Bauer K A, Kass B L, Beeler D L, Rosenberg R D
J Clin Invest. 1984 Dec;74(6):2033-41. doi: 10.1172/JCI111626.
We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the coagulation system at concentrations considerably less than 1.0 nM.
我们开发了一种针对十二肽的放射免疫分析方法（RIA），该十二肽是在蛋白C被结合于血管内皮上的血栓调节蛋白的凝血酶激活时从蛋白C释放出来的。蛋白C激活肽（PCP）采用梅里菲尔德的固相方法合成。用戊二醛将合成类似物与牛血清白蛋白偶联后免疫家兔制备抗血清。所得抗体群体与125I标记的酪氨酸化配体以及不同浓度的未标记PCP一起用于构建一种双抗体RIA，该方法能够检测低至10 pM的该成分。我们已经确定合成的十二肽与天然肽具有相同的免疫反应性，并且在摩尔基础上，蛋白C的反应性不到PCP的1/2000。正常个体中肽的水平极低，以及血浆成分对免疫反应信号的非特异性贡献，使得有必要开发一种能够从血浆中可重复提取PCP并将其浓缩约20倍的方法。这种方法使我们能够证明，17名正常供体的血浆PCP水平平均为6.47 pM，并且在有弥散性血管内凝血证据的个体中观察到水平升高至180 pM。在这两种情况下，RIA信号在反相高压液相色谱上的迁移方式与天然十二肽相同，这支持了这些蛋白C激活测量的有效性。我们还注意到，7名用华法林充分抗凝的患者的平均PCP浓度为2.61 pM。我们的研究还表明，PCP从灵长类动物血浆中清除的半衰期约为5分钟。鉴于活化蛋白C的半衰期估计为10 - 15分钟，后一种酶似乎在浓度远低于1.0 nM的情况下对凝血系统的活化辅因子发挥作用。
