Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan.
PLoS One. 2012;7(8):e43660. doi: 10.1371/journal.pone.0043660. Epub 2012 Aug 20.
Altered fibrin clot architecture is increasingly associated with cardiovascular diseases; yet, little is known about how fibrin networks are affected by small molecules that alter fibrinogen structure. Based on previous evidence that S-nitrosoglutathione (GSNO) alters fibrinogen secondary structure and fibrin polymerization kinetics, we hypothesized that GSNO would alter fibrin microstructure.
METHODOLOGY/PRINCIPAL FINDINGS: Accordingly, we treated human platelet-poor plasma with GSNO (0.01-3.75 mM) and imaged thrombin induced fibrin networks using multiphoton microscopy. Using custom designed computer software, we analyzed fibrin microstructure for changes in structural features including fiber density, diameter, branch point density, crossing fibers and void area. We report for the first time that GSNO dose-dependently decreased fibrin density until complete network inhibition was achieved. At low dose GSNO, fiber diameter increased 25%, maintaining clot void volume at approximately 70%. However, at high dose GSNO, abnormal irregularly shaped fibrin clusters with high fluorescence intensity cores were detected and clot void volume increased dramatically. Notwithstanding fibrin clusters, the clot remained stable, as fiber branching was insensitive to GSNO and there was no evidence of fiber motion within the network. Moreover, at the highest GSNO dose tested, we observed for the first time, that GSNO induced formation of fibrin agglomerates.
CONCLUSIONS/SIGNIFICANCE: Taken together, low dose GSNO modulated fibrin microstructure generating coarse fibrin networks with thicker fibers; however, higher doses of GSNO induced abnormal fibrin structures and fibrin agglomerates. Since GSNO maintained clot void volume, while altering fiber diameter it suggests that GSNO may modulate the remodeling or inhibition of fibrin networks over an optimal concentration range.
改变的纤维蛋白凝块结构与心血管疾病的关系越来越密切；然而，对于改变纤维蛋白原结构的小分子如何影响纤维蛋白网络，人们知之甚少。基于先前的证据表明 S-亚硝基谷胱甘肽（GSNO）改变纤维蛋白原二级结构和纤维蛋白聚合动力学，我们假设 GSNO 会改变纤维蛋白的微观结构。
方法/主要发现：因此，我们用 GSNO（0.01-3.75 mM）处理人血小板缺乏的血浆，并使用多光子显微镜对凝血酶诱导的纤维蛋白网络进行成像。使用定制的计算机软件，我们分析了纤维蛋白微观结构的变化，包括纤维密度、直径、分支点密度、交叉纤维和空隙面积等结构特征。我们首次报道 GSNO 剂量依赖性地降低纤维蛋白密度，直至完全抑制网络形成。在低剂量 GSNO 下，纤维直径增加 25%，保持约 70%的凝块空隙体积。然而，在高剂量 GSNO 下，检测到异常的不规则形状的纤维簇，具有高荧光强度核心，并且凝块空隙体积显著增加。尽管存在纤维簇，但凝块仍然稳定，因为纤维分支对 GSNO 不敏感，并且网络内没有纤维运动的证据。此外，在测试的最高 GSNO 剂量下，我们首次观察到 GSNO 诱导纤维蛋白聚集体的形成。
结论/意义：总之，低剂量 GSNO 调节纤维蛋白微观结构，产生纤维较粗的粗糙纤维蛋白网络；然而，更高剂量的 GSNO 诱导异常的纤维结构和纤维聚集体。由于 GSNO 保持凝块空隙体积，同时改变纤维直径，这表明 GSNO 可能在最佳浓度范围内调节纤维蛋白网络的重塑或抑制。
