Plasmin and the thrombin–thrombomodulin complex both contribute to thrombin‐activatable fibrinolysis inhibitor activation in whole blood model thrombi
Plasmin and the thrombin–thrombomodulin complex both contribute to thrombin‐activatable fibrinolysis inhibitor activation in whole blood model thrombi
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纤溶酶和凝血酶-血栓调节蛋白复合物均有助于全血模型血栓中凝血酶激活的纤溶抑制剂的激活
DOI：
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发表时间：
2013
影响因子：
10.4
通讯作者：
A. Gils
中科院分区：
文献类型：
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作者：
E. Vercauteren;N. Mutch;P. Declerck;A. Gils
活化凝血酶活化纤维蛋白溶解抑制剂（Activated fibrinolysis inhibitor, TAFIa）是由凝血酶单独或与凝血调节蛋白（T/TM复合物）以及纤溶酶激活TAFI而形成的[1,2]。体外血浆凝块溶解实验表明，T/TM复合物是TAFI[3]的主要激活剂。这一发现被一项体内研究证实，在大肠杆菌诱导的狒狒脓毒症中，TAFIa的产生被一种特异性抑制T/ tm介导的人TAFI[4]激活的单克隆抗体（MA）所抑制。我们实验室最近使用主要抑制纤溶酶介导的TAFI激活的特异性MAs的研究表明，纤溶酶在血浆凝块溶解过程中作为TAFI的激活剂发挥作用[5,6]。这在小鼠体内血栓栓塞模型中得到证实，所分析的MAs表现出强烈的纤原蛋白溶解作用[5]。在本研究中，我们进一步评估了TAFI在抑制纤溶中的作用，特别是不同的TAFI激活剂的重要性。在全血血栓溶解模型中评估了通过不同机制损害TAFI激活的MAs的影响[7,8]。分析的MAs有：(i)选择性抑制纤溶酶介导的人TAFI激活的MA-TCK11A9， （ii）选择性抑制T/ tm介导的人TAFI激活的MA-T12D11，以及（iii）抑制凝血酶、T/ tmin和纤溶酶介导的人TAFI激活的MA-TCK27A4[6,9]。如前所述，模型血栓在钱德勒循环系统中形成[7,8]。简单地说，将fitc标记的纤维蛋白原加入柠檬酸全血中，然后加入磷酸盐缓冲盐水（PBS）、马铃薯块茎羧肽酶抑制剂（PTCI, 25 lg mL; Calbiochem, Beeston, Notts， UK）或mas。TheMAs与TAFI的摩尔比为5倍（65 lg mL）形成血栓（假设血液中血浆含量为55%，人血浆中TAFI浓度为10 lg mL[10,11]）。将混合物重新钙化，在室温下以30rpm的剪切速率428 s旋转90 min，形成模型血栓。随后将产生的血栓浸泡在含有0.25 lg mL人组织纤溶酶原激活剂的PBS中。在37℃下孵育血栓，并在规定的时间内去除样品，以量化释放的荧光，反映纤维蛋白降解，从而反映纤维蛋白溶解。并与小分子TAFIa抑制剂PTCI进行了比较。本研究分析的所有TAFI抑制剂均导致荧光释放显著增加（P < 0.05；重复测量方差分析）（相对于无抑制剂；图1A）。有趣的是，抑制纤溶酶介导的TAFI激活（MA-TCK11A9）或通过所有TAFI激活剂抑制激活（MA-TCK27A4）的MAs导致比PTCI更快的裂解，即MA-TCK11A9和MA-TCK27A4的荧光释放分别增加1.92倍和1.90倍，而PTCI增加1.46倍。另一方面，仅抑制T/ tm介导的TAFI激活的MA-T12D11荧光释放仅增加1.34倍。这些数据表明，损害TAFI激活的MAs在并入血栓时具有纤原蛋白溶解作用。这表明TAFIa也可以在血栓环境中发挥抗纤溶作用，而不仅仅是像前面描述的那样在血栓-血浆界面。结果的差异很可能是由于在Mutch等人的研究中，与单独使用PTCI相比，这里使用的抑制性MAs具有高特异性。我们的观察还表明，在评估的TAFI抑制剂之间，荧光释放和因此的纤原蛋白溶解作用存在差异；与只抑制T/ tm介导的TAFI激活的MA （MA- t12d11）相比，发现抑制纤溶酶介导的TAFI激活的MA （MA- tck11a9和MA- tck27a4）的裂解速度更快。这表明，在模型血栓形成过程中，TAFI的激活在很大程度上是通过纤溶酶建立的，而T/TM复合物是一个不太突出的激活剂。这是当前关于各种TAFI活化剂的相对贡献的讨论中的一个重要发现（参见[12,13]）。通讯：Ann Gils，比利时鲁汶大学药学与药理科学系药物生物学实验室，Gasthuisberg校区，O&N2, pb824, Herestraat 49, B-3000 Leuven，比利时。电话：+32 16 32 34 36；传真：+32 16 32 34 60。电子邮件:ann.gils@pharm.kuleuven.be
Activated thrombin activatable fibrinolysis inhibitor (TAFIa) is formed upon activation of TAFI by thrombin, either alone or in complex with thrombomodulin (T/TM complex), and by plasmin [1,2]. In vitro clot lysis experiments in human plasma revealed that the T/TM complex is the predominant activator of TAFI [3]. This finding was substantiated by an in vivo study in which TAFIa generation during Escherchia coli-induced sepsis in baboons was inhibited by a monoclonal antibody (MA) specifically inhibiting the T/TM-mediated activation of human TAFI [4]. Recent studies from our laboratory using specific MAs that mainly inhibit plasmin-mediated TAFI activation revealed that plasmin functions as an activator of TAFI during plasma clot lysis [5,6]. This was confirmed in an in vivo mouse thromboembolism model in which the analyzed MAs exhibited a strong profibrinolytic effect [5]. In this study, we further evaluate the contribution of TAFIa in the inhibition of fibrinolysis and in particular the importance of the different activators of TAFI. The effect of MAs that impair TAFI activation via different mechanisms was assessed in a whole blood thrombus lysis model [7,8]. The MAs analyzed were (i) MA-TCK11A9 that selectively inhibits the plasmin-mediated human TAFI activation, (ii) MA-T12D11 that selectively inhibits the T/TM-mediated human TAFI activation and (iii) MA-TCK27A4 that inhibits the thrombin-, T/TMand plasmin-mediated human TAFI activation [6,9]. Model thrombi were formed in a Chandler loop system as previously described [7,8]. Briefly, FITC-labeled fibrinogen was added to citrated whole blood, followed by the addition of phosphate-buffered saline (PBS), potato tuber carboxypeptidase inhibitor (PTCI, 25 lg mL; Calbiochem, Beeston, Notts, UK) orMAs. TheMAs were incorporated into forming thrombi at a five-fold molar ratio (65 lg mL) over TAFI (assuming a plasma content of 55% in blood and a TAFI concentration of 10 lg mL in human plasma [10,11]). The mixture was recalcified and rotated at 30 rpm giving a shear rate of 428 s for 90 min at room temperature to form model thrombi. The resulting thrombi were subsequently bathed in PBS containing 0.25 lg mL human tissue plasminogen activator. Thrombi were incubated at 37 C and samples removed at defined times to quantify the released fluorescence that reflects fibrin degradation and thus fibrinolysis. The effect of the inhibitory MAs was compared with PTCI, a small molecule TAFIa inhibitor. All TAFI inhibitors analyzed in this study resulted in a significant increase (P < 0.05; repeated measures ANOVA) in fluorescence release (relative to no inhibitor; Fig. 1A). Interestingly, the MAs that inhibit either plasmin-mediated TAFI activation (MA-TCK11A9) or that inhibit activation through all TAFI activators (MA-TCK27A4) resulted in more rapid lysis than PTCI i.e. a 1.92-fold and 1.90-fold increase in fluorescence release for MA-TCK11A9 and MA-TCK27A4, respectively, compared with a 1.46-fold increase for PTCI. On the other hand, MA-T12D11 that exclusively inhibits the T/TM-mediated TAFI activation revealed only a 1.34-fold increase in fluorescence release. These data illustrate that MAs that impair TAFI activation are profibrinolytic when incorporated into thrombi. This suggests that TAFIa can also exert an antifibrinolytic effect within the environment of the thrombus and not just at the thrombus–plasma interface as previously described [7]. The difference in findings is most likely explained by the high specificity of the inhibitory MAs used here compared with the use of PTCI alone in Mutch et al. [7]. Our observations also indicate a difference in fluorescence release and thus the profibrinolytic effect between the evaluated TAFI inhibitors; with more rapid lysis observed on inclusion of MAs that inhibit plasmin-mediated TAFI activation (MA-TCK11A9 and MA-TCK27A4) compared with the MA that exclusively inhibits T/TM-mediated TAFI activation (MA-T12D11). This suggests that during model thrombus formation, TAFI activation is established to a significant extent via plasmin with the T/TM complex being a less prominent activator. This is an important finding in the current discussion regarding the relative contribution of the various TAFI activators (reviewed in [12,13]). Correspondence: Ann Gils, Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Campus Gasthuisberg, O&N2, PB 824, Herestraat 49, B-3000 Leuven, Belgium. Tel.: +32 16 32 34 36 ; fax: +32 16 32 34 60. E-mail: ann.gils@pharm.kuleuven.be
影响因子：
20.3
作者：
Bajzar, L;Nesheim, ME;Tracy, PB
通讯作者：
Tracy, PB