Background: Neutrophil extracellular traps (NETs) composed of DNA and proteins form a scaffold in thrombi, supplementing the fibrin matrix.Results: DNA and histones modify the structure of fibrin and render it resistant to mechanical and enzymatic destruction.Conclusion: NET components are essential factors in thrombus stability.Significance: Therapeutic strategies could be optimized to enhance fibrinolysis in clots containing DNA and histones.

SummaryIn response to various inflammatory stimuli, neutrophils secrete neutrophil extracellular traps (NETs), web-like meshworks of DNA, histones and granular components forming supplementary scaffolds in venous and arterial thrombi. Isolated DNA and histones are known to promote thrombus formation and render fibrin clots more resistant to mechanical forces and tissue-type plasminogen activator (tPA)-induced enzymatic digestion. The present study extends our earlier observations to a physiologically more relevant environment including plasma clots and NET-forming neutrophils. A range of techniques was employed including imaging (scanning electron microscopy (SEM), confocal laser microscopy, and photoscanning of macroscopic lysis fronts), clot permeability measurements, turbidimetric lysis and enzyme inactivation assays. Addition of DNA and histones increased the median fibre diameter of plasma clots formed with 16 nM thrombin from 108 to 121 and 119 nm, respectively, and decreased their permeability constant from 6.4 to 3.1 and 3.7×10−9 cm2. Histones effectively protected thrombin from antithrombin-induced inactivation, while DNA inhibited plasminogen activation on the surface of plasma clots and their plasmin-induced resolution by 20 and 40 %, respectively. DNA and histones, as well as NETs secreted by phorbol-myristate-acetate-activated neutrophils, slowed down the tPA-driven lysis of plasma clots and the latter effect could be reversed by the addition of DNase (streptodornase). SEM images taken after complete digestion of fibrin in NET-containing plasma clots evidenced retained NET scaffold that was absent in DNase-treated clots. Our results show that DNA and histones alter the fibrin architecture in plasma clots, while NETs contribute to a decreased lytic susceptibility that can be overcome by DNase.

Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/ function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domainagglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time. (Blood. 2011;117(2):661-668) IntroductionFibrin may be viewed as a substrate according to 2 distinct definitions of the word: (1) a surface or layer supporting biologic activity and (2) a substance acted on by an enzyme. The mechanism of tissue-type plasminogen activator (tPA) stimulation by fibrin is by "colocalization" of tPA and plasminogen on fibrin, which acts as a substrate (definition 1) or template; and the plasmin generated in this way is the enzyme that digests fibrin substrate (definition 2). Fibrinolysis encompasses both processes, which are distinct but overlapping. The role of fibrin structure in regulating the whole process of fibrinolysis is not completely understood, and there is disagreement over published results. For example, fibrin fiber diameter can be manipulated by thrombin, such that higher thrombin concentrations produce a fine network of thin fibers, whereas fibrin polymerization at lower thrombin concentration results in more lateral aggregation and produces clots composed of thicker fibers, as reviewed. 1 It has been observed from gross changes in turbidity of fibrin clots undergoing lysis 2,3 that clots made of thicker fibers appear to lyse more rapidly than clots made from fine fibers. However, this simple relationship has been questioned because clots made of thicker fibers are more turbid and lysis may appear to be faster when followed optically if turbidity is not normalized to correct for different starting values. 1 Furthermore, it has been noted from more detailed microscopic studies that thinner fibers are more susceptible to lysis than thick fibers, yet this relationship is not reflecte...

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.