In the present work we report a new fibrinogen variant with a missense mutation in the FGB
at g.3354 T>A, which at the protein level causes the replacement of the amino acid tyrosine for asparagine at Bβ41. This amino acid is located in the sequence Bβ 15–42, for which different roles in fibrin polymerization (27
), endothelial cell (EC) spreading (28
), and VE-cadherin receptor binding (14
) have been documented. One limitation of our findings is that the expression and quantification of the abnormal Bβ chains was not assessed. However, there was strong evidence from the fibrin polymerization and biophysical studies that the mutated Bβ chains were expressed.
During fibrin polymerization the newly exposed sequence, knob “A” and “B” interact with the hole “a” and “b”, respectively, forming the well known fibrin interactions A:a and B:b (29
). Unfortunately, knob A and B has not been visualized at atomic level, as hole “a” and “b”, because these regions appear to be flexible and do not show up in the crystal structure (29
). The crystal structures of the end regions of fibrinogens, called fragment D with various GPR or GHR containing peptides fit into holes “a” while GHR peptides fit into holes “b”. However, we do not know at present if the interactions of these peptides represent the entire binding sites or if these could be more extensive regions involved, as suggested by some studies (27
The plasma polymerization process of fibrinogen Caracas VIII was slightly impaired, as a mildly prolongation of the lag phase and a final turbidity increase was detected. However, when purified fibrinogen was used, a drastic change in the final turbidity, roughly twice that of the control, was observed. It seems likely that the replacement of the bulky aromatic side chain of tyrosine by the small polar uncharged side chain of asparagine probably altered the conformation of knob “B”, and consequently the B:b interactions. It is possible that this mutation decreases the rate of protofibril initiation, as can be inferred from and in Weisel and Nagaswami (31
The network stiffness is strongly dependent on fiber thickness and branchpoint concentration (32
). Fiber lengths and diameters decreased with increased branching (32
), contributing to increased clot rigidity. Fibrin is a viscoelastic polymer, which means that it has both elastic and viscous properties (34
). Thus, the mechanical properties of fibrin may be typified by the stiffness or storage modulus (representing its elastic properties) and creep compliance or loss modulus/loss tangent (representing its inelastic properties) (4
). The variant fibrinogen Bβ Y41N examined by us showed a diminished storage modulus and a slightly increased inelastic component, which means that its clot will deform more with applied mechanical stress than one with higher storage modulus and loss tangent. The elastic and inelastic properties of a clot are very sensitive to small changes that affect polymerization and clot structure. Clots ligated with Factor XIIIa usually show higher stiffness and a lower inelastic component of deformation (4
). We disregarded the effect of fibrin ligation by Factor XIIIa, as its value was normal (results not shown). The protein mutation largely affected the viscoelastic properties of the clot, highlighting the importance of this region in the clot mechanical properties. Recently, it has been demonstrated that the stiffness of clots formed from fibrinogen 325 (fibrinogen lacking the Bβ1-42, after digestion with Crotalus atrox
protease III), decreased by about 8-fold (35
). Although the clots formed with this degraded form of fibrinogen are not equivalent to those from the variant BβY41N, the former results confirmed the role of Bβ1-42 in oligomer formation and on the elastic properties of the fibrin clot.
We also wished to explore the structure of the patients’ fibrin clot formed on ECs, especially since the fibrinogen mutation presently reported was located in the fibrin binding site to VE-cadherin (14
), and this non-integrin receptor is likewise located on the adherens junction (zonula adherens) which allows calcium-dependent homophilic recognition between ECs (36
) that controls vascular permeability (37
lt has been reported elsewhere that fibrin near the EC surface is more organized and occurs in tighter bundles than fibrin 50 μm away from the surface (38
). When antibodies against αV
integrin subunits or the ligand-mimetic peptide d-RGDW were used (38
), the fibrin organization on the cells’ surface was lost, indicating that the endothelial receptors responsible for the fibrin arrangement were associated or formed part of the integrin αV
. In our attempt to explore what the in vivo
clot organization of fibrinogen Caracas VIII subjects would be like, it was found that the patient’s fibrin structure near the EC surface was much less tight than that of the control, closely resembling that observed farther away from the cells’ surface. It appears that this mutation upsets the normal association of fibrin with the integrin αV
EC receptor. Some researchers do not agree with the results of Jerome et al. (38
) on the effects of ECs on fibrin lysis; others have found that the fibrin structure near the cell surface depends on the thrombin generation, and that the presence of the ligand-mimetic peptide did not alter the fibrin structure in this zone, concluding that fibrin structure on the surface of ECs was not related to fibrin binding to the integrin αV
). Nonetheless, our results are in agreement with those of Jerome et al. (38
), and the addition of the mimetic peptide abrogated the tighter structure near the cell surface in the case of the control (results not shown). Evidently more research has to be carried out to discern these apparent discrepancies.
In conclusion, the amino acid exchange Bβ Y41N in the fibrinogen variant herein described has profound effects on clot structure, leading to increased fibrin fiber diameters and pore size, which impair the fibrin mechanical properties, increase dramatically the permeation through the clots and decrease the interaction between the fibrin and the surface of HMEC-1 cells. Furthermore, although this was not examined in the present work, it is to be expected that the patient’s fibrin interaction with the VE-cadherin receptor should be impaired. Further studies ought to address these issues and their interactions, in order to clarify the functional consequences on the barrier function of ECs.