The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent
The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent
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DOI：
10.1182/blood.v88.6.2093.bloodjournal8862093
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发表时间：
1996-09-15
期刊：
影响因子：
20.3
通讯作者：
Tracy, PB
中科院分区：
文献类型：
--
作者：
Bajzar, L;Nesheim, ME;Tracy, PB
凝血酶激活的纤溶抑制剂 (TAFI) 是与血浆原羧肽酶 B 相同的外肽酶的前体。在被凝血酶激活后，活化的 TAFI (TAFIa) 可能通过催化从部分降解的纤维蛋白中去除 C 末端赖氨酸来减弱纤溶作用。活化蛋白 C (APC) 通过蛋白水解使凝血酶原酶中必需的辅助因子 Va 因子失活，并限制凝血酶的形成和随后 TAFI 的激活，从而呈现纤维蛋白溶解原。 TAFI 能够在纯化系统中重建 Ape 依赖性裂解时间缩短；然而，在基于血浆的系统中，TAFI 在多大程度上参与 APC 的纤溶作用仍有待确定。为了帮助解决这个问题，针对纯化的 TAFI 制备了两种单克隆抗体（MoAbTAFI#16 和 #13）和一种多克隆抗体。 MoAbTAFI#16 显示可抑制 TAFI 激活，从而刺激纤维蛋白溶解。此外，还使用 MoAbTAFI#13 和多克隆抗体开发了酶联免疫吸附测定法。通过其使用，测定TAFI的血浆浓度为73nmol/L。此外，使用浊度测定法测定了APC对组织纤溶酶原激活剂诱导的正常人血浆（NHP）、TAFI免疫耗竭血浆（TdP）以及用纯化TAFI重构的TdP产生的凝块的纤维蛋白溶解的影响，Ape以可饱和且浓度依赖性的方式缩短了NHP产生的凝块的溶解时间。然而，在 80 nmol/L MoAbTAFI#16 存在的情况下，Ape 对 TdP 或 NHP 形成的凝块的裂解时间没有影响。通过添加纯化的 TAFI，可以在 TdP 中重建 APC 效应。 TdP 中的裂解时间以 TAFI 浓度依赖性方式从 50 分钟增加到 180 分钟。 EC(50) 为 15 nmol/L，在生理相关浓度 (60 nmol/L) 下接近饱和。 APC 的纤溶作用还与 MoAbTAFI#16 和两种竞争性抑制剂（从马铃薯块茎中纯化的羧肽酶 A 和 B 家族抑制剂以及 2-胍基乙基巯基琥珀酸 (GEMSA)）进行了比较。所有这些都能够将正常人血浆形成的凝块的溶解时间缩短90分钟，产生各自的EC(50)值5nmol/L、15nmol/L、50nmol/L和90μmol/L。因此，在体外血浆系统中，Ape 的大部分纤维蛋白溶解作用取决于 TAFI。由于 TAFIa 显着影响溶解时间，因此 TAFIa 或 TAFI 激活抑制剂可能被证明是溶栓治疗的重要佐剂。 (C) 1996 年，美国血液学会。
Thrombin-activatable fibrinolysis inhibitor (TAFI) is the precursor of an exopeptidase that is identical to plasma procarboxypeptidase B. Upon activation by thrombin, activated TAFI (TAFIa) attenuates fibrinolysis, presumably by catalyzing the removal of C-terminal lysines from partially degraded fibrin. Activated protein C (APC) proteolytically inactivates the essential cofactor in prothrombinase, factor Va, and limits both the formation of thrombin and subsequent activation of TAFI, thereby appearing profibrinolytic. TAFI is able to reconstitute an Ape-dependent shortening of lysis time in a purified system; however, it remained to be determined the extent to which TAFI is involved in the profibrinolytic affect of APC in a plasma-based system. To aid in addressing this question, two monoclonal antibodies (MoAbTAFI#16 and #13) and a polyclonal antibody were produced against purified TAFI. MoAbTAFI#16 was shown to inhibit TAFI activation and thereby appears to stimulate fibrinolysis. Furthermore,, an enzyme-linked immunosorbent assay was developed using MoAbTAFI#13 and the polyclonal antibody. Through its use, the plasma concentration of TAFI was determined to be 73 nmol/L. In addition, a turbidity assay was used to determine the effect of APC on tissue plasminogen activator-induced fibrinolysis of clots produced from normal human plasma (NHP), plasma immunodepleted of TAFI (TdP), and TdP reconstituted with purified TAFI, Ape shortened lysis time of clots produced from NHP in a saturable and concentration-dependent manner. However, Ape had no effect on lysis time of clots formed from either TdP or NHP in the presence of 80 nmol/L MoAbTAFI#16. The APC effect could be reconstituted in TdP by the addition of purified TAFI. The lysis time in TdP was increased from 50 to 180 minutes in a TAFI concentration-dependent manner. The EC(50) was 15 nmol/L and saturation was approached at physiologically relevant concentrations (60 nmol/L). The profibrinolytic effect of APC was also compared with that of MoAbTAFI#16 and two competitive inhibitors, an inhibitor of the carboxypeptidase A and B family purified from potato tubers and 2-Guanidinoethylmercaptosuccinic acid (GEMSA). All were able to reduce lysis time of clots formed from normal human plasma by 90 minutes, yielding respective EC(50) values of 5 nmol/L, 15 nmol/L, 50 nmol/L, and 90 mu mol/L. Therefore, the majority of the profibrinolytic effect of Ape, in an in vitro plasma system, is dependent on TAFI. Because TAFIa dramatically influences lysis time, inhibitors of TAFIa or TAFI activation may prove to be important adjuvants for thrombolytic therapy. (C) 1996 by The American Society of Hematology.