Yeaman M R, Puentes S M, Norman D C, Bayer A S
Department of Medicine, Harbor-UCLA Medical Center, Torrance 90509.
Infect Immun. 1992 Mar;60(3):1202-9. doi: 10.1128/iai.60.3.1202-1209.1992.
Thrombin-induced platelet microbicidal protein (PMP) is considered to play an important role in preventing an important role in preventing streptococcal endocarditis. However, the structural features and functions of PMPs have not been well characterized, and their antibacterial spectra against other common endocarditis pathogens, such as the staphylococci, are not known. Thrombin stimulation of washed rabbit platelets (10(8)/ml) yielded a PMP-rich preparation with a specific activity of approximately 25 U/mg of protein as determined by Bacillus subtilis bioassay. Twenty-eight clinical and laboratory Staphylococcus aureus isolates, exposed to a standardized PMP preparation (100 U/ml for 2 h at 37 degrees C), exhibited a Poisson-distributed heterogeneity to the bactericidal action of PMP, with approximately one-third designated as PMP resistant. Gel filtration chromatography (Sephadex G-50) identified the bioactive moiety within PMP preparations to be in the major protein elution peak; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) presumptively identified PMP as a low-molecular-weight (MW) (8,500) protein present only in such bioactive protein peaks. Both the bioactivity of PMP preparations and the low-MW protein band were removable by specific anionic membranes (e.g., cellulose-acetate/nitrate), as well as by a variety of anionic resins, further corroborating the suspected cationic charge of PMP. In addition, both PMP bioactivity and the low-MW protein band were recoverable by 1.5 M NaCl elution of the anionic membrane filters post-PMP adsorptive removal. Adsorption of bioactive PMP preparations by highly PMP-susceptible B. subtilis (10(8) CFU/ml, 30 min) resulted in a near-complete loss of residual bioactivity; in contrast, adsorption of bioactive PMP preparations with less PMP-susceptible S. aureus strains failed to reduce bioactivity. Significant lysozyme contamination of PMP-rich preparations was ruled out by determination of differences between bioactive PMP preparations and exogenous lysozyme as regards (i) relative heat stabilities; (ii) differential bactericidal activity versus B. subtilis and Micrococcus luteus; and (iii) SDS-PAGE protein profiles. These data show that the bioactive PMP protein moiety is of low MW, is heat stable, is probably cationic (similar to leukocyte-derived defensins), and possesses potent bactericidal activity against a significant percentage of S. aureus isolates.
凝血酶诱导的血小板杀菌蛋白（PMP）被认为在预防链球菌性心内膜炎中起重要作用。然而，PMP的结构特征和功能尚未得到充分表征，其对其他常见心内膜炎病原体（如葡萄球菌）的抗菌谱也不清楚。用凝血酶刺激洗涤过的兔血小板（10⁸/ml），通过枯草芽孢杆菌生物测定法测定，得到了一种富含PMP的制剂，其比活性约为25 U/mg蛋白质。28株临床和实验室金黄色葡萄球菌分离株，暴露于标准化的PMP制剂（37℃下100 U/ml，2小时），对PMP的杀菌作用表现出泊松分布的异质性，约三分之一被指定为PMP耐药。凝胶过滤色谱法（Sephadex G-50）确定PMP制剂中的生物活性部分位于主要蛋白质洗脱峰内；十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）推测PMP是一种仅存在于此类生物活性蛋白峰中的低分子量（MW）（8500）蛋白质。PMP制剂的生物活性和低MW蛋白带都可以通过特定的阴离子膜（如醋酸纤维素/硝酸纤维素）以及各种阴离子树脂去除，这进一步证实了PMP可能带阳离子电荷的推测。此外，通过对阴离子膜过滤器进行1.5 M NaCl洗脱，可以回收PMP吸附去除后的PMP生物活性和低MW蛋白带。高度易感PMP的枯草芽孢杆菌（10⁸ CFU/ml，30分钟）对生物活性PMP制剂的吸附导致残留生物活性几乎完全丧失；相比之下，用对PMP敏感性较低的金黄色葡萄球菌菌株吸附生物活性PMP制剂未能降低生物活性。通过测定生物活性PMP制剂与外源性溶菌酶在（i）相对热稳定性；（ii）对枯草芽孢杆菌和藤黄微球菌的不同杀菌活性；以及（iii）SDS-PAGE蛋白质谱方面的差异，排除了富含PMP的制剂中存在大量溶菌酶污染的可能性。这些数据表明，生物活性PMP蛋白部分分子量低，热稳定，可能带阳离子（类似于白细胞衍生的防御素），并且对相当比例的金黄色葡萄球菌分离株具有强大的杀菌活性。
