FIBRINOGEN--ROLE OF THE GAMMA VARIANT

申请人的广泛、长期目标是确定 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， 纤溶治疗的结果。 最近的研究结果表明， 纤维蛋白原的次要γ链变体直接结合到 因子XIII的酶原形式，一种稳定 纤维蛋白凝块，而γ变体作为载体蛋白 因子XIII。 这种载体功能可能会调节 纤维蛋白凝块的强度及其对溶解的抵抗力， 凝血因子XIII的局部浓度 具体目标 这一建议的目的是： I）确定γ纤维蛋白原在调节纤维蛋白原的强度中的作用。 纤维蛋白凝块 这将通过测量速率来实现， γ和γ-多聚体形成的程度，以及 测量α 2-纤溶酶抑制剂掺入 凝块 此外，对应于以下的合成肽的效果是显著的： γ-延伸，VRPEHPAETEYDSLYPE，批次裂解 将测量速率，以及肽对小鼠的IC 50。 γ-纤维蛋白原/因子XIII复合物形成。 的结合 γ-纤维蛋白原与活化和未活化的因子XIII的结合将 还可以测量凝血酶活化是否影响因子XIII 与γ-纤维蛋白原结合。 II）鉴定γ链中的关键氨基酸， 介导因子XIII结合。 这将通过 测定重组蛋白的平衡结合常数 γ-纤维蛋白原突变体与因子XIII，特别是 在位置γ ~418和γ ~422处的硫酸化Tyr残基。 此外，还将引入丙氨酸扫描替代 在整个20个氨基酸的γ-链延伸，以映射 关键的结合残基。 III）鉴定因子XIII中结合至多肽的肽序列。 γ链 这将通过结合蛋白水解的 将因子XIII亚基固定到固定化γ肽的树脂上， 鉴定结合肽片段。 本提案中的实验结果应提供 关于血液凝固过程的新知识， 导致调节体内纤维蛋白凝块稳定性和凝块溶解方法。

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.