Exposure of fibrinogen and thrombin to nitric oxide donor ProliNONOate affects fibrin clot properties

Abstract

Background Fibrin fibers form the structural backbone of blood clots. The structural properties of fibrin clots are highly dependent on formation kinetics. Environmental factors such as protein concentration, pH, salt and protein modification, to name a few, can affect fiber kinetics through altered fibrinopeptide release, monomer association and/or lateral aggregation. Objective To determine the effect of thrombin and fibrinogen exposed to nitric oxide on fibrin clot properties. Methods ProliNONOate (5 μM) was added to fibrinogen and thrombin before clot initiation and immediately following the addition of thrombin to the fibrinogen solution. Resulting fibrin fibers were probed with an atomic force microscope to determine their diameter and extensibility and fibrin clots were analyzed for clot density using confocal microscopy. Fiber diameters were also determined by confocal microscopy and the rate of clot formation was recorded using UV-vis spectrophotometry. Protein oxidation and S-nitrosation was determined by UV-vis, ELISA and chemiluminescence. Results The addition of ProliNONOate to fibrinogen or thrombin resulted a change in clot structure. ProliNONOate exposure produced clots with lower fiber density, thicker fibers and increased time to maximum turbidity. The effect of the exposure of nitric oxide to thrombin and fibrinogen were measured independently and indicated that each plays a role in altering clot properties. We detected thrombin S-nitrosation and protein carbonyl formation after nitric oxide exposure. Conclusions Our study reveals a regulation of fibrin clot properties by nitric oxide exposure and suggests a role of peroxynitrite in oxidative modifications of the proteins. These results relate NO bioavailability and oxidative stress to altered clot properties.

Erythrocytic bioactivation of nitrite and its potentiation by far-red light

“…To further explore potential applications of combined nitrite/far red light treatment, we examined clotting in platelet poor plasma. Although the main effects of NO on clotting are known to be through its action as an anti-platelet agent, a few reports have shown effects on thrombin and fibrinogen [62] , [63] . We did not observe any significant effects on the clot lysis time or maximum optical density (data not shown), but we did observe effects of nitrite and light on clotting lag time (time to reach 5% increase in OD, after initiating clotting with thrombin).…”

“…Motivated by the dramatic enhancement by far red light on RBC-mediated inhibition of platelet activation by nitrite, we explored whether nitrite and far red light treatment might have other potential benefits related to treating thrombosis. Potential of NO to treat thrombosis is primarily through its action in reducing platelet activation [5] , [75] , [76] , [77] , [78] , however, recent work suggests that it will also affect other aspects of clotting [62] . Previously, it was found that adding NO to thrombin and/or fibrinogen decreases the rate of fibrin fiber formation with consequentially lower fibrin fiber density and forming thicker fibers [62] .…”

“…Potential of NO to treat thrombosis is primarily through its action in reducing platelet activation [5] , [75] , [76] , [77] , [78] , however, recent work suggests that it will also affect other aspects of clotting [62] . Previously, it was found that adding NO to thrombin and/or fibrinogen decreases the rate of fibrin fiber formation with consequentially lower fibrin fiber density and forming thicker fibers [62] . In this work, we found that incubation of fibrinogen and thrombin with deoxygenated RBCs and nitrite results in a prolonged lag time in clotting measured using turbidity from fibrin polymerization ( Fig.…”

Clinical relevance of hypercoagulability and possible hypofibrinolysis associated with estrone and estriol

“…While Jana et al linked E 3 to increased fibrinolytic potential of platelet rich clots it should be noted that in our investigation platelet poor plasma was used where no platelets were present (Jana et al, ). Helms et al showed that exposure of fibrinogen and thrombin to a nitric oxide donor resulted in plasma clots with thicker fibers with a decreased density (Helms et al, ). This is contradictory to our results, therefore we can assume that the changes associated with E 1 and E 3 addition to platelet poor plasma are not to be attributed to nitric oxide influence or that these hormones can increase the fibrinolytic potential of fibrin clots in the absence of platelets.…”

Role of Fibrinogen in the Attenuation of the Ischemic Reperfusion Injury and in Remote Ischemic Preconditioning

“…In addition, the exposure of FB to ProliNONOate, a donor of NO, affects the structural formation of the fibrin clot, resulting in lower density, but thicker fibers [41]. Such structural variations can have significant impact on embolization and fibrinolysis, where the platelets can retract low density more than high density fibrin fibres [42].…”