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Background Neutrophil extracellular traps (NETs) containing DNA and histones are expelled from neutrophils in infection and thrombosis. Heparins, anticoagulant polyanions, can neutralize histones with a potential therapeutic advantage in sepsis. Polyphosphates, procoagulant polyanions, are released by platelets and microorganisms. Objectives To characterize the combined effects of NET components and polyanions on clot structure, mechanical properties and lytic susceptibility. Methods Scanning electron microscopy, pressure-driven permeation, turbidimetry, and oscillation rheometry were used for the characterization of the structure, viscoelasticity, and kinetics of formation and lysis of fibrin and plasma clots containing histones+/-DNA in combination with unfractionated heparin, its desulfated derivatives, low molecular weight heparin (LMWH), pentasaccharide, and polyphosphates of different sizes. Results Histones and DNA inhibited fibrin lysis by plasmin, but this behavior was not neutralized by negatively charged heparins or short polyphosphates. Rather, fibrin lysis was further inhibited by added polyanions. Histones inhibited plasma clot lysis by tissue plasminogen activator and the response to added heparin was size dependent. Unfractionated heparin, LMWH, and pentasaccharide had no effect, exacerbated, or reversed histone inhibition, respectively. Histones increased the mechanical strength of fibrin, which was exacerbated by smaller heparin and polyphosphate molecules. Histones increased fibrin diameter and pore size of fibrin clots and this effect was neutralized by all heparin variants but enhanced by polyphosphates. Conclusions Despite their common polyanionic character, heparins and polyphosphates exert distinct effects on fibrin mechanical and fibrinolytic stability. Anti-fibrinolytic effects of histones were more often enhanced by polyanions not counteracted. Careful selection of anti-histone strategies is required if they are to be combined with thrombolytic therapy.

Protease encapsulation and its targeted release in thrombi may contribute to the reduction of haemorrhagic complications of thrombolysis. We aimed to prepare sterically stabilized trypsin-loaded liposomes (SSLT) and characterize their structure and fibrinolytic efficiency. Hydrogenated soybean phosphatidylcholine-basedSSLTwere prepared and their structure was studied by transmission electron microscopy combined with freeze fracture (FF-TEM), Fourier transform infrared spectroscopy (FT-IR), and small-angle X-ray scattering (SAXS). Fibrinolytic activity was examined at 45, 37, or 24°C on fibrin or plasma clots with turbidimetric and permeation-driven lysis assays. Trypsin was shown to be attached to the inner surface of vesicles (SAXS and FF-TEM) close to the lipid hydrophilic/hydrophobic interface (FT-IR). The thermosensitivity ofSSLTwas evidenced by enhanced fibrinolysis at 45°C: time to reduce the maximal turbidity to 20% decreased by 8.6% compared to 37°C and fibrin degradation product concentration in the permeation lysis assay was 2-fold to 5-fold higher than that at 24°C.SSLTexerted its fibrinolytic action on fibrin clots under both static and dynamic conditions, whereas plasma clot dissolution was observed only in the permeation-driven assay. The improved fibrinolytic efficiency ofSSLTunder dynamic conditions suggests that they may serve as a novel therapeutic candidate for dissolution of intravascular thrombi, which are typically exposed to permeation forces.

SummaryThe formation of platelet-rich thrombi under high shear rates requires both fibrinogen and von Willebrand factor (VWF) as molecular adhesives between platelets. We attempted to describe the role of VWF as a potential substrate and modulator of the fibrinolytic system using binding assays, as well as kinetic measurements on the cleavage of fibrin(ogen) and a synthetic plasmin substrate (Spectrozyme-PL). The similar dissociation constants for the binding of plasminogen, plasmin, and active site-blocked plasmin onto immobilised VWF suggest that the primary binding site in plasmin(ogen) is not the active site. The progressive loss of clottability and generation of degradation products during fibrinogen digestion with plasmin were delayed in the presence of VWF at physiological concentrations, while VWF cleavage was not detectable. Determination of kinetic parameters for fibrinogen degradation by plasmin, miniplasmin and microplasmin showed that VWF did not modify the Km, whereas kcat values decreased with increasing VWF concentrations following the kinetic model of non-competitive inhibition. Inhibitory constants calculated for VWF were in the range of its physiological plasma concentration (5.4 μg/ml, 5.7 μg/ml and 10.0 μg/ ml for plasmin, miniplasmin and microplasmin, respectively) and their values suggested a modulating role of the kringle 5 domain in the interaction between VWF and (mini)plasmin. VWF had no effect on the amidolytic activity of plasmin on Spectrozyme-PL, or on fibrin dissolution by (mini)plasmin. Our data suggest that VWF, while a poor plasmin substrate relative to fibrinogen, protects fibrinogen against degradation by plasmin preserving its clottability in plasma and its adhesive role in platelet-rich thrombi.

Upon platelet activation, free fatty acids are released at the stage of thrombus formation, but their effects on fibrin formation are largely unexplored. Our objective was to characterize the kinetic effects of fatty acids on thrombin activity, as well as the structural and mechanical properties of the resultant fibrin clots. Thrombin activity on fibrinogen was followed by turbidimetry and detailed kinetic characterization was performed using a fluorogenic short peptide substrate. The viscoelastic properties of fibrin were measured with rotatory oscillation rheometer, whereas its structure was analyzed with scanning electron microscopy (SEM). In turbidimetric assays of fibrin generation, oleate and stearate at physiologically relevant concentrations (60-600 μM) produced a bell-shaped inhibitory dose response, increasing 10- to 30-fold the time to half-maximal clotting. Oleate inhibited thrombin activity on a short peptide substrate according to a mixed-type inhibitor pattern (a 9-fold increase of the Michaelis constant, Km and a 20% decrease of the catalytic constant), whereas stearate resulted in only a minor (15%) drop in the catalytic constant without any change in the Km. Morphometric analysis of SEM images showed a 73% increase in the median fiber diameter in the presence of stearate and a 20% decrease in the presence of oleate. Concerning the viscoelastic parameters of the clots, storage and loss moduli, maximal viscosity and critical shear stress decreased by 32-65% in the presence of oleate or stearate, but loss tangent did not change indicating decreased rigidity, higher deformability and decreased internal resistance to shear stress. Our study provides evidence that free fatty acids (at concentrations comparable to those reported in thrombi) reduce the mechanical stability of fibrin through modulation of thrombin activity and the pattern of fibrin assembly.

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.

The formation of platelet-rich thrombi under high shear rates requires both fibrinogen and von Willebrand factor (VWF) as molecular adhesives between platelets. We attempted to describe the role of VWF as a potential substrate and modulator of the fibrinolytic system using binding assays, as well as kinetic measurements on the cleavage of fibrin(ogen) and a synthetic plasmin substrate (Spectrozyme-PL). The similar dissociation constants for the binding of plasminogen, plasmin, and active site-blocked plasmin onto immobilised VWF suggest that the primary binding site in plasmin(ogen) is not the active site. The progressive loss of clottability and generation of degradation products during fibrinogen digestion with plasmin were delayed in the presence of VWF at physiological concentrations, while VWF cleavage was not detectable. Determination of kinetic parameters for fibrinogen degradation by plasmin, miniplasmin and microplasmin showed that VWF did not modify the Km, whereas kcat values decreased with increasing VWF concentrations following the kinetic model of non-competitive inhibition. Inhibitory constants calculated for VWF were in the range of its physiological plasma concentration (5.4 mg/ml, 5.7 mg/ml and 10.0 mg/ml for plasmin, miniplasmin and microplasmin, respectively) and their values suggested a modulating role of the kringle 5 domain in the interaction between VWF and (mini)plasmin. VWF had no effect on the amidolytic activity of plasmin on Spectrozyme-PL, or on fibrin dissolution by (mini)plasmin. Our data suggest that VWF, while a poor plasmin substrate relative to fibrinogen, protects fibrinogen against degradation by plasmin preserving its clottability in plasma and its adhesive role in platelet-rich thrombi.

Thrombi, which are dissolved primarily by plasmin (EC 3.4.21.7.), contain up to millimolar concentrations of fatty acids and these are known to affect the action of the protease. In the present study the modulation of plasmin activity was characterized quantitatively in a continuous amidolytic assay based on synthetic plasmin substrate (Spectrozyme-PL). A novel numerical procedure was applied for identification of kinetic parameters and their confidence intervals, with Monte Carlo simulation of the reaction progress curves, providing adequate grounds for discrimination of different models of the enzyme action. All three fatty acids caused a 10-20-fold increase in the Michaelis constant on Spectrozyme-PL (baseline value 5.9 mum). The catalytic constant decreased from 5.8.s(-1) to 2.4-2.8.s(-1) in the presence of arachidonate and oleate, but increased to 14.8.s(-1) in the presence of stearate, implying enhancement of plasmin activity at saturating substrate concentrations. However, based on the ratio of the catalytic and Michaelis constants, all three fatty acids acted as inhibitors of plasmin with various degrees of potency, showing concentration dependence in the range of 10-65 mum for oleate and arachidonate, and 115-230 mum for stearate. The reported effects of the three fatty acids require the presence of kringle 5 in the structure of the protease; miniplasmin (des-kringle 1-4 plasmin) is as sensitive to fatty acids as plasmin, whereas the activity of microplasmin (des-kringle 1-5 plasmin) is not affected.