Ravanat C, Archipoff G, Beretz A, Freund G, Cazenave J P, Freyssinet J M
INSERM U.311, Centre Régional de Transfusion Sanguine, Strasbourg, France.
Biochem J. 1992 Feb 15;282 ( Pt 1)(Pt 1):7-13. doi: 10.1042/bj2820007.
Annexin-V (PAP-I, lipocortin-V) acts as a potent anticoagulant in vitro by binding to negatively charged phospholipids with higher affinity than vitamin K-dependent proteins, with a Kd in the 10(-10) M range. The purpose of the present study was to use annexin-V as a probe to assess the catalytic potential of phospholipids in pro- and anti-coagulant reactions in purified systems and at the surface of endothelial cells in culture after stimulation. Procoagulant tissue factor and anticoagulant thrombomodulin activities were compared by using specific two-stage amidolytic assays performed with purified proteins. Procoagulant activity was estimated by the generation of Factor Xa by the Factor VII(a)-tissue factor complex. Anticoagulant activity was estimated by the generation of activated protein C by either the thrombin-thrombomodulin complex or Factor Xa. Annexin-V induced a decrease of 70% of thrombomodulin activity when thrombomodulin (5.4-214 nM) was reconstituted into phosphatidylcholine/phosphatidylserine (1:1, mol/mol) vesicles at 37.5 or 75 microM-phospholipid concentration, the apparent Ki being 0.5 microM at 75 microM-lipid. The saturating concentration of annexin-V was dependent on phospholipid concentration, but was independent of the phospholipid/thrombomodulin ratio. By contrast, when thrombomodulin was not reconstituted in vesicles, annexin-V had no effect. At 2 microM, annexin-V totally inhibited the generation of activated protein C by Factor Xa in the presence of 75 microM-lipid, the saturating inhibitory concentration being dependent on phospholipid concentration. At 0.1 microM, annexin-V totally inhibited tissue-factor activity present in crude brain thromboplastin. In the absence of stimulation, human endothelial cells in culture expressed significant thrombomodulin activity and no detectable tissue-factor activity. Basal thrombomodulin activity was only slightly inhibited (less than 15%) by 0.5 microM-annexin-V. Phorbol myristate acetate (PMA) induced the expression of tissue-factor activity and decreased thrombomodulin activity at the endothelial-cell surface. Annexin-V, at a concentration of 16 microM, caused an 80% decrease of tissue-factor activity induced by PMA at 10 ng/ml, whereas it inhibited thrombomodulin activity by only 15% on the same stimulated cells. Our results confirm that annexin-V inhibits, in vitro, procoagulant tissue-factor activity and anticoagulant activities (activation of protein C by the thrombin-thrombomodulin complex and by Factor Xa), through phospholipid-dependent mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)
膜联蛋白-V（PAP-I，脂皮质蛋白-V）在体外通过与带负电荷的磷脂结合发挥强效抗凝作用，其对磷脂的亲和力高于维生素K依赖性蛋白，解离常数在10^(-10) M范围内。本研究的目的是使用膜联蛋白-V作为探针，评估磷脂在纯化系统以及刺激后培养的内皮细胞表面的促凝和抗凝反应中的催化潜力。通过使用针对纯化蛋白进行的特定两步酰胺水解测定法，比较促凝组织因子和抗凝血栓调节蛋白的活性。促凝活性通过因子VII(a)-组织因子复合物生成因子Xa来估计。抗凝活性通过凝血酶-血栓调节蛋白复合物或因子Xa生成活化蛋白C来估计。当血栓调节蛋白（5.4 - 214 nM）在37.5或75 microM磷脂浓度下重构到磷脂酰胆碱/磷脂酰丝氨酸（1:1，摩尔/摩尔）囊泡中时，膜联蛋白-V使血栓调节蛋白活性降低70%，在75 microM脂质时表观抑制常数为0.5 microM。膜联蛋白-V的饱和浓度取决于磷脂浓度，但与磷脂/血栓调节蛋白比例无关。相比之下，当血栓调节蛋白未在囊泡中重构时，膜联蛋白-V没有作用。在2 microM时，膜联蛋白-V在存在75 microM脂质的情况下完全抑制因子Xa生成活化蛋白C，饱和抑制浓度取决于磷脂浓度。在0.1 microM时，膜联蛋白-V完全抑制粗制脑凝血活酶中存在的组织因子活性。在未受刺激的情况下，培养的人内皮细胞表达显著的血栓调节蛋白活性且未检测到组织因子活性。基础血栓调节蛋白活性仅被0.5 microM膜联蛋白-V轻微抑制（小于15%）。佛波酯（PMA）诱导组织因子活性表达并降低内皮细胞表面的血栓调节蛋白活性。在16 microM浓度下，膜联蛋白-V使10 ng/ml PMA诱导的组织因子活性降低80%，而在相同受刺激细胞上仅抑制血栓调节蛋白活性15%。我们的结果证实，膜联蛋白-V在体外通过磷脂依赖性机制抑制促凝组织因子活性和抗凝活性（凝血酶-血栓调节蛋白复合物以及因子Xa激活蛋白C）。（摘要截短于400字）
