SPECIFICITY OF ENZYMATIC PROTEIN CLEAVAGE IN GLYCEROL VS WATER

甘油与水中酶促蛋白质裂解的特异性

• 批准号: 6478951
• 负责人: ALEXANDER M KLIBANOV
• 金额: $ 5.36万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6478951
• 关键词: anesthesiology; biological products; biomedical resource; cardiovascular system; drug /agent; health care; hematology; spectrometry; surgery

Heparin a polyanionic glycosaminoglycan, remains the most commonly used anticoagulant in patients with acute coronary syndromes and in patients undergoing cardiovascular surgery. While the effects of heparin on the coagulation system have been well documented, abrupt cessation of heparin therapy can lead to a recrudescence of thrombosis and acute ischernia. In addition, clinically unexplained and catastrophic thrombotic complications occur in patients undergoing cardiovascular surgery despite adequate anticoagulation. Endothelial nitric oxide (NO) is an important endogenous inhibitor of platelet-mediated thrombosis; yet, biochemical studies examining the effect of heparin on NO production by the endothelium have heretofore been lacking. In an attempt to address heparin's effect on endothelial cell production of NO, confluent bovine aortic endothelial cells (BAEC) on microcarrier beads were incubated with media containing newborn calf serum and L-arginine, in the presence or absence of heparin. Results indicate that BAEC incubated for with heparin were less able to inhibit platelet aggregation than control cells, and that this effect correlated with a decrease in NO production by BAEC as determined by photolysis-chemiluminescence. A decrease in NO production also occurred after BAEC were exposed to an equimolar concentration of dextran sulfate, suggesting that the decrease in NO after heparin treatment is secondary to its negative charge rather than to a specific saccharide sequence. These data show that heparin, at concentrations achieved in the acute cardiovascular setting, increases platelet aggregation by decreasing endothelial NO production. These observations suggest a mechanism by which to explain, in part, the prothrombotic effects of heparin. The products of the reaction and the mechanism are being investigated by mass spectrometry. Results indicate that the exposure to NO leads to a structural modification of specific sulfated residues. The structural requirements and the modification are being probed with model compounds and stable isotope labeling.

肝素是一种聚阴离子糖胺聚糖， 在急性冠状动脉综合征患者和 接受心血管手术的患者。 虽然影响 肝素对凝血系统的影响已有充分的记录， 停止肝素治疗可能会导致血栓形成复发 急性缺血。 此外，临床上无法解释的， 灾难性的血栓并发症发生在接受 心血管手术，尽管充分抗凝。 内皮 一氧化氮（NO）是一种重要的内源性抑制剂， 血小板介导的血栓形成;然而，生化研究检查 肝素对内皮细胞产生NO的作用迄今为止 一直缺乏。 为了解决肝素对 内皮细胞产生NO，汇合的牛主动脉内皮细胞 将微载体珠上的细胞（BAEC）与培养基一起孵育 含有新生小牛血清和L-精氨酸，在存在或 没有肝素。 结果表明，BAEC与 肝素抑制血小板聚集能力低于对照组 细胞，并且这种效应与NO的减少有关。 通过光解-化学发光测定BAEC的产量。 一 BAEC暴露于一氧化氮后，NO产生也减少， 等摩尔浓度的硫酸葡聚糖，这表明 肝素治疗后NO的减少是继发于其阴性 电荷而不是特定的糖序列。 这些数据显示 肝素，在急性心血管疾病中达到的浓度 通过降低内皮NO增加血小板聚集 生产 这些观察结果表明了一种机制， 部分解释了肝素的促血栓作用。 产品 的反应和机理正在研究的质量 光谱法 结果表明，暴露于NO导致 特定硫酸化残基的结构修饰。 结构性 需求和修改正在探讨与模型 化合物和稳定同位素标记。