Role of calcium ions and the heavy chain of factor XIa in the activation of human coagulation factor IX

Sinha, Dipali; Seaman, F S; Walsh, Peter N.

doi:10.1021/bi00387a005

Cited by 61 publications

(61 citation statements)

References 25 publications

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“…Our conclusion that FXI zymogen activation is accompanied by major changes in domain structure is further supported by biochemical data indicating that the binding properties of FXIa differ from those of the zymogen with respect to its substrate, FIX, and the platelet surface (29)(30)(31). These observations are consistent with domain rearrangements exposing new binding sites upon conversion of FXI into FXIa.…”

Section: Domain Structure Of Fxi and Fxia Observed By Saxs And Emsupporting

confidence: 84%

Solution structure of the A4 domain of factor XI sheds light on the mechanism of zymogen activation

Samuel

Cheng

Riley

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Factor XI (FXI) is a homodimeric blood coagulation protein. Each monomer comprises four tandem apple-domain repeats (A1-A4) and a serine protease domain. We report here the NMR solution structure of the A4 domain (residues 272-361), which mediates formation of the disulfide-linked FXI dimer. A4 exhibits characteristic features of the plasminogen apple nematode domain family, including a five-stranded ␤-sheet flanked by an ␣-helix on one side and a two-stranded ␤-sheet on the other. In addition, the solution structure reveals a second ␣-helix at the C terminus. Comparison with a recent crystal structure of full-length FXI, combined with molecular modeling, suggests that the C-terminal helix is formed only upon proteolytic activation. The newly formed helix disrupts interdomain contacts and reorients the catalytic domains, bringing the active sites into close proximity. This hypothesis is supported by small-angle x-ray scattering and electron microscopy data, which indicate that FXI activation is accompanied by a major change in shape. The results are consistent with biochemical evidence that activated FXI cleaves its substrate at two positions without release of an intermediate.blood coagulation ͉ NMR ͉ plasminogen apple nematode domain ͉ small-angle x-ray scattering ͉ EM F actor XI (FXI) is a blood plasma protein in the intrinsic pathway of blood coagulation. In response to blood vessel injury, thrombin can convert the homodimeric FXI zymogen into its proteolytically active form, FXIa, which in turn cleaves its substrate, factor IX (FIX), resulting in a cascade of events leading to fibrin formation (1, 2). FXI may also play a role in protection of clots from fibrinolysis (3). The enzymatic activators of FXI (thrombin, FXIIa, or FXIa) cleave the Arg-369-Ile-370 bond in each monomer of FXI, yielding FXIa, which consists of a 369-residue heavy chain and a disulfide-linked serine protease domain of 238 residues. A substantial body of evidence indicates that binding interactions outside the protease domain (exosites) are important determinants of ligand and receptor interactions and of substrate affinity and specificity in coagulation reactions involving FXI/XIa (2).The FXI heavy chain comprises four apple-domain repeats, A1-A4, each of which contains six cysteine residues involved in intradomain disulfide bonds (1, 4). An additional cysteine in A4 (Cys-321) forms a physiologically important disulfide bridge with the corresponding Cys in the other subunit of the FXI dimer. The sequence identity among the four FXI apple domains ranges from 23% to 34%. They are members of the plasminogen apple nematode (PAN) domain family of proteins (5). Various domains of FXI have been modeled by using PAN domain structures as templates (6). However, the model could not predict the quaternary structure of the A4 domain, which mediates dimerization of FXI through both noncovalent interactions as well as an intersubunit disulfide bond (Cys-321-Cys-321Ј). A recent crystal structure of the FXI zymogen (7) showed that all of the contacts ...

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Section: Domain Structure Of Fxi and Fxia Observed By Saxs And Emsupporting

confidence: 84%

Solution structure of the A4 domain of factor XI sheds light on the mechanism of zymogen activation

Samuel

Cheng

Riley

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The wells were then incubated with 100 l/well of 1 g/ml of 5F4, a FXI light chain-specific monoclonal antibody, for 1 h at 37°C. Since the 5F4 monoclonal antibody binds with high affinity to FXIa (K d ϳ0.78 ϫ 10 Ϫ10 M) (37) and a saturating concentration of antibody (6.3 ϫ 10 Ϫ9 M) was used, this assay provides an excellent determination of bound FXIa. After washing, 100 l per well of a 1:1000 dilution of alkaline phosphatase-conjugated goat anti-mouse antibody was added for a 1-h incubation at 37°C.…”

Section: Methodsmentioning

confidence: 99%

A Binding Site for Heparin in the Apple 3 Domain of Factor XI

Ho¹,

Badellino²,

Baglia³

et al. 1998

Journal of Biological Chemistry

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Since heparin potentiates activated factor XI (FXIa) inhibition by protease nexin-2 by providing a template to which both proteins bind (Zhang, Y., Scandura, J. M., Van Factor XI (FXI) 1 is a zymogen that circulates in plasma in a non-covalent complex with high molecular weight kininogen (HMWK) (1, 2) and participates in the contact phase of blood coagulation (3). The active enzyme, activated factor XI (FXIa), is a trypsin-like serine protease that is generated when FXI is cleaved by FXIIa (3), thrombin, or FXIa (4, 5) at an internal Arg 369 -Ile 370 bond (2). Upon activation, FXIa is capable of converting FIX into its active form, FIXa (2, 6). FIXa, in the presence of FVIII and platelets, can activate FX (7,8), resulting in the generation of thrombin and, subsequently, a fibrin clot.The structure of FXI is unique among the plasma coagulation enzymes (9, 10), since it exists as a homodimer consisting of two subunits each of which contains 607 amino acids. During proteolytic activation, each of these subunits is cleaved to generate a heavy chain of 369 amino acids and a light chain or catalytic domain of 238 amino acids. The heavy chain of FXI provides binding surfaces for several blood coagulation proteins and is organized into four tandem repeat Apple domains designated A1, A2, A3, and A4 (9, 10). Each of these four domains contains 90 to 91 amino acids, the sequences of which are 23-34% identical. The A1 domain provides a binding site for HMWK (11), a protein that promotes the activation of FXI by FXIIa (12). Both the A2 domain (13) and the A3 domain (14) have been implicated in the binding of FXIa to its substrate, FIX. The A4 domain contains Cys 321 that is responsible for the dimerization of FXI (15,16). In addition, the A4 domain binds to FXIIa (10).Examination of the cellular site of FXI activation by FXIIa has resulted in evidence that platelets promote the proteolytic activation of FXI by FXIIa (17) and that FXI, in the presence of zinc ions, calcium ions, and HMWK, binds to activated platelets in a specific, reversible, and saturable manner with a dissociation constant (K d ) of ϳ10 nM (18). Previous work from this laboratory showed that a synthetic peptide (Asn 235 -Arg 266 ) from the A3 domain inhibited 125 I-FXI binding to platelets (inhibition constant (K i ) ϭ 10 nM) in the presence of HMWK, ZnCl 2 , and CaCl 2 (19,20). Hence, these studies indicate that the A3 domain mediates FXI binding to platelets.A second possible site of FXI activation is the endothelial cell surface. Berrettini et al. (21) demonstrated that FXI binds to endothelial cells in the presence of HMWK, CaCl 2 , and ZnCl 2 with a K d(app) ϳ4.5 nM, whereas FXIa binds with higher affinity (K d(app) ϳ1.5 nM). The binding of FXI to the endothelial cell surface may be of functional significance in localizing coagulation to the site of vascular injury since activation of FXI can occur on the endothelial cell surface (21), where proteoglycans such as heparan sulfate are known to be present. Both Naito and Fujikawa (4) and Gailani an...

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“…There is compelling evidence that one or more exosites (substrate binding sites distinct from the protease active site) on fXIa are required for normal fIX activation (17)(18)(19)(20)(21)(22)(23), but there is a lack of agreement on their locations. It is clear that Ca 2ϩ -dependent fIX activation requires the fXIa heavy chain (19,20).…”

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confidence: 99%

“…It is clear that Ca 2ϩ -dependent fIX activation requires the fXIa heavy chain (19,20). One report indicated that the A2 domain contains a fIX binding site (21), whereas others point to A3 (22,23).…”

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confidence: 99%