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

Abstract

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 ...

Molecular Mechanisms Affecting Fibrin Structure and Stability

“…Experiments with a variant fibrinogen, BβD432A, indicate that ‘B:b’ interactions per se influence the rate of clot lysis. This variant cannot form ‘B:b’ interactions, but the BβD432A fibrin network measured by turbidity and SEM is indistinguishable from normal 66. Nevertheless, lysis of BβD432A fibrin is enhanced relative to normal 67…”

Structural Basis of Interfacial Flexibility in Fibrin Oligomers

“…The D:D interface, which contains residues γ275–309 (Everse et al, 1998) is weak, yielding first to a pulling force upon stretching of fibrin(ogen) oligomers (Zhmurov et al, 2011, 2012). Studies of point mutations revealed that residues γ275, γ308, and γ309 are essential for D-D interactions and elongation of fibrin strands (Hirota-Kawadobora et al, 2004; Marchi et al, 2006; Bowley and Lord, 2009). Fibrin monomers can add longitudinally to form protofibrils, two-stranded oligomers that reach a certain length to begin lateral aggregation (Hantgan et al, 1980; Fowler et al, 1981; Medved et al, 1990).…”

Mechanisms of fibrin polymerization and clinical implications

“…12 On the other hand, fibrinogen variant BbD432A with impaired hole 'b' displays normal polymerization. 13 Therefore, it appears that B:b bonds can occur at least when A:a interactions are compromised, but their normal functional role is still mostly unknown. It is possible that B:b binding has an effect on the susceptibility of a clot to enzymatic destruction.…”