Flow cytometric analysis of microparticle phenotype and their role in thrombin generation.
BACKGROUND
Microparticles may be generated from a number of cell types and are known to play a role in haemostasis by a variety of mechanisms. We investigated the role of platelet, red cell, and leucocyte-derived microparticles in the measurement of thrombin generation.


METHODS
Four parameters of thrombin generation (the endogenous thrombin potential (ETP), lag time, time to peak, peak height) and microparticle content was determined in 35 plasma samples from normal individuals pre and post filtration to remove microparticles. Immunofluorescent flow cytometry was used to identify and enumerate platelet, leucocyte, monocyte and red cell derived microparticles in plasma samples based on the expression of CD42b, CD45, CD15, and Glycophorin A respectively. Expression of phosphatidylserine and tissue factor by microparticles was determined by Annexin V and anti CD142 binding. The pre and post filtration results were compared.


RESULTS
There was a significant decrease in ETP and Peak Height, and an increase in the time to peak post filtration (P < 0.001). A significant decrease in the number of CD42+, CD45+, CD15+, CD142+, and Annexin V+ microparticles was also observed. The change in CD42b+ microparticles correlated highly with the change in Annexin V+ microparticles (r = 0.68). Whilst the change in ETP correlated best with the change in CD15+ microparticles (r = 0.45) and the change in time to peak correlated with the change in Annexin V binding (r = 0.52) (P < 0.01).


CONCLUSIONS
The presence of micropartcles in plasma significantly affects thrombin generation.
Macey
M G
MG
Department of Haematology, The Royal London Hospital, Barts and the London NHS Trust, Pathology and Pharmacy Building, Whitechapel, London E1 2ES. marion.macey@bartsandthelondon.nhs.uk
Enniks
N
N
Bevan
S
S
D000328:Adult; D055252:Cell-Derived Microparticles / Q000235:genetics* / Q000502:physiology; D005260:Female; D005434:Flow Cytometry; D006021:Glycophorins / Q000032:analysis; D006801:Humans; D016130:Immunophenotyping; D017493:Leukocyte Common Antigens / Q000097:blood; D016256:Lewis X Antigen / Q000097:blood; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000032:analysis; D013917:Thrombin / Q000096:biosynthesis* / Q000235:genetics; D055815:Young Adult
D006021:Glycophorins; D016256:Lewis X Antigen; D019038:Platelet Glycoprotein GPIb-IX Complex; D017493:Leukocyte Common Antigens; D013917:Thrombin