Pidard D, Renesto P, Berndt M C, Rabhi S, Clemetson K J, Chignard M
INSERM U.353, Hôpital Saint-Louis, Paris, France.
Biochem J. 1994 Oct 15;303 ( Pt 2)(Pt 2):489-98. doi: 10.1042/bj3030489.
The proteolytic activity of the neutrophil serine-proteinase cathepsin G (CG) on platelet adherence receptors, the glycoprotein (GP) Ib-IX complex and the integrin alpha IIb beta 3, has been investigated. In the range 50 to 200 nmol/l, CG is a potent platelet agonist which induces shape change, granule exocytosis and aggregation. Investigation of the proteolysis of the receptors' subunits during the course of platelet activation by CG was performed by immunoblot analysis of platelet proteins using a panel of specific antibodies. Exposure of platelets for 3 min at 37 degrees C to CG at a concentration that induces full cell activation resulted in an extensive cleavage of the N-terminal region of the extracellular domain of GPIb alpha, the largest (relative molecular mass, M(r), 143,000) of the three subunits constituting the GPIb-IX complex. In contrast, no detectable proteolytic modification of the two other subunits, GPIb beta and GPIX, was detected. Similarly, we observed that neither of the two subunits of the alpha IIb beta 3 receptor were proteolytically modified by CG. Cleavage of GPIb alpha by CG leaves a remnant of the polypeptide chain with M(r) approx. 106,000 in the plasma membrane, while releasing into the extracellular milieu the N-terminal domain with M(r) in the range 40,000 to 46,000. N-terminal sequencing of the CG-derived fragments of GPIb alpha indicated that the Leu275-Tyr276 peptide bond was the primary cleavage site for this proteinase. Proteolysis of GPIb alpha was already detectable at concentrations of CG as low as 25 nmol/l, while with 200 nmol/l the cleavage was detected as soon as 10 s after exposure of platelets to the proteinase. Comparison of the kinetics and concentration dependency for the proteolysis of GPIb alpha and for the activation of platelets by CG showed that cleavage of the GPIb-IX receptor is an early event that accompanies exocytosis and aggregation. Quantitative evaluation of the conversion of GPIb alpha into its membrane fragment indicated that, under optimal conditions, a maximum of approx. 50% of the total GPIb alpha can be affected by proteolysis. However, this proteolysis was > 90% complete when platelets were in the presence of the potent antagonist prostacyclin, suggesting that cellular redistribution of the GPIb-IX receptor may also occur during activation by CG. These results thus indicate that the very early phase of platelet activation by CG is accompanied by extensive modifications in the structure and expression of the GPIb-IX receptor, an effect that might be of functional significance for the interaction of platelets with the vessel wall.
已对中性粒细胞丝氨酸蛋白酶组织蛋白酶G（CG）对血小板黏附受体、糖蛋白（GP）Ib-IX复合物和整合素αIIbβ3的蛋白水解活性进行了研究。在50至200 nmol/l范围内，CG是一种强效血小板激动剂，可诱导形态变化、颗粒胞吐和聚集。通过使用一组特异性抗体对血小板蛋白进行免疫印迹分析，研究了CG在血小板激活过程中对受体亚基的蛋白水解作用。将血小板在37℃下暴露于能诱导细胞完全激活的浓度的CG 3分钟，导致构成GPIb-IX复合物的三个亚基中最大的（相对分子质量，M(r)，143,000）GPIbα胞外结构域的N端区域发生广泛裂解。相比之下，未检测到另外两个亚基GPIbβ和GPIX有可检测到的蛋白水解修饰。同样，我们观察到αIIbβ3受体的两个亚基均未被CG进行蛋白水解修饰。CG对GPIbα的裂解在质膜上留下了一条M(r)约为106,000的多肽链残余物，同时将M(r)在40,000至46,000范围内的N端结构域释放到细胞外环境中。对GPIbα的CG衍生片段进行N端测序表明，Leu275-Tyr276肽键是该蛋白酶的主要裂解位点。在CG浓度低至25 nmol/l时即可检测到GPIbα的蛋白水解，而在200 nmol/l时，血小板暴露于该蛋白酶后10秒即可检测到裂解。比较GPIbα蛋白水解和CG激活血小板的动力学及浓度依赖性表明，GPIb-IX受体的裂解是伴随胞吐和聚集的早期事件。对GPIbα向其膜片段转化的定量评估表明，在最佳条件下，最多约50%的总GPIbα可受到蛋白水解的影响。然而，当血小板存在强效拮抗剂前列环素时，这种蛋白水解>90%完成，这表明GPIb-IX受体的细胞重新分布也可能在CG激活过程中发生。因此，这些结果表明，CG激活血小板的非常早期阶段伴随着GPIb-IX受体结构和表达的广泛修饰，这种作用可能对血小板与血管壁的相互作用具有功能意义。
