Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice
Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice
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DOI：
10.1160/th08-04-0242
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发表时间：
2008-12-01
影响因子：
6.7
通讯作者：
Bahou, Wadie F.
中科院分区：
文献类型：
--
作者：
Damon, Andrea L.;Scudder, Lesley E.;Bahou, Wadie F.
异位递送因子VIII(FVIII)到巨核细胞(Mk)是一种通过将FVIIIa/FixA酶-辅因子复合体浓缩到活化的血小板膜上来局部产生张力酶的可行方法。我们利用核心大鼠血小板因子4(PF4)启动子在血友病A小鼠(rPF4/hBDD/FVIII-/-)背景下Mk/血小板限制性表达人B区缺失(HBDD)FVIII。RPF4/hBDD/FVIII-/-小鼠的血小板含量类似于122mU FVIII：C/1×10(9)个/ml，未检测到血浆FVIII：C，对α颗粒衍生的血小板因子V/Va功能无影响。成对的Tenase分析(+/-凝血酶)证实，与血小板Fv不同，血小板(Pt)FVIII需要凝血酶裂解才能完全激活。RPF4/hBDD/FVIII-/-暴露于血小板生成素(TPO)刺激下的小鼠显示出摩尔ptFVIII活性显著降低66%，而总ptFVIII生物量没有显著降低。通过止血剪尾法评估，摩尔ptFVIII浓度降低与表型校正丧失相关。使用表达来自rPF4启动子的人淀粉样前体蛋白(HAβPP)的转基因小鼠的比较研究证实，HAβPP的表达减少，而不影响内源性α颗粒PF4，从而建立了这些观察结果的概括性。虽然Mk/血小板释放的ptFVIII(不同于pfv)在蛋白降解方面是无效的，但我们也得出结论，血小板刺激对ptFVIII的生物利用度和表型疗效产生负面影响，这一结果与摩尔ptFVIII浓度最相关，而不是系统可用的ptFVIII。
Ectopic delivery of factor VIII (FVIII) to megakaryocytes (Mk) represents a viable approach for localized tenase generation by concentrating the FVIIIa/FIXa enzyme-cofactor complex onto activated platelet membranes. We utilized a core rat platelet factor 4 (PF4) promoter for Mk/platelet-restricted expression of human B-domain-deleted (hBDD) FVIII within the background of a haemophilia A mouse (rPF4/hBDD/FVIII-/-). Platelets from rPF4/hBDD/FVIII-/- mice contained similar to 122 mU FVIII:C/1 x 10(9) platelets/ml with no detectable plasmatic FVIII:C, and with no effect on alpha-granule-derived platelet factorV/Va function. Paired tenase assays (+/- thrombin) confirmed that platelet (pt) FVIII (unlike platelet FV) required thrombin cleavage for complete activation. rPF4/hBDD/FVIII-/- mice exposed to a thrombocytotic stimulus (thrombopoietin,TPO) demonstrated a statistically-significant 66% reduction in molar ptFVIII activity with a non-significant reduction in total ptFVIII biomass. Decreased molar ptFVIII concentration correlated with loss of phenotypic correction as evaluated using a haemostatic tail-snip assay. Comparative studies using a transgenic mouse expressing human amyloid-precursor protein (hA beta PP) from the rPF4 promoter confirmed diminished hA beta PP expression without affecting endogenous alpha-granule PF4, establishing generalizability of these observations. While Mk/platelet-released ptFVIII (unlike pFV) is proteolytically inactive, we also conclude that thrombocytotic stimuli negatively affect ptFVIII bioavailability and phenotypic efficacy, results which correlate best with molar ptFVIII concentration, and not systemically available ptFVIII.