A novel fibrinogen variant (fibrinogen Seoul II; AαGln328Pro) characterized by impaired fibrin α-chain cross-linking

Abstract

We report a novel fibrinogen variant (fibrinogen Seoul II), which has a heterozygous point mutation from CAA to CCA leading to A␣Gln328Pro. The mutation site is among several glutamine residues that serve as ␣-chain cross-linking acceptor sites. Fibrinogen Seoul II was found in a 51-year-old male patient and his family in Seoul, Korea. The patient was diagnosed with myocardial infarction at age 43. Eight years later he was admitted to the emergency room due to recurrence of the disease, where he expired under treatment with tissue plasminogen activator (t-PA). Fibrin polymerization curves, made using purified fibrinogen from the patient's relatives, showed a decreased final turbidity, suggesting Seoul II fibrin clots are composed of thinner fibers. This supposition was verified using scanning electron microscopy. Alpha-polymer formation by the mutant fibrinogen upon thrombin treatment in the presence of factor XIII and calcium was distinctly impaired. This result confirms that the residue A␣328 plays a pivotal role in ␣ IntroductionFibrinogen, one of the critical plasma proteins, is a 340-kDa glycoprotein 1 synthesized in the liver 2,3 and has essential roles in both blood coagulation and platelet aggregation. 4 Fibrinogen is a dimer consisting of 2 identical pairs of A␣, B␤, and ␥ chains intertwined to form a trinodular molecule with 2 terminal D regions and a central E region. 5,6 The D region includes the carboxyl termini of the B␤ and ␥ chains, while that of the A␣ chain goes beyond the D region to form the ␣C domain, which is composed of residues A␣220-610 7 and normally interacts with the central E region.In the process of blood coagulation, thrombin cleaves fibrinopeptides A and B from A␣ and B␤ chains to form a fibrin monomer, exposing the GPR and GHRP sequences, respectively. Subsequently, protofibrils in a half-staggered array are formed, and the release of ␣C domains from the central E region is facilitated, allowing lateral aggregations through intermolecular associations between ␣C domains. 8,9 The final phase of fibrin clot formation involves the covalent modification of fibrin molecules by factor XIIIa. The factor XIIIa-mediated cross-linking process, where ␣-␣ cross-linking follows ␥-␥ cross-linking, contributes to stabilization of the fibrin clot and resistance to thrombolytic agents. 10,11 Until now, a variety of mutations in each of the fibrinogen chain genes have been reported in more than 350 families all over the world, with A␣-chain mutation as the most common form. 12 Those mutations usually have been associated with dysfibrinogenemia or hypofibrinogenemia, both of which feature decreased levels of plasma fibrinogen activity. Each A␣-chain has at least 2 glutamine acceptor sites located at amino acid residues 328 and 366 and 5 potential lysine donor sites between residues 518 and 584. 13 A␣ glutamine 328 is among several glutamine residues that serve as ␣-chain cross-linking acceptor sites, 10 for which only one fibrinogen variant has been described: compound heterozygotes characterized ...

Ranking reactive glutamines in the fibrinogen αC region that are targeted by blood coagulant factor XIII

“…Previous investigations, however, have shown that Q328 is an important reactive glutamine. 22,40 In our assay, we were able to characterize Q328 following a chymotrypsin digest. Our ability to monitor all three reactive glutamines simultaneously in aC (233-425) is enhanced by digesting each sample time point separately with chymotrypsin and GluC (Figure 1).…”

“…32,33,39 Fibrinogen Seoul II (Q328P mutation) exhibits impaired fibrin polymerization and a reduced extent of a-a crosslinking due in part to the loss of a reactive glutamine within the aC region. 40 Several studies have identified specific crosslinking sites in the aC region responsible for lateral aggregation, but little is known about the individual reactive glutamines and the roles played by the surrounding residues. 17,18,20,22,23,41,42 Antibody studies by Procyk et al identified a FXIIIa binding site within aC (242-424) and then narrowed the site more specifically to aC (389-402).…”

Characterizing reactive glutamines in fibrinogen and elucidating factor XIII substrate specificity.

“…Previous investigations, however, have shown that Q328 is an important reactive glutamine. 68,81 In this assay, we were able to characterize Q328 following a chymotrypsin digest. The ability to monitor all three reactive glutamines simultaneously in αC (233-425) is enhanced by digesting each sample time point separately with chymotrypsin and Glu C (Figure 23).…”

“…Truncations and mutations in this region have also been correlated with disease conditions associated with IDPs. 68,70 Furthermore, many of the reactive glutamines crosslinked by Factor XIIIa are located in loop and flexible regions.…”

“…With this mutation, fibrin γ-γ crosslinking occurs but α-α is impaired. 68 Considering our data, Q237 and Q366 would still be available in the Seoul II patients along with potential K residues in the more Cterminal portion of αC. Multiple crosslinks involving highly reactive Q237 may, however, not be sufficient to overcome the introduction of Q328P.…”