Alpha2-antiplasmin polymorphism and a new plasma enzyme

描述(申请人提供)：蛇毒蛋白α2-抗纤溶酶(A2AP)在调节纤溶中起主要作用。该提案的目标是(1)定义相互作用，以确定有多少a2AP被凝块结合以抑制纤溶酶；以及(2)确定调节a2AP的方法，以便在纤维蛋白沉积是主要因素的疾病中长期增强纤溶酶活性。A2AP主要由肝脏分泌到血液中，是一种由464个氨基酸组成的多肽。在循环中，分泌形式(“Pro”-a2AP)在Pro12-Asn13处被切割，得到“激活的”-a2AP(A2APact)，它被FX11a与纤维蛋白交联，速度大约是a2APpro的5倍，并提供了大约80%的纤维蛋白对纤溶酶的抵抗力。导致这种裂解的水解酶尚不清楚，但我们的发现表明，它是一种类似于细胞膜成纤维细胞激活蛋白酶(MFAP)的脯氨酸专一性丝氨酸蛋白酶，它没有已知的生理底物，不在血液中循环，而我们发现的一种有生理底物的丝氨酸蛋白酶被我们命名为CFAP。将进行研究以确定缺少mFAP的N-端23个残基的CFAP是否是mFAP的蛋白水解物。MFAP和CFAP将从人血浆或重组细胞培养物中纯化，并以2APPro或荧光肽为底物评估其动力学效率。将使用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文库将用于确定CFAP对每个肽的动力学效率，而高效的噬菌体展示文库将用于数据库搜索以确定其他生理底物。将努力通过根据亚基偏好替换特定残基来增强选定多肽的CFAP抑制特性。选择的CFAP重组变异体和FAP-抑制物复合体(ES)的结构域将进行结晶，进行X射线结构分析。我们的初步数据表明，a2APpro具有Arg6Trp多态，并且Arg6-Form被CFAP切割的速度大约是Trp6-Form的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值更高。将比较a2APPro(Arg6)、a2APproTrp6和a2APact作为XIIla因子底物和纤溶酶抑制物。最后，将在正常人群中检测CGG(Arg6)和TGG(Trp6)等位基因频率与血浆a2APact/a2APpro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.