血小板αIIbβ3 拮抗劑 TMV-7 與其衍生物之抗血栓作用和低出血風險

Abstract

臨床上αIIbβ3拮抗劑（如Abciximab、Eptifibatide和Tirofiban）廣泛應用於各種冠狀動脈候群（ACS）以防止血小板凝集和血栓形成，尤其是在急性心肌梗塞（AMI）病患進行冠狀動脈血管造形術（PTCA）時預防血小板凝集和血栓生成。然而，此類藥物結合後除了阻斷受體，同時也會造成受體結構改變，產生新的結合位置，能和病人內生性的抗體結合，進一步透過另一個受體FcγRIIA（CD32）活化血小板產生凝集並快速消耗病人體內血小板，而造成出血和血小板低下等副作用；另一方面此類藥物更抑制由talin所調控的Integrin outside-in signaling，此訊息傳遞路徑與正常生理止血功能有直接關係，這也是臨床上併發出血和thrombocytopenia等副作用的原因，使得藥物在使用上的侷限與困擾。 在本篇研究中，我們由龜殼花蛇毒純化得到兩種抗黏著蛋白TMV-2和TMV-7，兩者序列中皆含有Arg-Gly-Asp，其中TMV-7賦有獨特的結合方式不同於TMV-2以及臨床用藥Abciximab，TMV-7藉由結合到αIIb β-propeller domain不僅不會造成αIIbβ3受體活化而造成結構改變，也發現TMV-7與專一性αIIbβ3抗體-AP2在血小板製備中不會引發FcγRIIA所調控的血小板凝集與下游訊息傳遞路徑。另一方面TMV-7也能專一性地抑制Gα13所調控的outside-in signaling，而不影響talin所調控的inside-out signaling。基於此特性，TMV-7相對於TMV-2和Abciximab，較不影響正常生理止血，過程包含由talin所調控之初期inside-out signaling以及後期的clot retraction。在動物實驗中，TMV-2與TMV-7皆能有效地預防老鼠頸動脈以及腸繫膜中血栓的生成；但相較於TMV-2與臨床用藥eptifibatide會造成FcγRIIA基因轉入老鼠血小板數量低下而出現嚴重出血反應，TMV-7不僅不會造成老鼠血小板低下與出血時間延長，也不會影響人類全血之凝血指標。由此我們認為TMV-7與TMV-2及其他αIIbβ3拮抗劑作用於αIIbβ3不同之處可利於新一代αIIbβ3拮抗劑的研發。 我們更進一步以TMV-7 sequence當成模板，將RGD-domain中的ARGDNP點突變，改進為等電點（pI）較高的AKGDRR，衍生物RR有效地提高抑制血小板活化的效能 (inhibitory potency of platelet aggregation)至兩倍以上；在安全性方面，目前在抑制血小板凝集的IC50 (half maximal inhibitory concentration) 之1300倍劑量下仍沒有觀察到出血傾向，顯著地優化於TMV-7，將用藥安全性提升至60~70倍以上甚至更高。我們探究RR不造成出血副作用的機轉，發現其抑制血栓的作用機轉與TMV-7相同，兩者與一般蛇毒抗血栓Disintegrin不同之處為—兩拮抗劑結合到Integrin αIIbβ3皆不會造成αIIbβ3受體活化；另一方面TMV-7及RR作用 Integrin αIIbβ3皆不會影響由Talin所調控的Integrin inside-out signaling，故在使用上不影響正常生理止血功能。因此在動物活體實驗中，TMV-7及RR可有效抑制動脈血栓之產生，但有別於臨床用藥Abciximab、Eptifibatide及Tirofiban，即使投予高劑量仍不會造成出血時間延長，俱有較高的用藥安全性。 TMV-7及RR能專一性地抑制血小板凝集反應並抑制血栓生成，並解決目前抗血栓用藥存在出血風險的問題。目前亦進行化學修飾（PEGylation）以利後續發展成較穩定和長效或衍生小分子藥物之製劑，且同時進行大型動物體內試驗，以改善目前抗血栓藥物僅有靜脈注射製劑而尚未出現口服劑型以供長期使用。

The life-threatening thrombocytopenia, a common side effect of clinically available αIIbβ3 antagonists (e.g. abciximab, eptifibatide, and tirofiban), is associated with drug-dependent antibodies that recognize conformation-altered integrin αIIbβ3. Therefore, development of new antagonists with less tendency in causing bleeding is highly warranted. In these reports, we found that two disintegrins, TMV-2 and TMV-7, purified from snake venom of Trimeresurus mucrosquamatus, exhibit different antithrombotic properties. TMV-7 endows with a binding motif toward αIIb β-propeller domain of αIIbβ3, different from that of TMV-2, with advantages in that TMV-7 neither primed the platelets to bind ligand nor caused conformational change of αIIbβ3 identified by ligand-induced binding site (LIBS) mAb AP5. In contrast to eptifibatide and TMV-2, co-treatment of TMV-7 with AP2 did not induce FcγRIIa-mediated platelet aggregation and downstream activation cascade. Both TMV-2 and TMV-7 efficaciously prevented occlusive thrombosis in vivo. Notably, either eptifibatide or TMV-2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation-indexes, whereas TMV-7 did not impair the hemostatic capacity. These findings highlight the αIIbβ3 antagonist TMV-7 with minimal conformational effects may potentially help resolve the major problem associated with the current integrin antagonists that at doses where they exhibit high potency they also increase the risk of hemorrhage. Emerging evidence suggests that selective inhibition of outside-in signaling has the potential to have potent antithrombotic effects without causing bleeding. Our study also reveals that TMV-7 exhibits different binding epitope of αIIbβ3 from that of 7E3 and TMV-2, with advantages in that TMV-7 selectively inhibits Gα13 binding to integrin β3 without inhibiting talin binding to β3 in human and mouse platelets. Furthermore, TMV-2 and TMV-7 diminished activation of protein kinase c-Src and stimulated RhoA, consequently inhibiting platelet spreading. TMV-2 inhibited thrombin-induced clot retraction of platelet-rich plasma whereas TMV-7 did not. Notably, TMV-2 significantly prolonged the tail bleeding time and suppressed coagulation indexes as monitored by thromboelastography while TMV-7 did not as they were administered at efficacious antithrombotic doses. The study identifies the lead αIIbβ3 antagonist TMV-7 with unique binding epitopes efficaciously prevents thrombosis without affecting physiological hemostasis. In order to design an optimal anti-thrombotic agent with better safety profile, we mutated the RGD-domain of trimucrin from ARGDNP to AKGDRR, and found that this trimucrin AKGDRR mutant (RR) exhibits higher potency in inhibiting platelet aggregation and its safety index is raised to 70-time higher than TMV-7. RR prevented thrombosis by a mechanism similar to TMV-7 that decelerates αIIbβ3 ligation without causing conformational change and inducing little exposure of LIBs epitope on αIIbβ3, thus RR does not trigger FcγRII-mediated platelet aggregation. Furthermore, RR selectively inhibits Gα13-binding without affecting talin-binding to β3 in human thrombin-activated platelets. At efficacious antithrombotic doses, both TMV-7 and RR had little effect on tail-bleeding time even given at higher dose (i.e., 2.5 μg/g, 20-fold higher), indicating that they are efficacious antithrombotic αIIbβ3 antagonists with a greater safety profile than the current αIIbβ3 antagonists. Further examining the molecular interaction between TMV-7 and its derivative may provide valuable information for development of new anti-thrombotic with a better safety profile.