應用流式細胞分析儀與原子力顯微鏡探討血小板之受激收縮與聚集

Abstract

當傷口流血時，血小板會聚集在傷口處，並引發血纖維蛋白原 (fibrinogen) 聚合形成血纖維蛋白 (fibrin)，以造成凝血並止血。同時血小板也會收縮使血塊機械性質強化，進一步穩固血塊。臨床上發現敗血症病人之微血管中會產生異常凝血或所謂「急性瀰漫性血管內凝血 (disseminated Intravascular Coagulation, DIC)」，嚴重時可造成器官缺血的嚴重併發症。一般認為血小板、血纖維蛋白及紅血球為血塊之主要成份，但最近研究發現敗血症病人血管内形成的血栓可能與嗜中性球細胞 (neutrophil) 釋出網狀物質 (neutrophil extracellular trap, NET) 中的組織蛋白 (histone) 有密切關係。雖然有研究指出組織蛋白可引起血小板活化、貼附與聚集，但是組織蛋白是否能引起血小板的收縮仍未完全清楚。在此研究中，我們開發應用流式細胞儀 (flow cytometry) 探討人類血小板的形貌。光學及電子顯微影像顯示流式細胞儀可清楚分析凝血酵素 (thrombin) 與血纖維蛋白原刺激所引起之血小板聚集與收縮。我們也發現只有在組織蛋白刺激下，血小板會有小量聚集，但無顯著的收縮；而當組織蛋白與血纖維蛋白元同時存在時，血小板則會大量的聚集並同時產生收縮。為進一步探討血小板收縮之力學性質，我們應用原子力顯微鏡 (atomic force microscope, AFM) 的微探針 (microcantilever) 量測單一血小板之收縮力，結果顯示，當血纖維蛋白原存在時，組織蛋白和凝血酶引發血小板收縮所產生的力十分接近 (~ 9 nN)；而前者引起的收縮則有較高的收縮速度。控制實驗之初步結果也顯示，組織蛋白與血小板的作用與第二及第四型 toll-like receptor 有關係，似乎也存在一個有別於上述兩個受體且未被報導過之途徑或受體。我們的研究不但首次證明應用流式細胞儀分析血小板受激後形貌變化之可行性，上述方法結合原子力顯微鏡技術獲得的結果也幫助釐清組織蛋白在敗血症中血栓形成的角色。

As injury occurs, platelets tend to aggregate at the site of vascular injury. Fibrinogens are then induced to polymerize into fibrin and to form blood clots, and finally hemorrhage is prevented. In the meantime, platelets undergo contraction; the contraction of platelets reinforces the mechanical properties of clots and increases the stability of clots. While coagulation of platelets and thrombosis are important for hemostasis, abnormal microvascular thrombosis that occurs in the blood vessel of septic patients can result in ischemia of organs and become a deadly pathological condition. Blood clots are commonly thought to comprise mainly fibrins, platelets and erythrocytes, but results of recent studies indicate that histone, a major constituent of neutrophil extracellular trap (NET) might play an essential role in abnormal microvascular thrombosis associated with sepsis. While it has been shown that histone can cause adhesion, activation and aggregation of platelets, whether or not histone initiates platelet contraction remains an open question. Here we report a novel application of flow cytometry for the study of the morphological alteration of human platelets. Platelets were stimulated with fibrinogen and thrombin, and were sorted with flow cytometry. Optical and electron microscopy images obtained from the sorted samples show two distinct morphology features corresponding to contracted and aggregated platelets, respectively; this result indicates strongly a unique capability of flow cytometry for revelation of subtle morphological alteration of platelets under stimulation. We found also that co-treatment of histone and fibrinogen caused both significant aggregation and contraction, whereas the treatment of histone alone induced only moderate aggregation. To gain more mechanodynamics insight into the platelet contraction, we employed microcantilevers of atomic force microscopy (AFM) to determine quantitatively the contractile force and the contraction rate of single platelets under stimulation. Upon the treatment of histone, single platelets produced a drastic contractile force (~9 nN); the magnitude of the force is comparable to the force induced by the treatment of thrombin. Result of inhibitory assays show that this effect is related not only to toll-like receptor 2 and toll-like receptor 4, but also possibly to an unidentified receptors. We for the first time demonstrate an application of flow cytometry in the morphological analysis of platelets under stimulation, and confirm that histone can induce platelet contraction in the presence of fibrinogen. These results might help clarify the role of histone in the abnormal thrombosis during sepsis.