Rucinski B, Poggi A, James P, Holt J C, Niewiarowski S
Blood. 1983 Jun;61(6):1072-80.
Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6-0.9 M NaCl and at 1-1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000-7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno-precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.
通过肝素 - 琼脂糖亲和层析法将凝血酶刺激的血小板分泌的两种肝素中和蛋白纯化至同质。这些蛋白，分别称为猪血小板碱性蛋白（PBP）和猪血小板因子4（PF4），在0.6 - 0.9M NaCl和1 - 1.4M NaCl浓度下从肝素 - 琼脂糖柱上洗脱下来。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和氨基酸分析估计，猪血小板碱性蛋白的分子量为7000 - 7700道尔顿。该蛋白的等电点为pH 9.0。猪血小板碱性蛋白的氨基酸组成类似于人低亲和力血小板因子4（LA - PF4），只是猪蛋白不含酪氨酸。猪血小板因子4的分子量范围为10000（由氨基酸分析估计）至14000（由十二烷基硫酸钠聚丙烯酰胺凝胶电泳估计）。人血小板因子4和猪血小板因子4的氨基酸组成相似。在兔体内产生了针对猪血小板因子4和猪血小板碱性蛋白的单特异性抗体。竞争性放射免疫分析表明，人和猪血小板因子4之间以及猪血小板碱性蛋白与一组与人低亲和力结合肝素的分泌性血小板蛋白（β - 血小板球蛋白[βTG]和低亲和力血小板因子4）之间存在低但显著的免疫交叉反应性。用125I标记抗原进行直接免疫沉淀实验表明，所研究的所有四种蛋白（人血小板因子4、猪血小板因子4、人低亲和力血小板因子4或人β - 血小板球蛋白以及猪血小板碱性蛋白）具有共同的抗原决定簇。然而，具有相似肝素结合亲和力的异源抗原（人血小板因子4和猪血小板因子4）之间的免疫交叉反应程度高于具有不同结合亲和力的异源抗原（人血小板因子4和猪血小板碱性蛋白）之间的免疫交叉反应程度。总之，我们的发现表明这四种蛋白之间存在显著的结构同源性。
