Walsh Geraldine M, Sheehan David, Kinsella Anthony, Moran Niamh, O'Neill Sarah
Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin, Ireland.
Biochemistry. 2004 Jan 20;43(2):473-80. doi: 10.1021/bi0354536.
The molecular mechanisms involved in regulating the activation-dependent conformational switch in integrins are not known although recent evidence suggests that integrins are a direct target for redox modulation. We have identified an endogenous integrin thiol isomerase activity that may be responsible for regulating integrin activation states. The purpose of this study was to examine the effects of redox conditions elicited by nitric oxide and glutathione on the thiol isomerase activity of the platelet integrin alphaIIbbeta3 and also on the activation status of this integrin in intact platelets. The universal integrin activator, Mn2+, stimulates the thiol isomerase activity in purified alphaIIbbeta3. Kinetic analysis reveals that alphaIIbbeta3 is an allosteric enzyme which displays positive cooperativity in the presence of Mn2+ with an apparent Hill coefficient of 1.9. Also, addition of Mn2+ to platelets results solely in activation of the integrin as demonstrated by the binding of the antibody PAC-1. The addition of the nitric oxide donors SNP, SIN-1, and SNOAC in combination with glutathione can directly reverse the activation state of the platelet integrin induced by Mn2+. These compounds have no effect on platelet secretory responses indicating a direct effect on the integrin. In the presence of nitric oxide and glutathione, the enzymatic activity of alphaIIbbeta3 also displays positive cooperativity (apparent Hill coefficient of 1.9), and a significant increase in the saturability of the enzyme was observed. Thus, redox agents simultaneously modulate the thiol isomerase activity of purified alphaIIbbeta3 and its active conformation in intact platelets, suggesting a molecular mechanism for integrin regulation.
尽管最近有证据表明整合素是氧化还原调节的直接靶点，但调节整合素中激活依赖性构象转换的分子机制尚不清楚。我们已经鉴定出一种内源性整合素硫醇异构酶活性，它可能负责调节整合素的激活状态。本研究的目的是研究一氧化氮和谷胱甘肽引发的氧化还原条件对血小板整合素αIIbβ3硫醇异构酶活性的影响，以及对完整血小板中该整合素激活状态的影响。通用整合素激活剂Mn2+可刺激纯化的αIIbβ3中的硫醇异构酶活性。动力学分析表明，αIIbβ3是一种别构酶，在Mn2+存在下表现出正协同性，表观希尔系数为1.9。此外，向血小板中添加Mn2+仅导致整合素激活，如抗体PAC-1的结合所示。一氧化氮供体SNP、SIN-1和SNOAC与谷胱甘肽联合添加可直接逆转Mn2+诱导的血小板整合素的激活状态。这些化合物对血小板分泌反应没有影响，表明对整合素具有直接作用。在一氧化氮和谷胱甘肽存在下，αIIbβ3的酶活性也表现出正协同性（表观希尔系数为1.9），并且观察到酶的饱和度显著增加。因此，氧化还原试剂同时调节纯化的αIIbβ3的硫醇异构酶活性及其在完整血小板中的活性构象，提示了整合素调节的分子机制。
