Alpha2-antiplasmin polymorphism and a new plasma enzyme

描述（由申请人提供）：serpin alpha2-antiplasmin （a2AP）在调节纤维蛋白溶解中起主要作用。该提案的目标是：(1)确定相互作用，决定有多少a2AP成为抑制纤溶酶的凝块结合；(2)确定调节a2AP的方法，以便在以纤维蛋白沉积为主要因素的疾病中长期增强纤溶酶活性。a2AP是一种由464个氨基酸组成的多肽，主要由肝脏分泌进入血液。在循环过程中，分泌形式（“pro”-a2AP）在Pro12-Asn13处被切割，产生“活化的”-a2AP (a2APact)， FXlIla将其与纤维蛋白交联的速度约为a2APpro的5倍，并提供约80%的纤维蛋白对纤溶蛋白的抗性。导致这种切割的蛋白酶是未知的，但我们的研究结果表明，它是一种脯氨酸特异性丝氨酸蛋白酶，类似于细胞膜成纤维细胞激活蛋白酶（mFAP），它没有已知的生理底物，也不会在血液中循环，而我们发现的这种蛋白酶存在，我们称之为cFAP。缺少mFAP n端23个残基的cFAP是否为mFAP的蛋白水解衍生物还有待进一步研究。mFAP和cFAP将从人血浆或重组细胞培养物中纯化，并以a2APpro或荧光肽为底物评估其动力学效率。利用cFAP切割的Pro12-Asn13键周围序列的合成肽库来寻找cFAP的底物特异性和亚位点偏好。随机肽序列的噬菌体展示库将用于确定每个肽的cFAP动力学效率，效率高的噬菌体将用于数据库搜索以识别其他生理底物。我们将努力通过基于亚位偏好取代特定残基来增强选定肽的cFAP抑制特性。cFAP重组变体和fap -抑制剂复合物（es）的选定结构域将进行x射线结构分析的结晶。我们的实验数据表明a2APpro具有Arg6Trp多态性，并且Arg6-form被cFAP切割的速度比Trp6-form快约8倍。这与我们最初的发现相吻合，即在含有arg6形式的人血浆中，a2APact/a2APpro的比例更高。比较a2APpro（Arg6）、a2APpro（Trp6）和a2APact作为XIIla因子底物和纤溶酶抑制剂。最后，将在正常人群中检测CGG（Arg6）和TGG（Trp6）多态性的等位基因频率与血浆a2APact/a2APpro比值和内源性纤溶活性的相关性。

DESCRIPTION (provided by applicant): The serpin alpha2-antiplasmin (a2AP) has a major role in regulating fibrinolysis. This proposal's goal is to (1) define interactions that determine how much a2AP becomes clot-bound for inhibiting plasmin; and (2) identify means of regulating a2AP so that plasmin activity might be enhanced long-term in diseases where fibrin deposition is a major factor. a2AP is secreted mainly from liver into blood as a polypeptide of 464 amino acids. While circulating, the secreted form ("pro"-a2AP) is cleaved at Pro12-Asn13 to give "activated"- a2AP (a2APact) which is crosslinked to fibrin by FXlIla approximately 5-fold quicker than a2APpro and provides about 80% of fibrin's resistance to plasmin. The proteinase that makes this cleavage is unknown, but our findings suggest it is a proline-specific serine proteinase similar to cell membrane fibroblast activating proteinase (mFAP), which has no known physiologic substrate and does not circulate in blood, whereas the one we have found does, and is termed cFAP by us. Studies will be done to determine if cFAP, which lacks the N-terminal 23 residues of mFAP, is a proteolytic derivative of mFAP. mFAP and cFAP will be purified from human plasma or recombinant cell cultures and their kinetic efficiencies assessed with a2APpro or a fluorogenic peptide as substrate. Substrate specificity and subsite preferences of cFAP will be sought using a synthetic peptide library of sequences surrounding the Pro12-Asn13 bond cleaved by cFAP. A phage display library of random peptide sequences will be used to determine kinetic efficiency of cFAP for each peptide and those with high efficiency will be used in database searches to identify other physiological substrates. Efforts will be made to enhance cFAP inhibitory properties of selected peptides by substituting specific residues based on subsite preferences. Selected domains of cFAP recombinant variants and FAP-inhibitor complex(es) will be subjected to crystallization for x-ray structural analyses. Our pilot data indicate that a2APpro has an Arg6Trp polymorphism, and that Arg6-form is cleaved by cFAP approximately 8x faster than Trp6-form. This fits with our initial findings that a2APact/a2APpro ratios are higher in human plasma containing Arg6-form. Comparisons will be made between a2APpro(Arg6), a2APpro(Trp6) and a2APact as factor XIIla substrates and plasmin inhibitors. Lastly, allelic frequencies of the CGG(Arg6) and TGG(Trp6) polymorphisms will be examined in a normal population for correlation with plasma ratios of a2APact/a2APpro and endogenous fibrinolytic activity.