Helmond Sarah E, Catalfamo James L, Brooks Marjory B
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850, USA.
Am J Vet Res. 2013 Feb;74(2):207-15. doi: 10.2460/ajvr.74.2.207.
To measure platelet membrane-derived microparticle (PMP) content and thrombin-generating capacity of canine plasma subjected to specific processing and storage conditions.
31 clinically normal dogs (19 males and 12 females).
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.
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.
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.
测定经过特定处理和储存条件的犬血浆中血小板膜衍生微粒（PMP）含量及凝血酶生成能力。
31只临床正常犬（19只雄性，12只雌性）。
从每只犬采集的枸橼酸盐抗凝血样以2500×g离心以分离乏血小板血浆（PPP），然后将PPP以21000×g离心以分离无微粒血浆（MPF）和富含微粒血浆（MPEP）。全血以及新鲜和冻融后的PPP、MPF和MPEP配对样本进行双标记，用于流式细胞术检测膜CD61（组成性血小板抗原）和膜联蛋白V（指示磷脂酰丝氨酸外化）。使用荧光、尺寸校准微球对血小板和PMP进行计数。通过配置低组织因子和低磷脂浓度的动力学荧光测定法测量新鲜和冻融后的PPP、MPF和MPEP中的凝血酶生成。
初次离心得到的PPP中血小板数量不到全血的0.5%，雄性的PMP中位数计数为413个/μL，雌性为711个/μL。连续离心导致MPEP中PMP浓度增加10倍，而MPF中的PMP几乎耗尽。凝血酶生成取决于PMP含量，MPF、PPP和MPEP的内源性凝血酶潜力中位数分别为0、893和3650 nmol•min。冻融循环导致PMP计数和磷脂酰丝氨酸外化显著增加。
犬PMP是凝血酶生成能力的主要决定因素；分析前变量影响血浆PMP含量。本文所述的处理条件可为犬血栓形成临床研究中PMP的表征提供基础。
