In 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) cm(2). 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.
Lytic resistance of fibrin containing red blood cells Wohner, Nikolett; Sótonyi, Péter; Machovich, Raymund ...
Arteriosclerosis, thrombosis, and vascular biology,
2011-October, Letnik:
31, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Arterial thrombi contain variable amounts of red blood cells (RBCs), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. In this study, we ...evaluated the modulator role of RBCs in the lytic susceptibility of fibrin.
If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 to 96 nm at 40% (v/v) RBCs, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBCs prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6%, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein-labeled tPA and orange fluorescent fibrin showed that 20% to 40% (v/v) RBCs significantly slowed down the dissolution of the clots. The fluorescent tPA variant did not accumulate on the surface of fibrin containing RBCs at any cell count above 10%. The presence of RBCs in the clot suppressed the tPA-induced plasminogen activation, resulting in 45% less plasmin generated after 30 minutes of activation at 40% (v/v) RBCs.
RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions.
Abstract Introduction Platelet adhesion to collagen under high shear rates depends on the optimal size of the von Willebrand factor (VWF) multimers, which is determined by their limited proteolysis. ...The present study attempts to identify the role of hemostatic-fibrinolytic enzymes (thrombin, plasmin) and leukocyte-derived proteases (matrix metalloproteinase (MMP)-8, MMP-9, neutrophil elastase) in the cleavage of VWF and to characterize the effect of flow and platelets on this proteolysis and its functional consequences on platelet adhesion. Methods and results According to VWF immunoblots, plasmin, neutrophil elastase and thrombin at concentrations of in vivo relevance resulted in extensive degradation of VWF within several minutes. Platelets protected VWF against this proteolysis under static conditions, whereas perfusion of the proteases at 3350 s-1 shear rate over VWF immobilized on artery cross sections enhanced its degradation and blocked the protective effect of platelets. In parallel with VWF digestion, the examined proteases impaired the VWF-dependent platelet adhesion as reflected in the decreased surface-bound GpIIb/IIIa immunoreactivity following perfusion of collagen-coated surfaces or artery sections with blood and plasmin, neutrophil elastase or thrombin. Within the time frame of minutes no VWF cleavage could be detected under static or flow conditions after exposure to MMP-8 and MMP-9 at concentrations relevant to physiological neutrophil counts. Conclusion Our results indicate a shear- and platelet-dependent role for several proteases in the local modulation of the VWF function.
The structure of the fibrin network, the hemodynamic environment of the clot, the kinetic properties of the fibrinolytic enzymes and the balance of their formation and inactivation essentially ...determine the effectiveness of fibrinolysis in vivo. The fibrin structure and the action of proteases, however depend considerably on additional, apparently inert physiological and pathological factors, which are restricted to more or less transient compartments in fluid-solid interface, such as thrombus (fibrin with platelet membrane structures), endothelial cell surface, the environment of polymorphonuclear cells (PMN). In these compartments extreme changes in concentrations and rate enhancements are observed. Components released by endothelial cells, PMNs and platelets or molecules present in circulating blood create a heterogeneous milieu that modulates fibrinolysis. This review summarizes the effects, and where it is possible, explains the mechanism of modulators of the fibrinolytic processes, such as cell membrane and cellular contents of endothelium, PMN and platelets present in thrombi, the action of normal and pathological blood plasma- and extracellular matrix-components.
Protease‐activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. We have reported earlier that primary cultures of rat brain capillary ...endothelial (RBCE) cells express at least two receptors for thrombin: PAR‐1 and PAR‐3. In the present study we show that PAR‐2 activation by trypsin or by the PAR‐2 agonist peptide (SLIGRL) evokes Ca2+i signal in RBCE cells. Taking advantage of RBCE cells expressing PAR‐1 and PAR‐2, we show that trypsin activates both receptors. The relative agonist activity of trypsin and thrombin on PARs of RBCE cells compared with that of SLIGRL were 112% and 48%, respectively, whereas the potency of trypsin was 105‐fold higher than that of SLIGRL. Because under pathological conditions other proteases such as plasmin or leukocyte elastase may reach the cells of the blood–brain barrier, we investigated the effect of these proteases on RBCE cells. Elastase evoked a small increase in Ca2+i but preincubation of cells with elastase dose‐dependently reduced the trypsin‐induced Ca2+i signal. Plasmin had a 30% inhibitory effect on the trypsin‐induced response, and reduced the SLIGRL signal by 20%. It is concluded that PAR‐2 is functional in brain capillary endothelium, and that the main␣fibrinolytic proteases, plasmin and elastase, may regulate PAR‐2 signalling under pathological conditions.
Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin ...receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombin's cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in Ca2+i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in Ca2+i whereas, upon addition of active site-blocked thrombin or plasmin, Ca2+i remained unchanged. The Ca2+i signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the extracellular medium makes a contribution to the overall Ca2+i rise. The amplitude of the transient Ca2+i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in Ca2+i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the Ca2+i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented Ca2+i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced Ca2+i signals. Plasmin itself does not elevate Ca2+i but prevents the activation of receptors by thrombin.
Immunoglobulin G (IgG) isolated from normal human blood plasma stabilizes the structure of perfused crosslinked fibrin and prolongs the time for its dissolution with plasmin, when the fibrin surface ...is exposed to 500 s(-1) shear rate flow. The IgG from patients suffering in antiphospholipid syndrome with thrombotic complications exerts even stronger antifibrinolytic effect. A patient, whose IgG does not affect the fibrin dissolution with plasmin, displays a bleeding tendency. The shear stress-induced disassembly of the fibrin clots containing IgGs with antifibrinolytic potency occurs at a much more advanced stage of fibrin digestion, as evidenced by the electrophoretic pattern of the ureatreated samples. The antifibrinolytic effects are also produced under static conditions and these are caused by the variable portion of the IgG molecules (fragment Fab), whereas the constant part (fragment Fc) has no inhibitory effect. The IgGs with antifibrinolytic properties do not affect directly the plasmin activity in amidolytic assay, but the IgGs from APS patients obliterate the competition of the fibrin and the peptidyl-p-nitroanilide for the protease in the same assay system suggesting interference of the IgGs with the plasmin action on the fibrin substrate. Thus, the correlation of the clinical symptoms with the effect of the isolated IgG on the dissolution of perfused fibrin clots supports a physiological and a pathological role of IgG in the fibrinolytic process related to the variability of the cross-reactions of immunoglobulins with fibrin, fibrin degradation products or fibrin-plasmin complexes.
A new model has been introduced to characterize the action of a fluid phase enzyme on a solid phase substrate. This approach
is applied to evaluate the kinetics of fibrin dissolution with several ...proteases. The model predicts the rate constants for
the formation and dissociation of the protease-fibrin complex, the apparent order of the association reaction between the
enzyme and the substrate, as well as a global catalytic constant ( k
cat ) for the dissolution process. These kinetic parameters show a strong dependence on the nature of the applied protease and
on the structure of the polymerized substrate. The kinetic data for trypsin, PMN-elastase, and three plasminogen-derived proteases
with identical catalytic domain, but with a varied N-terminal structure, are compared. The absence of kringle 5 in des-kringle 1â5 -plasmin (microplasmin) is related to a markedly lower k
cat (0.008 s â1 ) compared with plasmin and des-kringle 1â4 plasmin (miniplasmin) (0.039 s â1 ). The essentially identical kinetic parameters for miniplasmin and plasmin with the exception of k
diss , which is higher for miniplasmin (81.8 s â1
versus 57.6 s â1 ), suggest that the first four kringle domains are needed to retain the enzyme in the enzyme-fibrin complex. Trypsin, a protease
of similar primary specificity to plasmin, but with a different catalytic domain, shows basically the same k
cat as plasmin, but its affinity to fibrin is markedly lower compared with plasmin and even microplasmin. The latter suggests
that in addition to the kringle domains, the structure of the catalytic domain in plasmin also contributes to its specificity
for fibrin. The thinner and extensively branched fibers of fibrin are more efficiently dissolved than the fibers with greater
diameter and lower number of branching points. When the polymer is stabilized through covalent cross-linking, the k
cat for plasmin and miniplasmin is 2â4-fold higher than on non-cross-linked fibrin, but the decrease in the association rate
constant for the formation of enzyme-substrate complex explains the relative proteolytic resistance of the cross-linked fibrin.
Thus, the functional evaluation of the discrete steps of the fibrinolytic process reveals new aspects of the interactions
between proteases and their polymer substrate.
Abstract Background Removal of C-terminal lysine residues that are continuously exposed in lysing fibrin is an established anti-fibrinolytic mechanism dependent on the plasma carboxypeptidase TAFIa, ...which also removes arginines that are exposed at the time of fibrinogen clotting by thrombin. Objective To evaluate the impact of alterations in fibrin structure mediated by constitutive carboxypeptidase activity on the function of fibrin as a template for tissue plasminogen activator-(tPA) induced plasminogen activation and its susceptibility to digestion by plasmin. Methods and results We used the stable carboxypeptidase B (CPB), which shows the same substrate specificity as TAFIa. If 1.5 – 6 μM fibrinogen was clotted in the presence of 8 U/mL CPB, a denser fibrin network was formed with thinner fibers (the median fiber diameter decreased from 138 – 144 nm to 89 – 109 nm as established with scanning electron microscopy). If clotting was initiated in the presence of 5 – 10 μM arginine, a similar decrease in fiber diameter (82 -95 nm) was measured. The fine structure of arginine-treated fibrin enhanced plasminogen activation by tPA, but slowed down lysis monitored using fluorescent tPA and confocal laser microscopy. However, if lysis was initiated with plasmin in CPB-treated fibrin, the rate of dissolution increased to a degree corresponding to doubling of the plasmin concentration. Conclusion The present data evidence that CPB activity generates fine-mesh fibrin which is more difficult to lyse by tPA, but conversely, CPB and plasmin together can stimulate fibrinolysis, possibly by enhancing plasmin diffusion.
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