Inosine抑制血小板凝集作用之機轉探討

1. Inosine抑制血小板凝集作用之機轉探討 • Inosine是人體內的內生性物質，它是adenosine的主要代謝物，主要經由adenosine deaminase，當組織缺血及產生發炎反應時，此物質會大量增加；然而報導證實它具有抗發炎的效果，能夠抑制cytokine的釋放，例如TNF-α、IL-1、IL-12、MIP-1α、MIP-2、IFN-α，同時，它也能夠抑制MPO (myeloperoxidase)的活性、MDA (malondialdehyde)的生成，暗示它在抗氧化也扮演著重要的角色。由於發炎反應 (inflammatory response)與動脈硬化症 (atherosclerosis)及血栓的形成 (thrombus formation)有很大的關聯性，所以我們想要進一步探討inosine能否調節血小板。本研究係探討inosine在血小板活化過程中對細胞內一些訊息傳遞的影響，亦即抑制血小板凝集的機轉。研究結果顯示：(1) 在in vitro實驗中已發現外加inosine確實具有抑制血小板凝集之能力。亦即在血小板凝集實驗中若外加inosine，則會隨著濃度之增加，有效地抑制由collagen (1 mg/ml)與arachidonic acid (60 mM)等血小板活化劑所引起的凝集作用，隨著血小板活化劑使用種類的不同，inosine 之IC50 約為1.5-3 mM，但對於thrombin (0.01 U/ml)與U46619 (1 mM)所引起的凝集並沒有抑制效果 (inosine>6 mM)。(2)同時，inosine可有效的抑制由collagen刺激之血小板活化所引起的細胞內鈣離子移動與 phosphoinositide breakdown。(3) inosine可促進 NO與cGMP之增加，抑制TxB2之形成，降低細胞內pH值，但並不影響cAMP的含量與細胞膜流動性。(4) 對於血小板中之47 kDa蛋白質磷酸化，這是一個標記protein kinase C活性的方法，在本實驗中我們分別使用collagen (5 mg/ml)和PDBu (0.15 mM)促進血小板47- kDa的蛋白質磷酸化；由研究結果顯示inosine只能抑制collagen所誘導47 kDa的蛋白質磷酸化，並不影響PDBu的作用。 • 由結果發現inosine抗血小板活性可能涉及以下路徑：(1) Inosine可能抑制 phospholipase C的活性，接著進一步抑制phosphoinositide breakdown，PKC磷酸化47 kDa protein和細胞內鈣離子的移動，進而降低phospholipase A2的活性，也抑制了thromboxane A2 (TxA2)的形成； (2) Inosine可能會經由活化血小板內NO synthetase產生NO，活化guanylate cyclase，使cGMP的含量增加；(3) Inosine可能抑制由thrombin所刺激之Na+/H+交換，導致細胞內DpH值的降低，進一步抑制細胞內鈣離子的增加；(4) Inosine可抑制hyroxyl radical (OH‧)的產生。藉由上述 (1)、(2)、(3)和(4)的作用，最後抑制了血小板凝集反應。

2. Mechanisms involved in the antiplatelet activity of inosine • Inosine, formed by the breakdown of adenosine by adenosine deaminase, is an endogenous substance. It is a major and stable metabolite of adenosine. In ischemic or inflammatory tissues, this substance would increase largely. However, there were evidences that inosine might posses anti-inflammatory effect. It could inhibit cytokine release, example of TNF-α, IL-1, IL-12, MIP-1α, MIP-2 and IFN-α. It could also inhibit myeloperoxidase (MPO) activity and malondialdehyde (MDA) formation. This implied that it had an important role of anti-oxidation. However, inflammatory responses, atherosclerosis and thrombus formation are highly relative, so we interestingly want to know whether inosine could regulate activity of platelets. Our study investigated that inosine involved in some signal transduction in the processes of platelet activation as well as mechanisms of anti-platelet activity. Our results showed that first it could inhibit platelet aggregation induced by collagen (1mg/ml) and AA (60 mM). The concentrations of IC50 are 1.5 and 3 mM, respectively. As to thrombin (0.01 U/ml) and U46619 (0.1 mM), inosine (> 6 mM) had no effect on them. Second, it could efficiently inhibit collagen-induced calcium mobilization and phosphoinositide breakdown. Third, it could stimulate the increase of NO and cGMP, inhibit thromboxane B2 (TxB2) formation and decrease intracellular pH value, but not change the amount of cAMP and cell membrane fluidity. Fourth, it could inhibit 47 kDa protein phosphorylation of protein kinase C (PKC) induced by collagen (5 mg/ml) but not PDbu (0.15 mM).Therefore, our results suggested that the anti-platelet activity of inosine might involve in the following pathways. (1) Inosine might inhibit phospholipase C (PLC), so that it further could inhibit phosphinositide breakdown, 47 kDa protein phosphorylation of PKC and intracellular calcium mobilization. Finally, Phospholipase A2 activity and thromboxane A2 (TxA2) formation also were reduced. (2) Inosine might activate NO synthetase (NOS), and then it could increase the amount of NO and cGMP. (3) Inosine might inhibit activity of Na+/H+ exchanger induced by thrombin, so decreased the intracellular pH value. This effect would enhance the inhibition of mobilization of intracellular calcium. (4) Inosine might diminish the production of hydroxy redcal induced by collagen. According to our results, inosine ultimately could inhibit platelet aggregation.