The effects of additional carbohydrate in the coiled-coil region of fibrinogen on polymerization and clot structure and properties: characterization of the homozygous and heterozygous forms of fibrinogen Lima (Aα Arg141→Ser with extra glycosylation)
Department of Cell & Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA
Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC)
John W. Weisel, Department of Cell & Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104–6058, USA. Tel.:+ 1 215 898 3573; fax:+ 1 215 898 9871; e-mail: firstname.lastname@example.org
Summary. Fibrinogen Lima is an abnormal fibrinogen with an Aα Arg141→Ser substitution resulting in an extra N-glycosylation at Aα Asn139, which seems to be responsible for the impairment of fibrin polymerization. We have studied the polymerization and properties of clots made from both plasma and purified fibrinogen of both the homozygous and heterozygous forms. The clot permeation studies with both plasma and purified protein revealed a normal flux through the network for the heterozygous form but very decreased permeation in the homozygous form. Consistent with turbidity results, the clot network of the homozygous form, seen by scanning electron microscopy, was tight and composed of thin fibers, with many branch points, while the appearance of clots from the heterozygous form was similar to that of control clots, but in both cases the fibers were more curved than those of control clots. The rheological properties of clots from the homozygous form were also altered, with rigidity being increased in plasma clots, but decreased in the purified system, a consequence of the balance between numbers of branch points and fiber curvature. From these results it seems that the extra carbohydrate moiety, located in the α coiled-coil region close to the βC domains, impairs the protofibril lateral association process, giving rise to thinner, more curved fibers, with the structural anomalies being most pronounced in the clots from the homozygous plasma. These studies support a model for fibrin polymerization in which the βC–βC interactions are involved in lateral aggregation.
Fibrinogen is a glycoprotein that circulates in plasma as the precursor of fibrin, which constitutes the scaffold of the blood clot. Fibrinogen is composed of two sets of three polypeptide chains, Aα, Bβ and γ, held together by disulfide bridges. The fibrinogen molecule is converted to fibrin monomer by the action of a serine protease, thrombin, which cleaves two small peptides in the N-terminal part of the Aα and Bβ chains. The fibrin monomers start to associate by electrostatic forces and form a three-dimensional polymer which in vivo is strengthened by the action of a transglutaminase, factor (F)XIIIa, which introduces isopeptide bonds between specific ε(γ-glutamyl) lysine residues located in the γ and α chains .
The fibrinogen molecule has approximately 3% carbohydrate consisting of NeuAc, Gal, Man, and GlcNAc  with a biantennary heterogeneous structure , linked to Asn Bβ364[4,5] and Asnγ52[6,7] as is shown in the following drawing:
± NeuAcα(2,6) Galβ(1,4) GlcNAcβ(1,2) Manα(1,3)
Manβ(1,4) GlcNAcβ(1,4) GlcNAcβ-Asn
± NeuAcα(2,6) Galβ(1,4) GlcNAcβ(1,2) Manα(1,6)
There are heterogeneities in the fibrinogen molecules introduced by different amounts of sialic acid in the terminal part of the oligosaccharides. The low-affinity Ca2+ binding sites are attributed mainly to the carbohydrate portion of the fibrinogen molecule and have a dissociation constant of approximately 1 mM . Different functions have been attributed to fibrinogen's carbohydrate, such as modulating the polymerization of fibrin monomer due to negative repulsive forces introduced by the sialic acid [9,10].
Sialic acid residues are removed by sialylases protruding from the surface of blood vessels; the exposed galactose residues are detected by the hepatic galactose/galactosamine binding lectins and could potentially mediate fibrinogen clearance in plasma . Most desialylated plasma glycoproteins are rapidly removed from circulation in this fashion, with the exception of transferrin . However, asialofibrinogen shows only a modest decrease in half-life compared with the intact protein in rabbits . An increase in NeuAc content has been reported for a number of fibrinogens from patients with inherited dysfibrinogenemias, chronic liver disease  and hepatomas .
Until now, six hereditary dysfibrinogens with an amino acid substitution that generates an Asn-X-Ser/Thr type sequence have been reported: Fibrinogen Pontoise at Bβ Asn333 , Asahi at γ Asn308 , Caracas II at Aα Asn434 , Kaiserslautern at γ Asn380 , Niigata at Bβ Asn158  and the present fibrinogen, fibrinogen Lima at Aα Asn139 .
Previous papers on fibrinogen Lima reported the clinical characterization of the patients, determination of the amino acid substitution, coagulation profile and some turbidity curves from fibrin monomer. The main functional findings from previous work on fibrinogen Lima were that plasmin generation is normal in clots of fibrin Lima  and the mutation has effects on crosslinked α-polymer formation in the homozygous form .
The aim of the present work was to study the polymerization, structure and mechanical properties of clots made from fibrinogen Lima in plasma and the purified system to understand better the molecular mechanisms of clot assembly. For this dysfibrinogenemia we have the distinct advantage that we could study both the heterozygous and homozygous forms.
Materials and methods
All chemicals used were of analytical grade, most of them purchased from Sigma Chemical Co. (St Louis, MO, USA). Human α-thrombin was from American Diagnostica (Greenwich, CT, USA). Lysine 4B and Gelatin 4B Sepharose® were from Pharmacia (Uppsala, Sweden).
We have studied the proposita (the homozygous form) and his father (the heterozygous form) of fibrinogen Lima using plasma and purified fibrinogen [19,20].
Fibrinogen was purified essentially as described elsewhere , by repeated 25% ammonium sulfate saturation after removal of plasminogen and fibronectin by passing the plasma through columns of lysine- and gelatin-Sepharose connected in tandem. The precipitate was dissolved in 50 mm Tris, 0.15 m NaCl buffer, pH 7.4, dialyzed against the same solution, and stored at − 80 °C until used. The clottability of the purified fibrinogens was 98% and no degraded fibrinogen chains (excluding the normal forms found in plasma and/or purified fibrinogen) were detected when the protein was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) . This high clottability means that there is the same amount of fibrin in these clots as in the control. The fibrinogen concentration was determined by gravimetric method .
Fibrin polymerization was carried out with purified fibrinogen solutions, the fibrinogen concentration was adjusted to 1 mg mL−1 with 50 mm Tris, 0.15 m NaCl pH 7.4 buffer, then 10 mm CaCl2 was added (final concentration) and incubated for 1 min at room temperature. Thrombin was added at 1 National Institutes of Health (NIH) units mL−1 (final concentration). The increase in optical density was recorded at a wavelength of 350 nm at 15-s intervals for 30 min in a Perkin-Elmer Lambda 4B spectrophotometer (Wellesley, MA, USA). Three replicates of each sample were examined. The lag period, the slope (the rate of increase in optical density with time) and final turbidity were calculated from the averaged values of the three curves.
Permeability and scanning electron microscopy
Permeation experiments were done with plasma (from a pool of healthy donors) and purified fibrinogen solutions, using a device designed in the laboratory. The clotting conditions used were the same as those for the fibrin polymerization studies, but plasma samples were recalcified with 20 mm CaCl2 (added to thrombin solution). After adding thrombin, the solutions were immediately transferred to pre-etched plastic tubes and the bottom part of the tube was sealed with parafilm. The columns were left in a moist environment at room temperature for 2 h, and then the top part of the column was filled with 50 mm Tris, 0.15 m NaCl pH 7.4 buffer. The columns were mounted in a support and connected to a reservoir at zero pressure containing the same buffer. The parafilm from the bottom of the tube was then removed, and the pressure was increased in order to start the flux through the clots. To wash out plasma proteins that had not been incorporated into the formed clots, these were perfused with buffer equivalent to three times its volume before starting the flux measurements. The flux (J) was calculated from the weight of the drops that percolated in a given time, which is equivalent to the volume of a drop with a density of approximately 1 g mL−1 (the density of water at room temperature). The permeation or Darcy constant  was calculated as follows:
where J = flux, cm3 s−1; η = viscosity of the buffer, 10−2 poise at room temperature; A = cross-section of the clot, cm2; L = length of the clot, cm; P = hydrostatic pressure, dyne cm−2. In our conditions, ηA/L = 0.286 dyne s−1 cm−3.
At the end, a dye (bromophenol blue in 50% glycerol) was passed through the column to evaluate the intactness of the clot structure, since reproducibility in the flux measurements does not necessarily mean that the clot structure is uniform.
The clots were then processed for scanning electron microscopy as described elsewhere [25,26]. Briefly, the clots were washed with a solution of cacodylate, fixed with 2% glutaraldehyde, and washed again with cacodylate buffer. The clots were then dehydrated with ethanol in serial dilutions from 30% to 100%, critical point dried and sputter-coated with gold-palladium and then observed using a Philips XL20 microscope. Digital images were saved and the thicknesses of the fibrin fibers were measured with the NIH ImageJ 1.18x program.
As a control for potential changes in clot structure that could occur during permeation experiments, some clots were prepared for scanning electron microscopy in shallow plexiglass buttons with holes drilled in the bottom to allow perfusion of fluid.
Plasma and purified fibrinogen clots were prepared as described above for permeation experiments. Immediately after adding thrombin, 120 µL of the mixture were transferred between 12-mm diameter glass cover slips in a Plazek torsion pendulum similar to that described by Janmey and others [27–29]. After 30 min, a momentary impulse was applied to the pendulum at room temperature, causing free oscillations with strains of less than a few percent; three records of each clot were taken. The dynamic storage modulus, G′, loss modulus, G′′, and loss tangent, tan δ, were calculated from recordings of the oscillations as follows :
The stiffness of the clot or elastic modulus:
where I = moment of inertia of pendulum arm, 545.2 g cm2; b = form factor, 1.92 cm3.
The loss modulus, the energy dissipated by viscous processes, G′′, dyne cm−2:
where Δ = ln (A/An+1), A = the amplitude of the nth oscillation;
The loss tangent, tan δ:
The fibrin polymerization process of the fibrinogen Lima homozygous form as monitored by turbidity was very impaired compared with both the control and the heterozygous form (Fig. 1). Although the lag phase was similar, the slope and the final turbidity were greatly decreased, 7- and 5-fold, respectively, less than that of the control (Table 1). In the turbidity curves of the heterozygous form, the lag phase was similar to that of the control, but the slope and final turbidity were quite decreased, approximately 1.3-fold (Fig. 1).
Table 1. Summary of the parameters measured from the fibrin polymerization curves
OD, Optical density.
Lag phase (s)
Slope (OD units min−1)
Final turbidity (OD units)
The permeation studies were done in plasma and in purified fibrinogen solutions; Table 2 summarizes the results. In plasma clots, the Darcy constant (Ks), reflecting the average pore size, of clots made from the fibrinogen Lima homozygous form was decreased 5-fold with respect to control values. Clots made with purified homozygous fibrinogen Lima were also much less permeable than control clots, approximately 8-fold. In the heterozygous form, clots made with both plasma and purified fibrinogen had Ks values that were similar to those of the controls.
Table 2. Permeability (Ks values) of clots formed in plasma and purified fibrinogen solutions
Fibrinogen Lima (homozygous)
Fibrinogen Lima (heterozygous)
Ks, Permeation or Darcy constant. In parentheses is the total number of determinations from different trials. Since clots from purified fibrinogen of the patients were more unstable, more trials and more determinations were done.
Fibrinogen (g L−1)
Ks (10−9 cm2)
4.2 ± 0.6 (18)
0.77 ± 0.07 (12)
3.7 ± 1 (18)
Ks (10−9 cm2)
12.2 ± 0.3 (18)
1.6 ± 0.1 (21)
10.6 ± 1.9 (36)
Scanning electron microscopy
After the permeation measurements were completed, these clots were processed for scanning electron microscopy. Plasma clots formed from the homozygous form were made up of very thin and curved fibers with many branch points, resulting in a reduced pore size (Fig. 2). The clot appearance of the heterozygous form was quite similar to that of the control, but fibers were less straight, there were more fibers with abrupt ends and the meshwork was more irregular than that of the control (Fig. 2). It is not likely that these fiber ends and curved fibers arise from broken fibers or other damage during specimen preparation, because our preparation procedures were developed over many years to minimize distortion and comparative experiments showed that permeation experiments did not damage the clots. Furthermore, measurements of the viscoelastic properties of these clots (see below), which are related to the clot's susceptibility to damage, show that these clots are not weak. Average fiber diameters were (homozygous, 72 ± 29 nm; heterozygous, 102 ± 48 nm; control, 120 ± 42 nm; n = 301 in all three cases), 1.4-fold less with respect to the heterozygous form and 1.7-fold less with respect to control. In the histogram of Fig. 3, it can be seen that the population of thin fibers in the homozygous form was greater with respect to both control and the heterozygous form. Also, there were no fibrin fibers in the homozygous form above 125–145 nm in diameter, although the number of fibers in the control and heterozygous forms in this range was high. The distribution of fibrin fiber diameters of the heterozygous form was quite similar to that of the control; only in the lower fibrin fiber diameter ranges (15–45 nm) did the heterozygous form have more thin fibers.
With purified fibrinogen, the proposita's clot characteristics were similar to those observed in plasma clots (Fig. 4). However, the fibers were thinner, as expected with the purified protein. The shape of the distribution of fibrin fiber diameters was different compared with that of plasma clots, with an increased number of thin fibers in all of them, including the control. The fibers of the homozygous clots were thinner than those of the control and those of the heterozygous clots (homozygous, 47 ± 19 nm; heterozygous, 55 ± 25 nm; control, 65 ± 32 nm; n = 300 in the three cases). In the histogram of the different fibrin fiber diameter ranges, it can be seen that the number of fibers at lower fibrin fiber diameter was less for the control, while that of the heterozygous clots was greater, and that of the homozygous form was greatest (Fig. 5). Only in the control clots were fibers seen above 137 nm, although very few. As found in plasma, the distribution of fibrin fiber diameters of the heterozygous form clots was quite similar to that of the control clots.
The viscoelastic properties were measured for clots from both plasma and purified fibrinogen. With plasma, it was found that the homozygous clots had increased rigidity values, approximately two times control values, while that of the heterozygous form was quite similar to control values (Table 3). It should be mentioned that the plasma fibrinogen concentration of the homozygous form was approximately 1 mg mL−1 higher than that of the control, while the plasma fibrinogen concentration of the heterozygous form was similar to that of the control. G′ is linearly related to the logarithm of fibrin concentration . The dependence of G′ on fibrinogen level in plasma in our conditions was proportional to the 2.2 power (2.2 ± 0.5) of the fibrinogen concentration. With such a correction of G′ for the different plasma fibrinogen concentrations, the expected value for G′ in the fibrinogen Lima homozygous form was lower (1619 dynes cm−2) than that observed (1997 dynes cm−2). In clots made from the heterozygous form, there was no difference in G′ compared with control values. On the other hand, when purified fibrinogen was used, the rigidity of the homozygous form clots was 1.8-fold less than that of the control and that of the heterozygous clots was similar to control values (see Table 3). The loss tangent of clots made from purified fibrinogen from both subjects was similar to that of the control. This result indicates that the relative inelastic or irreversible component of deformation is not affected by this mutation.
Table 3. Viscoelastic properties of clots made with plasma and purified fibrinogen solutions
Fibrinogen Lima (homozygous)
Fibrinogen Lima (heterozygous)
Fibrinogen (g L−1)
G′ (dyne cm−2)
514 ± 49 (6)
1084 ± 136 (12)
1997 ± 164 (12)
865 ± 118 (12)
G′′ (dyne cm−2)
40 ± 7
75 ± 9
113 ± 17
63 ± 10
0.079 ± 0.012
0.070 ± 0.004
0.056 ± 0.007
0.073 ± 0.005
Fibrinogen Lima (homozygous)
Fibrinogen Lima (heterozygous)
The numbers in parentheses represent the number of measurements from four clots (three recordings from each), except for the control (2.5 g L−1) where only two clots (three recordings from each) were measured.
The number in parentheses represents the number of measurements from three clots (three determinations from each).
G′ (dyne cm−2)
129 ± 26 (9)
71 ± 3 (9)
116 ± 25 (9)
G′′ (dyne cm−2)
9 ± 3
4 ± 1
8 ± 1
0.057 ± 0.013
0.063 ± 0.014
0.068 ± 0.008
In the fibrinogen molecule, the carbohydrate backbone itself, depending on its location, appears to interfere with the polymerization process, in addition to the repulsive forces from the sialic acid. Anomalies in the carbohydrate composition of fibrinogen have been reported from patients with inherited or acquired abnormal fibrinogens, chronic liver disease, hepatomas, and diabetes mellitus (in the form of glycosylated lysine residues). Of the abnormal fibrinogens described in the literature with extra glycosylation, severely impaired fibrin turbidity curves are reported for most of them [15–17,20]. In our case, the polymerization process was investigated with purified fibrinogen by measuring the turbidity curves in the presence of calcium; the results indicate that the extra carbohydrate present strongly inhibits lateral aggregation of protofibrils , resulting in clots with very thin fibers. Compared with the turbidity curves from other dysfibrinogens with extra carbohydrate insertions, in this case the lag phase is comparable to that of the normal fibrinogen curve, while, for example in fibrinogens Kaiserslautern, Niigata and Asahi, the lag phase is much longer than that of the control, especially in that of fibrinogen Asahi.
Other approaches have also been used in trying to establish the function of carbohydrate in the fibrin assembly process, either removing the sialic acid groups with neuraminidase or removing the whole carbohydrate by glycosidase [8,25,30]. In both cases, it was found that there was a dramatic increase in the rate of fibrin assembly, giving rise to more porous clots made up of thicker fibers that were less branched. These kinds of experiments were also carried out with some abnormal fibrinogens containing extra oligosaccharides, as in Kaiserslautern, Niigata and Lima. In the case of fibrinogen Kaiserslautern (heterozygous form) and Lima (homozygous form), the turbidity curves became similar to those of the control upon removal of the sialic acid, but in the homozygous form of fibrinogen Kaiserslautern the curve did not fully normalize. In fibrinogen Niigata, after glycosidase treatment, the turbidity curve became similar to that of control without enzymatic treatment. It should be emphasized that the mutations in these cases are located in different fibrinogen chains (Lima, in the Aα-chains, and Kaiserslautern and Asahi, in the γ-chains). The removal of the extra carbohydrate in the coiled-coil region of the fibrinogen molecule normalized the turbidity curve, and the point mutation did not have any effect on the polymerization process. However, in fibrinogens Kaiserslautern and Niigata, the molecules devoid of the extra sialic acids (Kaiserslautern, homozygous form) or the whole carbohydrate moieties in the latter case, did not normalize the polymerization process. In these two cases, the effect of the point mutation on the polymerization process is clearly evident. These experiments emphasize the importance of the C-terminal nodules of the γ-chains in fibrin monomer association.
Permeability is not a common technique used to study clots made from abnormal fibrinogens. Of the abnormal fibrinogens reported with extra carbohydrate, only in studies of clots from fibrinogens Caracas II  and Niigata was permeability measured. The clots made from fibrinogen Caracas II had an increased permeation constant and by scanning electron microscopy were characterized by the formation of large pores but thin fibers. In clots made from fibrinogen Niigata, the permeation constant was similar to that of the control. The published electron micrographs of fibrinogen Niigata clots seem to have thinner fibers and larger pores compared with those of control clots ; this combination could potentially account for the normal permeability in fibrinogen Niigata. In these three cases, the common features seem to be the formation of clots composed of thin fibers, although the porosity of the networks was greater for fibrinogen Caracas II because of the presence of larger pores. The position of the extra glycosylation in each case could probably explain the differences found in the clot structure, since in Caracas II the location is Aα Asn434, Niigata is Bβ Asn158, and Lima is Aα Asn139. The results from turbidity and permeation correlate well with the scanning electron microscope observations for clots from both homozygous and heterozygous forms of fibrinogen Lima. Clots from the homozygous form were composed of thin fibers, with increased fiber density, while the appearance of clots from the heterozygous form was closer to that of control clots. The fibers of the heterozygous fibrinogen Lima clots were thinner than those of the control clots, both in plasma and with purified fibrinogen, but the difference was not as great as with clots from the homozygous form. It seems that the presence of some normal α-chains is enough to correct partially for the impairment of clotting. In addition, we do not rule out the possibility that some of our results with purified heterozygous protein could be accounted for by selective loss of a fraction of the abnormal molecules during the fibrinogen purification process, although the bands of normal and abnormal chains on gels appear to be similar in density .
It has been found that the FXIII-induced covalent cross-linking within fibrin networks produces dramatic effects on clot rheological properties. Cross-linked clots exhibit mechanical stiffness five times greater than non-cross-linked ones [32–37]. The enhancement of rigidity in ligated clots has been attributed mainly to α-polymer formation, whereas γ-dimerization is thought to have a smaller or negligible effect on the network rigidity [34,36,37]. Also, clot stiffness is thought to be strongly dependent on fibrin fiber thickness, branch point density, and fibrin concentration [35–41]. The final clot stiffness seems to represent a combination of all these factors, structural features and degree of cross-linking .
We have studied the rheological properties of clots from fibrinogen Lima to determine the physical implications of the differences in clot structure. We could not compare the stiffness (G′) of plasma clots from control and the homozygous form directly, since the latter has a higher fibrinogen concentration than the control, but we can correct G′, knowing the relationship between G′ and the fibrinogen concentration. In the clots from the homozygous form, after taking into account the effects of differences in fibrinogen concentration, G′ was still higher than the value expected for a normal clot with this fibrinogen concentration. Clots from fibrinogen Lima have been reported previously to show decreased α-polymer formation , a characteristic that was again corroborated when plasma fibrin clots were analyzed by SDS–PAGE after viscoelastic measurements (results not shown). Although this decrease in crosslinking would be expected to result in a decrease in rigidity, the greatly increased number of branch points in these clots compensates, resulting in clots that are about twice as stiff as those from control plasma.
Clots from the purified protein of the homozygous form are less stiff than control clots. In this case, the number of branch points is also much greater compared with that of the control clots, but the effect of deficient cross-linking in the presence of only that FXIII present as a contaminant of the protein purification procedure is greater. In addition, the presence of many curved fibers would also contribute to decreased stiffness. The increase in the presence of curved fibers in clots from both homozygous and heterozygous forms suggests an impairment of the molecular packing in the fibers as a result of this mutation, probably a consequence of the bulk of the new carbohydrate. Although it is possible that some of these structural features arise from damage to the clots during permeation experiments, it seems unlikely because these clots are not weak and we have done many controls. Clots of the heterozygous form had stiffness similar to control clots, a reflection of the similarity in clot structure. With fibrinogen Caracas II (heterozygous form containing additional carbohydrate in a different location), the stiffness of the clot was also found to be normal, but the clots also contained many curved fibers.
To summarize, the presence of an extra carbohydrate moiety in the abnormal fibrinogen Lima impairs the lateral assembly. According to a recently published model on fibrin polymerization, the extra carbohydrate moiety of fibrinogen Lima is close to a proposed site of βC–βC lateral interactions . Our results provide empiric evidence for the involvement of this part of the molecule in lateral aggregation. Probably these associations are perturbed due to the insertion of the bulky carbohydrate moiety that impairs protofibril and fiber association, yielding clots with very thin, curved fibers in the homozygous form. Although clots from the homozygous form of fibrinogen Lima had decreased permeability, clots from the heterozygous form and other abnormal fibrinogens with an extra biantennary oligosaccharide had normal or augmented permeability, but all of the latter dysfibrinogens are heterozygous. The mechanical properties of the clots show the effects of changes in clot structure, including an increased number of branch points and curved fibers, as well as reduced cross-linking of α-chains. These studies demonstrate the utility of being able to study both homozygous and heterozygous forms to understand the effects of a mutation on polymerization and clot structure, providing conclusions that are likely to be applicable to the vast majority of dysfibrinogenemias that are heterozygous only. The comparison of clots from plasma and purified protein is useful because the former are more physiological while the latter contain fewer components, so that the results may be easier to interpret. Furthermore, the array of structural and biophysical studies employed here aid in the establishment of molecular mechanisms of polymerization.
We thank the personnel of the Blood Bank of Valencia, especially P. Requejo, for providing us with plasma from fibrinogen Lima patients. We are grateful to C. Zoila Carvajal and C. Nagaswami for technical assistance and helpful comments. This work was supported by grant HL30954 from the National Institutes of Health.