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

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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.