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Frelinger, Andrew

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Frelinger

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Andrew

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Frelinger, Andrew
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    P2Y12 Receptor Blockade Augments Glycoprotein IIb‐IIIa Antagonist Inhibition of Platelet Activation, Aggregation, and Procoagulant Activity
    (Blackwell Publishing Ltd, 2013) Berny‐Lang, Michelle A.; Jakubowski, Joseph A.; Sugidachi, Atsuhiro; Barnard, Marc R.; Michelson, Alan; Frelinger, Andrew
    Background: New antiplatelet agents that provide greater, more consistent inhibition of the platelet ADP receptor P2Y12 may be used in combination with glycoprotein (GP) IIb‐IIIa antagonists, but their combined effect on platelet function and procoagulant activity is not well studied. Therefore, the objective of this study was to evaluate the independent and complementary effects of P2Y12 and GPIIb‐IIIa inhibition on platelet function and procoagulant activity. Methods and Results: Healthy donor blood was treated with the active metabolite of prasugrel (R‐138727 5 μmol/L), GPIIb‐IIIa antagonists (abciximab 3 μg/mL or eptifibatide 0.9 μg/mL), and combinations thereof, exposed to physiologically relevant agonists (collagen and ADP) and then evaluated for markers of platelet activation and procoagulant activity. Significant interactions between R‐138727 and GPIIb‐IIIa antagonists were observed. R‐138727 and the GPIIb‐IIIa antagonists had additive inhibitory effects on collagen‐stimulated platelet aggregation and on the collagen plus ADP–stimulated level of activated platelet surface GPIIb‐IIIa. R‐138727 and abciximab each inhibited collagen plus ADP–stimulated platelet phosphatidylserine expression and prothrombin cleavage, and the combination produced greater inhibition than achieved with abciximab alone. In contrast, eptifibatide did not inhibit, but instead enhanced, collagen plus ADP–stimulated prothrombin cleavage. Addition of R‐138727 reduced prothrombin cleavage in eptifibatide‐treated samples, suggesting a novel mechanism for potential benefit from combined prasugrel and eptifibatide treatment. Conclusions: The complementary effects of abciximab and R‐138727 on platelet activation, aggregation, and procoagulant activity suggest their combined use may, to a greater degree than with either agent alone, reduce thrombus formation in vivo.
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    Antiplatelet Activity, P2Y1 and P2Y12 Inhibition, and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate
    (Public Library of Science, 2014) Chang, Hung; Yanachkov, Ivan B.; Dix, Edward J.; Yanachkova, Milka; Li, YouFu; Barnard, Marc R.; Wright, George E.; Michelson, Alan; Frelinger, Andrew
    Background: Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5′,5″″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers. Objective: To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma. Methods: We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma. Results: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers. Conclusions: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors.
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    Targeted inhibition of the serotonin 5HT 2A receptor improves coronary patency in an in vivo model of recurrent thrombosis
    (Wiley-Blackwell, 2010) PRZYKLENK, K.; Frelinger, Andrew; LINDEN, M. D.; WHITTAKER, P.; LI, Y.; BARNARD, M. R.; ADAMS, J.; MORGAN, M.; AL-SHAMMA, H.; Michelson, Alan
    BACKGROUND: Release of serotonin and activation of serotonin 5HT2A receptors on platelet surfaces is a potent augmentative stimulus for platelet aggregation. However, earlier-generation serotonin receptor antagonists were not successfully exploited as antiplatelet agents, possibly owing to their lack of specificity for the 5HT2A receptor subtype. OBJECTIVE: To assess whether targeted inhibition of the serotonin 5HT2A receptor attenuates recurrent thrombosis and improves coronary patency in an in vivo canine model mimicking unstable angina. METHODS: In protocol 1, anesthetized dogs were pretreated with a novel, selective inverse agonist of the 5HT2A receptor (APD791) or saline. Recurrent coronary thrombosis was then initiated by coronary artery injury+stenosis, and coronary patency was monitored for 3 h. Protocol 2 was similar, except that: (i) treatment with APD791 or saline was begun 1 h after the onset of recurrent thrombosis; (ii) template bleeding time was measured; and (iii) blood samples were obtained for in vitro flow cytometric assessment of platelet responsiveness to serotonin. RESULTS: APD791 attenuated recurrent thrombosis, irrespective of the time of treatment: in both protocols, flow-time area (index of coronary patency; normalized to baseline coronary flow) averaged 58-59% (P<0.01) following administration of APD791 vs. 21-28% in saline controls. Moreover, the in vivo antithrombotic effect of APD791 was not accompanied by increased bleeding, but was associated with significant and selective inhibition of serotonin-mediated platelet activation. CONCLUSION: 5HT2A receptor inhibition with APD791, even when initiated after the onset of recurrent thrombosis, improves coronary patency in the in vivo canine model.
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    Soluble CD40 Ligand in Aspirin-Treated Patients Undergoing Cardiac Catheterization
    (Public Library of Science, 2015) Gremmel, Thomas; Frelinger, Andrew; Michelson, Alan
    Plasma soluble CD40 ligand (sCD40L) is mainly generated by cleavage of CD40L from the surface of activated platelets, and therefore considered a platelet activation marker. Although the predictive value of sCD40L for ischemic events has been demonstrated in patients with acute coronary syndromes (ACS), studies on the association of sCD40L with cardiovascular outcomes in lower risk populations yielded heterogeneous results. We therefore sought to investigate factors influencing sCD40L levels, and the predictive value of sCD40L for long-term ischemic events in unselected, aspirin-treated patients undergoing cardiac catheterization. sCD40L was determined by a commercially available enzyme-linked immunosorbent assay in 682 consecutive patients undergoing cardiac catheterization. Two-year follow-up data were obtained from 562 patients. Dual antiplatelet therapy with aspirin and clopidogrel was associated with significantly lower levels of sCD40L and lower platelet surface expressions of P-selectin and activated GPIIb/IIIa compared to aspirin monotherapy (all p≤0.01). Hypertension was linked to lower plasma concentrations of sCD40L, whereas female sex, increasing high-sensitivity C-reactive protein, and hematocrit were associated with higher sCD40L concentrations (all p<0.05). sCD40L levels were similar in patients without and with the primary endpoint in the overall study population (p = 0.4). Likewise, sCD40L levels did not differ significantly between patients without and with the secondary endpoints (both p≥0.4). Similar results were obtained when only patients with angiographically-proven coronary artery disease (n = 459), stent implantation (n = 205) or ACS (n = 125) were analyzed. The adjustment for differences in patient characteristics by multivariate regression analyses did not change the results. ROC curve analyses did not reveal cut-off values for sCD40L for the prediction of the primary or secondary endpoints. In conclusion, plasma sCD40L levels are reduced by antiplatelet therapy with clopidogrel, but not associated with long-term ischemic outcomes in unselected consecutive aspirin-treated patients undergoing cardiac catheterization.
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    Assessment of whole blood thrombosis in a microfluidic device lined by fixed human endothelium
    (Springer US, 2016) Jain, Abhishek; van der Meer, Andries D.; Papa, Anne-Laure; Barrile, Riccardo; Lai, Angela; Schlechter, Benjamin; Otieno, Monicah A.; Louden, Calvert S.; Hamilton, Geraldine A.; Michelson, Alan; Frelinger, Andrew; Ingber, Donald
    The vascular endothelium and shear stress are critical determinants of physiological hemostasis and platelet function in vivo, yet current diagnostic and monitoring devices do not fully incorporate endothelial function under flow in their assessment and, therefore, they can be unreliable and inaccurate. It is challenging to include the endothelium in assays for clinical laboratories or point-of-care settings because living cell cultures are not sufficiently robust. Here, we describe a microfluidic device that is lined by a human endothelium that is chemically fixed, but still retains its ability to modulate hemostasis under continuous flow in vitro even after few days of storage. This device lined with a fixed endothelium supports formation of platelet-rich thrombi in the presence of physiological shear, similar to a living arterial vessel. We demonstrate the potential clinical value of this device by showing that thrombus formation and platelet function can be measured within minutes using a small volume (0.5 mL) of whole blood taken from subjects receiving antiplatelet medications. The inclusion of a fixed endothelial microvessel will lead to biomimetic analytical devices that can potentially be used for diagnostics and point-of-care applications. Electronic supplementary material The online version of this article (doi:10.1007/s10544-016-0095-6) contains supplementary material, which is available to authorized users.
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    Effect of adenosine A2 receptor stimulation on platelet activation–aggregation: Differences between canine and human models
    (Elsevier BV, 2008) Linden, Matthew D.; Barnard, Marc R.; Frelinger, Andrew; Michelson, Alan; Przyklenk, Karin
    INTRODUCTION: Adenosine A(2) agonists improve arterial patency in experimental models of recurrent thrombosis, an effect purportedly triggered by stimulation of platelet A(2) receptors and subsequent down-regulation of platelet function. However: (i) there is no direct evidence to substantiate this premise; and (ii) given the recognized differences among species in platelet signaling, it is possible that the mechanisms of A(2) receptor stimulation may be model-dependent. Accordingly, we applied an integrated in vivo and in vitro approach, using both canine and human models, to test the hypothesis that the anti-thrombotic effects of A(2) agonist treatment are due in part to inhibition of platelet activation. METHODS: In Protocol 1, recurrent coronary thrombosis was triggered in anesthetized dogs by application of a stenosis at a site of arterial injury. Coronary patency and flow cytometric indices of platelet activation (P-selectin expression; formation of heterotypic aggregates) were compared in dogs pre-treated with the A(2) agonist CGS 21680 versus controls. In Protocols 2 and 3, blood samples were obtained from dogs and human volunteers. In vitro aggregation and platelet activation (assessed by impedance aggregometry and flow cytometry, respectively) were quantified in paired aliquots pre-incubated with CGS versus vehicle. RESULTS: In the canine models, CGS improved in vivo coronary patency and attenuated in vitro aggregation but, contrary to our hypothesis, did not evoke a down-regulation in platelet activation. In contrast, in human blood samples, CGS attenuated both in vitro aggregation and flow cytometric markers of platelet activation-aggregation. CONCLUSION: The mechanisms contributing to the anti-thrombotic effect of A(2) agonist treatment are species-dependent: adenosine A(2) receptor stimulation inhibits platelet activation in human, but not canine, models.
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    Effects of in vitro adult platelet transfusions on neonatal hemostasis
    (Wiley-Blackwell, 2011) Ferrer-Marin, F.; Chavda, C.; Lampa, M.; Michelson, Alan; Frelinger, Andrew; Sola-Visner, Martha
    BACKGROUND: Thrombocytopenia is frequent among neonates, and 20-25% of affected infants are treated with platelet transfusions. These are frequently given for mild thrombocytopenia (platelets: 50-100 × 10(9) L(-1)), largely because of the known hyporeactivity of neonatal platelets. In tests of primary hemostasis, however, neonates have shorter bleeding and closure times (CTs) than adults. This has been attributed to their higher hematocrits, higher von Willebrand factor (VWF) concentrations, and predominance of longer VWF polymers. OBJECTIVE: To determine whether the 'transfusion' of adult (relatively hyperreactive) platelets into neonatal blood results in a hypercoagulable profile. METHODS: Cord blood (CB) and adult peripheral blood (PB) were separated (with a modified buffy coat method) to generate miniaturized platelet concentrates (PCs) and thrombocytopenic blood. PB-derived and CB-derived PCs (n = 7 per group) were then 'transfused'in vitro into thrombocytopenic CB and PB. The effects of autologous vs. allogeneic (developmentally mismatched) 'transfusions' were evaluated with whole blood aggregometry, a platelet function analyzer (PFA-100), and thromboelastography (TEG). RESULTS: Adult platelets aggregated significantly better than neonatal platelets in response to thrombin receptor-activating peptide, ADP, and collagen, regardless of the blood into which they were transfused. The 'transfusion' of adult platelets into thrombocytopenic CB resulted in shorter CTs-EPI (PFA-100) and higher clot strength and firmness (TEG) than 'transfusion' of neonatal autologous platelets. CONCLUSIONS: In vitro'transfusion' of adult platelets into neonatal blood results in shorter CTs than 'transfusion' with neonatal platelets. Our findings should raise awareness of the differences between the neonatal and adult hemostatic system and the potential 'developmental mismatch' associated with platelet transfusions for neonatal hemostasis.
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    The Human Endogenous Circadian System Causes Greatest Platelet Activation during the Biological Morning Independent of Behaviors
    (Public Library of Science (PLoS), 2011) Scheer, Frank; Michelson, Alan; Frelinger, Andrew; Evoniuk, Heather; Kelly, Erin E.; McCarthy, Mary; Doamekpor, Lauren A.; Barnard, Marc R.; Shea, Steven A.
    Platelets are involved in the thromboses that are central to myocardial infarctions and ischemic strokes. Such adverse cardiovascular events have day/night patterns with peaks in the morning (~9 AM), potentially related to endogenous circadian clock control of platelet activation. The objective was to test if the human endogenous circadian system influences (1) platelet function and (2) platelet response to standardized behavioral stressors. We also aimed to compare the magnitude of any effects on platelet function caused by the circadian system with that caused by varied standardized behavioral stressors, including mental arithmetic, passive postural tilt and mild cycling exercise. METHODOLOGY/PRINCIPAL FINDINGS: We studied 12 healthy adults (6 female) who lived in individual laboratory suites in dim light for 240 h, with all behaviors scheduled on a 20-h recurring cycle to permit assessment of endogenous circadian function independent from environmental and behavioral effects including the sleep/wake cycle. Circadian phase was assessed from core body temperature. There were highly significant endogenous circadian rhythms in platelet surface activated glycoprotein (GP) IIb-IIIa, GPIb and P-selectin (6-17% peak-trough amplitudes; p ≤ 0.01). These circadian peaks occurred at a circadian phase corresponding to 8-9 AM. Platelet count, ATP release, aggregability, and plasma epinephrine also had significant circadian rhythms but with later peaks (corresponding to 3-8 PM). The circadian effects on the platelet activation markers were always larger than that of any of the three behavioral stressors. CONCLUSIONS/SIGNIFICANCE: These data demonstrate robust effects of the endogenous circadian system on platelet activation in humans--independent of the sleep/wake cycle, other behavioral influences and the environment. The 9 AM timing of the circadian peaks of the three platelet surface markers, including platelet surface activated GPIIb-IIIa, the final common pathway of platelet aggregation, suggests that endogenous circadian influences on platelet function could contribute to the morning peak in adverse cardiovascular events as seen in many epidemiological studies.
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    Agonist and antagonist effects of diadenosine tetraphosphate, a platelet dense granule constituent, on platelet P2Y1, P2Y12 and P2X1 receptors
    (Elsevier BV, 2010) Chang, Hung; Yanachkov, Ivan B.; Michelson, Alan; Li, YouFu; Barnard, M.R.; Wright, George E.; Frelinger, Andrew
    INTRODUCTION: Diadenosine 5',5'''-P(1),P(4)- tetraphosphate (Ap(4)A) is stored in platelet dense granules, but its effects on platelet function are not well understood. METHODS AND RESULTS: We examined the effects of Ap(4)A on platelet purinergic receptors P2Y(1), P2Y(12) and P2X(1). Flow cytometry was used to measure the effects of Ap(4)A in the presence or absence of ADP on: a) P2Y(12)-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y(1)-mediated increase in platelet cytosolic Ca(2+), and c) P2X(1)-mediated intraplatelet entry of extracellular Ca(2+). ADP-stimulated platelet shape change (P2Y(1)-mediated) and aggregation (P2Y(1)- and P2Y(12)-mediated) were measured optically. Ap(4)A inhibited 3 microM ADP-induced: a) platelet aggregation (IC(50) 9.8+/-2.8 microM), b) P2Y(1)-mediated shape change, c) P2Y(1)-mediated increase in platelet cytosolic Ca(2+) (IC(50) 40.8+/-12.3 microM), and d) P2Y(12)-mediated decrease in VASP phosphorylation (IC(50)>250 microM). In the absence of added ADP, Ap(4)A had agonist effects on platelet P2X(1) and P2Y(12), but not P2Y(1), receptors. CONCLUSION: Ap(4)A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y(1) and P2Y(12) receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X(1) and P2Y(12) receptors.
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    Differences in Platelet Function in Patients With Acute Myeloid Leukemia and Myelodysplasia Compared to Equally Thrombocytopenic Patients With Immune Thrombocytopenia
    (Wiley-Blackwell, 2011) PSAILA, B.; BUSSEL, J. B.; Frelinger, Andrew; BABULA, B.; LINDEN, M. D.; LI, Y.; BARNARD, M. R.; TATE, C.; FELDMAN, E. J.; Michelson, Alan
    BACKGROUND: Severe thrombocytopenia is a major risk factor for hemorrhage, but platelet function and bleeding risk at low platelet counts are poorly understood, because of the limitations of platelet function testing at very low platelet counts. OBJECTIVES: To examine and compare platelet function in severely thrombocytopenic patients with acute myeloid leukemia (AML) or myelodysplasia (MDS) with that in patients with immune thrombocytopenia (ITP). METHODS: Whole blood flow cytometric measurement of platelet activation and platelet reactivity to agonists was correlated with the immature platelet fraction (IPF) and bleeding symptoms. RESULTS: Patients with AML/MDS had smaller platelets, lower IPF and substantially lower platelet surface expression of activated glycoprotein (GP)IIb-IIIa and GPIb, both with and without addition of ex vivo ADP or thrombin receptor-activating peptide, than patients with ITP. In both ITP and AML/MDS patients, increased platelet surface GPIb on circulating platelets and expression of activated GPIIb-IIIa and GPIb on ex vivo activated platelets correlated with a higher IPF. Whereas platelet reactivity was higher for AML/MDS patients with bleeding than for those with no bleeding, platelet reactivity was lower for ITP patients with bleeding than for those with no bleeding. CONCLUSIONS: AML/MDS patients have lower in vivo platelet activation and ex vivo platelet reactivity than patients with ITP. The proportion of newly produced platelets correlates with the expression of platelet surface markers of activation. These differences might contribute to differences in bleeding tendency between AML/MDS and ITP patients. This study is the first to define differences in platelet function between AML/MDS patients and ITP patients with equivalent degrees of thrombocytopenia.