Gregory S. Retzinger

Adsorption of Fibrinogen to Droplets of Liquid Hydrophobic Phases Functionality of the Bound Protein and Biological Implications

Fibrinogen adsorbs spontaneously from aqueous media containing that protein to droplets of liquid hydrophobic phases dispersed in those same media. Examples of such phases include mineral oils, straight-chain hydrocarbons, and various plant- and animal-derived oils. Lecithin preexisting on the surface of oil droplets reduces significantly the amount of fibrinogen that can otherwise bind to them. When bound, fibrinogen remains active in the classic sense of fibrin gelation. As a consequence, oil droplets coated with fibrinogen can participate in a host of biologically important adhesive processes in which the protein would be expected to participate. Certain polyanions, eg, heparin, pentosan polysulfate, dextran sulfate, and suramin, bind to adsorbed fibrin(ogen) and prevent thrombin-dependent adhesion of fibrinogen-coated surfaces. Thus, these polyanions can be used to prevent adhesion between fibrin(ogen)-coated oil droplets and other fibrin(ogen)-coated surfaces. Potential practical applications and biological implications of these phenomena are presented and discussed.

Adsorption and coagulability of fibrinogen on atheromatous lipid surfaces

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 lecithin bind little,if any, if 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.

Association of osteonecrosis in systemic lupus erythematosus with abnormalities of fibrinolysis

Patients with systemic lupus erythematosus (SLE) are at an increased risk of developing osteonecrosis (ON). Twenty-six patients with SLE were studied. Fifteen of these had ON and 11 did not. The latter were used as control subjects. Various coagulation analytes including antithrombin III (ATIII) activity, protein C activity, protein S activity, a2-antiplasmin activity, anticardiolipin antibodies (aCL), plasminogen activator inhibitor (PAI-1) activity and tissue type plasminogen activator (tPA) antigen were measured using citrated plasma samples from the patients. A significant proportion (80%) of patients had at least one laboratory abnormality that has been associated with a thrombotic predisposition. ON was significantly associated with elevated levels of PAI-1 activity; it was also associated with elevated PAI-1/tPA ratio. There was no association between ON of SLE and abnormalities of the other measured coagulation analytes. These results suggest that defective fibrinolysis seems to be operative in the pathogenesis of ON associated with SLE. The defect appears to involve an imbalance between tPA and its inhibitor, PAI-1. This imbalance could represent an important risk factor in the pathogenesis of ON.

A method for probing the affinity of peptides for amphiphilic surfaces

We have developed a rapid method for probing the affinity of peptides toward an amphiphilic surface. Hydrophobic polystyrene-divinylbenzene beads of 5.7 +/- 1.5 micron diameter are coated with a monomolecular film of egg lecithin to achieve the equilibrium spreading density of the phospholipid, 6 X 10(-3) molecule/A2. The coated beads are ideally suited for assessing the affinity of peptides for phospholipid surfaces: Large quantities of lipid-coated beads of known surface area can be prepared easily and rapidly. Within the pH range 2.0 to 9.0, the adsorbed phospholipids are relatively resistant to hydrolysis and remain bound indefinitely. Following incubation with peptide ligands, beads can be separated from the reaction mixture by centrifugation. Peptides, such as melittin, which destroy or cause fusion of single bilayer phospholipid vesicles, cannot disrupt lecithin-coated beads in a comparable way, and do not displace lecithin from the surface of beads. After incubating these beads in solutions of peptides and proteins, we have determined the parameters for the binding of several ligands to the phospholipid surface. The binding of many amphiphilic peptides obeys a Langmuir adsorption isotherm, i.e., saturable reversible binding to independent and equivalent sites on the bead. That the binding is a true reversible equilibrium is shown by desorption of the ligand upon dilution. From the isotherm, the surface areas occupied by the ligand molecules were calculated, and were observed to be similar to those observed in monolayers at the air-water interface. In comparing the binding of amphiphilic peptides to that of completely hydrophilic peptides, we observed that only the former bind at levels measurable by our techniques. Thus, this method can serve as a rapid assay for detecting amphiphilicity in peptides of putative amphiphilic character.

A turbidimetric method for measuring fibrin formation and fibrinolysis at solid-liquid interfaces☆

We have developed a rapid and sensitive method by which to quantitate proteolysis of fibrin(ogen) at interfaces. Microscopic polystyrene-divinylbenzene beads coated with a mixed monomolecular film of lecithin and fibrinogen aggregate in aqueous media following exposure to thrombin or enzymes of thrombin-like activity. This aggregation is a consequence of interbead association of fibrin. As an indirect measure of the rate of fibrin formation, the rate of aggregation of beads can be used advantageously to assay enzymes and enzyme regulators pertinent to coagulation. Since the apparent absorbance of monodisperse beads is greater than that of bead aggregates, determination of the rate of change of apparent absorbance of a stirred dispersion of beads following addition of enzyme or enzyme-regulator mixture is a convenient and simple means by which to quantitate the rate of bead aggregation. Using a simple spectrophotometer or aggregometer, the method can be used to quantitate as little as 0.0005 NIH unit of thrombin. Aggregates of fibrin-coated beads can be disaggregated by several proteinases, most notably plasmin. Thus, just as bead aggregation can be used to quantitate effectors of fibrin formation, dissociation of aggregates of fibrin-coated beads can be used to quantitate effectors of fibrinolysis. Using disaggregation as a measure of fibrinolysis, the method is sensitive to as little as 0.005 unit of plasmin. Fibrin(ogen)-coated beads should prove a useful tool for studying proteolysis of fibrin(ogen) in general, and adsorbed fibrin(ogen) in particular.

Elution of fibrinogen and other plasma proteins from unmodified and from lecithin-coated polystyrene—divinylbenzene beads

Abstract We have developed a method for the rapid, quantitative elution of fibrinogen adsorbed alone from aqueous solution to unmodified and to lecithin-coated polystyrene—divinylbenzene beads. More than 98% of adsorbed fibrinogen is eluted from these beads by a single exposure to an aqueous solution containing 4% (w/v) sodium dodecyl sulfate and 5% (v/v) 2-mercaptoethanol. The efficiency of elution is maintained even if beads have been exposed for 24 h at 37°C to solutions containing fibrinogen. In addition to fibrinogen, other proteins that adsorb from plasma to these beads are rapidly and efficiently removed using this formulation. Thus, the SDS 2-ME reagent, in conjunction with the bead system, can be used effectively to identify and quantitate proteins and regions of proteins that adsorb to surfaces in contact with blood, and to probe the role of surfaces in the biologic processing of these proteins.

Adsorbed fibrinogen regulates the behavior of human dendritic cells in a CD18-dependent manner.

The involvement of fibrinogen in inflammation has been considered by many, but the roles of the protein in that process have yet to be fully elucidated. The protein readily coats surfaces and is deposited at sites of inflammation. Furthermore, adsorbed fibrinogen influences many cells of the immune system, likely a result of increased receptor recognition upon ligand immobilization. To better understand adsorbed fibrinogens role in inflammation, we studied the effects of the protein, adsorbed to the surface of microscopic beads, on human dendritic cells. Adsorbed fibrinogen increased dendritic cell expression of IL-6, IL-8, MIP-1beta and MCP-1. In contrast, solution phase fibrinogen had no effect. Importantly, dendritic cells formed complexes with, and subsequently accumulated around, beads in fibrinogen-dependent fashion. Antibodies directed against CD18 significantly decreased cytokine/chemokine expression and bead-cell complexation. Epsilon-aminocaproic acid limited bead-cell complexation, suggesting fibrinogen degradation products modulate dendritic cell activity. In support of this proposal, fibrinogen fragment D also increased MCP-1 expression by human dendritic cells. Taken together our data indicate adsorbed fibrinogen and its degradation products directly influence human dendritic cell operation. We propose a model whereby adsorbed fibrinogen plays a distinct causatory role in inflammation through its beta(2) integrin-mediated interaction with dendritic cells.

Adsorption of plasma proteins on to poly(ethylene oxide)/poly(propylene oxide) triblock copolymer films: a focus on fibrinogen

Triblock copolymers of the form PEOαPPOβPEOα [where PEO is poly(ethylene oxide) and PPO is poly(propylene oxide)] have many biomedical applications, many of which depend on the surface properties of the copolymers and the influence that those properties have on the adsorption of proteins. As a tool to help us better understand, predict and exploit the influence of these triblock copolymers on protein adsorption, we developed a model system in which well‐defined monolayers of the copolymers are supported by solid, hydrophobic, microscopic beads. At the bead/water interface, the copolymers all form stable films in which the nominal molecular areas correspond to those of the molecules when they are packed rather tightly at the air/water interface. Beads coated with condensed films of copolymers that contain short PEO segments and elicit appreciable inflammation absorb appreciable quantities of plasma proteins, including fibrinogen, from aqueous solution. Beads coated with fibrinogen aggregate when they are stirred in the presence of thrombin, a consequence of interbead fibrin formation. Beads coated with condensed films of copolymers that contain long PEO segments and elicit little inflammation absorb little plasma protein, and they do not aggregate in the presence of thrombin. Our data and observations are consistent with the prevailing notion that the utility of triblock copolymers as agents for modifying the surface properties of blood‐contacting surfaces derives from the influence of the copolymers on the adsorption of plasma proteins. In this regard, the ability of the copolymers to influence fibrinogen‐mediated adhesive events may be particularly important. As to the mechanism of protein resistance, our data support the proposal that sibling PEO segments of copolymers in condensed films fold back across their parental PPO cores, limiting access of proteins to the hydrophobic cores themselves.

Fibrinogen-Coated Droplets of Olive Oil for Delivery of Docetaxel to a Fibrin(ogen)-Rich Ascites Form of a Murine Mammary Tumor

Micronized droplets of olive oil loaded with docetaxel and coated with functional fibrinogen were administered intraperitoneally to mice bearing the fibrin(ogen)-rich ascites form of the TA3/St mammary tumor. When compared with docetaxel administered intraperitoneally as its commercial formulation (i.e., Taxotere), docetaxel-loaded oil droplets coated with murine fibrinogen prolonged the median survival time of tumor-bearing mice from 14.5 to 29.5 days. Drug-free oil droplets provided no therapeutic benefit. Significantly more docetaxel was associated with tumor cells 24 and 48 hours after administration of the drug in fibrinogen-coated oil droplets than after its administration as Taxotere. Consistent with a role for thrombin in the retention of fibrinogen-coated oil droplets within the tumor microenvironment, hirudin significantly reduced the association of tumor cells with docetaxel delivered in fibrinogen-coated oil droplets and, at the same time, reduced the therapeutic efficacy of the droplets to that of Taxotere. Importantly, fibrinogen-coated oil droplets formed rosettes with tumor cells in vivo, a process prevented by hirudin. Although mice treated with oil droplets developed antifibrinogen antibodies, those antibodies seemed to be inconsequential. Taken together, our results and observations indicate fibrinogen-coated oil droplets markedly improve the therapeutic efficacy of docetaxel for the treatment of a mammary tumor grown in ascites form, a consequence of thrombin-mediated retention of the drug-loaded droplets within the tumor microenvironment.

Fibrinogen adsorbs from aqueous media to microscopic droplets of poly(dimethylsiloxane) and remains coagulable

Fibrinogen binds from aqueous media containing it to droplets of linear trimethylsilyl-terminated poly(dimethylsiloxane) (PDMS) dispersed in those same media. Once bound, fibrinogen elutes from emulsified droplets of PDMS only very slowly, even when incubated in buffer that contains a physiologic concentration of the protein. The bound fibrinogen is coagulable, as indicated by the thrombin-dependent agglutination of droplets. Thus fibrinogen bound to droplets of PDMS renders an adhesive potential to the surface of the droplets, a potential that may have relevance to the biologic processing of the polymer in vivo.