CN108159421A - 磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途 - Google Patents
磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途 Download PDFInfo
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Abstract
本发明公开了磷脂酰丝氨酸(PS)阻断剂在制备治疗血小板数量减少相关疾病药物中的用途。本发明首次通过实验证明了凋亡和活化血小板的PS暴露,促使血小板在肝脏中被清除。研究证明血小板减少症患者体内血小板不仅发生活化而且发生凋亡,凋亡和活化导致血小板暴露PS,使其被肝脏的巨噬细胞吞噬。封闭PS或阻断PS外翻可抑制血小板被清除,表明PS阻断剂可以参与血小板数量变化相关疾病的治疗过程,抑制外周循环血中血小板数量减少。因此,本发明揭示了PS外翻导致血小板被清除的机制,而且抑制PS暴露依赖性的血小板清除的阻断剂具有开发成新型血小板保护药物、治疗血小板减少性疾病药物的潜力,极具科研和经济价值。
Description
技术领域
本发明属于血小板相关药物领域,具体涉及磷脂酰丝氨酸(Phosphatidylserine,PS)阻断剂在制备治疗血小板数量减少相关疾病药物中的用途。
背景技术
磷脂酰丝氨酸(PS)外翻是血小板活化和凋亡后期常见的现象。目前研究发现,血小板凋亡在生理条件、血库储存血小板和许多常见疾病中存在;而血小板活化在许多常见的疾病如感染、癌症和心脏病中存在。在这些血小板活化或凋亡发生的常见疾病中,可出现引起致命出血的血小板减少症。然而,关于凋亡或活化血小板是如何从血液循环中清除并导致血小板减少的病理机制至今仍不清楚。
血小板数量减少是临床上常见的症状,可导致出血甚至致死性内出血的严重后果。血小板数量减少相关疾病包括免疫性血小板减少症、感染所致的血小板减少疾病、继发性血小板减少疾病、药物引起的血小板减少疾病、血小板生成缺欠疾病、非免疫性血小板减少疾病、血小板生成减少导致的疾病、血小板破坏增多导致的疾病或血栓性血小板减少性紫癜等等。这些不同病因导致的血小板减少,其发病机制都是由于凋亡和活化导致的血小板寿命缩短,而被清除。免疫血小板减少症(Immune Thrombocytopenia,ITP)是一种常见的自身免疫性疾病,其特点是血小板计数低,可导致危及生命的出血。在ITP患者中检测到两种针对血小板受体的自身抗体,分别是抗纤维蛋白原受体糖蛋白(glycoprotein,GP)GPIIb/IIIa和(或)抗Von Willebrand Factor(VWF)受体GPIb-IX复合物的抗体。目前认为,自身抗体结合的血小板通过脾脏内Fc-FcγR结合吞噬引起血小板清除。因此,ITP的主要治疗策略是免疫抑制、免疫调节和脾切除术。然而,抗GPIb-IX自身抗体的ITP患者出现更严重的血小板计数下降。此外,大多数抗GPIb-IX自身抗体介导的血小板减少症对传统疗法的反应较差,如静脉注射免疫球蛋白G(Intravenous Immunoglobuling,IVIG)和类固醇治疗,甚至脾切除术,提示抗GPIb-IX自身抗体诱导的血小板破坏可能涉及不同的病因。
GPIbɑ是GPIb-IX复合体的主要亚基,事实上,经研究发现抗GPIba单克隆抗体可以在体外激活血小板,并在体内引起血小板被清除[Yan, R. et al. Glycoprotein Ibalphaclustering inducesMacrophage-mediated platelet clearance in the liver(GPIbα诱导肝脏中巨噬细胞介导的血小板清除). Thrombhaemost(血栓和凝血疾病杂志)113, 107-117 (2015);Bergmeier, W. et al. Structural and functional characterization ofthe Mouse von Willebrand factor receptor GPIb-IX with novel Monoclonalantibodies(新型单克隆抗体对小鼠血管性血友病因子受体GPIb-IX的结构和功能鉴定).Blood(血液学杂志)95, 886-893 (2000);Becker, B.H. et al. Effects of anantiplatelet glycoprotein Ib antibody on hemostatic function in the guineapig(抗血小板糖蛋白Ib抗体对豚鼠凝血功能的影响). Blood(血液学杂志)74, 690-694(1989);Cadroy, Y. et al. Relative antithrombotic effects of Monoclonalantibodies targeting different platelet glycoprotein-adhesive Moleculeinteractions in nonhuman primates(在非人灵长类动物中针对不同血小板糖蛋白粘附分子相互作用的单克隆抗体的相对抗血栓作用). Blood(血液学杂志)83, 3218-3224(1994)]。
我们进一步证明抗GPIba抗体通过Fc-非依赖机制使血小板在肝脏中被吞噬,有一篇报道显示[Li, J. et al. Desialylation is a Mechanism of Fc-independentplatelet clearance and a therapeutic target in immune thrombocytopenia(在免疫性血小板减少症中去涎酸诱导Fc–非依赖的血小板清除). Nat Commun(自然通讯)6, 7737(2015)],抗小鼠GPIba单克隆抗体诱导Fc-非依赖的血小板激活和在肝的清除支持了我们的结论。GPIba的去涎酸作用使得肝细胞通过Ashwell-Morell受体依赖方式清除血小板。此外,剪切诱导GPIba机械感觉域解折叠也会引发血小板清除。因此,越来越多的证据表明抗GPIba抗体,不像抗GPIIb/IIIa的自身抗体,可能通过一种Fc-非依赖的方式引起血小板清除,而且抗GPIba抗体诱导血小板减少的机制仍不清楚。
GPIba包含了几个重要配体的结合位点,包括细胞外N端的VWF和凝血酶位点。VWF多聚体与GPIba的相互作用诱导了脂筏中GPIb-IX复合物的易位和聚集,触发信号级联反应,如Akt活化和钙离子动员,导致血小板活化和血栓形成。我们之前发现GPIba-VWF的相互作用也可以诱导血小板凋亡,但作用机制仍然未知。我们最近报道了PKA介导的血小板凋亡在病理生理条件中广泛存在[Zhao, L. et al. Protein kinase A determines plateletlife span and survival by regulating apoptosis(蛋白激酶A通过调节细胞凋亡来决定血小板的寿命和存活率). J Clin Invest(临床研究杂志)(2017)]。此外,越来越多的证据表明,各种病理刺激引起的血小板凋亡和活化导致许多常见疾病的血小板减少,如感染、癌症和糖尿病。然而,这些常见疾病中血小板减少的发病机制尚不完全清楚,从而导致与血小板减少疾病相关的药物研究无从下手。
发明内容
要解决的技术问题:不同病因导致的血小板减少,其发病机制都是由于凋亡和活化导致的血小板寿命缩短,而被清除。免疫血小板减少症(ITP)是一种常见的自身免疫性疾病,主要由抗GPIIb/IIIa和GPIb-IX自身抗体引起。抗GPIbα抗体的ITP患者对Fc依赖的治疗策略表现出难治性,而且其致病机制未明。我们要解决的技术问题是进一步研究抗GPIbα抗体诱导血小板减少的具体机制,进而公开磷脂酰丝氨酸阻断剂在制备治疗血小板数量变化相关疾病药物中的用途。
技术方案:针对上述问题,本发明公开了磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途。
进一步的,所述的磷脂酰丝氨酸阻断剂为无机阻断剂或有机阻断剂中的一种或几种。
优选的,所述的无机阻断剂为氢化物、氧化物、酸、碱、盐中的一种或几种。
优选的,所述的有机阻断剂为烃类、烃的衍生物、糖类、蛋白质、脂肪、核酸、合成高分子化合物中的一种或几种。
优选的,所述的烃类为烯烃、烷烃、炔烃、芳香烃中的一种或几种;所述的烃的衍生物为卤代烃、醇、酚、醛、酸、酯中的一种或几种;所述的糖类为单糖、二糖、低聚糖、多糖中的一种或几种;所述的蛋白质为氨基酸、多肽中的一种或几种;所述的核酸为脱氧核糖核酸、核糖核酸中的一种或几种。
进一步的,所述的磷脂酰丝氨酸阻断剂为磷脂酰丝氨酸结合物中的一种或几种。
优选的,所述的磷脂酰丝氨酸阻断剂为抗磷脂酰丝氨酸抗体、膜联蛋白(Annexin-V)、乳凝集素(Lactadherin)中的一种或几种。
进一步的,所述的血小板数量减少相关疾病包括免疫性血小板减少症、感染所致的血小板减少疾病、继发性血小板减少疾病、药物引起的血小板减少疾病、血小板生成缺欠疾病或非免疫性血小板减少疾病、血小板生成减少导致的疾病、血小板破坏增多导致的疾病或血栓性微血管病。
进一步的,所述的免疫性血小板减少症包括特发性血小板减少性紫癜。
进一步的,所述的感染所致的血小板减少疾病包括细菌感染血小板减少疾病或病毒感染血小板减少疾病。
进一步的,所述的继发性血小板减少相关疾病包括糖尿病病人体内的血小板减少疾病、肿瘤病人体内的血小板减少疾病、心脑血管疾病病人体内的血小板减少疾病、药物治疗过程中导致的血小板减少疾病、脾功能亢进疾病、妊娠过程中血小板减少疾病、继发于再生障碍性贫血的血小板减少疾病、继发于脾功能亢进的血小板减少疾病、继发于白血病的血小板减少疾病、继发于系统性红斑狼疮的血小板减少疾病、继发于干燥综合症的血小板减少疾病或继发于电离辐射的血小板减少疾病。
进一步的,所述的药物引起的血小板减少疾病中,该药物为抗肿瘤药物、抗疟药物、抗心律失常药物、抗凝剂、抗生素、抗惊厥药物中的一种或几种。
进一步的,所述的药物为奎宁、奎尼丁或肝素的一种或几种。
进一步的,所述的血小板生成缺欠疾病包括先天性血小板生成不良、无巨核细胞性血小板减少、范可尼综合征、血小板膜糖蛋白Ib-IX缺乏或功能异常引起的伯纳德-苏利耶综合征、灰色血小板综合征、湿疹血小板减少伴免疫缺陷综合征、再生障碍性贫血与骨髓增生异常综合征所引起的血小板减少疾病、获得性血小板生成不良、化疗药物所引起的血小板生成欠缺疾病或放射损伤引起的血小板生成欠缺疾病。
进一步的,所述的血小板生成减少导致的疾病包括慢性再生障碍性贫血、骨髓增生异常综合征、放疗引起的血小板生成减少疾病或化疗引起的血小板生成减少疾病;所述的血小板破坏增多导致的疾病包括自身免疫性疾病引起的血小板破坏增多疾病、抗磷脂综合征引起的血小板破坏增多疾病、人类免疫缺陷病毒引起的血小板破坏增多疾病或药物性血小板减少症引起的血小板破坏增多疾病,所述的血栓性微血管病包括血栓性血小板减少性紫癜。
进一步的,所述的药物为片剂、胶囊剂、颗粒剂、丸剂、缓释制剂、控释制剂、口服液或贴剂。
进一步的,所述的药物包含药学上有效剂量的磷脂酰丝氨酸阻断剂和药学上可接受的载体。
进一步的,所述的药物通过口服、注射、喷雾吸入或经胃肠道进行给药。
有益效果:本发明公开了磷脂酰丝氨酸阻断剂在制备治疗血小板数量变化相关疾病药物中的用途。本发明首次通过实验证明了凋亡和活化血小板的PS暴露促使血小板在肝脏中被清除。研究证明抗GPIbα抗体的ITP患者血小板不仅发生活化而且发生凋亡,而且抗GPIbα抗体诱导Akt调控磷酸二酯酶介导的PKA活化依赖的血小板凋亡。凋亡和活化血小板暴露PS,使其被肝脏的巨噬细胞吞噬。阻断PS外翻可阻止结合抗体的血小板被清除,表明PS阻断剂可以参与血小板数量变化相关疾病的治疗过程,抑制外周循环血中血小板数量减少。因此,我们的研究结果对血小板活化或凋亡的疾病提供了一种病理机制,而且抑制PS暴露依赖性的血小板清除的阻断剂具有开发成新型血小板保护药物和新型治疗血小板减少性疾病药物的潜力,极具科研和经济价值。
附图说明
图1 为抗GPIba自身抗体的ITP患者的血小板发生活化和凋亡的指标表征图。其中a为P-选择素的含量变化图,b为线粒体膜电位(Mitochondrial TransmembranePotential,ΔΨm)去极化图,c为PS外翻表征图,d为检测半胱天冬酶Caspase-3酶切的蛋白质印迹(Western blot)结果图,e为依据图d得到的酶切Caspase-3的统计结果图。
图2 为抗GPIba抗体AN51、SZ2、HIP1不同时间诱导血小板凋亡和活化的指标表征图。其中a为P-选择素的表征图,b为PAC-1检测活化GPIIb/IIIa的表征图,c为线粒体膜电位(ΔΨm)去极化表征图,d为PS外翻表征图。
图3 为抗GPIba抗体R300在不同时间诱导血小板凋亡和活化的指标表征图。其中a为P-选择素的表征图,b为JON/A检测活化GPIIb/IIIa的表征图,c为线粒体膜电位(ΔΨm)去极化表征图,d为PS外翻表征图。
图4 为PS结合物抑制抗GPIba抗体诱导血小板被清除的血小板数量变化图。
图5 为PS外翻敲除小鼠的抗GPIba抗体诱导血小板清除减弱表征图。其中a为TMEM16F基因敲除小鼠的表征图,b为WT小鼠和TMEM16F+/-小鼠抑制血小板清除的表征图。
具体实施方式
1、试剂与材料:
JC-1购于中国碧云天公司,FITC-human CD62P、PE-human CD41、PE-mouse CD41购于美国Biolegend公司;R300、FITC-mouse CD62P、PE-JON/A购于德国Emfret公司;FITC-PAC1购于美国BD Biosciences公司;抗Human-caspase-3抗体购于美国Biolegend公司;Fermentas蛋白Marker(10~170KD)购自Thermo公司;FITC-lactadherin购于美国HaematologicTechnologies公司;NormalMouse IgG、Normal rat IgG购于美国Santa cruz公司;AN51和SZ2由苏州大学附属第一医院血研所提供。
2、实验用小鼠:
野生型(Wild Type,WT)C57BL/6小鼠,购自上海斯莱克实验动物有限责任公司。所有实验动物相关福利均严格按照中华人民共和国《实验动物管理条例》执行。动物实验经苏州大学医学伦理委员会批准。
3、洗涤血小板:
健康成人志愿者自肘正中静脉采血。献血者无烟酒等不良生活习惯,献血前2周内未服用任何影响血小板功能的药物。女性献血者处于非月经期。献血者均知情同意并签署协议书。实验方案经苏州大学附属第一医院伦理委员会批准,符合赫尔辛基宣言。
抽取一定量健康人静脉血,用1/7体积的柠檬酸葡萄糖缓冲液(Acid-citratedextrose,ACD)(2.5%柠檬酸三钠,2.0%葡萄糖,1.5%柠檬酸)抗凝剂抗凝。抗凝后的全血在300g下离心10-15min,离心后下层为红细胞,上层为富含血小板的血浆。小心的将上层液体吸出至新的离心管。富含血小板的血浆(Platelet-rich Plasma,PRP)在1500g下离心10min,沉淀为血小板,上层液体为乏血小板血浆。弃上清后,将血小板重悬于与富含血小板血浆等体积的柠檬酸葡萄糖盐水(Citrateglucose Saline,CGS)缓冲液(0.123MNaCl,0.033M葡萄糖,0.013M柠檬酸三钠,pH6.5)中,在1500g下离心5min以洗去血浆蛋白。重复此洗涤步骤一次。沉淀的血小板最终重悬于一定体积的改良Tyrode缓冲液(2.5mMhepes,150mM NaCl,2.5mM KCl,12mM NaHCO3,5.5mM D-glucose,1mM CaCl2,1mM MgCl2,pH7.4),浓度为3´108/mL。重悬后的洗涤血小板在室温下放置1h以使其恢复至生理状态后用于后续实验。
4、流式检测血小板活化和凋亡指标
病人及正常人全血经3.8%柠檬酸钠抗凝后,1500g离心分离得到乏血小板血浆(Platelet-poor Plasma,PPP),将得到的病人PPP与正常人血小板在37℃温育8h,分别用JC1(2µg/mL)检测线粒体膜电位去极化,FITC-lactadherin(10µg/mL)标记PS,FITC-humanCD62P(20µg/mL)标记P选择素以及FITC-PAC-1(20µg/mL)或FITC-JON/A(20µg/mL)标记活化GPIIb/IIIa。
将步骤3获得的人洗涤血小板分别与对照同型抗体小鼠IgG(10µg/mL)、AN51(10µg/mL)、SZ2(10µg/mL)、HIP1(10µg/mL)在37℃孵育,并在不同时间点(2h、4h、6h、8h、10h),分别用JC1(2µg/mL)检测线粒体膜电位去极化,FITC-lactadherin(10µg/mL)标记PS,FITC-human CD62P(20µg/mL)标记P选择素以及FITC-PAC-1(20µg/mL)或FITC-JON/A(20µg/mL)标记活化GPIIb/IIIa。
小鼠下腔静脉血3.8%柠檬酸钠抗凝后,1100rpm、11min离心后得PRP,静息20min后,与R300(5µg/mL)在37℃共孵育。在体外阻断血小板活化和凋亡后被清除的实验中,小鼠PRP分别提前与Annexin-V(100mM)、Lactadherin(100µM)在37℃下孵育,Annexin-V和Lactadherin均分别孵育15min和30min后,在不同组中均加入R300(5µg/mL)于37℃下孵育8h,并通过血小板计数检测血小板含量的变化。
5、Western blot分析Caspase-3蛋白
人洗涤血小板分别与正常志愿者或是ITP病人PPP在37℃共孵育8h后,加入等体积等体积的2×细胞裂解液(含2mM PMSF,2mM NaF,2mM Na3VO4,以及蛋白酶阻断剂)冰上裂解30min后,加入蛋白上样缓冲液,99℃,5min后,-80℃保存;Western blot分析,检测caspase-3蛋白水平。
6、TMEM16F基因敲除小鼠模型的构建
利用CRISPR/Cas9基因组编辑技术在TMEM16F的第二个外显子上诱导双链断裂从而建立TMEM16F小鼠突变模型,Cas9MRNA在体外进行转录,靶向于TMEM16F第二个外显子的两个单导向sgRNAs为5'-ACAATTGTCTGCCCCACCTTTGG-3'和5'-CTGATTCTCCAGTGATCCAAAGG-3'。单导向RNA在体外进行转录,将体外转录的Cas9MRNA和单导向sgRNAs注射到C57BL/6J小鼠受精卵的细胞质中,并转移到假孕受体中进行增值。然后通过PCR扩增对突变体进行筛选,PCR扩增所使用的引物为F-5'-TTTGACCTCTGGCTCATCTATTC-3',R-5'-CCTAGTCCTTCTGGGGTTGC-3',将突变体的PCR产物进行Sanger法测序来确定特定的突变。将突变体小鼠与C57BL/6J野生型小鼠进行共同繁殖,从而产生杂合的TMEM16F突变小鼠和纯合的TMEM16F突变小鼠。并通过Western-blot检测血小板中TMEM16F的缺失。按照与步骤5相同的实验条件将R300注入TMEM16F-/+小鼠体内后,检测TMEM16F-/+小鼠体内循环血小板数量的变化。
7、钙黄绿素标记小鼠血小板
(1)20-25g C57小鼠由2%戊巴比妥钠腹腔注射麻醉后,下腔静脉取血,经1/7小鼠ACD抗凝后混匀;
(2)小鼠全血生理盐水稀释一倍后,每管5mL稀释后全血,200g,11min,去上层乳白色液体,为小鼠富血小板血浆;
(3)小鼠PRP经1200g,2min离心后,去上清,CGS buffer重悬;
(4)室温600g,2min,去上清,CGS buffer重悬;
(5)室温600g,2min,去上清,MTB buffer重悬;
(6)上述洗涤血小板调至1×109/mL,与钙黄绿素-AM(calcein-AM)5µg/mL室温下孵育15min;
(7)加入等体积含20µg/mL PGI2 CGS buffer稀释;
(8)室温600g,2min离心,去上清,CGS(含10µg/mL PGI2)buffer重悬;
(9)室温600g,2min离心,去上清,MTB重悬;
(10)小鼠洗涤血小板调整至1×109/mL,加入1mM CaCl2,1mM MgCl2后静息2h备用。
8、血小板回输模型
上述calcein标记的小鼠血小板分别与对照抗体兔源IgG(2µg/mL)或是R300(2µg/mL)室温下孵育1h,在抑制实验中,提前5min向受体小鼠注射对照溶剂或AnnexinV(10µg/mL);向受体小鼠通过眼眶静脉注射上述R300孵育的血小板1×108个。在回输后0min、15min、30min通过眼眶静脉采血,经3.8%柠檬酸钠抗凝后,全血用PE-小鼠CD41在室温下标记15min,1mL PBS稀释后流式检测。所有的血小板(受体小鼠自己的血小板和回输血小板)均能够被PE-小鼠CD41标记,而回输的血小板还被calcein标记,因此用FL1和FL2双阳性血小板/FL2单阳血小板比例绘制回输血小板清除曲线。
9、统计学分析
试验数据以Prism 5.1统计软件(GraphPad)进行分析。数据经正态性检验符合正态分布,以±s表示,采用不配对Student’s t检验进行组间比较。分组数据经方差齐性检验后,采用单因素方差分析(one-way analysis of variance,ANOVA)进行组间比较;方差不齐者采用Kruskal-Wallis检验。p<0.05作为显著性差异界值。
10、实验结果:
(1)抗GPIba自身抗体的ITP患者的血小板发生活化和凋亡
为了研究抗GPIba抗体诱发血小板减少症的发病机制,我们用微球蛋白筛选了23例有抗GPIba抗体的ITP患者。从图1得出,正常血小板与抗GPIba自身抗体血浆孵育后,抗GPIba自身抗体血浆明显诱导血小板P-选择素(图1a)和PS外翻(图1c),使血小板活化。
从图1b得出,抗GPIba自身抗体血浆启动血小板内线粒体调控的凋亡程序,使血小板发生线粒体膜电位(ΔΨm)去极化。从图1d、e得出,通过直接检测caspase-3活性和检测caspase-3底物酶切情况,抗GPIba自身抗体血浆使血小板内caspase-3活性明显升高。这些数据表明,抗GPIba自身抗体血浆在体外可诱导血小板活化和凋亡。
(2)抗GPIba抗体诱导血小板凋亡和活化
为了进一步探明抗GPIba抗体对血小板的影响,并避免来自血浆的非特异性效应,我们选择了抗GPIba单克隆抗体AN51、SZ2、R300、HIP1。从图2和图3中得出抗GPIba抗体AN51、SZ2、R300诱导血小板P-选择素表达量增高(图2a和图3a),活化的GPIIb/IIIa活性升高(图2b和图3b),PS暴露水平提高(图2d和图3d),促使血小板活化,同时AN51、SZ2、R300显著诱导血小板ΔΨm去极化(图2c和图3c),这些数据进一步证明抗GPIba抗体可引起血小板活化和凋亡现象,抗GPIba抗体HIP1不能诱导血小板凋亡和活化。血小板凋亡与活化,单独每种情况或两者情况均可导致血小板在体内被清除。
(3)抗GPIba抗体诱导血小板PS暴露使其被清除
为了证明PS暴露是血小板活化和凋亡后导致血小板清楚的关键因素,因此,研究了PS在体内血小板被吞噬中的作用,在R300加入之前加入Annexin V后,Annexin V能够与PS结合,从图4中得出,与未加入Annexin V组相比血小板的含量明显升高,Annexin V能够与PS结合,有效的保护抗GPIba抗体诱导的血小板被清除。然而加入另外一种与PS结合的物质乳凝集素(Annexin V),加入乳凝集素(Annexin V)组的血小板的含量呈现降低的趋势,因此乳凝集素(Annexin V)不能抑制由PS诱导的血小板清除现象。以上结果表明,PS在抗GPIba抗体诱导血小板清除中具有重要的作用。
在血小板活化过程中,PS暴露需要TMEM16F。为了进一步证实PS暴露在血小板清除中的作用,我们建立了TMEM16F基因敲除小鼠。从图5a中得出,使用Western blot检测证实了血小板中TMEM16F的缺失。从图5b中得出,将R300注入WT小鼠和TMEM16F+/-小鼠体内后,注入R300的TMEM16F+/-小鼠与WT小鼠相比体内循环血小板数量明显升高。这些数据表明,PS暴露对于抗GPIba抗体诱导血小板清除至关重要。
PS外翻是是抗GPIba抗体诱导血小板活化和凋亡后血小板被清除的关键。因此,阻断PS可以抑制抗GPIba抗体诱导的血小板活化和凋亡后的清除。实验结果显示,膜联蛋白V确实明显减少了抗GPIba抗体诱导的血小板清除,因此膜联蛋白V可以作为PS阻断剂以降低血小板数量的减少。
综上可知,以上实验结果表明抗GPIba抗体诱导血小板活化和凋亡,活化和凋亡后的血小板暴露PS,使其被肝脏的巨噬细胞吞噬。封闭PS或者基因敲除阻断PS外翻均可降低血小板被清除。本发明揭示了PS外翻导致血小板被清除的机制,为自身抗体或其他致病因素引起的血小板减少症提出了新的治疗策略,极具科研和经济价值。
Claims (18)
1.磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途。
2.根据权利要求1所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的磷脂酰丝氨酸阻断剂为无机阻断剂或有机阻断剂中的一种或几种。
3.根据权利要求2所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的无机阻断剂为氢化物、氧化物、酸、碱、盐中的一种或几种。
4.根据权利要求2所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的有机阻断剂为烃类、烃的衍生物、糖类、蛋白质、脂肪、核酸、合成高分子化合物中的一种或几种。
5.根据权利要求4所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的烃类为烯烃、烷烃、炔烃、芳香烃中的一种或几种;所述的烃的衍生物为卤代烃、醇、酚、醛、酸、酯中的一种或几种;所述的糖类为单糖、二糖、低聚糖、多糖中的一种或几种;所述的蛋白质为氨基酸、多肽中的一种或几种;所述的核酸为脱氧核糖核酸、核糖核酸中的一种或几种。
6.根据权利要求1所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的磷脂酰丝氨酸阻断剂为磷脂酰丝氨酸结合物中的一种或几种。
7.根据权利要求6所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的磷脂酰丝氨酸阻断剂为抗磷脂酰丝氨酸抗体、膜联蛋白、乳凝集素中的一种或几种。
8.根据权利要求1至7任一所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的血小板数量减少相关疾病包括免疫性血小板减少症、感染所致的血小板减少疾病、继发性血小板减少疾病、药物引起的血小板减少疾病、血小板生成缺欠疾病或非免疫性血小板减少疾病、血小板生成减少导致的疾病、血小板破坏增多导致的疾病或血栓性微血管病。
9.根据权利要求8所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的免疫性血小板减少症包括特发性血小板减少性紫癜。
10.根据权利要求8所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的感染所致的血小板减少疾病包括细菌感染血小板减少疾病或病毒感染血小板减少疾病。
11.根据权利要求8所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的继发性血小板减少相关疾病包括糖尿病病人体内的血小板减少疾病、肿瘤病人体内的血小板减少疾病、心脑血管疾病病人体内的血小板减少疾病、药物治疗过程中导致的血小板减少疾病、脾功能亢进疾病、妊娠过程中血小板减少疾病、继发于再生障碍性贫血的血小板减少疾病、继发于脾功能亢进的血小板减少疾病、继发于白血病的血小板减少疾病、继发于系统性红斑狼疮的血小板减少疾病、继发于干燥综合症的血小板减少疾病或继发于电离辐射的血小板减少疾病。
12.根据权利要求8所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的药物引起的血小板减少疾病中,该药物为抗肿瘤药物、抗疟药物、抗心律失常药物、抗凝剂、抗生素、抗惊厥药物中的一种或几种。
13.根据权利要求12所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的药物为奎宁、奎尼丁或肝素的一种或几种。
14.根据权利要求8所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的血小板生成缺欠疾病包括先天性血小板生成不良、无巨核细胞性血小板减少、范可尼综合征、血小板膜糖蛋白Ib-IX缺乏或功能异常引起的伯纳德-苏利耶综合征、灰色血小板综合征、湿疹血小板减少伴免疫缺陷综合征、再生障碍性贫血与骨髓增生异常综合征所引起的血小板减少疾病、获得性血小板生成不良、化疗药物所引起的血小板生成欠缺疾病或放射损伤引起的血小板生成欠缺疾病。
15.根据权利要求8所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的血小板生成减少导致的疾病包括慢性再生障碍性贫血、骨髓增生异常综合征、放疗引起的血小板生成减少疾病或化疗引起的血小板生成减少疾病;所述的血小板破坏增多导致的疾病包括自身免疫性疾病引起的血小板破坏增多疾病、抗磷脂综合征引起的血小板破坏增多疾病、人类免疫缺陷病毒引起的血小板破坏增多疾病或药物性血小板减少症引起的血小板破坏增多疾病,所述的血栓性微血管病包括血栓性血小板减少性紫癜。
16.根据权利要求1至7任一所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的药物为片剂、胶囊剂、颗粒剂、丸剂、缓释制剂、控释制剂、口服液或贴剂。
17.根据权利要求1至7任一所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的药物包含药学上有效剂量的磷脂酰丝氨酸阻断剂和药学上可接受的载体。
18.根据权利要求1至7任一所述的磷脂酰丝氨酸阻断剂在制备治疗血小板数量减少相关疾病药物中的用途,其特征在于,所述的药物通过口服、注射、喷雾吸入或经胃肠道进行给药。
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