Probing the γ2 Calcium-Binding Site:  Studies with γD298,301A Fibrinogen Reveal Changes in the γ294-301 Loop that Alter the Integrity of the “a” Polymerization Site<sup>,</sup>

Kostelansky, M.S.; Lounes, Karim C.; Ping, Li Fang; Dickerson, Sarah K; Gorkun, Oleg V.; Lord, Susan T.

doi:10.1021/bi602607a

Cited by 13 publications

(16 citation statements)

References 44 publications

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“…The partial protection from plasmin for both variants with calcium and GPRP together suggests that they act synergistically to affect each other's binding site conformation. These results are consistent with the previous studies using the engineered recombinant fibrinogens of γD318,320A (altered γ1-calcium binding site) (24) or γD298,301A (altered γ2-calcium binding site) (25).…”

Section: Discussionsupporting

confidence: 93%

Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

Park¹,

Ping²,

Song³

et al. 2012

Thromb Haemost

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SummaryThe fibrinogen γ-module has several important sites relating to fibri-nogen function, which include the high affinity calcium binding site, hole ‘a’ that binds with knob ‘A’, and the D:D interface. Residue γAla341, which is located in the vicinity of these sites, is altered in three variant fibrinogens: fibrinogen Seoul (γAla341Asp), Tolaga Bay (γAla341Val), and Lyon III (γAla341Thr). In order to investigate the impaired polymerisation of fibrinogens γAla341Asp and γAla341Val to understand the role of γAla341 in fibrin polymerisation and fibrinogen synthesis, we have expressed γAla341Asp and γAla341Val in Chinese hamster ovary (CHO) cells, purified these fibrinogens from the culture media and performed biochemical tests to elucidate their function. Expression in CHO cells was similar for these variants. For both variants the kinetics of thrombin-catalysed FpA release was not different from normal fibrinogen, while FpB release was slower than that of normal. Thrombin-catalysed polymerisation of both variants was dependent on the calcium concentration. At physiologic calcium (1 mM) the variants showed impaired polymerisation with a longer lag period and a slower Vmax than normal fibrinogen. Scanning electron micrographs showed the clots were less organised than normal, having thicker and more twisted fibers, and larger pores. Analysis by SDS-PAGE showed that factor XIIIa-catalysed γ and α chain cross-linking was delayed, and plas-min-catalysed lysis was not reduced by the presence of 5 mM calcium or 5 mM GPRP (Gly-Pro-Arg-Pro). Our data indicate that fibrinogen residue γAla341 is important for the proper conformation of the γ-module, maintaining calcium-binding site and ‘A-a’ interactions.

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Section: Discussionsupporting

confidence: 93%

Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

Park¹,

Ping²,

Song³

et al. 2012

Thromb Haemost

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“…The γ2 sites are located in the loops γ294–301 (Everse et al 1999). Impairment of these sites by mutating residues γAsp298 and γAsp301 caused only moderate effects on the crystal structure and functional properties of fibrinogen (Kostelansky et al 2007). It is likely that the γ2 sites are formed as a result of molecular rearrangements induced by crystal packing (Kostelansky et al 2004a, b).…”

Section: 2 Biochemistry Of Fibrinogen the Precursor To Fibrinmentioning

confidence: 99%

Fibrin Formation, Structure and Properties

Weisel

Litvinov

2017

Subcellular Biochemistry

556

498

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Fibrinogen and fibrin are essential for hemostasis and are major factors in thrombosis, wound healing, and several other biological functions and pathological conditions. The X-ray crystallographic structure of major parts of fibrin(ogen), together with computational reconstructions of missing portions and numerous biochemical and biophysical studies, have provided a wealth of data to interpret molecular mechanisms of fibrin formation, its organization, and properties. On cleavage of fibrinopeptides by thrombin, fibrinogen is converted to fibrin monomers, which interact via knobs exposed by fibrinopeptide removal in the central region, with holes always exposed at the ends of the molecules. The resulting half-staggered, double-stranded oligomers lengthen into protofibrils, which aggregate laterally to make fibers, which then branch to yield a three-dimensional network. Much is now known about the structural origins of clot mechanical properties, including changes in fiber orientation, stretching and buckling, and forced unfolding of molecular domains. Studies of congenital fibrinogen variants and post-translational modifications have increased our understanding of the structure and functions of fibrin(ogen). The fibrinolytic system, with the zymogen plasminogen binding to fibrin together with tissue-type plasminogen activator to promote activation to the active proteolytic enzyme, plasmin, results in digestion of fibrin at specific lysine residues. In spite of a great increase in our knowledge of all these interconnected processes, much about the molecular mechanisms of the biological functions of fibrin(ogen) remains unknown, including some basic aspects of clotting, fibrinolysis, and molecular origins of fibrin mechanical properties. Even less is known concerning more complex (patho)physiological implications of fibrinogen and fibrin.

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“…on April 7, 2019. by guest www.bloodjournal.org From fibrinogen ␥D298,301A showed modest impairment in polymerization, suggesting that ␥2 does not significantly modulate polymerization. 26 …”

Section: Structure Of Rfd-b␤d432a؉ghmentioning

confidence: 99%

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

Bowley¹,

Lord

2009

Blood

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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 ...

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Probing the γ2 Calcium-Binding Site: Studies with γD298,301A Fibrinogen Reveal Changes in the γ294-301 Loop that Alter the Integrity of the “a” Polymerization Site^,

Cited by 13 publications

References 44 publications

Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

Fibrin Formation, Structure and Properties

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

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Probing the γ2 Calcium-Binding Site: Studies with γD298,301A Fibrinogen Reveal Changes in the γ294-301 Loop that Alter the Integrity of the “a” Polymerization Site,

Cited by 13 publications

References 44 publications

Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

Fibrin Formation, Structure and Properties

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

Contact Info

Product

Resources

About

Probing the γ2 Calcium-Binding Site: Studies with γD298,301A Fibrinogen Reveal Changes in the γ294-301 Loop that Alter the Integrity of the “a” Polymerization Site^,