Stern D M, Nawroth P P, Kisiel W, Handley D, Drillings M, Bartos J
J Clin Invest. 1984 Dec;74(6):1910-21. doi: 10.1172/JCI111611.
Previous studies have demonstrated the binding of Factors IX and IXa to cultured bovine aortic endothelial cells. The present study examines the interaction of Factors IX, IXa, and Xa with the luminal surface of calf aortas, shown by microscopic examination to have a continuous layer of endothelium. Radioimmunoassay of Factor IX showed that 74 fmol/10(6) cells of Factor IX could be eluted from freshly prepared aortic segments. Binding of 3H-Factors IX and IXa to aortic segments was saturable, and comparable to binding in previous studies using cultured endothelial cells. Preincubation of aortic segments with 3H-Factor IXa and von Willebrand factor (VWF)/Factor VIII, followed by washing and addition of Factor X, resulted in formation of Factor Xa. The addition of prothrombin to these activation mixtures resulted in formation of thrombin. Exogenous phospholipid and Factor V were not required for Factor X and prothrombin activation on the intact native endothelium. Incubation of 125I-Factor Xa with the vessel segments resulted in most of the tracer being complexed with antithrombin III originally present on the aortic segment (3.8 pmol antithrombin III/10(6) cells). The Factor Xa-antithrombin III complex was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis exclusively in the supernatants. 125I-Factor Xa not complexed with antithrombin III bound specifically to the vessel segment. The time course of binding was biphasic, consisting of an initial more rapid reversible phase followed by a slower irreversible phase. The latter phase correlated with the formation of a covalent complex (Mr, 76,000) between 125I-Factor Xa and a vessel-localized protein presumably distinct from antithrombin III. The activation of prothrombin by vessel-bound Factor Xa was inhibited by anti-bovine Factor V IgG, suggesting that there is interaction of Factor Xa with a Factor V-like molecule provided by the endothelial cell surface. Addition of antibody to antithrombin III prevented formation of Factor Xa-antithrombin III and thrombin-antithrombin III complexes in the supernatant and increased apparent thrombin activity 30-50-fold. These studies demonstrate that freshly obtained vessels with a continuous layer of native endothelium can support activation of Factor X and prothrombin: vessel-bound Factor IXa can activate Factor X in the presence of VWF/Factor VIII. Factor Xa can also bind to the vessel and participate in the activation of prothrombin. The apparent efficiency of prothrombin activation, however, is dampened by the presence of functional antithrombin III on the vessel wall.
以往的研究已证实因子IX和IXa与培养的牛主动脉内皮细胞结合。本研究检测了因子IX、IXa和Xa与小牛主动脉腔面的相互作用，显微镜检查显示其具有连续的内皮细胞层。因子IX的放射免疫分析表明，从新鲜制备的主动脉段中可洗脱74 fmol/10⁶个细胞的因子IX。³H-因子IX和IXa与主动脉段的结合是可饱和的，与以往使用培养内皮细胞的研究中的结合情况相当。用³H-因子IXa和血管性血友病因子（VWF）/因子VIII预孵育主动脉段，然后洗涤并加入因子X，导致因子Xa形成。向这些激活混合物中加入凝血酶原导致凝血酶形成。在完整的天然内皮上，因子X和凝血酶原的激活不需要外源性磷脂和因子V。用¹²⁵I-因子Xa与血管段孵育，结果大部分示踪剂与最初存在于主动脉段的抗凝血酶III结合（3.8 pmol抗凝血酶III/10⁶个细胞）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳仅在上清液中观察到因子Xa-抗凝血酶III复合物。未与抗凝血酶III结合的¹²⁵I-因子Xa特异性地与血管段结合。结合的时间过程是双相的，包括最初较快的可逆阶段，随后是较慢的不可逆阶段。后一阶段与¹²⁵I-因子Xa与一种可能不同于抗凝血酶III的血管定位蛋白之间形成共价复合物（分子量76,000）相关。血管结合的因子Xa对凝血酶原的激活受到抗牛因子V IgG的抑制，这表明因子Xa与内皮细胞表面提供的一种因子V样分子存在相互作用。加入抗凝血酶III抗体可防止上清液中因子Xa-抗凝血酶III和凝血酶-抗凝血酶III复合物的形成，并使表观凝血酶活性增加30 - 50倍。这些研究表明，新鲜获得的具有连续天然内皮细胞层的血管能够支持因子X和凝血酶原的激活：血管结合的因子IXa在VWF/因子VIII存在的情况下可激活因子X。因子Xa也可与血管结合并参与凝血酶原的激活。然而，血管壁上功能性抗凝血酶III的存在会降低凝血酶原激活的表观效率。
