背景:白细胞-血小板聚集体包含止血和免疫之间的致病联系,但是它们形成的先决条件和机制仍然不清楚。
目的:为了量化地层,composition,在各种细胞激活剂的影响下,体外白细胞-血小板聚集体的形态。
方法:Phorbol-12-肉豆蔻酸-13-乙酸酯(PMA),脂多糖(LPS),凝血酶受体激活肽(TRAP-6),和二磷酸腺苷(ADP)被用作细胞活化剂。流式细胞术用于鉴定和定量人全血和富血小板血浆中的聚集体。使用针对适当细胞标记的荧光标记抗体鉴定细胞类型和细胞聚集体,并且通过适当的表面标志物的表达来评估细胞活化。对于共聚焦荧光显微镜,标记细胞膜和细胞核。使用扫描电子显微镜研究中性粒细胞-血小板聚集体。
结果:在存在PMA的情况下,ADP或TRAP-6,约17-38%的嗜中性粒细胞和61-77%的单核细胞与全血中的血小板形成聚集体,而LPS由于不能激活血小板而不诱导中性粒细胞或单核细胞的血小板聚集。当向富含血小板的血浆中加入分离的嗜中性粒细胞时,获得了类似的结果。参与异型聚集的所有细胞类型均表达活化的分子标记。聚集体的荧光和电子显微镜显示,主要的血小板/白细胞比率为1:1和2:1。
结论:白细胞-血小板聚集体的形成取决于细胞活化剂的性质及其细胞活化能力的谱。白细胞-血小板聚集体形成的一个必不可少的条件是所有细胞类型的激活,包括血小板。这是限制性的步骤。
BACKGROUND: Leukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood.
OBJECTIVE: To quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators.
METHODS: Phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP-6), and adenosine diphosphate (ADP) were used as cellular activators. Flow cytometry was utilized to identify and quantify aggregates in whole human blood and platelet-rich plasma. Cell types and cellular aggregates were identified using fluorescently labeled antibodies against the appropriate cellular markers, and cell activation was assessed by the expression of appropriate surface markers. For confocal fluorescent microscopy, cell membranes and nuclei were labeled. Neutrophil-platelet aggregates were studied using scanning electron microscopy.
RESULTS: In the presence of PMA, ADP or TRAP-6, about 17-38 % of neutrophils and 61-77 % of monocytes formed aggregates with platelets in whole blood, whereas LPS did not induce platelet aggregation with either neutrophils or monocytes due the inability to activate platelets. Similar results were obtained when isolated neutrophils were added to platelet-rich plasma. All the cell types involved in the heterotypic aggregation expressed molecular markers of activation. Fluorescent and electron microscopy of the aggregates showed that the predominant platelet/leukocyte ratios were 1:1 and 2:1.
CONCLUSIONS: Formation of leukocyte-platelet aggregates depends on the nature of the cellular activator and the spectrum of its cell-activating ability. An indispensable condition for formation of leukocyte-platelet aggregates is activation of all cell types including platelets, which is the restrictive step.