Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences St. Petersburg, Russia.
Center for Theoretical Problems of Physico-Chemical Pharmacology, Russian Academy of Sciences, Moscow, Russia.
Thromb Res. 2018 Nov;171:22-30. doi: 10.1016/j.thromres.2018.09.047. Epub 2018 Sep 13.
Arachidonic acid induced aggregation is a generally accepted test for aspirin resistance. However, doubts have been raised that arachidonic acid stimulated aggregation can be regarded as reliable testing for aspirin resistance. Arachidonic acid, in addition to platelet activation, can induce phosphatidylserine translocation on the outer surface of platelet membrane which could be mediated by apoptosis pathways or transformation of platelets to the procoagulant state.
We explored effects of arachidonic acid over a vast range of concentrations and a wide range of read-outs for human platelet activation, procoagulant activity, and platelet viability. Additionally we tested whether cAMP- or cGMP-dependent protein kinase activation can inhibit procoagulant activity or platelet viability.
Arachidonic acid-induced washed platelet activation was detected at low micromolar concentrations during the first 2 min of stimulation. After longer incubation and/or at higher concentrations arachidonic acid triggered platelet procoagulant activity and reduced platelet viability. At the same time, arachidonic acid stimulated adenylate cyclase mediated protein phosphorylation which correlated with reduced platelet activation. Moreover, additional stimulation of cAMP- or cGMP-dependent protein kinase inhibited only platelet activation, but did not prevent pro-coagulant activity and platelet death.
While arachidonic acid induces platelet activation at low concentrations and during short incubation time, higher concentrations and lasting incubation evokes adenylate cyclase activation and subsequent protein phosphorylation corresponding to reduced platelet activation, but also enhanced pro-coagulant activity and reduced viability. Our observations provide further proof for the complex fine tuning of platelet responses in a time and agonist concentration dependent manner.
花生四烯酸诱导的聚集通常被认为是阿司匹林抵抗的检测方法。然而，人们对花生四烯酸刺激的聚集是否可以被视为可靠的阿司匹林抵抗检测方法产生了怀疑。花生四烯酸除了能激活血小板外，还能诱导血小板膜外表面的磷脂酰丝氨酸易位，这一过程可能通过凋亡途径或血小板向促凝状态的转化来介导。
我们探索了花生四烯酸在广泛浓度范围和多种血小板激活、促凝活性和血小板活力检测方法中的作用。此外，我们还测试了环磷酸腺苷（cAMP）或环鸟苷酸（cGMP）依赖性蛋白激酶的激活是否可以抑制促凝活性或血小板活力。
在刺激的前 2 分钟内，低微摩尔浓度的花生四烯酸即可诱导被洗涤的血小板激活。在更长的孵育时间和/或更高的浓度下，花生四烯酸会触发血小板的促凝活性并降低血小板的活力。与此同时，花生四烯酸刺激腺苷酸环化酶介导的蛋白磷酸化，与血小板激活减少相关。此外，cAMP 或 cGMP 依赖性蛋白激酶的额外刺激仅抑制血小板的激活，但不能阻止促凝活性和血小板死亡。
虽然低浓度和短孵育时间的花生四烯酸会诱导血小板激活，但更高的浓度和更长的孵育时间会引发腺苷酸环化酶的激活和随后的蛋白磷酸化，对应于血小板激活的减少，但也会增强促凝活性和降低血小板活力。我们的观察结果进一步证明了血小板反应在时间和激动剂浓度依赖性方面的复杂精细调节。
