Mechanism of the synergistic effect between oversulfated chondroitin-6-sulfate and lysine or 6-aminohexanoic acid in enhancing the in-vitro activation of glutamic plasminogen by tissue plasminogen activator or urokinase.
Mechanism of the synergistic effect between oversulfated chondroitin-6-sulfate and lysine or 6-aminohexanoic acid in enhancing the in-vitro activation of glutamic plasminogen by tissue plasminogen activator or urokinase.
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过硫酸化6-硫酸软骨素与赖氨酸或6-氨基己酸之间的协同作用增强组织纤溶酶原激活剂或尿激酶体外激活谷氨酸纤溶酶原的机制。
DOI：
10.1097/mbc.0b013e328337b436
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Doctor,Vasant
中科院分区：
文献类型：
--
作者：
Kouemo,Stella;McMillan,Erinn;Doctor,Vasant
早期的研究表明，加入天然硫酸多糖，包括普通肝素，可显著增强组织型纤溶酶原激活剂(t-PA)或尿激酶型纤溶酶原激活剂(u-PA)对谷氨酸纤溶酶原(Glu-PLG)的体外激活作用。然而，在缓冲液中添加生理浓度的氯化钠(0.9%)则逆转了这种增强。为了克服这一逆转，人们试图过多地硫酸盐化合物，并重新评估它们的生物学特性。用氯磺酸-吡啶络合物对硫酸软骨素-6-硫酸酯(N-2)进行过硫酸盐化，并将其分离为钠盐。红外和1H-核磁共振研究表明，过硫化化合物引入了新的硫酸盐基团，形成了60%的软骨素-4-6-二硫酸盐。在含0.9%氯化钠的0.05mol/L Tris缓冲液(pH 7.3)中，比较过硫酸软骨素-6-硫酸软骨素(S-2)、天然化合物(N-2)和普通肝素对t-PA或u-PA激活Glu-PLG的影响。以HD-Glu-Phe-Lys-PNA(S-2403)为底物，通过形成纤溶酶来测定t-PA或u-PA对Glu-PLG激活的促进作用。2.86mmolg/mlS-2可使t-PA的活性增加一倍，32.4mmoL/L的赖氨酸或5.4mmoL/L的6-AH可使t-PA的活性增加4~6倍，S-2与赖氨酸或S-2与6-AH合用可使t-PA的活性增加14~16倍，两者有协同作用，而单纯肝素无增强作用，且与赖氨酸或6-AH联用时无明显增强作用。使用u-PA代替t-PA的类似研究得出了相同的结果。在Glu-PLG活化为纤溶酶的过程中，赖氨酸纤溶酶原(Lys-PLG)被认为是一种中间体。因此，我们研究了S-2、赖氨酸和6-AH在Lys-PLG激活纤溶酶中的作用。结果表明，S-2能增强谷氨酸-PLG的激活，而赖氨酸或6-AH对谷氨酸-PLG的激活无促进作用，说明赖氨酸或6-AH的增强作用部位在谷氨酸-PLG的起始阶段。T-PA加或不加S-2和赖氨酸激活Glu-PLG的双倒易图显示，Km无变化，而K-cat增加10倍，提示这两种辅因子的作用均为模板机制。
Earlier studies of addition of naturally sulfated polysaccharides including unfractionated heparin showed a significant enhancement of the in-vitro activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA). However, supplementing of physiological concentration of NaCl (0.9%) to the buffer reversed the enhancement. To overcome this reversal attempts were made to oversulfate the compounds and re-evaluate their biological properties. Chondroitin-6-sulfate (N-2) was oversulfated using chlorosulfonic acid–pyridine complex and isolated as the sodium salt. Infrared and 1 H-NMR studies of the oversulfated compound showed introduction of new sulfate groups with the formation of 60% of chondroitin-4-6-disulfate. In-vitro studies were conducted on comparing the effect of oversulfated chondroitin-6-sulfate (S-2) with native compound (N-2) and unfractionated heparin in enhancing the activation of Glu-Plg by t-PA or u-PA using 0.05 mol/l Tris buffer (pH 7.3) containing 0.9% of NaCl. The enhancement of activation of Glu-Plg by t-PA or u-PA was measured by formation of plasmin using HD-Glu-Phe-Lys-pNA (S-2403) as the substrate. The activation by t-PA was enhanced two-fold by 2.86 μg/ml of S-2, 4–6-fold by addition of 32.4 mmol/l of lysine or 5.4 mmol/l of 6-aminohexanoic acid (6-AH) and 14–16-fold enhancement by addition of both S-2 and lysine or S-2 and 6-AH showing a synergistic effect, whereas unfractionated heparin alone gave no enhancement and in conjunction with lysine or 6-AH gave no additional enhancement. Similar studies using u-PA in place of t-PA gave identical results. During the activation of Glu-Plg to plasmin, lysine plasminogen (Lys-Plg) is reported to be an intermediate. Therefore we investigated the role of S-2, lysine and 6-AH in the activation of Lys-Plg to plasmin. The results showed that S-2 enhanced this activation, whereas lysine or 6-AH which were active in enhancing the activation of Glu-Plg were not active using Lys-Plg indicating that the site of enhancement by lysine or 6-AH was during the initial phase. Double reciprocal plot of Glu-Plg activation by t-PA with or without S-2 and lysine showed no change in K m but a 10-fold increase of K cat suggesting a template mechanism for the attenuation when both cofactors are used.