Inosine 抑制血小板凝集作用之機轉探討

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 deaminas e ，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 (ather osclerosis) 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 in osine 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 inosin e 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 ph osphoinositide 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 Dp H 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。

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Inosine, formed by the breakdown of adenosine by adenosine deaminase, is an endogenous substance. It i s a major and stable metabolite of adenosine. In ischemic or inflammatory tissues, this substance would in crease largely. However, there were evidences that inosine might posses anti-inflammatory effect. It coul d 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 imp ortant role of anti-oxidation. However, inflammatory responses, atherosclerosis and thrombus formation a re highly relative, so we interestingly want to know whether inosine could regulate activity of platelets. O ur study investigated that inosine involved in some signal transduction in the processes of platelet activati on as well as mechanisms of anti-platelet activity. Our results showed that first it could inhibit platelet ag gregation 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 breakdo wn. Third, it could stimulate the increase of NO and cGMP, inhibit thromboxane B2 (TxB2) formation an d 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/m l) but not PDbu (0.15 mM).

Therefore, our results suggested that the anti-platelet activity of inosine might involve in the following pa thways. (1) Inosine might inhibit phospholipase C (PLC), so that it further could inhibit phosphinositide b reakdown, 47 kDa protein phosphorylation of PKC and intracellular calcium mobilization. Finally, Phosp holipase 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 a ctivity of Na+/H+ exchanger induced by thrombin, so decreased the intracellular pH value. This effect wo uld enhance the inhibition of mobilization of intracellular calcium. (4) Inosine might diminish the product ion of hydroxy redcal induced by collagen. According to our results, inosine ultimately could inhibit plate let aggregation.

Mechanisms involved in the antiplatelet activity of

inosine