DataSheet_1_Platelet-Depletion of Whole Blood Reveals That Platelets Potentiate the Release of IL-8 From Leukocytes Into Plasma in a Thrombin-Dependent Manner.docx

ObjectiveIn a recent study, we found an elevated level of interleukin 8 (IL-8) in response to bacterial incubation in thrombin-sufficient human whole blood anticoagulated by the fibrin polymerization blocking peptide GPRP. Whether thrombin directly activated leukocytes or mediated the release via thrombin-dependent activation of platelets remains unresolved. Herein, we addressed the role of thrombin and platelets in IL-8 release.
MethodsWe separated platelets from whole blood using a combination of 0.7% (w/v) citrate and GPRP for attenuating the hemostatic response during the separation of platelets. Cytokine responses were compared in whole blood and platelet-depleted blood upon Escherichia coli incubation. Cytokine responses were also profiled with and without reconstitution of either platelets or the supernatant from activated platelets.
ResultsPlatelets were not activated during the separation process but responded to stimuli upon re-calcification. Plasma levels of IL-1β, IL-1Ra, IL-6, IL-8, IP-10, MIP-1α, and MIP-1β were significantly reduced in platelet-depleted blood compared to whole blood, but recovered in the presence of platelets, or with the supernatant of activated platelets. The leukocyte fraction and platelets were each found to contribute to the elevation of IL-8 at around 5 ng/ml; however, if combined, the release of IL-8 increased to 26 ng/ml. This process was dependent on thrombin since the levels of IL-8 remained at 5 ng/ml in whole blood if thrombin was blocked. Intracellular staining revealed that monocytes were the main source for IL-8 expression.
ConclusionOur findings suggest that the release of IL-8 is mediated by the leukocytes, mainly monocytes, but potentiated via thrombin-dependent activation of platelets.

### 研究目的
既往研究中，我们发现采用纤维蛋白聚合阻断肽GPRP（fibrin polymerization blocking peptide GPRP）抗凝的凝血酶充足的人全血（human whole blood）体系内，经细菌孵育（bacterial incubation）后，白细胞介素8（interleukin 8, IL-8）水平显著升高。目前尚未明确凝血酶（thrombin）是直接激活白细胞，还是通过依赖凝血酶的血小板活化途径介导细胞因子释放。本研究旨在探讨凝血酶与血小板在IL-8释放过程中的作用。

### 实验方法
本研究采用0.7%（质量体积比，w/v）柠檬酸盐联合GPRP的方案分离血小板，以弱化血小板分离过程中的止血级联反应。我们将经大肠杆菌（Escherichia coli）孵育的全血与去血小板全血（platelet-depleted blood）的细胞因子应答水平进行对比；此外，我们还通过设置添加血小板、添加活化血小板上清液以及不添加的对照组，对细胞因子应答谱进行了分析。

### 实验结果
血小板在分离过程中未被激活，但经复钙化处理后可对刺激产生应答。与全血组相比，去血小板全血组的IL-1β、IL-1Ra、IL-6、IL-8、干扰素诱导蛋白10（IP-10）、巨噬细胞炎症蛋白1α（MIP-1α）与巨噬细胞炎症蛋白1β（MIP-1β）血浆水平均显著降低，而添加血小板或活化血小板上清液后，上述细胞因子水平可恢复至正常水平。研究发现，白细胞组分与血小板各自可使IL-8水平升高至约5 ng/ml；若二者联合作用，IL-8释放量可升至26 ng/ml。该过程依赖凝血酶：若阻断凝血酶活性，全血组IL-8水平将维持在5 ng/ml。细胞内染色实验证实，单核细胞（monocytes）是IL-8表达的主要来源。

### 研究结论
本研究结果表明，IL-8的释放由白细胞（主要为单核细胞）介导，而依赖凝血酶的血小板活化可增强该过程。