Abstract
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.
Highlights
Atherothrombotic lesions of stenosed intracranial and extracranial vessels may lead to microembolization resulting in multiple small cerebral infarcts and progressive cognitive impairment [1,2,3]
Our study revealed that free oleic and stearic acids at biologically relevant concentrations modify the kinetics of fibrin formation and the ultrastructure of the fibrin network, resulting in clots that can be mechanically disassembled at shear stress of magnitude corresponding to the hydrodynamic conditions in partially occluded coronaries [27]
Two different kinetic assays were applied to investigate the influence of free fatty acids on thrombin activity
Summary
Atherothrombotic lesions of stenosed intracranial and extracranial vessels may lead to microembolization resulting in multiple small cerebral infarcts and progressive cognitive impairment [1,2,3]. In addition to the release of a broad spectrum of hemostatic proteins and signal molecules during platelet activation, the increase in the cytosolic calcium concentration leads to activation of the cytosolic phospholipase-A2. This enzyme hydrolyses membrane phospholipids releasing free fatty acids and lysophospholipid [9]. The remodeling of phospholipids within thrombi raises the concentrations of oleic and stearic acid above their plasma levels (180 and 70 μM, respectively [14]) Being present both at the clotting phase and the breakdown of thrombi, free fatty acids are potential modulators of thrombus formation and thrombolysis. The presence of fatty acids in the clot is known to directly affect the activity of proteases involved in fibrin degradation [12,15,16], but their effects on the enzyme activities in fibrin formation (e.g. thrombin) and the resultant clot structure and stability have hardly been investigated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
