Mark I. Furman

Increased Platelet Reactivity and Circulating Monocyte-Platelet Aggregates in Patients with Stable Coronary Artery Disease

OBJECTIVES We sought to examine whether patients with stable coronary artery disease (CAD) have increased platelet reactivity and an enhanced propensity to form monocyte-platelet aggregates. BACKGROUND Platelet-dependent thrombosis and leukocyte infiltration into the vessel wall are characteristic cellular events seen in atherosclerosis. METHODS Anticoagulated peripheral venous blood from 19 patients with stable CAD and 19 normal control subjects was incubated with or without various platelet agonists and analyzed by whole blood flow cytometry. RESULTS Circulating degranulated platelets were increased in patients with CAD compared with control subjects (mean [+/- SEM] percent P-selectin-positive platelets: 2.1 +/- 0.2 vs. 1.5 +/- 0.2, p < 0.01) and were more reactive to stimulation with 1 micromol/liter of adenosine diphosphate (ADP) (28.7 +/- 3.9 vs. 16.1 +/- 2.2, p < 0.01), 1 micromol/liter of ADP/epinephrine (51.4 +/- 4.6 vs. 37.5 +/- 3.8, p < 0.05) or 5 micromol/liter of thrombin receptor agonist peptide (TRAP) (65.7 +/- 6.8 vs. 20.2 +/- 5.1, p < 0.01). Patients with stable CAD also had increased circulating monocyte-platelet aggregates compared with control subjects (percent platelet-positive monocytes: 15.3 +/- 3.0 vs. 6.3 +/- 0.9, p < 0.01). Furthermore, patients with stable CAD formed more monocyte-platelet aggregates than did control subjects when their whole blood was stimulated with 1 micromol/liter of ADP (50.4 +/- 4.5 vs. 28.1 +/- 5.3, p < 0.01), 1 micromol/liter of ADP/epinephrine (60.7 +/- 4.3 vs. 48.0 +/- 4.8, p < 0.05) or 5 micromol/liter of TRAP (67.6 +/- 5.7 vs. 34.3 +/- 7.0, p < 0.01). CONCLUSIONS Patients with stable CAD have circulating activated platelets, circulating monocyte-platelet aggregates, increased platelet reactivity and an increased propensity to form monocyte-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.

Platelet GP IIIa Pl(A) polymorphisms display different sensitivities to agonists

BACKGROUND Both inherited predisposition and platelet hyperreactivity have been associated with ischemic coronary events, but mechanisms that support genetic differences among platelets from different subjects are generally lacking. Associations between the platelet Pl(A2) polymorphism of GP IIIa and coronary syndromes raise the question as to whether this inherited variation may contribute to platelet hyperreactivity. METHODS AND RESULTS In this study, we characterized functional parameters in platelets from healthy donors with the Pl(A) (HPA-1) polymorphism, a Leu (Pl(A1)) to Pro (Pl(A2)) substitution at position 33 of the GP IIIa subunit of the platelet GP IIb/IIIa receptor (integrin alpha(IIb)beta(3)). We studied 56 normal donors (20 Pl(A1,A1), 20 Pl(A1,A2), and 16 Pl(A2,A2)). Compared with Pl(A1,A1) platelets, Pl(A2)-positive platelets showed a gene dosage effect for significantly greater surface-expressed P-selectin, GP IIb/IIIa-bound fibrinogen, and activated GP IIb/IIIa in response to low-dose ADP. Surface expression of GP IIb/IIIa was similar in resting platelets of all 3 genotypes but was significantly greater on Pl(A2,A2) platelets after ADP stimulation (P=0.003 versus Pl(A1,A1); P=0.03 versus Pl(A1,A2)). Pl(A1,A2) platelets were more sensitive to inhibition of aggregation by pharmacologically relevant concentrations of aspirin and abciximab. CONCLUSIONS Pl(A2)-positive platelets displayed a lower threshold for activation, and platelets heterozygous for Pl(A) alleles showed increased sensitivity to 2 antiplatelet drugs. These in vitro platelet studies may have relevance for in vivo thrombotic conditions.

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 ≈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.

Twenty-two year (1975 to 1997) trends in the incidence, in-hospital and long-term case fatality rates from initial q-wave and non-q-wave myocardial infarction: a multi-hospital, community-wide perspective

OBJECTIVES The goal of this study was to examine long-term trends in the incidence, in-hospital and long-term mortality patterns in patients with an initial non-Q-wave myocardial infarction (NQWMI) as compared with those with an initial Q-wave myocardial infarction (QWMI). BACKGROUND Limited data are available describing trends in the incidence and mortality from an initial QWMI and NQWMI from a multi-hospital community-wide perspective. METHODS Our study was an observational study of 5,832 metropolitan Worcester, Massachusetts residents (1990 census = 437,000) hospitalized with validated initial acute MI in all greater Worcester hospitals during 11 annual periods between 1975 and 1997. RESULTS The incidence of QWMI progressively decreased between 1975/78 (incidence rate = 171/100,000 population) and 1997 (101/100,000 population). In contrast, the incidence of NQWMI progressively increased between 1975/78 (62/100,000 population) and 1997 (131/100,000 population). Hospital death rates were 19.5% for patients with QWMI and 12.5% for those with NQWMI. After controlling for various covariates, patients with QWMI remained at significantly increased risk for hospital mortality (adjusted odds ratio = 1.63; 95% confidence interval: 1.35, 1.97). While the hospital mortality of QWMI has progressively declined over time (1975/78 = 24%; 1997 = 14%), the in-hospital mortality for NQWMI has remained the same (1975/78 = 12%; 1997 = 12%). These trends remained after adjusting for potentially confounding prognostic factors. The multivariable adjusted two-year mortality after hospital discharge declined over time for patients with QWMI and NQWMI. CONCLUSIONS Despite impressive declines in the incidence, in-hospital and long-term mortality associated with QWMI, NQWMI is increasing in frequency and has the same in-hospital mortality now as it did 22 years ago.

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 ≥3 days) patients. Platelet function was tested by (1) serum thromboxane B2; (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 B2 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 B2 >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 B2 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.

Clinical study: myocardial infarctionTwenty-two year (1975 to 1997) trends in the incidence, in-hospital and long-term case fatality rates from initial q-wave and non-q-wave myocardial infarction: a multi-hospital, community-wide perspective☆

OBJECTIVES The goal of this study was to examine long-term trends in the incidence, in-hospital and long-term mortality patterns in patients with an initial non-Q-wave myocardial infarction (NQWMI) as compared with those with an initial Q-wave myocardial infarction (QWMI). BACKGROUND Limited data are available describing trends in the incidence and mortality from an initial QWMI and NQWMI from a multi-hospital community-wide perspective. METHODS Our study was an observational study of 5,832 metropolitan Worcester, Massachusetts residents (1990 census = 437,000) hospitalized with validated initial acute MI in all greater Worcester hospitals during 11 annual periods between 1975 and 1997. RESULTS The incidence of QWMI progressively decreased between 1975/78 (incidence rate = 171/100,000 population) and 1997 (101/100,000 population). In contrast, the incidence of NQWMI progressively increased between 1975/78 (62/100,000 population) and 1997 (131/100,000 population). Hospital death rates were 19.5% for patients with QWMI and 12.5% for those with NQWMI. After controlling for various covariates, patients with QWMI remained at significantly increased risk for hospital mortality (adjusted odds ratio = 1.63; 95% confidence interval: 1.35, 1.97). While the hospital mortality of QWMI has progressively declined over time (1975/78 = 24%; 1997 = 14%), the in-hospital mortality for NQWMI has remained the same (1975/78 = 12%; 1997 = 12%). These trends remained after adjusting for potentially confounding prognostic factors. The multivariable adjusted two-year mortality after hospital discharge declined over time for patients with QWMI and NQWMI. CONCLUSIONS Despite impressive declines in the incidence, in-hospital and long-term mortality associated with QWMI, NQWMI is increasing in frequency and has the same in-hospital mortality now as it did 22 years ago.

Mortality following placement of drug-eluting and bare-metal stents for ST-segment elevation acute myocardial infarction in the Global Registry of Acute Coronary Events

AIMS To assess mortality after drug-eluting stent (DES) or bare-metal stent (BMS) for ST-segment elevation myocardial infarction (STEMI). METHODS AND RESULTS In this multinational registry, 5093 STEMI patients received a stent: 1313 (26%) a DES and 3780 (74%) only BMS. Groups differed in baseline characteristics, type, or timing of percutaneous coronary intervention, with a higher baseline risk for patients receiving BMS. Two-year follow-up was available in 55 and 60% of the eligible BMS and DES patients, respectively. Unadjusted mortality was lower during hospitalization, similar for the first 6 months after discharge, and higher from 6 months to 2 years, for DES patients compared with that of BMS patients. Overall, unadjusted 2-year mortality was 5.3 vs. 3.9% for BMS vs. DES patients (P = 0.04). In propensity- and risk-adjusted survival analyses (Cox model), post-discharge mortality was not different up to 6 months (P = 0.21) or 1 year (P = 0.34). Late post-discharge mortality was higher in DES patients from 6 months to 2 years (HR 4.90, P = 0.01) or from 1 to 2 years (HR 7.06, P = 0.02). Similar results were observed when factoring in hospital mortality. CONCLUSION The observation of increased late mortality with DES vs. BMS suggests that DES should probably be avoided in STEMI, until more long-term data become available.

Laboratory markers of platelet activation and their clinical significance

Whole blood flow cytometry is a powerful new laboratory technique for assessment of platelet activation and function. Flow cytometry can be used to measure platelet hyperreactivity, circulating activated platelets, leukocyte-platelet aggregates, and procoagulant platelet-derived microparticles in a number of clinical settings, including acute coronary syndromes, angioplasty, cardiopulmonary bypass, acute cerebrovascular ischemia, peripheral vascular disease, diabetes mellitus, preeclampsia, and Alzheimers disease. Clinical applications of whole blood flow cytometric assays of platelet function in these diseases may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of glycoprotein IIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.

Effect of Elevated Leukocyte Count on In-Hospital Mortality Following Acute Myocardial Infarction *

The peripheral white blood cell count on presentation with acute myocardial infarction directly correlates with short-term in-hospital mortality. This association is independent of other prognostic factors, including extent and size of the acute myocardial infarction.