Influence of ' Fibrinogen Splice Variant on Fibrin Physical Properties and Fibrinolysis Rate
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《动脉硬化血栓血管生物学》
From the Department of Cardiology (J.P.C., G.M.), Pitié-Salpêtrière Hospital, Paris, France (AP-HP); Department of Cell and Developmental Biology (J.P.C., C.N., J.W.W.), University of Pennsylvania School of Medicine, Philadelphia, PA; and Department of Pathology (D.H.F.), Oregon Health & Sciences University, Portland, OR.
Correspondence to J. W. Weisel, Department of Cell and Developmental Biology, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104. E-mail weisel@mail.cellbio.upenn.edu
Abstract
Objective— A splice variant of fibrinogen, ', has an altered C-terminal sequence in its gamma chain. This A/' fibrin is more resistant to lysis than A/A fibrin. Whether the physical properties of ' and A fibrin may account for the difference in their fibrinolysis rate remains to be established.
Methods and Results— Mechanical and morphological properties of cross-linked purified fibrin, including permeability (Ks, in cm2) and clot stiffness (G', in dyne/cm2), were measured after clotting A and ' fibrinogens (1 mg/mL). '/' fibrin displayed a non-significant decrease in the density of fibrin fibers and slightly thicker fibers than A/A fibrin (12±2 fiber/10-3nm3 versus 16±2 fiber/10-3nm3 and 274±38 nm versus 257±41 nm for '/' and A/A fibrin, respectively; P=NS). This resulted in a 20% increase of the permeability constant (6.9±1.7 10-9 cm2 versus 5.5±1.9 10-9 cm2, respectively; P=NS). Unexpectedly, ' fibrin was found to be 3-times stiffer than A fibrin (72.6±2.6 dyne/cm2 versus 25.1±2.3 dyne/cm2; P<0.001). Finally, there was a 10-fold decrease of the fibrin fiber lysis rate.
Conclusions— Fibrinolysis resistance that arises from the presence of A/' fibrinogen in the clot is related primarily to an increase of fibrin cross-linking with only slight modifications of the clot architecture.
Key Words: coagulation ? fibrin ? fibrinogen ? fibrinolysis ? thrombosis
Introduction
High plasma fibrinogen is an independent predictor of cardiovascular events.1 The resulting hypercoagulable state and the decrease of fibrinolysis rate related to the greater amount of fibrin formed are thought to be the major underlying mechanisms of thrombotic events.2–4 Abnormal fibrin structure in vitro has been related to premature coronary artery disease in young patients and to severe thromboembolic disease, irrespective of the plasma fibrinogen concentration.5–8 This so-called thrombogenic fibrin consists of numerous thin fibers organized in a tight three-dimensional network in which resistance to lysis arises directly from its architecture.7,9,10
Recent in vitro investigations have revealed an association between defective fibrinolysis and the amount of A/' fibrinogen incorporated into the clot.4 This variant fibrinogen contains an altered A chain termed ' and constitutes approximately 7% to 15% of the total fibrinogen found in plasma.11 The ' chain, which arises from alternative processing of the A chain mRNA, serves as a carrier for factor XIII.12,13 Therefore, it has been suggested that A/' affects the stability of the clot formed in vitro by concentrating and increasing the rate of factor XIII activation, which catalyzes the formation of isopeptide bonds between and chains of polymerizing fibrin strands.4,13 The resulting extensive cross-linking is thought to be responsible for the lysis resistance and may account in part for the role of A/' fibrinogen as an independent risk factor for coronary artery disease.14,15 In addition, a recent study of A/' fibrinogen levels in patients undergoing coronary angiography also showed that A/' fibrinogen levels were higher, on average, in coronary artery disease patients than in patients without coronary artery disease; this association was independent of total fibrinogen levels.15 More recently, clotting of A/A and A/' fibrinogens purified from plasma has been studied.16 There was delayed release of fibrinopeptide B from A/' compared with A/A, and turbidity and scanning electron microscopy showed that clots formed from plasma A/' fibrinogen were made of thinner fibers with more branch points than those from A/A fibrinogen.
In the present study, we compared the physical characteristics, including mechanical and morphological properties, of A/A and '/' fibrin. A dynamic and structural approach using confocal microscopy was also used to assess fibrinolysis of A/A and '/' fibrin.
Methods
Human thrombin was purchased from Enzyme Research Laboratories (South Bend, IN) and stored at 1000 IU/mL. Unconjugated colloidal gold solution for light microscopy was from British Biocell International (UK). Average particle size was 5 nm and the particle concentration was 5x1013/ mL. Recombinant tissue plasminogen activator (rtPA) was purchased from Boehringer Ingelheim (Germany). Glu-plasminogen was purchased from American Diagnostica (USA). Calcium chloride was from Sigma. Purified factor XIII was a generous gift from Dr O. Gorkun (University of North Carolina, Chapel Hill). Recombinant A and ' fibrinogens were prepared as described.17
Preparation of Fibrin Clots
To a volume of 0.10 mL of purified fibrinogen (4.4 10-6 Mol/L final concentration in 20 mmol/L HEPES, pH 7.4, 0.15mol/L NaCl), 10 μL of a concentrated CaCl2 solution was added to obtain a 5-mmol/L final concentration, and activated XIII was added to obtain a final concentration of 1 IU/mL. After 1 minute of incubation, 10 μL of thrombin from the stock solution was added to obtain a final concentration of 0.9 IU/mL. Mixing and incubation were conducted in polypropylene tubes at room temperature. This solution was either soaked up into glass microchambers designed for flow measurements or placed between the coverslips of a torsion pendulum. Clotting was allowed for at least 40 minutes in a moist atmosphere at room temperature.
Scanning Light and Electron Microscopy
Fibrin clots in the microchambers were permeated with 5-nm (diameter) colloidal gold particles at a final concentration of 2.5x1012/mL dissolved in buffer containing 0.15 mol/L NaCl, 0.01 mol/L Tris/HCl, pH 7.4. The excess beads were washed out with 500 μL of the same buffer.10 Labeled specimens were scanned with LSM 510 interactive laser cytometer (Carl Zeiss) linked to a Zeiss inverted microscope equipped with a Zeiss 63x water immersion objective using the reflection mode. Optical sections were then projected and combined into one image, which generated three-dimensional reconstructed images.10
For scanning electron microscopy, the same clots were fixed, dehydrated, critical-point dried, and coated with gold palladium.18 Specimens were observed and photographed digitally using a Philips XL20 scanning electron microscope (Philips Electron Optics, Eindhoven, The Netherlands).
Morphological Analysis
Average fibrin fiber diameter and fiber and branching point densities were determined on reconstructed images of laser scanning confocal microscopy taken at high and low magnification, respectively.10 Fiber diameters were measured on digitized micrographs obtained from the scanning electron microscopy experiments.
Clot Mechanical Properties
Permeability index (Ks) of fibrin clots was measured using the permeation technique. Briefly, clots formed in thin glass microchambers (250 μm) were permeated with 0.15 mol/L NaCl, 0.01 mol/L Tris/HCl, pH 7.4, at different gradients of pressure. The calculated Ks index (cm2) provides information on the fibrin network architecture (shape and size of the pores). It represents the surface of the gel allowing flow and is obtained with the following equation: Ks=QxLx/AxPxt, where Q is the volume of liquid (mL) having the viscosity (10-2 poise) flowing through the fibrin gel with length (L) (2.2 cm) and cross-section (A) (0.03 cm2) in a given time (t) (seconds) under a differential pressure (P) (range: 4000 to 10 000 dyne/cm2).19
Clots of a constant width of 1 mm were formed between two 12-mm-diameter glass coverslips in a torsion pendulum device as previously described.20 A momentary impulse was carefully applied to the torsion pendulum arm (air pressure), causing free oscillations of this arm with strains <3%. The frequency of these free oscillations and the rate at which they are damped are functions of the elastic and viscous properties of the clots and are independent of the amplitude of the initial displacement of the arm. The rigidity index G' in dyne/cm2, which reflects the clot’s elastic properties, was calculated from the recordings of these oscillations on a chart recorder. The loss modulus (G''), which represents the energy dissipated by non-elastic viscous processes was measured from damping of the oscillations. The loss tangent (G''/G'), which is a measure of the energy dissipated by non-elastic viscous processes relative to the energy stored by elastic processes, was also assessed. This investigation was performed with and without activated factor XIII.
Lysis Experiments
At the edge of the gold-labeled fibrin clots in the glass microchambers,10 μL of a solution containing rt-PA (150 nmol/L) and Glu-plasminogen (2.5 μg/mL) was loaded. After 15 minutes of incubation in a moist atmosphere during which rt-PA and Glu-plasminogen were allowed to diffuse within the fibrin network, the edge of the thrombus was scanned with the laser scanning confocal microscope set in the reflection mode. Scanning was performed at low magnification every minute, and 20 scans were performed 1 μm apart along the z-axis for each time point. The lysis front velocity and the rate of fiber digestion were determined for each type of fibrin using reconstructed images.10
Statistical Analysis
Conventional tests were used for calculation of means and standard deviations. Group differences in continuous variables between A/A and '/' fibrins were determined by ANOVA (Version 5.0; Abacus Concepts). A risk of error of 0.05 was accepted to evaluate the statistical significance.
Results
Fibrin Physical Properties
Although the clots were formed under similar fibrinogen concentrations and clotting conditions, dramatic differences were demonstrated between the physical properties of A/A and '/' fibrin.
Both types of fibrin network consisted of straight rod-like elements organized in a three-dimensional network. The spatial organization of the fibrin fibers was found to be homogeneous in both types of fibrin clots using laser scanning confocal microscopy and scanning electron microscopy (Figure 1). However, '/' fibrin structure appeared less compact than A/A fibrin, with a 25% decrease of the fibrin fiber density (P=NS) and a 7% increase of the average fibrin fiber diameter (P=NS) (Table 1). Interestingly, a 3-fold difference in the average fiber diameter was found between laser scanning confocal microscopy and scanning electron microscopy, indicating a possible shrinkage of the clots during processing for scanning electron microscopy and/or an overestimation of the fiber diameter related to the limitation of the optical microscopy spatial resolution (Table 1). Consistent with morphological studies, permeability of '/' fibrin was increased by 20% compared with AA fibrin, indicating a lower fibrin fiber density and larger pores (Table 2).
Figure 1. Three-dimensional reconstruction images of A/A (A) and '/' (C) fibrin networks obtained by scanning confocal laser microscopy. Scanning electron micrographs of A (B) and '(D) fibrin networks. Magnification: A and C, 30x30x30 μm3; B and D, x10,000.
TABLE 1. Morphological Properties of Recombinant A/A and '/' Fibrin Using Confocal Microscopy
TABLE 2. Mechanical Properties of Recombinant A/A and '/' Fibrin
Besides these slight structural differences, striking differences were demonstrated between viscoelastic properties of A/A and '/' fibrin (Table 2). The '/' fibrin was found to be 3-times stiffer than A/A fibrin, as shown by the difference in the storage moduli G' (dyne/cm2). The energy dissipated by non-elastic viscous processes (loss modulus G'') was 1.6-times lower in A/A fibrin compared with '/' fibrin. As a consequence, the energy dissipated by viscous processes relative to the energy stored by elastic processes (tan =G''/G') was decreased by 40% in '/' fibrin compared with A/A fibrin. Of interest, there was no significant difference in the storage moduli G' of uncross-linked A/A and '/' fibrin (10.1±2.1 dyne/cm2 versus 11.8±1.9 dyne/cm2; P=NS).
Fibrinolysis
The dynamic and structural approaches of laser scanning confocal microscopy were used to assess fibrinolysis of both A/A and '/' clots.10 Lysis was found to progress as a straight and sharp front moving across the entire area of scanning (Figure 2). However, unexpectedly, the lysis front velocity of '/' fibrin was found to be 8-fold slower than for A/A fibrin, although only slight morphological differences were found between A/A and '/'. At the fiber level, similar changes were observed in both types of fibrin. Fibers underwent progressive disaggregation by transverse cutting with a transient increase in their diameter. The fiber lysis rate of A/A fibrin was 10-times faster than '/' fibrin (Table 3).
Figure 2. Series of micrographs showing the dynamic lysis of A/A (A, B, C) and '/' fibrin with rtPA (150 nmol) and glu-plasminogen (2.5 μg/mL). Lysis-front motion is visualized every minute on the top panel and every 4 minutes (D, E, F). Lysis front progresses as a straight and sharp line with a higher velocity in A/A fibrin as compared with '/' fibrin. Each micrograph is a reconstruction from optical sections representing a volume of 146x146x20 μm3.
TABLE 3. Fibrinolysis Rate of Recombinant A/A and '/' Fibrin
Discussion
A variant form of the chain, the ' chain, is found in 7% to 15% plasma fibrinogen and is present as a heterodimer with the more common A chain in A/' fibrinogen.21,22 The plasma level of the A/' fibrinogen has been shown to be an independent risk factor for coronary artery disease.14,15,23 In the process of investigating the mechanism responsible for the thrombogenicity of A/' fibrinogen, it has been shown that A/' fibrin was more resistant to lysis than A/A fibrin because of the more extensive cross-linking in A/' fibrin.4 This has been related to a higher rate of factor XIII activation by A/' fibrinogen as compared with A/A fibrinogen.13 Fibrin architecture and fibrinolysis rate of cross-linked plasma fibrin have been shown to be closely related,10 and whether structural differences between A/A and A/' fibrin may account for the difference of fibrinolysis rate between A/A and A/' fibrin is an important issue. The present investigation shows that the higher density of cross-links in A/' previously reported accounts for the dramatic reduction in the fibrinolysis rate of A/' fibrin compared with A/A fibrin.
Fibrin actively regulates its self-dissolution through numerous interactions with fibrinolytic and anti-fibrinolytic components. In particular, fibrin fiber diameter and the spatial distribution of fibers are determinants in regulating rtPA binding and fibrinolysis speed.10 The fibrinolysis resistance of the so-called thrombogenic tight fibrin conformation, made of a high density of thin fibers organized in a tight three-dimensional network, is primarily related to a decrease of rt-PA binding as compared with the coarse fibrin conformation made of fewer fibers that are thicker.9,10 In particular, the tight fibrin conformation has been found in young myocardial infarction patients and in patients with severe venous thrombotic disorder.5,6 In those situations, the resulting hypofibrinolysis is suspected to favor fibrin accumulation and thrombosis.
In contrast to our results showing relatively small differences in the structure of clots made from recombinant homozygous '/' fibrinogen in comparison with A/A fibrinogen, a recent study of purified plasma A/' and A/A found significant differences in structure.16 There are several possible reasons for these differences. There are many heterogeneities in plasma fibrinogen, including -chain splice variants, genetic polymorphisms, and posttranslational modifications such as glycosylation, nitration, oxidation, sulfation, phosphorylation, and proteolytic degradation. The recombinant fibrinogens used in the present study were not subject to these heterogeneities, although the amount of protein available was certainly a limitation in that there was not enough protein for all studies that we wanted to perform. In recognition of the heterogeneities of pooled plasma, Cooper et al did perform some experiments with A/' and A/A fibrinogens purified from a single donor.16 An alternative explanation may be that the homozygous '/' fibrinogen polymerizes differently than the heterozygous form. However, Cooper et al did not check whether there was some factor XIII in their preparations. This is an important issue, because it is established that '/A preparations have a lot more factor XIII than A/A preparations, because factor XIII binds the ' chain.24 Because we know that the effect of factor XIII will be to produce clots with thinner fibers,25 it is therefore possible that their findings are not a direct consequence of the presence of the ' chain but rather an indirect result caused by factor XIII that is present without their realizing it.
In the present investigation, recombinant ' fibrin was used so that only interactions between identical variant molecules occur rather than the more complex interactions between the more common A/'molecules. In this case, only slight differences between the morphological properties of '/' and A/A fibrin network were observed. Indeed, '/' fibrin displayed a slight increase of the fibrin fiber diameter and a slight decrease of the number of fibrin fibers per volume compared with A/A fibrin. This is further supported by the permeation experiments. In addition, scanning electron microscopy and laser scanning confocal microscopy showed the same trend, although there was a 3-fold difference in the average fibrin fiber diameter between laser scanning confocal microscopy and scanning electron microscopy. It is obvious that shrinkage of the clots may have occurred during sample processing for the scanning electron microscope, whereas fully hydrated and non-damaged fibers were visualized with the laser scanning confocal microscope. It is also likely that the limitation of the optical resolution of the laser scanning confocal microscope might have overestimated these measurements.
Given our data from both morphological analysis and permeation experiments, one would have expected '/' fibrin clots to be less stiff than A/A clots. However, clot mechanical properties or clot stiffness do not result solely from a balance between a high degree of branching and thicker fibers. These characteristics enhance the network rigidity but are antithetical, because more branching leads to thinner fibers and thicker fibers yield less branching.26 It should be emphasized that besides fibrin network architecture, fibrinogen concentration and factor XIIIa-induced cross-linking are critical factors in the regulation of fibrinolysis.26 The present investigation provides a good support for this statement. The 3-fold increase of stiffness of '/' fibrin compared with A/A fibrin is explained neither by the clotting conditions nor by the morphological properties of the three-dimensional network. Fibrinogen concentration and clotting conditions were similar, and one would have expected a 100-fold increase in both fibrin fiber and branch point densities to account for such a difference in fibrin stiffness.26 In addition, no difference was found between the stiffness of uncross-linked '/' and A/A fibrin. As a consequence, the only plausible explanation is a substantial increase in the extent of cross-linking as previously reported.13
A unique opportunity provided by laser scanning confocal microscopy was the possibility of measuring the fiber lysis rate with respect to the fibrin network configuration. It has been reported using this kind of dynamic and structural approach to fibrinolysis that fibrin network architecture rather than fibrin diameter regulates both the distribution of the fibrinolytic components and the fibrinolysis speed.10 In particular, although thicker fibers are digested at a slower rate than thin fibers, networks made of a high density of thin fibrin fibers are much more resistant to lysis than those made of thicker fibers with a lower fibrin fiber density. Our present investigation is contrary to these previous findings. One would have expected only a slight increase in fibrinolysis speed of '/' fibrin compared with A/A fibrin, whereas the lysis rate was greatly decreased. The present study shows that in addition to fibrin configuration, the extent of fibrin cross-linking is critical and may overwhelm the impact of fibrin architecture itself on fibrinolysis speed. It is important to emphasize that if there is a similar increase in cross-linking of plasma A/' clots, together with the reported change in fibrin architecture,16 the effects on lysis rates should be even more dramatic.
Several important issues should be addressed in the future. One of the most important questions is whether fibrinogen ' affects the lysis rate of platelet-rich clots. The architecture of platelet-rich clots plays a critical role in determining fibrinolysis speed.27,28 This issue is especially relevant given the fact that plasma A/' fibrinogen levels are a marker of arterial thrombotic activity, and also given that fibrinogen ' chain displays much less binding to the platelet fibrinogen receptor, glycoprotein IIb/IIIa.17,29 Finally, other issues are the interaction between fibrin architecture and the plasma A/' fibrinogen level, as well as the factor XIIIa Val34Leu polymorphism, which affects both the physical properties of the fibrin network and platelet deposition in vitro and confers a protective effect on subjects for the occurrence of myocardial infarction.25,30
In conclusion, this investigation provides new insights into the mechanism of the fibrinolysis resistance of '/' fibrin compared with A/A fibrin and confirms previous findings regarding the critical role of the extensive cross-linking in '/' fibrin.4,13
Acknowledgments
This work was supported in part by National Institutes of Health grant HL-30954 (to J.W.W.), by a grant from Parke-Davis (to J.P.C.), and by National Institutes of Health grants HL-53997 and HL-04215 and American Heart Association, Northwest Affiliate grant 0256337Z (to D.H.F.).
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Lovely RS, Falls LA, Al-Mondhiry HA, Chambers CE, Sexton GJ, Ni H, Farrell DH. Association of A/' fibrinogen levels and coronary artery disease. Thromb Haemost. 2002; 88: 26–31.
Cooper AV, Standeven KF, Ariens RA. Fibrinogen gamma chain splice variant {gamma}' alters fibrin formation and structure. Blood. 2003; 102: 535–542.
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Correspondence to J. W. Weisel, Department of Cell and Developmental Biology, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104. E-mail weisel@mail.cellbio.upenn.edu
Abstract
Objective— A splice variant of fibrinogen, ', has an altered C-terminal sequence in its gamma chain. This A/' fibrin is more resistant to lysis than A/A fibrin. Whether the physical properties of ' and A fibrin may account for the difference in their fibrinolysis rate remains to be established.
Methods and Results— Mechanical and morphological properties of cross-linked purified fibrin, including permeability (Ks, in cm2) and clot stiffness (G', in dyne/cm2), were measured after clotting A and ' fibrinogens (1 mg/mL). '/' fibrin displayed a non-significant decrease in the density of fibrin fibers and slightly thicker fibers than A/A fibrin (12±2 fiber/10-3nm3 versus 16±2 fiber/10-3nm3 and 274±38 nm versus 257±41 nm for '/' and A/A fibrin, respectively; P=NS). This resulted in a 20% increase of the permeability constant (6.9±1.7 10-9 cm2 versus 5.5±1.9 10-9 cm2, respectively; P=NS). Unexpectedly, ' fibrin was found to be 3-times stiffer than A fibrin (72.6±2.6 dyne/cm2 versus 25.1±2.3 dyne/cm2; P<0.001). Finally, there was a 10-fold decrease of the fibrin fiber lysis rate.
Conclusions— Fibrinolysis resistance that arises from the presence of A/' fibrinogen in the clot is related primarily to an increase of fibrin cross-linking with only slight modifications of the clot architecture.
Key Words: coagulation ? fibrin ? fibrinogen ? fibrinolysis ? thrombosis
Introduction
High plasma fibrinogen is an independent predictor of cardiovascular events.1 The resulting hypercoagulable state and the decrease of fibrinolysis rate related to the greater amount of fibrin formed are thought to be the major underlying mechanisms of thrombotic events.2–4 Abnormal fibrin structure in vitro has been related to premature coronary artery disease in young patients and to severe thromboembolic disease, irrespective of the plasma fibrinogen concentration.5–8 This so-called thrombogenic fibrin consists of numerous thin fibers organized in a tight three-dimensional network in which resistance to lysis arises directly from its architecture.7,9,10
Recent in vitro investigations have revealed an association between defective fibrinolysis and the amount of A/' fibrinogen incorporated into the clot.4 This variant fibrinogen contains an altered A chain termed ' and constitutes approximately 7% to 15% of the total fibrinogen found in plasma.11 The ' chain, which arises from alternative processing of the A chain mRNA, serves as a carrier for factor XIII.12,13 Therefore, it has been suggested that A/' affects the stability of the clot formed in vitro by concentrating and increasing the rate of factor XIII activation, which catalyzes the formation of isopeptide bonds between and chains of polymerizing fibrin strands.4,13 The resulting extensive cross-linking is thought to be responsible for the lysis resistance and may account in part for the role of A/' fibrinogen as an independent risk factor for coronary artery disease.14,15 In addition, a recent study of A/' fibrinogen levels in patients undergoing coronary angiography also showed that A/' fibrinogen levels were higher, on average, in coronary artery disease patients than in patients without coronary artery disease; this association was independent of total fibrinogen levels.15 More recently, clotting of A/A and A/' fibrinogens purified from plasma has been studied.16 There was delayed release of fibrinopeptide B from A/' compared with A/A, and turbidity and scanning electron microscopy showed that clots formed from plasma A/' fibrinogen were made of thinner fibers with more branch points than those from A/A fibrinogen.
In the present study, we compared the physical characteristics, including mechanical and morphological properties, of A/A and '/' fibrin. A dynamic and structural approach using confocal microscopy was also used to assess fibrinolysis of A/A and '/' fibrin.
Methods
Human thrombin was purchased from Enzyme Research Laboratories (South Bend, IN) and stored at 1000 IU/mL. Unconjugated colloidal gold solution for light microscopy was from British Biocell International (UK). Average particle size was 5 nm and the particle concentration was 5x1013/ mL. Recombinant tissue plasminogen activator (rtPA) was purchased from Boehringer Ingelheim (Germany). Glu-plasminogen was purchased from American Diagnostica (USA). Calcium chloride was from Sigma. Purified factor XIII was a generous gift from Dr O. Gorkun (University of North Carolina, Chapel Hill). Recombinant A and ' fibrinogens were prepared as described.17
Preparation of Fibrin Clots
To a volume of 0.10 mL of purified fibrinogen (4.4 10-6 Mol/L final concentration in 20 mmol/L HEPES, pH 7.4, 0.15mol/L NaCl), 10 μL of a concentrated CaCl2 solution was added to obtain a 5-mmol/L final concentration, and activated XIII was added to obtain a final concentration of 1 IU/mL. After 1 minute of incubation, 10 μL of thrombin from the stock solution was added to obtain a final concentration of 0.9 IU/mL. Mixing and incubation were conducted in polypropylene tubes at room temperature. This solution was either soaked up into glass microchambers designed for flow measurements or placed between the coverslips of a torsion pendulum. Clotting was allowed for at least 40 minutes in a moist atmosphere at room temperature.
Scanning Light and Electron Microscopy
Fibrin clots in the microchambers were permeated with 5-nm (diameter) colloidal gold particles at a final concentration of 2.5x1012/mL dissolved in buffer containing 0.15 mol/L NaCl, 0.01 mol/L Tris/HCl, pH 7.4. The excess beads were washed out with 500 μL of the same buffer.10 Labeled specimens were scanned with LSM 510 interactive laser cytometer (Carl Zeiss) linked to a Zeiss inverted microscope equipped with a Zeiss 63x water immersion objective using the reflection mode. Optical sections were then projected and combined into one image, which generated three-dimensional reconstructed images.10
For scanning electron microscopy, the same clots were fixed, dehydrated, critical-point dried, and coated with gold palladium.18 Specimens were observed and photographed digitally using a Philips XL20 scanning electron microscope (Philips Electron Optics, Eindhoven, The Netherlands).
Morphological Analysis
Average fibrin fiber diameter and fiber and branching point densities were determined on reconstructed images of laser scanning confocal microscopy taken at high and low magnification, respectively.10 Fiber diameters were measured on digitized micrographs obtained from the scanning electron microscopy experiments.
Clot Mechanical Properties
Permeability index (Ks) of fibrin clots was measured using the permeation technique. Briefly, clots formed in thin glass microchambers (250 μm) were permeated with 0.15 mol/L NaCl, 0.01 mol/L Tris/HCl, pH 7.4, at different gradients of pressure. The calculated Ks index (cm2) provides information on the fibrin network architecture (shape and size of the pores). It represents the surface of the gel allowing flow and is obtained with the following equation: Ks=QxLx/AxPxt, where Q is the volume of liquid (mL) having the viscosity (10-2 poise) flowing through the fibrin gel with length (L) (2.2 cm) and cross-section (A) (0.03 cm2) in a given time (t) (seconds) under a differential pressure (P) (range: 4000 to 10 000 dyne/cm2).19
Clots of a constant width of 1 mm were formed between two 12-mm-diameter glass coverslips in a torsion pendulum device as previously described.20 A momentary impulse was carefully applied to the torsion pendulum arm (air pressure), causing free oscillations of this arm with strains <3%. The frequency of these free oscillations and the rate at which they are damped are functions of the elastic and viscous properties of the clots and are independent of the amplitude of the initial displacement of the arm. The rigidity index G' in dyne/cm2, which reflects the clot’s elastic properties, was calculated from the recordings of these oscillations on a chart recorder. The loss modulus (G''), which represents the energy dissipated by non-elastic viscous processes was measured from damping of the oscillations. The loss tangent (G''/G'), which is a measure of the energy dissipated by non-elastic viscous processes relative to the energy stored by elastic processes, was also assessed. This investigation was performed with and without activated factor XIII.
Lysis Experiments
At the edge of the gold-labeled fibrin clots in the glass microchambers,10 μL of a solution containing rt-PA (150 nmol/L) and Glu-plasminogen (2.5 μg/mL) was loaded. After 15 minutes of incubation in a moist atmosphere during which rt-PA and Glu-plasminogen were allowed to diffuse within the fibrin network, the edge of the thrombus was scanned with the laser scanning confocal microscope set in the reflection mode. Scanning was performed at low magnification every minute, and 20 scans were performed 1 μm apart along the z-axis for each time point. The lysis front velocity and the rate of fiber digestion were determined for each type of fibrin using reconstructed images.10
Statistical Analysis
Conventional tests were used for calculation of means and standard deviations. Group differences in continuous variables between A/A and '/' fibrins were determined by ANOVA (Version 5.0; Abacus Concepts). A risk of error of 0.05 was accepted to evaluate the statistical significance.
Results
Fibrin Physical Properties
Although the clots were formed under similar fibrinogen concentrations and clotting conditions, dramatic differences were demonstrated between the physical properties of A/A and '/' fibrin.
Both types of fibrin network consisted of straight rod-like elements organized in a three-dimensional network. The spatial organization of the fibrin fibers was found to be homogeneous in both types of fibrin clots using laser scanning confocal microscopy and scanning electron microscopy (Figure 1). However, '/' fibrin structure appeared less compact than A/A fibrin, with a 25% decrease of the fibrin fiber density (P=NS) and a 7% increase of the average fibrin fiber diameter (P=NS) (Table 1). Interestingly, a 3-fold difference in the average fiber diameter was found between laser scanning confocal microscopy and scanning electron microscopy, indicating a possible shrinkage of the clots during processing for scanning electron microscopy and/or an overestimation of the fiber diameter related to the limitation of the optical microscopy spatial resolution (Table 1). Consistent with morphological studies, permeability of '/' fibrin was increased by 20% compared with AA fibrin, indicating a lower fibrin fiber density and larger pores (Table 2).
Figure 1. Three-dimensional reconstruction images of A/A (A) and '/' (C) fibrin networks obtained by scanning confocal laser microscopy. Scanning electron micrographs of A (B) and '(D) fibrin networks. Magnification: A and C, 30x30x30 μm3; B and D, x10,000.
TABLE 1. Morphological Properties of Recombinant A/A and '/' Fibrin Using Confocal Microscopy
TABLE 2. Mechanical Properties of Recombinant A/A and '/' Fibrin
Besides these slight structural differences, striking differences were demonstrated between viscoelastic properties of A/A and '/' fibrin (Table 2). The '/' fibrin was found to be 3-times stiffer than A/A fibrin, as shown by the difference in the storage moduli G' (dyne/cm2). The energy dissipated by non-elastic viscous processes (loss modulus G'') was 1.6-times lower in A/A fibrin compared with '/' fibrin. As a consequence, the energy dissipated by viscous processes relative to the energy stored by elastic processes (tan =G''/G') was decreased by 40% in '/' fibrin compared with A/A fibrin. Of interest, there was no significant difference in the storage moduli G' of uncross-linked A/A and '/' fibrin (10.1±2.1 dyne/cm2 versus 11.8±1.9 dyne/cm2; P=NS).
Fibrinolysis
The dynamic and structural approaches of laser scanning confocal microscopy were used to assess fibrinolysis of both A/A and '/' clots.10 Lysis was found to progress as a straight and sharp front moving across the entire area of scanning (Figure 2). However, unexpectedly, the lysis front velocity of '/' fibrin was found to be 8-fold slower than for A/A fibrin, although only slight morphological differences were found between A/A and '/'. At the fiber level, similar changes were observed in both types of fibrin. Fibers underwent progressive disaggregation by transverse cutting with a transient increase in their diameter. The fiber lysis rate of A/A fibrin was 10-times faster than '/' fibrin (Table 3).
Figure 2. Series of micrographs showing the dynamic lysis of A/A (A, B, C) and '/' fibrin with rtPA (150 nmol) and glu-plasminogen (2.5 μg/mL). Lysis-front motion is visualized every minute on the top panel and every 4 minutes (D, E, F). Lysis front progresses as a straight and sharp line with a higher velocity in A/A fibrin as compared with '/' fibrin. Each micrograph is a reconstruction from optical sections representing a volume of 146x146x20 μm3.
TABLE 3. Fibrinolysis Rate of Recombinant A/A and '/' Fibrin
Discussion
A variant form of the chain, the ' chain, is found in 7% to 15% plasma fibrinogen and is present as a heterodimer with the more common A chain in A/' fibrinogen.21,22 The plasma level of the A/' fibrinogen has been shown to be an independent risk factor for coronary artery disease.14,15,23 In the process of investigating the mechanism responsible for the thrombogenicity of A/' fibrinogen, it has been shown that A/' fibrin was more resistant to lysis than A/A fibrin because of the more extensive cross-linking in A/' fibrin.4 This has been related to a higher rate of factor XIII activation by A/' fibrinogen as compared with A/A fibrinogen.13 Fibrin architecture and fibrinolysis rate of cross-linked plasma fibrin have been shown to be closely related,10 and whether structural differences between A/A and A/' fibrin may account for the difference of fibrinolysis rate between A/A and A/' fibrin is an important issue. The present investigation shows that the higher density of cross-links in A/' previously reported accounts for the dramatic reduction in the fibrinolysis rate of A/' fibrin compared with A/A fibrin.
Fibrin actively regulates its self-dissolution through numerous interactions with fibrinolytic and anti-fibrinolytic components. In particular, fibrin fiber diameter and the spatial distribution of fibers are determinants in regulating rtPA binding and fibrinolysis speed.10 The fibrinolysis resistance of the so-called thrombogenic tight fibrin conformation, made of a high density of thin fibers organized in a tight three-dimensional network, is primarily related to a decrease of rt-PA binding as compared with the coarse fibrin conformation made of fewer fibers that are thicker.9,10 In particular, the tight fibrin conformation has been found in young myocardial infarction patients and in patients with severe venous thrombotic disorder.5,6 In those situations, the resulting hypofibrinolysis is suspected to favor fibrin accumulation and thrombosis.
In contrast to our results showing relatively small differences in the structure of clots made from recombinant homozygous '/' fibrinogen in comparison with A/A fibrinogen, a recent study of purified plasma A/' and A/A found significant differences in structure.16 There are several possible reasons for these differences. There are many heterogeneities in plasma fibrinogen, including -chain splice variants, genetic polymorphisms, and posttranslational modifications such as glycosylation, nitration, oxidation, sulfation, phosphorylation, and proteolytic degradation. The recombinant fibrinogens used in the present study were not subject to these heterogeneities, although the amount of protein available was certainly a limitation in that there was not enough protein for all studies that we wanted to perform. In recognition of the heterogeneities of pooled plasma, Cooper et al did perform some experiments with A/' and A/A fibrinogens purified from a single donor.16 An alternative explanation may be that the homozygous '/' fibrinogen polymerizes differently than the heterozygous form. However, Cooper et al did not check whether there was some factor XIII in their preparations. This is an important issue, because it is established that '/A preparations have a lot more factor XIII than A/A preparations, because factor XIII binds the ' chain.24 Because we know that the effect of factor XIII will be to produce clots with thinner fibers,25 it is therefore possible that their findings are not a direct consequence of the presence of the ' chain but rather an indirect result caused by factor XIII that is present without their realizing it.
In the present investigation, recombinant ' fibrin was used so that only interactions between identical variant molecules occur rather than the more complex interactions between the more common A/'molecules. In this case, only slight differences between the morphological properties of '/' and A/A fibrin network were observed. Indeed, '/' fibrin displayed a slight increase of the fibrin fiber diameter and a slight decrease of the number of fibrin fibers per volume compared with A/A fibrin. This is further supported by the permeation experiments. In addition, scanning electron microscopy and laser scanning confocal microscopy showed the same trend, although there was a 3-fold difference in the average fibrin fiber diameter between laser scanning confocal microscopy and scanning electron microscopy. It is obvious that shrinkage of the clots may have occurred during sample processing for the scanning electron microscope, whereas fully hydrated and non-damaged fibers were visualized with the laser scanning confocal microscope. It is also likely that the limitation of the optical resolution of the laser scanning confocal microscope might have overestimated these measurements.
Given our data from both morphological analysis and permeation experiments, one would have expected '/' fibrin clots to be less stiff than A/A clots. However, clot mechanical properties or clot stiffness do not result solely from a balance between a high degree of branching and thicker fibers. These characteristics enhance the network rigidity but are antithetical, because more branching leads to thinner fibers and thicker fibers yield less branching.26 It should be emphasized that besides fibrin network architecture, fibrinogen concentration and factor XIIIa-induced cross-linking are critical factors in the regulation of fibrinolysis.26 The present investigation provides a good support for this statement. The 3-fold increase of stiffness of '/' fibrin compared with A/A fibrin is explained neither by the clotting conditions nor by the morphological properties of the three-dimensional network. Fibrinogen concentration and clotting conditions were similar, and one would have expected a 100-fold increase in both fibrin fiber and branch point densities to account for such a difference in fibrin stiffness.26 In addition, no difference was found between the stiffness of uncross-linked '/' and A/A fibrin. As a consequence, the only plausible explanation is a substantial increase in the extent of cross-linking as previously reported.13
A unique opportunity provided by laser scanning confocal microscopy was the possibility of measuring the fiber lysis rate with respect to the fibrin network configuration. It has been reported using this kind of dynamic and structural approach to fibrinolysis that fibrin network architecture rather than fibrin diameter regulates both the distribution of the fibrinolytic components and the fibrinolysis speed.10 In particular, although thicker fibers are digested at a slower rate than thin fibers, networks made of a high density of thin fibrin fibers are much more resistant to lysis than those made of thicker fibers with a lower fibrin fiber density. Our present investigation is contrary to these previous findings. One would have expected only a slight increase in fibrinolysis speed of '/' fibrin compared with A/A fibrin, whereas the lysis rate was greatly decreased. The present study shows that in addition to fibrin configuration, the extent of fibrin cross-linking is critical and may overwhelm the impact of fibrin architecture itself on fibrinolysis speed. It is important to emphasize that if there is a similar increase in cross-linking of plasma A/' clots, together with the reported change in fibrin architecture,16 the effects on lysis rates should be even more dramatic.
Several important issues should be addressed in the future. One of the most important questions is whether fibrinogen ' affects the lysis rate of platelet-rich clots. The architecture of platelet-rich clots plays a critical role in determining fibrinolysis speed.27,28 This issue is especially relevant given the fact that plasma A/' fibrinogen levels are a marker of arterial thrombotic activity, and also given that fibrinogen ' chain displays much less binding to the platelet fibrinogen receptor, glycoprotein IIb/IIIa.17,29 Finally, other issues are the interaction between fibrin architecture and the plasma A/' fibrinogen level, as well as the factor XIIIa Val34Leu polymorphism, which affects both the physical properties of the fibrin network and platelet deposition in vitro and confers a protective effect on subjects for the occurrence of myocardial infarction.25,30
In conclusion, this investigation provides new insights into the mechanism of the fibrinolysis resistance of '/' fibrin compared with A/A fibrin and confirms previous findings regarding the critical role of the extensive cross-linking in '/' fibrin.4,13
Acknowledgments
This work was supported in part by National Institutes of Health grant HL-30954 (to J.W.W.), by a grant from Parke-Davis (to J.P.C.), and by National Institutes of Health grants HL-53997 and HL-04215 and American Heart Association, Northwest Affiliate grant 0256337Z (to D.H.F.).
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