Lori A. Krueger

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.

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

Summary.  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.

Immunophenotypic Analysis of Platelets

Platelets are the smallest cellular component in the peripheral circulation. Their primary role is maintenance of hemostasis. The evaluation of platelets by flow cytometry has proven beneficial in the investigation of many disease states, including inherited defects, cardiovascular disease, stroke, and many other inflammatory processes. In the absence of an added exogenous platelet agonist, the activation state of in vivo circulating platelets can be measured. Inclusion of an exogenous agonist in the assay enables analysis of the reactivity of circulating platelets in vitro. This unit describes protocols to evaluate platelet surface glycoproteins, platelet activation status, and platelet responsiveness to agonist. Detailed discussion of appropriate sample handling, reagent preparation, flow cytometric set‐up, and data interpretation are provided for three independent assays.

Whole Blood Analysis of Leukocyte‐Platelet Aggregates

In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. This leukocyte-platelet aggregate formation is initiated primarily through platelet surface expression of P-selectin (CD62P), following activation-dependent degranulation of α-granules, binding to its constitutively expressed counter-receptor, P-selectin glycoprotein ligand 1 (PSGL-1), on leukocytes. Monocyte-platelet aggregates are a more sensitive marker of platelet activation than platelet surface P-selectin. Detection of leukocyte-platelet aggregates is relatively simple by whole-blood flow cytometry. Light scatter and at least one leukocyte-specific antibody are used to gate the desired population, and the presence of associated platelets is detected by immunostaining for abundant platelet-specific markers.

UNIT 6.15 Whole Blood Analysis of Leukocyte-Platelet Aggregates

In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. The platelet binding is initiated primarily through platelet surface expression of P‐selectin (CD62P) following activation‐dependent degranulation. The levels of P‐selectin involved can be low enough to make direct measurement difficult, but detection of leukocyte‐platelet aggregates is relatively simply by whole‐blood flow cytometry. Light scatter and at least one leukocyte‐specific antibody are used to gate the desired population, and the presence of associated platelets is detected by immunostaining for abundant platelet‐specific markers.

Laser scanning cytometry: a novel method for the detection of platelet--endothelial cell adhesion

BACKGROUND Adherence of platelets to endothelial cells may be a significant event in the development of vascular thrombosis. Existing models, which examine platelet-endothelial cell interactions, compromise endothelial cell integrity or use radioactivity to identify platelets that adhere to endothelial cells. We report a novel method for in vitro detection of platelet-endothelial cell adhesion that allows endothelial cells to remain as an intact monolayer and for visualization of individual platelets. METHODS Fluorescently labeled platelets were incubated with a confluent monolayer of endothelial cells. Laser scanning cytometry (LSC) identified platelets bound to endothelial cells based on their fluorescent signals. RESULTS LSC detection of platelets reliably reproduced well-described findings of thrombin-induced platelet-endothelial cell adhesion. Results demonstrating reduced adhesion with a glycoprotein IIb-IIIa-specific blocking monoclonal antibody confirmed the specificity of the LSC detection of platelet-endothelial cell adhesion. CONCLUSIONS LSC is a novel method for detecting platelet--endothelial cell adhesion. Its advantages over other methods are: (a) endothelial cells remain undisturbed and adherent throughout; (b) the ability to detect individual bound platelets and subpopulations; (c) the ability to store images and slides and then relocate, revisualize, and reanalyze individual cells or cell populations of interest; and (d) no radioactivity.