Mobarrez Fariborz, Sjövik Carolina, Soop Anne, Hållström Lars, Frostell Claes, Pisetsky David S, Wallén Håkan
Department of Clinical Sciences, Karolinska Institutet, Danderyd Hospital, Division of Cardiovascular Medicine , Stockholm , Sweden .
Platelets. 2015;26(5):486-90. doi: 10.3109/09537104.2014.932339. Epub 2014 Jun 25.
CD40 ligand (CD40L) is a transmembrane protein that is mainly expressed on activated T cells and platelets. This protein, however, may also be shed from cells and circulate in the blood in a soluble form. "Soluble CD40L" has attracted interest as a biomarker as it can interact with CD40 and elicit cellular responses involved in the pathophysiology of various thrombotic and inflammatory conditions. As platelets can release microvesicles following activation, we investigated the expression of CD40L on circulating microvesicles as well as CD40L in plasma, in an experimental model of inflammation in healthy volunteers (i.e., intravenous lipopolysaccharide administration). We studied CD40L quantified as CD40L-positive platelet microvesicles by flow cytometry, and as CD40L in plasma ("soluble CD40L") by an ELISA. Results of these studies showed that levels of CD40L exposed on platelet microvesicles were significantly increased after lipopolysaccharide administration. ELISA measurements of CD40L in plasma ("soluble CD40L") did not show any significant increase in plasma levels over time. Separation of soluble and vesicle-bound CD40L by high-speed centrifugation indicated that the ELISA can also detect CD40L on microvesicles, as a trend toward increased concentrations were observed in the pellet of high-speed centrifuged samples (i.e., in samples in which microvesicles are enriched). Together, these findings suggest that platelet microvesicles are a source of CD40L in the circulation and that CD40L exposure on platelet microvesicles increases following experimentally induced inflammation. Our data also suggest that determining levels of CD40L on microvesicles in plasma samples may provide a more sensitive detection of changes in CD40L expression than measurement of "soluble CD40L" in plasma with an ELISA. In addition, information regarding the cellular source of CD40L can be obtained with a flow cytometry-based microvesicle assay in a way not possible with an ordinary ELISA.
CD40配体（CD40L）是一种跨膜蛋白，主要表达于活化的T细胞和血小板上。然而，这种蛋白也可能从细胞上脱落，并以可溶性形式在血液中循环。“可溶性CD40L”作为一种生物标志物引起了人们的关注，因为它可以与CD40相互作用，并引发参与各种血栓形成和炎症性疾病病理生理学的细胞反应。由于血小板在激活后可以释放微泡，我们在健康志愿者的炎症实验模型（即静脉注射脂多糖）中，研究了循环微泡上CD40L的表达以及血浆中CD40L的情况。我们通过流式细胞术将CD40L定量为CD40L阳性血小板微泡，并通过酶联免疫吸附测定法（ELISA）将其定量为血浆中的CD40L（“可溶性CD40L”）。这些研究结果表明，脂多糖给药后，血小板微泡上暴露的CD40L水平显著增加。ELISA法测定血浆中的CD40L（“可溶性CD40L”）显示，血浆水平并未随时间出现任何显著增加。通过高速离心分离可溶性和与微泡结合的CD40L表明，ELISA法也可以检测微泡上的CD40L，因为在高速离心样品的沉淀中（即微泡富集的样品）观察到浓度有增加的趋势。总之，这些发现表明血小板微泡是循环中CD40L的一个来源，并且在实验性诱导的炎症后，血小板微泡上CD40L的暴露会增加。我们的数据还表明，与用ELISA法测量血浆中的“可溶性CD40L”相比，测定血浆样品中微泡上的CD40L水平可能对CD40L表达变化的检测更为敏感。此外，通过基于流式细胞术的微泡检测可以获得有关CD40L细胞来源的信息，而这是普通ELISA法无法做到的。
