Electron microscopy of fine fibrin clots and fine and coarse fibrin films. Observations of fibers in cross-section and in deformed states.

Fine fibrin clots and coarse and fine fibrin films (both ligated and unligated), formed by shrinkage of clots in one dimension, were examined by electron microscopy. Specimens of clots were prepared by critical point drying and by embedding and sectioning; specimens of films were prepared by embedding and sectioning only. In the fine clots, network junctions appeared to be formed by fiber segments in which two or more protofibrils were gently twisted around each other for distances of the order of 200 nm and then diverged to give trifunctional branch points. This topology appeared to be preserved in the fine films. It is proposed that the strength of the junctions is primarily provided by the twisting topology, though reinforced by non-covalent bonding involving the B sites uncovered by thrombin. In coarse films, bundles of protofibrils, lying primarily in the film plane, had diameters of 40 to 200 nm and were gently twisted around each other to form thicker cables. Uniaxial stretching, up to 100%, of either fine or coarse film before fixing caused suprisingly extensive orientation of the protofibrils or bundles. However, random orientation was recovered if a stretched ligated film was allowed to retract to its original dimensions before fixing. In a stretched coarse film sectioned perpendicular to the stretch direction, fiber bundles could be seen in cross-section; these were roughly circular with scalloped edges. The changes with stretching and recovery are discussed in relation to possible mechanisms of deformation and elastic energy storage.

The clinical utility of prostate-specific antigen and bone scintigraphy in prostate cancer follow-up.

To assess the value of serum prostate-specific antigen (PSA) in prostate cancer follow-up, we prospectively studied 107 consecutive patients with: (1) pathologically confirmed prostate cancer; (2) definitive prostatectomy and/or radiation therapy greater than or equal to 3 mo prior to bone scanning; and (3) one bone scan and serum PSA sampling within 3 mo of each other. The mean and range of patient follow-up since definitive therapy was 1.6 and 0.5-8 yr, respectively. Abnormal bone scans were correlated with pertinent radiographs. Of 107 bone scans, 16 demonstrated metastatic bone disease. A PSA value of less than or equal to 8 ng/ml excluded bone metastases with a predictive value of a negative test of 98.5%. Without radiographic correlation, abnormal bone scans rarely represented metastases if the PSA value was less than or equal to 8 ng/ml. In summary, serum PSA concentration determines the need for follow-up bone scanning and assists in scan interpretation in patients status post definitive therapy for prostate cancer.

Thermally induced conformational changes in fibrin film.

Fibrin film is prepared by compaction in one dimension of a fibrin clot (pH 6.3, ionic strength 0.15, fibrin concentration about 0.5%) by expulsion of fluid to reach a fibrin concentration of about 15%. Strips of film, equilibrated in the same buffer with very slowly increasing temperature, shrink in length in a narrow temperature range, as reported in 1962 by Loeb and Scheraga. The transition temperature was found to be 54 +/- 2 degrees C independently of whether the film was unligated or ligated (cross-linked) by Factor XIIIa and whether the film had previously undergone stretching with about 50% stress relaxation at a relative length of 1.23 to 1.44 and subsequent stress-free retraction. The percentage of linear shrinkage in buffer was about 32%. The transition corresponds to that observed calorimetrically by Mihalyi and Donovan in both fibrinogen and fibrin and by Medved' and Privalov in fibrinogen, localized in the D fragment. It is attributed to unfolding of structures in the D domain.

Strain enhancement of elastic modulus in fine fibrin clots.

Fine fibrin clots, prepared at pH 8.5, ionic strength 0.45, with minimal lateral aggregation of protofibrils, and ligated (cross-linked) by factor XIIIa, were subjected to constant static shear strain (gamma) with superposed small oscillating strains. The incremental shear modulus (dynamic storage modulus) measured in the oscillating deformations was strain-independent at small static strains (up to about 0.1) and approximately equal to the static modulus. At higher static strains, it increased rapidly, up by a factor of 5 to 8 at gamma = 0.35. Comparison with earlier data on unligated clots showed that the enhancement of stiffness was independent of ligation except at very high strains. The enhancement is attributed to additional forced contacts between network fibers as the strands are bent and oriented. When the static strain was maintained for up to one day, in a clot ligated by factor XIIIa the enhanced incremental modulus remained constant or decreased slightly, and after removal of stress the clot returned almost to its original shape. This contrasts with the behavior of unligated clots, where most of the enhancement was progressively lost as the incremental modulus fell toward its small-strain value, and there was a substantial permanent deformation after the removal of stress. The latter behavior has been attributed to gradual severance of network strands at high strains, followed by their rejoining in relaxed configurations, but leaving some structural damage that is only very slowly recovered in the resting state. Ligation of protofibrils evidently eliminates the possibility of strand rupture.

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. III. Shear creep and creep recovery of fine ligated and coarse unligated closts.

Creep and creep recovery of human fibrin clots in small shearing deformations have been investigated over a time scale from 24 to 10(4) s. Coarse, unligated clots and fine clots ligated by fibrinoligase in the presence of calcium ions were studied to suppliement 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 6.2%. 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 closts there is comparatively little creep subsequent to the initial deformation; ligation (in this case involving mostly the gamma chains) reduces the creep to nearly zero. For coarse unligated clots, there is substantially more creep under constant stress, and creep recovery is not complete. Ligation (in this case involving both camma and alpha chains) alrgely supresses the creep and causes the recovery to be complete. If the structure if fully formed before creep begins, tests of creep recovery by the Boltzmann superposition principle show adherence to linear visoelastic 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 clot 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 alpha chains of the fibrin.

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.

Modification of shear modulus and creep compliance of fibrin clots by fibronectin.

Shear moduli and creep compliances have been measured for four types of clots of human fibrin (about 7 mg/ml) clotted with and without human plasma fibronectin (usually 1.2 mg/ml). Fine clots (with little lateral aggregation of the fibrin protofibrils) were found at pH 8.5, ionic strength 0.45; coarse clots (with substantial lateral aggregation) were formed at pH 7.5, ionic strength 0.15; in both cases with and without ligation by fibrinoligase. In fine clots, the addition of fibronectin without ligation scarcely affected the shear modulus; with ligation, the modulus was decreased by a factor of 0.48. In coarse clots, the shear modulus was increased by addition of fibronectin. The increase was by a factor of 2.0 without ligation and by a factor of 2.4 with ligation. Creep and creep recovery in clots formed with and without fibronectin were similar except for the scale factor represented by the change in modulus.

Complex viscosity of bovine red blood cells in suspensions.

The complex viscosity eta* has been measured of bovine red blood cells suspended in a medium of isotonic NaCl solutions including dextran and buffered with potassium phosphate at pH 7.0. A multiple lumped resonator apparatus was used at the frequencies of 144, 572, 1491, 3742, and 8026 Hz at 20.0 degrees C. Due to the high molecular weight of dextran the medium also exhibited some visco-elasticity eta s*. So we adopted the complex specific viscosity eta sp* = (eta*-eta s*)/[eta s*]. At 20.0 degrees C eta sp* decreased with the frequency where the hematocrit was 0.233 and eta s 0.34 poise. The measurements were made for the medium with different viscosity at 5.0 degrees C and 25.0 degrees C. The results are compared with the theory of elastic shells.

Clots of beta-fibrin. Viscoelastic properties, temperature dependence of elasticity, and interaction with fibrinogen-binding tetrapeptides.

Clots of human beta-fibrin, in which only (or predominantly) the B fibrinopeptide is released, were formed at 14 degrees C by copperhead venom procoagulant enzyme (CVE or venzyme), at pH 8.5, ionic strength 0.45. The shear modulus of elasticity increased slowly and after several days attained a constant value, which was lower than those of alpha-fibrin or alpha beta-fibrin under the same conditions. Before studying the temperature dependence of elasticity, the CVE was then inhibited by introducing phenyl methyl sulfonyl chloride (PMSF) by diffusion. With increasing temperature, the modulus decreased progressively from 5 degrees C to nearly zero at 35 degrees and was essentially reversible with temperature change; recovery of elasticity after change from 34.5 degrees to 14 degrees required approximately 2 d but was considerably faster than the initial buildup of elasticity by CVE at 14 degrees. Creep and creep recovery measurements on unligated clots showed creep rates and irrecoverable deformation that were similar in magnitude to those of alpha-fibrin clots formed with batroxobin and much larger than those of alpha beta-fibrin clots formed with thrombin, under the same conditions. During creep and creep recovery, the differential modulus or compliance remained constant, showing that there was no permanent structural damage, and if network strands are severed in slow flow, they must rejoin in new configurations. Introduction (by diffusion) of the tetrapeptides Gly-His-Arg-Pro (GHRP) and Gly-Pro-Arg-Pro (GPRP), which resemble the B and A binding sites on the E domain of fibrin respectively, reduced the shear modulus and increased the creep rate of beta-fibrin clots to an extent similar to the effect of GPRP on alpha beta-fibrin, much more than that of GHRP on alpha beta-fibrin, but much less than that of GPRP on a-fibrin. A ligated beta-fibrin clot formed with Factor XIIIa (in which the activating thrombin had been neutralized by hirudin) showed essentially perfect elastic behavior, with no creep and with complete recovery after removal of stress, and was inert to GHRP.

Small-angle X-ray scattering studies of fibrin film: comparisons of fine and coarse films prepared with thrombin and ancrod.

Measurements of small-angle x-ray scattering have been made on films prepared from fine and coarse (i.e., formed at high and low, respectively, pH and ionic strength) clots of bovine fibrin by osmotic shrinkage or compression in one dimension. Intensity profiles were obtained with pinhole geometry on films stretched up to a stretch ratio of 1.43. In unstretched coarse films, repeat spacings were seen at about 245, 120, and 77-80 A. These peaks can probably be identified with the first, second, and third orders of the well-known fibrin repeat of 225 A. In unstretched fine films, only the 77-80 A spacing was seen. In this case, the first two orders may be weak because the half-staggered arrangement of monomer units giving rise to the 225 A reflection is not reinforced by lateral aggregation of protofibrils; the third order may be strong since the molecular subdomains appear to divide the repeat roughly into thirds. After stretching, the 77-80 A spacing persisted in the meridional direction but almost disappeared in the equatorial. Experiments on unstretched films prepared with ancrod substituted for thrombin gave similar results.

Entanglement coupling in linear polymers demonstrated by networks crosslinked in States of strain.

Linear 1,2-polybutadiene is crosslinked at 0 degrees by gamma-irradiation while strained in simple extension, with extension ratios from 1.3 to 2.0. During irradiation times up to several hours, entanglement slippage is slight, since the temperature is only slightly above the glass transition. Subsequently, samples are released and reach their equilibrium states of ease at room temperature. From the extension ratio at state of ease, the ratio of nu(x) (effective network strands terminated by crosslinks introduced) to nu(N) (effective network strands terminated by entanglements) is calculated by composite network theories of Flory and others; and from the extension ratios together with the modulus, measured at small extensions, nu(N) is calculated explicitly. It appears that nu(x) increases approximately proportional to irradiation time; nu(N) is approximately independent of irradiation, and it corresponds to a molecular weight between effective entanglement loci of about 13,000. This figure, however, which is larger than that deduced from rheological properties of the uncrosslinked polymer, is subject to future downward correction for partial entrapment of the entanglements and other refinements.

Effects of fibrinogen-binding tetrapeptides on mechanical properties of fine fibrin clots.

The tetrapeptides Gly-Pro-Arg-Pro and Gly-His-Arg-Pro, analogs of the amino termini of the alpha and beta chains of fibrin monomer, respectively, were introduced by diffusion into fine unligated fibrin clots. Gly-Pro-Arg-Pro decreased the shear modulus of elasticity progressively and at a concentration of 5.8 mM the clot was eventually liquefied. The decrease in elastic modulus was accompanied by enormously enhanced viscoelastic creep under shear stress and irrecoverable deformation after removal of stress. However, the differential compliance (or modulus) for clots containing the tetrapeptide remained constant during creep and creep recovery, so the structure rearranged under stress without any permanent damage. Ligation with factor XIIIa and calcium largely eliminated these effects. From these changes in mechanical properties, it appears that Gly-Pro-Arg-Pro competes for binding sites, with consequent depolymerization. The tetrapeptide Gly-His-Arg-Pro at comparable concentrations decreased the modulus and increased the creep to a lesser degree; when combined with Gly-Pro-Arg-Pro it enhanced the effectiveness of the latter.

Kinetics of ligation of fibrin oligomers.

Human fibrinogen was treated with thrombin in the presence of fibrinoligase and calcium ion at pH 8.5, ionic strength 0.45, and the ensuring polymerization was interrupted at various time intervals (t) both before and after the clotting time (tc) by solubilization with a solution of sodium dodecyl sulfate and urea. Aliquots of the solubilized protein were subjected to gel electrophoresis on polyacrylamide gels after disulfide reduction by dithiothreitol and on agarose gels without reduction. The degree of gamma-gamma ligation was determined from the former and the size distribution of ligated oligomers, for degree of polymerization x from 1 to 10, from the latter. The degree of gamma-gamma ligation was calculated independently from the size distribution with the assumption that every junction between two fibrin monomers remaining intact after solubilization is ligated, and this agreed well with the direct determination. The size distribution at t/tc = 1.3 to 1.6 differed somewhat from that calculated by the classical theory of linear polycondensation on the assumption that all reactive sites react with equal probability and rate. Analysis of the difference suggests that ligation of a fibrin digomer is not a random process; the probability of ligation of a given junction between two monomers increases with the oligomer length. The number-average degree of polymerization, xn, of ligated oligomers increases approximately linearly with time up to a value of 1.6.

Viscoelastic properties of very dilute paramyosin solutions.

The storage and loss shear moduli, G' and G'', have been measured for dilute solutions of paramyosin from the clam Mercenaria mercenaria in water and glycerol--water mixtures containing potassium chloride and phosphate buffer. The Birnboim--Schrag multiple-lumped resonator was used in the frequency range from 150 to 8100 Hz; the concentration range was 0.7 to 2 X 10(-3) g/mL and the temperature range was 0.0 to 6.0 degrees C. The intrinsic moduli were obtained by extrapolation to infinite dilution. When compared with predictions of Yamakawa for a rigid cylindrical molecule, they agreed at low frequencies but diverged at high frequencies. Excellent agreement was obtained with calculations for a hybrid model whose relaxation times are attributed to rigid-body end-over-end rotation together with some internal modes of motion, probably flexural. The rotational relaxation time agreed rather well with that determined by DeLaney and Krause from electrical birefringence measurements. From the ratio of the rotational to the longest flexural relaxation time, the flexural rigidity and Young's modulus of the paramyosin molecule were estimated by relations derived by Wada and collaborators; the modulus was 1.2 X 10(10) dyn/cm2.

Conformational states of fibronectin. Effects of pH, ionic strength, and collagen binding.

Human plasma fibronectin was enzymatically labeled with dansylcadaverine using plasma Factor XIIa. Fluorescence polarization studies of dansylcadaverine-labeled fibronectin indicate that fibronectin has a significant degree of chain flexibility in physiologic solution and that there is an increase in chain flexibility at high pH or ionic strength. Binding of a collagen peptide to dansylcadaverine-fibronectin results in a decrease in fluorescence polarization, suggesting that such binding causes a conformational change which also results in increased chain flexibility.l Quasielastic light scattering and intrinsic viscosity measurements of fibronectin were performed under physiologic conditions and at high pH and ionic strength. Shape calculations based on these data indicate that fibronectin is in an elongated configuration under physiologic conditions and further unfolds at high pH or ionic strength into a very flexible, strand-like configuration. Light scattering studies of fibronectin after binding of a collagen fragment indicate that such binding results in a decrease in the diffusion coefficient, suggesting that collagen binding also results in a partial unfolding of fibronectin. These results suggest that published electron micrographs of fibronectin showing a long, strand-like molecule do not reflect the conformation of plasma fibronectin under physiologic conditions; fibronectin, however, may assume an unfolded conformation upon binding to collagen in the tissue matrix.