Department of Medical Biochemistry, Semmelweis University, 1094 Budapest, Tűzoltó utca 37-47, Hungary.
Department of Biological Nanochemistry, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1117 Budapest, Magyar Tudósok körútja 2, Hungary.
Matrix Biol. 2017 Nov;63:55-68. doi: 10.1016/j.matbio.2016.12.008. Epub 2016 Dec 19.
Hyaluronic acid (HA) is a large, non-sulfated glucosaminoglycan abundantly present at sites where fibrin is also formed (during wound healing, in arterial restenotic lesions and eroded atherosclerotic plaques). The aim of the present study was to characterize the structure of composite fibrin-HA clots with scanning electron microscopy (SEM), pressure-driven permeation and small-angle X-ray scattering (SAXS) and their viscoelastic properties with an oscillation rheometer. In addition the efficiency of fibrinolysis in these clots was investigated by kinetic turbidimetric and chromogenic assays for dissolution of fibrin and plasminogen activation by tissue-type plasminogen activator (tPA). Fibrin formed in the presence of native (1500kDa) HA and its 500kDa fragments had thicker fibers and larger pores according to the SEM and clot permeation data, whereas the 25kDa HA fragments had only minor effects. SAXS evidenced a mild disarrangement of protofibrils. These structural alterations suggest that HA modifies the pattern of fibrin polymerization favouring lateral association of protofibrils over formation of branching points. Rheometer data showed softer fibrin structures formed with 1500kDa and 500kDa HA and these clots presented with lower dynamic viscosity values and lower critical stress values at gel/fluid transition. tPA-catalysed plasminogen activation was markedly inhibited by HA, both in free solution and on the surface of fibrin clots, in the presence and in the absence of 6-aminohexanoate suggesting a kringle-independent mechanism. HA of 1500 and 500kDa size prolonged clot lysis with both plasmin and tPA and this inhibition was kringle-mediated, because it was abolished by 6-aminohexanoate and was not observed with des-(kringle1-4)-plasmin. Our data suggest that HA size-dependently modifies the pattern of fibrin polymerization with consequent inhibition of fibrinolysis. At sites of tissue injury and inflammation, HA could stabilize fibrin through modification of its structure and lysibility.
透明质酸（HA）是一种大量的、非硫酸化的氨基葡聚糖，大量存在于纤维蛋白形成的部位（在伤口愈合、动脉再狭窄病变和侵蚀性动脉粥样硬化斑块中）。本研究的目的是通过扫描电子显微镜（SEM）、压力驱动渗透和小角 X 射线散射（SAXS）来描述复合纤维蛋白-HA 血栓的结构，并使用振荡流变仪研究其粘弹性特性。此外，还通过动力学浊度法和发色底物法测定纤维蛋白溶解和组织型纤溶酶原激活物（tPA）激活纤溶酶原的效率来研究这些血栓中的纤维蛋白溶解效率。在天然（1500kDa）HA 及其 500kDa 片段的存在下形成的纤维蛋白具有更厚的纤维和更大的孔，根据 SEM 和血栓渗透数据，而 25kDa 的 HA 片段只有较小的影响。SAXS 证明原纤维的轻度排列紊乱。这些结构改变表明，HA 改变了纤维蛋白聚合的模式，有利于原纤维的侧向缔合而不是分支点的形成。流变仪数据显示，与 1500kDa 和 500kDa HA 形成的纤维蛋白结构更柔软，这些血栓在凝胶/流体转变时具有较低的动态粘度值和较低的临界应力值。tPA 催化的纤溶酶原激活在 HA 的存在和不存在 6-氨基己酸的情况下，无论是在游离溶液中还是在纤维蛋白血栓表面，都被明显抑制，这表明存在一种kringle 非依赖性机制。1500 和 500kDa 大小的 HA 延长了纤溶酶和 tPA 的血栓溶解，并通过kringle 介导抑制，因为它被 6-氨基己酸消除，并且在没有 des-(kringle1-4)-纤溶酶的情况下观察不到。我们的数据表明，HA 依赖于大小改变纤维蛋白聚合的模式，从而抑制纤维蛋白溶解。在组织损伤和炎症部位，HA 可以通过改变纤维蛋白的结构和可溶度来稳定纤维蛋白。
