Binding of human blood-coagulation Factors IXa and X to phospholipid membranes

Mertens, Koen; Cupers, R; Wijngaarden, A van; Bertina, Rogier M.

doi:10.1042/bj2230599

Cited by 71 publications

(72 citation statements)

References 37 publications

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“…FVIII A2 domain was isolated from FVIIIa employing S-Sepharose chromatography as described (24). Plasma-derived FX and FIX were purified as outlined previously (25). Monoclonal antibody CLB-FIX 14 has been described previously (23) and was purified from culture medium employing protein A-Sepharose as recommended by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

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The Connecting Segment between Both Epidermal Growth Factor-like Domains in Blood Coagulation Factor IX Contributes to Stimulation by Factor VIIIa and Its Isolated A2 Domain

Celie¹,

Stempvoort²,

Fribourg³

et al. 2002

Journal of Biological Chemistry

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The light chain of activated factor IX comprises multiple interactions between both epidermal growth factor-like domains that contribute to enzymatic activity and binding of factor IXa to its cofactor factor VIIIa. To investigate the association between factor IXa-specific properties and surface-exposed structure elements, chimeras were constructed in which the interconnection between the modules Leu 84 -Thr 87 and the factor IX-specific loop Asn 89 -Lys 91 were exchanged for corresponding regions of factor X and factor VII. In absence of factor VIIIa, all chimeras displayed normal enzymatic activity. In the presence of factor VIIIa, replacement of loop Asn 89 -Lys 91 resulted in a minor reduction in factor IXa activity. However, chimeras with substitutions or insertions in the spacer between the epidermal growth factor-like domains showed a major defect in response to factor VIIIa. Of these chimeras, some displayed a normal response to isolated factor VIII A2 domain as a cofactor in factor X activation. Surprisingly, chimeras containing elongated inter-domain spacers from factor X or VII displayed reduced response to both complete factor VIIIa and the isolated A2 domain. Moreover, these chimeras still displayed effective association with immobilized A2 domain as assessed by surface plasmon resonance. We conclude that both sequence and length of the junction Leu 84 -Thr 87 between both epidermal growth factor-like domains contribute to the enhancement of factor IXa enzymatic activity that occurs upon assembly with factor VIIIa. Factor IX (FIX)1 is a vitamin K-dependent serine protease precursor that participates in the process of blood coagulation (1, 2). Factor IX is converted into an active serine protease upon cleavage by either factor XIa (FXIa) or the factor VIIa (FVIIa)-tissue factor complex (3, 4). Proteolysis occurs at restricted cleavage sites and results in the formation of the two-chain enzyme factor IXa (FIXa). FIXa is composed of a light chain that contains an N-terminal Gla domain followed by a short hydrophobic region and two epidermal growth factor (EGF)-like domains (5). The heavy chain comprises the protease domain with the catalytic center. FIXa shares its typical domain structure with related serine proteases of the blood coagulation cascade, including FVIIa, factor Xa (FXa), and activated protein C (6, 7). All of these enzymes contain two EGF-like domains that display considerable sequence homology with the EGF-like modules in factor IXa (8), indicating that these domains are similar in structure and function. Indeed, FIXa, FXa, and FVIIa are similar in that calcium binding to the first EGF-like module is essential for conserving the linkage between its N-terminal half and the N-terminal Gla domain in these proteins (for review, see Ref. 9). However, with respect to the relative orientations of the two EGF-like modules, the three-dimensional crystal structures reveal that this is different in each of these proteases. The connection between these modules is flexible within the structures of F...

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Section: Methodsmentioning

confidence: 99%

“…FVIIIa concentrations were determined using a 1-stage clotting assay (16). The concentration of FX was determined as described previously (25) assuming that 1 unit/ml corresponds to 0.14 M FX. Concentrations of wt-FIXa and FIXa chimeras were measured by active site titration using antithrombin as described (19).…”

Section: Methodsmentioning

confidence: 99%

The Connecting Segment between Both Epidermal Growth Factor-like Domains in Blood Coagulation Factor IX Contributes to Stimulation by Factor VIIIa and Its Isolated A2 Domain

Celie¹,

Stempvoort²,

Fribourg³

et al. 2002

Journal of Biological Chemistry

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“…Plasma factor VIII differs at the protein level from heterodimeric des-(713-1637)-factor VIII in that the latter lacks the heavy-chain sequence Lys713 -Arg740. Concentrations of factor IXa and factor Xa [24], factor X [25], and a-thrombin [26] were determined by active site titration [25]. Proteins were homogenous as assessed by SDSPAGE.…”

Section: Methodsmentioning

confidence: 99%

Kinetics of Factor VIII Light‐Chain Cleavage by Thrombin and Factor Xa

Donath

Lenting

Mourik

et al. 1996

European Journal of Biochemistry

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Activation and limited proteolysis of factor VIII have been investigated with respect to the role of the heavy-chain region Lys713-Arg740. The kinetics of factor VIII activation have been analyzed in a system consisting of human factor VIII, factor IXa, factor X, phospholipids, and thrombin or factor Xa. Plasma-derived factor VIII is activated by thrombin with a second-order rate constant of 3.3 t O.3X1O6 M-' s-l, which proved to be slightly higher than for activation by factor Xa. The second-order rate constant of activation by thrombin of plasma-derived factor VIII in the presence of a monoclonal antibody against the sequence Lys713 -Arg740 is markedly reduced. The same result was obtained for activation by thrombin and factor Xa of factor VIII with a deletion including the sequence Lys713-Arg740, des-(713 -1637)-factor VIII. This suggests that the region Lys713 -Arg740 promotes factor VIII activation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and light chains was analyzed quantitatively. These studies indicated that heavy-chain cleavage of des-(713 -1637)-factor VIII is similar to that of plasma-derived factor VIII. In contrast, cleavage of the light chain of des-(713-1637)-factor VIII is clearly reduced. Furthermore, the secondorder rate constant (0.2 % 0.1 X106 M-' s-') of des-(713-1637)-factor VIII light-chain cleavage by thrombin was reduced tenfold compared with that of plasma-derived factor VIII. Proteolysis by factor Xa yielded similar results. The rate of des-(713 -1637)-factor VIII light-chain cleavage by thrombin is similar to that of isolated light-chain, but isolated light-chain is cleaved by factor Xa 20-fold more efficiently than the light chain in des-(713-1637)-factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is closely associated with light-chain cleavage. Furthermore, within the factor VIII heterodimer, the heavy-chain sequence Lys713 -Arg740 promotes both activation and light-chain proteoly sis.

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“…Factor VIII is a phosphatidyl-L-serine (PS) 1 binding cofactor (1,2) for the vitamin K-dependent serine protease, factor IXa, that also binds to PS-containing membranes (3,4). The membrane-bound factor VIIIa-factor IXa complex cleaves the zymogen, factor X, to factor Xa, which is then responsible for catalyzing prothrombin activation (5).…”

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confidence: 99%

Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs

Gilbert¹,

Kaufman²,

Arena³

et al. 2002

Journal of Biological Chemistry

110

148

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Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a ␤-hairpin turn, and also by net positive charge and specific interactions with phospho-L-serine. , and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipidlimiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced ϳ20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced ϳ5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipidbinding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.Factor VIII is a phosphatidyl-L-serine (PS) 1 binding cofactor (1, 2) for the vitamin K-dependent serine protease, factor IXa, that also binds to PS-containing membranes (3, 4). The membrane-bound factor VIIIa-factor IXa complex cleaves the zymogen, factor X, to factor Xa, which is then responsible for catalyzing prothrombin activation (5). The importance of this enzyme complex is illustrated by hemophilia, a disease in which a deficiency of either factor VIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. Factor IXa gains more than 100,000-fold greater efficiency in activating factor X by assembling with factor VIIIa on a PS-containing membrane than when free in solution (6). We have recently found that the predominant effect of PS-containing membranes on the factor VIIIa-factor IXa complex is to increase the k cat by more than 1000-fold (7). These membranes also increase the affinity of factor IXa for factor VIIIa and for factor X. The central importance of the membrane binding function of factor VIII motivates studies to define the membrane-binding motif.Factor VIII, with M r 280,000, is homologous to another procoagulant protein, factor V, in amino acid sequence (8 -10) and in function, as a membrane-bound enzyme cofactor (5, 11-13). The proteins share a repeating domain structure of A1-A2-B-A3-C1-C2 in which the A domains are homologous with ceruloplasmin, the B domain is unique to each protein, and the C domains are homologous with discoidin I, a phospholipid-binding lectin (14), and with a murine milk fat globule membrane ...

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Binding of human blood-coagulation Factors IXa and X to phospholipid membranes

Cited by 71 publications

References 37 publications

The Connecting Segment between Both Epidermal Growth Factor-like Domains in Blood Coagulation Factor IX Contributes to Stimulation by Factor VIIIa and Its Isolated A2 Domain

The Connecting Segment between Both Epidermal Growth Factor-like Domains in Blood Coagulation Factor IX Contributes to Stimulation by Factor VIIIa and Its Isolated A2 Domain

Kinetics of Factor VIII Light‐Chain Cleavage by Thrombin and Factor Xa

Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs

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