Identification of fibrin oligomers in sonicated fibrin clots

Clots of bovine fibrin, with both coarse and fine structure, and ligated to different extents by fibrinoligase, have been broken up by ultrasonic agitation and the sonicates have been examined by ultracentrifugal sedimentation. Sonication is followed by gross aggregation of the fragments unless guanidine hydrochloride is introduced (order of 1 M). In that case, sonicates of gamma-ligated fine clots contain two species whose sedimentation coefficients correspond to fibrin monomer and an oligomer with twice the monomer cross-section area and at least 20 monomer units, presumably with the structure of lateral dimerization with staggered overlapping. If the gamma ligation is incomplete, shorter oligomers are identified. The monomer and oligomer with degree of polymerization greater than 20 appear also in sonicates of coarse clots, but in smaller amounts, the principal product consisting of larger aggregates. The implications of these results with respect to metastability of the fine clot and the pattern of polymerization are discussed.     

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.     

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.     

Interaction of fibrinogen-binding tetrapeptides with fibrin oligomers and fine fibrin clots

The polymerization of fibrin, at pH 8.5 and ionic strength 0.45, and under conditions where the action of thrombin on fibrinogen was the rate-determining step, was interrupted by inactivating thrombin with p-nitrophenyl-p′-guanidinobenzoate (NPGB). Addition of the tetrapeptide Gly-Pro-Arg-Pro (GPRP) partially dissociated the fibrin oligomers as shown by subsequent ligation with Factor XIIIa and calcium ion followed by denaturation and gel electrophoresis; polyacrylamide gel electrophoresis with reduction showed a decrease in the proportion of γ-γ ligation compared with controls untreated by GPRP, and agarose gel electrophoresis showed a shift in the distribution of oligomer sizes. The dissociation was accomplished within 15 min and its extent was consistent with establishment of an equilibrium in which two molecules of GPRP react to sever an oligomer. When GPRP was introduced into fine unligated fibrin clots by diffusion, there was some dissociation as shown by differences in the degree of γ-γ ligation after treatment by Factor XIIIa; but the action of GPRP was much slower and less complete than on soluble oligomers. However, even a small amount of dissociation affected the mechanical properties of fine clots profoundly. The shear modulus (measured 25 s after application of stress) decreased progressively with increasing concentration of GPRP introduced by diffusion. The rate of shear creep under constant stress and the proportion of irrecoverable deformation also increased enormously. If the steadystate creep rate is interpreted in terms of an effective viscosity, the latter is decreased by up to three orders of magnitude by the presence of GPRP. In terms of transient network theories of viscoelasticity, the average lifetime of a network strand is greatly diminished. However, the total density of strands remains constant during creep and creep recovery as shown by constancy of the differential modulus or compliance. Removal of GPRP by diffusion only partially restores the original shear modulus and creep behavior of the original clot. Some limited data on the effect of the tetrapeptide Gly-His-Arg-Pro are also reported.     

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.     

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.     

The course and prerequisites of Lys-plasminogen formation during fibrinolysis

Plasmin-catalyzed modification of the native plasma zymogen Glu1-plasminogen to its more reactive Lys78 form has been shown to be enhanced in the presence of fibrin. The aim of the present work has been to characterize the influence of fibrinopeptide release, fibrin polymerization, and plasmin cleavage of fibrin on the rate of Lys78-plasminogen formation. 125I-Labeled Glu1- to Lys78-plasminogen conversion was catalyzed by performed Lys78-plasmin, or by plasmin generated during plasminogen activation with tissue plasminogen activator or urokinase. The two forms of plasminogen were quantitated following separation by polyacrylamide gel electrophoresis in acetic acid/urea. Plasmin generated by plasminogen activator was monitored by a fixed-time amidolytic assay. The rate of Lys78-plasminogen formation was correlated, in separate experiments, to the simultaneous, plasmin-catalyzed cleavage of 125I-labeled fibrinogen or fibrin to fragments X, Y, and D. The radiolabeled components were quantitated after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results show that the formation of both bathroxobin-catalyzed des-A-fibrin and thrombin-catalyzed des-AB-fibrin leads to marked stimulation of Lys78-plasminogen formation, whereas inhibition of fibrin polymerization, with Gly-Pro-Arg-Pro, abolishes the stimulatory effect. The rate of Lys78-plasminogen formation varies markedly in the course of fibrinolysis. The apparent second-order rate constant of the reaction undergoes a transient increase upon transformation of fibrin to des-A(B) fragment X polymer and decreases about 10-fold to the level observed during fibrinogenolysis upon further degradation to soluble fragments Y and D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous measurement of lysis of radioactively marked fibrin clots

Methods for continuous measurement of dissolution of experimentally induced radioactively labelled thrombi were described. They are suited for the use in artificial circulating systems and in animal experiments. The radioactivity can be measured continuously in a circulating system by fitting a flow through cell with a well scintillator. In order to measure thrombotic processes in vivo, we developed a specially adapted single hole collimator. By this device changes in radioactivity over a defined occluded area of the vessel could be detected. The usefulness of the methods was demonstrated by means of a thrombolytic agent.     

Adherence of Candida species to fibrin clots in vitro

The adherence of six Candida species to fibrin clots was studied using a simple, in vitro technique. Yeast suspensions were incubated with fibrin clots and the number of adherent organisms quantified as follows: after washing, the clots were subjected to vortex mixing and the number of CPU's which subsequently grew on Sabourauds medium were counted. Adhesion was directly proportional to the concentration of Candida species in the suspension (r=0.99 p<0.001). C. albicans and C. tropicalis exhibited marked adherence whereas C. krusei, C. gulliermondi and C. glabrata adhered less readily. C. parapsilosis was intermediate in its ability to adhere.     

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.     

Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves

Summary The sink-source transition in tobacco leaves was studied noninvasively using transgenic plants expressing the green-fluorescent protein (GFP) under control of theArabidopsis thaliana SUC2 promoter, and also by imaging transgenic plants that constitutively expressed a tobacco mosaic virus movement protein (MP) fused to GFP (MP-GFP). The sink-source transition was measured on intact leaves and progressed basipetally at rates of up to 600 m/h. The transition was most rapid on the largest sink leaves. However, leaf size was a poor indicator of the current position of the sink-source transition. A quantitative study of plasmodesmatal frequencies revealed the loss of enormous numbers of simple plasmodemata during the sink-source transition. In contrast, branched plasmodesmata increased in frequency during the sink-source transition, particularly between periclinal cell walls of the spongy mesophyll. The progression of plasmodesmal branching, as mapped by the labelling of plasmodesmata with MP-GFP fusion, occurred asynchronously in different cell layers, commencing in trichomes and appearing lastly in periclinal cell walls of the palisade layer. It appears that dividing cells retain simple plasmodesmata for longer periods than nondividing cells. The rapid conversion of simple to branched plasmodesmata is discussed in relation to the capacity for macromolecular trafficking in developing leaf tissues.     

Rheological studies of creep and creep recovery of unligated fibrin clots: comparison of clots prepared with thrombin and ancrod

Mechanical creep and creep recovery in small shearing deformations have been studied in unligated clots formed with both thrombin and ancrod. In thrombin clots, both A binding sites (which interact with “a” sites to link monomer units within a protofibril) and B sites (which interact with “b” sites to form links between protofibrils) are exposed to enable formation of linkages; in ancrod clots, only the A sites are exposed. Fine clots (with minimal lateral aggregation of protofibrils), coarse clots (with substantial aggregation of fibril bundles), and clots of intermediate coarseness were compared. Fine thrombin clots showed less creep at short times but more creep at long times than coarse or intermediate clots and had more irrecoverable deformation relative to the initial elastic deformation. Ancrod clots had greater irrecoverable deformation than the corresponding thrombin clots, both fine and coarse. The permanent deformation in fine ancrod clots was enormous, corresponding almost to fluid character; the rate of permanent deformation was larger than that in fine thrombin clots by more than two orders of magnitude. For all types of clots, differential measurements of compliance (or its reciprocal, elastic modulus), as well as the applicability of the Boltzmann superposition principle to calculation of creep recovery, showed that the overall density of structure remained constant throughout the mechanical history; i.e., if structural elements were breaking, they were reforming at the same rate in different configurations. The possibility that the weakness of ancrod clots is attributable to partial degradation of α-chains rather than absence of Bb linkages was eliminated by comparisons of clots made with thrombin, ancrod, and ancrod plus thrombin; the last two showed identical partial degradation of α-chains (by gel electrophoresis), but the first and third had essentially identical initial elastic moduli and creep behavior. Two alternative mechanisms for irrecoverable deformation in fine clots are discussed, involving rupture of protofibrils and slippage of twisted segments, respectively.     

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.     

The effect of fibrin structure on fibrinolysis.

Fibrin structure contributes to the regulation of the fibrinolytic rate. As the fibrin fiber size is decreased, the fibrinolytic rate also decreases. Fibrin structure was altered by either changing the ratio of thrombin to fibrinogen, i.e. altering the assembly rate or by adding a fibrin assembly inhibitor, iopamidol. Changes in the fibrinolytic rate were followed by measuring the time dependence of the decrease in the fiber mass/length ratio during fibrinolysis. A measure of the overall fibrinolytic rate was determined from the decrease in the mass/length ratio versus time. An 8-fold reduction in the fibrinolytic rate was seen on decreasing the mass/length ratio from 2.7 x 10(12) daltons/cm to 0.5 x 10(12) daltons/cm. It is shown that thin fibrin fibers have a decreased rate of conversion of plasminogen to plasmin by tissue plasminogen activator and that thin fibrin fibers are lysed more slowly than thick fibrin fibers.     

Rheology of fibrin clots. IV. Darcy constants and fiber thickness.

Measurements of small oscillatory deformations of a fibrin clot by axial motion of a rod in a closed tube reveal an anomalous mechanical loss due to permeation of fluid through the clot structure. The Darcy constant for permeation can be calculated from data at the frequency where the apparent storage and loss shear moduli are equal, without the necessity of measurements at much lower frequencies as previously employed. From the Darcy constant, the average number of fibrin monomer units (v) per cross-section of a fibrous element of the clot can be calculated; it ranges from 4 to several hundred. In the range of fibrin concentration(c) from 3 to 14 milligrams, v is approximately proportional to c-2 for clots of coarse structure and to c-0.5 for clots of fine structure.