Effects of Aspirin on Clot Structure and Fibrinolysis Using a Novel In Vitro Cellular System

Abstract

Objectives-The purpose of this study was to investigate the direct effects of aspirin on fibrin structure/function. Methods and Results-Chinese Hamster Ovary cell lines stably transfected with fibrinogen were grown in the absence (0) and presence of increasing concentrations of aspirin. Fibrinogen was purified from the media using affinity chromatography, and clots were made from recombinant protein. Mean final turbidity [OD(ϮSEM)] was 0.083(Ϯ0.03), 0.093(Ϯ0.002), 0.101(Ϯ0.005), and 0.125(Ϯ0.003) in clots made from 0, 1, 10, and 100 mg/L aspirin-treated fibrinogen, respectively (PϽ0.05). Permeability coefficient (Ks cm 2 ϫ10 Ϫ8 ) was 1.68(Ϯ0.29) and 4.13(Ϯ0.33) comparing fibrinogen produced from cells grown with 0 mg/L and 100 mg/L aspirin respectively (PϽ0.05). Scanning electron microscopy confirmed a looser clot structure and increased fiber thickness of clots made from aspirin-treated fibrinogen, whereas rheometer studies showed a significant 30% reduction in clot rigidity. Fibrinolysis was quicker in clots made from aspirin-treated fibrinogen. Ex vivo studies in 3 normal volunteers given 150 mg aspirin daily for 1 week demonstrated similar changes in clot structure/function. Conclusion-Aspirin directly altered clot structure resulting in the formation of clots with thicker fibers and bigger pores, which are easier to lyse. This study clearly demonstrates an alternative mode of action for aspirin, which should be considered in studies evaluating the biochemical efficacy of this agent. Key Words: -fibrinogen Ⅲ aspirin Ⅲ fibrin polymerization Ⅲ fibrin structure Ⅲ acetylation Ⅲ fibrinolysis T he use of antiplatelet agents for prevention of atherothrombotic events is now well established, and aspirin is often the first line agent used in individuals with stable disease. 1 Acetylation of platelet cyclo-oxygenase-1 (COX-1) by aspirin results in irreversible inhibition of thromboxane A 2 (TXA 2 ) production and reduced platelet aggregation potential. This is regarded as the main mechanism for the cardioprotective activity of this agent. However, another mode of action for aspirin, which is not widely considered, is its potential direct effect on clotting factors, including fibrinogen and factor (F) XIII, modulating in the process fibrin clot formation and fibrinolysis. 2 A key event in clot formation is the production of thrombin, which mediates the conversion of fibrinogen into a 3-dimensional network of fibrin fibers, and this is further cross-linked and stabilized by thrombin-activated factor (F)XIII. 3 Clot structure has a role in atherothrombotic disease; clots with thin fibers, small pores, and compact structure are associated with the development of premature and more severe coronary artery disease, which may be related to slower clot lysis of clots. 4,5 Although the ability of aspirin to acetylate fibrinogen has been known for 4 decades, 6 little work has been conducted to study the functional effect of fibrinogen-aspirin interactions, with poor characterization of fibrin gel properties. Limited in vit...

Fibrin Clot Structure and Function

“…97 In an in vitro cellular model of fibrinogen acetylation by aspirin, fibrin clots were produced that were less compact, with thicker fibers. 98 Acetylation reduced clot rigidity by 30% and enhanced clot lysis, which was confirmed with fibrinogen purified from healthy individuals receiving 150 mg/day aspirin for 7 days. 98 Bjornsson et al demonstrated that in vitro acetylation of purified fibrinogen impairs the ability of fibrin to polymerize and decreases clot stability.…”

“…98 Acetylation reduced clot rigidity by 30% and enhanced clot lysis, which was confirmed with fibrinogen purified from healthy individuals receiving 150 mg/day aspirin for 7 days. 98 Bjornsson et al demonstrated that in vitro acetylation of purified fibrinogen impairs the ability of fibrin to polymerize and decreases clot stability. 99 An inverse correlation exists between the extent of fibrinogen acetylation and clot lysis.…”

Plasma fibrin clot structure and thromboembolism: clinical implications

“…Administration of low -dose aspirin was shown to be associated with improved clot properties largely through fibrinogen acetylation. 76,77 Fibrinogen is acetylated at several lysine residues, which also are involved in the FXIII -mediated cross linking of fibrin. 77 In vivo, aspirin concentration of 37.5, 320, and 640 mg/d increased K s by 44%, 31%, and 21%, respectively, and treatment with low -dose aspirin led to formation of thicker fibrin fibers with larger pores.…”

Oxidative Modification of Fibrinogen Is Associated With Altered Function and Structure in the Subacute Phase of Myocardial Infarction

“…Actually, clot structure is determined by several factors. 27,28 In most purified systems, as fiber size increases, pore size increases too. Fibrin networks composed of thin fiber strands have small pores and are more rigid and less permeable.…”