Rezaie A R
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Blood. 1998 Jun 15;91(12):4572-80.
A recent study indicated that negatively charged substances such as heparin and dextran sulfate accelerate thrombin activation of coagulation factor XI by a template mechanism. Because the serine proteinase of the natural anticoagulant pathway, activated protein C, can bind heparin, it was reasonable to think that these compounds may also bind protein C (PC) and accelerate its activation by thrombin or other heparin binding plasma serine proteinases by a similar mechanism. To test this, PC activation by thrombin and factor Xa (fXa) was studied in the presence of these polysaccharides. With thrombin in the absence of thrombomodulin (TM), these polysaccharides markedly reduced the Km for PC and Gla-domainless PC (GDPC) activation in the presence of Ca2+. With TM containing chondroitin sulfate, heparin did not influence PC activation by thrombin, but with TM lacking chondroitin sulfate, the characteristic high-affinity PC interaction at low Ca2+ (approximately 50 to 100 micromol/L) was largely eliminated by heparin. In EDTA, heparin enhanced thrombin activation of GDPC by reducing the Km, but it inhibited PC activation by increasing the Km. PC activation in EDTA was insensitive to the presence of heparin if the exosite 2 mutant, R93,97,101A thrombin, was used for activation. These results suggest that, when the Gla-domain of PC is not fully stabilized by Ca2+, it interacts with the anion binding exosite 2 of thrombin and that heparin binding to this site prevents this interaction. Additional studies indicated that, in the presence of phospholipid vesicles, heparin and dextran sulfate dramatically accelerate PC activation by fXa by also reducing the Km. Interestingly, on phospholipids containing 40% phosphatidylethanolamine, the activation rate of near physiological PC concentrations ( approximately 80 nmol/L) by fXa in the presence of dextran sulfate was nearly comparable to that observed by the thrombin-TM complex. The biochemical and potential therapeutical ramifications of these findings are discussed.
最近的一项研究表明，带负电荷的物质，如肝素和硫酸葡聚糖，通过模板机制加速凝血因子XI的凝血酶激活。由于天然抗凝途径的丝氨酸蛋白酶，活化蛋白C，能结合肝素，因此有理由认为这些化合物也可能结合蛋白C（PC），并通过类似机制加速其被凝血酶或其他肝素结合血浆丝氨酸蛋白酶激活。为了验证这一点，在这些多糖存在的情况下研究了凝血酶和因子Xa（fXa）对PC的激活作用。在没有血栓调节蛋白（TM）的情况下，对于凝血酶，这些多糖在Ca2+存在时显著降低了PC和无Gla结构域PC（GDPC）激活的Km值。对于含有硫酸软骨素的TM，肝素不影响凝血酶对PC的激活，但对于缺乏硫酸软骨素的TM，肝素在低Ca2+（约50至100微摩尔/升）时显著消除了PC的特征性高亲和力相互作用。在乙二胺四乙酸（EDTA）中，肝素通过降低Km增强了GDPC的凝血酶激活，但通过增加Km抑制了PC激活。如果使用外位点2突变体R93,97,101A凝血酶进行激活，EDTA中的PC激活对肝素的存在不敏感。这些结果表明，当PC的Gla结构域未被Ca2+完全稳定时，它与凝血酶的阴离子结合外位点2相互作用，而肝素与该位点的结合阻止了这种相互作用。进一步的研究表明，在磷脂囊泡存在的情况下，肝素和硫酸葡聚糖也通过降低Km显著加速了fXa对PC的激活。有趣的是，在含有40%磷脂酰乙醇胺的磷脂上，在硫酸葡聚糖存在下，接近生理PC浓度（约80纳摩尔/升）时fXa的激活速率与凝血酶-TM复合物观察到的激活速率几乎相当。讨论了这些发现的生化和潜在治疗意义。
