Fibrinogen Maracaibo: Hypo-Dysfibrinogenemia Caused by a Heterozygous Mutation in the Gen that Encodes for the Fibrinogen Aα Chain (G.1194G>A: P.Gly13>Glu) with Diminished Thrombin Generation

Marchi R1*, Rojas H2, Echenagucia M3, Meyer M4, Acosta M5, Apitz R3 and Ruiz-Sáez A3 1Centro de Medicina Experimental, IVIC, Caracas, República Bolivariana de Venezuela 2Instituto de Inmunología, Universidad Central de Venezuela. República Bolivariana de Venezuela 3CNH-Banco Municipal de Sangre, DC, República Bolivariana de Venezuela 4University of Applied Sciences, Department of Medical Engineering and Biotechnology, Jena, Germany 5Banco de Sangre del Estado Zulia. República Bolivariana de Venezuela


Introduction
Fibrinogen or coagulation factor I (FI) is a glycoprotein of 340 k Da present in plasma at 2-4 mg/mL, is synthesized predominantly in the liver and its levels increase under inflammatory stimuli [1,2]. Fibrinogen is secreted into the bloodstream as a hexamer composed by three pairs of identical chains (Aα, Bβ and γ)2, joined together by 29 disulphide bonds that form a dimer. Each fibrinogen chain is encoded by paralogous genes (FGA, FGB, and FGG for Aα, Bβ and γ chains, respectively) [3]. In the last step of the coagulation cascade, thrombin cleaves the bonds at Aα-Arg16 and Bβ-Arg14 removing short electronegative peptides (fibrinopeptides A and B, respectively). These modified fibrinogen molecules (fibrin monomers) polymerize spontaneously and form the 3-dimentional clot network that is further stabilized by activated factor XIII (FXIIIa) [4].
Inherited fibrinogen disorders affect either the quantity (hypofibrinogenemia, fibrinogen levels <150 mg/dL) and afibrinogenemia, characterized by the complete deficiency of fibrinogen or the quality of the circulating fibrinogen (dysfibrinogenemia) or both (hypo-dysfibrinogenemia) [5]. Up to date, approximately 115 mutations have been reported that cause dysfibrinogenemia, 67 hypofibrinogenemia, 75 afibrinogenemia, and 13 hypodysfibrinogenemia; 101 in the Aα, 63 in the Bβ and 93 in the γ chain. About 50% of approximately more than 600 cases reported in the literature are silent [6,7]. Thrombin binds to its substrate, fibrinogen, and remains bound to the product, fibrin, after fibrinopeptides are removed [8,9]. Different studies have established that Asp7 to Val20, particularly residues on the N-terminal side P1 to P10 (nomenclature is that suggested by Abramovitz [10]) are required for the binding of fibrinogen´s fibrinopeptides to thrombin [11]. Within the sequence of fibrinopeptide A there are both critical (nonvariable) residues and those that can be modified without impairs thrombin catalytic activity [12]. The amino acid sequence of FpA between Asp7 and Arg16 is highly conserved among mammalian species, suggesting that this region is critical for thrombin binding [13,14]. Several abnormal fibrinogens Laser scanning confocal microscopy of fibrin clot: Fibrin clots were formed inside the eight wells LabTek chambers (Invitrogen, Nalge Nunc International, Rochester, NY, USA). The plasma sample was mixed with Alexa Fluor 488-labeled fibrinogen (4 µg/215 µl final sample volume), then clotted with a thrombin -CaCl 2 solution (0.14 U/mL and 19 mM, respectively, final concentration). The chambers were placed in a moist environment for 2 h at 37°C for complete fibrin polymerization. The fibrin clots were observed in an Olympus laser scanning confocal microscopy (LSCM) system, Model FV1000, with an argon ion laser (473 nm excitation and 520/540 nm for emission). The objective used was UPLSAPO 60X W NA: 1.20 water immersions with a work distance of 0.28. The acquisition pinhole was set to 100 µm. The images were acquired with a field of view of 212 × 212 µm (0.331 µm/ pixel). One z-stack of 30 μm thick (1 µm/slice) and one volume render was made for each field. Image analysis was performed as described elsewhere [24].

Statistical Analysis
The data obtained from the different assays are represented as the mean ± standard deviation (SD). Statistical analysis was done using Origin Pro version 8.1. Purified fibrinogen polymerization, Ks, and LSCM results were compared using the Student's t-test and a p<0.05 was considered statistically significant.

Case report
A four-year-old boy was referred to the Banco Municipal de Sangre of Caracas due to low functional fibrinogen levels, found during preoperative examination for hernia and hydrocele repair. There was not personal or family history of haemorrhagic diathesis. The mother of the propositus was a 31 year-old woman with normal menstrual flow, and no haemorrhagic complications during teeth extraction, orthopedic surgery, and caesarian. She told that only an aunt of her mother had a venous thromboembolism episode at the age of 40. Coagulation screening tests revealed a prolonged thrombin time +10.9 and +10.1 sec for the propositus and his mother, respectively, and low functional fibrinogen concentration determined by Clauss [26]. Antigenic factor von Willebrand, factor VII, protein C and S, and antithrombin levels were normal. In Table 1 the coagulation screening have been reported with mutations in this region: Asp7>Asn [15], Phe8>Cys [6], Leu9>Pro [16], Glu11>Gly [17], Gly12>Val, and Gly13>Glu in fibrinogen Olovnice [18] and Krakow II [19].
Fibrinogen Maracaibo is a new venezuelan abnormal fibrinogen with an asymptomatic phenotype discovered during preoperative examination in a four-year-old boy. The Aα Gly13>Glu delayed fibrin formation; however, normal clot morphology was observed.

Blood collection and routine coagulation tests
Blood was collected in citrate (1 volume of 0.13 mol/l trisodium citrate and 9 volumes of blood) and immediately centrifuged at 2500 × g and 4°C, during 20 min. Plasma was aliquoted and kept frozen until use. Routine coagulation tests were performed with citrated plasma on coagulation analyzer STA Compact  , Stago, France. Fibrinogen level was determined by Clauss (Laboratoire Stago, Asnière, France) and clot weight method [20].

Mutation analysis
Genomic DNA was isolated using the Invisorb Spin Blood Mini Kit (Invitek GmbH, Berlin, and Germany) according to the manufacturer´s protocol. Sequences comprising all exons and exonintron boundaries from the three fibrinogen genes: FGA, FGB, and FGG were amplified by the polymerase chain reaction (PCR) according to standard protocols. After purification of the PCR products using the Invisorb Spin PCRapid Kit  (Invitek, Berlin, FRG), direct DNA cycle sequencing was performed, applying the Big Dye kit from Applied Biosystems (Foster City, CA, USA), according to the manufacturer´s recommendations.

Fibrin network characterization
Fibrin polymerization: Fibrin polymerization was examined in plasma and purified fibrinogen. One hundred µL of plasma was mixed with 10 µL of bovine thrombin -CaCl 2 solution (0.6 units/mL and 20 mM, final, respectively); samples were run by triplicate. Purified fibrinogen (obtained by β-alanine precipitation [21]) at 1 mg/mL in Tris -buffered saline (50 mM Tris, 0.15 M NaCl), pH 7.4 was incubated for 1 min with 5 mM of CaCl 2 (final concentration), then clotted with 1 units/mL of thrombin (final concentration); samples were run by triplicate in three independent experiments. Changes in absorbance were followed during 1 h every 15 sec at 37ºC in a Tecan Infinite M200 microplate reader (Vienna, Austria). The lag time, slope and maximum absorbance (MaxAbs) were calculated for each curve and averaged.
Permeation: Permeation through plasma clots was performed essentially as described elsewhere [22]. The clotting conditions used were 0.6 unit/mL of thrombin and 20 mM CaCl 2 (final concentrations). The buffer percolated through the column was Tris-buffered saline (50 mM Tris, 0.15 M NaCl, pH 7.4). In general, six clots were used and one measurement of each was taken. Experiments were done by triplicate except for the propositus where only 7 clots were run due to the scarcity of his plasma.
The permeation coefficient or Darcy constant (Ks) was calculated using the following equation [23]:

Ks= QLη/tAP
Where Q= volume of the buffer (cm 3 ), having a viscosity η (poise), flowing through a column of height L (cm) and area A (cm 2 ) in a given time (sec), under a hydrostatic pressure P (dyne/cm 2 ). tests are summarized. DNA analysis revealed a heterozygous missense mutation in the fibrinogen gen that encoded for the Aα chain (FGA g.1194G>A: p.Gly13>Glu) in the propositus and his mother, close to the thrombin cleavage site at Aα Arg16/Gly17. Informed consent was obtained from the propositus´ mother. We have named this new hypodysfibrinogenemia as fibrinogen Maracaibo.

Fibrin network characterization
The patients´ fibrin kinetic formation was slower than control. The parameter more affected was the slope (the stage of fibrin fibers formation and association), both in plasma and purified fibrinogen, approximately two times less than control ( Figure 1 and Table 1). The permeation coefficient (Ks) of the propositus was similar to control, while that of his mother was approximately 1.6 times less than control (p<0.05). By laser scanning confocal microscopy the patients´ fibrin meshwork had normal fibrin morphology (Figure 2).

Haemostasis global tests
Thromboelastography showed only prolonged INTEM -CT both in the propositus and his mother (Table 2). Interestingly, the propositus had impaired thrombin generation. Peak thrombin and ETP were approximately 3 and 2.5 times less than control (Table 3).

Discussion
In the clinical practice dysfibrinogenemia is suspected when the thrombin time is prolonged and a low ratio between clottable fibrinogen to its antigen is found. However, the ultimate diagnosis is established based on molecular fibrinogen defect tests [27]. A new case of dysfibrinogenemia was found in a venezuelan family with an Aα Gly13>Glu mutation, molecular defect already described in fibrinogen Olovnice [18] and Krakow II [19], reported as mild bleeders. However, the carriers of fibrinogen Maracaibo were asymptomatic. This mutation was first reported by Gaja et al. [28] in a Czech Republic´ family, one carrier had thrombosis and three were asymptomatic.
The N-terminal part of the Aα and Bβ fibrinogen chains are cleaved by thrombin at Arg 16/Gly17 and Arg14/Gly15, respectively, initiating clot formation. The thrombin -fibrinogen interaction is very specific. The change of glycine, a neutral and small amino acid, by glutamic acid, an acidic and larger one at AαGly13 impairs the substrate binding in the thrombin active site [29], decreasing the efficiency of fibrinopeptides A and B release [18], lengthening clot formation but without affecting clot´s morphology, as was observed in fibrinogen Olovnice and Krakow II.
The permeation coefficient or Darcy constant is a measure of the surface available for flow [30]. Under a pressure-driven system the quantity of buffer that percolates through the fibrin clot is quantified as fibrin meshwork porosity. In our experiments, the Ks was not related to fibrinogen molecular defect, since it was normal in the propositus and decreased in his mother. Clots made with plasma are rather a complex system, and it is well known the effects of other plasma proteins in fibrin structure [31,32]. In contrast, in fibrinogen Krakow II the Ks of three family members were consistently increased.
It is important to remark that the fibrin molecules that make up the clots are normal, since FpAs are released. This could explain the almost normal clot morphology. By computer modeling, a lengthening in FpAs release predicts an increase in MaxAbs [33].
By thromboelastography the patients´ maximum clot firmness (MCF) was normal. Since clot elastic properties are related to the clot structure, these results confirmed the normality of clot morphology observed by LSCM. Interestingly, the propositus´ thrombin generation was decreased. Probably this fact was due to the subject´s young age, since it has been reported diminished thrombin generation in childhood [34].  In conclusion, the molecular fibrinogen defect Aα Gly13>Glu lengthened fibrin formation but did not alter clot structure. The differences found in thrombin generation between the propositus and his mother highlights the utility of global assays for therapy individualization. Table 2: Thromboelastographic results performed using whole blood, the extrinsic and intrinsic coagulation pathway were analyzed.