Interaction of fibrinogen and its derivatives with fibrin

The binding between complementary polymerization sites of fibrin monomers plays an essential role in the formation of the fibrin clot. One set of polymerization sites involved in the interaction of fibrin monomers is believed to pre-exist in fibrinogen, while the complementary set of binding sites is exposed after the cleavage of fibrinopeptides from fibrinogen. The polymerization sites present in fibrinogen and its derivatives mediate their binding to fibrin. Although the binding of fibrinogen and its derivatives to fibrin have been qualitatively studied, there has been no systematic, quantitative investigation of their interaction with forming or preformed clots. In the present study, the binding of fibrinogen and fragments DD, D1, and E1 was measured using a sonicated suspension of plasminogen- and thrombin-free human cross-linked fibrin as a model of a preformed clot. Dissociation constants of 0.056, 0.19, and 2.44 microM, and the number of binding sites corresponding to 0.10, 0.21, and 0.13/fibrin monomer unit of fibrin polymer were found for fibrinogen, fragment DD, and fragment D1, respectively. Fragment E1 did not bind to sonicated noncross-linked or cross-linked fibrin suspensions. However, it was bound to forming fibrin clots as well as to fibrin-Celite, suggesting that the binding sites on fibrin involved in the interaction with fragment E1 may have been altered upon sonication. Affinity chromatography of various fibrinogen derivatives on a fibrin-Celite column showed that only part of the bound fragment DD was displaced by arginine, whereas fragments D1 and E1 were completely eluted under the same conditions. The results indicate that interaction of fibrinogen with the preformed fibrin clots is characterized by affinity in the nanomolar range and that binding between fibrin monomers, in the process of clot formation, could be characterized by even a higher affinity.     

Interaction of fibrin(ogen) with fibronectin: further characterization and localization of the fibronectin-binding site

The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) alphaC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D-D:E(1) complex and a recombinant alphaC fragment (residues Aalpha221-610) corresponding to the alphaC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D-D:E(1) complex or the immobilized alphaC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the alphaC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the alphaC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH(2) terminal connector region of the alphaC domain since in ELISA, the immobilized recombinant Aalpha221-391 sub-fragment bound fibronectin well while the immobilized recombinant Aalpha392-610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aalpha221-391 region of the fibrinogen alphaC domain that is not accessible in fibrinogen but becomes exposed in fibrin.     

Influence of the subendothelial basement membrane components on fibrin assembly. Evidence for a fibrin binding site on type IV collagen

Effective repair of a vascular injury depends on establishment of a stable fibrin patch at the injury site. Data presented in this study demonstrate that structural modification of fibrin occurs as a result of fibrin interaction with naturally occurring components of the vascular basement membrane and subendothelial structures. Of the basement membrane components, type IV collagen produces the greatest structural modification, generating thick fibrin fibers; a 3-fold increase in the fiber mass/length ratio occurs when type IV collagen is increased from 0 to 100 ng/ml. Laminin and dermatan sulfate decrease the fibrin fiber mass/length ratio resulting in thinner fibers. However, the overall effect of the basement membrane on fibrin is to increase the fibrin fiber diameter. Electrophoretic light scattering and the binding of type IV collagen by fibrinogen-Sepharose further establish the interaction between type IV collagen and fibrinogen. Incorporation of laminin with type IV collagen onto coated surfaces decreases the ability of type IV collagen to bind fibrinogen. These studies emphasize that the final fibrin structure is influenced by the milieu in which the clot is assembled.     

Features of the interaction of Glu- and Lys-forms of plasminogen with native and partially hydrolyzed fibrin]

Glu- and Lys-plasminogen interaction with native and desAABB-fibrin obtained from fibrinogen partially hydrolyzed by plasmin was studied. It was found that native fibrin adsorbs 6 times more Lys-plasminogen as compared to the native form of the proenzyme. The range of the Lys-plasminogen binding does not change, if part of the fibrinogen molecules hydrolyze down to X-fragments. At the same time, the appearance in the system of 1% Xi-fragments leads to a 6-fold increase in the Glu-plasminogen binding. The amount of adsorbed Glu-plasminogen reaches the level of Lys-plasminogen adsorption both in the native and partially hydrolyzed fibrin. It was found that kringle K 1-3 or 6-aminohexanoic acid at saturating for high-affinity lysine-binding sites concentrations do not influence the Glu-plasminogen binding to native fibrin but inhibit it when the partially purified form is used. It is assumed that the manyfold increase of the Glu-plasminogen binding to partially hydrolyzed fibrin is due to the alteration of the proenzyme conformation at the initial steps of fibrin hydrolysis during the formation of Xi fragments.     

Mechanism of functioning of specific sites of interdomain D-E binding of fibrin molecules: interaction of fibrinogen with the N-terminal disulfide node of fibrin

The intermolecular noncovalent binding of complementary fibrin polymerization sites localized in fibrin domains D and E was investigated in the model system. In this system fibrinogen molecules represent the active D domains and the N-terminal disulphide knot of fibrin (N-DSK) represents the active E domain. Quantitative definition of insoluble fibrinogen and N-DSK copolymer and light scattering data of their mixtures before the appearance of visible precipitate show that complexing of these structures decreases with an increase of the temperature and ionic strength. The character of this dependence permits certain conclusions to be made on the functioning mechanism for two types of the D-E binding sites. These conclusions are based on an idea of their different affinity. The interdomain binding is primarily realized by D1-E1 sites which are characterized by a high affinity and work mainly on the basis of electrostatic forces. This binding directs the D2-E2 binding which is characterized by lower affinity and which determines the final degree of fibrinogen and N-DSK complexing. These sites function mainly by the H-binding.     

Analysis of soluble fibrin complexes by agarose gel chromatography and protamine sulfate gelation.

The molecular makeup of soluble fibrin complexes was studied by gel exclusion chromatography using radio-labelling to characterize individual components in protein mixtures. Products of limited plasmin degradation of fibrinogen and mixtures of fibrinogen and "early" fibrinogen digests formed high molecular weight soluble fibrin complexes upon incubation with thrombin. Purified, nonclottable fragment Y did not incorporate into soluble fibrin complexes, nor could we demonstrate incorporation of fragments D and E as previously described from our laboratory. Thus, under the conditions of these experiments, soluble fibrin complexes have two identifiable components, fibrin monomer and clottable fragment X monomer, although incorporation of native fibrinogen or fragment X unreacted by thrombin into soluble fibrin complexes cannot be excluded. Individual fractions of thrombin-treated early fibrinogen digests isolated by agarose gel chromatography were treated with protamine sulfate at 37 degrees C resulting in precipitation-gelation of greater than 90 per cent of high molecular weight soluble fibrin complexes; whereas, less than 10 per cent of lower molecular weight fibrinogen degradation products precipitated by protamine sulfate. These findings do not support the widely held concept that soluble fibrin complexes incorporate nonclottable degradation products of fibrinogen proteolysis, nor do they support the notion that the so-called paracoagulation reaction induced by protamine sulfate results from the splitting of complexes between fibrin monomer and nonclottable fibrinogen degradation products.     

Complexes of the N-terminal disulfide branch point of fibrin with fibrinogen

Investigation of fibrin N-terminal disulphide knot (N-DSK) binding with fibrinogen (F) showed, that the F-N-DSK-complex represents growing polymer structure which is soluble at early polymerization stage and forms a solid phase during the further growth. This complex is characterized by constant stoichiometry expressed by formula F (N-DSK)2. A model of the complex structure as a regular copolymer of fibrinogen and N-DSK is proposed, in which neighbouring fibrinogen molecules are clamped with two N-DSK molecules. Such copolymer was never described. Since its formation is caused by specific D-E interdomain binding, it may be considered as a peculiar analogue of fibrin polymer.     

Proteolytic activity of the Glu-plasminogen complex with fibrinogen fragment E

Glu-plasminogen interaction with fibrinogen fragment E results in the alteration of its adsorptive capacity. During this interaction in the absence of plasmin and tissue activator of plasminogen, Glu-plasminogen is transformed into a partly degraded form. Glu-plasminogen complexes with soluble and immobilized fibrinogen fragment E. contain a serine proteinase-specific activity which is inhibited by diisopropylfluorophosphate. The complexes under study are active towards fibrin and the plasmin-specific tripeptide substrate, D-Val-L-Leu-L-Lys-p-nitroanilide. It is concluded that fibrinogen fragment E induces structural changes in the enzyme molecule which eventually result in the formation of an active center.     

Effect of peptides--structural analogs of NH2-terminal sites of fibrin alpha- and beta-chains--on specific binding of the NH2-terminal disulfide bond of fibrin with fibrinogen

Peptides Gly-Pro-Arg-Pro and Gly-His-Arg-Pro (fibrin alpha- and beta-chain NH2-terminal analogs, respectively) are studied for their effect on fibrinogen (F) and fibrin NH2-terminal disulphide knot (N-DSK) specific binding. Both peptides are found to inhibit the formation of soluble and insoluble F-N-DSK-complexes through inhibition of the interdomain D-E-binding. Gly-Pro-Arg-Pro is much more potent inhibitor than Gly-His-Arg-Pro. Lowering the insoluble F-N-DSK-copolymer quantity by concentration-dependent way these peptides do not change its composition described by the formula [F(N-DSK)2]n. Invariability of fibrinogen and N-DSK copolymer structure is asserted. In this structure neighbouring fibrinogen molecules are bound by two N-DSK molecules via the D1-E1 and D2-E2 binding sites.     

Ozone-induced damage of fibrinogen molecules: identification of oxidation sites by high-resolution mass spectrometry

Fibrinogen is highly susceptible to oxidation compared to other plasma proteins. Fibrinogen oxidation damages its structure and affects the protein function. Ozone-induced oxidative modifications of the fibrinogen Aα, Bβ, and γ polypeptide chains upon addition of various amounts of the oxidiser were studied by mass spectrometry. Amino acid residues located on all three chains and main structural parts of the protein were revealed to be involved in oxidation. The αC-connector was shown to be most vulnerable to oxidation as compared to other structural parts while the E region turned out to be the most protected area of the protein. For the first time, it was established that numerous amino acid residues responsible for the conversion of fibrinogen to fibrin remain unaffected upon fibrinogen oxidation. The data obtained in this study indicate that none of the identified residues, which are considered crucial for the binding of both hole “a” and hole “b” to knob “A” and knob “B”, respectively, as well as those responsible for the thrombin binding to fibrinogen E region, have been subjected to chemical alterations under moderate oxidation. The data on fibrinogen oxidation acquired in the current study enable one to assume that some of the structural fibrinogen parts and easily oxidisable residues could be endowed with antioxidant properties. New findings presented here could be essential for the detection of adaptive molecular mechanisms capable of mitigating the detrimental action of reactive oxygen species (ROS) on the functioning of oxidatively damaged fibrinogen. Data are available via ProteomeXchange with identifier PXD012046.

• Highlights

• Various oxidative modifications were detected in fibrinogen by mass spectrometry

• αC-connector has been shown to be most susceptible to oxidation

• E region proved to be least vulnerable to the action of the oxidising agent

• Some of the Met residues in the fibrinogen structure could operate as ROS scavengers

     

The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin

Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains ("A" and "B," respectively) in the central portion of the molecule, and exposure of a noncatalytic "secondary" thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the B beta 1-42 sequence ("des-(B beta 1-42)-fibrinogen"), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by B beta 1-42 in the polymerization of alpha-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (mu, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(B beta 1-42)-alpha-fibrin, thus indicating that the B beta 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(B beta 1-42)-alpha-fibrin polymerized even more slowly than thrombin-activated des-(B beta 1-42)-alpha-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent alpha-fibrin monomers. Thrombin-activated des-(B beta 1-42)-alpha-fibrin polymerized more rapidly at low ionic strength (mu, 0.04) than did native alpha,beta-fibrin, a reversal of their behavior at physiological ionic strength (mu, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(B beta 1-42)-fibrinogen (t1/2, 36.5 versus 21.5 min) and marked slowing from native fibrinogen (t1/2, 138 versus 22.2 min). This behavior correlated with increased thrombin binding to native alpha,beta-fibrin at low ionic strength, coupled with weak thrombin binding to des-(B beta 1-42)-alpha-fibrin, and indicates that secondary thrombin binding plays an important role in regulating thrombin diffusion and catalytic activity. Des-(B beta 1-42)-fibrinogen lacks or has a markedly defective secondary thrombin-binding site, from which we conclude that the B beta 15-42 sequence in fibrin plays a major role in forming or providing this site.     

Analysis of soluble fibrin complexes by agarose gel chromatography and protamine sulfate gelation

The molecular makeup of soluble fibrin complexes was studied by gel exclusion chromatography using radio-labelling to characterize individual components in protein mixtures. Products of limited plasmin degradation of fibrinogen and mixtures of fibrinogen and “early” fibrinogen digests formed high molecular weight soluble fibrin complexes upon incubation with thrombin. Purified, nonclottable fragment Y did not incorporate into soluble fibrin complexes, nor could we demonstrate incorporation of fragments D and E as previously described from our laboratory. Thus, under the conditions of these experiments, soluble fibrin complexes have two identifiable components, fibrin monomer and clottable fragment X monomer, although incorporation of native fibrinogen or fragment X unreacted by thrombin into soluble fibrin complexes cannot be excluded. Individual fractions of thrombin-treated early fibrinogen digests isolated by agarose gel chromatography were treated with protamine sulfate at 37 °C resulting in precipitation-gelation of greater than 90 per cent of high molecular weight soluble fibrin complexes; whereas, less than 10 per cent of lower molecular weight fibrinogen degradation products precipitated by protamine sulfate. These findings do not support the widely held concept that soluble fibrin complexes incorporate nonclottable degradation products of fibrinogen proteolysis, nor do they support the notion that the so-called paracoagulation reaction induced by protamine sulfate results from the splitting of complexes between fibrin monomer and nonclottable fibrinogen degradation products.     

Binding phenomena of isolated unique plasmic degradation products of human cross-linked fibrin.

Proteolysis of human cross-linked fibrin by plasmin results in the formation of a DD . E complex, and Fragments DD and E as the major degradation products. Three species of Fragment E, which differ both in molecular weights (E1, Mr = 60,000; E2, Mr = 55,000; E3, Mr = 50,000) and in charge, have been isolated from a digest of cross-linked fibrin. Each Fragment E species reacts with monospecific anti-E antiserum. Fragments E1 and E2 bind with Fragment DD to form a DD . E complex but Fragment E3 is inactive. This binding is specific since these Fragments E do not bind to fibrinogen or to degradation products of fibrinogen or of noncross-linked fibrin. Fragments E1 and E2 incubated with plasmin are degraded to Fragment E3, suggesting that the three species represent sequential degradation products. Plasmin-treated Fragments E1 and E2 no longer bind with Fragment DD; therefore, it appears that the peptides cleaved from Fragment E2 by plasmin contain or modify the sites responsible for complex formation. On the other hand, Fragment DD binds not only to Fragments E1 and E2, but also to fibrinogen, Fragments X (Stage 1), X (Stage 2), Y, and NH2-terminal disulfide knot, but only after thrombin treatment, suggesting that Fragment DD binds to complementary sites on the NH2-terminal region of fibrinogen which are exposed after thrombin treatment.     

Conversion of fibrinogen to fibrin: mechanism of exposure of tPA- and plasminogen-binding sites

Conversion of fibrinogen into fibrin results in the exposure of cryptic interaction sites and modulation of various activities. To elucidate the mechanism of this exposure, we tested the accessibility of the Aalpha148-160 and gamma312-324 fibrin-specific epitopes that are involved in binding of plasminogen and its activator tPA, in several fragments derived from fibrinogen (fragment D and its subfragments) and fibrin (cross-linked D-D fragment and its noncovalent complex with the E(1) fragment, D-D. E(1)). Neither D nor D-D bound tPA, plasminogen, or anti-Aalpha148-160 and anti-gamma312-324 monoclonal antibodies, indicating that their fibrin-specific epitopes were inaccessible. The Aalpha148-160 epitope became exposed only upon proteolytic removal of the beta- and gamma-modules from D. At the same time, both epitopes were accessible in the D-D.E(1) complex, indicating that the DD.E interaction resulted in their exposure. This exposure was reversible since the dissociation of the D-D.E(1) complex made the sites unavailable, while reconstitution of the complex made them exposed. The results indicate that upon fibrin assembly, driven primarily by the interaction between complementary sites of the D and E regions, the D regions undergo conformational changes that cause the exposure of their plasminogen- and tPA-binding sites. These changes may be involved in the regulation of fibrin assembly and fibrinolysis.     

Immunochemical mapping of the conformation of human fibrinogen. The gamma 95-264 segment in inaccessible to antibody in native fibrinogen but progressively exposed by plasmic cleavage

The accessibility of the gamma 95-264 sequence to specific antibody probes in the native fibrinogen molecule and its plasmic cleavage fragments have been investigated. The gamma 95-264 segment was generated by cyanogen bromide cleavage of the gamma chain and isolated by gel filtration and ion exchange chromatography. Rabbit antisera to this peptide and to gamma chain recognized at least five antigenic loci uniformly distributed throughout this segment. In primary binding assays, antibodies to gamma 95-264 bound gamma 95-264, free gamma chain, and fibrinogen fragment D, but not native fibrinogen. Also, gamma 95-264 was bound by antibodies to gamma chain and fibrinogen fragment D, but not by antibodies generated to native fibrinogen. Thus, the gamma 95-264 sequence was not accessible to antibody in the native structure. In competitive equilibrium radioimmunoassays, neither native fibrinogen nor highly soluble fibrinogen fraction I-9 inhibited the binding of gamma 95-264 by its antiserum or anti-gamma chain. With plasmic cleavage, however, the gamma 95-264 sequence became accessible to antibody and the series of fragments D greater than Y greater than D:E = X describes the relative reactivity of the gamma chain sequence in fibrinogen degradation products. Differential expression of gamma 95-264 antigenic loci was also observed with D fragments differing in molecular weight. Plasmic cleavage of cross-linked and noncross-linked fibrin generated D fragments which did not express gamma 95-264 as well as fibrinogen D derivatives, indicating that the D domains of fibrinogen and fibrin are immunochemically distinguishable. These findings indicate that the central segment of the gamma chain is inaccessible to antibody in native fibrinogen, but that proper surface orientation is achieved upon plasmic degradation.     

Differences between the complexes formed by monomeric fibrin with fragment D and dimer D

The paper is concerned with studies in formation of monomeric fibrin (fm) complexes with fragment D (D) of fibrinogen and dimer D (DD) of stabilized fibrin. The complexes are shown to be essentially different. The fm-D complexes are unstable, their composition is a function of D concentration in the mixture, the ultimate molar D/fm ratio is equal to 3. The fm-DD complexes are quite stable, their composition is constant: the molar DD/fm ratio is equal to 1. In mixtures containing fm, DD and different amounts of D complexes of different composition are formed but the total number of D-units in them approaches 3. A model is suggested showing interaction of fm molecules in protofibril formation with allowance for the retention of binding centres which provide the lateral link between protofibrils.     

The interaction of human platelet thrombospondin with fibrinogen. Thrombospondin purification and specificity of interaction

Human platelet thrombospondin (TSP) was purified to homogeneity by chromatography on fibrinogen coupled to cyanogen bromide-activated Sepharose. The yield of TSP was 1.3 mg or approximately 22% of that present in platelet-rich plasma as determined by radioimmunoassay. It analyzed on discontinuous sodium dodecyl sulfate gels as a single band having apparent molecular weights of 180,000 and greater than 400,000 under reducing and nonreducing conditions, respectively. Amino acid analysis gave results similar to previously published values. Antibodies raised in rabbits were monospecific as evaluated by radioimmunoassay. In double immunodiffusion tests, these antibodies gave one line of identity against TSP purified by this procedure and TSP purified by published procedures, confirming the identity of the material isolated. The protein possesses no lectin-like activity. The specificity of the TSP-fibrinogen interaction was investigated. TSP binding to fibrinogen-Sepharose occurred in the presence of EDTA, indicating that calcium and magnesium ions are not required for interaction of TSP with fibrinogen. The binding of TSP to fibrinogen-Sepharose was quantitatively blocked by pretreatment with an antibody to the cyanogen bromide cleavage fragment composed of residues 241-476 of the carboxyl-terminal end of the alpha chain of fibrinogen. Antibodies against the D and E domains of fibrinogen had no effect on the binding. Excess fibrinogen (30 mg/ml) added to platelet extract quantitatively inhibited binding of TSP to fibrinogen-Sepharose. TSP preferentially bound to uncross-linked fibrin, suggesting that the TSP-fibrinogen binding site is unavailable in cross-linked fibrin. These results indicate that TSP binds specifically to immobilized fibrinogen or uncross-linked fibrin through determinants present in the carboxyl-terminal portion of the alpha chain and that these interactions do not require calcium or magnesium ions.     

Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins.

In vitro experiments employing the soluble proteins from Escherichia coli reveal that about half of them, in their unfolded or partially folded states, but not in their native states, can form stable binary complexes with chaperonin 60 (groEL). These complexes can be isolated by gel filtration chromatography and are efficiently discharged upon the addition of Mg.ATP. Binary complex formation is substantially reduced if chaperonin 60 is presaturated with Rubisco-I, the folding intermediate of Rubisco, but not with native Rubisco. Binary complex formation is also reduced if the transient species that interact with chaperonin 60 are permitted to progress to more stable states. This implies that the structural elements or motifs that are recognized by chaperonin 60 and that are responsible for binary complex formation are only present or accessible in the unfolded states of proteins or in certain intermediates along their respective folding pathways. Given the high-affinity binding that we have observed in the present study and the normal cellular abundance of chaperonin 60, we suspect that the folding of most proteins in E. coli does not occur in free solution spontaneously, but instead takes place while they are associated with molecular chaperones.     

Influence of fibrinogen on fibrin polymerization. Ultracentrifugation studies

During the transformation of fibrinogen to fibrin, excess fibrinogen suppresses further polymerization of fibrin, thereby enabling the nascent fibrin to be transported in a soluble form in blood. The question of possible complex formation between fibrin and fibrinogen was addressed by analyzing fibrin/fibrinogen (1:20, mol/mol) mixtures in the presence of calcium ions in stable linear sucrose density gradients by ultracentrifugation at 37 degrees C. During the period of ultracentrifugation in independent experiments, 40% of desAA-fibrin and 30% of desAABB-fibrin, respectively, precipitated without the participation of fibrinogen. The desAABB-fibrin, recovered in the gradient fractions, appeared as high-molecular-weight polymers (22 S), whereas the recovered desAA-fibrin exhibited only a slight increase in molecular weight (9 S) compared to fibrinogen (8 S). In contrast to this finding, both types of fibrin were totally recovered in gradient fractions provided that fibrinogen was present in the gradient at a uniform concentration of 2 mg/ml. In addition, the presence of fibrinogen but not human serum albumin reduced the size of desAABB-fibrin polymers (17 S). However, stable fibrin-fibrinogen complexes could not be demonstrated, since cosedimentation of differently labelled desAABB-fibrin and fibrinogen was not detectable. These studies suggest a specific but weak interaction of the solubilizing fibrinogen with the soluble fibrin polymers as demonstrated by a rapid exchange of both macromolecules.     

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 M of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10⁶. When endothelial cells were activated by different concentrations of thrombin (0–0.1 NIH units ml^–1), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml^–1, no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm^–2 for fibrinogen and 4350±235 cells cm^–2 for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm^–2 min^–1 for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.