Download A new method to detect causative mutations in fibrinogen

A new method to detect causative mutations in fibrinogen.
Pablo García de Frutos
Department of Cell Death and Proliferation
Institute of Biomedical Research of Barcelona (IIBB-CSIC, IDIBAPS)
c/ Rosellón 161, 6p
08036 Barcelona, SPAIN
Fibrinogen plays a central role in coagulation and therefore has been studied in
great detail since its discovery as coagulation’s factor I. Structure-function studies have
deciphered minute details on how this molecule works, and the extend of the studies is
not surprising, as fibrinogen contains all the “knobs”, “holes”, loops and binding sites to
interact with the machinery that activates the formation of the clot mesh, while also the
ability to eliminate it by controlled proteolysis (1). The result of this research provides a
picture of one of the most astonishing molecular machines in biology: a soluble protein
that is capable of forming an enormous polymeric network that confers the capacity to
stop bleeding. Fibrinogen does this task with efficacy and also with elegance by rapidly
forming dimers and then oligomers that pack in protofibrils and fibrils to then branch
and form the network that nests platelets and erythrocytes, bringing consistency and
flexibility to the clot and avoiding the loss of blood from an injured vessel, which could
otherwise be lethal in a short time. Fibrinogen is so admirably fitted for its function that
structure/function studies are especially rewarding in terms of how they help us to
understand the way this molecule works.
A field of research that has been more fruitful in this regard is the study of
naturally occurring mutations that are detected in the three genes responsible of the
synthesis of fibrinogen. In Thrombosis and Haemostasis, several recent studies are
good examples of this research. For a long time it has been known that some common
genetic variants in are correlated with fibrinogen levels (2). These variants could define
the concentration of plasma fibrinogen, and they correlate with hematological
parameters. Still, a full span of effects on fibrinogen could be observed, from functional
defects without affecting the total amount of fibrinogen to the complete or almost
complete absence of fibrinogen in plasma in cases of afibrinogenaemia (3). Because
most mutations are found in patients and/or families suffering from diseases of
haemostasis, they allow us to study the relationship of the detected genetic variants
with their manifestation in a given phenotype. Although most mutations in fibrinogen
manifest as an increased bleeding tendency, some mutations are associated with
thrombophilia, i.e. an increased risk of thrombus formation. This is due to the subtle
relation between structure and function that occurs in the fibrinogen molecule, where a
mutation in specific residues could affect not only its synthesis but also the formation of
the polymer, its stability, or the capacity of the fibrinolytic system to degrade it. For
instance, fibrinogen Perth causes a partial deletion in the αC domain and codes for a
free cysteine that is associated with tighter and thinner clots and impaired fibrinolysis,
resulting in familial thrombophilia (4). Similar results are observed in mutations
affecting the tPA binding site associated with thrombosis, (5), but also in mutations
where the main effect seems to be structural rather than regulatory (6). An interesting
addition to the array of effects studied is provided by a mutation in the coiled-coil
module of Aα chain, that was shown to affect platelet aggregation (7). The calcium
binding site affected in the fibrinogen Tolaga Bay mutation showed an effect on the
stabilization of the clot through delaying the factor XIIIa-catalysed γ and α chain crosslinking (8). Interestingly, Park et al show that the direct mutation of one of the factor
XIIIa crosslinking acceptor sites (fibrinogen Seoul II) affected polymerization but
alternative sites could substitute for factor XIIIa crosslinking (9). Finally, the study of
Marchi et al shows that some fibrinopathies could manifest with bleeding and
thrombotic episodes (10).
Although advances in DNA sequencing technology have facilitated the
identification of numerous mutations in hereditary dysfibrinogenaemia, one of the
persisting problems is to determine the genetic changes that are really the molecular
basis of a disease is not mutation detection, but deciding whether or not an identified
sequence variation is responsible for the disease phenotype. In the present issue of
Thrombosis and Haemostasis, the study of Brenner et al describes a simple method to
analyze mutations in fibrinogen with extreme simplicity. The method uses a small
amount of blood processed through HPLC and time of flight (TOF) mass spectrometry.
To test the methodology, they analyzed a patient with hypofibrinogenaemia where they
could detect three mutations associated with three mass changes in the Bβ and 
chains. They could determine the relative amount of protein derived from each allele,
and, together with modelling information and sequence preservation, determine which
is the mutation causative of hypofibrinogenaemia in the patient.
New technologies as the one described here will facilitate the faster
determination of mutations and its correlation with protein data, providing new insights
on how the different parts of fibrinogen function. Also this research will allow us to
continue admiring the sheer beauty of this superb molecule.
References
1.
Weisel JW, Litvinov RI. Mechanisms of fibrin polymerization and clinical
implications. Blood 2013; 121: 1712–9.
2.
Jeff JM, Brown-Gentry K, Crawford DC. Replication and characterisation of
genetic variants in the fibrinogen gene cluster with plasma fibrinogen levels and
haematological traits in the Third National Health and Nutrition Examination
Survey. Thromb Haemost 2012; 107:458–67.
3.
Zhang J, Zhao X, Wang Z, et al. A novel fibrinogen B beta chain frameshift
mutation causes congenital afibrinogenaemia. Thromb Haemost 2013; 110: 76–
82.
4.
Westbury SK, Duval C, Philippou H, et al. Partial deletion of the αC-domain in
the Fibrinogen Perth variant is associated with thrombosis, increased clot
strength and delayed fibrinolysis. Thromb Haemost 2013; 110: 1135–44.
5.
Brennan SO, Chitlur M. Hypodysfibrinogenaemia and thrombosis in association
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deleted). Thromb Haemost 2013; 109: 1180–2.
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Brennan SO, Zebeljan D, Ho LL. Thrombosis in association with a novel
substitution (γ346Gly→Val) at an absolutely conserved site in the fibrinogen γ
chain. Thromb Haemost 2013; 109: 757–8.
7.
Riedelová-Reicheltová Z, Kotlín R, Suttnar J, et al. A novel natural mutation
AαPhe98Ile in the fibrinogen coiled-coil affects fibrinogen function. Thromb
Haemost 2014; 111: 79–87.
8.
Park R, Ping L, Song J, et al. Fibrinogen residue γAla341 is necessary for
calcium binding and “A-a” interactions. Thromb Haemost 2012; 107: 875–83.
9.
Park R, Ping L, Song J, et al. An engineered fibrinogen variant AαQ328,366P
does not polymerise normally, but retains the ability to form α cross-links.
Thromb Haemost 2013; 109: 199–206.
10.
Marchi R, Walton BL, McGary CS, et al. Dysregulated coagulation associated
with hypofibrinogenaemia and plasma hypercoagulability: implications for
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516–26.