Hindered dissolution of fibrin formed under mechanical stress

Abstract

BackgroundRecent data indicate that stretching forces cause a dramatic decrease in clot volume accompanied by gross conformational changes of fibrin structure.ObjectiveThe present study attempts to characterize the lytic susceptibility of fibrin exposed to mechanical stress as a model for fibrin structures observed in vivo.Methods and resultsThe relevance of stretched fibrin models was substantiated by scanning electron microscopic (SEM) evaluation of human thrombi removed during surgery, where surface fibrin fibers were observed to be oriented in the direction of shear forces, whereas interior fibers formed a random spatial meshwork. These structural variations were modeled in vitro with fibrin exposed to adjustable mechanical stress. After two- and three-fold longitudinal stretching (2 × S, 3 × S) the median fiber diameter and pore area in SEM images of fibrin decreased two- to three-fold. Application of tissue plasminogen activator (tPA) to the surface of model clots, which contained plasminogen, resulted in plasmin generation which was measured in the fluid phase. After 30-min activation 12.6 ± 0.46 pmol mm−2 plasmin was released from the non-stretched clot (NS), 5.5 ± 1.11 pmol mm−2 from 2 × S and 2.3 ± 0.36 pmol mm−2 from 3 × S clot and this hampered plasmin generation was accompanied by decreased release of fibrin degradation products from stretched fibrins. Confocal microscopic images showed that a green fluorescent protein-fusion variant of tPA accumulated in the superficial layer of NS, but not in stretched fibrin.ConclusionMechanical stress confers proteolytic resistance to fibrin, which is a result of impaired plasminogen activation coupled to lower plasmin sensitivity of the denser fibrin network.

Time-Dependent Thrombus Resolution After Tissue-Type Plasminogen Activator in Patients With Stroke and Mice

“…This results in lower penetration of tPA, limiting its fibrinolytic action within the thrombus. 7,22,23 Previous semiquantitative histological analyses of thrombi obtained by thromboaspiration from the coronary artery have shown that the fibrin content of the thrombus increases over several hours in patients with ST-segmentelevation myocardial infarction. The time-dependent changes in the thrombus may affect the efficacy of thrombolytic treatment, as observed in our results.…”

Fractal Kinetic Behavior of Plasmin on the Surface of Fibrin Meshwork

“…The parameters Scanning electron microscope (SEM) imaging of fibrin-Fibrinogen (5.9 M in 25 mM Naphosphate pH 7.4 buffer containing 75 mM NaCl) was clotted for 1 h at 37 °C with 5 or 100 nM thrombin in Eppendorf tubes (pre-treated with 25 v/v% Triton X-100 solution for 1 h and thoroughly washed with water). Clots were fixed, dehydrated, sputter coated with gold and examined as described in (15). Preparation of patterned fibrinogen substrate surface with micro-contact printing and monitoring of plasmin distribution with AFM-Fibrinogen was printed on mica using microcontact printing (25,26).…”

“…Thus, any model that is based on diffusion, binding and reaction rate parameters gained in homogeneous dilute solutions is necessarily just an approximation of the real situation in fibrin, where an additional level of complexity is rendered by the variability of fibrin meshwork structure (e.g. in cell-free areas of in vivo human thrombi the fiber diameter varies in the range 100 -200 nm and the pore diameter in the range 160 -380 nm) (15). The third type of kinetic models of interfacial proteolysis in fibrin overcomes the limitations related to the deviation from the homogeneity assumptions by introducing a microscale stochastic approach, which operates with single enzyme molecules rather than enzyme concentrations in deterministic equations (16).…”

Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

“…30 This has downstream implications in terms of thrombus stability and susceptibility to lysis 29,31 and can influence penetration of cells into thrombi. Models incorporating physiological flow rates have helped define the processes governing thrombus formation.…”