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

Samuel, Dharmaraj; Cheng, Hong; Riley, Paul; Canutescu, Adrian A.; Nagaswami, Chandrasekaran; Weisel, John W.; Bu, Zimei; Walsh, Peter N.; Röder, Heinrich

doi:10.1073/pnas.0703080104

Cited by 24 publications

(24 citation statements)

References 34 publications

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“…This value is also very close to that previously determined (K d ϳ 45 nM) for the isolated A4 domain of FXI (29). These results provide the basis for a high level of confidence that the totality of the binding energy mediating noncovalent dimer formation of FXI resides within the A4 domain, as indicated by x-ray crystallography (19,29,36).…”

Section: Discussionsupporting

confidence: 73%

Factor XI Homodimer Structure Is Essential for Normal Proteolytic Activation by Factor XIIa, Thrombin, and Factor XIa

Sinha

Shikov

et al. 2008

Journal of Biological Chemistry

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Coagulation factor XI (FXI) is a covalent homodimer consisting of two identical subunits of 80 kDa linked by a disulfide bond formed by Cys-321 within the Apple 4 domain of each subunit. Because FXI C321S is a noncovalent dimer, residues within the interface between the two subunits must mediate its homodimeric structure. The crystal structure of FXI demonstrates formation of salt bridges between Lys-331 of one subunit and Glu-287 of the other subunit and hydrophobic interactions at the interface of the Apple 4 domains involving Ile-290, Leu-284, and Tyr-329. FXI C321S , FXI C321S,K331A , FXI C321S,E287A , FXI C321S,I290A , FXI C321S,Y329A , FXI C321S,L284A , FXI C321S,K331R , and FXI C321S,H343A were expressed in HEK293 cells and characterized using size exclusion chromatography, analytical ultracentrifugation, electron microscopy, and functional assays. Whereas FXI C321S and FXI C321S,H343A existed in monomer/ dimer equilibrium (K d ϳ 40 nM), all other mutants were predominantly monomers with impaired dimer formation by analytical ultracentrifugation (K d ‫؍‬ 3-38 M). When converted to the active enzyme, FXIa, all the monomeric mutants activated FIX similarly to wild-type dimeric FXIa. In contrast, these monomeric mutants could not be activated efficiently by FXIIa, thrombin, or autoactivation in the presence of dextran sulfate. We conclude that salt bridges formed between Lys-331 of one subunit and Glu-287 of the other together with hydrophobic interactions at the interface, involving residues Ile-290, Leu-284, and Tyr-329, are essential for homodimer formation. The dimeric structure of FXI is essential for normal proteolytic activation of FXI by FXIIa, thrombin, or FXIa either in solution or on an anionic surface but not for FIX activation by FXIa in solution.Factor XI (FXI), 3 the zymogen form of a serine protease coagulation enzyme that is essential for normal hemostasis, is activated either by FXIIa or by thrombin or by autoactivation (1, 2). Once converted to FXIa, the enzyme recognizes its natural macromolecular substrate, FIX, the Ca 2ϩ -dependent activation of which requires the exposure of a substrate-binding site within the Apple 2 (A2) and/or Apple 3 (A3) domains of FXIa and the ␥-carboxyglutamic acid domain of FIX, as well as an extended, macromolecular substrate-binding exosite in the protease domain of FXIa (3-9). The activation of FIX to FIXa␤ involves two cleavages by FXIa, one after Arg-145 and the other after Arg-180, thereby releasing an 11-kDa activation peptide (3,4,10). FIX is also activated to FIXa␤ by the tissue factorFVIIa complex (11).FXI and plasma prekallikrein (PK) are 58% identical in their amino acid sequences, and the domain structures of the two molecules are very similar, with each molecule containing four homologous apple (A1-A4) domains (12). The high homology between the heavy chain of FXI and PK indicates a common origin of these two zymogens, in contrast to FXII and other coagulation factors (13,14). However, FXI is a homodimer of two identical subunits joined by a disu...

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Section: Discussionsupporting

confidence: 73%

Factor XI Homodimer Structure Is Essential for Normal Proteolytic Activation by Factor XIIa, Thrombin, and Factor XIa

Sinha

Shikov

et al. 2008

Journal of Biological Chemistry

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“…In the crystal structure of zymogen FXI, the catalytic domains are at opposite ends of the molecule, and would be unable to interact simultaneously with one FIX mol- ecule (38). However, work on the structure of the FXI A4 domain by Samuel et al (16) suggests that conformational changes occur during FXI activation that bring the catalytic domains into closer proximity. In the structure for FXI A4 dimer, an ␣-helix not present in the zymogen crystal structure is observed.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, little intermediate appears to be generated during activation by FXIa (8,15). It has been proposed that the dimeric structure of FXIa may account for its capacity to activate FIX without intermediate formation (15,16), with the two protease domains each cleaving one FIX activation site prior to releasing FIXa␤.…”

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confidence: 99%

Characterization of Novel Forms of Coagulation Factor XIa

Smith

Verhamme

Sun

et al. 2008

Journal of Biological Chemistry

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Factor XI is the zymogen of a dimeric plasma protease, factor XIa, with two active sites. In solution, and during contact activation in plasma, conversion of factor XI to factor XIa proceeds through an intermediate with one active site (1/2-FXIa). Factor XIa and 1/2-FXIa activate the substrate factor IX, with similar kinetic parameters in purified and plasma systems. However, factor IX␣ accumulated during activation by the factor XIa catalytic domain, demonstrating the importance of the factor XIa heavy chain. Fluorescence titration of active site-labeled factor XIa revealed a binding stoichiometry of 1.9 ؎ 0.4 mol of factor IX/mol of factor XIa (K d ‫؍‬ 70 ؎ 40 nM). The results indicate that two forms of activated factor XI are generated during coagulation, and that each half of a factor XIa dimer behaves as an independent enzyme with respect to factor IX.Factor XI (FXI), 3 the zymogen of the blood coagulation serine protease factor XIa (FXIa), is comprised of two identical disulfide-linked 80-kDa subunits (1-5). All other coagulation serine proteases are monomers (1, 2). The proteases factor XIIa (FXIIa) and ␣-thrombin are likely physiologic activators of FXI, cleaving the Arg 369 -Ile 370 bond to form FXIa (3-7). Whereas it is assumed that FXIa generated during coagulation has both subunits of the dimer cleaved (two active sites per molecule), this has not been studied in detail, and, hypothetically, FXI with only one cleaved subunit may be generated. Here, we report that FXI activation by factor XIIa or ␣-thrombin proceeds through an intermediate in which only one subunit of the dimer is cleaved, and that this intermediate species is formed in plasma during contact activation-induced coagulation. We purified and characterized the intermediate and used it, along with other forms of FXIa with one active site per molecule, to address the importance of the dimeric structure of FXIa to FIX activation. EXPERIMENTAL PROCEDURESMaterials-FXIIa, FIXa␣, high molecular weight kininogen (HK) and corn trypsin inhibitor were from Enzyme Research Labs (South Bend, IN). Human ␣-thrombin, FVIIa, FXIa, FIX, FIXa␤, and fluorescein-Phe-Pro-Arg-CH 2 Cl were from Hematologic Technologies (Essex Junction, VT). Hirudin (Lepirudin) was from Berlex (Wayne, NJ), soybean trypsin inhibitor-agarose from Sigma, and benzamidine-Sepharose from Amersham Biosciences. L-Pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) was from DiaPharma (West Chester, OH), 1,5-Dansyl-Glu-Gly-Arg-CH 2 Cl was from Calbiochem (La Jolla), and methyl-sulfonyl-D-cyclo-hexyl-glycyl-glycyl-arginine-p-nitroanilide (S-299) from American Diagnostics (Greenwich, CT).Purification of Plasma FXI-Frozen plasma (2 liters) collected in acid-citrate-dextrose was thawed at 4°C, and supplemented with benzamidine (20 mM). FXI was purified from the cryosupernatant by affinity chromatography using the anti-human FXI antibody 1G5.12 (17). After loading, the column was washed with 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM benzamidine and eluted with 2 M NaSCN in the same buffer...

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“…15 fXI, 1/2-fXIa, and fXIa migrate slightly differently on nonreducing SDS-PAGE ( Figure 1 and 4) despite similar molecular masses, which is consistent with activation-induced conformational changes. 30 FXI activation by fXIIa ( Figure 4A) or a-thrombin ( Figure 4B) is a slow process in the absence of a polyanion. Rates of conversion of plasma fXI to 1/2-fXIa were measured at early time points prior to appearance of fully activated fXIa ( Figure 4C and 4D).…”

Section: Activation Of Fxi In the Absence Of A Polyanionmentioning

confidence: 99%

The dimeric structure of factor XI and zymogen activation

Geng

Verhamme

Smith

et al. 2013

Blood

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Key Points• FXI must be a dimer for normal activation by fXIIa but not for activation by thrombin or autoactivation.• Poly-P is a cofactor for activation of coagulation fXI by fXIIa and thrombin and supports fXI autoactivation.Factor XI (fXI) is a homodimeric zymogen that is converted to a protease with 1 (1/2-fXIa) or 2 (fXIa) active subunits by factor XIIa (fXIIa) or thrombin. It has been proposed that the dimeric structure is required for normal fXI activation. Consistent with this premise, fXI monomers do not reconstitute fXI-deficient mice in a fXIIa-dependent thrombosis model. FXI activation by fXIIa or thrombin is a slow reaction that can be accelerated by polyanions. Phosphate polymers released from platelets (poly-P) can enhance fXI activation by thrombin and promote fXI autoactivation. Poly-P increased initial rates of fXI activation 30-and 3000-fold for fXIIa and thrombin, respectively. FXI monomers were activated more slowly than dimers by fXIIa in the presence of poly-P. However, this defect was not observed when thrombin was the activating protease, nor during fXI autoactivation. The data suggest that fXIIa and thrombin activate fXI by different mechanisms. FXIIa may activate fXI through a trans-activation mechanism in which the protease binds to 1 subunit of the dimer, while activating the other subunit. For activation by thrombin, or during autoactivation, the data support a cis-activation mechanism in which the activating protease binds to and activates the same fXI subunit. (Blood. 2013;121(19):3962-3969)

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

Cited by 24 publications

References 34 publications

Factor XI Homodimer Structure Is Essential for Normal Proteolytic Activation by Factor XIIa, Thrombin, and Factor XIa

Factor XI Homodimer Structure Is Essential for Normal Proteolytic Activation by Factor XIIa, Thrombin, and Factor XIa

Characterization of Novel Forms of Coagulation Factor XIa

The dimeric structure of factor XI and zymogen activation

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