Articles

Adsorption and Coagulability of Fibrinogen on Atheromatous Lipid Surfaces

Gregory S. Retzinger
https://doi.org/10.1161/01.ATV.15.6.786
Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:786-792
Originally published June 1, 1995

Jump to

Abstract

Abstract Fibrinogen, the precursor of the blood clot matrix and a major constituent of atherosclerotic lesions, is shown to adsorb with high affinity to hydrophobic beads coated with cholesteryl oleate, cholesterol, or loosely packed lecithin. The quantity of fibrinogen that binds to cholesterol- or lecithin-coated beads decreases as the surface concentration of the lipid increases; densely packed films of lecithin bind little, if any, of the protein. In sharp contrast, the appreciable quantity of fibrinogen that binds to cholesteryl oleate–coated beads is indifferent to the surface concentration of that lipid. Not unexpectedly, the quantity of fibrinogen that binds to beads coated with mixtures of cholesteryl oleate and lecithin increases with increasing concentration of the cholesteryl ester. When bound, fibrinogen can be converted by thrombin to fibrin and nucleate clot formation as manifested by the aggregation of stirred beads. These results indicate that hydrophobic, atheromatous lipid surfaces, particularly those rich in cholesteryl esters, may be predisposed to thrombosis by virtue of their inherent capacity to bind functional fibrinogen.

  • Received October 26, 1994.
  • Accepted March 16, 1995.

Like cholesterol and its esters, fibrinogen, the precursor of the blood clot matrix, is a major component of atherosclerotic lesions, and the fibrinogen concentration of these lesions appears to correlate positively with their lipid content.1 2 3 4 Like cholesterol, fibrinogen is also a major risk factor for atherosclerosis.5 6 7 Indeed, most of the morbid and mortal consequences of atherosclerosis derive from fibrinogen-dependent thrombotic events intimately related to the development and growth within the arterial wall of lipid deposits, ie, plaques, that are rich in cholesterol and cholesteryl esters.8 Importantly, therapies mitigating the effects of atherosclerosis most frequently include anticoagulation. Thus, the accumulation of fibrinogen on or about atherosclerotic lesions does not appear to be incidental and may, in fact, contribute to processes leading to plaque formation and progression.1 2 3 4 It would be worthwhile, therefore, to elucidate the mechanism by which fibrinogen becomes incorporated into the lipid-rich lesions that define atherosclerosis.

Evidence indicates that clots in vivo are initiated on newly formed surfaces, in part because fibrinogen accumulates on these surfaces.9 10 11 This last phenomenon is of particular interest to biomedical materials researchers who seek to understand the basis for the relatively selective uptake of fibrinogen by any synthetic, hydrophobic or amphiphilic surface in contact with blood. This interest is certainly warranted since, once bound from plasma,12 fibrinogen can nucleate clot formation, thus potentially leading to thrombosis and a host of biological processes attending thrombosis, including retraction of endothelial cells and recruitment and adhesion of platelets and inflammatory cells.13 14 15 16 Indeed, recognition of these potentialities has guided the formulation and screening of polymeric materials for use in circulatory prosthetics since materials that do not bind fibrinogen are, in general, considered “hemocompatible.”17 18 19 20 But, as shown by the deposition on atherosclerotic lesions, fibrinogen deposition from blood is not unique to the surface of synthetic materials. Fibrinogen accumulates in vivo not only on or near atherosclerotic lesions but also on many other naturally occurring surfaces including the cytoplasmic membrane of certain cells,15 21 22 eg, platelets, macrophages, and tumor cells. While in most instances the binding of fibrinogen to cells appears to be mediated by specific proteins expressed on the outer surface of the cell membrane, the binding of fibrinogen to relatively acellular, hydrophobic sites, eg, atherosclerotic plaques, need not be mediated by such receptors. More likely, adsorption of fibrinogen to such sites is a consequence of physicochemical processes similar to those accounting for adsorption of the protein to the synthetic, hydrophobic materials used in the manufacture of circulatory prosthetics. These sites would then be subject to and participate in many of the fibrinogen-mediated processes that characterize the biological responses to the synthetic materials.

Since physicochemical properties of surfaces play such an important role in the adsorption of fibrinogen, it is not unreasonable to propose that molecular features dictating the capacity and affinity of some biological surfaces for fibrinogen derive from particular properties of lipids. Because not all lipid deposits are atheromatous and bind fibrinogen, something peculiar to the lipid of atherosclerotic lesions must confer on these lesions their capacity for this adhesive protein. Consistent with the observed affinity of synthetic, hydrophobic materials for fibrinogen, this “peculiarity” might derive from the hydrophobicity of cholesteryl esters, the major lipids of atherosclerotic lesions.23

A rigorous test of such a proposal might involve the formulation and use of well-defined lipid surfaces. Such well-defined lipid surfaces have already been prepared and characterized; they simply involve coating microscopic, polystyrene-divinylbenzene beads with lipids.24 25 These beads can then be used advantageously to assess the influence of the lipid microenvironment on the adsorption and subsequent polymerization and proteolyis of fibrin(ogen).26 27 28 29 This article reports on the use of hydrophobic beads for exploring the interactions of fibrinogen with lecithin-, cholesterol-, and/or cholesteryl oleate–coated surfaces. The data indicate that the identity and/or packing density of the lipid influences the adsorption of fibrinogen to the coated surface. In particular, surfaces coated with cholesteryl oleate adsorb fibrinogen avidly, even from plasma. As a consequence of this adsorption, such surfaces should be expected to function as clot nucleation sites and thus influence a host of fibrin(ogen)-mediated processes.

Methods

Reagents and Chemicals

Human fibrinogen, grade L, from Kabi AB was dialyzed exhaustively against deionized water, divided into aliquots, and stored at −20°C until use. According to the manufacturer, this commercial fibrinogen contains only trace amounts of factor XIII and no more than 0.9% by weight of plasmin(ogen)-related materials. Before use, a frozen aliquot of fibrinogen solution was first thawed to room temperature, diluted to a desired concentration by using an appropriate buffered salt solution, and then heated to 37°C to dissolve any residual cryoprecipitate. The fibrinogen concentration of stock solutions was determined by using the molar absorptivity of the protein at 280 nm, 5.12×105 L · mol−1 · cm−1.30 Fibrinogen was uniformly labeled by using Na125I from Amersham and Iodo-Gen from Pierce.31 Fatty acid–free bovine serum albumin was from Miles Biochemicals. Egg yolk l-α-lecithin from Avanti Polar Lipids was dissolved in absolute ethanol and stored under nitrogen at −20°C until use. This lecithin (Mr, 786) contains predominantly C16 and C18 fatty acid esters, of which approximately half are monounsaturated or diunsaturated. Cholesteryl oleate and anhydrous cholesterol were from Sigma. 1-Palmitoyl-2-[1-14C]oleoyl-l-3-phosphatidylcholine (specific activity, 2.11 GBq/mmol), [14C]cholesterol (2.16 GBq/mmol), and [1α,2α(n)-3H]cholesteryl oleate (1.67 TBq/mmol) were from Amersham. Human thrombin (>3000 U/mg) was from Sigma. Polystyrene-divinylbenzene beads of 6.4±1.9 μm diameter and beads of 0.945±0.0064 μm diameter were from Seragen Diagnostics. Prior to coating with lipids and/or fibrinogen, the larger beads were washed.24 Water was deionized and then distilled by using an all-glass apparatus. Organic solvents were of a grade suitable for high-performance liquid chromatography. All other chemicals were of the highest quality available commercially. Fresh plasma was prepared from the blood of a healthy donor known to have a “low normal” plasma concentration of fibrinogen. For this purpose, blood was drawn directly into evacuated siliconized glass tubes containing sodium citrate, yielding a final concentration of the anticoagulant of 0.013 mol/L (Becton Dickinson). Blood cells were pelleted by centrifugation at 1500g for 10 minutes, and the citrated plasma was then aspirated into plastic tubes and stored at −20°C until use.

Coating Beads With Lipids and/or Fibrinogen

Beads were first washed and lyophilized.24 Beads (up to 350 mg) were placed in a glass tube containing the desired lipid(s) in 3.0 mL hexane/ethanol (9:1, vol/vol). The beads were dispersed briefly by using an ultrasonic water bath and then dried with a stream of nitrogen. The dry beads were redispersed in 3.0 mL glass-distilled water, pelleted by centrifugation at 300g for 3 minutes, washed twice with 3.0 mL water, and used immediately after dispersion in 0.02 mol/L Tris-HCl, pH 7.40, containing 0.15 mol/L KCl. Radiolabeled lipids were used to determine the amount of lipid that bound to a known amount of beads.25

Adsorption isotherms of 125I-fibrinogen were generated by using both uncoated and lipid-coated beads.25 In brief, beads were dispersed in 0.02 mol/L Tris-HCl, pH 7.40, containing 0.15 mol/L KCl and a predetermined concentration of 125I-fibrinogen. The radioactivity bound to the beads was then determined. The final bead concentration of the fibrinogen binding assays was 6.7 mg/mL.

Thrombin-Inducible Aggregation of Beads as a Measure of the Functionality of Bound Fibrinogen

When stirred in the presence of thrombin, beads coated with an appropriate layer of fibrinogen aggregate as a consequence of interbead fibrin formation, and thus, thrombin-inducible aggregation is a convenient measure of the functionality of the bead-bound fibrinogen. While thrombin quantitatively liberates both fibrinopeptides A and B from fibrinogen adsorbed to beads, the liberation of fibrinopeptide A alone is necessary and sufficient for bead aggregation to occur.32 A detailed description of the aggregation assay is available.32 When using an aggregometer, a typical aggregation assay is performed at room temperature as follows. A 0.5-mL dispersion containing 0.02 mol/L Tris-HCl, pH 7.40, 1.0 mg/mL bovine serum albumin, and 1.0 mg/mL beads, ie, ≈8.07×106 beads/mL, is added to the cylindrical, glass sample cuvette (internal diameter, 6 mm). This dispersion has an apparent absorbance of 1.0 at 500 nm when a 1.0-cm path length cuvette is used. As reference material, a dispersion of beads of diameter 0.945±0.0064 μm is routinely used. The apparent absorbance of this reference dispersion is 0.5 at 500 nm when using a cuvette of 1.0-cm path length. Once the baseline signal of the stirred (1000 rpm) test sample is established, 20 μL of an aqueous solution containing 0.5 NIH U of human thrombin is added to the reaction cuvette. The relative absorbance of the sample as a function of time is then recorded. A value of 1.0 is arbitrarily assigned to the maximal change of the aggregometry signal that occurs after the addition of 0.5 U thrombin to beads coated with a mixed film of lecithin (0.08 μg/cm2) and fibrinogen (0.38 μg/cm2). When measured at 500 nm by using a spectrophotometer and a 1.0-cm cuvette, this change in apparent absorbance corresponds to 0.85 absorbance unit.

Removal and Analysis of Proteins From Beads

Proteins were eluted from beads by using an aqueous solution containing sodium dodecyl sulfate (SDS; 4%, wt/vol) and, as necessary, 2-mercaptoethanol (5%, vol/vol).12 These polypeptides were then separated by using polyacrylamide gel electrophoresis in the presence of SDS.33 The stacking gel was 4% polyacrylamide and the resolving gel, 10% polyacrylamide. Prior to electrophoresis, samples were diluted with an equal volume of 0.125 mol/L Tris-HCl, pH 6.80, and glycerol 20% (vol/vol). They were then heated to 80°C for 10 minutes. Materials eluted from beads were either stained in gels directly by using a commercial silver stain (Pierce) or electrophoretically transferred to polyvinylidene difluoride membranes for limited N-terminal sequence analysis and/or amino acid analysis by personnel of the Protein and Nucleic Acid Shared Facility of the Medical College of Wisconsin. The identities of sequences were determined by using a computer-facilitated search of the most recent update of the Protein Sequence Database of the Protein Identification Resource, the National Biomedical Research Foundation, Washington, DC.

Results

Adsorption of Fibrinogen to Lipid-Coated Beads

Once coated onto polystyrene-divinylbenzene beads, lecithin, cholesterol, and/or cholesteryl oleate remain associated with the beads even if the coated beads are stored in citrated plasma for more than 2 weeks. Binding of fibrinogen to beads coated with any of the lipids is a saturable process27 29 32 (Fig 1⇓). As is generally true for the adsorption of proteins onto hydrophobic surfaces,9 10 11 the adsorption of fibrinogen onto beads coated with any of the lipids of this study is irreversible and thus precludes rigorous analysis of the binding data according to a conventional equilibrium scheme. The saturation value, however, does provide an excellent estimate of the capacity of the lipid-coated beads for the protein. Additionally, because desorption is so slow, the surface concentration of fibrinogen remains essentially constant during the course of most experiments, and, consequently, these beads can be used to assess the role(s) of the bound lipid on both adsorption and proteolysis of the protein.26 27 28 29

Figure 1.
Figure 1.

Line graph showing that adsorption of fibrinogen to microscopic, lipid-coated, polystyrene-divinylbenzene beads is a saturable process. Beads were coated with a loosely packed lecithin monolayer of nominal surface concentration 0.11 μg/cm2, ie, ≈120 Å2/molecule. The binding of fibrinogen was monitored,25 and the paired values of [Fibrinogen]free (Ff) and [Fibrinogen]bound (Fb) were fit empirically to the equation Fb=(α×Ff)/(β+Ff), where α is the capacity of the surface for fibrinogen and β is an apparent “equilibrium” constant. In fact, little protein dissociated from beads during the time course of these experiments.11 12 32

The adsorption of fibrinogen to beads coated with either lecithin or cholesterol decreases in linear fashion with increasing lipid concentration (Fig 2⇓). The minimum amount of fibrinogen bound to these beads approaches but does not reach zero. Instead, the profiles become discontinuous at ≈0.16 and ≈0.21 molecule/Å2 for lecithin and cholesterol, respectively, indicating that a constant amount of protein binds to the otherwise hydrophobic surface once the limiting area of these lipids is reached.34 The minimum, nominal surface concentration of fibrinogen on cholesterol-coated beads is ≈0.25 μg/cm2, or about tenfold higher than that of lecithin-coated beads. From these data, it can be inferred that the binding capacity of the otherwise hydrophobic surface of the bead is reduced by an amount proportional to the area occupied by the hydrophilic head group of the adsorbed lipid. As the area per lipid approaches that of the head group, which does not bind protein, the capacity of the surface for the protein is reduced accordingly. In the case of lecithin, which is more compressible than cholesterol,34 the limiting area is approximately equivalent to that of the choline head group. Thus, at high coverage of the surface by lecithin, little protein binds. As determined by using Corey-Pauling-Kolton space-filling models, the lone hydroxyl of cholesterol, however, accounts for no more than 50% of the limiting area of the molecule. Thus, even at high surface coverage by cholesterol, appreciable hydrophobic space will be available for protein adsorption. Beads coated with cholesteryl oleate are distinctly different from those coated with either lecithin or cholesterol in that binding of fibrinogen to these beads is independent of the surface concentration of the lipid. Such a result reflects the absence of a hydrophilic function of the cholesteryl ester. A diagrammatic representation of the hypothetical condition existing on the surface of beads coated maximally with the individual lipids is given in Fig 3⇓.

Figure 2.
Figure 2.

Graph showing the capacity (ie, the α of Fig 1⇑) for binding fibrinogen of beads coated with various concentrations of lecithin (•), cholesterol (□), or cholesteryl oleate (○). The initial concentration of fibrinogen in solution was 7.40×10−7 mol/L, a concentration that ensures saturation of the lipid-coated surface given the conditions of the binding assay.28 29

Figure 3.
Figure 3.

Diagram showing the hypothetical condition existing on the surface of hydrophobic beads when coated maximally with the individual lipids of this study. Fibrinogen and lipids are not drawn to scale. Evidence indicates that in dense films of fibrinogen the long axis of the protein is nearly normal to the interface.12 28 29

Removal and Identification of Proteins Adsorbed to Lipid-Coated Beads

An aqueous solution of SDS and 2-mercaptoethanol elutes proteins from both unmodified and lipid-coated beads.12 Significant differences in the capacities of the various lipid-coated beads for fibrinogen are obvious from silver-stained gels containing materials eluted from the beads after their exposure to buffer containing fibrinogen alone as protein (Fig 4A⇓). These same differences are observed for plasma proteins in general if beads coated with the individual lipids are exposed to citrated plasma (Fig 4B⇓). As expected,9 10 11 12 despite the low normal concentration of fibrinogen in this plasma, that protein is still the dominant protein of those in the adsorbed film. Albumin and immunoglobulin are also represented in these adsorbed films.12 An as yet unidentified material of Mr >94 000 (probably close to Mr ≈150 000) is conspicuous in the eluate from cholesterol-coated beads but is only barely visible in the eluates from beads coated with the other two lipids individually. A limited, N-terminal sequence of this material12 has no significant homology with any polypeptide now catalogued in the Protein Identification Resource.

Figure 4.
Figure 4.

Silver-stained gels of reduced and denatured proteins eluted from beads coated with condensed films of either lecithin, cholesterol, or cholesteryl oleate. A, Lipid-coated beads (10 mg) were dispersed in 2.0 mL 0.02 mol/L Tris-HCl, pH 7.40, containing 2.9×10−6 mol/L fibrinogen. After washing with buffer, the bound fibrinogen was eluted from beads by using an aqueous mixture of sodium dodecyl sulfate (4%, wt/vol) and 2-mercaptoethanol (5%, vol/vol). The intensity of staining of the materials of the gel reflects the relative capacities of the various lipid-coated beads for fibrinogen. a indicates fibrinogen α-chain; b, fibrinogen β-chain; and c, fibrinogen γ-chain. B, Lipid-coated beads (20 mg) were dispersed in 4.0 mL citrated plasma obtained from a healthy donor and then incubated in this medium for an additional 20 minutes. The fibrinogen concentration of this plasma was ≈4.4×10−6 mol/L, ie, ≈151 mg/dL (151 mg%). The intensity of staining of the materials in the gel reflects the relative capacities of these lipid-coated surfaces for plasma proteins, fibrinogen in particular. a indicates a polypeptide of Mr>94 000 and of as yet undetermined identity12 ; b, dimeric immunoglobulin heavy chain; c, fibrinogen α-chain and albumin; d, fibrinogen β-chain; and e, fibrinogen γ-chain and monomeric immunoglobulin heavy chain.

Thrombin-Inducible Aggregation of Lipid-Coated Beads That Bind Fibrinogen

In addition to yielding valuable quantitative information on the binding of fibrinogen to lipid films, the bead system can be used to assess rapidly and conveniently the coagulability of the adsorbed fibrinogen. In the presence of thrombin or enzymes of thrombin-like specificity, beads coated with a monolayer of fibrinogen aggregate when stirred in an aqueous phase, a consequence of interbead fibrin dimerization.32 This aggregation can be monitored turbidimetrically by using a platelet aggregometer or even a spectrophotometer equipped with a stirred cell. The decrease in absorbance that follows the addition of thrombin to a stirred dispersion of fibrinogen-coated beads corresponds to the formation of bead aggregates of increasing size (Fig 5⇓). The maximal slope of the tracing, ie, the maximal rate of aggregation, is proportional to thrombin activity and can be used to quantify the enzyme.32 For a fixed amount of thrombin, the rate and extent of aggregation depend on the concentration of adsorbed fibrinogen.

Figure 5.
Figure 5.

Absorbance tracing. If coated with fibrinogen to a sufficiently high surface concentration, microscopic polystyrene-divinylbenzene beads aggregate in aqueous medium when stirred in the presence of thrombin or enzymes of thrombin-like specificity.32 Beads were coated with a mixed film of fibrinogen (0.38 μg/cm2) and “low-density” (0.08 μg/cm2) lecithin. Addition of thrombin is indicated by the vertical arrow. This aggregation, which is a consequence of interbead fibrin dimerization, can be monitored turbidimetrically by using a platelet aggregometer or any comparable photometric device.32 A decrease in the apparent absorbance of the stirred dispersion corresponds to an increase in the size of bead aggregates (insets).

The rate of aggregation of beads that are coated with the various lipids and then exposed to fibrinogen reflects the capacity of the lipid-coated particles for the protein (Figs 2⇑ and 6⇓). Beads coated with a liquid-expanded film of lecithin (≈160 Å2/molecule) or even a condensed film of cholesterol or cholesteryl oleate all bind ≥0.25 μg/cm2 fibrinogen and, consequently, aggregate when stirred in the presence of thrombin. In marked contrast, beads coated with a membrane-mimetic, condensed film of lecithin bind almost no fibrinogen and do not aggregate when stirred in the presence of thrombin. With the notable exception of beads coated with a condensed film of cholesterol, these same tendencies are observed when citrated plasma is used as the coating medium instead of buffered fibrinogen (Fig 6B⇓). The inability of cholesterol-coated beads to aggregate after exposure to plasma and then thrombin may be a consequence of these beads having bound insufficient fibrinogen to stabilize developing bead aggregates under the conditions of the turbidimetric assay. This reduced capacity for fibrinogen may, in turn, be a consequence of occupancy of the cholesterol-coated surface by the unidentified polypeptide of apparent Mr >94 000 (Fig 4⇑).

Figure 6.
Figure 6.

Absorbance tracings showing aggregation profiles of various lipid-coated beads after exposure of these beads to fibrinogen-containing medium. Addition of thrombin is indicated by the vertical arrows. A, Lipid-coated beads (25 mg) were dispersed in 5.0 mL 0.02 mol/L Tris-HCl, pH 7.40, containing 2.90×10−6 mol/L fibrinogen. After the beads were washed with buffer, they were used according to the routine aggregation assay.32 Beads coated with condensed films (nominal area, ≈40 Å2/molecule) of cholesterol and cholesteryl oleate bind fibrinogen and aggregate when stirred in the presence of thrombin. In contrast, beads coated with a condensed film of lecithin (lecithin-40; nominal area, ≈40 Å2/molecule) bind little fibrinogen and thus do not aggregate in the presence of the enzyme. Beads coated with a liquid-expanded film of lecithin (lecithin-160; nominal area, ≈160 Å2/molecule) bind fibrinogen and aggregate just as do the beads coated with condensed films of cholesterol and cholesteryl oleate. B, Lipid-coated beads (25 mg) were dispersed in 5.0 mL fresh, citrated plasma and then incubated in this medium for an additional 20 minutes. The fibrinogen concentration of this citrated plasma was ≈4.4×10−6 mol/L, ie, ≈151 mg%. These beads were then washed with buffer and used according to the routine aggregation assay. When coated with an expanded film of lecithin or a condensed film of cholesteryl oleate, the beads aggregated in the presence of thrombin. Beads coated with a densely packed film of either lecithin or cholesterol did not aggregate in the presence of thrombin after they were exposed to citrated plasma.

Mixed Films of Lecithin and Cholesteryl Oleate

Having delineated the relations between fibrinogen binding and the surface concentration of lecithin, cholesterol, and cholesteryl oleate bound individually to beads, an assessment was next made of the adsorption of fibrinogen to surfaces coated with mixed films of lecithin and cholesteryl oleate, since such films would more closely mimic the lipid composition of an atherosclerotic lesion.23 The amount of fibrinogen that binds to a surface coated with these mixed films is a function of both the lipid packing density and, for a given packing density, the ratio of the two lipids (Fig 7⇓). Increasing the cholesteryl ester content increased the capacity of the surface for fibrinogen. This increasing capacity for fibrinogen was reflected, again, in the thrombin-induced aggregability of the particles (Fig 8⇓).

Figure 7.
Figure 7.

Graph showing capacity for binding fibrinogen of beads coated with mixed films of lecithin and cholesteryl oleate. Binding of fibrinogen was determined as described in Fig 2⇑. Capacity is a function of both the nominal lipid packing density and, for a given packing density, the ratio of the two lipids. The amount of fibrinogen that binds increases with increasing concentrations of cholesteryl ester.

Figure 8.
Figure 8.

Absorbance tracing showing that capacity for binding fibrinogen of beads coated with a condensed (nominal area, ≈40 Å2/molecule), mixed film of lecithin and cholesteryl oleate is reflected in the thrombin-inducible aggregation of these beads. Addition of thrombin is indicated by the vertical arrow. Aggregation becomes obvious once the mole fraction of the cholesteryl ester exceeds ≈0.2.

Discussion

Taken together, these results argue that the capacity of a lipid surface for binding fibrinogen and, consequently, the thrombogenic potential of the surface depend on the availability of hydrophobic domains exposed at the interface: lipid surfaces with appreciable hydrophobic character will bind fibrinogen and, because of this binding, will be thrombogenic. This principle is relevant, mechanistically, to a host of disease processes that involve lipid surfaces and thrombosis, an important example of which is atherosclerosis. Atherosclerotic lesions are rich in fibrin(ogen) and both intracellular and extracellular lipids.1 2 3 4 23 Of these lipids, cholesteryl esters are the most conspicuous, accounting for as much as 80% to 90% of the lipid of atherosclerotic lesions most associated with overt thrombosis, ie, advanced plaques.23 Given the results presented here, it is reasonable to assume that the high affinity of cholesteryl ester–rich surfaces for fibrinogen accounts in part for the thrombotic predisposition of atherosclerotic lesions, and measures that reduce either fibrinogen concentration or the capacity of the lesions for the protein should reduce this predisposition. Such notions are eminently consistent with current opinions on the importance of both cholesterol/cholesteryl esters and fibrinogen in atherosclerosis1 2 3 4 5 6 7 8 and, in fact, provide a rationale unifying these opinions.

While this work relates to the interactions of fibrinogen with deposits of extracellular lipid, it may also relate to the interactions of fibrinogen with living cells. It is tempting to speculate that, in certain instances, the deposition/adsorption of fibrinogen onto cells, including platelets, macrophages, tumor cells, and erythrocytes,35 is driven by membrane alterations that yield hydrophobic patches on the surface of these cells. Such alterations might include redistribution36 37 38 or chemical modification39 40 of existing membrane lipids or a change in membrane lipid composition.41 42 Having bound fibrinogen, these cells would then be primed to participate in all the processes involving the protein, including adhesion, inflammation,43 metastasis,44 45 and thrombosis.

Acknowledgments

This work was supported by a grant from the Lucille P. Markey Charitable Trust; the author is a Lucille P. Markey Scholar. He thanks Ruth Mary Retzinger for inspiration.

References

  1. ↵
    Smith EB, Keen GA, Grant A, Stirk C. Fate of fibrinogen in human arterial intima. Arteriosclerosis. 1990;10:263-275.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Kunz F, Pechlaner C, Erhart R, Zwierzina W-D, Kemmler G. Increased lipid binding to thrombi in coronary artery disease: findings in patients without premedication in native (not anticoagulated) test systems. Arterioscler Thromb. 1992;12:1516-1521.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Fatah K, Hamsten A, Blombäck B, Blombäck M. Fibrin gel network characteristics and coronary heart disease: relations to plasma fibrinogen concentration, acute phase protein, serum lipoproteins and coronary atherosclerosis. Thromb Haemost. 1992;68:130-135.
    OpenUrlPubMed
  4. ↵
    Lassila R, Peltonen S, Lepäntalo M, Saarinen O, Kauhanen P, Manninen V. Severity of peripheral atherosclerosis is associated with fibrinogen and degradation of cross-linked fibrin. Arterioscler Thromb. 1993;13:1738-1742.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Kannel WB, Wolf PA, Castelli WP, D’Agostino RB. Fibrinogen and risk of cardiovascular disease. JAMA. 1987;258:1183-1186.
    OpenUrlCrossRefPubMed
  6. ↵
    Ernst E. The role of fibrinogen as a cardiovascular risk factor. Atherosclerosis. 1993;100:1-12.
    OpenUrlCrossRefPubMed
  7. ↵
    Heinrich J, Balleisen L, Schulte H, Assman G, van de Loo J. Fibrinogen and factor VII in the prediction of coronary risk: results from the PROCAM study in healthy men. Arterioscler Thromb. 1994;14:54-59.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488-500.
    OpenUrlCrossRefPubMed
  9. ↵
    Baier RE, Dutton RC. Initial events in interactions of blood with a foreign surface. J Biomed Mater Res. 1969;3:191-206.
    OpenUrlCrossRefPubMed
  10. ↵
    Vroman L, Adams AL, Klings M, Fischer GC, Munoz PC, Solansky RP. Reactions of formed elements of blood with plasma proteins at interfaces. Ann N Y Acad Sci. 1977;283:65-76.
    OpenUrlCrossRef
  11. ↵
    Brash JL. Protein adsorption at the solid-solution interface in relation to blood-material interactions. In: Brash JL, Horbett TA, eds. Proteins at Interfaces: Physicochemical and Biochemical Studies. Washington, DC: American Chemical Society; 1987:490-506.
  12. ↵
    Cook BC, Retzinger GS. Elution of fibrinogen and other plasma proteins from unmodified and from lecithin-coated polystyrene-divinylbenzene beads. J Colloid Interface Sci. 1992;153:1-12.
    OpenUrlCrossRef
  13. ↵
    Rowland FN, Donovan MJ, Picciano PT, Wilner GC, Kreutzer DL. Fibrin-mediated vascular injury: identification of fibrin peptides that mediate endothelial cell retraction. Am J Pathol. 1984; 117:418-428.
  14. ↵
    Kay AB, Pepper DS, McKenzie R. The identification of fibrinopeptide B as a chemotactic agent derived from human fibrinogen. Br J Haematol. 1974;27:669-675.
    OpenUrlCrossRefPubMed
  15. ↵
    Colvin RB, Dvorak HF. Fibrinogen/fibrin on the surface of macrophages: detection, distribution, binding requirements, and possible role in macrophage adherence phenomena. J Exp Med. 1975;142:1377-1390.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Salzman EW, Lindon J, Brier D, Merrill EW. Surface-induced platelet adhesion, aggregation, and release. Ann N Y Acad Sci. 1977;283:114-127.
    OpenUrlCrossRef
  17. ↵
    Baier RE. Selected methods of investigation for blood-contact surfaces. Ann N Y Acad Sci. 1987;516:68-77.
    OpenUrlPubMed
  18. ↵
    Hoffman AS. Modification of material surfaces to affect how they interact with blood. Ann N Y Acad Sci. 1987;516:96-101.
    OpenUrlPubMed
  19. ↵
    Andrade JD, Hlady V. Plasma protein adsorption: the big twelve. Ann N Y Acad Sci. 1987;516:158-172.
    OpenUrlCrossRefPubMed
  20. ↵
    Wurzinger LJ, Schmid-Schönbein H. Surface abnormalities and conduit characteristics as a cause of blood trauma in artificial internal organs: the interaction of fluid-dynamic physicochemical and cell biological reactions in thrombus formation. Ann N Y Acad Sci. 1987;516:316-332.
    OpenUrlPubMed
  21. ↵
    Marguerie GA, Plow EF, Edgington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem. 1979;254:5357-5363.
    OpenUrlFREE Full Text
  22. ↵
    Dvorak HF, Orenstein NS, Carvalho AC, Churchill WH, Dvorak AM, Galli SJ, Feder J, Bitzer AM, Rypysc J, Giovinco P. Induction of a fibrin gel investment: an early event in line 10 hepatosarcoma growth mediated by tumor-secreted products. J Immunol. 1979;122:166-174.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Small DM. Progression and regression of atherosclerotic lesions. Arteriosclerosis. 1988;8:103-129.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Retzinger GS, Meredith SC, Hunter RL, Takayama K, Kézdy FJ. The role of surface in the biological activities of trehalose 6,6’-dimycolate: surface properties and development of a model system. J Biol Chem. 1981;256:8208-8216.
    OpenUrlFREE Full Text
  25. ↵
    Retzinger GS, Meredith SC, Lau SH, Kaiser ET, Kézdy FJ. A method for probing the affinity of peptides for amphiphilic surfaces. Anal Biochem. 1985;150:131-140.
    OpenUrlCrossRefPubMed
  26. ↵
    Retzinger GS, Cook BC, Smith RE, McGinnis MC. Quantitation of plasma factor XIIIa activity using fibrin-coated microscopic latex beads. Anal Biochem. 1991;195:18-23.
    OpenUrlCrossRefPubMed
  27. ↵
    Retzinger GS, Chandler LJ, Cook BC. Complexation with heparin prevents adhesion between fibrin-coated surfaces. J Biol Chem. 1992;267:24356-24362.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Cook BC, Retzinger GS. Lipid microenvironment influences the processivity of adsorbed fibrin(ogen): enzymatic processing and adhesivity of the bound protein. J Colloid Interface Sci. 1994:162:171-181.
  29. ↵
    Retzinger GS, Cook BC, DeAnglis AP. The binding of fibrinogen to surfaces and the identification of two distinct surface-bound species of the protein. J Colloid Interface Sci. 1994;168:514-521.
    OpenUrlCrossRef
  30. ↵
    Mihalyi E. Physicochemical studies of bovine fibrinogen. IV: ultraviolet absorption and its relation to the structure of the molecule. Biochemistry. 1968;7:208-223.
    OpenUrlCrossRefPubMed
  31. ↵
    Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem Biophys Res Commun. 1978;80:849-857.
    OpenUrlCrossRefPubMed
  32. ↵
    Retzinger GS, McGinnis MC. A turbidimetric method for measuring fibrin formation and fibrinolysis at solid-liquid interfaces. Anal Biochem. 1990;186:169-178.
    OpenUrlCrossRefPubMed
  33. ↵
    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685.
    OpenUrlCrossRefPubMed
  34. ↵
    Gaines GL. Insoluble Monolayers at Liquid-Gas Interfaces. New York, NY: Interscience; 1966:254-264.
  35. ↵
    Fahraeus R. The suspension-stability of the blood. Acta Med Scand. 1921;55:3-228.
    OpenUrl
  36. ↵
    Zwaal RFA, Hemker HC. Blood cell membranes and haemostasis. Haemostasis. 1982;11:12-39.
    OpenUrlPubMed
  37. ↵
    Allen AM, Williamson P, Schlegel RA. Phosphatidylserine as a determinant of reticuloendothelial recognition of liposome models of the erythrocyte surface. Proc Natl Acad Sci U S A. 1988;85:8067-8071.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Connor J, Bucana C, Fidler IJ, Schroit AJ. Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation. Proc Natl Acad Sci U S A. 1989;86:3184-3188.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Yau TM, Buckman T, Hale AH, Weber MJ. Alterations in lipid acyl group composition and membrane structure in cells transformed by Rous sarcoma virus. Biochemistry. 1976;15:3212-3219.
    OpenUrlCrossRefPubMed
  40. ↵
    Mandel G, Shimizu S, Gill R, Clark W. Alteration of the fatty acid composition of membrane phospholipids in mouse lymphoid cells. J Immunol. 1978;120:1631-1636.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Hildebrand J, Marique D, Vanhouche J. Lipid composition of plasma membranes from human leukemic lymphocytes. J Lipid Res. 1975;16:195-199.
    OpenUrlAbstract
  42. ↵
    Searls DB, Edidin M. Lipid composition and lateral diffusion in plasma membranes of teratocarcinoma-derived cell lines. Cell. 1981;24:511-517.
    OpenUrlCrossRefPubMed
  43. ↵
    Dunn CJ, Willoughby DA. Leukocyte and macrophage migration inhibitory activities in inflammatory exudates: involvement of the coagulation system. Lymphokines. 1981;4:231-269.
  44. ↵
    Laki K. Fibrinogen and metastases. J Med. 1974;5:32-37.
    OpenUrlPubMed
  45. ↵
    Zacharaski LR. Basis for selection of anticoagulant drugs for therapeutic trials in human malignancy. Haemostasis. 1986;16: 300-320.
View Abstract

This Issue

Jump to

Article Tools

Share this Article

  • Email
  • Adsorption and Coagulability of Fibrinogen on Atheromatous Lipid Surfaces
    Gregory S. Retzinger
    Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:786-792, originally published June 1, 1995
    https://doi.org/10.1161/01.ATV.15.6.786
    Permalink:
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...