Clot Stiffness Research Articles

Fibrin Gel Architecture Influences Endogenous Fibrinolysis and May Promote Coronary Artery Disease

An altered fibrin network architecture has been associated with premature coronary artery disease (CAD).1,2 Hypofibrinolysis, ie, impaired dissolution of fibrin in blood clots, is another common finding in such patients. Hypofibrinolysis is associated with elevated activity of inhibitors of the fibrinolytic process, particularly of plasminogen activator inhibitor-1 (PAI-1)3 and thrombin activatable fibrinolysis inhibitor (TAFI),4 but it is also influenced by the characteristics of the fibrin network itself.5,6 However, previous studies on fibrin architecture, its regulation, and implications for CAD have been hampered by imperfect and/or incomplete methodology. Thus, the relationships between fibrin structure, fibrinolytic function, and premature CAD warrant further thorough examination. This issue of Arteriosclerosis, Thrombosis, and Vascular Biology features a comprehensive investigation of physical and viscoelastic characteristics of fibrin clots formed ex vivo from plasma samples, their relationships to fibrinolysis rate, and potential role in CAD.7 See page 2567 Both the morphology and mechanical properties of fibrin influence susceptibility to fibrinolysis. Fibrin structure is generally assessed by using liquid permeation, light scattering, scanning electron microscopy, and confocal microscopy, from which variables such as the fiber thickness, length and density, and the number of branch points and porosity of the network are derived.8 Blunted fibrinolysis is associated with a tight fibrin structure composed of thin and short fibers with increased number of branch points, and small pores.5,6 Individual thick fibers are actually lysed at a slower rate,9 but tight network configurations display a significantly higher fiber density …

The αC domains of fibrinogen affect the structure of the fibrin clot, its physical properties, and its susceptibility to fibrinolysis

The functions of the alphaC domains of fibrinogen in clotting and fibrinolysis, which have long been enigmatic, were determined using recombinant fibrinogen truncated at Aalpha chain residue 251. Scanning electron microscopy and confocal microscopy revealed that the fibers of alpha251 clots were thinner and denser, with more branch points than fibers of control clots. Consistent with these results, the permeability of alpha251 clots was nearly half that of control clots. Together, these results suggest that in normal clot formation, the alphaC domains enhance lateral aggregation to produce thicker fibers. The viscoelastic properties of alpha251 fibrin clots differed markedly from control clots; alpha251 clots were much less stiff and showed more plastic deformation, indicating that interactions between the alphaC domains in normal clots play a major role in determining the clot's mechanical properties. Comparing factor XIIIa cross-linked alpha251 and control clots showed that gamma chain cross-linking had a significant effect on clot stiffness. Plasmin-catalyzed lysis of alpha251 clots, monitored with both macroscopic and microscopic methods, was faster than lysis of control clots. In conclusion, these studies provide the first definitive evidence that the alphaC domains play an important role in determining the structure and biophysical properties of clots and their susceptibility to fibrinolysis.

Influence of gamma' fibrinogen splice variant on fibrin physical properties and fibrinolysis rate.

A splice variant of fibrinogen, gamma', has an altered C-terminal sequence in its gamma chain. This gammaA/gamma' fibrin is more resistant to lysis than gammaA/gammaA fibrin. Whether the physical properties of gamma' and gammaA fibrin may account for the difference in their fibrinolysis rate remains to be established. Mechanical and morphological properties of cross-linked purified fibrin, including permeability (Ks, in cm2) and clot stiffness (G', in dyne/cm2), were measured after clotting gammaA and gamma' fibrinogens (1 mg/mL). gamma'/gamma' fibrin displayed a non-significant decrease in the density of fibrin fibers and slightly thicker fibers than gammaA/gammaA fibrin (12+/-2 fiber/10(-3) nm3 versus 16+/-2 fiber/10(-3) nm3 and 274+/-38 nm versus 257+/-41 nm for gamma'/gamma' and gammaA/gammaA fibrin, respectively; P=NS). This resulted in a 20% increase of the permeability constant (6.9+/-1.7 10(-9) cm2 versus 5.5+/-1.9 10(-9) cm2, respectively; P=NS). Unexpectedly, gamma' fibrin was found to be 3-times stiffer than gammaA fibrin (72.6+/-2.6 dyne/cm2 versus 25.1+/-2.3 dyne/cm2; P<0.001). Finally, there was a 10-fold decrease of the fibrin fiber lysis rate. Fibrinolysis resistance that arises from the presence of gammaA/gamma' fibrinogen in the clot is related primarily to an increase of fibrin cross-linking with only slight modifications of the clot architecture.

A monoclonal antibody to the fibrinogen γ-chain alters fibrin clot structure and its properties by producing short, thin fibers arranged in bundles

We previously reported that hamster monoclonal antibody 7E9, which reacts with the C-terminus of the gamma-chain of mouse fibrinogen, inhibits factor (F)XIIIa-mediated cross-linking, platelet adhesion to fibrinogen, and platelet-mediated clot retraction; in addition, it facilitates thrombolysis. To understand the mechanism(s) by which 7E9 acts, we have now studied the effect of 7E9 IgG, 7E9 F(ab')2, and 7E9 Fab on fibrin clot structure using electron microscopy and measurements of clot physical properties. By transmission electron microscopy, 7E9 IgG was found to bind primarily to the ends of the fibrinogen molecule. 7E9 IgG and 7E9 F(ab')2, both of which are bivalent, were capable of binding to two fibrinogen molecules simultaneously. Scanning electron microscopy of clots formed in the presence of equimolar concentrations of fibrinogen and 7E9 IgG demonstrated the presence of very short and thin fibers (63% reduction in fiber diameter) arranged in unusual bundles, surrounding large pores. Clots formed in the presence of 7E9 demonstrated a marked increase in permeation (approximately 25-fold increase in perfusion rate at constant pressure), an approximately 50% reduction in dynamic storage modulus (G'; a reflection of decreased clot stiffness), and an approximately 38% increase in loss tangent (tan delta; a reflection of the clot's ability to undergo irreversible deformation). These clots also showed decreased absorbance at 350 nm, reflecting the clot structure produced by 7E9 IgG. The effects of 7E9 IgG were not observed with control hamster IgG, 7E9 F(ab')2, or 7E9 Fab fragments, indicating requirements for both the binding properties and mass of 7E9 IgG. These data indicate that 7E9 antibody affects fibrin clot structure in a way that is consistent with the enhanced fibrinolysis we reported previously. Together with our previous observations, we conclude that 7E9 is directed at a strategically important region of fibrinogen with regard to platelet function, FXIIIa-mediated cross-linking, clot retraction, fibrin structure, and fibrinolysis. Thus targeting this region of fibrinogen may have antithrombotic therapeutic potential.

Functional properties of a prototype rheolytic catheter for percutaneous thrombectomy. In vitro investigations.

The performance of a rheolytic catheter designed to provide rapid fragmentation and evacuation of debris was evaluated. Specifically, fragmentation and evacuation efficiency was investigated in vitro. Fragmentation, aspiration, and recanalization of large thrombotic occlusions (8-cm length, 6-mm inner diameter) composed of clot (n = 11) or collagen gels (n = 20) of different stiffness were performed with different jet pressures at rates optimized by visual observation and were recorded on videotape for subsequent analysis. The size and number of the resulting particles in the evacuated fluid and those remaining in the artificial vessel were determined using filtration and laser light scattering techniques. The rate of fragmentation, mean particulate size, and efficiency of evacuation were evaluated as a function of operating parameters and "clot stiffness." Complete fragmentation of fresh thrombus occurred at 11,000 psi by direct action of the 6 radially disposed jets, which created a trapping vortex from which most of the particles (80%) were evacuated by suction. At lower pressure (5,500 psi), fragmentation efficiency was unchanged for fresh thrombus and softer 5% gels. Ninety-five percent of the thrombus was fragmented in very small particles with a mean diameter of 0.8 +/- 0.4 micron, while 5% of the mass of the fragmented thrombus was in particles > 40 microns. At 11,000 psi, the maximal rate of advancement associated with complete material fragmentation decreased with stiffness. For very stiff gels or heavily cross-linked old thrombus, complete fragmentation and removal of material was not achieved. The high-pressure rheolytic catheter rapidly fragmented and evacuated large thrombi much larger than the catheter diameter--features with potential for treating large thrombi, such as acute pulmonary emboli or deep vein thrombi. However, this rapid forceful disruption may damage vessel walls and invariably creates a broad range of particulate sizes, which makes evacuation of almost all of the particulate material imperative to minimize clinically relevant distal embolization.

The Effects of Prostacyclin on the Coagulation of Whole Blood

The effects of prostacyclin (PGI2) on mechanical properties of forming clots were investigated by testing human blood samples on a Thrombelastograph. Concentrations greater than 50 ng/ml (blood) caused a biphasic development of clot stiffness. During the first phase, PGI2 partially inhibited the platelet involvement in coagulation causing initial clot formation at a normal time but with reduced clot stiffness. The second phase occurred after neutralization of PGI2 activity and was characterized by recovery of platelet activity to produce a final clot with normal shear modulus. The duration of the inhibitory effects depended on PGI2 concentration and hematocrit. With a normal hematocrit, a PGI2 concentration of 60 ng/ml caused an inhibition for about 40 min whereas a concentration of 100 ng/ml caused inhibition for about 75 min.