Melanie M. White

Effect of Ca2+ on GP IIb-IIIa Interactions With Integrilin Enhanced GP IIb-IIIa Binding and Inhibition of Platelet Aggregation by Reductions in the Concentration of Ionized Calcium in Plasma Anticoagulated With Citrate

BACKGROUND Integrilin (eptifibatide), a potent inhibitor of the fibrinogen binding function of GP IIb-llla, has been shown to reduce the thrombotic complications of angioplasty and of acute coronary syndromes. The present study was designed to determine whether the reduced Ca2+ concentrations in plasma anticoagulated with citrate affect Integrilin binding to GP IIb-IIIa and the ex vivo pharmacodynamic measurements for this drug. METHODS AND RESULTS Lower concentrations of Integrilin were found to inhibit platelet aggregation in plasma anticoagulated with citrate (for ADP, mean+/-SD IC(50)=140+/-40 nmol/L, n=6; Ca2+ =40 to 50 micromol/L) than with PPACK (IC(50)=570+/-70 nmol/L, P<.0001, n=6; Ca2+ approximately 1 mmol/L). Chelation of Ca2+ with EDTA or citrate caused a similar degree of enhancement in the inhibitory activity of Integrilin. Measurements of D3 LIBS epitope expression showed that the enhanced inhibitory activity was caused by enhanced GP IIb-IIIa occupancy by Integrilin. Citrate anticoagulation decreased the amounts of Integrilin required to inhibit the binding of PAC1, a monoclonal antibody that mimics the GP IIb-IIIa binding activity of fibrinogen. Reduced Ca2+ also increased Integrilin inhibition of the binding of biotinylated fibrinogen to purified, immobilized GP IIb-IIIa. CONCLUSIONS These data suggest that citrate anticoagulation removes Ca2+ from GP IIb-IIIa and enhances the apparent inhibitory activity of Integrilin. This finding indicates that the inhibitory activity of Integrilin is overestimated in blood samples collected with citrate, suggesting that it may be possible to achieve greater antithrombotic efficacy beyond that observed in clinical trials to date with Integrilin.

Expression of ligand-induced binding sites on glycoprotein IIb/IIIa complexes and the effect of various inhibitors

Glycoprotein (GP) IIb/IIIa is a therapeutic target for the blockade of platelet aggregation during acute coronary syndromes. Peptides, peptidomimetics, and antibodies are generated that competitively block the binding of fibrinogen or von Willebrand factor to the activated GPIIb/IIIa complex. Binding of these receptor blockades to GPIIb/IIIa effectively inhibits the formation of the platelet aggregate because ligand binding to the activated GPIIb/IIIa is the final common pathway to thrombus formation. In addition, bound antagonists induce a conformational change in the receptor. This conformational change, also called a ligand-induced binding site, can be used as a marker for receptor occupancy by GPIIb/IIIa receptor blockades. Using the binding properties of the D3 monoclonal antibody that binds with high affinity to the antagonist bound GPIIb/IIIa, we have developed a new method for monitoring the extent of receptor blockade by GPIIb/IIIa antagonists. This method has specific advantages over the interpatient variability of the aggregation assay and provides a method for the evaluation of appropriate target levels of GPIIb/IIIa blockade.

Cocaine-induced platelet defects.

Background and Purpose Numerous studies have demonstrated an association between acute cardiac events, cerebrovascular accidents, and cocaine use. The underlying mechanisms leading to these complications have not been well defined. Using various in vitro model systems, it has been reported that cocaine, up to or greater than an order of magnitude of the lethal dose, causes either inhibitory or proaggregatory effects on platelet function. Methods To address these reported discrepancies, we examined the effect of cocaine and its carrier on the activation and aggregation of human platelets in vitro. Results We found that cocaine inhibited platelet aggregation when platelets were challenged with ADP, collagen, or arachidonic acid. This inhibition was due to a direct effect on fibrinogen binding to the activated platelet. Cocaine also caused the dissociation of preformed platelet aggregates. At these same concentrations, cocaine did not inhibit agonist-mediated increases in cytosolic calcium or inhibit platelet shape change, suggesting that its effect on platelet aggregation was a selective process and not due to a total destruction of platelet function. Interestingly, the organization of the cytoskeleton of activated platelets, a secondary event critical to cell receptor clustering and clot retraction, was disrupted by cocaine treatment. In addition, alterations in platelet protein electrophoretic patterns were observed on preincubation of platelets with cocaine. Conclusions We conclude that cocaine may have a direct inhibitory effect on the ability of platelets to participate in thrombus formation. The contribution of this effect as an underlying mechanism of sudden death in cocaine abusers is unknown.

The use of the point of care Helena ICHOR/Plateletworks and the Accumetrics Ultegra RPFA for assessment of platelet function with GPIIB-IIIa antagonists.

AbstractObjective: To evaluate a newly modified rapid platelet function analysis system (ICHOR/ Plateletworks®) and to compare the results obtained with those of traditional light transmission aggregometry (LTA), and the Ultegra/RPFA® system. Background: Anti-platelet therapy is standard of care for patients as an adjunct to percutaneous coronary intervention (PCI) or for medical management of non-ST elevation acute coronary syndromes (NSTE ACS). Recent clinical trial results suggest that the three currently approved platelet GPIIb-IIIa receptor antagonists, eptifibatide, tirofiban and abciximab, may vary in extent of inhibition of platelet aggregation (IPA) at the approved doses. Thus, pharmacodynamic evaluations of these agents to determine the extent of platelet function inhibition, especially during the periprocedural time of a cardiac intervention, are necessary. A rapid measurement method as a surrogate for LTA, the current gold standard, would be ideal in order to have the option for dose monitoring or adjustment prior to or during an intervention. The Helena ICHOR/ Plateletworks® may be useful for point of care testing. Methods: Blood samples collected in D-Phe-Pro-Arg-chloromethyl ketone dihydrochloride (PPACK) anticoagulant were treated with increasing concentrations of eptifibatide, tirofiban or abciximab. LTA was carried out in conjunction with the ICHOR/Plateletworks®, using a modified method, and Accumetrics Ultegra® with RPFA cartridges. Results: This study demonstrated that platelet inhibition measured by the ICHOR/Plateletworks® mirrored the level of IPA obtained with LTA. In contrast, the Ultegra® system had less correlation when compared to LTA at inhibition levels < 90%. Conclusions: Based on these data, the ICHOR/ Plateletworks® utilized under modified guidelines may serve as a surrogate for LTA when rapid measurements are necessary.Abbreviated AbstractA rapid platelet function measurement method as a surrogate for light transmission aggregometry (LTA), the current gold standard, is ideal in order to have the option for GPIIb-IIIa antagonist dose monitoring or adjustment prior to or during a coronary intervention. A newly modified rapid platelet function analysis system (ICHOR/Plateletworks® was evaluated and compared to the results obtained with traditional light transmission aggregometry (LTA), and the Ultegra/RPFA® system. Blood samples collected in D-Phe-Pro-Arg-chloromethyl ketone dihydrochloride (PPACK) anticoagulant were treated with increasing concentrations of eptifibatide, tirofiban or abciximab. LTA was carried out in conjunction with the ICHOR/Plateletworks®, using a modified method, and Accumetrics Ultegra® with RPFA cartridges. Based on these data, the ICHOR/Plateletworks® utilized under modified guidelines may serve as a surrogate for LTA when rapid measurements are necessary.

Variability of platelet aggregate dispersal with glycoprotein IIb–IIIa antagonists eptifibatide and abciximab

Summary.  Background: Utilization of glycoprotein IIb–IIIa (GPIIb–IIIa) inhibitors improves outcomes of patients with acute coronary syndromes (ACS), including those undergoing percutaneous coronary intervention (PCI). These results may be related to the ability of the inhibitors to destabilize coronary thrombi, reduce microembolization, and restore vessel patency. Objective: To evaluate in vitro the ability of GPIIb–IIIa antagonists, abciximab and eptifibatide, to promote the disaggregation of platelet‐rich thrombus. Methods: Antagonist‐induced disaggregation was assayed in plasma by aggregometry, as well as in whole blood by point of care and capillary perfusion systems. Fibrinogen dissociation from the platelet surface was quantified by flow cytometry. Results: Significant disaggregation of 5 μm ADP‐induced aggregates was observed after addition of either agent. The maximum extent and rate of disaggregation were significantly higher with eptifibatide than with abciximab. Both antagonists also dispersed 2 μg mL−1 collagen‐induced aggregates, again with eptifibatide having a greater effect. Eptifibatide, but not abciximab (up to 10 μg mL−1), was efficient at dissociating aggregates to single platelets in whole blood and dispersing aggregates that had been aged for 30 min before treatment. Eptifibatide also reduced existing thrombus burden in the perfusion model under arterial flow conditions. A key mechanism of aggregate dispersal was antagonist‐induced displacement of platelet‐bound fibrinogen, which was greater with eptifibatide, a competitive inhibitor of fibrinogen binding, than with the noncompetitive inhibitor, abciximab. Conclusions: These results suggest that drug concentration and residence time, along with thrombus extent and age, may be critical determinants in promoting timely recanalization.

Chinese Hamster Ovary Cell Motility to Fibronectin Is Modulated by the Second Extracellular Loop of CD9 IDENTIFICATION OF A PUTATIVE FIBRONECTIN BINDING SITE

CD9, a member of the tetraspanin family of proteins, is characterized by four transmembrane domains and two extracellular loops. Surface expression of CD9 on Chinese hamster ovary (CHO) cells dramatically enhances spreading and motility on fibronectin. To elucidate the mechanistic basis of CD9-fibronectin interaction, binding to fibronectin was investigated using purified and recombinant forms of CD9. The affinity of fibronectin for CD9 in enzyme-linked immunosorbent assay was 81 ± 25 nm. The binding of fibronectin to immobilized CD9 was enhanced by Ca2+ ions. Protein binding and peptide competition studies demonstrated that peptide 6 derived from CD9 extracellular loop 2 (amino acids 168–192) contained part of the fibronectin-binding domain. Additionally, enhanced adhesion of CD9-CHO-B2 cells to fibronectin was significantly reduced by peptide 6. CD9-CHO cells had a 5-fold increase in motility to fibronectin as compared with mock-transfected controls, an effect that correlated with CD9 cell surface density. Truncation of CD9 extracellular loop 2 and peptide 6 caused inhibition of CD9-CHO cell motility to fibronectin. Deletion of CD9 extracellular loop 1 had no significant effect on CHO cell motility. These findings demonstrate a critical role for CD9 extracellular loop 2 in cell motility to fibronectin and clarify the mechanism by which CD9-fibronectin interaction modulates cell adhesion and motility.

Regulation of CD40L (CD154) and CD62P (p-selectin) Surface Expression upon GPIIb-IIIa Blockade of Platelets from Stable Coronary Artery Disease Patients

INTRODUCTION The aim of this study was to further characterize the effect of the antiplatelet agents, aspirin and eptifibatide, on the surface expression of CD40L and CD62P on platelets from patients with stable coronary artery disease. MATERIALS AND METHODS Platelet function was evaluated using standard light transmission aggregometry. Measurements of CD62P and CD40L were carried out by flow cytometry and ELISA assays. RESULTS All patients had the expected level of platelet aggregation inhibition in response to 20 muM ADP in the presence of increasing eptifibatide concentrations. Platelet activation by adenosine diphosphate (ADP) or thrombin agonist peptide (TRAP) increased CD62P and CD40L surface density in the presence of aspirin by 1.9 - 2.8 -fold. Aspirin treatment did not prevent either CD62P or CD40L expression. Eptifibatide pretreatment at pharmacologically relevant concentrations blocked agonist-induced increases in CD62P platelet surface density. A marked percentage of platelets still expressed low levels of surface CD62P suggesting slight platelet activation even with potent platelet inhibition. Eptifibatide also blocked agonist-induced increases in CD40L surface expression and decreased the percent of platelets positive for surface CD40L. Decreased expression of CD40L was due to an inhibition of CD40L translocation and not caused by enhanced shedding from the surface, as soluble CD40L (sCD40L). Eptifibatide concentrations that effectively blocked platelet aggregation correlated with total inhibition of increased CD62P and CD40L surface density. CONCLUSION Blockade of the GPIIb-IIIa receptor on platelets from coronary artery disease patients may have significant bearing on reducing proinflammatory and procoagulant events mediated by CD62P and sCD40L.

Role of in vitro cholesterol depletion in mediating human platelet aggregation

Summary.  We investigated the direct role of cholesterol lowering on human platelet aggregation by in vitro cholesterol depletion using methyl‐β‐cyclodextrin. Collagen and thrombin receptor agonist peptide induced maximal aggregation was significantly decreased in cholesterol depleted platelets. In contrast, anti‐CD9 antibody, mAb7, or anti‐β3 antibody, D3, induced percent maximal aggregation was unaffected by cholesterol depletion. Surface and total αIIbβ3 levels were equivalent in both groups. Morphological and ultrastructural analysis of collagen induced aggregates revealed that normal and cholesterol depleted platelets changed shape and aggregated; however, cholesterol depletion impaired microtubule ring formation and aggregate size. Cholesterol depletion also diminished the extent of the open canalicular system and collagen induced platelet ATP release. These data suggest cholesterol depletion impairs platelet aggregation by altering platelet ultrastructure critical in mediating secretion. Temporal differences and differences in tyrosine phosphoprotein levels following collagen stimulation were observed, thereby indicating that platelet signaling was concurrently affected by cholesterol depletion.

Platelet p24/CD9, a Member of the Tetraspanin Family of Proteinsa

p24/CD9 has a wide yet selective distribution in both hematopoietic and nonhematopoietic cells. The first description of this antigen was reported by Kersey et a/.’ who described the production of a monoclonal antibody (mAb) raised against NALM-6 cells, a pre-B form of ALL. This antibody, BA-2, precipitated a 24,000Da protein from solubilized lymphoid leukemia cell proteins. This antigen was detectable on the surface of bone marrow lymphohemopoietic precursors and in most cases of non-T, non-B ALL. Since 1981, there have been numerous reports of antibodies generated from lymphoid cell origin or platelet origin that bind to a 24,000-Da protein and cause the activation of human platelets. At the Third Workshop on Human Leukocyte Antigens held at Oxford in 1986, 14 such antibodies were reported.2 Through the use of immunofluorescent techniques, the CD9 antigen has been detected mainly on platelets, lymphoid progenitor cells, and mitogenstimulated T and B cells. p24/CD9 is found on the surface of 90% of non-Tlymphoid leukemias, about 20% of T-acute-lymphoid leukemias, and all megakaryocytic leukemia^.^ It has been suggested that the antigen is weakly expressed in myeloid and chronic lymphoid leukemias. Thus, because this antigen is present on nucleated cells which are highly proliferative, it is possible that p24/CD9 may contribute to the process of cell proliferation or other normal physiologic processes such as cell trafficking. All anti-p24/CD9 mAbs described thus far cause platelet activation. This activation response is apparently due to the interaction of the bound CD9 mAb and the Fc receptor, FcyRII. It has been demonstrated that the activating activity of antip24/CD9 mAb is inhibited by the preincubation of platelets with a mAb to the Fc receptor on platelets, IV-3.4-6 Monovalent Fab fragments generated from CD9 mAbs have had different effects ranging from no effect to that of enhancing the aggregation response of subthreshold levels of other agonists such as collagen or ADP.’v8 Thus, activation by anti-p24/CD9 Fab fragments or the augmentation of platelet response by anti-p24/CD9 might be due to contamination of the preparations by intact mAb or, on the other hand, may represent some effector function

Platelet function recovery following exposure to triple anti-platelet inhibitors using an in vitro transfusion model

INTRODUCTION Dual anti-platelet therapy (DAPT) with aspirin and a P2Y12 antagonist is standard of care to reduce risk of thrombosis, but does not directly target thrombin-dependent platelet activation. Therefore, PAR-1 antagonist addition to DAPT (i.e., triple anti-platelet therapy; TAPT) may improve the efficacy of treatment, though at the expense of an increase in bleeding risk. Using an in vitro transfusion model, we evaluated if platelet function loss associated with TAPT can be remedied by the addition of drug-naïve platelets. METHODS To mimic TAPT, platelet-rich plasma (PRP) prepared from consented DAPT patients (DPRP) was incubated with a vorapaxar at therapeutic plasma levels (TPRP). To simulate platelet transfusions, TPRP was mixed with increasing proportions of drug-naïve PRP (NPRP). Platelet function recovery was assessed by light transmission aggregometry (LTA), aggregate morphology, and P-selectin expression. RESULTS LTA results demonstrated that 20% NPRP was required to restore the ADP aggregation response in TPRP to the response observed in DPRP and 40% NPRP recovered aggregation to >65%. Higher NPRP fractions (60%) were required to restore the platelet reactivity using TRAP-6 (SFLLRN) or arachidonic acid (AA). PAR-4 aggregation was unaffected by platelet antagonists. A decrease in single, free platelets and incorporation of mepacrine-labeled naïve platelets into aggregates occurred with increasing NPRP portions. Upon agonist activation, the surface density and percent of P-selectin positive platelets increased linearly upon addition of NPRP. CONCLUSION This in vitro model demonstrated that administration of drug-naïve platelets can be a useful strategy for reversing overall platelet inhibition observed with TAPT.