CHARACTERIZATION OF HUMAN-PLATELET BASIC-PROTEIN, A PRECURSOR FORM OF LOW-AFFINITY PLATELET FACTOR-IV AND BETA-THROMBOGLOBULIN
CHARACTERIZATION OF HUMAN-PLATELET BASIC-PROTEIN, A PRECURSOR FORM OF LOW-AFFINITY PLATELET FACTOR-IV AND BETA-THROMBOGLOBULIN
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DOI：
10.1021/bi00356a023
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发表时间：
1986-04-22
期刊：
影响因子：
2.9
通讯作者：
NIEWIAROWSKI, S
中科院分区：
文献类型：
--
作者：
HOLT, JC;HARRIS, ME;NIEWIAROWSKI, S
采用离子交换、亲和、分子筛、高效液相色谱等方法，从凝血酶刺激、洗涤的人血小板上清液中分离纯化出血小板碱性蛋白(PBP)。用自动Edman降解法测定NH2末端氨基酸序列，发现9个唯一残基，紧随其后的是已建立的低亲和力血小板因子4/β-血栓球蛋白(LA-PF4/βTG)序列的10个残基。在9个碱基中有3个碱基，这是PBP等电点较高的原因。前体状态的其他证据包括所有三个物种的免疫交叉反应以及纤溶酶和胰酶从PBP产生电荷、疏水性和大小类似于βTG的物种的能力。PBP和LA-PF4的反相高效液相色谱图非常相似，并且从每个蛋白中分离出一个肽，其氨基酸组成与βTG的COOH末端胰解肽预测的氨基酸组成相同。正常血小板主要含有LA-PF4，其PBP占总βTG抗原的10%到30%。即使用三氯乙酸裂解新鲜的血小板，以提供最完全和最快速的蛋白分解活性抑制，这也是正确的。在这种情况下或在受刺激的血小板的释放上清液中从未检测到βTG本身，只有在未保护的裂解物中很少检测到。与早期的结果一致，PBP粗品对3T3细胞具有促有丝分裂作用，但高纯度的PBP和LA-PF4制剂不具有这一活性。
Platelet basic protein (PBP) was purified from the supernatant of thrombin-stimulated, washed human platelets by ion-exchange, affinity, molecular sieve, and high-performance liquid chromatography (HPLC). The NH2-terminal amino acid sequence was determined by automated Edman degradation, revealing 9 unique residues followed by 10 residues of the established low-affinity platelet factor 4/.beta.-thromboglobulin (LA-PF4/.beta.TG) sequence. Among the nine were three basic residues, accounting for the high isoelectric point of PBP. Additional evidence for precursor status includes the immunological cross-reactivity of all three species and the ability of plasmin and trypsin to produce from PBP a species resembling .beta.TG in charge, hydrophobicity, and size. Tryptic peptide maps of PBP and LA-PF4 obtained by reverse-phase HPLC were very similar, and from each protein, a peptide was isolated which showed the amino acid composition predicted for the COOH-terminal tryptic peptide of .beta.TG. Normal platelets contained predominantly LA-PF4, with PBP ranging from 10% to 30% of total .beta.TG antigen. This was true even when fresh platelets were lysed with trichloroacetic acid in order to provide the most complete and rapid inhibition of proteolytic activity. .beta.TG itself was never detected in this situation or in the release supernatant of stimulated platelets, and only rarely in unprotected lysates. In agreement with earlier results, crude preparations of PBP were mitogenic for 3T3 cells, but highly purified preparations of PBP and LA-PF4 were free of this activity.