Piccardoni P, Evangelista V, Piccoli A, de Gaetano G, Walz A, Cerletti C
Istituto di Ricerche Farmacologiche, Mario Negri, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy.
Thromb Haemost. 1996 Nov;76(5):780-5.
Thrombin-activated human platelets release substance(s) of a proteic nature which induce an increase in the intracellular calcium concentration in polymorphonuclear leukocytes (PMN). Aim of this study was to characterize the platelet released product(s) responsible for PMN stimulation. PMN-stimulating activity was isolated from platelet supernatant by FPLC and HPLC. The N-terminal sequence analysis revealed that the purified fractions consisted in 90% of a peptide of 73 amino acids and in 10% of a peptide of 74 amino acids; both are truncated forms of the connective tissue-activating peptide III (CTAP-III), a platelet alpha-granule product, and have 3 and 4 additional amino acids at the N-terminus compared with the neutrophil-activating peptide 2 (NAP-2): Asp-Leu-Tyr and Ser-Asp-Leu-Tyr, respectively. Treatment of platelet supernatant (previously depleted of PMN-activating nucleotides) with Affi-gel heparin resulted in the disappearance of PMN-stimulating effects, suggesting that NAP-2 variants, which are heparin-binding proteins, account for ATP-independent PMN-stimulating activity of the supernatant. Cross-desensitization between rNAP-2 and the platelet supernatant and inhibition by the anti-NAP-2 antibody are in agreement with this conclusion. Although NAP-2 and its variants are reportedly generated from the inactive precursors, CTAP-III and platelet basic protein, through a proteolytic cleavage, NAP-2 variants were not generated in our system by proteases deriving from platelets or contaminating leukocytes. Indeed, treatment of intact platelet suspensions with different protease inhibitors failed to modify the calcium stimulating activity of the resulting supernatants. In conclusion, thrombin-activated platelets release NAP-2 variants which are not generated outside the platelets by proteolytic processing but are released in an active form. This finding enhances our understanding of platelet-PMN interaction in thrombosis and inflammation.
凝血酶激活的人血小板释放出具有蛋白质性质的物质，这些物质会导致多形核白细胞（PMN）细胞内钙浓度升高。本研究的目的是鉴定负责刺激PMN的血小板释放产物。通过快速蛋白质液相色谱（FPLC）和高效液相色谱（HPLC）从血小板上清液中分离出刺激PMN的活性物质。N端序列分析显示，纯化后的组分中90%是由73个氨基酸组成的肽段，10%是由74个氨基酸组成的肽段；这两种肽段都是结缔组织激活肽III（CTAP-III，一种血小板α颗粒产物）的截短形式，与中性粒细胞激活肽2（NAP-2）相比，在N端分别多了3个和4个氨基酸：分别为天冬氨酸-亮氨酸-酪氨酸和丝氨酸-天冬氨酸-亮氨酸-酪氨酸。用肝素琼脂糖凝胶处理血小板上清液（先前已去除刺激PMN的核苷酸）后，刺激PMN的效应消失，这表明作为肝素结合蛋白的NAP-2变体是上清液中不依赖ATP刺激PMN活性的原因。重组NAP-2（rNAP-2）与血小板上清液之间的交叉脱敏以及抗NAP-2抗体的抑制作用均与该结论一致。尽管据报道NAP-2及其变体是由无活性前体CTAP-III和血小板碱性蛋白通过蛋白水解裂解产生的，但在我们的系统中，血小板或污染白细胞来源的蛋白酶并未产生NAP-2变体。实际上，用不同的蛋白酶抑制剂处理完整的血小板悬液，未能改变所得上清液的钙刺激活性。总之，凝血酶激活的血小板释放出NAP-2变体，但这些变体并非通过蛋白水解加工在血小板外产生，而是以活性形式释放。这一发现增进了我们对血栓形成和炎症中血小板与PMN相互作用的理解。
