phase 2. However, phase 2 of aggregation was not inhibited by two synthetic peptides (alone or in combination) analogous to the two principal fibrinogen-binding domains on the platelet glycoprotein (GP) IIb/IIIa integrin receptor: (i) a recognition site on the IIIa molecule for the Arg-Gly-Asp (RGD) sequence of the fibrinogen alpha-chain and (ii) a recognition site on the IIb molecule for a dodecapeptide sequence of the fibrinogen delta-chain. This differs from ADP-induced platelet aggregation, which relies on an intact platelet GP IIb/IIIa receptor with an accessible RGD sequence and dodecapeptide recognition site for fibrinogen. Furthermore, a monoclonal antibody directed against the RGD recognition site on rabbit platelet GP IIb/IIIa receptors failed to inhibit rabbit platelet aggregation by S. aureus. Collectively, these data suggest that S. aureus-induced platelet aggregation requires bacterial binding to fibrinogen but is not principally dependent upon the two major fibrinogen-binding domains on the platelet GP IIb/IIIa integrin receptor, the RGD and dodecapeptide recognition sites.     

Rapid platelet-function assay: an automated and quantitative cartridge-based method

BACKGROUND: The platelet glycoprotein (GP) IIb/IIIa receptor is important in mediating platelet thrombus formation, and the GP IIb/IIIa antagonist abciximab (c7E3 Fab; ReoPro) is effective in preventing thrombotic ischemic cardiovascular complications of unstable angina and percutaneous coronary interventions. Small-molecule antagonists of GP IIb/IIIa based on the Arg-Gly-Asp (RGD) sequence show similar benefit, and some of these agents are orally active. However, there may be significant interindividual variation in response to such antagonists, especially with chronic oral therapy. It will be essential to balance the beneficial antithrombotic effect of these drugs with their potential for causing bleeding. In response to this need, we have developed a rapid platelet-function assay (RPFA), a point-of-care system that provides a quantitative measure of the competence of the GP IIb/IIIa receptor as reflected in the ability of platelets to agglutinate fibrinogen-coated beads. METHODS AND RESULTS: Polystyrene beads were coated with fibrinogen and placed in a cartridge along with a lyophilized peptide that activates the thrombin receptor. Anticoagulated whole blood was added to the cartridge, and then a microprocessor-controlled operation mixed the reagents and detected agglutination between platelets and coated beads. Quantitative digital results were displayed within 3 minutes. Because there is no dilution of the blood, the assay can be used to measure platelet activity in samples that have been treated with GP IIb/IIIa antagonists with high dissociation rates. RPFA results of whole-blood samples treated with different GP IIb/IIIa antagonists correlated well with both conventional turbidimetric platelet aggregation (r2=0.95) and the percentage of free GP IIb/IIIa molecules in the sample (r2=0.96). The mean difference in measurements between RPFA and aggregometry was -4% (+/-4% SD), and the mean difference in measurements between RPFA and free GP IIb/IIIa receptors was -2% (+/-6% SD). CONCLUSIONS: The RPFA provides rapid information on platelet function that mirrors turbidimetric platelet aggregation and reflects GP IIb/IIIa receptor blockade.     

Pharmacodynamic efficacy, clinical safety, and outcomes after prolonged platelet Glycoprotein IIb/IIIa receptor blockade with oral xemilofiban: results of a multicenter, placebo-controlled, randomized trial

BACKGROUND: Parenteral administration of platelet glycoprotein IIb/IIIa (GP IIb/IIIa) receptor blockers can reduce ischemic complications of coronary angioplasty. Orally active GP IIb/IIIa blockers may allow more sustained receptor antagonism with the potential for long-term secondary prevention. The pharmacodynamic efficacy, clinical safety, and outcomes after prolonged receptor blockade with an orally active GP IIb/IIIa antagonist are not known. The Oral Glycoprotein IIb/IIIa Receptor Blockade to Inhibit Thrombosis (ORBIT) Trial is a multicenter, placebo-controlled, randomized trial of xemilofiban, an oral platelet GP IIb/IIIa blocking agent, administered to patients after percutaneous coronary intervention. METHODS AND RESULTS: After successful elective percutaneous coronary intervention, 549 patients were randomized to receive either placebo or xemilofiban in a dose of 15 or 20 mg. Stented patients randomized to placebo also received ticlopidine 250 mg orally BID for 4 weeks. Patients who received abciximab during the coronary intervention and who were randomized to receive xemilofiban were administered a reduced dosage (10 mg TID for 2 weeks) followed by the randomized maintenance dose of 15 or 20 mg BID for 2 more weeks. All patients received 325 mg aspirin PO QD. Ex vivo platelet aggregation in response to 20 micromol/L ADP and 4 microg/mL collagen was measured over time after the initial dose of study drug and at days 14 and 28 of long-term therapy in 230 patients. All patients were followed clinically for 90 days. Xemilofiban inhibited platelet aggregation to both ADP and collagen with peak levels of inhibition that were similar at 14 and 28 days of long-term oral therapy. Plasma levels of xemilofiban correlated with the degree of platelet inhibition. Peak platelet inhibition on day 1 correlated with the subsequent occurrence of insignificant or mild bleeding events. Although this study was not powered to evaluate differences in clinical outcomes, a trend (P=0.04) was observed for reduction of cardiovascular events at 3 months in patients not treated with abciximab who received the highest dose (20 mg) of xemilofiban studied. CONCLUSIONS: Xemilofiban inhibited platelet aggregation and was well tolerated during 28 days of long-term oral therapy. The observed trend in reduction of cardiovascular events in follow-up awaits confirmation in the larger-scale phase III study (EXCITE trial) currently in progress.     

Comparative studies of glycoprotein Ib in heparin coated and nonheparin coated extracorporeal circulation circuits

Contact between blood and artificial materials has various effects on blood. Impairment of platelet function is an especially important and well known effect, but its precise mechanism is not clearly understood. The authors constructed a circulation model to investigate the effect of extracorporeal circulation on platelet membrane glycoproteins (GPs), especially GP Ib, and to compare the changes in GP Ib in heparin coated (group C) and nonheparin coated (group N) circuits. As determined by flow cytometry, GP Ib in both groups decreased on initiating circulation, but the decrease in group N was significantly larger than that in group C. There was no observed change in GP IIb/IIIa levels in either group. The extent of shear stress induced platelet aggregation significantly decreased during circulation in both groups. Decreases in the extent of shear stress induced platelet aggregation were significantly less with the use of heparin coated circuits. In addition, the amount of GP Ib in the high speed pellet decreased progressively during circulation in both groups. In contrast, the amount of GP Ib in the Triton insoluble (low speed) pellet increased dramatically during circulation. However, expression of GP Ib in the Triton soluble platelet fraction was low in both groups. From the results, it was concluded that the cause of the decrease in platelet function during extracorporeal circulation is attributable to the internalization of GP Ib from the platelet surface inside the platelet. It also can be said that a heparin coated circuit is one effective means of controlling this change.     

Expression of inducible nitric oxide synthase after endothelial denudation of the rat carotid artery: role of platelets

There is functional evidence suggesting that endothelial denudation stimulates inducible nitric oxide synthase (iNOS) activity in the vascular wall. In vitro studies have shown that iNOS expression in smooth muscle cells is reduced by endothelial cells. In the present study we have analyzed the time course of iNOS protein expression in the arterial wall after in vivo deendothelialization. Endothelial denudation was performed in the left carotid artery of Wistar rats, and the right carotid artery was used as control. Whereas iNOS protein was weakly expressed 6, 24, and 48 hours after endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after vascular damage. Because platelet adhesion and aggregation occur early after endothelial damage, we studied the role of activated platelets in the negative modulation of iNOS protein expression during the first 2 days after endothelial denudation. Early after in vivo endothelial injury, platelet-depleted rats showed a marked iNOS protein expression in the vascular wall. Similar results were obtained by blocking the platelet glycoprotein (GP) IIb/IIIa. Although iNOS protein is present in the arterial wall several days after endothelial denudation, early after arterial wall injury iNOS protein is weakly expressed. Platelets play a crucial role in preventing iNOS protein expression early after endothelial damage, an effect that can be avoided with GP IIb/IIIa blockers. Although iNOS protein was weakly expressed in vivo in the rat carotid artery wall 6, 24, and 48 hours after balloon endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after arterial damage. iNOS expression could be increased early after endothelial injury by removing circulating platelets and by an antibody against the GP IIb/IIIa. In conclusion, platelets prevent iNOS protein expression early after endothelial balloon damage, an effect that can be avoided with GP IIb/IIIa blocking agents.     

High performance in refolding of Streptomyces griseus trypsin by the aid of a mutant of Streptomyces subtilisin inhibitor designed as trypsin inhibitor

The platelet glycoprotein (GP) IIb/IIIa receptor antagonist appears to reduce the need for revascularization after coronary angioplasty. However, since the effect of GP IIb/IIIa receptor antagonist on the in-stent neointimal thickening has not been clarified, we examined it in the canine model. The beagle dogs were assigned to the control (n=7) or the GP IIb/IIIa receptor antagonist FK633 group (n=7). FK633 was administered by subcutaneous osmotic pumps (0.2 mg. kg-1. h-1) and an intravenous bolus injection (1 mg/kg) before stenting. A coil stent was implanted in the left circumflex coronary arteries. The platelet aggregation capability was significantly (<5%) and consistently reduced by FK633 except for the mild elevation (10% to 30%) on the next day of stenting. Hearts were excised 3 months after stent implantation. The area of intima and media and the area stenosis were obtained from the sections of the stented arteries. The area of intima and media and the area stenosis (1.3+/-0.2 mm2, 41.8+/-7.5% and 1.3+/-0.2 mm2, 33.9+/-6.7% in the FK633 and the control group, respectively) were not different between the groups. We conclude that, although GP IIb/IIIa antagonist FK633 prevented the platelet aggregation significantly and consistently, it could not prevent the neointimal thickening after stent implantation in canine coronary artery.     

Administration of abciximab during percutaneous coronary intervention reduces both ex vivo platelet thrombus formation and fibrin deposition: implications for a potential anticoagulant effect of abciximab

Abciximab (c7E3 Fab, ReoPro), a platelet glycoprotein (GP) IIb/IIIa inhibitor, decreases acute ischemic complications after percutaneous coronary interventions. Recently, abciximab was shown to decrease thrombin generation in vitro in a static system. To assess whether abciximab can decrease fibrin formation in blood from patients, we quantified both platelet thrombi and fibrin deposition by using an ex vivo flow chamber model. We prospectively studied 18 consecutive patients who underwent percutaneous interventions for unstable coronary syndromes. Blood was perfused directly from the patient through an ex vivo perfusion chamber at a high shear rate, thus mimicking mildly stenosed coronary arteries. Perfusion chamber studies were performed when patients were being treated with heparin plus aspirin before the procedure (baseline) and then repeated after the procedure, when patients were on either aspirin plus heparin alone (group 1, no abciximab, control) or aspirin plus heparin plus abciximab (group 2, abciximab treated). Each patient served as his or her own control. Specimens were stained with combined Masson's trichrome-elastin and antibodies specific for fibrinogen, fibrin, and platelet GP IIIa. Total thrombus area and areas occupied by platelet aggregates and fibrin layers were quantified by planimetry. Group 1 demonstrated no significant change in thrombus area before versus after the procedure; in contrast, treatment with abciximab reduced total thrombus area by 48% in group 2 (after the procedure versus baseline, P=0.01). This decline was due to significant reductions in both platelet aggregates (55%, P=0.005) and fibrin layers (45%, P=0.03). The addition of abciximab to heparin and aspirin in patients undergoing coronary interventions significantly decreases ex vivo thrombus formation on an injured vascular surface. Treatment with abciximab appears to reduce both the platelet and the fibrin thrombus components. This finding supports a potential role for GP IIb/IIIa receptor blockade in decreasing fibrin formation in addition to inhibition of platelet aggregation. Thus, potent inhibitors of GP IIb/IIIa may also act as anticoagulants.     

Prospective use of platelet glycoprotein IIb/IIIa receptor blockers in the emergency department setting

Platelets play a pivotal role in the pathophysiology of acute coronary syndromes (ACS) and thus are logical therapeutic targets for treatment of this disease process. Platelet glycoprotein (GP IIb/IIIa receptor antagonists, which interrupt the final common pathway of platelet aggregation, have been proven to reduce the 30-day incidence of death, acute myocardial infarction (MI), and urgent revascularization in both high-risk and low-risk patients undergoing percutaneous intervention procedures. Three-year follow-up has indicated that these benefits appear durable. Recent large-scale randomized trials have demonstrated the value of GP IIb/IIIa receptor inhibitors in reducing the risk of death and MI in unstable angina/non-Q-wave MI patients receiving pharmacologic management. And, emerging evidence suggests a future role for GP IIb/IIIa receptor inhibitors as an adjunct to low-dose fibrinolytic therapy in patients with acute MI. As the list of indications for GP IIb/IIIa receptor antagonists expands to encompass the full spectrum of ACS, there is increasing interest in the potential use of these agents in the emergency department setting. The integration of GP IIb/IIIa receptor inhibitors into emergency department protocols will ultimately depend largely on whether these drugs prove to be safe and effective regardless of the direction of ST-segment deviation, and irrespective of whether definitive therapy will be invasive or conservative.     

Increased platelet sensitivity toward platelet inhibitors during physical exercise in patients with coronary artery disease

Generalized atherosclerosis and coronary artery disease (CAD) are associated with endothelial dysfunction and during acute myocardial ischemia platelet activation has been reported. Activated platelets exert activated fibrinogen receptors (GP IIb/IIIa) and express CD 62p being regarded as reliable marker for platelet activation. Patients with angiographically proven CAD performed a bicycle exercise test until the onset of angina or ST-segment depression. We studied the ischemia-induced alterations in fibrinogen binding to activated platelet GP IIb/IIIa receptors and CD 62p expression. Therefore, the basal fibrinogen binding to GP IIb/IIIa and CD 62p expression and the thrombin-concentration for half-maximal platelet activation before and after exercise testing were determined. Additionally, inhibition of thrombin-induced platelet activation by increasing concentrations of the prostacyclin-analog iloprost and the NO-donor SIN-1 was examined. In patients with CAD, a significantly reduced basal activation and a highly significant reduction in sensitivity towards thrombin was measured. The thrombin-induced expression of GP IIb/IIIa and CD 62p was significantly diminished in patients with CAD after physical exercise and their platelets were significantly more sensitive towards the inhibitory effects of iloprost and SIN-1. These data demonstrate a significant reduction in platelet activation in response to physical exercise in patients with CAD and advanced atherosclerosis. Despite exercise induced myocardial ischemia as evidenced by angina and ECG-changes, the platelets are not generally activated, as it could be expected. Thus, patients with myocardial ischemia experienced a reduced platelet activity and enhanced sensitivity towards prostacyclin (PGI2) and nitric oxide, probably due to an augmented release of endogenous platelet inhibitory mediators.     

Harnessing the platelet

Appreciation of the critical role of platelets in cardiovascular disease came when it was shown that aspirin, by virtue of its ability to block platelet aggregation, reduced the combined incidence of MI, stroke, and vascular death by 25%. Understanding the key role played by platelets in acute thrombotic vascular events prompted the development of a new class of drugs to control platelet action. Platelet aggregation is mediated exclusively by the platelet fibrinogen receptor GP IIb/IIIa. The binding of the receptor with fibrinogen is the final common pathway leading to platelet aggregation and thrombus formation. Abciximab, the first GP IIb/IIIa platelet receptor inhibitor, effectively reduces the thrombotic complications in acute coronary vascular events. The newer GP IIb/IIIa inhibitors, the synthetic peptide antagonists, have been shown to be more specific, to be nonimmunogenic, and to cause less bleeding. It is predictable that an oral GP IIb/IIIa inhibitor will become part of the standard repertoire in patients with unstable angina. The platelet has taken center stage in the battle against arterial thrombosis. The direction of our medical attack on acute coronary events is clear: harness the platelet.     

Increase in beta-galactosidase activity in a non-isothermal bioreactor utilizing immobilized cells of Kluyveromyces fragilis: fundamentals and applications

Heparin-induced thrombocytopenia is an increasingly common side effect associated with heparin usage. In the more severe manifestation of the syndrome, patients can develop thrombosis; a 10% mortality is associated with heparin induced thrombocytopenia. To date, the therapeutic options for patients with heparin-induced thrombocytopenia are limited. Glycoprotein IIb/IIIa inhibitors have been shown to block platelet aggregation induced by a wide variety of agonists. The ability of antibody and synthetic small molecule inhibitors of glycoprotein IIb/IIIa to block in vitro activation and aggregation of platelets in response to heparin-induced thrombocytopenia positive serum/heparin was examined using flow cytometry, platelet aggregometry, and luminescence aggregometry. Abciximab, YM 337, and SR 121566A were each found to inhibit platelet microparticle formation and P-selectin expression in whole blood, in response to heparin-induced thrombocytopenia positive serum/heparin. In a platelet rich plasma system, the platelet aggregation response was inhibited by all three agents. The IC50 for inhibition of heparin-induced thrombocytopenia positive serum/heparin induced platelet aggregation by SR 121566A was 18 nM, a concentration which was 4 to 8 fold lower than that observed for collagen and arachidonic acid induced aggregation. Adenosine triphosphate release from activated platelets, as measured by luminescence aggregometry, was concentration-dependently inhibited by SR 121566A. These results suggest that glycoprotein Ilb/IIIa inhibitors may be beneficial in the management of heparin-induced thrombocytopenia and warrant further investigation.     

Inhaled nitric oxide inhibits human platelet aggregation, P-selectin expression, and fibrinogen binding in vitro and in vivo

BACKGROUND: Recent data suggest that inhaled NO can inhibit platelet aggregation. This study investigates whether inhaled NO affects the expression level and avidity of platelet membrane receptors that mediate platelet adhesion and aggregation. METHODS AND RESULTS: In 30 healthy volunteers, platelet-rich plasma was incubated with an air/5% CO2 mixture containing 0, 100, 450, and 884 ppm inhaled NO. ADP- and collagen-induced platelet aggregation, the membrane expression of P-selectin, and the binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor were determined before (t0) and during the 240 minutes of incubation. In addition, eight patients suffering from severe adult respiratory distress syndrome (ARDS) were investigated before and 120 minutes after the beginning of administration of 10 ppm inhaled NO. In vitro, NO led to a dose-dependent inhibition of both ADP-induced (3+/-3% at 884 ppm versus 70+/-6% at 0 ppm after 240 minutes; P<.001) and collagen-induced (13+/-5% versus 62+/-5%; P<.01) platelet aggregation. Furthermore, P-selectin expression (36+/-7% of t0 value; P<.01) and fibrinogen binding (33+/-11%; P<.01) were inhibited. In patients with ARDS, after two who did not respond to NO inhalation with an improvement in oxygenation had been excluded, an increase in plasma cGMP, prolongation of in vitro bleeding time, and inhibition of platelet aggregation and P-selectin expression were observed, and fibrinogen binding was also inhibited (19+/-7% versus 30+/-8%; P<.05). CONCLUSIONS: NO-dependent inhibition of platelet aggregation may be caused by a decrease in fibrinogen binding to the platelet GP IIb/IIIa receptor.     

Glycoprotein IIb/IIIa receptor antagonists in the management of cardiovascular diseases

Recent studies of the effects of glycoprotein (GP) IIb/IIIa receptor antagonists on the clinical outcomes of patients with cardiovascular diseases are reviewed. The GP IIb/IIIa receptor antagonists studied include abciximab (a murine monoclonal antibody); eptifibatide (a synthetic peptide); and tirofiban, lamifiban, xemilofiban, sibrafiban, and lefradafiban (synthetic nonpeptides). A majority of clinical trials of GP IIb/IIIa receptor antagonists have been performed in patients with unstable angina or acute myocardial infarction and in patients undergoing percutaneous coronary interventions in whom an intracoronary thrombus may lead to ischemic complications. There is abundant evidence that GP IIb/IIIa receptor antagonists reduce the risk of death, acute myocardial infarction, and urgent revascularization procedures in high- and low-risk patients undergoing percutaneous coronary interventions. Abciximab remains the most studied of these agents in interventional settings. Data are accumulating on synthetic peptide and nonpeptide GP IIb/IIIa receptor antagonists that also demonstrate lower rates of death and ischemic complications in the treatment of acute coronary syndromes. In patients who have had a successful response to intravenous GP IIb/IIIa receptor antagonists, oral agents may represent an option for secondary prevention. Additional studies are required in order to determine further uses for these agents. A growing body of evidence supports the role of GP IIb/IIIa receptor antagonists in invasive and pharmacologic treatment approaches to acute coronary syndromes.     

SC-49992--a potent and specific inhibitor of platelet aggregation

Fibrinogen binding is required for platelet aggregation and subsequent thrombus formation. SC-49992 (SC), an RGDF mimetic, is a potent and specific inhibitor of the binding of fibrinogen to its receptor on activated platelets, glycoprotein IIb/IIIa (IC50 0.7 microM). SC was more potent (1-5 microM) than either RGDS, RGDF or the gamma chain dodecapeptide in blocking platelet aggregation to a variety of agonists in both dog and human platelet rich plasma. SC was more potent as an inhibitor of GP IIb/IIIa on platelets than it was against other integrin and non-integrin receptors, including the RGD-dependent vitronectin receptor and other non-RGD-dependent integrins such as CDII/CD18. SC had little effect on ristocetin induced agglutination. SC blocked ex vivo collagen induced aggregation in dogs and collagen induced thrombocytopenia in rats. These data suggest that elimination of the Arg-NH2 and the Arg-Gly amide bond of RGDF provided increased inhibitory potency and specificity. This structural modification may be of value in the development of other more potent RGDF mimetics for the inhibition of platelet aggregation.     

Detection of platelets activated during acetylcholine-induced coronary vasospasm

Although platelet activation may play a role in coronary artery spasm, platelets activated following coronary vasospasm have not been clinically detected. We performed flow cytometric analysis of activation-dependent granular proteins, CD62P (P-selectin), CD63, PAC-1 (activated glycoprotein [GP] IIb/IIIa) and thrombospondin on the platelet plasma membrane in patients who exhibited acetylcholine-induced coronary vasospasm and compared findings with those in control patients without vasospasm. We simultaneously investigated the plasma levels of thrombin anti-thrombin III complex (TAT), plasmin alpha2-plasmin inhibitor complex (PIC), and thrombomodulin. In patients with vasospasm, the expression of CD62P, CD63 and PAC-1 on the platelet membrane surface increased in coronary sinus blood samples following coronary vasospasm, although the expression in aortic samples did not change. The TAT level also increased in the coronary sinus after vasospasm. Platelets might be activated by coronary vasospasm within the coronary circulation. The platelet activation process may be modulated by thrombin generation.     

Use of platelet glycoprotein IIb/IIIa receptor inhibitors in acute coronary syndrome and coronary intervention

New strategies in the treatment of acute coronary syndromes have focused on the potential for blocking platelet aggregation through the use of platelet surface membrane glycoprotein (GP) IIb/IIIa receptor inhibitors. The benefits of GPIIb/IIIa inhibition in preventing ischemic complications of interventional treatment have been well defined in patients with unstable angina. In the future, major therapeutic applications for this class of agents may include the stabilization of patients with unstable angina and potentially as single medical therapy, as several recently completed trials have suggested. This article attempts to review recently published clinical trials regarding the use of GPIIb/IIIa receptor inhibitors, and clarify current problems in the use of this agent and directions for future investigation.