The influence of intermittent hypoxemia on platelet activation in obese patients with obstructive sleep apnea.

OBJECTIVES: Literature regarding platelet function in obstructive sleep apnea (OSA) has considerable limitations. Given the central role of platelets in atherothrombosis and the known cardiovascular risk of OSA, we hypothesized that OSA severity is predictive of platelet function, independent of known comorbidities. DESIGN: Obese subjects, without comorbidities, underwent overnight, in-lab polysomnography. The following morning, 5 biomarkers of platelet activation were measured by whole-blood flow cytometry at baseline and in response to agonists (no stimulation, stimulation with 5 µM ADP agonist, and stimulation with 20 µM ADP agonist): platelet surface P-selectin, activated glycoprotein (GP) IIb/IIIa, and GPIb receptor expression, platelet-monocyte aggregation (PMA) and platelet-neutrophil aggregation (PNA). RESULTS: Of the 77 subjects, 47 were diagnosed with OSA (median apnea-hypopnea index [AHI] of 24.7 ± 28.1/h in subjects with OSA and 3.0 ± 3.9/h in subjects without OSA, p < 0.001). The groups were matched for body mass index, with a mean body mass index of 40.3 ± 9.6 kg/m(2) in subjects with OSA and 38.9 ± 6.0 kg/m(2) in subjects without OSA (p = 0.48). A comparison of time spent with an oxygen saturation of less than 90% showed that subjects who had 1 minute or more of desaturation time per hour of sleep had lower GPIb fluorescence in circulating platelets, as compared with those subjects who had less than 1 minute of desaturation time per hour of sleep; similar findings were observed following 5 µM and 20 µM of ADP stimulation, as compared with control vehicle, suggesting higher levels of circulating platelet activity. In multivariate analyses, only nocturnal hypoxemia and female sex predicted agonist response. Platelet surface P-selectin, platelet surface-activated GPIIb/IIIa, PMA, and PNA were not significantly correlated with markers of OSA. CONCLUSIONS: In obese patients with OSA, platelet activation is associated with greater levels of oxygen desaturation, compared with matched control subjects. Metrics other than AHI (e.g., hypoxemia) may determine OSA-related thrombotic risk.

Heparin-associated anti-Xa activity and platelet-derived prothrombotic and proinflammatory biomarkers in moderate to high-risk patients with acute coronary syndrome.

Heparin compounds, to include fractionated and unfractionated preparations, exert both antithrombotic and antiinflammatory effects through combined inhibition of factor Xa and thrombin. The contribution of modulated platelet activity in vivo is less clearly defined. The SYNERGY library was a prospectively designed repository for candidate clinical, hemostatic, platelet, and molecular biomarkers from patients participating in SYNERGY--a large-scale, randomized clinical trial evaluating the comparative benefits of unfractionated heparin (UFH) and enoxaparin in high-risk patients with acute coronary syndrome (ACS). Samples were collected from 201 patients enrolled at 26 experienced, participating sites and shipped to established core laboratories for analysis of platelet, endothelium-derived, inflammatory and coagulation activity biomarkers. Tissue factor pathway inhibitor (TFPI)--a vascular endothelial cell-derived factor Xa regulatory protein-correlated directly with plasma anti-Xa activity (unadjusted: r = 0.23, P < 0.0001; adjusted: ß = 0.10; P = 0.001), as did TFPI-fXa complexes (unadjusted: r = 0.34, P < 0.0001; adjusted: ß = 0.38; P = < 0.0001). In contrast, there was a direct and inverse relationship between anti-Xa activity and two platelet-derived biomarkers-plasminogen activator inhibitor-1 (unadjusted: r = -0.18, P = 0.0012; adjusted: ß = -0.10; P = 0.021) and soluble CD40 ligand (unadjusted: r = -0.11, P = 0.05; adjusted: ß = -0.13; P = 0.049). All measured analyte relationships persisted after adjustment for age, creatinine clearance, weight, sex, and duration of treatment. Differences in biomarkers between patients receiving UFH and those randomized to enoxaparin were not observed. The ability of heparin compounds to affect the prothrombotic and proinflammatory states which characterize ACS may involve factor Xa-related modulation of platelet activation and expression. Whether this potentially beneficial effect is direct or indirect and achieved, at least in part, through the release of endothelial cell-derived coagulation regulatory proteins will require further investigation.

Association of cyclooxygenase-1-dependent and -independent platelet function assays with adverse clinical outcomes in aspirin-treated patients presenting for cardiac catheterization.

BACKGROUND: Poor clinical outcome in aspirin-treated patients has been termed aspirin resistance and may result from inadequate inhibition of platelet cyclooxygenase-1 (COX-1) by aspirin. The objectives of this study were to determine prospectively whether COX-1-dependent and other platelet function assays correlate with clinical outcomes in aspirin-treated patients. METHODS AND RESULTS: Blood was collected before percutaneous coronary intervention from 700 consecutive aspirin-treated (81 or 325 mg for > or =3 days) patients. Platelet function was tested by (1) serum thromboxane B(2); (2) arachidonic acid-stimulated platelet surface P-selectin and activated glycoprotein IIb/IIIa and leukocyte-platelet aggregates; and (3) platelet function analyzer (PFA)-100 collagen-epinephrine and collagen-ADP closure time (CT). Adverse clinical outcomes of all-cause death, cardiovascular death, and major adverse cardiovascular events (cardiovascular death, myocardial infarction, hospitalization for revascularization, or acute coronary syndrome) were assessed by telephone interview and/or medical record review. Clinical outcomes information was obtained at 24.8+/-0.3 months after platelet function testing. By univariate analysis, COX-1-dependent assays, including serum thromboxane B(2) level, were not associated with adverse clinical outcomes, whereas the COX-1-independent assay, PFA-100 collagen-ADP CT <65 seconds, was associated with major adverse cardiovascular events (P=0.0149). After adjustment for covariables (including sex, aspirin dose, Thrombolysis in Myocardial Infarction risk score, clopidogrel use), both serum thromboxane B(2) >3.1 ng/mL and PFA-100 collagen-ADP CT <65 seconds were associated with major adverse cardiovascular events. In contrast, indirect measures of platelet COX-1 (arachidonic acid-stimulated platelet markers, shortened PFA-100 collagen-epinephrine CT) were not significantly associated with adverse clinical outcomes even after adjustment for covariables. CONCLUSIONS: In this prospective study of 700 aspirin-treated patients presenting for angiographic evaluation of coronary artery disease, residual platelet COX-1 function measured by serum thromboxane B(2) and COX-1-independent platelet function measured by PFA-100 collagen-ADP CT, but not indirect COX-1-dependent assays (arachidonic acid-stimulated platelet markers, shortened PFA-100 collagen-epinephrine CT), correlate with subsequent major adverse cardiovascular events. This study suggests that multiple mechanisms, including but not confined to inadequate inhibition of COX-1, are responsible for poor clinical outcomes in aspirin-treated patients, and therefore the term aspirin resistance is inappropriate.

Assessing the current role of platelet function testing.

In vitro platelet function tests are commonly applied in research and offer justification for using antiplatelet therapy. However, studies assessing the ability of standardized platelet function tests to predict patients' clinical response to aspirin or clopidogrel have generated contradictory results. At this time, there is no standardized definition for resistance to antiplatelet therapy, and the appropriate treatment of patients who are hyporesponsive to these agents is not known. Although such tests have a role in research, their place in guiding therapy remains to be established, and prospective trials are urgently needed. The ideal platelet function test for clinical practice would be rapid, easy-to-use, inexpensive, and reliable.

The problem of persistent platelet activation in acute coronary syndromes and following percutaneous coronary intervention.

Platelets play a central role in the atherosclerotic inflammatory response, thrombotic vascular occlusion, microembolization, vasoconstriction, and plaque progression. Persistent platelet activation poses a serious problem among patients with acute coronary syndromes (ACS) and those who have undergone percutaneous coronary intervention (PCI), placing them at risk for ischemic events and subacute stent thrombosis. Patients undergoing PCI are at risk for further ischemic events because of procedure-related platelet activation as well as the inherent persistent platelet hyperreactivity and enhanced thrombin generation associated with ACS. Persistent platelet activation following an acute coronary event and/or PCI supports incorporating antiplatelet strategies into the standard medical management of such patients. In this clinical setting, antiplatelet therapies are capable of improving outcomes. Aspirin, thienopyridines, and glycoprotein IIb/IIIa inhibitors, the 3 major pharmacologic approaches to persistent platelet activation, target various levels of the hemostatic pathways and thrombus formation.

Antiplatelet strategies: evaluating their current role in the setting of acute coronary syndromes.

Numerous clinical trials have established the value of antiplatelet therapies for acute coronary syndromes (ACS). Aspirin (ASA), thienopyridines (i.e., clopidogrel and ticlopidine) and GP IIb/IIIa antagonists comprise the major classes of antiplatelet therapies demonstrated to be of benefit in the treatment of ACS and for the prevention of thrombotic complications of percutaneous coronary intervention (PCI). Clopidogrel is beneficial when administered before and after PCI, and is more effective when combined with either ASA or GP IIb/IIIa inhibitors in preventing post-PCI complications, coronary subacute stent thrombosis, and thrombotic events in general. It is currently unclear whether a higher loading dose of clopidogrel (600 mg) is better than the standard loading dose (300 mg), how long therapy should continue, and which maintenance dose is optimal. The role of the GP IIb/IIIa antagonists in ACS is less clear due to conflicting data from several studies with different patient populations. Currently, it appears that the use of GP IIb/IIIa antagonists might be most beneficial in high-risk ACS patients scheduled to undergo PCI, who demonstrate non-ST-segment elevation myocardial infarction and elevated troponin levels.

Investigating the mechanisms of hyporesponse to antiplatelet approaches.

Hyporesponsiveness, or resistance, to antiplatelet therapy may be a major contributor to poorer outcomes among cardiac patients and may be attributed to an array of mechanisms--both modifiable and unmodifiable. Recent evidence has uncovered clinical, cellular, and genetic factors associated with hyporesponsiveness. Patients with severe acute coronary syndromes (ACS), type 2 diabetes, and increased body mass index appear to be the most at risk for hyporesponsiveness. Addressing modifiable mechanisms may offset hyporesponsiveness, while recognizing unmodifiable mechanisms, such as genetic polymorphisms and diseases that affect response to antiplatelet therapy, may help identify patients who are more likely to be hyporesponsive. Hyporesponsive patients might benefit from different dosing strategies or additional antiplatelet therapies. Trials correlating platelet function test results to clinical outcomes are required. Results from these studies could cause a paradigm shift toward individualized antiplatelet therapy, improving predictability of platelet inhibition, and diminishing the likelihood for hyporesponsiveness.

Clinical considerations with the use of antiplatelet therapy in patients undergoing percutaneous coronary intervention.

Despite the proven benefits of using antiplatelet therapy in patients undergoing percutaneous coronary intervention (PCI), a number of key questions remain to be answered. In recent years, clopidogrel dosing strategies among such patients have evolved considerably, with newer approaches involving loading doses prior to PCI and increases in the time interval and loading dosage in an effort to overcome variable responsiveness/hyporesponsiveness to platelet inhibition. Further, the role of glycoprotein (GP) IIb/IIIa antagonists in elective stenting continues to be defined, with recent evidence suggesting that most appropriate use of these agents is in high-risk patients with elevated troponin levels. There appears to be a relationship between the use of GP IIb/IIIa antagonists with clopidogrel loading and attenuation of early inflammatory and cardiac marker release. Strategies to minimize the chance of late stent thrombosis in patients who receive drug-eluting stents (DES) are also under intense investigation. Among some patients receiving sirolimus and paclitaxel DES, current standard long-term antiplatelet strategies may be insufficient. Patient nonadherence to treatment and premature discontinuation and underutilization of antiplatelet therapies by physicians remain important clinical problems with potentially dire consequences.

The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin.

The novel thienopyridine prodrug prasugrel, a platelet P2Y(12) ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation, there are few data on the direct effects of the prasugrel's active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates, platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent inhibition of these ADP-stimulated thrombo-inflammatory markers. These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC(50) values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y(12)-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y(12). In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y(12)-mediated up-regulation of thrombo-inflammatory markers of platelet activation. This inhibition is enhanced in the presence other blood cells and calcium, but not aspirin.

Measuring antiplatelet drug effects in the laboratory.

This review discusses the advantages and disadvantages of currently available tests for the monitoring of antiplatelet therapy (especially aspirin and clopidogrel). Many tests of platelet function are now available for clinical use, and some of these tests have been shown to predict clinical outcomes after antiplatelet therapy. However, in most of these studies, the number of major adverse clinical events was low. No published studies address the clinical effectiveness of altering therapy based on the results of monitoring antiplatelet therapy. Therefore, the correct treatment, if any, of "resistance" to antiplatelet therapy is unknown and, other than in research trials, monitoring of antiplatelet therapy in patients is not generally recommended. A clinically meaningful definition of "resistance" to antiplatelet drugs needs to be developed, based on data linking drug-dependent laboratory tests to clinical outcomes in patients.

'Postconditioning' the human heart: multiple balloon inflations during primary angioplasty may confer cardioprotection.

Growing evidence from experimental models suggests that relief of myocardial ischemia in a stuttering manner (i.e., 'postconditioning' [PostC] with brief cycles of reperfusion-reocclusion) limits infarct size. However, the potential clinical efficacy of PostC has, to date,been largely unexplored. Using a retrospective study design, our aim was to test the hypothesis that creatine kinase release (CK: clinical surrogate of infarct size) would be attenuated in ST-segment elevation myocardial infarction (STEMI) patients requiring multiple balloon inflations-deflations during primary angioplasty versus STEMI patients who received minimal balloon inflations and/or direct stenting. To investigate this concept, we reviewed the records of all STEMI patients with single vessel occlusion who presented to our institution from November 2004 - April 2006 for primary angioplasty. Exclusion criteria were: previous MI, cardiogenic shock, patients resuscitated from cardiac arrest, or pre-infarct angina. Patients were prospectively divided into two subsets: those receiving 1-3 balloon inflations (considered the minimum range to achieve patency and stent placement) versus those in whom 4 or more inflations were applied. Peak CK release was significantly lower in patients requiring > or =4 versus 1-3 inflations (1655 versus 2272 IU/L; p<0.05), an outcome consistent with the concept that relief of sustained ischemia in a stuttered manner (analogous to postconditioning) may evoke cardioprotection in the clinical setting.

Current options in platelet function testing.

The variable response to antiplatelet therapy has led to the use of platelet function tests to monitor the effects of antiplatelet drugs in cardiovascular diseases. The goal is to guide antiplatelet therapy to the optimal dose for the prevention or treatment of thrombosis while minimizing hemorrhagic side effects. The bleeding time is no longer recommended for use because of its nonspecificity and lack of clinical correlations. The current de facto "gold standard" test of platelet function is turbidometric platelet aggregometry. Although this method has been successful in measuring the aggregation of platelets in a glycoprotein (GP) IIb/IIIa (integrin alpha(IIb)beta(3))-dependent manner, it has several limitations, including poor reproducibility, high sample volume, requirement for sample preparation, length of assay time, requirement for a skilled technician, and cost. Therefore, new options for platelet function testing have been developed to address these disadvantages and to meet the need for point-of-care testing that can be performed at or near a patient's bedside without requiring a high degree of technical expertise. The new tests include VerifyNow (Accumetrics, San Diego, CA); Plateletworks (Helena Laboratories, Beaumont, TX); Thrombelastograph PlateletMapping System (Haemoscope Corporation, Niles, IL); Impact cone and plate(let) analyzer (DiaMed, Cressier, Switzerland); and Platelet Function Analyzer 100 (PFA-100; Dade Behring, Newark, DE). In patients treated with antiplatelet drugs, the degree of platelet inhibition, as determined by several of these new platelet function assays, has been shown to predict major adverse cardiac events.

Residual arachidonic acid-induced platelet activation via an adenosine diphosphate-dependent but cyclooxygenase-1- and cyclooxygenase-2-independent pathway: a 700-patient study of aspirin resistance.

BACKGROUND: Thrombotic events still occur in aspirin-treated patients with coronary artery disease. METHODS AND RESULTS: To better understand aspirin "resistance," serum thromboxane B2 (TXB2) and flow cytometric measures of arachidonic acid-induced platelet activation (before and after the ex vivo addition of aspirin and indomethacin) were analyzed in 700 consecutive aspirin-treated patients undergoing cardiac catheterization. In 680 of 682 evaluable patients, serum TXB2 concentrations were reduced compared with nonaspirinated healthy donors. Twelve patients had serum TXB2 that was lower than nonaspirinated healthy donors but >10 ng/mL. Arachidonic acid stimulated greater platelet activation in patients with high serum TXB2 (>10 ng/mL) than in patients with low serum TXB2. Addition of ex vivo aspirin reduced arachidonic acid-induced platelet activation to similar levels regardless of serum TXB2 concentrations, which suggests that patients with high residual serum TXB2 concentrations were either noncompliant or underdosed with aspirin. Among the remaining 98% of patients, ex vivo administration of either aspirin or indomethacin failed to prevent platelet activation across all degrees of arachidonic acid-induced platelet activation and aspirin doses. Although the patients were not randomized with respect to clopidogrel treatment, multivariate analysis showed that arachidonic acid-induced platelet activation was less in patients receiving clopidogrel. CONCLUSIONS: There is a residual arachidonic acid-induced platelet activation in aspirin-treated patients that (1) is caused by underdosing and/or noncompliance in only approximately 2% of patients and (2) in the remaining patients, occurs via a cyclooxygenase-1 and cyclooxygenase-2 independent pathway, in direct proportion to the degree of baseline platelet activation, and is mediated in part by adenosine diphosphate-induced platelet activation.

Application of flow cytometry to platelet disorders.

Flow cytometry is a powerful and versatile tool that can be used to yield definitive information regarding the phenotypic status of platelets. The method provides a quantitative assessment of the physical and antigenic properties of platelets (e.g., surface expression of receptors, bound ligands, components of granules, or interactions of platelets with other platelets, other blood cells, or components of the plasma coagulation system), thereby facilitating the diagnosis of inherited or acquired platelet disorders (e.g., Bernard-Soulier syndrome, Glanzmann thrombasthenia, storage pool disease), the pathological activation of platelets (e.g., in the setting of acute coronary syndromes, cerebrovascular ischemia, peripheral vascular disease, cardiopulmonary bypass), and changes in the ability of platelets to activate via specific stimuli (e.g., efficacy of antiplatelet therapies). Accordingly, this review summarizes the key technical and methodologic components of flow cytometric analysis of platelets, as well as specific examples of its application to diagnosis and patient care.

The role of platelet inhibition in the drug-eluting stent era.

The use of antiplatelet agents in the management of patients undergoing percutaneous coronary interventions involves the administration of aspirin, thienopyridines, and in high-risk patients, GPIIb-IIIa antagonists. Drug-eluting stents now account for greater than 50% of stenting procedures. This review focuses on the limited available data describing the use of antiplatelet agents in patients undergoing drug-eluting stent implantation.

Release of soluble CD40L from platelets is regulated by glycoprotein IIb/IIIa and actin polymerization.

OBJECTIVES: The purpose of this study was to examine the effects of glycoprotein (GP) IIb/IIIa antagonists (abciximab, eptifibatide, and tirofiban) and other inhibitors on translocation of CD40L from intraplatelet stores to the platelet surface and on the release of soluble CD40L (sCD40L) from platelets. BACKGROUND: CD40L is a proinflammatory and prothrombotic ligand in the tumor necrosis factor family. METHODS: Platelet surface CD40L was measured by flow cytometry, and sCD40L was measured by enzyme-linked immunosorbent assay. RESULTS: Translocation of CD40L from intraplatelet stores to the platelet surface was not inhibited by GP IIb/IIIa antagonists. However, release of sCD40L from the surface of activated platelets was inhibited by GP IIb/IIIa antagonists in a dose-dependent manner, in concert with inhibition of PAC1 binding to platelets (a surrogate marker for fibrinogen binding). Release of sCD40L from activated platelets was also markedly reduced in Glanzmann platelets (deficient in GP IIb/IIIa). Ethylenediaminetetraacetic acid was an effective inhibitor of sCD40L release, but only when added before platelet activation. Both cytochalasin D (an inhibitor of actin polymerization) and GM6001 (an inhibitor of matrix metalloproteinases [MMPs]) inhibited the release of sCD40L from platelets when added before, as well as 3 min after, platelet activation. However, neither cytochalasin D nor GM6001 affected translocation of CD40L to the platelet surface. CONCLUSIONS: The GP IIb/IIIa antagonists inhibit release of sCD40L from activated platelets. Release of sCD40L from platelets is regulated, at least in part, by GP IIb/IIIa, actin polymerization, and an MMP inhibitor-sensitive pathway. In addition to their well-characterized inhibition of platelet aggregation, GP IIb/IIIa antagonists may obviate the proinflammatory and prothrombotic effects of sCD40L.

Elevated leukocyte count and adverse hospital events in patients with acute coronary syndromes: findings from the Global Registry of Acute Coronary Events (GRACE).

OBJECTIVE: To examine the association between elevated leukocyte count and hospital mortality and heart failure in patients enrolled in the multinational, observational Global Registry of Acute Coronary Events (GRACE). BACKGROUND: Elevated leukocyte count is associated with adverse hospital outcomes in patients presenting with acute myocardial infarction (AMI). The association of this prognostic factor with hospital mortality and heart failure in patients with other acute coronary syndromes (ACS) is unclear. METHODS: We examined the association between admission leukocyte count and hospital mortality and heart failure in 8269 patients presenting with an ACS. This association was examined separately in patients with ST-segment elevation AMI, non-ST-segment elevation AMI, and unstable angina. Leukocyte count was divided into 4 mutually exclusive groups (Q): Q1 <6000, Q2 = 6000-9999, Q3 = 10,000-11,999, Q4 >12,000. Multiple logistic regression analysis was performed to examine the association between elevated leukocyte count and hospital events while accounting for the simultaneous effect of several potentially confounding variables. RESULTS: Increasing leukocyte count was significantly associated with hospital death (adjusted odds ratio [OR] 2.8, 95% CI 2.1-3.6 for Q4 compared to Q2 [normal range]) and heart failure (OR 2.7, 95% CI 2.2-3.4) for patients presenting with ACS. This association was seen in patients with ST-segment elevation AMI (OR for hospital death 3.2, 95% CI 2.1-4.7; OR for heart failure 2.4, 95% CI 1.8-3.3), non-ST-segment elevation AMI (OR for hospital death 1.9, 95% CI 1.2-3.0; OR for heart failure 1.7, 95% CI 1.1-2.5), or unstable angina (OR for hospital death 2.8, 95% CI 1.4-5.5; OR for heart failure 2.0, 95% CI 0.9-4.4). CONCLUSION: In men and women of all ages with the spectrum of ACS, initial leukocyte count is an independent predictor of hospital death and the development of heart failure.