Van Oost B A, Smith J B, Holmsen H, Vladutiu G D
Proc Natl Acad Sci U S A. 1985 Apr;82(8):2374-8. doi: 10.1073/pnas.82.8.2374.
Thrombin induces partial secretion (up to 60%) of beta-N-acetyl-D-hexosaminidase (EC 3.2.1.52) from untreated platelets. Preincubation of platelets with 10 mM NH4Cl for up to 2 hr resulted in a time-dependent and marked stimulation of thrombin-induced secretion of both this enzyme and other acid glycosidases from platelets. The enhancement of the thrombin-induced secretion was not due to cell lysis, and NH4Cl alone did not cause leakage of lysosomal enzymes into the medium. The effect could be reversed by reincubating the platelets in NH4Cl-free medium. Stimulation of thrombin-induced secretion also was produced by a series of aliphatic primary amines from methylamine to butylamine, and by micromolar concentrations of chloroquine. The effect of weak bases on platelets appeared to be quite specific for enhancing lysosomal enzyme secretion. Thrombin-induced secretion of adenine nucleotides from dense granules and of beta-thromboglobulin from alpha granules was slightly enhanced by NH4Cl but was slightly inhibited by methylamine. The only direct effect of the weak bases on platelets was the displacement of serotonin from dense granules. Accumulation of weak bases in acidic pools in the platelets (e.g., lysosomes) might, therefore, be responsible for the enhanced secretion of lysosomal enzymes. By using controlled digitonin-induced platelet lysis, it was found that preincubation of platelets with NH4Cl lowered the digitonin concentration required for enzyme solubilization. We suggest that loading of lysosomes with weak bases dissociates already bound enzyme inside the lysosomes, resulting in a more effective discharge upon stimulation by thrombin.
凝血酶可诱导未处理的血小板分泌部分β-N-乙酰-D-己糖胺酶（EC 3.2.1.52）（分泌量高达60%）。将血小板与10 mM氯化铵预孵育长达2小时，会导致凝血酶诱导的该酶及其他酸性糖苷酶从血小板中分泌的时间依赖性显著增强。凝血酶诱导分泌的增强并非由于细胞裂解，且单独的氯化铵不会导致溶酶体酶泄漏到培养基中。通过在无氯化铵的培养基中重新孵育血小板，这种效应可以逆转。从甲胺到丁胺的一系列脂肪族伯胺以及微摩尔浓度的氯喹也能刺激凝血酶诱导的分泌。弱碱对血小板的作用似乎对增强溶酶体酶分泌具有相当的特异性。氯化铵略微增强了凝血酶诱导的致密颗粒中腺嘌呤核苷酸以及α颗粒中β-血小板球蛋白的分泌，但甲胺则略微抑制了这种分泌。弱碱对血小板的唯一直接作用是使致密颗粒中的5-羟色胺移位。因此，弱碱在血小板酸性区室（如溶酶体）中的积累可能是溶酶体酶分泌增强的原因。通过使用受控的洋地黄皂苷诱导的血小板裂解，发现用氯化铵预孵育血小板可降低酶溶解所需的洋地黄皂苷浓度。我们认为，溶酶体中加载弱碱会使溶酶体内已结合的酶解离，从而在凝血酶刺激时导致更有效的释放。
