Effects of platelet binding on whole blood flow cytometry assays of monocyte and neutrophil procoagulant activity
Effects of platelet binding on whole blood flow cytometry assays of monocyte and neutrophil procoagulant activity
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DOI：
10.1111/j.1538-7836.2005.01603.x
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发表时间：
2005-11-01
影响因子：
10.4
通讯作者：
Michelson, AD
中科院分区：
文献类型：
--
作者：
Barnard, MR;Linden, MD;Michelson, AD
背景：单核细胞和中性粒细胞最初通过血小板表面p -选择素与白细胞表面p -选择素糖蛋白配体-1 （PSGL-1）的结合与血小板形成异型聚集体。由此产生的细胞内信号传导导致白细胞表面组织因子的表达和白细胞表面Mac-1(整合素α (M) β (2), CD11b/CD18)的激活。单核细胞表面Mac-1的激活依赖性构象变化导致凝血因子Xa （FXa）和/或纤维蛋白原与Mac-1的结合。本研究的目的是建立这些促凝剂活性的全血流细胞测定法，并研究血小板与单核细胞和中性粒细胞结合的影响。方法：用抗CD14 （PECy5）单克隆抗体孵育柠檬酸盐或d - ph - pro -精氨酸-氯甲基酮（PPACK）抗凝全血。CD42a (PE)， fitc偶联试验抗体和激动剂，然后用FACS裂解酶固定。平行制备合适的同型阴性对照。使用BID FACSCalibur分析基于CD14荧光、正向和90度光散射鉴定的中性粒细胞和非中性粒细胞。这些人群进一步被门控为cd42a阳性（血小板结合）和cd42a阴性（血小板无）。收集每个亚群的几何平均荧光和百分比阳性数据，以测量针对CD42a、组织因子、凝血FXa、结合纤维蛋白原、活化Mac-1和CD11b的测试抗体的结合。在研究之前，对6名正常供体进行了补偿控制，这些设置在10名供体研究中一直使用。阴性对照证实了串扰的缺乏，特别是在量化的FITC和PE参数中。结果：生理性激动剂胶原和ADP增加单核细胞-血小板和中性粒细胞-血小板聚集，增加白细胞表面Mac-1/CD11b和表面结合组织因子、FXa和纤维蛋白原。Mac-1/CD11b的升高主要不依赖于白细胞-血小板结合，而表面结合组织因子、FXa和纤维蛋白原的升高主要依赖于白细胞-血小板结合。结论：(i)我们开发了新的全血流式细胞术检测方法来测量单核细胞和中性粒细胞表面的结合组织因子、凝血FXa、纤维蛋白原、活化的Mac-1和CD11b，允许独立分析有或没有表面粘附血小板的单核细胞和中性粒细胞。（ii）组织因子、FXa和纤维蛋白原的单核细胞和中性粒细胞表面结合主要依赖于血小板对单核细胞和中性粒细胞的粘附，而CD11b和活化的Mac-1的单核细胞和中性粒细胞表面表达主要不依赖于血小板对单核细胞和中性粒细胞的粘附。
Background: Monocytes and neutrophils form heterotypic aggregates with platelets initially via engagement of platelet surface P-selectin with leukocyte surface P-selectin glycoprotein ligand-1 (PSGL-1). The resultant intracellular signaling causes the leukocyte surface expression of tissue factor and activation of leukocyte surface Mac-1 (integrin alpha(M)beta(2), CD11b/CD18). The activation-dependent conformational change in monocyte surface Mac-1 results ill the binding of coagulation factor Xa (FXa) and/or fibrinogen to Mac-1. The aim of this study was to develop whole blood flow cytorrietry assays of these procoagulant activities and to investigate the effects of platelet binding to monocytes and neutrophils. Methods: Citrate or D-Phe-Pro-Arg-chloromethylketone (PPACK) anticoagulated whole blood was incubated with monoclonal antibodies against CD14 (PECy5). CD42a (PE), FITC-conjugated test antibody and an agonist, and then fixed with FACS lyse. Appropriate isotype negative controls were prepared in parallel. A BID FACSCalibur Was used to analyze rnonocytes and neutrophils, which were identified based on CD14 fluorescence, forward and 90 degrees light scatter. These populations were further gated into CD42a-positive (platelet-bound) and CD42a-negative (platelet-free). Geometric mean fluorescence and per cent positive data were collected for each subpopulation to Measure the binding of test antibodies directed at CD42a, tissue factor, coagulation FXa, bound fibrinogen, activated Mac-1, and CD11b. Compensation controls were prepared on six normal donors prior to the Study and these settings were used throughout the 10 donor Study. Negative controls verified the lack of cross talk, particularly in the quantified FITC and PE parameters. Results: The physiologic agonists collagen and ADP increased monocyte-platelet and neutrophil-platelet aggregates and increased leukocyte surface Mac-1/CD11b and surface-bound tissue factor, FXa and fibrinogen. Whereas the increases in Mac-1/CD11b were mainly independent of leukocyte-platelet binding, the increases in surface-bound tissue factor, FXa and fibrinogen were mainly dependent on leukocyte-platelet binding. Conclusions: (i) We have developed novel whole blood flow cytometry assays to measure bound tissue factor, coagulation FXa, fibrinogen, activated Mac-1 and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface-adherent platelets. (ii) The monocyte and neutrophil Surface binding of tissue factor, FXa and fibrinogen is mainly dependent on platelet adherence to monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac-1 is mainly independent of platelet adherence to monocytes and neutrophils.