Functional investigations into the dominant-negative patho-molecular mechanisms underlying FXIII heterozygous missense mutations of emerging clinical importance

凝血因子XIII（FXIII）酶原作为由两个FXIIIA和两个FXIIIB亚基组成的异源四聚体在血浆中循环。在被凝血酶和钙激活后，FXIIIB亚基从异源四聚体解离以形成活化的FXIIIA 2 * 转氨酶，其交联预先形成的纤维蛋白凝块以增加对降解的抗性。缺乏FXIII可导致出血倾向，可能具有遗传或获得性起源。遗传性严重凝血因子XIII缺乏症是一种罕见的凝血功能障碍，患病率为1-4/100万，其特征是典型的严重出血素质。较不为人所知但更常见的形式，即轻度遗传性FXIII缺乏症（FXIII水平在20-60%之间），是由F13 A1和F13 B基因（分别为FXIIIA和FXIIIB亚基）中的孤立杂合突变引起的。轻度FXIII缺乏症较难检测和表征，因为受影响的个体通常无症状或仅在激发（即手术和/或物理创伤）时才显示症状，因此目前报告不足。在过去的十年中，我们的研究小组已经发现并报告了一些F13 A1和F13 B基因突变的个体患有这种形式的轻度缺乏症。然而，这些突变的临床相关性和意义尚不清楚，因为其主要表现为无症状。因此，在缺乏明确的症状相关性的情况下，需要对这些突变进行详细的功能分析，以a）澄清它们是否对携带它们的个体存在现实的临床显著影响/出血风险，以及B）在分子水平上解释它们在本质上是常染色体Recombinant遗传性疾病（即，病理学上明确的重度遗传形式）中的显性负效应。我们的建议旨在表达，纯化和富集的几个杂合蛋白变体（从F13 A1和F13 B基因，我们以前报道）使用双顺反子双标签策略。纯化的变体随后将在多个功能和结合平台上进行测试，并进行离体测试（FXIII缺陷小鼠全血），以详细表征其病理分子效应。我们的研究不仅将澄清这些杂合突变的作用，而且还将揭示凝血因子XIII在创伤和创伤样情况下的参与。

The coagulation Factor XIII (FXIII) pro-enzyme circulates in plasma as a heterotetramer composed of two FXIIIA and two FXIIIB subunits. Upon activation by thrombin and calcium, the FXIIIB subunits dissociate from the heterotetramer to form activated FXIIIA2* transglutaminase that cross-links pre-formed fibrin clots to increase resistance to degradation. Deficiency of FXIII can result in a bleeding predisposition that can have inherited or acquired origins. Inherited severe FXIII deficiency is a rare coagulation disorder with a prevalence of 1-4 in a million and characterized by typically severe bleeding diathesis. The lesser known but more frequent form, i.e. mild inherited FXIII deficiency (FXIII level between 20-60%), is caused by isolated heterozygous mutations in F13A1 and F13B genes (FXIIIA and FXIIIB subunits respectively). Mild FXIII deficiency is harder to detect and characterize since the affected individual can often be asymptomatic or shows symptoms only upon provocation (i.e. surgery and/or physical trauma) and therefore it is currently underreported. Our group, in the past decade has detected and reported a number of mutations in F13A1 and F13B genes in individuals with this form of mild deficiency. The clinical correlation and significance of these mutations however remains unclear owing to its primarily asymptomatic presentation. Therefore, in the absence of clear cut symptomatic correlation, these mutations need to be functionally analyzed in detail to a) clarify if they present a realistic clinically significant impact/bleeding risk on the individuals carrying them, and b) explain at a molecular level their dominant negative effect in what is essentially a autosomal recessively inherited disorder (i.e. the symptomatically clear severe inherited form). Our proposal aims to express, purify and enrich for the several heterozygous protein variants (from both F13A1 and F13B genes that we have previously reported) using a bi-cistronic double tag strategy. The purified variants will subsequently be tested on multiple functional and binding platforms and also tested ex vivo (FXIII deficient mice whole blood) in order to characterize in detail their pathomolecular effect. Our investigations will clear the air on not only the role of these heterozygous mutations but will also shed light on the involvement of coagulation FXIII in trauma and trauma like situations.