Comparison of the effect of different platelet GPIIb/IIa antagonists on the dynamics of platelet/fibrin-mediated clot strength induced using thromboelastography

The effect of various platelet glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists on the dynamics of platelet-fibrin clot formation and strength induced by various stimuli was measured by thromboelastography (TEG). GPIIb/IIIa antagonists with high affinity for resting and activated platelets and with slow rates of dissociation from GPIIb/IIIa (Class I antagonists) demonstrated potent and comparable inhibition of platelet aggregation and tissue factor (TF), lipopolysaccharide (LPS), Factor Xa, and thrombin-induced clot strength, in contrast to antagonists that dissociate rapidly from GPIIb/IIIa (Class II antagonists). For example, the Class I antagonist XV459 (the free acid form of roxifiban) inhibited TF, endotoxin, Factor Xa, and thrombin-induced maximal clot strength and platelet aggregation with an IC(50)=30-70 nM, whereas the IC(50) of the Class II antagonist YZ211 (the free acid form of sibrafiban) for altering clot formation and strength was 0.3-4.7 microM. Moreover, the IC(50)'s of sibrafiban, and another Class II antagonist, orbofiban, for inhibiting platelet-fibrin clot formation and strength were substantially greater than their clinically achievable concentrations. Further, although aspirin treatment improved the efficacy of all GPIIb/IIIa antagonists, it did not alter the differences between Classes I and II antagonists. Thus, these data indicate that there are differences in the efficacy of various GPIIb/IIIa antagonists in inhibiting platelet-fibrin clot formation and strength. They also suggest that inhibiting platelet aggregation may not be the sole determinant for the in vivo efficacy of various GPIIb/IIIa antagonists.     

Effects of glycoprotein IIb/IIIa antagonists on platelet activation: development of a transfer method to mimic peak to trough receptor occupancy

Several oral glycoprotein (GP) IIb/IIIa antagonists, Sibrafiban, Orbofiban and Lotrafiban, have been studied in large phase III trials; each has failed to provide efficacy and has been associated with increased mortality. Roxifiban has pharmacokinetic and pharmacodynamic properties believed to be more favorable than the earlier oral agents. Here, we revisit the controversial hypothesis of platelet activation liabilities of GP IIb/IIIa antagonists. The effects of site occupancy by four fibans (Roxifiban, Sibrafiban, Orbofiban and Lotrafiban) on platelet activation was assessed using P-selectin expression, fibrinogen binding and microaggregate formation. All four fibans inhibited ADP and TRAP-stimulated fibrinogen binding and microaggregate formation in a concentration-dependent manner, whereas P-selectin expression was relatively unaltered. To more vigorously test for activation liabilities, the effects of transition from peak to trough receptor occupancy upon platelet stimulation was analyzed. The high affinity of Roxifiban for resting platelets precluded reduction of site occupancy by dialysis or gel filtration. A method was developed that takes advantage of the rapid equilibrium of Roxifiban between platelets and soluble GPIIb/IIIa. The platelet occupancy is controlled by the ratio of platelet GPIIb/IIIa to soluble GPIIb/IIIa. This method allows in vitro investigation of peak/trough transitions on platelet activation. A decrease in site occupancy from peak to trough of Roxifiban or Sibrafiban did not result in increased activation of platelets. The loss of platelet-bound antagonist upon incubation with purified soluble GPIIb/IIIa returned fibrinogen binding/microaggregate formation to no drug levels. In conclusion, these studies do not provide evidence for an activation liability of GPIIb/IIIa antagonists in vitro.     

Comparative study of antiplatelet drugs in vitro: distinct effects of cAMP-elevating drugs and GPIIb/IIIa antagonists on thrombin-induced platelet responses.

Among various categories of antiplatelet drugs, cAMP-elevating agents and GP IIb/IIIa antagonists have been reported to inhibit platelet aggregation stimulated by a wide variety of platelet agonists. To clarify the qualitative difference between these two agents, their effects on various platelet responses in washed platelets evoked by thrombin (0.05 U/mL) were compared in vitro. Two types of cAMP-elevating drugs, cilostazol (a phosphodiesterase III inhibitor) and prostaglandin E1 (an adenylate cyclase activator), both inhibited platelet aggregation, thromboxane A2 formation, and platelet factor 4 release in a concentration-dependent manner. In addition, both agents suppressed intracellular Ca++ elevation induced by thrombin. However, two classes of GP IIb/IIIa antagonists, abciximab (Fab fragment of antibody) and tirofiban (a synthetic compound), showed no inhibitory effects against thromboxane A2 formation and platelet factor 4 release, although these drugs inhibited platelet aggregation. Essentially the same results were obtained in platelet-rich plasma stimulated with high concentration (100 microM) of thrombin receptor activating peptide. In contrast to these different profiles on thromboxane A2 formation and release reaction, both cAMP-elevating agents and GP IIb/IIIa antagonists potently suppressed procoagulant activity in thrombin-stimulated platelets. These results suggest that the development of platelet procoagulant activity induced by thrombin is exclusively dependent on platelet aggregation or aggregation-dependent processes. These observations also indicate that cAMP-elevating agents possess wider inhibitory effects on platelet responses evoked by strong agonists than GP IIb/IIIa antagonists.     

Differential inhibitory effects of platelet glycoprotein IIb/IIIa antagonists on aggregation induced by procoagulant agonists

INTRODUCTION: In addition to mediating the final common pathway of aggregation, the glycoprotein (GP) IIb/IIIa receptor participates in the activation of coagulation on the platelet surface. High-affinity conformation of GP IIb/IIIa in response to collagen-induced inside-out signalling seems to be mediated by GP VI(-FcRgamma) and reinforced by release of soluble mediators. METHODS: We assessed the effects of the three currently available GP IIb/IIIa antagonists--abciximab, tirofiban and eptifibatide--on platelet aggregation induced by various procoagulant and GP VI-related agonists, i.e. collagen-related peptide (CRP), convulxin and collagen fibrils, in PPACK-anticoagulated platelet-rich plasma. RESULTS: At concentrations that equally inhibited 80% of ADP-induced maximal aggregation abciximab-inhibited GP VI-mediated platelet responses to CRP or convulxin significantly more than the low-molecular-weight antagonists (CRP: abciximab 75+/-18%, tirofiban 41+/-7% and eptifibatide 41+/-6%; convulxin: abciximab 90+/-6%, tirofiban 64+/-20%, eptifibatide 61+/-14%, p<0.01 for all). In contrast, aggregation induced by collagen was equally abolished with all antagonists under the similar conditions. During CRP- or convulxin-triggered platelet activation, inhibition of fibrin polymerisation with GPRP potentiated the antiaggregatory effects of tirofiban and eptifibatide to reach that of abciximab. GPRP as such did not affect platelet aggregation. CONCLUSIONS: GP IIb/IIIa antagonists exhibit distinct inhibition profiles in platelet aggregation, depending on fibrin polymerization and calcium. Specifically, the ability of procoagulant platelet agonists to expose pre-activated and ligand-bound GP IIb/IIIa from the internal pool seems important.     

Peptides and monoclonal antibodies which bind to platelet glycoproteins IIb and/or IIIa inhibit clot retraction

The rate of clot retraction in platelet-rich plasma was decreased by synthetic peptide analogues of fibrinogen and monoclonal antibodies each of which bind to the platelet plasma membrane glycoproteins IIb and/or IIIa. These and related data demonstrate that intact complexes of the glycoproteins IIb and IIIa are required for platelet-mediated clot retraction.     

ClfA(221-550), a fibrinogen-binding segment of Staphylococcus aureus clumping factor A, disrupts fibrinogen function

Clumping factor A (ClfA) is a surface protein of Staphylococcus aureus bacteria known for its ability to bind the C-terminus of plasma fibrinogen gamma chain, which participates in mediating fibrinogen-platelet interaction and fibrin cross-linking, resulting in thrombus formation. With an aim to develop agents that block fibrinogen gamma chain C-terminus, the fibrinogen-binding segment of ClfA locating at residues 221-550 was produced by recombinant technology and tested for its ability to inhibit platelet functions and fibrin clot formation. Recombinant ClfA(221-550) bound fibrinogen and blocked fibrinogen-platelet interaction, resulting in the inhibition of both ADP- and collagen-induced platelet aggregations. ClfA(221-550) also affected fibrin clot formation, in which factor XIIIa-mediated cross-linking of fibrinogen gamma chains was abrogated by ClfA(221-550) leaving the release of fibrinopeptides A and B from fibrinogen by thrombin unaltered, indicating that ClfA(221-550) interfered with fibrin clot formation without affecting thrombin's catalytic activity. Platelet-mediated clot retraction depends on both platelet-fibrinogen interaction and fibrin clot formation, which makes platelet thrombus less susceptible to fibrinolysis. At the concentration that reduced platelet aggregation by 40%, ClfA(221-550) prevented platelet-mediated clot retraction, whereas the glycoprotein IIb/IIIa antagonist tirofiban needed a higher concentration in inhibiting clot retraction than inhibiting platelet aggregation. By virtue of the multiple effects of ClfA(221-550) on platelet aggregation, fibrin clot formation and platelet-mediated clot retraction, the binding of ClfA(221-550) to fibrinogen merits further investigation for its potential as a new antithrombotic agent.     

A new variant of thrombasthenia with abnormally glycosylated GP IIb/IIIa

A 15 year-aged Japanese girl with a life long mild purpura was found as a variant type of thrombasthenia. Basic tests revealed prolonged bleeding time, border-line clot retraction, no coagulation defect, no giant platelets and mild thrombocytopenia (70,000-110,000/microliters). Neither ADP, epinephrine nor collagen aggregated her platelet rich plasma. Thrombin (0.1U/ml) caused slightly decreased aggregation of her washed platelets and about 20% normal production of thromboxane B2. PAS-stained SDS-PAGE of her whole platelets showed markedly decreased GP IIb and IIIa. However, crossed immunoelectrophoresis (CIE) against anti-whole platelets antibody showed a normal amount of GP IIb/IIIa complex in her whole platelets solubilized with 1% Triton X-100. CIE using monospecific anti-GP IIb/IIIa complex antibody showed normal dissociation of the patient's GP IIb/IIIa complex into two new bands in the presence of EDTA. Crossed affino-immunoelectrophoresis with the first dimension containing Concanavalin A revealed that the patient's GP IIb/IIIa was much less shifted to the cathode than controls. Immunoprecipitation lines of her GP IIb/IIIa complex were excised from unstained CIE using anti-GP IIb/IIIa antibody and subjected to the silver-stained reduced SDS-PAGE, which showed two protein bands with molecular weights of 125KD and 108KD, corresponding to GP IIb alpha and GP IIIa, respectively. These results suggest that the platelets of this apparently thrombasthenic patient have an antigenically normal but abnormally glycosylated GP IIb/IIIa complex, which is functionally abnormal because of abnormal glycosylation.     

Platelet-monocyte complex formation: effect of blocking PSGL-1 alone, and in combination with alphaIIbbeta3 and alphaMbeta2, in coronary stenting

BACKGROUND: Binding of platelet P-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) is an initial event in the interactions between platelets and monocytes. Platelet-monocyte complexes (PMCs) have been implicated in several vascular disease processes, including acute coronary syndromes (ACS) and complications after percutaneous coronary intervention (PCI). We investigated the effect of ex vivo blockade of PSGL-1, alone and in combination with blockade of the alphaMbeta(2) (Mac-1) and alpha(IIb)beta(3) (GP IIb/IIIa) integrins, on PMC formation. METHODS AND RESULTS: Dual-label flow cytometry was used to detect PMCs in the blood of 10 volunteers and 10 patients undergoing PCI who received intravenous GP IIb/IIIa antagonists. PSGL-1 blockade, both prior to and after platelet stimulation, markedly reduced the formation of PMCs. Concomitant ex vivo blockade of the alphaMbeta(2) and alpha(IIb)beta(3) integrins did not result in further decreases of PMCs compared to PSGL-1 blockade alone. Antagonism of PSGL-1 also led to near elimination of leukocyte-platelet interactions under flowing conditions. CONCLUSION: Blockade of PSGL-1 alone is sufficient to inhibit and reverse the formation of PMCs following platelet stimulation. Concurrent antagonism of PSGL-1 and the alpha(IIb)beta(3) and alphaMbeta(2) integrins was not more effective than inhibition of PSGL-1 alone. These results suggest that platelet-monocyte complex formation is mostly dependent on PSGL-1.     

Platelet GPIIb/IIIa receptor occupancy studies using a novel fluoresceinated cyclic Arg-Gly-Asp peptide

DMP 728 is a potent and specific platelet GPIIb/IIIa antagonist. Like all GPIIb/IIIa antagonists, DMP 728 has a steep dose-response relationship in inhibiting platelet aggregation. In this study the relationships between receptor occupancy, platelet aggregation and bleeding time was determined in anesthetized dogs after intravenous infusion of DMP 728 (0.01 and 0.1 mg/kg/2h). Receptor occupancy was determined by flow cytometry using XL086, a novel fluorescent cyclic RGD peptide that binds to GPIIb/IIIa with high specificity and affinity (k_d ~55 nM). Mean number of GPIIb/IIIa as determined by flow cytometric assay was ~53,800 and 79,000 on unactivated and ADP-activated platelets respectively. After DMP 728 intravenous infusion, there was a dose and time-dependent increase in receptor occupancy, inhibition of platelet aggregation and bleeding time. The two methods of receptor occupancy determination correlate with each other with an r² = 0.78. The present data suggest that blockade of only 40–60% (~40,000 receptors) of the total platelet GPIIb/IIIa was required to achieve >90% inhibition of platelet aggregation and >15 min bleeding time. Our results showed the potential clinical utility of this approach in the study of GPIIb/IIIa dose-response relationship.     

Inhibition of Platelet-Mediated Clot Retraction by Integrin Antagonists

The effects of the platelet IIbβ3 integrin (GPIIb/IIIa) antagonists XV459 (non-peptide), c7E3 (Fab monoclonal antibody) and DMP728 (cyclic peptide) as well as the vβ3 integrin antagonists, LM609 (monoclonal antibody) and XT199 (non-peptide) on clotting and platelet-mediated clot retraction were examined. While 30 nM of XV459 had no significant effect on the kinetics of coagulation, platelet-mediated clot retraction was nearly fully inhibited at this concentration (Relative Retraction Rate = 0.09). XV459 resulted in a concentration related-response curve. Other experiments demonstrated that platelet aggregation was maximally inhibited at XV459 concentrations ranging from 30–50 nM. Similarly, c7E3 demonstrated comparable inhibitory efficacy in inhibiting either clot retraction or platelet aggregation. In contrast, DMP728, an equally potent anti-aggregatory agent with an IC₅₀ of 20–50 nM in inhibiting platelet aggregation induced by various agonists, was found to be a less potent inhibitor of platelet-mediated clot retraction with a half-maximal inhibition of clot retraction at 0.7 μM, and maximum effects at concentrations of 10 μM. The vβ3 integrin antagonists, LM609 or XT199 were without any significant effects on either platelet-mediated clot retraction or platelet aggregation. In conclusion, these data suggest a differential efficacy among different GPIIb/IIIa antagonists in inhibiting platelet-mediated clot retraction in spite of the equivalent anti-aggregatory potency. Additionally, the vβ3 integrin antagonists do not affect platelet-mediated clot retraction or aggregation. Further studies with the previously described IIbβ3 integrin antagonists as well as others revealed a distinct correlation between the Kd to resting and activated platelets and the efficacy in inhibiting platelet-mediated clot retraction.     

血小板糖蛋白Ⅱb/Ⅲa受体拮抗剂治疗急性缺血性卒中的研究进展

血小板活化在血栓形成中起着重要作用,虽然阿替普酶（alteplase,rtPA）静脉溶栓是治疗 急性缺血性卒中（acute ischemic stroke,AIS）的标准治疗,但国内外一直在探索联合或单独使用各类 抗血小板药物在治疗AIS中的作用。抑制血小板聚集过程最后共同通路的新型抗血小板药物血小板 糖蛋白（GP）Ⅱb/Ⅲa受体拮抗剂,在急性冠脉综合征（acute coronary syndrome,ACS）等心血管疾病 中的安全性和有效性已得到验证,但在AIS中的作用尚存争议,本文拟就相关临床研究作一综述。     

A defined peptide that inhibits the formation of the glycoprotein IIb and IIIa complex

Collagen–platelet interaction plays an important role in hemostasis and pathological thrombosis. The proposed mechanism of the interaction was the activation of platelets→releasing of contents from granules→aggregation. The common end point is the platelets and fibrin aggregates. Platelet glycoprotein (GP) IIb/IIIa (the IIbβ3 integrin) complexes serve as a receptor for the binding of fibrinogen to form firmed aggregates. Blockading of GP IIb/IIIa has been proposed to prevent platelet aggregation independent of the substance(s) responsible for activating the platelets. The development of various forms of GP IIb/IIIa inhibitor has resulted in the inhibition of platelet aggregation, although studies of IIbβ3 receptor function and various GP IIb/IIIa inhibitors have demonstrated the potential for these agents to produce effects on other aspects of platelet function as well as having nonplatelet effects. This study investigated platelet inhibition provided by blocking the GP IIb/IIIa complex formation by using a peptide derived from the GP IIIa molecule. The peptide inhibits both types I and III collagen-induced platelet aggregation in a dose-dependent manner. The defined peptide interferes with the formation of the GP IIb/IIIa complex by inhibiting the binding of FITC–PAC-1 onto ADP-, type I collagen-, and type III collagen-activated platelets. However, P-selectin secretion is not affected by the peptide. In addition, the peptide is not interfering with the binding of FITC–PAC-1 to platelets that were preincubated with indomethacin. Results from this study may suggest that the defined peptide is an effective agent to block the interaction of types I and III collagen with platelets.     

Upregulation of GP IIb/IIIa receptors during platelet activation: influence on efficacy of receptor blockade

INTRODUCTION: GP IIb/IIIa inhibitor doses high enough to inhibit platelet macroaggregation may not completely prevent formation of microaggregates. Platelets contain an internal pool of GP IIb/IIIa receptors which externalizes with activation and supports microaggregation. This study assesses microaggregation in the presence of the two GP IIb/IIIa inhibitors abciximab and tirofiban. METHODS: Citrated whole blood was preincubated with abciximab (5 microg/ml), tirofiban (50 ng/ml), or saline as control and activated with TRAP (5 microM) or ADP (2 microM) at 37 degrees C under constant stirring. Microaggregate formation and receptor expression were determined with flow cytometry. RESULTS: Within few seconds after TRAP-activation the platelet count dropped from 266,000/microl to 20,000/microl, the number of microaggregates increased from 3700/microl to 10,100/microl and the mean number of GP IIb/IIIa receptors increased from 53,000 to 65,000/platelet. With TRAP+abciximab the platelet count dropped from 259,000 to 113,000/microl, microaggregates increased from 2500 to 9300/microl, GP IIb/IIIa receptors from 56,000 to 77,000/platelet. Platelet microaggregate formation was reversible. With TRAP and tirofiban platelet count dropped to only 190,000/microl, there was no increase in platelet microaggregates, receptors increased to 66,000/platelet. Platelet activation with ADP gave similar results. CONCLUSIONS: During the early phase of activation additional GP IIb/IIIa receptors externalize to the platelet surface. Abciximab does not block these new receptors sufficiently to prevent microaggregate formation. However, the number of unblocked receptors is not high enough to maintain a stable aggregate, microaggregation is reversible. With tirofiban there is no microaggregate formation, possibly because the inhibitor rapidly binds to newly externalized receptors.     

Monoclonal antibodies against platelet membrane glycoproteins IIb/IIIa and Ibalpha inhibit platelet dependent thrombin generation by different mechanisms

The antithrombotic effect of antiplatelet agents is principally due to their anti-aggregatory action, but these agents may also interfere with coagulation. We have investigated the effect of monoclonal antibodies (MAb) to platelet membrane glycoproteins (GP) IIb/IIIa and Ibalpha on thrombin generation. Antibodies to platelet membrane glycoprotein IIb/IIIa (RFGP56 and c7E3) were shown to inhibit platelet-mediated thrombin generation stimulated by both intrinsic and extrinsic methods. An antibody to GP Ibalpha (RFGP37) also inhibited thrombin generation in these systems. FITC-annexin V was used to determine the effect of these antibodies on the exposure of procoagulant phospholipids on the platelet membrane, and it was found that the anti-IIb/IIIa antibodies reduced this, whereas the anti-Ibalpha antibody caused an increase. We conclude that our monoclonal antibodies against platelet membrane glycoproteins IIb/IIIa and Ibalpha inhibit platelet dependent thrombin generation by different mechanisms.     

Ischemic brain tissue salvaged from infarction by the GP IIb/IIIa platelet antagonist tirofiban

In an open pilot study, the authors tested whether the nonpeptide glycoprotein (GP) IIb/IIIa antagonist tirofiban, a highly effective and selective blocker of platelet aggregation, prevents the transition of ischemic brain tissue into the infarct proper as defined by MRI (perfusion-weighted/T2-weighted) in patients with acute ischemic stroke. The infarct volume (T2 lesion after 1 week) was smaller in treated patients (n = 10) compared with matched control subjects (n = 10; p = 0.029) with similar initial perfusion deficit (TTP-maps). The authors conclude that GP IIb/IIIa antagonists have therapeutic potential in acute stroke therapy.     

Intrinsic activating properties of GP IIb/IIIa blockers

Potential intrinsic activating properties are probably the most controversially discussed issues with respect to GP IIb/IIIa blockers, especially since clinical trials with oral GP IIb/IIIa blockers revealed disappointing results. Based on the finding that currently clinically used GP IIb/IIIa blockers are ligand mimetics, experimental data are discussed, demonstrating an intrinsic activating effect of ligand mimetic GP IIb/IIIa blockers that potentially results in fibrinogen binding to alpha(IIb)beta(3) and in platelet aggregation. Furthermore, the inhibitory effect of aspirin on GP IIb/IIIa blocker-induced platelet aggregation is discussed as a clinically relevant finding. Finally, the potential association of GP IIb/IIIa blocker-induced thrombocytopenia with platelet activation is described.     

CD32-dependent platelet activation by a drug-dependent antibody to glycoprotein IIb/IIIa antagonists

Thrombocytopenia is observed with a frequency of up to 2% in patients treated with glycoprotein (GP) IIb/IIIa antagonists. We recently provided evidence that thrombocytopenia is caused by antibody binding to drug-induced conformational changes in GP IIb/IIIa. Here, we report that a murine monoclonal antibody binds to GP IIb/IIIa in an antagonist-dependent manner and activates platelets. Platelet stimulation is associated with a disruption of the phospholipid asymmetry, resulting in the assembly of catalytic active intrinsic Xase and prothrombinase complexes. Further mechanistic studies revealed that this response is (I) mediated in cis, (II) not associated with the formation of prothrombotic microparticles, and (III) requires intact platelet signaling and (IV) is blocked by increases in cAMP. The prothrombotic response is not observed using F(ab')2 fragments and is blocked by incubation of platelets with neutralizing antibodies to the platelet FcgammaRIIa receptor (CD 32).Taken together, these observations suggest that GPIIb/IIIa antagonist-dependent antibody binding to the platelet fibrinogen receptor has the propensity to lead to CD32-mediated platelet activation and accelerated platelet clearance, leading to thrombocytopenia.     

The stratification of platelet reactivity and activation in patients with stable coronary artery disease on aspirin therapy

Heightened platelet reactivity may affect the occurrence of ischemic events in patients with coronary artery disease on aspirin therapy. However, a definition to stratify platelet reactivity in this group of patients has not been previously reported. We studied platelet reactivity and activation by measuring platelet aggregation and the expression of p-selectin, total GP IIb/IIIa and active GP IIb/IIIa (n=96). Patients were divided into quartiles by each of the markers; correlations were made between the markers; and a definition of heightened platelet reactivity was proposed. Marked variability in activation and reactivity were observed despite aspirin therapy. BACKGROUND: Heightened platelet reactivity and activation may affect the occurrence of ischemic events in patients with coronary artery disease on aspirin therapy. However, a definition to stratify platelet reactivity has not been previously reported. METHODS AND RESULTS: Platelet aggregation (5 and 20 micromol/l ADP), total GP IIb/IIIa, active GP IIb/IIIa and the expression of maximally stimulated p-selectin were measured in patients about to undergo elective coronary stenting (n=96). All patients had received aspirin (325 mg). There was marked variability in platelet reactivity and activation as measured by all markers. The highest quartile was defined by 77+/-1% and 98+/-1% aggregation by 5 and 20 micromol/l ADP, respectively; 65+/-2% p-selectin positivity; 508+/-15 MFI for total GP IIb/IIIa; and 23.0+/-1.8 MFI for active GP IIb/IIIa. CONCLUSIONS: There is a wide range in platelet reactivity and activation as measured by multiple markers in stable coronary disease patients on aspirin therapy. From these indices, we can define those patients at the extremes of reactivity and activation and thus, the greatest potential risk of thrombosis and bleeding. These indices will serve as a guide to future studies investigating the relationships of platelet reactivity, activation, drug-induced inhibition and clinical outcomes.     

Abciximab therapy is associated with increased platelet activation and decreased heparin dosage in patients with acute myocardial infarction

The inhibition of the glycoprotein (GP) IIb/IIIa receptor for reducing periprocedural ischemic events in patients undergoing coronary intervention is known to influence platelet reactivity. Suboptimal doses of GP IIb/IIIa antagonists have been suggested to be prothrombotic and proinflammatory. This study was performed to observe platelet activation markers, whole blood aggregation and the dosage of unfractionated heparin (UFH) in the presence or absence of the GP IIb/IIIa inhibitor abciximab. Patients with acute myocardial infarction undergoing percutaneous coronary intervention were treated with (n = 15) or without (n = 15) abciximab. Platelet activation markers were flow cytometrically measured before and after PCI. Whole blood platelet aggregation was tested by a platelet function assay. The patients with abciximab showed a significant increase in platelet activation markers (P-selectin: 7.12 +/- 0.36 AU vs 11.05 +/- 0.79 AU) and a lower requirement of UFH to prolong aPTT > 60 sec during the infusion. 12 hours after infusion P-selectin level decreased (7.20 +/- 0.58 AU), whereas whole blood aggregation was increasing again. After stopping abciximab, requirement of UFH to prolong aPTT increased in the treated group to a greater extent to a level similar to the untreated group even when most of the platelets were still inhibited. The increased platelet activation found at the end of abciximab treatment points to a procoaguable condition that should be carefully monitored and treated by adapting anticoagulation and antiplatelet drugs.     

Heparin potentiation of collagen-induced platelet aggregation is related to the GPIIb/GPIIIa receptor and not to the GPIb receptor, as tested by whole blood aggregometry.

To determine whether heparin potentiation of platelet aggregation is related to platelet GP IIb/IIIa and GP Ib receptors, four series of experiments were performed on blood from normal volunteers. In the first experiment pretreatment with the monoclonal antibody 7E3 (MAb 7E3), which antagonizes at the GP IIb/IIIa receptor, potently inhibited the collagen-induced platelet aggregation (p less than 0.001). With heparin added to blood pretreated with MAb 7E3, the aggregation increased (p less than 0.005) to an extent similar to that when only saline was used for pretreatment. In the second experiment, monoclonal antibody 10E5 (MAb 10E5) and peptide RGDS, substances which also antagonize at the GP IIb/IIIa receptor, decreased collagen-induced platelet aggregation to an extent similar to that after pretreatment with MAb 7E3. Following pretreatment with RGDS, heparin increased platelet aggregation (p less than 0.03), while after pretreatment with antibody MAb 10E5 heparin did not enhance platelet aggregation. In the third experiment aurin, an inhibitor of von Willebrand factor and its interaction with the platelet GPIb receptor, decreased platelet aggregation dose-dependently. In the fourth experiment heparin enhanced platelet aggregation to a similar extent (p less than 0.005), regardless of pretreatment of the blood with saline, aurin or monoclonal antibody 6D1 (MAb 6D1), the latter an antagonist at the GP Ib receptor. In conclusion, the potentiation of collagen-induced platelet aggregation by heparin was not inhibited by MAb 7E3, RGDS, aurin or MAb 6D1, but was abolished by MAb 10E5, implying that the heparin effect is related to activation of the platelet GP IIb/IIIa receptor complex.