Common variation in the C-terminal region of the fibrinogen β-chain: effects on fibrin structure, fibrinolysis and clot rigidity

Abstract

Fibrinogen B␤Arg448Lys is a common polymorphism, positioned within the carboxyl terminus of the B␤-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. IntroductionAn arginine to lysine substitution in the coding region of the ␤-chain of fibrinogen, B␤Arg448Lys, is a relatively common polymorphism, of which the Lys448 allele has a frequency of 15% to 20% in whites. 1,2 A previous study has shown an association between B␤Arg448Lys polymorphism and the severity of coronary artery disease as assessed by angiography, 3 with an increased frequency of the Lys448 allele in patients with triple-vessel disease compared with patients having single-vessel or double-vessel disease. The polymorphism has also been implicated in predisposition to stroke in female patients, an effect that was independent of fibrinogen levels, suggesting a functional role for this polymorphism. 2 We have previously shown that genetic factors contribute to the ultrastructure of the fibrin clot and that more than one-third of the variation in fibrin structure is determined by genes. 4 Clot structure has a role in determining the predisposition to atherothrombotic disease, as clots composed of tightly packed, thin fibers and small pores are associated with increased risk of cardiovascular disease. 5 Furthermore, changes in clot structure have been reported to affect interactions with endothelial cells and fibroblasts, whereby dense fibrin structures with small pores impaired reorganization of these cells into microtubules, 6 implicating effects on angiogenesis, wound healing, and atherosclerosis. 7 Fibrin clot formation initiates with cleavage of fibrinopeptide (Fp)A from the fibrinogen A␣-chain by thrombin, exposing a binding site in the E-region that interacts with a complementary binding site on the ␥-chain in the D-region, allowing formation of protofibrils. Thrombin cleavage of fibrinopeptide (Fp)B from the B␤-chain occurs at a slower rate and is thought to also expose a binding site, this time for a complementary region on the ␤-chain, again located in the D-region. The precise function of this latter interaction is still a matter for debate. Interactions of the ␤-chain have been proposed ...

A novel mechanism for hypofibrinolysis in diabetes: the role of complement C3

“…Turbidity assay These were performed as described elsewhere [6]. Briefly, pooled purified fibrinogen from diabetic and control participants (n=9 each) was used at 2.94 μmol/l, C3 at 0.5 g/l (or BSA at 2 mg/ml), in the presence of 10 μg/ml Factor XIII (FXIII) and 6 μg/ml plasminogen.…”

“…Scanning electron microscopy Clots were prepared using purified fibrinogen as previously described, in the presence and absence of C3 [6]. Fibrinogen was used at 1.44 μmol/l, C3 at 0.245 g/l and samples clotted in the presence of 0.5 U/ml thrombin, 2.5 mmol/l CaCl 2 and 4.9 μg/ml FXIII.…”

“…Fibrinogen was purified by affinity chromatography using calcium-dependent IF-1 monoclonal antibody as previously described [6].…”

“…Fibrin network structure plays a crucial role in determining predisposition to atherothrombotic events as clots with thin fibres and small pores are associated with premature and more severe cardiovascular disease [2][3][4]. This is due to more difficult lysis of clots composed of such compact structure [5], which in turn is related to differential incorporation of various plasma proteins [6]. Previous work has shown more compact fibrin clots in type 1 diabetic patients compared with healthy controls [7], and limited evidence suggests a thrombotic and hypofibrinolytic state in individuals with type 1 diabetes by mechanisms that are not entirely understood [8].…”

The prothrombotic state in paroxysmal nocturnal hemoglobinuria: a multifaceted source

“…102 Moreover, clots with densely packed fibers are stiffer and more resistant to mechanical deformation. 103 Due to these structural and functional changes, dense clots with smaller pores are associated with an increased risk of thrombosis. There have been no studies to date that have investigated clot structure in patients with PNH.…”