Department of Biological Sciences, University of North Texas, 1510 Chestnut, Denton TX 76203, USA.
Blood Cells Mol Dis. 2012 Mar 15;48(3):188-96. doi: 10.1016/j.bcmd.2011.12.008. Epub 2012 Feb 3.
Hemostasis is a defense mechanism that protects an organism from bleeding in the event of injury. We have previously demonstrated the utility of the zebrafish as a model to study human hemostasis. However, there are no studies on the role of microparticles in hemostasis in early vertebrates. Studying microparticles in zebrafish may provide insight into the evolution of microparticle function in hemostasis and may lead to direct observation of these microparticles in zebrafish larvae due to transparency of the vessels. In this investigation we demonstrate the presence of cellular microparticles in fish blood by both immunostaining as well as by using zebrafish whose thrombocytes are labeled with green fluorescent protein. Further investigation showed that microparticles were also labeled by fluorescein isothiocyanate annexin V, suggesting that these particles are derived via apoptosis. A portion of the fluorescein isothiocyanate annexin V labeled microparticles was also labeled by DiI-C18. Labeling by DiI-C18 suggests that some microparticles are derived from young thrombocytes. Additionally, GpIIb antibody labels almost all thrombocyte-derived microparticles and a greater percentage of microparticles are labeled by GpIIb antibody than by DiI-C18. This suggests that thrombocyte microparticles are derived from both young and mature thrombocytes. Furthermore, the increase of microparticles by adding excessive microparticles into blood in vitro and through intravenous injections led to an increased hemostatic response. In addition, treatment with tumor necrosis factor alpha resulted in an increased number of thrombocyte microparticles and enhanced hemostasis; in contrast, treatment with zVAD-FMK, a caspase inhibitor, resulted in a decrease in thrombocyte microparticles and decreased hemostasis. We also found that thrombocyte microparticles agglutinate, along with other cells and cellular microparticles, in the presence of an excess of either ristocetin or ultra-large von Willebrand factor. Also, stimulation of von Willebrand factor release in vivo resulted in clusters of thrombocyte microparticles in the veins. Moreover, thrombocyte microparticles were the first to appear at the site of arterial injury. We found that thrombocyte microparticles are functionally equivalent to platelet microparticles. The microparticles initiate arterial thrombus formation in a von Willebrand factor-dependent manner and further enhance thrombus formation by forming clusters of microparticles in venous thrombosis. This finding may have applications for understanding the role of platelet microparticles in humans and may have diagnostic applications.
止血是一种保护机体在受伤时不出血的防御机制。我们之前已经证明了斑马鱼作为研究人类止血的模型的实用性。然而，在早期脊椎动物中，关于微粒在止血中的作用还没有研究。研究斑马鱼中的微粒可能有助于深入了解微粒在止血中的功能进化，并且由于血管透明，可能会直接观察到斑马鱼幼虫中的这些微粒。在这项研究中，我们通过免疫染色以及使用血小板用绿色荧光蛋白标记的斑马鱼来证明鱼类血液中存在细胞微粒。进一步的研究表明，微粒也被异硫氰酸荧光素 Annexin V 标记，表明这些颗粒是通过细胞凋亡产生的。一部分异硫氰酸荧光素 Annexin V 标记的微粒也被 DiI-C18 标记。DiI-C18 的标记表明，一些微粒来源于年轻的血小板。此外，GpIIb 抗体标记几乎所有由血小板衍生的微粒，并且 GpIIb 抗体标记的微粒百分比大于 DiI-C18 标记的微粒百分比。这表明血小板微粒来源于年轻和成熟的血小板。此外，通过在体外将过量的微粒添加到血液中以及通过静脉注射，增加了微粒的数量，从而导致止血反应增加。此外，肿瘤坏死因子α的处理导致血小板微粒的数量增加和止血作用增强；相比之下，半胱天冬酶抑制剂 zVAD-FMK 的处理导致血小板微粒减少和止血作用降低。我们还发现，在存在过量的瑞斯托菌素或超大 von Willebrand 因子的情况下，血小板微粒与其他细胞和细胞微粒一起聚集。此外，体内刺激 von Willebrand 因子释放导致静脉中血小板微粒簇的形成。而且，血小板微粒是首先出现在动脉损伤部位的。我们发现血小板微粒在功能上与血小板微粒等效。微粒通过 von Willebrand 因子依赖性方式引发动脉血栓形成，并通过在静脉血栓形成中形成微粒簇进一步增强血栓形成。这一发现可能有助于理解血小板微粒在人类中的作用，并具有诊断应用。
