BβGlu397 and BβAsp398 but Not BβAsp432 Are Required for “B:b” Interactions ,

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 ...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

Ajjan

Lim

Standeven

et al. 2008

“…8 Fibrinogen, which is composed of 2 fragment D domains and 1 fragment E domain, is a heterodimer composed of pairs of a, b, and g chains. 9 Ab42 binds b-chain residues b366-414 within fragment D. 7 This region is in close proximity to the b-hole of fibrinogen, 10 which is involved in the lateral aggregation of fibrin protofibrils. 11,12 Two different types of therapeutics targeting Ab-fibrinogen association have been investigated.…”

Section: Introductionmentioning

confidence: 99%

Biochemical and structural analysis of the interaction between β-amyloid and fibrinogen

Zamolodchikov¹,

Berk-Rauch²,

Oren

et al. 2016

• Binding to fibrinogen is mediated by the central region of Ab42 and is enhanced by its C-terminal residues.• Ab42 binds the aC region of fibrinogen, delaying plasminmediated fibrin cleavage and generating a persistent aC degradation product.The majority of patients with Alzheimer disease (AD) suffer from impaired cerebral circulation. Accumulating evidence suggests that fibrinogen, the main protein component of blood clots, plays an important role in this circulatory dysfunction in AD. Fibrinogen interacts with b-amyloid (Ab), forming plasmin-resistant abnormal blood clots, and increased fibrin deposition is found in the brains of AD patients and mouse models. In this study, we investigated the biochemical and structural details of the Ab-fibrinogen interaction. We identified the central region of Ab42 as the most critical region for the interaction, which can be inhibited by specific antibodies against the central region of Ab and by naturally occurring p3 peptides, Ab17-40 and Ab17-42. X-ray crystallographic analysis revealed that Ab42 binding to fragment D of fibrinogen induced a structural change in the C-terminal region of the fibrinogen b-chain (b384-393). Furthermore, we identified an additional Ab-binding site within the aC region of fibrinogen. Ab binding to this aC region blocked plasmin-mediated fibrin cleavage at this site, resulting in the generation of increased levels of a plasmin-resistant fibrin degradation fragment. Overall, our study elucidates the Ab-fibrinogen interaction and clarifies the mechanism by which Ab-fibrinogen binding delays fibrinolysis by plasmin. These results may facilitate the development of effective therapeutics against the Ab-fibrinogen interaction to treat cerebrovascular abnormalities in

“…The rfD-B␤D398AϩGH structure showed that GHRP was not bound to hole "b" but was bound to hole "a" in the ␥-chain forming noncanonical "B:a" interactions. 10 As with the rfD-B␤D398AϩGH structure, comparison of the 2 molecules in the asymmetric unit of rfD-B␤D432AϩGH showed 2 different structures for the 2 "B:a" interactions with calcium present in the ␥2 site in only one of the molecules (Figure 2). The ␥2 calcium is located in the ␥294-301 loop, which is in close proximity to hole "a."…”

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confidence: 80%

“…Previously published data on B␤D432A fibrinogen, a variant with mutation in hole "b," showed normal polymerization. 10 Without structural data for B␤D432A, it is reasonable to anticipate that B␤Asp432 may not be critical in binding knob "B" and that there may be alternative and new sets of interactions in B␤D432A that would still allow "B:b" interactions to occur. We also based this hypothesis on recent studies with ␥D364A variant fibrinogen.…”

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confidence: 99%

Fibrinogen variant BβD432A has normal polymerization but does not bind knob “B”

Bowley¹,

Lord

2009

Self Cite

Fibrinogen residue B␤432Asp is part of hole "b" that interacts with knob "B," whose sequence starts with Gly-His-ArgPro-amide (GHRP). Because previous studies showed B␤D432A has normal polymerization, we hypothesized that B␤432Asp is not critical for knob "B" binding and that new knob-hole interactions would compensate for the loss of this Asp residue. To test this hypothesis, we solved the crystal structure of fragment D from B␤D432A. Surprisingly, the structure (rfD-B␤D432A؉GH) showed the peptide GHRP was not bound to hole "b." We then re-evaluated the polymerization of this variant by examining clot turbidity, clot structure, and the rate of FXIIIa crosslinking. The turbidity and the rate of ␥-␥ dimer formation for B␤D432A were indistinguishable compared with normal fibrinogen. Scanning electron microscopy showed no significant differences between the clots of B␤D432A and normal, but the thrombin-derived clots had thicker fibers than clots obtained from batroxobin, suggesting that cleavage of FpB is more important than "B:b" interactions. We conclude that hole "b" and " IntroductionThe fibrinogen molecule consists of 3 pairs of nonidentical polypeptide chains: A␣, B␤, and ␥. These chains are folded into 3 distinct structural regions: 2 distal D regions and 1 central E region, linked by coiled-coil connectors forming a trinodular structure. 1 Each D region contains polymerization holes "a" and "b" located in the C-terminus of the ␥-and B␤-chains, respectively. The central E region contains 2 sets of polymerization knobs "A" and "B" that are cryptic in fibrinogen but become exposed in fibrin after thrombin cleaves fibrinopeptide A (FpA) and fibrinopeptide B (FpB) from the N-terminus of the A␣-and B␤-chains, respectively. 1,2 The exposed knob "A" binds to hole "a" of another fibrin molecule forming "A:a" interactions that lead to a double-stranded protofibril with a half-staggered overlap between molecules in different strands. Cleavage of FpB occurs primarily during protofibril growth, leading to the binding of knob "B" to hole "b" forming "B:b" interactions. 1 The location of the binding holes and possible models for knob-hole interactions are known from X-ray crystallographic studies using 2 synthetic peptide analogs of knobs "A" and "B." When fibrinogen fragment D and double-D are crystallized in the presence of both peptide analogs, the knob "A" peptide mimic, Gly-Pro-Arg-Pro-amide (GPRP), forms H-bond interactions with residues ␥364Asp, ␥330Asp, ␥329Gln, and ␥340His found in hole "a." [3][4][5] In the same manner, the knob "B" peptide mimic, Gly-His-Arg-Pro-amide (GHRP), interacts with residues B␤397Glu, B␤398Asp, and B␤432Asp in hole "b." 3,5,6 The functional role of "A:a" interactions was demonstrated in several studies with reported dysfibrinogenemias, 7,8 as well as studies with ␥364Asp variant fibrinogens. 9 Although the importance of "A:a" interactions have been well documented, the role of "B:b" interactions is still unclear. Previously published data on B␤D432A fibrinogen, a variant with mutation ...