Siska Samantha, Sake Cara, Neeves Keith B, Boyle Nanette
Depeartment of Chemical and Biological Engineering, Colorado School of Mines.
Department of Bioengineering, University of Colorado Denver, Anschutz Medical Campus; Department of Pediatrics, Section of Hematology, Oncology, and Bone Marrow Transplant, University of Colorado Denver, Anschutz Medical Campus.
J Vis Exp. 2025 Jan 10(215). doi: 10.3791/67639.
Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%-40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.
血小板是血细胞，在止血和先天性免疫反应中发挥着不可或缺的作用。血小板活性过高和过低都与代谢紊乱有关，会增加血栓形成和出血的风险。血小板的激活和代谢紧密相连，测量前者的方法众多，而测量后者的方法相对较少。为了在不受其他血细胞和血浆成分干扰的情况下研究血小板代谢，必须分离血小板，由于血小板对剪切敏感且具有不可逆激活的能力，这一过程并非易事。本文介绍了一种血小板分离（洗涤）方案，该方案可产生对血小板激动剂刺激敏感的静息血小板。通过连续离心步骤并添加血小板抑制剂，从全血中分离出血小板，并将其重悬于可控的等渗缓冲液中。该方法可从全血中可重复地获得30%-40%的血小板回收率，且颗粒分泌和整合素活性标志物测量显示其激活程度较低。血小板计数和燃料浓度可精确控制，以便使用者探究各种代谢情况。
