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.     

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.     

Formation of lethal blood clots in fishes

Evidence is presented of lethal blood clot formation in fishes. A variety of factors commonly encountered under aquacultural conditions may generate such clots.     

Protofibrin clots induced by calcium and zinc

During the course of studies with fibrin protofibrils, produced by adding hirudin to thrombin-activated fibrinogen prior to the onset of gelation, turbid clots were observed to be generated merely by adding Ca(II) or Zn(II) to protofibrils. The rate of gelation (CT) and turbidity of the “protofibrin” clots increases with cation levels in a concentration-dependent manner, with Zn(II) much more potent than Ca(II). For example, 50 μM Zn(II) generated a more turbid protofibrin clot than 0.5 mM Ca(II). In combination, levels of Zn(II) and Ca(II), which individually have no effect, induce protofibril gelation. The generation of protofibrin clots by Zn(II) is decreased at increasing ionic strength. Apparently, the underlying electrostatic forces that bind the monomers in fibrin and protofibrin gels are similar. SEM micrographs show that Ca(II)- or Zn(II)-induced protofibrin clots (600–1500Å thick) are essentially indistinguishable from those formed directly from fibrinogen and thrombin with divalent cation. The protofibrin fibers induced by the cations are thicker than the fibers formed directly from fibrinogen and thrombin in the absence of divalent cation. Branching appears brought about the the divalent cation-sensitive lateral association of different protofibril strands. These findings describe simple experimental methods for separately studying the early and late stages of fibrin gelation.     

Finger-like lysing patterns of blood clots.

One-dimensional modeling of fibrinolysis (Senf, 1979; Zidansek and Blinc, 1991; Diamond and Anand, 1993) has accounted for the dissolution velocity, but the shape of the lysing patterns can be explained only by two- or three- drug-induced blood clot dissolution patterns obtained by proton nuclear magnetic resonance imaging, which can be described by the enzyme transport-limited system of fibrinolytic chemical equations with diffusion and perfusion terms (Zidansek and Blinc, 1991) in the reaction time approximation if the random character of gel porosity is taken into account. A two-dimensional calculation based on the Hele-Shaw random walk models (Kadanoff, 1985; Liang, 1986) leads to fractal lysing patterns as, indeed, is observed. The fractal dimension of the experimental lysing patterns changes from 1.2 at the beginning of the experiments to a maximum of approximately 1.3 in the middle and then decreases toward one when the clot is recanalized.     

Fibrinogen Dusart: electron microscopy of molecules, fibers and clots, and viscoelastic properties of clots.

Ultrastructural perturbations resulting from defects in polymerization of fibrinogen Dusart, a congenital dysfibrinogenemia with the amino acid substitution A alpha 554 arginine to cysteine, were investigated by a variety of electron microscope studies. Polymerization of this mutant fibrinogen on addition of thrombin is impaired, producing clots with decreased porosity and increased resistance to fibrinolysis, resulting in thrombotic complications in the family members with this dysfibrinogenemia. Electron microscopy of rotary-shadowed individual molecules revealed that, in contrast to control fibrinogen, most of the alpha C domains of fibrinogen or fibrin Dusart appeared to be free-swimming appendages that do not exhibit intra- or intermolecular interactions either with each other or with the central domains. The location of albumin on the alpha C domains was demonstrated by electron microscopy using anti-albumin antibodies. Electron microscopy of negatively contrasted fibrin Dusart fibers indicated that they were less ordered than control fibers and had additional mass visible. Electron microscopy of freeze-dried, unidirectionally shadowed fibers showed that they were twisted with a shorter pitch. Scanning electron microscopy revealed that intact clots were made up of thin fibers with many branch points and very small pore sizes. The viscoelastic properties of Dusart fibrin clots measured with a torsion pendulum indicated a marked increase in stiffness consistent with the structural observations.     

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.     

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.     

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.     

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.     

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.     

Activated protein C--an anticoagulant that does more than stop clots

Activated protein C (APC) is a glycoprotein derived from its precursor, protein C and formed by the cleavage of an activation peptide by thrombin bound to thrombomodulin. Originally thought to be synthesized exclusively by the liver, recent reports have shown that protein C is synthesized by endothelial cells, keratinocytes and some hematopoietic cells.APC functions as a physiological anticoagulant with cytoprotective, anti-inflammatory and anti-apoptotic properties. In vitro and preclinical data have revealed that APC exerts its protective effects via an intriguing mechanism requiring endothelial protein C receptor and the thrombin receptor, protease-activated receptor-1. Remarkably, even though APC cleaves this receptor in an identical fashion to thrombin, it exerts opposing effects.Recently approved as a therapeutic agent for severe sepsis, APC is now emerging as a potential treatment for a number of autoimmune and inflammatory diseases including lung disorders, spinal cord injury and chronic wounds. The future pharmacologic use of APC holds remarkable promise.