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

凝血因子XIII （FXIII）前酶作为由两个FXIIIA和两个FXIIIB亚基组成的异源四聚体在血浆中循环。在凝血酶和钙的激活下，fxiiiib亚基从异源四聚体中分离出来，形成活化的FXIIIA2*转谷氨酰胺酶，该酶交联预先形成的纤维蛋白凝块，以增加对降解的抵抗力。缺乏FXIII可导致出血倾向，可能有遗传或获得性起源。遗传性严重FXIII缺乏症是一种罕见的凝血障碍，发病率为百万分之一至四分之一，其特征是典型的严重出血。鲜为人知但更常见的形式，即轻度遗传性FXIII缺乏症（FXIII水平在20-60%之间），是由F13A1和F13B基因（分别为FXIIIA和FXIIIB亚基）的分离杂合突变引起的。轻度FXIII缺乏症更难检测和表征，因为受影响的个体通常无症状或仅在刺激（即手术和/或身体创伤）时才出现症状，因此目前报告不足。在过去的十年中，我们的研究小组已经检测并报告了F13A1和F13B基因在这种轻度缺陷个体中的一些突变。然而，由于其主要无症状表现，这些突变的临床相关性和意义尚不清楚。因此，在没有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)阐明它们是否对携带它们的个体具有现实的临床显著影响/出血风险，b)在分子水平上解释它们在常染色体隐性遗传疾病（即症状明确的严重遗传形式）中的主要负面影响。我们的建议旨在使用双顺反子双标签策略表达，纯化和富集几种杂合蛋白变体（来自我们之前报道的F13A1和F13B基因）。纯化的变体随后将在多个功能和结合平台上进行测试，并在体外（FXIII缺陷小鼠全血）进行测试，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，还将阐明凝血FXIII在创伤和类似创伤情况中的作用。

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.