血栓调节素进展

血 栓 调 节 素™

摘要：自从发现血栓调节素™启动蛋白C(PC)抗凝系统的重要作用后[1,2]，其生物化学性质、物理结构和基因小鼠的研究提示它具有新的以及部分不依赖于PC和凝血酶如纤溶、炎症和胚胎发生等方面功能。本文献回顾TM分子最近文献关于其结构和功能的研究，阐明其基因多态性与人类疾病的关系，以及关于它在血栓形成、休克、动脉粥样硬化和癌症等状态下的功能变化，另外对其最新发现如炎症和胚胎发生中的作用也作以详细介绍。
关键词：炎症 胎盘 血栓调节素 血栓

TM的结构和功能

TM的氨基酸序列、二级结构、构象以及部分晶体结构均已解决[3-8]。TM是Ⅰ型跨膜糖蛋白，含557个氨基酸的，而无内在酶活性。大约一半在膜外，其结构域含与稍微有点似C型动物凝集素的N末端球形区[9-11]。此结构域对于受体内吞[12]、肿瘤生长调控[13]和炎症调节中内皮的作用[14]等具有重要作用。其他胞外区部分由6个表皮生长因子(EGF)模块组成的茎样结构组成[15]，其中EGF模块1-4、6具有1-3、2-4及5-6二硫键的原型[16]；EGF模块4-6含有功能重要的Ca2+结合位点[17-19]。富含丝/苏氨酸区在第六EGF模块和跨膜区之间，且其中含几个转录后N-及O-型糖链修饰和加上硫酸软骨素靶位点[20，21]。但从尿中分离的TM及其他重组可溶性的TM不含此糖链[22-28]。TM胞内区较短，而无类似于已知蛋白结构域在信号传导或蛋白间相互作用的任何功能。
TM的抗凝作用由凝血酶和PC的相互作用介导(见综述[3,5,29-31])。内皮细胞膜表面结合TM与凝血酶形成高亲和力的复合物，抑制凝血酶(Kd ~0.5nmolL-1)与纤溶酶和经凝血酶exosite1的蛋白酶活化受体(PAR-1)等的作用。相反地，TM-凝血酶复合物可促进两倍以上的凝血酶依赖的PC活化，是潜在的PC活化剂。由于微血管中含丰富的TM，其中产生的大量凝血酶被TM固定在局部范围。组成性抑制凝血酶和活化PC通过蛋白酶作用水解凝血因子Ⅴa和Ⅷa阻止凝血酶的大量产生是其重要的抗凝机制。与活化PC的TM辅因子功能一样，TM-凝血酶复合物也可活化血浆羧肽酶B，如凝血酶活化纤溶酶抑制物(TAFI)[32-34]。该酶有使纤维蛋白的羧基末端精氨酸和赖氨酸残基失去的作用，这些残基可使纤溶酶与纤维蛋白结合，因此它们一旦去除后血凝块则抵抗纤溶。现在知道TAFI也作用于其它底物，包括补体C5a和C3a[35,36]。TAFI从C5a去除羧基末端精氨酸可能是调控补体总活性的显性生理机制。单链尿激酶型纤溶酶原活化剂(uPA)为TM-凝血酶复合物的另一作用底物[37-40]，提示TM调控纤溶的广泛性。
凝血酶通过exosite1结合EGF5,6[3,5,8]。PC的结合以及PC活化辅因子表达需要第4个EGF模块和富丝/苏氨酸结构域的存在[41]。在体内，TM-凝血酶复合物的底物—PC很可能存在于内皮细胞受体(EPCR)的复合物中[42,43]，但未见TM与EPCR生理性接触的实验结果报道。TAFI与TM-凝血酶复合物结合位点不同PC活化，如TM的EGF模块，而且TAFI和PC竞争结合，从而形成与凝血酶不连续的表面接触[44-47]。连接到TM富含丝/苏氨酸结构域的硫酸软骨素提供另一凝血酶结合位点，参与PC活化，形成凝血酶-抗凝血酶复合物[48,49]，并提供疟原虫的结合位点[50-52]。TM也有其它不需与PC、TAFI或凝血酶相互作用的生物学功能，但需通过似凝集素样结构域[14]和EGF模块介导[53]。

图1 TM结构域和多种突变的定位
图1 TM结构示意图。TM由似C型动物凝集素的N-末端球形结构域、6个EGF模块茎样结构、转录后加N-型糖链及O-型糖链修饰靶位的富丝/苏氨基酸区、跨膜结构域和较短的胞内尾巴组成。EGF结构域5、6（红色）结合凝血酶；EGF结构域4活化PC（红/灰色）。EGF结构域3（灰/黑色）与TAFI作用。TM分子中氨基酸多肽性（一个硷基变化）、TM分子内定位、与表型的关系及突变对其功能的作用显示在右侧。TM增强子的核苷酸多肽性以负号标明。具体见正文。
MI，心梗；TE，血栓性疾病；PC，妊并发症；VST，静脉窦血栓；VT，静脉血栓。

人类疾病中TM基因的多态性

TM基因的多态性特征见图1。它们与血栓形成或动脉粥样硬化的关系是研究的焦点。TM基因5’末端调控元件的两个多态性使其转录减弱，从而使TM分子表达减少。在一部分有心梗(MI)史的患者鉴定出TM基因上游133C/A多态性[54]。亚洲人种上游3G/A相当常见(12%-15%)，并且与心梗史或者颈动脉粥样硬化有关[55-60]。它与静脉血栓的关系或胎儿晚期流产的关系不明确[47,53,54]。似凝集素结构域中A25T的多态性发生MI的风险高于正常两倍，这种多态性与获得性危险性的正相关已得到证明[61-63]。点突变并不改变TM活化PC的能力[64]，提示这种多态性与另一种突变的联系或者不依赖于PC的凝集素样TM结构域破坏[14]。第四EGF模块Arg385Ser多态性使TM表达减少两倍，TM辅因子活性减少4倍[64]。这个突变与连接第四、第五EGF模块的3’氨基酸端相近。结构域内突变减少TM辅因子活性的抗凝活性[65,66]。人群中Arg385Ser多态性的频率不清楚。对于心梗或静脉血栓和在第六EGF模块Ala455Val关系的研究结果不一致[54,67-70]，提示这种关联性较小或者仅限于少部分人群。然而，在非洲-美洲人群中这种多态性和心梗的关系有很强的相关性[68]。人群中使TM完全无活性的突变仅在一有肺梗塞、脑静脉窦血栓和休克病史的年轻杂合子携带者中发现[64]。10碱基缺失(del791-801, Arg246)在TM第二EGF模块位点306产生一不成熟终止密码子，此突变使血浆中TM减少50%，体外抑制蛋白的表达。
这些流行病学结果说明TM基因多态性和动脉粥样硬化或心梗的密切关联性，而与静脉血栓无此关联性。同样地，一病例组分析(ARIC研究)指出血液中高水平可溶性TM可减少冠状动脉性疾病(CAD)的发生[71]。考虑到类似破坏PC途径的Leiden V突变倾向于增加静脉而不是动脉血栓形成的危险性，这些研究的发现多少有点令人奇怪[72]。这些研究结果不一部分反应了TM可以依赖于PC活化的方式调控炎症发生、补体活化以及纤溶等现象。在整个人群中导致TM活性的实质性降低很少，可能因为功能缺失性突变干扰再生（见下文）、或者使宿主对细菌感染过程产生严重不利影响（见下文）。

TM和血栓形成

TM功能降低导致血栓形成的最有力证据来自于动物研究。给予TM可减轻在小鼠和大鼠凝血酶诱导的血栓形成产生的后果，而注射抑制TM依赖PC活化的抗体反而加剧凝血酶注射引起的后果[73，74]。我们过去建了三个不同程度TM缺陷基因工程小鼠细胞系。基本上纯合子突变TM缺陷小鼠由于在胎盘发育的缺陷而死于子宫中[75，76]（见下文）。仅表达50%正常TM（杂合子）的小鼠不发生自发血栓形成，但长期缺氧状态下在肺中纤维蛋白形成增多[75]。野生型和TM缺陷型胚胎干细胞嵌合小鼠在正常TM表达邻近区血管内皮产生完全TM缺陷的不连续区域[77]。大于100 mm2的血管管腔上纤维蛋白沉积仅发生TM缺陷区，说明象发生在动脉粥样硬化或者治疗性内皮损伤一样局部TM缺陷可致局部凝血发生和血栓形成[78，79]。另一品系小鼠（TM Pro小鼠）在EGF模块之间携带一点突变(Glu387Pro)[80]。其他学者显示此突变能够TM促进PC活化的能力[66]，也可能是解除TAFI的作用[47]。TM Pro突变动物尽管PC活化降低到5%以下，但能正常发育、存活，而细胞表面的TM表达仅为正常的1/3。这种小鼠出现血管床特异性纤维蛋白沉积的高凝状态，轻微促进形成血小板血栓形成，特别易发生止血血栓。TM Pro突变小鼠的血栓依赖于小鼠品系特异的基因修饰[81]。与纯合子Leiden V小鼠相反[82]，TMPro 突变动物不会自发发生血栓形成。TM Pro突变小鼠联合纤溶缺陷（如tPA缺失）可发生心肌微小血栓形成[83]。TM Pro突变小鼠的IL-1b和IL-6基础水平不变，但脂多糖诱导的内毒素血症死亡率高且细胞因子水平高[81]。因而TM Pro突变小鼠TM辅因子功能的实质性缺乏仅产生轻微高凝状态，而且明显血栓形成也只发生于合并有其他基因缺陷的病理状态。纯合子TM Pro突变小鼠凝血酶差不多与杂合子Leiden V小鼠相近，但明显低于纯合子Leiden V小鼠（作者未发表的研究结果）。总的说来，TM促进PC活化的能力几乎完全丧失仅稍微增加血栓形成的危险性，这些研究确实令人惊讶，但如有其他危险因子的参与才可致血栓形成。通过内皮细胞中敲除TM基因建立第三种小鼠模型[84]，这种操作并不妨碍其胚胎发育，但动物出生后短期内可出现大的、自发性的动静脉血栓，导致多脏器损坏，最后死于消耗性凝血障碍，华法林抗凝治疗可完全阻止此病理变化，表明其主要病理机制是通过凝血而起作用的。

TM和动脉粥样硬化

内皮层发生动脉粥样硬化病变[79]的TM局部缺失使PC活化功能发生局部损伤。分析血浆TM水平与动脉粥样硬化疾病的临床横向研究的结果仍不一致[85-88]，但可能反映内皮TM表达血浆量的基础水平与CAD的发生呈负相关[71]。从此研究尚不清楚对动脉粥样硬化这种抑制作用是否由细胞表面相关TM的增加介导，还是可溶性TM的抗炎作用[14,89–91]。TM也可调控发生再狭窄或移植静脉粥样硬化血管壁的病理改变[92–94]。通过逆转录病毒方法把TM基因导到机械扩张的兔股动脉可减轻血栓作用、神经水肿形成、炎症细胞浸润和细胞基质降解[92]。这种作用部分归于TM依赖性平滑肌增殖[95，96]，可能通过改变凝血酶依赖的细胞间信号传导过程。在内皮细胞中也可以观察到TM的抗增殖作用，TM改变MAPK/ERK途径的凝血酶受体依赖的细胞内信号传导[97]。相反地，仅有6个EGF模块的重组可溶性TM分子（因而缺乏似Lectin结构域）通过不明途径增强纤维母细胞和平滑肌细胞的增殖[53，94]。

TM与休克

一项研究观察到出血性休克和血浆TM水平的负相关，这项有趣的发现有待进一步证实[98]。与其它血管床相比，脑部毛细血管TM的表达很低，并且在大脑其它部位可能缺如[99–103]。体外研究提示脑血管TM分子的表达为星性胶质细胞来源TGFb所抑制[104,105]，而且抵御凝血酶诱导的经由APC依赖机制引起的神经细胞死亡[108]。一项有趣的动物实验显示用TM处理可大大地改善脊髓损伤的后果[109]。

TM与癌症

人类的各种肿瘤[110–114]中TM的表达和调查TM表达与肿瘤生长的临床研究[115–119]提示TM可以调控肿瘤生长和转移的可能性。在肺组织、食管或者口腔鳞状细胞癌中高水平的TM表达与原发病灶的分化较好及长生存相关[115,116,118,119]。肝细胞癌中TM表达水平低发生肝内转移高，容易腹膜转移[117]。原发性乳腺癌表达TM水平低与其复发率高相关[113]。实验研究提示肿瘤细胞中TM的水平与肿瘤增殖或体外浸润负相关[13,120,121]。TM对肿瘤浸润至少部分通过似Lectin结构域介导[121]，而TM的抗增殖作用也需要其跨膜和/或胞内结构域的存在[13]。

TM在炎症中的作用

PC系统是宿主炎症反应中对感染的重要调节因子[122,123]。PC系统的抗炎作用，至少部分与其抑制凝血酶生成途径无关；而是，该系统调节内皮细胞和免疫细胞对炎症刺激的反应。特别地，由凝血酶-TM复合物产生的APC抑制单核细胞分泌细胞因子，可下调内皮细胞中促炎胞内信号传导途径，并且启动内皮细胞保护性基因表达的程序[124–126]。体现APC作用的非抗凝作用的两种机制也有阐述。一项最近研究结果[127]表明APC-EPCR复合物选择性地活化内皮细胞中PAR-1受体。相反地，APC-EPCR复合物活化纤维母细胞中PAR-1及PAR-2。经APC-EPCR复合物由PAR-1参与内皮细胞反应与由凝血酶或特异性的PAR-1受体激动剂引发的反应极为类似，而且可复制出内皮细胞暴露于APC 的抗凋亡和抗炎症基因表达的病理现象。TM的功能显然是PC结合于EPCR后发生活化，但是有待于证实细胞特异性APC–EPCR复合物选择性对PAR-1作用某种程度上是否由TM调节。再者， APC改变内皮细胞基因表达的PAR非依赖途径可能牵涉到APC–EPCR复合物的核内移位[128]。EPCR或者PC–EPCR复合物均不定位于核内，但是可以很快观察到APC和EPCR之间的复合物在细胞核定位。与PAR-1介导的信号相似，凝血酶与TM结合引起无活性的PC-EPCR复合物转化为有活性的APC–EPCR复合物。Conway等学者[14]研究结果得出结论，TM可通过不依赖于凝血酶和PC的第三机制调控炎症的发生。小鼠TM基因经修饰转导产生表达缺失N-末端似lectin结构域的动物。重要的是体内似lectin结构域的缺失不会改变APC的功能。这些小鼠呈现一系列异常：基础状态下吸入脂多糖(LPS)后白细胞聚集在肺组织中、内毒素产生败血症的死亡率高、LPS增加TNFa和IL-1b的表达以及实验性心梗/再灌注后对心脏损害更严重。突变小鼠分离得到内皮细胞细胞间粘附分子(ICAM-1) 强表达和ICAM-1介导的白细胞黏附功能增强。MAP/ERK激酶信号传导途径体现在调控黏附受体的表达以及经LPS处理的突变小鼠心脏中ERK1/2磷酸化的确实得到了增强。重要的是，含似Lectin N-末端结构域的可溶性TM分子足以抑制TNFa诱导的ERK磷酸化和恢复正常白细胞黏附于突变内皮细胞。另外重组类似Lectin结构域的TM使培养内皮细胞免于无血清导致的死亡，可能系通过NF-kB途径调控。介导TM的似Lectin结构域作用的分子机制尚不清楚。补体活性的调控可能是TM不依赖于APC的宿主先天性对感染反应的第二个例子。在实验肾小球肾炎大鼠模型，给与重组可溶性TM阻止疾病的发展，使血浆羧肽酶升高，减轻肾小球白细胞/中性粒细胞浸润[129]。很明显，TAFI的抑制可明显干扰重组可溶性TM对白细胞浸润的影响。不幸的是，在需要TM时或需要表达的地方TM表达却下调。很多与炎症有关的状态，如覆于内皮下层的粥样硬化和移植静脉内皮等内皮细胞的TM表达下降[79,93,130]。TM功能丧失可通过几种机制发生，包括细胞因子诱导的TM基因转录抑制[131–133]、胞外TM结构域重要功能氨基酸的氧化[65]或者内皮细胞膜表面中性粒细胞弹性蛋白酶依赖的可溶性TM蛋白水解[134]。这些疾病状态下如给予重组可溶性TM可使TM功能恢复，是一很有前景的治疗方法。保留或恢复内皮细胞的活性也可以达到这一目的，但为细胞因子所抑制的TM基因转录并不完全清楚，而且可能牵涉到多种途径，包括内皮细胞Smad蛋白等[135]。

TM在小鼠胚胎形成中的作用

从胚胎干细胞中敲除TM基因导致胚胎宫内死亡[75]。TM缺陷(TM/-)胚胎交媾后8.5天不能生存（交媾后8.5天；小鼠怀孕周期大约18天），24小时后完全吸收。发育失败时，TM不仅表达于发育的血管系统，而且在胎盘的滋养层细胞也有表达[136]。TM/-胚胎的流产归于胎盘滋养层细胞TM的缺失，因为当TM在胎盘选择性表达时突变胚胎能够正常发育[76]，或者内皮细胞仅缺乏TM基因的表达[84]。小鼠以及人胎盘发育，供血给胎儿母体血管的完整性受到破坏，因而胚胎滋养层细胞与母体血接触。人们可能希望TM在胚胎滋养层表达具有与内皮细胞中表达同样的作用，也就是阻止形成血凝快和胎盘梗塞。据我们对TM基因敲除小鼠的分析可产生意想不到的结果[137]：胚胎TM缺乏与明显的血凝快的存在无关，或者在胎盘中有纤维蛋白沉积。TM缺乏小鼠反而表现为：(i)二倍体滋养层细胞保持增殖的作用完全缺乏；(ii)形成胚胎组织外层直接与母体血接触的多倍体滋养层细胞发生凋亡。对孕妇进行体内抗凝或去除纤维蛋白原（用纤维蛋白原缺失小鼠的生殖实验[138]）可阻止滋养层细胞死亡及TM缺失小鼠胚胎的很快吸收，但不能克服其成长缺陷。给孕妇用纤溶抑制剂如氨甲环酸效果与抗凝一样，并且可达到与完全去除纤维蛋白原抑制滋养层细胞死亡同样的效果，但不能阻止缺失TM胚胎的生长抑制。相反地，通过TM缺陷小鼠杂交产生抗凝作用可彻底缺乏组织因子(TF)[139]（该因子由小鼠滋养层细胞分泌），或者仅弱表达TF活性[140]，后者显示TF明显的活性缺乏使TM完全缺乏的小鼠辛免于死而且可以发育长大。TM缺失小鼠胚胎死亡的病理机制有两个方面（图2）。一方面，滋养层细胞表达TF抑制导致纤维蛋白沉积的凝血酶形成，局部纤维蛋白降解产物的沉积可引起巨大滋养层细胞的凋亡。体外实验结果提示，仅有纤维蛋白降解产物，而不是凝血酶，PAR激动多肽，纤维蛋白原或纤维蛋白诱导滋养层细胞凋亡。另一方面，TF也能启

图2 胎儿母体之间TM功能的模式图
胎盘中母体和胎儿组织之间形成滋养层细胞，直接与母体血液接触。小鼠该细胞组成性表达凝血启动因子——组织因子（TF）、凝血调控因子如TM和EPCR以及凝血因子活化受体PAR1, 2及4。TM缺乏凝血不能抑制可能经由PAR 2 和/或4参与阻止滋养层前体细胞生长而获得。纤维蛋白形成和随后发生的纤溶介导滋养层细胞凋亡和TM缺陷胚胎恢复，但是不影响胎盘生长。发生细胞凋亡和TM缺陷，而TM缺陷小鼠胚胎的生长状况不因抗凝治疗、去除或者抑制纤溶酶原而阻止。去除或减少TF活性克服了生长缺陷和凋亡及恢复的发生。红色箭头示凝血活化后滋养层细胞增殖调控的潜在途径。
动引起二倍体细胞生长抑制的过程，该二倍体细胞由产生巨大滋养层的前体细胞组成。TF诱导生长抑制的机制还没完全阐明，但是体外实验提示PAR-2或PAR-4活化可能是其机制。确实，大约凝血酶浓度为0.1–10 nmol /L可抑制体外培养二倍体滋养层细胞的增殖，但是不抑制巨大滋养层细胞。通过PAR-2和PAR-4激动肽可产生这种效应。相反地，PAR-1可促进增殖。TM凝血酶复合物生成APC，当胎儿与母体血接触时APC EPCR复合物活化PAR-1[127]能够促进胎盘生长。然而，这种PAR-1依赖的机制单独不能解释TM缺乏时生长缺陷，因为PAR-1敲除胚胎只表现为一过性生长缺陷[141]。因而，胎盘发育中TM功能呈现出象已知凝血和纤溶抑制剂叠加现象。无论是TM的N-末端似Lectin结构域还是其胞内区均不能介导胚胎发生中必要的功能；通过遗传工程手段表达TM突变小鼠无发育异常[14，142]。在胎盘中TM的发生功能是否牵涉到EPCR还是APC形成仍不清楚。在小鼠滋养层细胞有EPCR大量表达[143]，小鼠EPCR基因的破坏可使其胚胎发生损伤[144]。不管如何，EPCR和TM缺陷并不完全相同。EPCR缺陷小鼠损伤的发生要稍微晚于TM缺陷鼠，EPCR功能丧失可引起胎儿和母体维系区纤维蛋白沉积，而且，最为重要的是，抗凝治疗可挽救EPCR缺失鼠。另外，人为形成的小鼠TM点突变使APC（TM Pro，见上文）减少到至少象EPCR功能完全丧失的程度，但是TMPro鼠并无发育异常[80]。上述结果的不一致提示有TM和EPCR功能有重叠的可能性，而且在发育中有独特的功能。不考虑EPCR和TM的详细功能，人胎盘滋养层以及人/鼠胎盘形成EPCR和TM的表达强烈提示EPCR和TM对于人类再生也是重要的。

结论

本综述主要阐明TM的功能以及TM-PC途径的总的生理学方面的进展。TM对羧肽酶原/TAFI活化作用的发现和与似Lectin结构域相关抗炎效应的阐明提供了强有力的证据，TM发挥部分不依赖于其与PC和凝血酶作用的生物学活性。这些发现揭示了TM在调控纤溶中重要作用，并且强调了TM调控炎症的更为强大的作用。确实，Lectin结构域的抗炎和细胞保护作用，为TAFI潜在地抑制补体活性，以及已证实APC的抗炎作用均有可能一起保护微血管床内皮的完整性。很显然，TM在凝血和炎症的信号传导事件中发挥额外的作用，因为APC介导对内皮细胞细胞保护和抗炎途径，也就是，EPCR–APC复合物和EPCR–APC复合物的核内定位中PAR-1的参与，由通过TM-凝血酶复合物活化PC而启动。这些新的对凝血因子参与特异细胞受体能力的见解提示TM是重要的因子和凝血蛋白酶信号的调节剂，其意义远远超出血管的止血作用。上述生化和动物研究的文献复习说明一方面缩短了生化和物质结构研究的差距，另一方面关于TM功能缺陷或改变导致的体内实验出奇地少。至少小鼠TM或者EPCR功能的完全丧失与胚胎发育不平衡的解释了整个人群中有害的TM突变相当少事实。也许并不奇怪，TM和EPCR在胎盘滋养层的发育作用很多方面可能类似于活化内皮细胞调节凝血活化和凝血因子依赖细胞信号事件。总之，应该承认，尽管大约20年前就已提出TM这个名词，但是将来其研究仍有若干新的和有趣的假说优待于验证。
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