Rheology of fibrin clots. I. Dynamic viscoelastic properties and fluid permeation

The storage and loss shear moduli (G', G″) of human fibrin clots have been measured in small oscillating deformations over a frequency range of 0.01 to 160 Hz with the modified Birnboim transducer apparatus. Most clots were prepared by the action of thrombin on purified fibrinogen, under various conditions of pH and ionic strength to produce networks ranging from coarse to fine structure; some were liaated by fibrinoligase. The fine, unligated clot showed very little mechanical loss or frequency dependence of G' over the experimental frequency range, though loss mechanisms evidently appear at higher frequencies; G' was proportional to the 1.5 power of fibrin concentration. The coarse, unligated clot showed a slight increase of G' with frequency, reflecting some relaxation mechanisms with time constants whose reciprocals lie in the experimental frequency range. Ligation did not greatly affect the magnitude of G'. However, clots prepared by dilution of solutions of fibrin monomer in 1 M sodium bromide had smaller moduli by a factor of ten than corresponding clots prepared by the action of thrombin of fibrinogen. Oscillatory measurements in the Birnboim apparatus with closed-end (annular pumping) geometry revealed a low-frequency anomaly which was shown to be due to permeation of fluid through the clot structure, and from these measurements the Darcy constants for coarse clots were calculated. From the Darcy constants, the average thicknesses of the fibrous elements of the structures were estimated to be from 300 to 700 A.     

Analysis of fibrous network fluid permeation data using the theory of ultracentrifugation: application to fibrin gels

A new and alternative method for calculating the strand diameter of fibrous gel networks from fluid permeation data is developed and used to analyze and compare previous Darcy constant measurements of fibrin gels. The calculated diameters from the various sets of experimental data using this method gives for a coarse fibrin clot a strand diameter of approximately 1000 A and for a fine fibrin clot a strand diameter of 170 A.     

Gel formation by fibrin oligomers without addition of monomers

Soluble fibrin oligomers were formed by reacting fibrinogen with thrombin under fine clotting conditions where the action of thrombin is the rate-determining step for polymerization, and by inhibiting the reaction shortly before gelation. Oligomeric fibrin was separated from unreacted fibrinogen and small oligomers by gel permeation chromatography. Electron microscopy revealed that the largest soluble fibrin oligomers resemble the protofibrils present in fine clots, but are somewhat shorter and entirely lack the twisted, trifunctional junctions that contribute to the elastic properties of fine clots. When thrombin was added to the soluble fibrin oligomers, polymerization resumed and clots were formed at a more rapid rate than from fibrinogen at the same concentration and resulted in a less-opaque clot under coarse clotting conditions. The results confirm a prediction of a theory for the polymerization of fibrin and provide additional evidence that the final state of a coarse fibrin clot depends on the mobility of protofibrils during its formation.     

A study of the protection of plasmin from antiplasmin inhibition within an intact fibrin clot during the course of clot lysis

Previous work using soluble fibrin surrogates or very dilute fibrin indicate that inhibition of plasmin by antiplasmin is attenuated by fibrin surrogates; however, this phenomenon has not been quantified within intact fibrin clots. Therefore, a novel system was designed to measure plasmin inhibition by antiplasmin in real time within an intact clot during fibrinolysis. This was accomplished by including the plasmin substrate S2251 and a recombinant fluorescent derivative of plasminogen (S741C-fluorescein) into clots formed from purified components. Steady state plasmin levels were estimated from the rates of S2251 hydrolysis, the rates of plasminogen activation were estimated by fluorescence decrease over time, and residual antiplasmin was deduced from residual fluorescence. From these measurements, the second order rate constant could be inferred at any time during fibrinolysis. Immediately after clot formation, the rate constant for inhibition decreased 3-fold from 9.6 x 10(6) m(-1) s(-1) measured in a soluble buffer system to 3.2 x 10(6) m(-1) s(-1) in an intact fibrin clot. As the clot continued to lyse, the rate constant for inhibition continued to decrease by 38-fold at maximum. To determine whether this protection was the result of plasmin exposure of carboxyl-terminal lysine residues, clots were formed in the presence of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). In the presence of TAFIa, the initial protective effect associated with clot formation occurred; however, the secondary protective effect associated with lysine residue exposure was delayed in a TAFIa concentration-dependent manner. This latter effect represents another mechanism whereby TAFIa attenuates fibrinolysis.     

Incorporation of thrombospondin into fibrin clots

Thrombospondin is a major platelet glycoprotein which is released from platelets during blood coagulation. We examined the interaction of thrombospondin with polymerizing fibrin. Thrombospondin, purified from human platelets and labeled with 125I, became incorporated into clots formed from both plasma and purified fibrinogen. Plasma clots contained somewhat less thrombospondin than clots formed from equivalent concentrations of fibrinogen. In plasma clots and fibrin clots formed in the presence of factor XIII, thrombospondin was cross-linked in the clot; thrombospondin in the supernatant remained largely monomeric. Cross-linking of thrombospondin by factor XIII, however, only slightly increased the amount of thrombospondin which was incorporated into the clot. In contrast, incorporation of 125I-fibronectin into clots was dependent upon cross-linking. Most of the incorporation of 125I-thrombospondin occurred during fibrin polymerization as judged by parallel studies of the incorporation of 125I-fibrinogen. The amount of thrombospondin incorporated into a clot was directly related to thrombospondin concentration and was only weakly dependent on fibrinogen concentration. Incorporation was not saturated at thrombospondin:fibrin (mol/mol) ratios as high as 2/1. Thrombospondin, however, modified the final structure of fibrin clots in a concentration-dependent manner as monitored by opacity. When tryptic digests of 125I-thrombospondin were studied, the 270-kilodalton core became incorporated into fibrin whereas the 30-kilodalton heparin binding fragment was excluded. These results indicate that thrombospondin specifically co-polymerizes with fibrin during blood coagulation and may be an important modulator of clot structure.     

Pro-thrombotic state induced by post-translational modification of fibrinogen by reactive nitrogen species

Formation of nitric oxide-derived oxidants has been linked to development of atherosclerosis and associated thrombotic complications. Although systemic levels of protein nitrotyrosine predict risk for coronary artery disease, neither specific proteins targeted for modification nor functional consequences that might contribute to disease pathogenesis have been defined. Here we report a selective increase in circulating levels of nitrated fibrinogen in patients with coronary artery disease. Exposure of fibrinogen to nitrating oxidants, including those produced by the myeloperoxidase-hydrogen peroxide-nitrite system, significantly accelerates clot formation and factor XIII cross-linking, whereas exposure of fibrinogen to non-nitrating oxidants decelerates clot formation. Clots formed with fibrinogen exposed to nitrating oxidants are composed of large bundles made from twisted thin fibrin fibers with increased permeation and a decrease in storage modulus G' value, suggesting that these clots could be easily deformed by mechanical stresses. In contrast, clots formed with fibrinogen exposed to non-nitrating oxidants showed decreased permeation with normal architecture. Fibrinogen modified by exposure to physiologic nitration systems demonstrated no difference in the rate of plasmin-induced clot lysis, platelet aggregation, or binding. Thus, increased levels of fibrinogen nitration may lead to a pro-thrombotic state via acceleration in formation of fibrin clots. The present results may account, in part, for the association between nitrative stress and risk for coronary artery disease.     

Rheology of fibrin clots: III. Shear creep and creep recovery of fine ligated and coarse unligated clots

Creep and creep recovery of human fibrin clots in small shearing deformations have been investigated over a time scale from 24 to 10⁴ s. Coarse, unligated dots and fine dots ligated by fibrinoligase in the presence of calcium ions were studied to suppllement previous data on coarse ligated and fine unligated clots. Stress was found to be proportional to strain up to at least a maximum shear strain (in torsion geometry) of 2.6%. The initial modulus (25 s after imposition of stress) is proportional to approximately the 1.5 power of concentration for fine ligated and coarse unligated clots. For fine unligated clots, there is comparatively little creep subsequent to the initial deformation; ligation (in this case involving mostly the γ chains) reduces the creep to nearly zero. For coarse unligated dots, there is substantially more creep under constant stress, and creep recovery is not complete. legation (in this casa involving both γ and α chains) largely suppresses the creep and causes the recovery to be complete. If the structure is fully formed before creep begins, tests of creep recovery by the Boltzmann superposition principle show adherence to linear viscoelastic behavior for all four clot types. Otherwise, the Boltzmann test fails and the recovery is much less than calculated. For fine ligated clots, the observed recovery agrees well with that calculated on the basis of a dual structure model in which an additional independent structure is built up in the deformed state, so that the state of ease after removal of stress is a balance between two structures deformed in opposite senses, it is postulated that the coherence and elastic modulus of the fine ligated dot are largely due to steric blocking of long protofibrils with a high flexural stiffness. In the coarse clot, it is proposed that the structure involves extensive branching of thick bundles of protofibrils, which become permanently secured by the ligation of the α chains of the fibrin.     

Flow-induced permeation of non-occlusive blood clots: an MRI study and modelling

The success of clot thrombolysis very much depends on efficient clot permeation with blood plasma carrying the thrombolytic agent. In this paper clot permeation was studied by dynamic magnetic resonance imaging (MRI) on artificial non-occlusive blood clots inserted in an artificial circulation system filled with blood plasma to which an MRI contrast agent was added. The MRI results revealed that clot permeation is much faster and more efficient at the entrance of the flow channel across the clot. Clot permeation with fluid was simulated numerically as well. The simulation was based on numerical solution of Navier-Stokes equations for the flow in the channel and within the clot. The clot was considered as a porous material with known permeability and porosity. Based on the calculated velocity profiles, concentration profiles of fluid in the clot were modelled. These agreed well with the MRI results. The presented model of clot permeation with fluid may also serve as a useful extension to numerical modelling of dissolution of non-occlusive blood clots during thrombolytic therapy.     

Polydispersion in the diameter of fibers in fibrin networks: Consequences on the measurement of mass–length ratio by permeability and turbidity

The diameters of the fibrin fibers in a clot are not uniform. Morphometric analysis of transmission electron micrographs show a bimodal distribution. The effect of polydispersity of fiber diameter on mass–length ratio calculated from the turbidity and permeability of a clot has been investigated with the aid of a two network model, the networks being called Major and Minor. The fibers in the Major network are many times thicker than those in the Minor network. In a model in which Major network fibers are 10 times thicker than fibers of the Minor network, the fibers in the Minor network make a negligible contribution to the turbidity of the clot. However, they may have a marked effect on its permeability. Experiments with clots made from human fibrinogen show that the Minor network is stabilized by α-polymer and γ-γ linkages and that without such linkages it is washed away during permeation. It remains relatively intact in crosslinked clots. In agreement with the theoretical model, when mass–length ratios calculated from the turbidity are compared with those calculated from the permeability, the latter were reduced in crosslinked clots with an intact Minor network.     

Cross-linking of cold-insoluble globulin by fibrin-stabilizing factor.

Cold-insoluble globulin (CI globulin) was purified from human plasma and identified on the basis of its sedimentation coefficient, electrophoretic mobility, and concentration in normal plasma. CI globulin was distinguished from antihemophilic factor (AHF) by amino acid analysis, position of elution from 4% agarose, and electrophoretic migration in polyacrylamide gels in the presence of sodium dodecyl sulfate without prior reduction. CI globulin and AHF could not be distinguished by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction and probably have very similar subunit molecular weights. CI globulin apparently consists of two polypeptide chains, each of molecular weight 2.0 x 10(5), held together by disulfide bonds. CI globulin was a substrate for activated fibrin-stabilizing factor (FSF, blood coagulation factor XIII). FSF catalyzed the incorporation of a fluorescent primary amine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide, into CI globulin and also catalyzed the cross-linking of CI globulin into multimers, as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction. In the presence of fibrin, cross-linking of CI globulin by FSF occurred without the formation of CI globulin multimers. Instead, polypeptides with apparent molecular weights of 2.6 x 10(5) and 3.0 x 10(5) were seen. The formation of these polypeptides coincided with the loss of the alpha chain of fibrin and CI globulin. The polypeptides were not seen when fibrin alone was cross-linked. The formation of the polypeptides was greater in fine clots than in coarse clots, and greater in clots incubated at 0 degrees than in clots incubated at 37 degrees. In clots made from purified fibrinogen, CI globulin, and FSF, the concentration of CI globulin in the clot liquor was greater if either FSF or calcium ion was omitted and cross-linking did not take place. These observations suggest that CI globulin is enzymically cross-linked to one of the chains of fibrin, most likely the alpha chain, and is thus covalently incorporated into the fibrin clot. CI globulin is very similar to a protein in the plasma membrane of fibroblasts. The cross-linking of CI globulin to itself and to fibrin may typify reactions also involving the fibroblast membrane protein.     

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.     

Features of the structure of fibrin clots,connected with conditions of gel formation

Fibrinogen to fibrin conversion and then fibrin clot three-dimensional network formation is one of the final steps in the coagulation system activation. Different factors, such as the environment temperature and pH, ions, so on, render an effect on the fibrin gel formation. Recently, the presence or absences of interface between two phases influence on the fibrin gel structure during its formation have been shown. Studies of fibrin gel structure peculiarities formed at different conditions (between two phases and without one phase) are demonstrated in this article. The plasmin enzymatic hydrolysis of fibrin clots both with surface film and without it was investigated. Experimental data allow to make a conclusion that the fibrin clot structure changes depend on its essential influence on the plasmin hydrolysis process of these clots.     

Requirements for alpha(5)beta(1) integrin-mediated retraction of fibronectin-fibrin matrices.

Retraction of the blood clot by nucleated cells contributes both to hemostasis and to tissue remodeling. Although plasma fibronectin (FN) is a key component of the clot, its role in clot retraction is unclear. In this report, we demonstrate that the incorporation of FN into fibrin matrices significantly improves clot retraction by nucleated cells expressing the integrin alpha(5)beta(1). Further, we show that FN-fibrin clots support increased cell spreading when compared with fibrin matrices. To determine the structural requirements for FN in this process, recombinant FN monomers deficient in ligand binding or fibrin cross-linking were incorporated into fibrin clots. We show that recombinant FN monomers support clot retraction by Chinese hamster ovary cells expressing the integrin alpha(5)beta(1). This process depends on both the Arg-Gly-Asp (RGD) and the synergy cell-binding sites and on covalent FN-fibrin binding, demonstrating that cross-linking within the clot is important for cell-FN interactions. These data show that alpha(5)beta(1) can bind to FN within a clot to promote clot retraction and support cell shape change. This provides strong evidence that alpha(5)beta(1)-FN interactions may contribute to the cellular events required for wound contraction.     

Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers

Fibrinogen, the major structural precursor of blood clots, was deglycosylated by peptide-N-(N-acetyl-beta-glucosaminyl)asparagine amidase without denaturation of the polypeptide chains. Deglycosylated fibrinogen behaved normally in clinical coagulation assays, although it is less soluble than normal fibrinogen. However, the turbidity of clots formed from deglycosylated fibrinogen always rose faster and higher than that of clots from normal fibrinogen. Scanning and transmission electron microscopy demonstrated that fibrin made from clots of deglycosylated fibrinogen consisted of thicker, less-branched fiber bundles in a more porous network. Moreover, the degree of lateral aggregation was directly related to clot turbidity and inversely related to branching. Deglycosylation promoted turbidity development, lateral aggregation, and porosity of clots under all conditions tested. All other steps in the coagulation pathways appeared to be unaffected by the absence of carbohydrate. These results suggest that carbohydrate constitutively affects the behavior of deglycosylated fibrinogens by 1) contributing a repulsive force that promotes fibrinogen solubility and limits fibrin assembly and 2) sensitizing fibrin to conditions that influence assembly and clot structure.     

Effects of free fatty acids on fibrinolytic activity.

A novel method for the estimation of fibrinolytic activity is proposed. In this method, a fibrin clot suspension is used as a substrate (fibrin is known to be a physiological substrate of plasmin). The fibrin clot suspension was prepared by homogenization of human fibrin clots. With this method, we found that free fatty acids inhibited the plasmin activity, and long-chain, unsaturated free fatty acids had a particularly strong inhibitory action on plasmin. As regards the mechanism of the inhibitory action, free fatty acids may not inhibit complex formation between plasmin and fibirin, but may make it impossible for plasmin to act on fibrin due to deformation of the surface of the fibrin clot.     

Similarities between platelet contraction and cellular motility during mitosis: role of platelet microtubules in clot retraction.

The effects of inhibitors of mitosis, energy metabolism and protein synthesis on clot retraction were investigated. The results show that (1) Incubation of colchicine (0-01-0-1 mM) with platelet-rich plasma (PRP) inhibits the subsequent retraction of clots derived from diluted PRP. (2) Inhibition of clot retraction by high concentrations of colchicine (up to 40 mM) can be overcome by increasing the platelet concentration in the system. (3) Incubation of clots in colchicine or 80% D2O solutions inhibits their retraction. Exposure of partially retracted clots to these agents is without effect. (4) Hydrostatic pressure retards clot retraction. (5) Incubation of PRP with either 2-deoxy-D-glucose or antimycin alone does not affect clot retraction, but a combination of these agents is inhibitory. (6) Clot retraction is not inhibited by puromycin or cycloheximide. (7) Platelets in retracting clots have constricted regions containing microfilaments and pseudopods containing microtubules. Fibrin strands are progressively condensed around the constricted regions as retraction advances. (8) The development of platelet constriction, platelet pseudopods and the intracellular microfilaments are delayed in colchicinized clots, corresponding to the retardation of retraction. Following the initial delay of retraction colchicinized clots, like controls, show condensation of fibrin strands adjacent to these constricted areas of platelets containing microfilaments. The formation of pseudopods is impaired and no microtubules are found in platelets in the presence of colchicine. The above results suggest that the thrombin-induced platelet contraction during clot retraction is a coordinated movement, which, under optimal conditions involves both microtubules and microfilaments. The contraction of microfilaments produces the constriction of platelets and brings about clot retraction by reducing the angle between fibrin strands. Platelet microtubules are related to the development of pseudopods and play a supplementary role in facilitating microfilament-mediated cellular constriction. The similarities between platelet contraction and cellular motility in mitosis is discussed.     

Rheology of fibrin clots. v. shear modulus,creep, and creep recovery of fine unligated clots

Creep and creep recovery in small shearing deformations have been studied in fibrin clots at pH 8.5 and ionic strength 0.45, where the fine, transparent clot is formed with very little lateral aggregation of protofibrils. The initial shear modulus G₁ was measured 25 s after deformation on clots aged long enough for complete development of structure. For both human and bovine fibrin, the data were approximately described by log G₁ = 1.45 + 1.90 log c, where c is concentration in $\text{g}\text{l}$ and G₁ is in $\text{dyn}\text{cm}{}^2$, over a range of c from 4 to 13 $\text{g}\text{l}$. For bovine clots with completely developed structure, creep and creep recovery showed substantial irrecoverable deformation but the differential modulus G_Δ measured at intervals agreed with G₁ and did not change during the course of the experiment; it also agreed with the value calculated from the initial recovery after removal of stress. Moreover, several tests showed that the course of recovery conformed closely to the Boltzmann superposition principle. Thus the irrecoverable strain was associated with a structural rearrangement which caused no permanent damage. The irrecoverable deformation relative to the initial deformation was proportional to the elapsed time during creep in the early stages with a proportionality constant that decreased somewhat with increasing clot age prior to imposition of stress; it corresponded to a pseudo-viscosity of the order of 10⁷ poise. However, the irrecoverable deformation does not represent viscous flow and appears to approach a limiting value at long times. Experiments on clots without completely developed structure, i.e., with imposition of stress at an earlier clot age, showed an increase in the differential modulus G_Δ during creep. The irrecoverable deformation was greater and a portion of it could be attributed to the balance between two structures formed in the unstrained and strained states. However, unlike the case of ligated clots strained before complete development of structure, where the irrecoverable deformation is entirely due to a two-structure balance, there is also a contribution from structural rearrangement. Experiments with reverse creep and creep recovery showed that the structural rearrangement is symmetrical with respect to direction of deformation. The interpretation of these results in terms of clot structure and internal motions of protofibrils is discussed.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

Ultrastructure of clots during isometric contraction

We explored the retraction or contraction of platelet-fibrin clots under isometric conditions. In the presence of micromolar calcium clots of normal platelet-rich plasma developed tension at an initial rate of 0.1 to 0.2 g/min per cm2 (initial cross-sectional area). Electron microscopy of clots fixed after attaining a force of 1.6 g/cm2 revealed platelets with elongated bodies and pseudopods in close apposition to fibrin strands which were oriented in cablelike fashion in the direction of tension. The development of tension could not be explained simply on the basis of platelet-platelet association and interaction alone. First, factor XIII-dependent cross-linking of fibrin fibers was critical to normal isometric contraction. Second, tension decreased linearly, rather than exponentially, when the platelet count in the platelet-fibrin clot was decreased, suggesting that platelets must be interacting with another component (i.e. fibrin). Thrombasthenic platelets, deficient in fibrinogen receptors, failed to develop tension or to align fibrin strands or pseudopods in the clot. Platelet-fibrin clots treated with vincristine to disassemble microtubules or cytochalasin B to disrupt microfilaments failed to develop tension and relaxed if these agents were added after tension had developed. Relaxation under these conditions, however, was not associated with loss of orientation of fibrin strands. Our findings suggest that platelet-fibrin interaction in clots under isometric conditions leads to orientation of fibrin strands and platelets in the direction of force generation. Tension develops as platelets simultaneously attach to and spread along fibrin strands, and contract. The contraction draws some fibrin into platelet-fibrin clumps and aligns other strands in the long axis of tension. The achievement and maintenance of maximum tension appears to depend on the development of platelet-fibrin attachments and extension of platelet bodies and long pseudopods containing bundles of microfilaments and microtubules along the oriented fibrin fibers.