人體傷口的癒合分為四個階段，其中凝血期(Hemostasis)是利用凝血機轉活化纖維蛋白酶原成纖維蛋白產生凝血塊所達到的止血效應。常見的凝血方法有添加促進纖維蛋白形成的凝血因子或是使用提高纖維蛋白酶原濃度的血漿。而近年來大氣微電漿產生活性物種並應用於促進血液凝血已有相當的成效，其優勢為利用物理效應凝血可避免感染或排斥的現象。因此，本研究目的為評估並探討微電漿照射低血小板血漿(platelet-poor blood plasma, PPP)內纖維蛋白酶原的活化以及可能的機制。
本研究以射頻供應器為氬氣微電漿系統激發源，在4 mm工作距離下對低血小板血漿進行照射。隨微電漿暴露時間不同探討血漿的凝血效果，含凝血分析儀偵測血漿凝血時間、掃描式電子顯微鏡(SEM)觀察血漿凝血塊的纖維蛋白直徑、螢光染色分析纖維蛋白結構緻密度，及微量盤偵測系統量測凝血塊纖溶時間。另外藉光學放射光譜儀監控(OES)微電漿激發時的反應性物種分佈，並使用微量盤偵測系統量測照射後血漿內產生的氧活性物種(reactive oxygen species, ROS)濃度變化，最後在傅立葉轉換紅外光譜顯微鏡(FTIR-microscopy)下分析微電漿照射後血漿蛋白質官能基變化。
研究結果顯示，PPP部分凝血酶原時間(aPTT)較控制組經微電漿照射90 sec在正常範圍內(27~35 sec)，照射180及270 sec則各顯著延長3.8及16.6 sec。PPP凝血塊纖維蛋白直徑經微電漿照射90及180 sec各顯著縮小0.045及0.064 μm，而270 sec照射則無顯著差異。以螢光染色纖維蛋白其結構緻密度經微電漿照射90、180及270 sec各顯著增加11%、13%及5%。PPP經微電漿照射90及180 sec生成的凝血塊加入纖溶素後其纖溶時間各延長15以及21 min，而照射270 sec後纖溶時間無差異。
由從OES光譜中得知微電漿主要產生O*與OH*的含氧物種，這些物種溶於PPP產生ROS濃度增加，並且於FTIR圖譜中觀察到，ROS濃度與FTIR偵測到含氧官能基特性峰強度有正相關。因此，PPP經微電漿照射90~180 sec產生的ROS不會延長aPTT的情況下，ROS會氧化內含纖維蛋白酶原而生成較緻密的纖維蛋白結構以達到延長纖溶時間的目的，此種電漿作用未來可能應用於在傷口癒合階段中的加速及穩定凝血塊的形成。

There are four stages in wound healing, and among them, Hemostasis is the step that transforms the fibrinogen to fibrin in order to stops bleeding by activating the coagulation cascade. There are several methods of pro-coagulation during the would healing, like the addition of coagulation factors or the use of the blood plasma in high concentration of fibrinogen. Recently, atmospheric micro-plasma generates abundant reactive plasma species (RPS) and provides a potential technique to apply for blood coagulation. The primary advantage is the physically inducing blood coagulation without the risk of infection. So in this study, we evaluated the activation of fibrinogen in the platelet-poor plasma and discussed the possible mechanism.
In this study, radio frequency-induced argon micro-plasma system exposed to PPP with 4 mm working distance from the plasma nozzle and the exposure time of micro-plasma was 0~270 sec. The coagulation effect in PPP, including coagulation time was detected by coagulometer, fibrin diameter was observed by scanning electron microscopy (SEM), fibrin density was analyzed by fluorescent image, and fibrinolytic time was measured by microplate readers. In addition, an optical emission spectroscope (OES) was utilized to detect the RPS during plasma excitation process, Microplate readers was also applied to measure the intensity of reactive oxygen species (ROS) in PPP, and FTIR-microscopy was used to analyze the change of functional group of proteins in PPP.
The results showed that the PPP of varied micro-plasma exposure groups were compared with control group. Activated partial thromboplastin time (aPTT) significantly prolonged 3.8 and 16.6 sec after micro-plasma exposure for 180 and 270 sec, respectively, and noticed that there was no change after 90 sec micro-plasma exposure. In addition, the diameter of fibrin in PPP clot significantly decreased 0.045, 0.063, and 0.017 μm after micro-plasma exposure for 90, 180, 270 sec, respectively. Moreover, the density of fibrin increased 11%, 13%, and 5% after micro-plasma exposure for 90, 180, and 270 sec. Finally, the fibrinolytic time of PPP clot prolonged 15, 21 min after micro-plasma exposure for 90 and 180 sec, respectively, and noticed that there was no effect after 270 sec micro-plasma exposure.
This study provided that micro-plasma mostly generated RPS, especially O* and OH*, and these RPS dissolved in PPP and continuously generated ROS to oxidize fibrinogen by decreasing the residues of the peptides. And within 90~180 sec micro-plasma exposure, the PPP could form denser fibrin structure by oxidized fibrinogen without prolonging aPTT, and resulted in longer fibrinolytic time compared with control group. This effect by micro-plasma might apply for accelerating and stabilizing the formation of the clot during the would healing.

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