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.
Highlights
Pharmacological thrombolysis is based on administration of plasminogen activators, which attack a solid-phase thrombus from the fluid phase [reviewed in 1]
In the course of this therapy a series of biochemical reactions is initiated, which can be simplified to a two-stage process: activation of plasminogen initially present in the thrombus and digestion of fibrin by the generated plasmin
In all cases the core of the thrombi contained fibrin forming a random 3D network, whereas in four thrombi the gel pores on the surface were elongated in one direction resulting in longitudinal alignment of the fibers accompanied by their tighter packing in the transverse direction
Summary
Pharmacological thrombolysis is based on administration of plasminogen activators, which attack a solid-phase thrombus from the fluid phase [reviewed in 1]. Mechanical forces acting in one direction on pre-formed fibrin clots result in longitudinal orientation of the fibers, in which the unfolding of separate domains in the extended single fibrin monomers is accompanied by exposure of hydrophobic domains and expulsion of water out of the fibers with consequent reduction of the fibrin volume. In light of these gross alterations in fibrin structure, the present study was undertaken in an attempt to understand the relationship
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