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 αMβ2, 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° 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 neu