Michelson A D, Barnard M R, Hechtman H B, MacGregor H, Connolly R J, Loscalzo J, Valeri C R
Departments of Pediatrics and Surgery, University of Massachusetts Medical School, Worcester 01655, USA.
Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11877-82. doi: 10.1073/pnas.93.21.11877.
To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.
为了验证循环中表面P选择素阳性（脱颗粒）血小板会被迅速清除这一假说，我们开发了在体内追踪血小板和测量血小板功能的新方法。从非人类灵长类动物（狒狒）制备的洗涤血小板用PKH2（一种亲脂性荧光染料）标记，经凝血酶激活、洗涤后，再回输到同一狒狒体内。采用三色全血流式细胞术同时进行：（i）用针对糖蛋白（GP）IIb-IIIa（整合素α11bβ3）的单克隆抗体识别血小板；（ii）通过PKH2荧光区分输入的血小板；（iii）用单克隆抗体分析血小板功能。输入自体凝血酶激活的血小板（P选择素阳性，PKH2标记）两小时后，循环中PKH2标记的血小板95±1%（平均值±标准误，n = 5）变为P选择素阴性。与输注前未用凝血酶激活的血小板相比，这些循环中PKH2标记、P选择素阴性的血小板在输注后24小时的回收率相似，在输注后48小时仅略低。血小板表面P选择素的丢失完全是由于可溶性P选择素血浆浓度增加了67.1±16.7 ng/ml。循环中PKH2标记、P选择素阴性的血小板在体内仍能发挥功能，这通过以下方面得以确定：（i）参与出血时间伤口处形成的血小板聚集体；（ii）在动静脉分流中与涤纶结合；（iii）单克隆抗体PAC1（针对GPIIb-IIIa上的纤维蛋白原结合位点）的结合；（iv）促凝血小板衍生微粒的生成。总之，（i）循环中的脱颗粒血小板会迅速将表面P选择素释放到血浆池中，但仍继续循环并发挥功能；（ii）我们开发了新的三色全血流式细胞术方法用于在体内追踪血小板和测量血小板功能。
