The basis examination of leukocyte-platelet aggregates with CD45 gating as a novel platelet activation marker.
BACKGROUND
Platelet activation in circulation is considered to be associated with thrombosis and inflammation; thus, sensitive and easy-to-use markers are necessary. In this study, we established a simple and rapid protocol to clinically examine leukocyte-platelet aggregate formation associated with activated platelets in circulation.


METHODS
Whole blood was stained with PC5-conjugated anti-CD45 monoclonal antibody and fluorescent isothiocyanate-conjugated anti-CD41 monoclonal antibody for leukocyte-platelet aggregate analysis. For platelet activation, 5 μm thrombin receptor-activated peptide (TRAP) or 2 μg/mL collagen was added. Samples were analyzed by EPICS XL (Beckman Coulter, Miami, FL, USA). Monocytes, neutrophils, and lymphocytes were gated based on differences in CD45 fluorescence intensity and side scatter. For each gate, the percentage (%) of platelets expressing CD41 was analyzed. Same drawing sample was stained with anti-CD62P monoclonal antibody. Platelet CD62P expression was then analyzed with gating for platelet cell population.


RESULTS
We analyzed leukocyte-platelet aggregates and platelet CD62P expression in 18 healthy individuals. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, increased when platelets were activated by platelet agonists. Monocyte-platelet aggregates and neutrophil-platelet aggregates also increased over time with mild platelet activation.


CONCLUSIONS
Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, appear to be a sensitive marker of platelet activation in circulation.
Graduate School of Health Sciences, Hokkaido University, Sapporo, Hokkaido, Japan.
A
A
Nagasawa
K
K
Matsuno
S
S
Tamura
K
K
Hayasaka
C
C
Shimizu
T
T
Moriyama
D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D003094:Collagen / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism*; D016131:Lymphocyte Subsets / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D019007:P-Selectin / Q000378:metabolism; D015539:Platelet Activation / Q000502:physiology*; D018179:Receptors, Thrombin / Q000378:metabolism; D013997:Time Factors; D055815:Young Adult
D015415:Biomarkers; D019007:P-Selectin; D018179:Receptors, Thrombin; D003094:Collagen; D017493:Leukocyte Common Antigens
Monocyte-platelet aggregates; flow cytometry; neutrophil-platelet aggregates; platelet CD62P expression; platelet activation