凝血酶引起血管内皮细胞渗透性改变的机制
Mechanism of Thrombin-Induced Changes in Vascular Endothelial Cell Permeability

作者: 叶浩文 , 李 丽 :暨南大学附属广州市红十字会医院,广东 广州;

关键词: 凝血酶血管内皮细胞渗透性Thrombin Vascular Endothelial Cell Permeability

摘要: 完整的内皮屏障功能与机体正常的生理活动密不可分,血管内皮屏障功能受损,会出现血管渗漏、水肿等一系列病理变化,而凝血酶正是目前经典的内皮屏障功能破坏剂。从18世纪至今,人们追寻于研究凝血酶诱导内皮渗透性升高的相关机制,希望能从中找到对抗凝血酶破坏屏障功能的方法。本文将针对研究中凝血酶诱导血管内皮细胞渗透性增加的机制,包括凝血酶及其受体的相关特性,以及Ca2+、PKC、Rho GTPases家族、酪氨酸激酶/磷酸酶途径等在凝血酶诱导EC渗透性增加中的作用,结合近年来有关S1P及自噬相关通路的作用作一综述。

Abstract: Intact endothelial barrier function is closely related to body physiological activities. Vascular leak-age, edema and a series of pathological changes will arise if vascular endothelial barrier function is damaged. Thrombin is the most common breaker of endothelial barrier function. Since the 18th century, researchers have investigated the mechanism of thrombin-induced increase in endothelial permeability, seeking a way to ameliorate thrombin-induced barrier disruption. This article is a summary focus on recent studies on the mechanism of thrombin inducing endothelial cell permea-bility, including the characteristics of thrombin and its receptor, and the role of Ca2+, PKC, Rho GTPases families, tyrosine kinase/phosphatase pathway, combined with the function of S1P and autophagy-related pathways.

1. 引言

血管内皮细胞层是血液和组织交换物质的一个半透性动态屏障,与体内物质渗透、炎症反应、血液凝固及其他生理病理活动密切相关 [1] [2]。正常血管内皮细胞(Endothelial Cell, EC)之间通过肌动蛋白–肌球蛋白偶联的内皮细胞收缩机制及肌动蛋白细胞骨架锚定的粘附连接(Adheren Junction, AJ),即细胞骨架蛋白通过连环蛋白与VE-钙黏蛋白相连来调节血管屏障功能 [2] [3];诸如凝血酶、组胺、血管内皮生长因子(Vascular Endothelial Growth Factor, VEGF)的炎性介质会通过改变细胞连接的状态来影响EC渗透性变化 [4] [5],当血管EC屏障功能受损时,就会出现血管渗漏、水肿等一系列病理变化,同时也是动脉粥样硬化的损伤因素之一 [6] [7],其中以凝血酶对于血管EC渗透性改变最为经典。从80年代至今的科学研究中,凝血酶已被明确证实具有升高血管EC渗透性的作用,但其具体作用机制仍未完全明确,本文将总结当前凝血酶引起血管EC渗透性改变的相关机制,为防治凝血酶诱导的血管EC渗透性升高提供参考。

2. 凝血酶及其受体

凝血酶分子量大小为37 kda,是由A和B两条多肽链组成的异二聚体,轻链A由36个残基组成,通过残基C1和C122之间的二硫键共价连接到重链B上,重链B则有259个残基,其中含有3个链内二硫键 [8] [9] [10]。作为凝血级联中的关键效应蛋白酶,凝血酶本质上是一种Na+激活的变构丝氨酸蛋白酶 [9] [10],其上Na+结合位点的占据控制了凝血酶的某些活性,对于凝血酶的变构作用至关重要 [10]。当血管受损后,通过内源性与外源性凝血途径,FX被激活,生成凝血酶原酶复合物,促进凝血酶原激活从而形成凝血酶 [9]。凝血酶在血液循环中存在的时间很短,这是因为抗凝血酶(肝脏与血管内皮细胞产生)的存在,可与之结合从而灭活凝血酶,同时,凝血酶在产生后,也会通过激活蛋白质C,负反馈抑制凝血酶自身的产生 [9],凝血酶的短暂存在及快速灭活,与凝血酶对于血管内皮渗透性短暂升高后恢复有一定关系。除了参与生成纤维蛋白单体、激活F XIII以及激活蛋白质C系统等凝血相关过程外,凝血酶与血管内皮细胞屏障功能密切相关。

凝血酶的绝大多数细胞效应,是通过激活与异三聚体G蛋白相偶联的蛋白酶激活受体(Protease Activated Receptor, PARs)超家族来发挥的 [11] [12]。PARs是一种独特的G蛋白偶联受体(G Protein Coupled Receptor, GPCR),其独特之处在于它们能被细胞外的丝氨酸蛋白酶水解PARs的N末端序列,从而产生新的末端,称之为“栓系配体(Tethered Ligand)”,而被凝血酶特定区域所激活,进而产生信号转导。PARs在血管内皮细胞、血小板,平滑肌细胞和其他细胞类型中表达。目前在人及鼠中已知的PAR家族有PAR1、PAR2、PAR3、PAR4,而人的PAR1、PAR3及PAR4能够被凝血酶激活,PAR2则是被胰蛋白酶激活,不能被凝血酶激活。凝血酶的四个受体中,又以PAR1最为主要,PAR1的激活,涉及G12/13、Gq、Gi蛋白通路,其被蛋白水解的N末端位于第41位精氨酸(Arg41)与42位丝氨酸之间(Scr42) [9] [13]。

与野生型小鼠相比,G12/13或Gq缺乏的小鼠,表现出明显的凝血酶诱导后MLC磷酸化水平降低,表明G蛋白以及偶联的PARs在当中的重要性 [14]。在抑制PAR1或PAR3后,凝血酶诱导的内皮细胞渗透性受到明显抑制 [15] [16],同时PAR1具有与PAR3形成异源二聚体的能力,该异源二聚体很容易在生理条件下形成,PAR3可以促进凝血酶与PAR1的结合作用,从而增强凝血酶的效应 [17],提示PARs在凝血酶破坏EC屏障功能中发挥着重要作用。凝血酶引起的血管EC渗透性改变涉及多种通路机制,而在众多的通路当中,PARs占据着核心位置,研究表明,其中包括凝血酶刺激后胞内Ca2+升高、PKC的激活、RhoA信号的诱导在内途径,皆与PARs (特别是PAR1)的激活密切相关 [18]。

3. 凝血酶的酶活性对内皮细胞单层渗透性的影响

早期学者认为,凝血酶作为一种丝氨酸蛋白酶,其本身具有酶活性,除了通过受体介导机制外,凝血酶可通过其本身的酶活性直接影响血管EC单层渗透性 [19] [20] [21]。α-凝血酶的衍生物可分为:δ-凝血酶(具有89%凝血酶活性的凝血酶制剂,因此有结合活性及酶促活性)、γ-凝血酶(具有酶促活性的胰酶制剂,据报道该酶缺乏受体结合活性)、DIP-α-凝血酶(Diisopropylphosphorofluoridate-α-thrombin)以及PPACK-α-凝血酶(D-Phe-Pro-Arg-chloromethylketone-α-thrombin),后两者酶活性不高,但具有高亲和的受体结合活性) [19]。使用Transwell小室渗透研究模型,在单用δ-凝血酶或γ-凝血酶处理牛肺动脉内皮细胞时,可见125I-白蛋白渗透率明显升高,而在该用DIP-α-凝血酶及PPACK-α-凝血酶这两种非酶促形式的衍生物时,并不能增加125I-白蛋白渗透率 [19] [20]。

另外,有研究表明,α-凝血酶及γ-凝血酶都能激活磷脂酶C (Phospholipase C, PLC),导致1,4,5-三磷酸肌醇(Inositol Triphosphate 3, IP3)的产生及蛋白激酶C (Protein Kinase C, PKC)的激活,从而通过影响Ca2+浓度来发挥升高渗透性的作用,而DIP-α-凝血酶及PPACK-α-凝血酶是无酶活性的,在人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cell, HUVEC)中,该两者并不能增加胞内Ca2+浓度 [21],上述情况表明,缺乏酶活性、单有受体结合活性的凝血酶衍生物,并不能发挥影响通透性的作用,这提示酶活性在凝血酶对内皮细胞单层渗透性的作用上是必不可少的。当前关于凝血酶与EC渗透性的研究,很少提及酶活性的作用,至于酶活性本身通过增强受体结合作用还是酶活性本身通过其他相关机制来作用于内皮屏障功能,目前尚未有明确定论。

4. Ca2+在凝血酶诱导的内皮细胞渗透性升高的作用

作为常见的胞内信使物质,Ca2+很早就被证实与凝血酶诱导的屏障功能息息相关 [22]。凝血酶通过上述机制水解与G蛋白相偶联的凝血酶受体,激活磷脂酰肌醇(Phosphatidylinositol, PI)依赖的PLC催化水解磷脂酰肌醇4,5-二磷酸(Phosphatidylinositol 4,5-diphosphate, PIP2),并生成两个重要的第二信使,分别是1,2-二酰基甘油(Diacylglycerol, DAG)和IP3。DAG可激活PKC,IP3更是经典的促Ca2+分泌剂,可激活Ca2+内流,而这引起的持续激活的PKC以及内流Ca2+的增加,与骨架蛋白、连接蛋白的改变密切相关 [21] [23] [24]。

内皮细胞在静息状态下,细胞浆维持着非常低的游离Ca2+浓度,范围为30~100 nmol/l,在细胞膜两侧的Ca2+浓度大概为胞浆的20,000倍,而胞浆低Ca2+浓度的维持,跟细胞膜上的跨膜通道(如浆膜钙ATP酶通道,Plasma Membrane Calcium ATPase, PMCA)及内质网膜上的跨膜通道(如sarco/ER Ca2+-ATPase通道)密切相关 [25] [26]。凝血酶作用于EC后,会通过刺激GPCR或者酪氨酸激酶受体(Receptor Tyrosine Kinase, RTK),激活PLC,进而引起IP3生成增加,IP3会先介导内质网膜上的钙离子释放,而后待内质网钙离子储存耗竭后,再通过钙库依赖性阳离子通道(Store-Operated Cation Channels, SOCs)启动胞外Ca2+内流 [24] [27]。据研究报道,凝血酶也可以通过水解G蛋白偶联受体而产生的DAG来激活受体依赖性阳离子通道(Receptor-Operated Cation Channels, ROCs)来直接促进胞外Ca2+内流,而无需先耗尽内质网钙库 [28]。另外,有实验表明,凝血酶也可通过介导内质网钙离子浓度感受器(stim1)来实现钙库依赖性Ca2+内流(Store-Operated Calcium Entry, SOCE),而该过程涉及到钙释放激活钙通道蛋白1 (Orai1)的参与 [27]。

研究表明,升高的Ca2+与钙调蛋白(Calmodulin, CaM)结合后,会引起CaM构象发生改变,使CaM的疏水面暴露,从而使下游蛋白质与CaM相结合,如特异性钙调蛋白激酶(CaM-Kinases, CaMK)——肌球蛋白轻链激酶(Myosin Light-Chain Kinase, MLCK),导致肌球蛋白轻链(Myosin Light-Chain, MLC)磷酸化,细胞骨架重排,同时,VE-钙黏蛋白之间的连接分解,细胞骨架收缩力大于细胞间黏附力,细胞间隙增加,渗透性升高 [24]。多功能CaMK-CaMKII被证明对于内皮屏障功能有明显的影响。例如,凝血酶增加了CaMKII的活化,而用KN-93抑制CaMKII的活化则减弱了凝血酶介导的内皮渗透性的增加 [29]。在凝血酶诱导的EC高渗透性过程中,升高的胞内Ca2+除了通过Ca2+/CaM-CaMK-pMLC来介导屏障功能的变化,还会去影响PKC、Rho、cAMP信号来发挥屏障受损作用(见下文描述)。

Ca2+也会通过影响Ca2+结合蛋白的作用来影响屏障作用,如膜联蛋白A2,具有将VE-钙黏蛋白复合物连接至细胞膜表面及肌动蛋白丝的能力,有研究得出,A2的缺乏,显示出微血管内皮渗透性升高的表现 [30],这也进一步说明Ca2+在整个EC屏障功能中的重要作用。

5. PKC在凝血酶诱导的内皮细胞渗透性升高的作用

PKC属于丝氨酸/苏氨酸激酶家族,目前至少存在11种同工型,按照不同的特性,其同工型可分为三个亚家族:经典型PKC (cPKC,包括α,βI,βII,γ)、新型PKC (nPKC,包括δ,ϵ,η和θ)和非经典型PKC (aPKC,ζ,λ),其中,经典型PKC是钙离子依赖性的,且受DAG、磷脂酰丝氨酸(Phosphatidylserine,PS)、佛波酯(Phorbol Myristate Acetate, PMA)等的调节,新型PKC受DAG或PMA双重调节,但不受钙离子激活 [31],而非经典型目前其确切的激活机制尚不清楚,人们只知道这两种亚型的激活不依赖于Ca2+、DAG及PMA,但依赖于PS并可被磷酸肌醇-3激酶(Phosphatidylinositol 3 Kinase, PI3K)的两种产物(PIP2和PIP3)激活 [31],这提示aPKC可能是PI3K的下游靶标。

PIP2经水解后产生的DAG会激活PKC的产生,虽然PKC会负反馈抑制PI-PLC的激活,但PKC的激活与钙离子的增加对凝血酶诱导的磷脂酶A2 (Phospholipase A2, PLA2)以及磷脂酶D (Phospholipase D, PLD)水解膜磷脂酰胆碱(卵磷脂,Phosphatidylcholine, PC)产生花生四烯酸(Arachidonate, AA)和磷脂酸(Phosphatidic Acid, PA)的释放至关重要 [23],据国外研究报道,PA很可能作为细胞内的第二信使直接激活PKC或促进磷脂酸磷酸酯酶水解为产生DAG,从而产生PKC的持续激活 [21] [23]。另外,凝血酶引起的胞内高钙,也会进一步促进PKC的激活 [23]。

凝血酶刺激后可引起Caldesmon77和波形蛋白的磷酸化来增加血管内皮渗透性,而在使用PKC抑制剂星形胞菌素后,可明显看到上述两种蛋白磷酸化减少,这也进一步证实了PKC在其中的重要性 [32]。同时,PKC同样介导凝血酶诱导的肌动蛋白、肌球蛋白、Ca2+/钙调蛋白结合蛋白及中间丝蛋白磷酸化,综合影响AJ的多个相关蛋白 [32]。

另外,PKC还与凝血酶刺激后p120-连环蛋白及VE-钙黏蛋白的解离、钙黏蛋白复合物稳定性减弱密切相关 [33] [34]。研究表明,凝血酶诱导的PKCα活化,可经过Rho-GDI (鸟嘌呤核苷酸解离抑制剂)的磷酸化或者p115RhoGEF磷酸化途径激活RhoA,进而降低HUVECs的跨上皮电阻(Trans-epithelium electrical resistant, TER) [35]。

新型PKC与Ca2+调控无关,而在使用PKCδ抑制剂或者沉默PKCδ后,皆显示出凝血酶诱导的TER及MLC磷酸化降低,提示PKCδ同样参与到凝血酶作用通路当中,且不受胞浆内Ca2+的影响 [36]。

6. RhoA、Rac1及cAMP在凝血酶诱导的内皮细胞渗透性升高的作用

Rho家族有RhoA、RhoB及RhoC三种同工型 [37],目前国内外研究当中,RhoA已被证实参与到凝血酶升高内皮细胞通透性的机制当中 [38] [39],凝血酶在刺激PAR1受体后,G12及G13会结合鸟嘌呤交换因子(Rho Guanine Exchange Factors, Rho GEFs),诱导RhoA短暂快速的升高,但不引起Rac1的升高 [40],激活Rho激酶(Rho-Associated Kinase,ROCK,RhoA的下游靶效应蛋白),导致蛋白磷酸酶-1M (Protein Phosphatase-1M, PP1M)失活,从而通过MLCP来调节肌球蛋白磷酸化、应力纤维形成以及细胞间隙增大等的改变,来发挥影响内皮屏障功能的作用 [38]。在凝血酶影响MLC的过程中,主要通过Ca2+-MLCK及Rho-MLCP两个方式来调节MLC磷酸化。另外,有研究表明,单独敲低RhoA和RhoB基因,内皮屏障功能明显受到影响,而单独敲低RhoC对内皮屏障功能影响不大 [39],这提示,对于凝血酶诱导的内皮细胞渗透性改变的过程中,主要是由RhoA和RhoB介导的,而RhoC则被证实促进了Rac1依赖性的内皮屏障功能的恢复,Rac1与凝血酶快速引起内皮屏障功能受损后屏障功能的恢复有关 [39]。除了通过Rho GEFs途径来激活RhoA,Ca2+途径也可激活RhoA,但RhoA对于凝血酶诱导的内皮胞浆Ca2+似乎没有影响 [41],这表明,RhoA在凝血酶Ca2+通路中,位于Ca2+下游。

单层内皮细胞屏障功能与细胞间连接和骨架蛋白磷酸化及重排密切相关,作为经典的调节钙黏蛋白及骨架蛋白的相关调节剂,Ras超家族之一的小鸟嘌呤核苷酸三磷酸酶(Rho GTPases)参与了凝血酶影响内皮细胞渗透性改变 [5]。目前Rho GTPases家族以Rho家族、Rac家族及Cdc42研究最为广泛,其中,Rac1通过介导肌动蛋白重构保护内皮屏障功能,RhoA则通过诱导细胞骨架改变导致内皮渗透性升高。有报道Cdc42与凝血酶诱导的应力纤维形成有关,但却不影响内皮细胞渗透性情况 [42]。

另外,RhoA跟另一个调节血管内皮屏障功能的第二信使cAMP存在交互作用。细胞内cAMP水平对于EC渗透性的维持及恢复起着重要作用,cAMP水平的升高可以显著增加内皮完整性,凝血酶诱导后,细胞的cAMP水平会出现短暂的降低,这也是凝血酶影响内皮完整性的通路之一 [43] [44]。而凝血酶导致的cAMP降低,通过PARs途径刺激磷酸二酯酶3 (Phosphodiesterase3,PDE3,可水解cAMP)进而降低胞浆cAMP水平或者PARs来直接抑制腺苷酸环化酶(AC,可合成cAMP)来减少cAMP的生成 [45] [46],凝血酶刺激的AC主要为AC6 (AC的其中一种亚型,为Ca2+抑制性亚型),为Ca2+依赖相关,但不需要CaM,与Ca2+依赖的PLA2以及COX和前列腺素合成酶介导的前列腺素有关,有研究发现,HUVEC与前列腺素受体拮抗剂共孵育,可显著降低凝血酶诱导的cAMP减少 [47],这表明前列腺素受体参与凝血酶破坏内皮完整性的机制当中。

在介导凝血酶诱导渗透性的过程中,cAMP主要有两种下游效应蛋白发挥作用,通过Epac1/Rap1通路来激活Rac1 [48],而Rac1会经LIM激酶起作用,通过磷酸化并抑制“丝切蛋白(Cofilin)”的形成,从而阻止Cofilin介导的皮层肌动蛋白解聚来发挥保护内皮完整性的作用 [49];另外,除了Epac1/Rap1的非PKA信号依赖性信号通路,cAMP还会经PKA途径来抑制RhoA活化 [50],经ROCK磷酸化MLC来影响肌球蛋白的收缩。综合上述,凝血酶会短暂性降低胞浆cAMP,通过Rac1和RhoA双重作用导致内皮完整受损。当前研究表明,在大血管内皮细胞中,与微血管细胞相比,发现PKA在调节内皮完整性方面占主导地位,而在微血管细胞中,PKA和Epac均起作用 [51]。但有趣的是,凝血酶刺激引起的cAMP降低,是短暂的,而后会出现缓慢的升高,这似乎可以解释凝血酶对于内皮细胞渗透性短暂影响后的恢复现象。

7. 酪氨酸激酶/磷酸酶途径在凝血酶诱导的内皮细胞渗透性升高的作用

随着对内皮渗透性研究的深入,发现蛋白质的酪氨酸磷酸化在其中扮演着重要角色。蛋白质磷酸酶可分为三种:丝氨酸/苏氨酸特异性蛋白磷酸酶、双特异性蛋白磷酸酶(能使丝氨酸、苏氨酸和酪氨酸残基上的蛋白质底物脱磷酸化)和蛋白质酪氨酸磷酸酶(Protein Tyrosine Phosphatases, PTP)。在细胞当中,PTP是含量最丰富的一类磷酸酶,可分为受体型及非受体型两种 [52]。当前研究中,包含SH2域的非受体PTP,即SHP2,被证实与凝血酶诱导的渗透性相关 [52] [53]。众所周知,VE-钙黏蛋白与p120连环蛋白以及β、γ-连环蛋白组合成复合物,构成AJ的重要部分,VE-钙黏蛋白复合物酪氨酸磷酸化与内皮通透性的变化密切相关,其中与VE-钙黏蛋白复合物相关PTP包括:PTP-μ,VE-PTP、PTP1B和SHP2,前三者被认为跟内皮细胞旁渗透性密切相关 [53] [54] [55]。SHP2包含两个串联的SH2域,紧接其后是PTP域及一个带有两个酪氨酸残基的C末端。凝血酶可诱导SHP2酪氨酸磷酸化并从VE-钙黏蛋白复合物中解离出来,同时,SHP2的损失也会引起VE-钙黏蛋白复合物中的几种连环蛋白酪氨酸磷酸化急剧增加,从而导致与α-连环蛋白脱节,AJ收到破坏,内皮细胞渗透性改变,而凝血酶对于SHP2的失活,被证实与PARs相关 [54]。有趣的是,在使用Src激酶抑制剂PP2和SU6656后,可以阻止凝血酶刺激后SHP2活性的增加,同时也会使凝血酶刺激后屏障功能恢复延迟 [54],这表明,Src激酶与凝血酶诱导SHP2变化有关。

另外,有研究表明,凝血酶会迅速刺激内皮细胞中粘着斑(Focal Adhesion, FA)的形成,其中涉及到RhoA以及ROCK通路,经凝血酶刺激后,FAK以RhoA依赖性的方式被酪氨酸磷酸化,与Src协同调节FA的解离装配 [56],同时,GIT1,一种Src激酶的底物,会磷酸化易位至FAs当中,共同调节FA的改变,从而影响渗透性 [57]。

8. S1P在凝血酶诱导的内皮细胞渗透性升高的作用

1-磷酸鞘氨醇(Sphingosine 1-Phosphate, S1P)是一种脂质介质,参与多种细胞生理反应,在维持正常的内皮细胞屏障功能中具有重要作用。S1P通过作用于位于内皮细胞表面的受体(S1PR)发挥对EC屏障功能的影响,S1P有5种特异性受体(S1PR1-5),其中在血管组织中,以S1PR1-3表达为主,当前研究认为,S1P在调节EC屏障功能方面具有双向作用,这取决于S1PR1-3几种受体表达和激活之间的平衡 [58] [59];生理浓度下(0.5~1 μmol/L),S1P主要与S1PR1结合,激活Rac家族,通过影响骨架蛋白来发挥EC屏障保护作用,而当浓度较高时(如超过10 μmol/L),则以结合S1PR2为主,介导RhoA依赖的屏障损伤作用,S1PR3研究相对较少,但被证明与保护EC屏障功能相关 [59]。研究表明,S1P-S1PR轴是PAR-1的下游信号 [60],而凝血酶通过PAR-1引起的EC屏障破坏途径以及S1P-S1PR1的EC屏障保护途径存在交叉 [61],凝血酶刺激内皮细胞后,通过PKCα途径,可以上调鞘氨醇激酶1 (Sphingosine Kinases 1, SphK1),诱导S1P生成增加,增加的S1P会激活1-磷酸鞘氨醇受体1 (Sphingosine-1-Phosphate Receptor-1, S1PR1),进而通过激活Rac1通路来减弱及恢复凝血酶诱导的内皮损伤及血管渗漏,而凝血酶诱导的S1P增加以及S1PR1激活Rac1通路的过程似乎与凝血酶快速升高EC渗透性后的恢复有关。在敲除SphK1或者S1PR1的小鼠中,凝血酶刺激后会显示出比对照组小鼠更为严重的血管渗透反应 [62] [63] [64] [65],另外,凝血酶诱导的S1P升高,是否有S1PR2及S1PR3的参与,目前仍未有确切定论。

9. 自噬在凝血酶诱导的内皮细胞渗透性升高的作用

自噬是细胞对胞内物质的包被、吞噬后在溶酶体中降解的过程,当中涉及到“隔离膜(Isolation Membrane)”囊泡及自噬体的形成、自噬体结合溶酶体、溶酶体内降解等过程 [66]。有学者对凝血酶诱导的血管内皮通透性与自噬之间的关系做了研究,分别使用多个阶段的自噬抑制剂(3-MA——PI3K通路抑制剂,干扰自噬体形成,NAC——活性氧ROS清除剂,抑制自噬的启动,BafA1——阻止自噬体和溶酶体结合和CQ——阻止自噬体和溶酶体结合)处理HMEC-1和HUVEC,皆显示出凝血酶诱导的的内皮渗透性减弱 [67],同时,凝血酶在处理10 min后明显可见LC3转化,这表明,自噬确实参与到凝血酶诱发的内皮渗透性损伤当中。巨噬细胞迁移抑制因子(Macrophage Migration Inhibitory Factor, MIF)也被证实参与了凝血酶触发的自噬和内皮细胞通透性升高当中 [67],在凝血酶诱导的内皮渗透性过高的过程中,细胞内MIF与MLC沿肌动蛋白应激纤维周围共定位,从而导致MIF分泌 [68],使用MIF抑制剂(ISO-1,p425)可以明显降低凝血酶诱导的内皮自噬及渗透性变化 [66]。有学者通过沉默自噬相关基因Beclin1,发现可以减少凝血酶诱导后的黏附连接处VE-钙粘着蛋白的丢失、肌动蛋白解聚蛋白Cofilin1的磷酸化/失活、RelA/p65核转运所需的肌动蛋白应激纤维形成,从而改善凝血酶引起的渗透性升高现象 [69];也有相关学者沉默ATG7 (一种必须的自噬调节剂)后得到同样类似的结果 [70]。这也进一步证实了自噬在凝血酶诱导内皮渗透性过程当中的重要性,但是自噬涉及的相关蛋白较多,凝血酶究竟通过何种机制影响自噬来调控内皮屏障功能,目前尚未得到有效证实。

10. 结论

凝血酶诱导EC渗透性升高的机制有很多,诸如Ca2+升高、PKC激活以及RhoA升高等通路,都被证实围绕着PAR1来实现,可见PAR1在凝血酶与内皮屏障之间的重要性;另外,关于S1PR以及自噬在发挥凝血酶升高EC渗透性的过程当中是否有PAR1的调节,目前还需证实。随着对凝血酶与EC渗透性的深入研究,阻断或者干扰当中的通路分子,或许能为临床上防治异常升高凝血酶所导致的EC渗透性增加提供帮助。

NOTES

*通讯作者。

文章引用: 叶浩文 , 李 丽 (2021) 凝血酶引起血管内皮细胞渗透性改变的机制。 生物医学, 11, 96-105. doi: 10.12677/HJBM.2021.112013

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