Stief T W, Jeske W P, Walenga J, Schultz C, Kretschmer V, Fareed J
Department of Clinical Chemistry and Molecular Diagnostics, Hospital of Philipps University, Marburg, Germany.
Clin Appl Thromb Hemost. 2001 Jul;7(3):219-24. doi: 10.1177/107602960100700307.
Major mediators of activated polymorphonuclear leukocytes (PMN) are the oxidants HOCl and chloramine, which are a source for the nonradical photon-emitting oxidant singlet oxygen (1O2). We were interested in a possible platelet-modulating activity of 1O2. As a stable 1O2 source we chose the mild oxidant chloramine T (CT), which mimics the natural chloramine N-chloro-taurine. Freshly drawn native whole blood from donors (n = 5) was incubated at 0 to 3 mM CT for 1 minute at 37 degrees C. Then saline. 10 microM adenosine diphosphate (ADP), 5 microg/mL collagen, or 6.25 microM thrombin receptor activator peptide (TRAP) were added and the mixtures were allowed to incubate for 3 minutes at 37 degrees C. Aliquots of activated blood were fixed in 1% para-formaldehyde. After removal of the fixative, platelets were labeled with anti-CD61-FITC and anti-CD62P-PE antibodies and analyzed by flow cytometry. An oxidant concentration-dependent decrease in the expression of P-selectin appeared (at 3 mM CT to 39, 23, and 20% of the 100% saline control level for ADP, collagen, and TRAP, respectively). There was also an oxidant concentration-dependent decrease in the formation of platelet aggregates (at 3 mM CT to 8, 12, and 13% of the 100% saline control level for ADP, collagen, and TRAP, respectively; the 50% effective dose was 1.0 to 1.5 mM chloramine). In ADP- and TRAP-stimulated platelets, an oxidant-mediated increase in platelet fragments appeared (at 3 mM CT: three- to fourfold of the initial value). The addition to the blood of 30 mM of the oxyradical scavenger mannitol in contrast to excess methionine did not antagonize these oxidative modulations of platelet activation. The results were confirmed using equimolar concentrations of NaOCI and N-chloro-taurine. This study shows that 1O2 inhibits platelets, decreasing the expression of CD62P and the formation of platelet aggregates. Activated PMN might modulate hemostasis, shifting it into an antithrombotic state. The physiologic signal action and the direct anticoagulant action of 1O2 (released by chloramines such as vancomycin) might be a new principle for pharmacologic intervention in atherothrombosis.
活化的多形核白细胞（PMN）的主要介质是氧化剂次氯酸（HOCl）和氯胺，它们是非自由基发光氧化剂单线态氧（1O2）的来源。我们对1O2可能的血小板调节活性感兴趣。作为稳定的1O2来源，我们选择了温和的氧化剂氯胺T（CT），它模拟天然氯胺N-氯代牛磺酸。从供体（n = 5）采集的新鲜全血在37℃下于0至3 mM CT中孵育1分钟。然后加入生理盐水、10 microM二磷酸腺苷（ADP）、5 microg/mL胶原蛋白或6.25 microM凝血酶受体激活肽（TRAP），混合物在37℃下孵育3分钟。取活化血液的等分试样用1%多聚甲醛固定。去除固定剂后，血小板用抗CD61-FITC和抗CD62P-PE抗体标记，并通过流式细胞术分析。出现了氧化剂浓度依赖性的P-选择素表达降低（在3 mM CT时，对于ADP、胶原蛋白和TRAP，分别为100%生理盐水对照水平的39%、23%和20%）。血小板聚集体的形成也有氧化剂浓度依赖性的降低（在3 mM CT时，对于ADP、胶原蛋白和TRAP，分别为100%生理盐水对照水平的8%、12%和13%；50%有效剂量为1.0至1.5 mM氯胺）。在ADP和TRAP刺激的血小板中，出现了氧化剂介导的血小板碎片增加（在3 mM CT时：为初始值的三至四倍）。与过量的甲硫氨酸相比，向血液中加入30 mM的氧自由基清除剂甘露醇并不能拮抗这些血小板活化的氧化调节。使用等摩尔浓度的NaOCI和N-氯代牛磺酸证实了结果。这项研究表明，1O2抑制血小板，降低CD62P的表达和血小板聚集体的形成。活化的PMN可能调节止血，使其转变为抗血栓状态。1O2（由诸如万古霉素的氯胺释放）的生理信号作用和直接抗凝作用可能是动脉粥样硬化血栓形成药物干预的新原则。
