Douglas J. Christie

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.

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.

The effect of antiplatelet drugs, heparin, and preanalytical variables on platelet function detected by the platelet function analyzer (PFA-100).

The platelet function analyzer (PFA)-100® is a newly developed instrument that provides a rapid, in vitro, quantitative measurement of platelet adhesion and aggregation in whole blood flowing through a small aperture under high shear conditions. Thirty patients undergoing percutaneous transluminal coronary angioplasty (PTCA) and ten normal in dividuals were included in this study. In vitro and in vivo studies were conducted to discern the effect of combinations of antiplatelet drugs (aspirin, ticlopidine, abciximab) and heparin on the performance of the device as well as the effects of preanalytical variables, such as method of sample collection and ex vivo anticoagulants. Studies were also conducted ex amining the effect of aperture size (standard 150 μm vs. smaller 120 μm) on the ability of the device to detect the effect of antiplatelet drugs. There was no difference in mean PFA- 100® closure time with citrate versus PPACK anticoagulants or with venipuncture vs. sheath sampling. Closure times did not vary with heparin administration. Closure times were slightly longer for patients taking aspirin plus ticlopidine compared to aspirin alone (p = NS). In contrast adenosine disphosphate (ADP) induced platelet aggregation was significantly less in patients that took aspirin plus ticlopidine vs. aspirin alone (p = .0005). In vitro, there was a dose-dependent increase in closure time for both aperture sizes with increasing abciximab concen tration. Although both cartridges showed infinite closure times at an abciximab concentration of 2.25 μg/mL, there was a slight benefit to using the 120 μm aperture cartridges at abcix imab concentrations of 1.75 to 2.0 μg/mL. In ten patients who were followed during abciximab therapy to assess the effect of aperture size, the PFA-100® was able to detect in vivo platelet inhibition by abciximab, but detection of recovery from abcix imab-induced platelet dysfunction was slightly better for the PFA-100® with the 120 μm aperture compared to the standard 150 μm aperture collagen/ADP cartridge.

COMPARISON OF FOUR COMMERCIAL CITRATE BLOOD COLLECTION SYSTEMS FOR PLATELET FUNCTION ANALYSIS BY THE PFA-100TM SYSTEM1.

The PFA-l OOTMsystem is a recently developed in vitro test system for measurement of platelet fi.mction in whole blood anticoagulated with sodium citrate. The test system is based on the principle originally described by Kratzer and Born (l), and has been characterized elsewhere (2,3). In this system, a motor-driven syringe aspirates blood samples under steady-flow conditions through a small aperture (approximately 150 pm in diameter) cut into a membrane placed in the test cartridge. The membrane is coated with type I collagen and either epinephrine (Col/Epi) or adenosine-5’-diphosphate (Col/ADP). Progressively, platelets attach to the area surrounding the aperture and eventually form a platelet plug that occludes it. The instrument records the time necessary for the occlusion of the aperture, defined as the Closure Time (CT), which is indicative of platelet function in the blood sample.

Hypersensitivity of platelets to adenosine diphosphate in patients with stable cardiovascular disease predicts major adverse events despite antiplatelet therapy.

Enhanced platelet activity correlates with early markers of myocardial damage in patients with cardiovascular disease. However, the extent to which enhanced platelet function signals subsequent adverse clinical outcomes in patients with cardiovascular disease is unknown. Blood from patients with stable cardiovascular disease receiving aspirin (325 mg/day) as the only antiplatelet therapy was tested for closure time (CT) with the Dade® PFA-100® Platelet Function Analyzer system collagen/adenosine diphosphate (ADP) [CADP] cartridge and platelet aggregometry using 10 µM ADP. This study intentionally focused on those patients defined as aspirin sensitive by previously established criteria of arachidonic acid- and ADP-induced platelet aggregometry, and separately by collagen/epinephrine (CEPI) CT using the PFA-100®. Follow up averaged 22 months for the adverse clinical events of death, myocardial infarction or cerebrovascular accident. For aspirin sensitivity determined by aggregometry, patients with CADP CT < 90 seconds (125/296 = 42.2%) had a composite endpoint rate of 19.2% (24/125), while those with CADP CT ≥90 seconds (171/296 = 57.8%) had an endpoint rate of 5.3% (9/171). Patients with CADP CT <90 seconds had a relative risk (RR) of 3.65 (95% CI.: 1.76–7.57) for recurrent events and 6.56 (95% CI.: 1.93–22.35) for death compared to patients with CADP CT ≥ 90s. Nearly identical results were obtained when patients were categorized as aspirin sensitive by CEPI CT. Platelet aggregometry with 10 µM ADP yielded no significant RR for the selected outcomes. Platelet function testing using the PFA-100® system appears to identify a subgroup of stable cardiovascular disease patients with increased risk of major adverse events that is associated with hypersensitivity to ADP, regardless of apparently effective aspirin therapy.

Comparison of optical and mechanical clot detection for routine coagulation testing in a large volume clinical laboratory.

Hemostasis instrumentation has rapidly advanced and laboratories are demanding fully automated coagulation systems. Two distinct technological families exist based on optical and mechanical clot detection methodologies. Until now, there have been no comprehensive studies to determine whether one methodology is superior to the other. In order to answer this question, we conducted a large clinical study performing standard coagulation testing on more than 2000 clinical samples randomly chosen from a high-volume laboratory in a tertiary care hospital. Results demonstrated that photo-optical clot detection and electro-mechanical detection systems were highly correlated (r-squared values ≥ 0.96 for all assays) Correlation between the two clot detection systems was maintained even when measuring turbid samples (r-squared values ≥ 0.98 for all assays).

Evaluation of the Stratus CS Acute Care D-dimer assay (DDMR) using the Stratus CS STAT Fluorometric Analyzer: a prospective multisite study for exclusion of pulmonary embolism and deep vein thrombosis.

BACKGROUND D-dimer testing is an integral part of the diagnostic algorithm in excluding patients with venous thromboembolism. In this study, we prospectively evaluated the Stratus DDMR D-dimer test in patients suspected of pulmonary embolism (PE) and deep vein thrombosis (DVT). METHODS Patients suspected of venous thromboembolism were prospectively enrolled at four different clinical sites, with sodium citrate and lithium heparin plasma was tested using the DDMR D-dimer test on the Stratus CS analyzer. RESULTS 1,012 patients were enrolled for analysis, with 85/603 (14.1%) patients with PE and 80/443 (18.1%) with DVT, and four of the patients (0.4%) with PE and DVT. For the samples collected in 3.2% sodium citrate, the DDMR method had a sensitivity, specificity, and negative predictive value for VTE of 98.0%, 38.1%, and 99.1%, respectively. For the samples collected in lithium heparin, the DDMR method had a sensitivity, specificity, and negative predictive value (NPV) for VTE of 98.9%, 28.8%, and 99.4%, respectively. In PE, DDMR testing on citrate plasma had a sensitivity, specificity, and NPV of 98.8%, 39.5%, and 99.6%, respectively, while heparin samples had a sensitivity, specificity, and NPV for PE of 98.0%, 28.4%, and 99.1%, respectively. In DVT, citrate plasma had a sensitivity, specificity, and NPV for DVT of 97.5%, 32.0%, and 98.3%, respectively, while heparin samples had a sensitivity, specificity, and NPV for DVT of 100%, 27.8%, and 100%, respectively. CONCLUSION The Stratus CS DDMR D-dimer can be used in those patients with non-high clinical pre-test probability for the exclusion of PE.

Response to Letter Regarding Article, “Association of Cyclooxygenase-1–Dependent and –Independent Platelet Function Assays With Adverse Clinical Outcomes in Aspirin-Treated Patients Presenting for Cardiac Catheterization”

Our conclusion that “poor clinical outcomes of aspirin-treated patients is due in part to incomplete cyclooxygenase-1 (COX-1) inhibition, but is also due in part to COX-1–independent platelet hyperreactivity” is based on the independent association of both high residual serum thromboxane B2 (TXB2) and shortened platelet function analyzer (PFA-100) collagen/ADP closure time (CADP CT; indicative of COX-1–independent platelet hyperreactivity) with adverse outcomes in aspirin-treated patients.1 These findings help define factors that contribute to the risk for adverse events in …