A caspase-6-cleaved fragment of Glial Fibrillary Acidic Protein as a potential serological biomarker of CNS injury after cardiac arrest

Blood levels of Glial Fibrillary Acidic protein (GFAP) reflect processes associated with different types of CNS injury. Evidence suggests that GFAP is cleaved by caspases during CNS injury, hence positioning GFAP fragments as potential biomarkers of injury-associated processes. We set out to develop an assay detecting the neo-epitope generated by caspase-6 cleavage of GFAP (GFAP-C6), and to assess the ability of GFAP-C6 to reflect pathological processes in patients suffering a cardiac arrest and subsequent global cerebral ischemia. Anti-GFAP-C6 antibodies recognized their specific target sequence, and dilution and spike recoveries in serum were within limits of ±20% reflecting high precision and accuracy of measurements. Intra- and inter-assay CVs were below limits of 10% and 15%, respectively. Serological levels of GFAP-C6 were significantly elevated 72 hours after CA (Mean±SD) (20.39±10.59 ng/mL) compared to time of admission (17.79±10.77 ng/mL, p<0.0001), 24 hours (17.40±7.99 ng/mL, p<0.0001) and 48 hours (17.87±8.56 ng/mL, p<0.0001) after CA, but were not related to neurological outcome at day 180. GFAP-C6 levels at admission, 24, 48, and 72 hours after cardiac arrest correlated with two proteolytic fragments of tau, tau-A (r = 0.30, r = 0.40, r = 0.50, r = 0.53, p < 0.0001) and tau-C (r = 54, r = 0.48, r = 0.55, r = 0.54, p < 0.0001), respectively. GFAP-C6 levels did not correlate with other markers of CNS damage; total tau, NSE and S100B. In conclusion, we developed the first assay detecting a caspase-6 cleaved fragment of GFAP in blood. Increased levels at 72 hours after cardiac arrest as well as moderate correlations between GFAP-C6 and two other blood biomarkers of neurodegeneration suggest the ability of GFAP-C6 to reflect pathological processes of the injured brain. Investigations into the potential of GFAP-C6 in other types of CNS injury are warranted.

血液中胶质纤维酸性蛋白（Glial Fibrillary Acidic Protein, GFAP）的水平可反映与不同类型中枢神经系统（Central Nervous System, CNS）损伤相关的病理过程。已有研究表明，GFAP在中枢神经系统损伤过程中会被半胱天冬酶（caspases）切割，因此GFAP片段有望成为损伤相关病理过程的潜在生物标志物。本研究拟开发一种检测半胱天冬酶6（caspase-6）切割GFAP所产生的新表位（neo-epitope，GFAP-C6）的检测方法，并评估GFAP-C6反映心脏骤停及后续全脑缺血患者病理过程的能力。抗GFAP-C6抗体可特异性识别其靶序列，血清中的稀释及加标回收率处于±20%的范围内，体现了该检测方法较高的精密度与准确度。批内与批间变异系数分别低于10%与15%。与入院时（17.79±10.77 ng/mL，p<0.0001）、心脏骤停后24小时（17.40±7.99 ng/mL，p<0.0001）及48小时（17.87±8.56 ng/mL，p<0.0001）相比，心脏骤停后72小时的GFAP-C6血清水平显著升高（平均值±标准差：20.39±10.59 ng/mL），但该水平与患者发病后180天的神经功能预后无相关性。心脏骤停入院时、骤停后24、48及72小时的GFAP-C6水平，分别与tau蛋白的两个水解片段tau-A（相关系数r分别为0.30、0.40、0.50、0.53，p<0.0001）及tau-C（相关系数r分别为0.54、0.48、0.55、0.54，p<0.0001）存在中等程度的相关性；但GFAP-C6水平与其他中枢神经系统损伤标志物——总tau、神经元特异性烯醇化酶（Neuron-Specific Enolase, NSE）及S100B蛋白——无显著相关性。综上，本研究开发了首个检测血液中半胱天冬酶6切割型GFAP片段的检测方法。心脏骤停后72小时GFAP-C6水平升高，且其与另外两种神经退行性变血液生物标志物存在中等程度的相关性，提示GFAP-C6可反映受损大脑的病理过程。未来有必要进一步探究GFAP-C6在其他类型中枢神经系统损伤中的应用潜力。