Flow cytometric detection and procoagulant activity of circulating canine platelet-derived microparticles.
OBJECTIVE
To measure platelet membrane-derived microparticle (PMP) content and thrombin-generating capacity of canine plasma subjected to specific processing and storage conditions.


METHODS
31 clinically normal dogs (19 males and 12 females).


METHODS
Citrate-anticoagulated blood samples obtained from each dog were centrifuged at 2,500 × g to isolate platelet-poor plasma (PPP), then PPP was centrifuged at 21,000 × g to isolate microparticle-free plasma (MPF) and microparticle-enriched plasma (MPEP). Whole blood and paired samples of fresh and frozen-thawed PPP, MPF, and MPEP were dual labeled for flow cytometric detection of membrane CD61 (constitutive platelet antigen) and annexin V (indicating phosphatidylserine externalization). Platelets and PMPs were enumerated with fluorescent, size-calibrated beads. Thrombin generation in fresh and frozen-thawed PPP, MPF, and MPEP was measured via kinetic fluorometric assays configured with low tissue factor and low phospholipid concentrations.


RESULTS
Initial centrifugation yielded PPP with < 0.5% the platelets of whole blood, with median counts of 413 PMPs/μL for males and 711 PMPs/μL for females. Sequential centrifugation resulted in a 10-fold concentration of PMPs in MPEP and virtually depleted PMPs from MPF. Thrombin generation depended on PMP content, with median endogenous thrombin potential of 0, 893, and 3,650 nmol•min for MPF, PPP, and MPEP, respectively. Freeze-thaw cycling caused significant increases in PMP counts and phosphatidylserine externalization.


CONCLUSIONS
Canine PMPs were major determinants of thrombin-generating capacity; preanalytic variables influenced plasma PMP content. Processing conditions described here may provide a basis for characterization of PMPs in clinical studies of thrombosis in dogs.
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850, USA.
Sarah E
SE
Helmond
James L
JL
Catalfamo
Marjory B
MB
Brooks
D000818:Animals; D001774:Blood Chemical Analysis / Q000379:methods* / Q000662:veterinary; D001792:Blood Platelets / Q000166:cytology*; D001800:Blood Specimen Collection / Q000379:methods* / Q000662:veterinary; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D004285:Dogs; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000662:veterinary; D008297:Male; D010718:Phosphatidylserines / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D010718:Phosphatidylserines; D013917:Thrombin