Do immature platelet levels in chest pain patients presenting to the emergency department aid in the diagnosis of acute coronary syndrome?

INTRODUCTION: Early and accurate identification of acute coronary syndrome (ACS) vs. noncardiac chest pain in patients presenting to the emergency department (ED) is problematic and new diagnostic markers are needed. Previous studies reported that elevated mean platelet volume (MPV) is associated with ACS and predictive of cardiovascular risk. MPV is closely related to the immature platelet fraction (IPF), and recent studies have suggested that IPF may be a more sensitive marker of ACS than MPV. The objective of the present study was to determine whether the measurement of IPF assists in the diagnosis of ACS in patients presenting to the ED with chest pain. METHODS: In this single-center, prospective, cross-sectional study, adult patients presenting to the ED with chest pain and/or suspected ACS were considered for enrollment. Blood samples from 236 ACS-negative and 44 ACS-positive patients were analyzed in a Sysmex XE-2100 for platelet count, MPV, IPF, and the absolute count of immature platelets (IPC). RESULTS: Total platelet counts, MPV, IPF, and IPC were not statistically different between ACS-negative and ACS-positive patients. The IPF was 4.6 ± 2.7% and 5.0 ± 2.8% (mean ± SD, P = 0.24), and the IPC was 10.0 ± 4.6 and 11.5 ± 7.5 × 10(3) /µL (P = 0.27) for ACS-negative and ACS-positive patients, respectively. CONCLUSION: In 280 patients presenting to the ED with chest pain and/or suspected ACS, no differences in IPF, IPC or MPV were observed in ACS-negative vs. ACS-positive patients, suggesting that these parameters do not assist in the diagnosis of ACS.

Modified diadenosine tetraphosphates with dual specificity for P2Y1 and P2Y12 are potent antagonists of ADP-induced platelet activation.

BACKGROUND: Diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A), a natural compound stored in platelet dense granules, inhibits ADP-induced platelet aggregation. Ap(4)A inhibits the platelet ADP receptors P2Y(1) and P2Y(12), is a partial agonist of P2Y(12), and is a full agonist of the platelet ATP-gated ion channel P2X1. Modification of the Ap(4)A tetraphosphate backbone enhances inhibition of ADP-induced platelet aggregation. However, the effects of these Ap(4)A analogs on human platelet P2Y(1), P2Y(12) and P2X1 are unclear. OBJECTIVE: To determine the agonist and antagonist activities of diadenosine tetraphosphate analogs towards P2Y(1), P2Y(12), and P2X1. METHODS: We synthesized the following Ap(4)A analogs: P(1),P(4)-dithiotetraphosphate; P(2),P(3)-chloromethylenetetraphosphate; P(1)-thio-P(2),P(3)-chloromethylenetetraphosphate; and P(1),P(4)-dithio-P(2),P(3)-chloromethylenetetraphosphate. We then measured the effects of these analogs on: (i) ADP-induced platelet aggregation; (ii) P2Y(1)-mediated changes in cytosolic Ca(2+); (iii) P2Y(12)-mediated changes in vasodilator-stimulated phosphoprotein phosphorylation; and (iv) P2X1-mediated entry of extracellular Ca(2+). RESULTS: Ap(4)A analogs with modifications in the phosphate backbone inhibited both P2Y(1) and P2Y(12), and showed no agonist activity towards these receptors. The dithio modification increased inhibition of P2Y(1), P2Y(12), and platelet aggregation, whereas the chloromethylene modification increased inhibition of P2Y(12) and platelet aggregation, but decreased P2Y(1) inhibition. Combining the dithio and chloromethylene modifications increased P2Y(1) and P2Y(12) inhibition. As compared with Ap(4)A, each modification decreased agonist activity towards P2X1, and the dual modification completely eliminated P2X1 agonist activity. CONCLUSIONS: As compared with Ap(4)A, tetraphosphate backbone analogs of Ap(4)A have diminished activity towards P2X1 but inhibit both P2Y(1) and P2Y(12) and, with greater potency, inhibit ADP-induced platelet aggregation. Thus, diadenosine tetraphosphate analogs with dual receptor selectivity may have potential as antiplatelet drugs.

Differences in platelet function in patients with acute myeloid leukemia and myelodysplasia compared to equally thrombocytopenic patients with immune thrombocytopenia.

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.

Agonist and antagonist effects of diadenosine tetraphosphate, a platelet dense granule constituent, on platelet P2Y1, P2Y12 and P2X1 receptors.

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.

Targeted inhibition of the serotonin 5HT2A receptor improves coronary patency in an in vivo model of recurrent thrombosis.

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.

Aspirin 'resistance': role of pre-existent platelet reactivity and correlation between tests.

BACKGROUND: Aspirin 'resistance' is a widely used term for hyporesponsiveness to aspirin in a platelet function test. Serum thromboxane (TX) B(2) is the most specific test of aspirin's effect on platelets. OBJECTIVES: (i) To examine the role of pre-existent platelet hyperreactivity in aspirin 'resistance'. (ii) To determine the correlation between aspirin resistance defined by serum TXB(2) and other assays of platelet function. METHODS: To enable pre-aspirin samples to be drawn, platelet function was measured in normal subjects (n = 165) before and after aspirin 81 mg daily for seven days. RESULTS: The proportion of the post-aspirin platelet function predicted by the pre-aspirin platelet function was 28.3 +/- 7.5% (mean +/- asymptotic standard error) for serum TXB(2), 39.3 +/- 6.8% for urinary 11-dehydro TXB(2), 4.4 +/- 7.7% for arachidonic acid-induced platelet aggregation, 40.4 +/- 7.1% for adenosine diphosphate-induced platelet aggregation, 26.3 +/- 9.2% for the VerifyNow Aspirin Assay, and 45.0 +/- 10.9% for the TEG PlateletMapping System with arachidonic acid. There was poor agreement between aspirin-resistant subjects identified by serum TXB(2) vs. aspirin-resistant subjects identified by the other five assays, irrespective of whether the analysis was based on categorical or continuous variables. Platelet count correlated with pre-aspirin serum TXB(2) and VerifyNow Aspirin Assay, but not with any post-aspirin platelet function test. CONCLUSIONS: (i) Aspirin 'resistance' (i.e. hyporesponsiveness to aspirin in a laboratory test) is in part unrelated to aspirin but is the result of underlying platelet hyperreactivity prior to the institution of aspirin therapy. (ii) Aspirin resistance defined by serum TXB(2) shows a poor correlation with aspirin resistance defined by other commonly used assays.

The active metabolite of prasugrel inhibits adenosine diphosphate- and collagen-stimulated platelet procoagulant activities.

BACKGROUND: Prasugrel is a novel antiplatelet prodrug of the same thienopyridine class as clopidogrel and ticlopidine. Metabolism of prasugrel generates the active metabolite R-138727, an antagonist of the platelet P2Y(12) adenosine diphosphate (ADP) receptor, leading to inhibition of ADP-mediated platelet activation and aggregation. ADP also enhances the platelet response to collagen, and these two agonists contribute to the generation of platelet procoagulant activity. We therefore examined whether R-138727 inhibits ADP- and collagen-triggered platelet procoagulant activities. METHODS AND RESULTS: As shown by whole blood flow cytometry, R-138727 inhibited surface phosphatidylserine expression on ADP plus collagen-stimulated platelets and tissue factor (TF) expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte-platelet aggregates. R-138727 reduced monocyte-platelet aggregate formation, thereby further inhibiting TF expression. ADP, collagen, and ADP plus collagen accelerated the kinetics of thrombin generation in recalcified whole blood and R-138727 significantly inhibited this acceleration. Clot strength in a modified thromboelastograph system was also inhibited by R-138727 (IC50 0.7 +/- 0.1 microM). CONCLUSIONS: In addition to its previously known inhibitory effects on platelet activation and aggregation, the active metabolite of prasugrel, R-138727, inhibits platelet procoagulant activity in whole blood (as determined by phosphatidylserine expression on platelets and TF expression on monocyte-platelet aggregates), resulting in the functional consequences of delayed thrombin generation and impaired clot development.

Indices of platelet activation and the stability of coronary artery disease.

AIM: To determine whether indices of platelet activation are associated with the stability of coronary artery disease (CAD). METHODS: Platelet function was examined in 677 consecutive aspirin-treated patients presenting for cardiac catheterization. Patients were grouped into recent myocardial infarction (MI), no MI but angiographically documented CAD (non-MI CAD) and no angiographically detectible CAD (no CAD), as well as additional subgroups. RESULTS: Compared with non-MI CAD or no CAD patients, more patients with recent MI had a shortened platelet function analyzer (PFA)-100 collagen-epinephrine closure time (CT) and increased circulating monocyte-platelet aggregates, neutrophil-platelet aggregates, activated platelet surface GPIIb-IIIa and plasma soluble CD40 ligand (sCD40L). More patients with non-MI CAD had shortened PFA-100 CTs and increased monocyte-platelet aggregates compared with patients with no CAD. Platelet surface P-selectin did not differ among the groups. Subgroup analysis revealed that decreasing PFA-100 CT correlated with the stability of CAD. CONCLUSIONS: Indices of platelet activation, especially the PFA-100 CT, are associated with the stability of CAD, and may reflect plaque instability, an ongoing thrombotic state and/or reduced responsiveness to aspirin.

Evidence that pre-existent variability in platelet response to ADP accounts for 'clopidogrel resistance'.

BACKGROUND: Clopidogrel is a widely used antithrombotic agent that inhibits the platelet P2Y(12) adenosine diphosphate (ADP) receptor. There is increasing interest in 'clopidogrel resistance'. OBJECTIVES: To determine whether 'clopidogrel resistance' is accounted for by a pre-existent variability in platelet response to ADP. METHODS: Platelet response to 20 microm ADP was analyzed by four independent whole blood flow cytometric assays: platelet surface activated GPIIb-IIIa, platelet surface P-selectin, monocyte-platelet aggregates and neutrophil-platelet aggregates. In 25 consecutive, non-aspirin-treated healthy subjects, we studied platelet response before and after clopidogrel administration. In addition, we studied the platelet response in 613 consecutive aspirinated patients with or without coronary artery disease (CAD, as determined by angiography) who had or had not been treated with clopidogrel. In these patients, we tested for homogeneity of variance across all durations of clopidogrel exposure and severity of CAD by estimating the 'goodness of fit' of two independent models. RESULTS: In the healthy subjects, pre-clopidogrel response to ADP predicted post-clopidogrel response to ADP. In the patients, clopidogrel, as expected, inhibited the platelet response to ADP. However, irrespective of the duration of clopidogrel administration, the severity of CAD, and the dose of aspirin, clopidogrel did not increase the variance in the platelet response to ADP in any of the four assays of platelet response. CONCLUSIONS: These studies provide evidence that 'clopidogrel resistance' is accounted for by a pre-existent variability in platelet response to ADP and this variability is not increased by clopidogrel administration.

Preconditioning ischemia attenuates molecular indices of platelet activation-aggregation.

BACKGROUND: Previous studies have shown that ischemic preconditioning (PC) not only limits infarct size, but also improves arterial patency in models of recurrent thrombosis. We hypothesize that this enhanced patency is presumably because of a PC-induced attenuation of platelet-mediated thrombosis. However, there is, at present, no direct evidence that PC acts on the platelets per se and favorably down-regulates platelet reactivity. OBJECTIVES: Our goal was to test the concept that PC ischemia attenuates molecular indices of platelet activation-aggregation. METHODS: Anesthetized dogs were randomly assigned to receive 10 min of PC ischemia followed by 10 min of reperfusion or a time-matched control period. Spontaneous recurrent coronary thrombosis was then initiated in all dogs by injury + stenosis of the left anterior descending coronary artery. Coronary flow was monitored for 3 h poststenosis, and molecular indices of platelet activation-aggregation were quantified by whole blood flow cytometry. RESULTS: Coronary patency was, as expected, better-maintained following injury + stenosis in the PC group vs. controls (53% +/- 5%* vs. 23% +/- 5% of baseline flow, respectively; *P < 0.05). Moreover, PC was accompanied by: (i) a significant down-regulation of platelet-fibrinogen binding and formation of neutrophil-platelet aggregates (112% +/- 14%* vs. 177% +/- 21% and 107% +/- 8%* vs. 155% +/- 19% of baseline values in PC vs. control groups); and (ii) a trend towards a reduction in platelet P-selectin expression (148% +/- 12% vs. 190% +/- 21% of baseline; *P < 0.05 and P = 0.09 vs. control). CONCLUSION: These data provide novel, direct evidence in support of the concept that ischemic PC attenuates molecular indices of platelet activation-aggregation.

Effects of platelet binding on whole blood flow cytometry assays of monocyte and neutrophil procoagulant activity.

BACKGROUND: Monocytes and neutrophils form heterotypic aggregates with platelets initially via engagement of platelet surface P-selectin with leukocyte surface P-selectin glycoprotein ligand-1 (PSGL-1). The resultant intracellular signaling causes the leukocyte surface expression of tissue factor and activation of leukocyte surface Mac-1 (integrin alphaMbeta2, CD11b/CD18). The activation-dependent conformational change in monocyte surface Mac-1 results in the binding of coagulation factor Xa (FXa) and/or fibrinogen to Mac-1. The aim of this study was to develop whole blood flow cytometry assays of these procoagulant activities and to investigate the effects of platelet binding to monocytes and neutrophils. METHODS: Citrate or D-Phe-Pro-Arg-chloromethylketone (PPACK) anticoagulated whole blood was incubated with monoclonal antibodies against CD14 (PECy5), CD42a (PE), FITC-conjugated test antibody and an agonist, and then fixed with FACS lyse. Appropriate isotype negative controls were prepared in parallel. A BD FACSCalibur was used to analyze monocytes and neutrophils, which were identified based on CD14 fluorescence, forward and 90 degrees light scatter. These populations were further gated into CD42a-positive (platelet-bound) and CD42a-negative (platelet-free). Geometric mean fluorescence and per cent positive data were collected for each subpopulation to measure the binding of test antibodies directed at CD42a, tissue factor, coagulation FXa, bound fibrinogen, activated Mac-1, and CD11b. Compensation controls were prepared on six normal donors prior to the study and these settings were used throughout the 10 donor study. Negative controls verified the lack of cross talk, particularly in the quantified FITC and PE parameters. RESULTS: The physiologic agonists collagen and ADP increased monocyte-platelet and neutrophil-platelet aggregates and increased leukocyte surface Mac-1/CD11b and surface-bound tissue factor, FXa and fibrinogen. Whereas the increases in Mac-1/CD11b were mainly independent of leukocyte-platelet binding, the increases in surface-bound tissue factor, FXa and fibrinogen were mainly dependent on leukocyte-platelet binding. CONCLUSIONS: (i) We have developed novel whole blood flow cytometry assays to measure bound tissue factor, coagulation FXa, fibrinogen, activated Mac-1 and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface-adherent platelets. (ii) The monocyte and neutrophil surface binding of tissue factor, FXa and fibrinogen is mainly dependent on platelet adherence to monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac-1 is mainly independent of platelet adherence to monocytes and neutrophils.

Survival of baboon biotin-X-N-hydroxysuccinimide and (111)In-oxine-labelled autologous fresh and lyophilized reconstituted platelets.

BACKGROUND AND OBJECTIVES: In accordance with Food and Drug Administration (FDA) regulations, platelets can be stored in the liquid state at 22 degrees C for only 5 days. Platelets frozen with 6% dimethylsulphoxide (DMSO) can be stored at -80 degrees C for 2 years, and platelets frozen with 5% DMSO can be stored at -150 degrees C for 3 years. Studies are being conducted to determine the effects of lyophilization of platelets. In the present study, we assessed the survival of autologous lyophilized-reconstituted platelets in the baboon. MATERIALS AND METHODS: We studied fresh baboon platelets and baboon platelets that had been treated with paraformaldehyde, frozen, lyophilized, thawed and reconstituted. Aliquots of these platelets were labelled with (111)In-oxine or biotin-X-N-hydroxysuccinimide (biotin-X-NHS) before autotransfusion, and measurements were made of the in vivo recovery and lifespan. We also evaluated the response of fresh and lyophilized platelets to in vitro agonists by measuring the level of platelet surface markers and heterotypic aggregates in the peripheral blood following the autotransfusions. RESULTS: The (111)In-oxine- or biotin-X-NHS-labelled lyophilized, reconstituted platelets exhibited survival times of less than 15 min. These platelets did not respond to stimulation with agonists to decrease platelet GPIb and increase platelet P-selectin and platelet GPIIb-IIIa levels 1 min post-transfusion and they accumulated more procoagulant factor V than did the fresh platelets. CONCLUSIONS: Lyophilized reconstituted baboon platelets labelled with (111)In-oxine or biotin-X-NHS before autotransfusion exhibited an in vivo circulation time of less than 15 min. Further study of the lyophilized, reconstituted platelets is required to evaluate their haemostatic function.

GPIIb-IIIa antagonists reduce thromboinflammatory processes in patients with acute coronary syndromes undergoing percutaneous coronary intervention.

OBJECTIVE: To investigate the effects of abciximab, eptifibatide and no GPIIb-IIIa antagonist (control) on soluble CD40 ligand (sCD40L) and the formation of leukocyte-platelet aggregates (LPA) in 98 ACS patients undergoing percutaneous coronary intervention (PCI). BACKGROUND: sCD40L and LPA are increased in patients with ACS. METHODS: sCD40L was measured by enzyme-linked immunosorbent assay (ELISA) and LPA by whole blood flow cytometry. RESULTS: There were no baseline differences between the three groups in sCD40L and LPA. At the end of PCI, sCD40L was unchanged in the controls, decreased by 30% (P < 0.001) in the abciximab group and by 11% (P < 0.02) in the eptifibatide group. Eighteen to 24 h after PCI, sCD40L was unchanged in the controls, reduced 30% (P < 0.001) in the abciximab-treated group and 9% (P < 0.01) in the eptifibatide-treated group. At the end of PCI, circulating monocyte-platelet aggregates (MPA) were reduced by 12% (P = NS) in the abciximab-treated group, 13% in the eptifibatide-treated group (P = NS), but slightly increased in the controls (P = NS). Eighteen to 24 h after PCI, MPA were reduced by 41% (P < 0.001) compared to baseline in the abciximab-treated group, by 23% (P = NS) in the eptifibatide-treated group, and 15% (P = NS) in the controls. In contrast to control patients presenting while on clopidogrel, control patients presenting not on clopidogrel demonstrated a reduction in sCD40L and LPA 18-24 h post-PCI (P = NS). At low receptor occupancy, GPIIb-IIIa antagonists did not augment the release of sCD40L or the number of circulating LPA. CONCLUSIONS: GPIIb-IIIa antagonists reduce circulating sCD40L and LPA formation in patients with ACS undergoing PCI. At low receptor occupancy, GPIIb-IIIa antagonists do not activate platelets.

In vitro testing of fresh and lyophilized reconstituted human and baboon platelets.

BACKGROUND: Studies have been performed on human fresh, liquid-preserved, and cryopreserved platelets (PLTs) to assess PLT-adhesive surface receptors, PLT membrane procoagulant activity, PLT aggregation, and thromboxane production. Lyophilization has been developed as a method to preserve PLTs. This study was performed to evaluate these measurements on human and baboon fresh and lyophilized reconstituted PLTs. STUDY DESIGN AND METHODS: In both human and baboon fresh and lyophilized PLTs, aggregation response and PLT production of thromboxane A2 were measured after stimulation, and PLT surface markers P-selectin, glycoprotein (GP) Ib, GPIIb-IIIa, and factor (F) V were measured before and after stimulation. RESULTS: Fresh PLTs responded to the dual agonists arachidonic acid and adenosine diphosphate (ADP) to aggregate and produce thromboxane A2, and in both the PLT surface markers P-selectin and GPIIb-IIIa increased and GPIb decreased after stimulation. Neither human nor baboon lyophilized reconstituted PLTs aggregated to dual agonists, and neither produced thromboxane A2, increased PLT surface markers P-selectin or GPIIb-IIIa, or decreased PLT GPIb after stimulation. Nevertheless, after recalcification the lyophilized reconstituted PLTs accumulated FV to a significantly greater degree than fresh PLTs. CONCLUSIONS: Lyophilized reconstituted PLTs exhibited modification of the PLT membrane that interfered with aggregation and thromboxane production, prevented increases in PLT P-selectin and GPIIb-IIIa and decreases in GPIb after stimulation, and increased FV accumulation after recalcification. The in vitro data suggest that lyophilized PLTs may have reduced in vivo survival. In vivo studies are needed to determine the survival and function of lyophilized PLTs.

Platelet FcgammaRIIA His131Arg polymorphism and platelet function: antibodies to platelet-bound fibrinogen induce platelet activation.

The His131Arg polymorphism of platelet FcgammaRIIA affects the binding affinity of certain IgG subclasses. The Arg131 allele has been associated with (auto)immune thrombocytopenia and heparin-induced thrombocytopenia in some studies. Because FcgammaRIIA can transmit platelet activation signals, we studied platelet responsiveness from 73 healthy donors to determine if this polymorphism modulated platelet function. Platelet function was studied by agonist and shear-induced activation, and standard aggregation. FcgammaRIIA was genotyped by allele-specific PCR. Compared with His131, the Arg131 allele was associated with significantly greater binding of activation-dependent antibodies. This effect was most prominent for the receptor-induced binding site (RIBS) antibodies F26 (P < 0.0001) and RIBS1 (P = 0.0057), and the ligand-induced binding site antibody LIBS1 (P = 0.0367). Unexpectedly, Arg131-positive platelets did not show greater fibrinogen binding, platelet aggregation or shear-induced platelet activation. We considered whether enhanced Fc binding and FcgammaRIIA cross-linking were responsible for those discrepancies. The increased binding of the two RIBS antibodies to the Arg131 isoform was abolished by blocking FcgammaRIIA, and the FcgammaRIIA genotype effect on F26 IgG binding was lost when F26 F(ab')2 fragments were used. Furthermore, intact F26 and RIBS1 IgG directly and specifically induced P-selectin expression, and this effect was greatest in Arg131-positive platelets. We concluded that (a) the His131Arg polymorphism of FcgammaRIIA does not affect intrinsic platelet reactivity; (b) RIBS antibodies are able to cross-link FcgammaRIIA and activate platelets, and this activation has a modest effect on Arg131 platelets; and (c) flow cytometric based platelet assays may need to compensate for this FcgammaRIIA His131Arg effect on platelet activation.

Leukocyte-platelet aggregation, platelet surface P-selectin, and platelet surface glycoprotein IIIa after percutaneous coronary intervention: Effects of dalteparin or unfractionated heparin in combination with abciximab.

BACKGROUND: Plaque disruption with resultant platelet activation and leukocyte-platelet aggregation is a pathophysiologic process common to both acute coronary syndromes and percutaneous coronary interventions. Unfractionated heparin is a standard antithrombotic therapy in patients with both acute coronary syndromes and in those undergoing percutaneous coronary interventions. Low-molecular-weight heparins have been reported to cause less platelet activation than unfractionated heparin. METHODS: Monocyte-platelet aggregates, neutrophil-platelet aggregates, platelet surface P-selectin, and platelet surface glycoprotein (GP) IIIa were measured serially by whole blood flow cytometry in 40 patients with unstable angina (randomly assigned to either unfractionated heparin 70 U/kg or the low-molecular-weight heparin dalteparin 60 IU/kg) undergoing coronary intervention with planned abciximab administration (in 2, one-half-dose boluses). Assays were performed at baseline, 5 minutes after administration of either type of heparin, 10 minutes after the first bolus of abciximab, 10 minutes after second bolus of abciximab, and 8 to 10 and 16 to 24 hours after administration of either heparin. RESULTS: No significant differences in clinical outcomes were observed between patients receiving either unfractionated heparin or dalteparin. The number of circulating P-selectin-positive platelets was increased by unfractionated heparin but not dalteparin, and abciximab reversed this increase. The number of circulating P-selectin-positive platelets was reduced below baseline levels in both treatment groups 8 to 10 and 16 to 24 hours after study drug administration. At 8 to 10 and 16 to 24 hours after administration of study drug, platelet degranulation in response to iso-thrombin receptor agonist peptide 1.5 mmol/L was significantly reduced by almost 50% (compared with immediately after study drug administration). Both unfractionated heparin and dalteparin significantly increased the numbers of circulating monocyte-platelet and neutrophil-platelet aggregates, which were subsequently reduced to baseline levels after administration of the second abciximab bolus and to below baseline at both 8 to 10 and 16 to 24 hours in all patients. After both unfractionated heparin and dalteparin administration, platelet surface GP IIIa expression was significantly increased compared with baseline at both 8 to 10 and 16 to 24 hours. CONCLUSIONS: Dalteparin in combination with abciximab in patients with unstable angina undergoing coronary intervention appears to be safe. Unfractionated heparin, but not dalteparin, degranulates platelets in patients with unstable angina. Both heparins increase the number of circulating monocyte-platelet and neutrophil-platelet aggregates. Abciximab therapy during coronary interventions rapidly reduces the number of degranulated platelets and leukocyte-platelet aggregates.

Circulating monocyte-platelet aggregates are an early marker of acute myocardial infarction.

OBJECTIVES: We investigated whether elevated levels of circulating monocyte-platelet aggregates (MPA) can be used to identify patients with acute myocardial infarction (AMI). BACKGROUND: Commonly used blood markers of AMI reflect myocardial cell death, but do not reflect the earlier pathophysiologic processes of plaque rupture, platelet activation and resultant thrombus formation. Circulating MPA form after platelet activation. METHODS: In a single center between October 1998 and November 1999, we measured circulating MPA in a blinded fashion by whole blood flow cytometry in 211 consecutive patients who presented to the emergency department (ED) with chest pain and were admitted to rule out AMI. Acute myocardial infarction was diagnosed by a CK-MB fraction greater than three times control. RESULTS: Patients with AMI (n = 61), as compared with those without AMI (n = 150), had significantly higher numbers of circulating MPA (11.6 +/- 11.4 vs. 6.4 +/- 3.6, mean +/- SD, p < 0.0001). After controlling for age, the adjusted odds of developing AMI for patients in the 2nd, 3rd and 4th quartiles of MPA, in comparison with patients in the lowest quartile (odds ratio = 1.0), were 2.1 (95% confidence interval [CI]: 0.7, 6.8), 4.4 (95% CI: 1.5, 13.1) and 10.8 (95% CI: 3.6, 32.0), respectively. The number of circulating MPA in patients with AMI presenting within 4 h of symptom onset (14.4) was significantly greater than those presenting after 4 h (9.4) and after 8 h (7.0), (p < 0.001). Of the 61 patients with AMI, 35 (57%) had a normal creatine kinase isoenzyme ratio at the time of presentation to the ED, but had high levels of circulating MPA (13.3). CONCLUSIONS: Circulating MPA are an early marker of AMI.

Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction.

BACKGROUND: Platelet surface P-selectin is considered the "gold standard" marker of platelet activation. Degranulated, P-selectin-positive platelets, however, aggregate with leukocytes in vitro and rapidly lose surface P-selectin in vivo. METHODS AND RESULTS: Flow cytometric tracking of autologous, biotinylated platelets in baboons enabled us to directly demonstrate for the first time in vivo that (1) infused degranulated platelets very rapidly form circulating aggregates with monocytes and neutrophils, and (2) 30 minutes after infusion of the degranulated platelets, the percentage of circulating monocytes aggregated with infused platelets persist at high levels, whereas the percentage of circulating neutrophils aggregated with infused platelets and the platelet surface P-selectin of nonaggregated infused platelets return to baseline. We therefore performed 2 clinical studies in patients with acute coronary syndromes. First, after percutaneous coronary intervention (n=10), there was an increased number of circulating monocyte-platelet (and to a lesser extent, neutrophil-platelet) aggregates but not P-selectin-positive platelets. Second, of 93 patients presenting to an Emergency Department with chest pain, patients with acute myocardial infarction (AMI) (n=9) had more circulating monocyte-platelet aggregates (34.2+/-10.3% [mean+/-SEM]) than patients with no AMI (n=84, 19.3+/-1.4%, P<0.05) and normal control subjects (n=10, 11.5+/-0.8%, P<0.001). Circulating P-selectin-positive platelets, however, were not increased in chest pain patients with or without AMI. CONCLUSIONS: As demonstrated by 3 independent means (in vivo tracking of activated platelets in baboons, human coronary intervention, and human AMI), circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin.

Dissociation of glycoprotein IIb/IIIa antagonists from platelets does not result in fibrinogen binding or platelet aggregation.

BACKGROUND: The primary mechanism of action of glycoprotein (GP) IIb/IIIa antagonists is inhibition of the final common pathway of platelet aggregation: fibrinogen binding to the GP IIb/IIIa complex. However, it has been reported that induction of fibrinogen binding and platelet aggregation is an intrinsic prothrombotic property of low-dose GP IIb/IIIa antagonists. These apparently paradoxical results have been extensively referenced in the cardiology literature. METHODS AND RESULTS: By platelet aggregation and flow cytometry, we demonstrate that (1) dissociation of GP IIb/IIIa antagonists (abciximab, tirofiban, eptifibatide, or xemilofiban) from platelets does not result in platelet aggregation; (2) tirofiban and eptifibatide can induce a fibrinogen-binding-competent conformation of the GP IIb/IIIa receptor, but stable fibrinogen binding does not occur without fixation; (3) the slow off-rate of abciximab exposes only a small proportion of unblocked GP IIb/IIIa receptors at any time, and these also fail to stably bind fibrinogen; and (4) the GP IIb/IIIa antagonist-induced fibrinogen binding in some previously reported experiments was probably the result of artifactual thrombin generation. CONCLUSIONS: Under physiological conditions, GP IIb/IIIa antagonists currently in clinical use do not have an intrinsic activating property that results in platelet aggregation or stable fibrinogen binding to GP IIb/IIIa.

Serial determinations of platelet counts in mice by flow cytometry.

Elucidation of the pathophysiological basis of platelet disorders in murine models requires a reliable method for the frequent determinations of platelet counts in individual mice. Here, we present a rapid, reproducible and accurate flow cytometric method for enumeration of platelets that involves fluorescent staining of platelets in whole blood with specific antibody and the addition of known numbers of fluorescent beads for standardization of the sample volume. Analysis of platelets obtained by tail bleeding indicated that this sampling procedure did not activate platelets, and that only five microliters of blood were required for platelet counting. Using this method, we followed platelet counts in mice infected with the relapsing fever spirochete Borrelia turicatae for 26 days, and found that this bacterium induces thrombocytopenia, a common manifestation of human relapsing fever. Therefore, this method can be used to follow the number and the activation state of circulating platelets from individual mice over extended periods of time and is applicable to a wide range of murine models of platelet disorders.