导致血浆代用品凝血障碍和有效血容量降低的关键明胶活性肽分离与作用机制研究

Gelatin plasma substitutes were commonly used blood volume expanders in clinic, but they still get some deficiency against blood coagulation and blood volume expanding effectiveness. To address this challenge, we put forward that the RGD bioactive peptides and fibronectin (Fn) binding peptides may play an important role in blood coagulation function and blood volume expanding capacity. The proposed research is motivated by our previous work on preparation and blood volume expanding capacity/anti-inflammatory investigation of genipin cross-linked gelatin as a candidate for plasma substitute, and the main focus of this proposal is: (1) the separation and identification of RGD and Fn binding peptides in gelatin, (2) the improvement investigation of blood coagulation function and blood volume expanding capacity of genipin cross-linked gelatin not containing RGD and Fn peptides, (3) the coagulation disturbance and blood volume decreasing mechanisms of RGD and Fn binding peptides. The objectives of this research are to explore the feasibility of the specific peptides removed genipin cross-linked gelatin used as a plasma substitute with decreased side effect and increased blood volume, and to unravel the effect mechanism of RGD and Fn binding peptides. The research will induce valuable scientific theory foundation and practical experience for further research of gelatin plasma substitute and utilization of gelatin materials.

明胶血浆代用品是用于血容量扩充的常用血液替代药物，但导致凝血障碍的副作用和扩容有效性不足的问题，是阻碍明胶血浆代用品在临床更广泛应用的关键。本项目在制备京尼平交联明胶，并验证其作为兼具扩容和抗炎活性的新型血浆代用品有效性的工作基础上，提出了明胶中的RGD肽和Fn结合肽，是影响机体凝血功能和导致有效循环血量降低重要潜在因素的新思路。基于此，本项目拟重点研究：① 明胶中RGD活性肽与Fn结合肽的分离与鉴定；② "去活"（去除RGD活性肽与Fn结合肽）型京尼平交联明胶的体内促凝血与增强扩容效果；③ RGD活性肽与Fn结合肽体内"抑制凝血功能/减低有效血容量"作用机制。通过上述研究，探索去除特征活性肽的京尼平交联明胶对降低血浆代用品凝血副作用和提升扩容有效性的可行性，明确RGD活性肽与Fn结合肽对凝血功能和血容量的影响机制，为血浆代用品的进一步研究与明胶的高值化利用，提供重要理论科学依据和实践经验

明胶血浆代用品作为临床广泛应用的血容量扩充用药，具有血液替代、使用方便和体内兼容性好等突出优势，但仍存在扩容有效性待进一步提升、对凝血系统有一定干扰等问题待改进。本项目提出，明胶中的RGD肽和Fn结合肽，是影响机体凝血功能和有效循环血量降低的重要因素。本项目通过酶膜耦合法制备了分子量均一的明胶多肽，并从中分离鉴定了RGD活性肽与Fn结合肽，二者的整体分离效率分别为67%和87%。利用去除大部分相关活性肽的明胶为原料、以京尼平为交联剂，制备了具有适宜理化性质的京尼平交联明胶，通过体内外实验考查了京尼平交联明胶作为血浆代用品的有效性和安全性，确证了去除RGD活性肽和Fn结合肽的京尼平交联明胶血浆代用品对改善凝血功能和增强扩容效果的理论预测，探索了分离过程中的明胶的凝胶层析保留特征及其光吸收规律、明胶活性肽与Fn结合规律及结合特征、炎性细胞因子对Fn和胶原蛋白的调节作用等。这些研究为血浆代用品的进一步提升提供了一种新的手段和思路，也为明胶和胶原蛋白的更深层次开发利用奠定了基础。