百步蛇蛇毒蛋白 Agkistin 抗血栓活性與作用機轉探討

Abstract

蛇毒蛋白可抑制或活化血小板。多種蛇毒蛋白成份會專一性結合在血小板上的αIIbβ3，α5β1和αVβ3。另種蛇毒蛋白則作用於醣蛋白Ib和他主要的生理配體，von Willebrand factor (vWF)。 Snaclecs（蛇毒C型凝集素）具有與兩個α和β，通常由雙硫鍵共價連接異源二聚體的結構。 Agkistin是一個從百步蛇毒中純化而得的C型類凝血素蛋白家族之成員，其具有典型的α及β次單元。Snacle主要會作用在GPIb, GPVI, a2b1, Factor IX，而Agkistin是作用在醣蛋白Ib上。 本實驗中，透過離子交換的分析方式，從百步蛇原毒中純化出Agkistin，Agkistin皆能專一性的抑制由ristocetin所引發的醣蛋白Ib相關之血小板凝集。GPIb試驗的活化劑ristocetin卻無法在小鼠富含血小板血漿中引起凝集現象。因此我們使用了Gramicetin 替代，Gramicetin為一種vWF非依賴性的GPIb活化劑，也是純化自蛇毒，希望藉此作為小鼠模式之活化劑。不論在人類血小板懸浮液或富含血小板血漿中Agkistin 皆可以有效的抑制由Gramicetin所引起的血小板凝集現象，不過在於小鼠富含血小板血漿中Gramicetin僅呈現微小的活化現象，這一反應也可以藉由加入Agkistin 所抑制，除此之外，Agkistin也能部分抑制低濃度thrombin所引起的血小板凝集，也會抑制低濃度PAR1所引起的血小板凝集。 在小鼠體內的實驗測試，我們發現Agkistin會造成小鼠體內血小板減少的情形，也會延長小鼠尾部出血的時間，在重症聯合免疫缺陷小鼠( SCID mice)的體內也仍觀察到血小板減少的情形，在排除了免疫細胞T和B細胞的交互作用，Agkistin所引發的血小板低下，可能與血管中其他細胞所表現的受體產生的交互作用有關。在氯化鐵造成的血栓形成的動物模式中，我們觀察到Agkistin有濃度依賴性的延長血栓形成的時間，推論可能具有抗血栓的作用。 總之，Agkistin 是作用在血小板醣蛋白Ib上的拮抗劑，抑制血小板活化，它能部分抑制低濃度thrombin和完全抑制低濃度PAR-1 agonist造成的血小板活化。血栓形成的動物模式中，Agkistin有濃度依賴性延長血栓形成的時間，這些實驗結果解釋了Agkistin有抗血栓的作用機轉。但是它和其他醣蛋白Ib的拮抗劑一樣都會造成血小板低下，而原因需要進一步的探討，因此，藉由研究Agkistin，GPIb的拮抗劑在出血風險可以接受的範圍下，具有抗血栓活性與作用探討。

One of the major targets for snake venom proteins is haemostasis. Snake venom proteins weaken the prey and help with swallowing and digestion. The venom proteins act either on coagulation factors or on platelets. Snake venom proteins often adapt physiological mechanisms to inhibit or activate platelets. The inhibitory mechanisms of these components are mainly directed against integrins, called disintegrin, which represent a family of low molecular weight, cysteine-rich polypeptides that bind specifically to integrins αIIbβ3, α5β1, and αvβ3 expressed on platelets and other cells. The other target is glycoprotein Ib, and its main physiological ligand, von Willebrand factor(vWF). Snaclecs (Snake venom C-type lectins) have a typical heterodimeric structure with two subunits, α and β, usually linked covalently, via a disulphide bond. Agkistin, a heterodimer protein purified from the snake venom of Agkistrodon acutus, is highly homologous to those of C-type lectin GPIb-binding proteins. The molecular weight of Agkstin was determined as 32,512Da. As additional glycoprotein targets have received a fair amount of attention in the drive to develop novel approaches for antiplatelet intervention. GPIb-binding proteins may be the target to be concerned. In vitro study, Agkstin concentration dependently inhibited platelet aggregation in human platelet-rich plasma and washed platelet suspension triggered by Ristocetin in the presence of vWF. Agkistin specifically inhibited human platelet aggregation and agglutination triggered by Ristocetin in the presence of vWF in vitro by acting as the platelet GPIb antagonist. Agkistin concentration-dependently inhibited platelet aggregation in mice PRP in response to Gramicetin. Agkstin also exhibited partial inhibition and decreased the oscillation and prolonged the latent period caused by thrombin (0.02 or 0.03U/mL). Moreover, Agkistin inhibited platelet aggregation caused by low concentration of PAR-1 agonist. Furthermore, in order to evaluate Agkistin effect in vivo, we measured the platelet count and bleeding time of mice. It caused a dose-dependent decrease in platelet counts, however it didn’t cause a marked bleeding. We observed thrombocytopenia caused by Agkistin in the SCID mice, suggesting that the reduction in platelet count may not be related to T or B cells. In a FeCl3-injured thrombosis model, we measured carotid artery blood flow after ferric chloride injury, and found that Agkistin dose-dependently prolonged occlusion time. However, Agkistin did not cause a persistent bleeding (>10 min) side effect under the dosr of 30ng/g. In summary, Agkistin, a GPIb antagonist, inhibits platelet agglutination. Agkistin exhibited partial inhibition and decreased the oscillation and prolonged the latent period caused by low concentration of thrombin (0.02-0.03U/mL) or PAR-1 agonist, and dose-dependently prolonged occlusion time in FeCl3-injured carotid artery in mice model. These data suggested that Agkistin has an antithrombotic effect. However, Agkistin rapidly causes thrombocytopenia like other GPIb-binding snake venoms in mice. The clear mechanism needs further investigation. Therefore, Agksitn may provide valuable information for the future development of GPIb antagonist.