Flow cytometric detection and procoagulant activity of circulating canine platelet-derived microparticles

Sarah E. Helmond Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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James L. Catalfamo Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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Marjory B. Brooks Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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Abstract

Objective—To measure platelet membrane–derived microparticle (PMP) content and thrombin-generating capacity of canine plasma subjected to specific processing and storage conditions.

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

Procedures—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 and Clinical Relevance—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.

Abstract

Objective—To measure platelet membrane–derived microparticle (PMP) content and thrombin-generating capacity of canine plasma subjected to specific processing and storage conditions.

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

Procedures—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 and Clinical Relevance—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.

Contributor Notes

Dr. Helmond's present address is Sage Centers for Veterinary Specialty and Emergency Care, 1410 Monument Blvd, Ste 100, Concord, CA 94520.

Supported in part by the College of Veterinary Medicine Clinical Fellowship Program.

Address correspondence to Dr. Brooks (mbb9@cornell.edu).
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