Chuang J L, Schleef R R
92037, USA.
J Cell Biochem. 2001;82(2):277-89. doi: 10.1002/jcb.1113.
Platelet plasminogen activator inhibitor I (PAI-1), a trace alpha-granule protein, is a key physiological regulator of fibrinolysis. Because information on the packaging of PAI-1 into alpha-granules during megakaryocytopoiesis may reveal novel approaches for controlling hemostasis, this study investigated basal, plasmid-mediated, and alphavirus-mediated PAI-1 packaging into alpha-granules-like structures in the megakaryocytic cell line MEG-01. Differentiation of MEG-01 cells with phorbol myristate acetate (PMA) was observed to result in a four-fold increase in both secreted and cell-associated PAI-1 antigen over a four day period. Subcellular fractionation of PMA-treated MEG-01 cells on 45% self-forming Percoll gradients was employed to separate low density membrane and Golgi-rich fractions from a high density granule-containing region. A subsequent 30-60% pre-formed Percoll gradient was employed to remove contaminating lysosomes from the PAI-1/glycoprotein IIbIIIa-containing granules. Electron microscopy showed that these MEG-01 granules share a similar size distribution (350-600 nm) and morphology to platelet alpha-granules. PAI-1 (40 ng/mg protein) in isolated MEG-01 storage granules was approximately 10% of the levels present in isolated platelet alpha-granules. To elevate PAI-1 production/storage, two expression systems were investigated. Experiments with plasmids encoding PAI-1 and beta-galactosidase resulted in low transfection efficiency (0.001%). In contrast, Semliki Forest virus (SFV)-mediated gene transfer increased cellular PAI-1 by 31-fold (1,200 ng/10(6) cells at 10 MOI) in comparison to mock-infected cells. Pulse-chase experiments demonstrated that SFV/PAI-1 mediated gene expression could enhance PAI-1 storage 6-9-fold, reaching levels present within platelets. To document the ability of PAI-1 to be stored in a rapidly releasable form in MEG-01 cells, we isolated platelet-like particles from the media conditioned by the cells and examined secretagogue-induced release of PAI-1. Particles from SFV/PAI-1 infected cells display a 5-fold enhanced secretion of PAI-1 following treatment with ADP in comparison to particles incubated in the absence of secretagogue. These results suggest that SFV mediated gene expression in MEG-01 cells provides a useful framework for analyzing the production and storage of alpha-granule proteins.
血小板纤溶酶原激活物抑制剂I（PAI-1）是一种微量α-颗粒蛋白，是纤维蛋白溶解的关键生理调节因子。由于关于巨核细胞生成过程中PAI-1包装到α-颗粒中的信息可能揭示控制止血的新方法，本研究调查了基础状态、质粒介导和甲病毒介导的PAI-1包装到巨核细胞系MEG-01中的α-颗粒样结构中。观察到用佛波酯（PMA）诱导MEG-01细胞分化，在四天时间内，分泌型和细胞相关型PAI-1抗原均增加了四倍。采用45%自形成的Percoll梯度对经PMA处理的MEG-01细胞进行亚细胞分级分离，以从富含高密度颗粒的区域中分离出低密度膜和富含高尔基体的部分。随后采用30%-60%预形成的Percoll梯度从含PAI-1/糖蛋白IIbIIIa的颗粒中去除污染的溶酶体。电子显微镜显示，这些MEG-01颗粒与血小板α-颗粒具有相似的大小分布（350-600nm）和形态。分离的MEG-01储存颗粒中的PAI-1（40ng/mg蛋白）约为分离的血小板α-颗粒中PAI-1水平的10%。为了提高PAI-1的产生/储存，研究了两种表达系统。用编码PAI-1和β-半乳糖苷酶的质粒进行的实验导致转染效率较低（0.001%）。相比之下，与模拟感染的细胞相比，辛德毕斯病毒（SFV）介导的基因转移使细胞内PAI-1增加了31倍（在10MOI时为1200ng/10^6细胞）。脉冲追踪实验表明，SFV/PAI-1介导的基因表达可使PAI-1的储存增加6-9倍，达到血小板内的水平。为了证明PAI-1在MEG-01细胞中能够以快速释放的形式储存，我们从细胞条件培养基中分离出血小板样颗粒，并检测促分泌剂诱导的PAI-1释放。与未添加促分泌剂孵育的颗粒相比，来自SFV/PAI-1感染细胞的颗粒在用ADP处理后PAI-1的分泌增加了5倍。这些结果表明，SFV介导的MEG-01细胞基因表达为分析α-颗粒蛋白的产生和储存提供了一个有用的框架。
