Evidence that fibrinogen γ′ directly interferes with protofibril growth: implications for fibrin structure and clot stiffness
Evidence that fibrinogen γ′ directly interferes with protofibril growth: implications for fibrin structure and clot stiffness
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DOI：
10.1111/j.1538-7836.2012.04717.x
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发表时间：
2012-06-01
影响因子：
10.4
通讯作者：
Ariens, R. A. S.
中科院分区：
文献类型：
--
作者：
Allan, P.;de Willige, S. Uitte;Ariens, R. A. S.
背景资料：纤维蛋白原含有选择性剪接的γ链（γ '），其主要作为具有共同γ A链的异源二聚体（γ A/γ'）存在。已报道纤维蛋白原γ '抑制凝血酶并调节纤维蛋白结构，但其潜在机制尚不清楚。目的：我们的目的是研究γ '对纤维蛋白聚合、结构和粘弹性影响的分子机制。方法：采用阴离子交换层析法分离γ A/γ A和γ A/γ '纤维蛋白原。用纯化的FXIIIa和合成抑制剂控制交联。通过浊度分析纤维蛋白聚合，并使用凝血仪测量胶凝点时间。我们使用原子力显微镜（AFM）的图像原纤维形成，而最终的凝块结构进行了评估，通过共聚焦和扫描电子显微镜。使用磁性显微流变仪测量凝块粘弹性。结果：γ A/γ '纤维蛋白形成的寡聚体比γ A/γ A形成的寡聚体更短，而且γ A/γ A形成的寡聚体更早。与γ A/γ A凝块的粗纤维的均匀排列相比，γ A/γ A凝块显示出细纤维的非均匀排列。凝块结构的这些差异不是由于凝血酶抑制，如用立止血制成的凝块所示。非交联的γ A/γ A纤维蛋白比γ A/γ '硬约2.7倍。通过FXIIIa交联增加了两种纤维蛋白变体的硬度;然而，硬度的差异增加到约4.6x（γ A/γ A对γ A/γ ′）。结论：纤维蛋白原γ '与由细纤维组成的机械性较弱、不均匀的凝块的形成有关。这是由γ直接破坏原纤维形成引起的。
Background: Fibrinogen contains an alternatively spliced gamma-chain (gamma'), which mainly exists as a heterodimer with the common gamma A-chain (gamma A/gamma'). Fibrinogen gamma' has been reported to inhibit thrombin and modulate fibrin structure, but the underlying mechanisms are unknown. Objective: We aimed to investigate the molecular mechanism underpinning the influence of gamma' on fibrin polymerization, structure and viscoelasticity. Methods: gamma A/gamma A and gamma A/gamma' fibrinogens were separated using anion exchange chromatography. Cross-linking was controlled with purified FXIIIa and a synthetic inhibitor. Fibrin polymerization was analyzed by turbidity and gel-point time was measured using a coagulometer. We used atomic force microscopy (AFM) to image protofibril formation while final clot structure was assessed by confocal and scanning electron microscopy. Clot viscoelasticity was measured using a magnetic microrheometer. Results: gamma A/gamma' fibrin formed shorter oligomers by AFM than gamma A/gamma A, which in addition gelled earlier. gamma A/gamma' clots displayed a non-homogenous arrangement of thin fibers compared with the uniform arrangements of thick fibers for gamma A/gamma A clots. These differences in clot structure were not due to thrombin inhibition as demonstrated in clots made with reptilase. Non-cross-linked gamma A/gamma A fibrin was approximately 2.7 x stiffer than gamma A/gamma'. Cross-linking by FXIIIa increased the stiffness of both fibrin variants; however, the difference in stiffness increased to approximately 4.6 x (gamma A/gamma A vs. gamma A/gamma'). Conclusions: Fibrinogen gamma' is associated with the formation of mechanically weaker, non-uniform clots composed of thin fibers. This is caused by direct disruption of protofibril formation by gamma'.