FIBRINOGEN--ROLE OF THE GAMMA VARIANT

申请者的广泛、长期目标是定义 纤维蛋白原在止血和血栓形成中的作用。这项建议 解决了纤维蛋白凝块强度的问题，这是 纤溶治疗的结果。最近的研究结果表明， 纤维蛋白原的次要伽马链变体直接与 凝血因子XIII的酶原形式，一种稳定的谷氨酰胺转氨酶 纤维蛋白凝块，而伽马变异体作为载体蛋白 对于因子XIII。这种载波功能可能会调制 通过增加纤维蛋白凝块的强度和抗溶解能力 凝块中凝血因子XIII的局部浓度。具体目标 这项建议的目的是： I)确定纤维蛋白原在调节血管紧张素转换酶的强度中的作用 纤维蛋白凝块。这将通过测量速率和 伽马和伽马~多聚体形成的程度，以及 检测α2-纤溶酶抑制物在细胞内的掺入 血块。此外，合成的多肽对应于 关于批次分解的Gamma~扩张，VRPEHPAEYDSLYPEDDL 将测量Rate，以及多肽在 γ~纤维蛋白原/凝血因子XIII复合体形成。的约束性 激活和未激活的凝血因子XIII的γ~Fg 同时测量凝血酶活性是否影响凝血因子XIII 与γ~纤维蛋白原结合。 Ii)确定伽马~链中的关键氨基酸 中介因子XIII结合。这将通过以下方式实现 重组人的平衡结合常数的测定 具有凝血因子XIII的γ~纤维蛋白原突变体，特别是在 在Gamma~418和Gamma~422位置硫化的Tyr残留物。 此外，还将引入丙氨酸扫描替代方案 纵观二十个氨基酸的伽马~链延伸图 关键结合残基。 Iii)鉴定凝血因子XIII中与 伽马~链。这将通过结合蛋白水解物来完成 凝血因子XIII亚基到树脂上的固定化伽马多肽 鉴定结合肽片段(S)。 本提案中的实验结果应提供 对凝血过程的新认识可能会 导致体内调节纤维蛋白凝块稳定性和凝块溶解的途径。

The broad, long-term objectives of the applicant are to define the roles of fibrinogen in hemostasis and thrombosis. This proposal addresses the issue of fibrin clot strength, a critical factor in the outcome of fibrinolytic therapy. Recent findings indicate that the minor gamma chain variant of fibrinogen binds directly to the zymogen form of factor XIII, a transglutaminase that stabilizes fibrin clots, and that the gamma variant serves as a carrier protein for factor XIII. This carrier function is likely to modulate the strength of the fibrin clot and its resistance to lysis by increasing the local concentration of factor XIII at the clot. The specific aims of this proposal are to: I) Determine the role of y fibrinogen in modulating the strength of fibrin clots. This will be accomplished by measuring the rate and extent of gamma and gamma~ multimer formation, and by measuring the incorporation of alpha2-plasmin inhibitor into the clot. In addition, the effect of a synthetic peptide corresponding to the gamma~ extension, VRPEHPAETEYDSLYPEDDL, on lot lysis rates will be measured, as well as the IC50 of the peptide on gamma~ fibrinogen/factor XIII complex formation. The binding of gamma~ fibrinogen to both activated and unactivated factor XIII will also be measured to see if thrombin activation affects factor XIII binding to gamma~ fibrinogen. II) Identify the critical amino acids in the gamma~ chain that mediate factor XIII binding. This will be accomplished by measuring the equilibrium association constant of recombinant gamma~ fibrinogen mutants with factor XIII, particularly mutants in the sulfated Tyr residues at positions gamma~418 and gamma~422. In addition, alanine-scanning substitutions will be introduced throughout the twenty amino acid gamma~ chain extension to map the critical binding residues. III) Identify the peptide sequences in factor XIII that bind to the gamma~ chain. This will be accomplished by binding proteolyzed factor XIII subunits to a resin of immobilized gamma~ peptide to identify the binding peptide fragment(s). The results from the experiments in this proposal should provide new knowledge about the process of blood coagulation that may lead to way of modulating fibrin clot stability and clot lysis in vivo.