Purification and characterization of an abnormal factor IX (Christmas factor) molecule. Factor IX Chapel Hill.

Chung, Kyung Soo; Madar, D A; Goldsmith, Jonathan C.; Kingdon, Henry S.; Roberts, Harold R.

doi:10.1172/jci109213

Cited by 33 publications

(19 citation statements)

References 40 publications

(44 reference statements)

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“…8). The (14,15,35,36). Factor lXzvh,, variant has an abnormally high molecular weight (-90,000) and an apparent reduced affinity for Ca2" (14); additionally, the variant molecule could not be cleaved by Factor XIa (14).…”

Section: Resultsmentioning

confidence: 99%

“…Another variant of Factor IX, Factor IXChapel Hill, is well characterized. This variant does not undergo normal activation by Factor XIa; during activation only Arg"'0-Val"'8 bond is cleaved (15,16). The inability of Factor XIa to cleave Arg'45-Ala14 bond in Factor IXChp1 Hill is due to the substitution of histidine for arginine at position 145 (17).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

Usharani¹,

Warn-Cramer²,

Kasper³

et al. 1985

J. Clin. Invest.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

Usharani¹,

Warn-Cramer²,

Kasper³

et al. 1985

J. Clin. Invest.

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“…In an earlier report from this laboratory some of the properties of Factor IXChapel Hill (Factor IXCH) isolated from a patient with clinically mild hemophilia B were described (11 chased from Johns-Manville, Denver, Colo. All other chemicals were of at least reagent grade or better. Human Factor XIa preparation.…”

Section: Introductionmentioning

confidence: 99%

“…Both proteins were prepared essentially as described (11 (11). Concentration of Factor XIa was based on the extinction coefficient 13.4 (6).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the defect in activation of factor IX Chapel Hill by human factor XIa.

Braunstein¹,

Noyes²,

Griffith³

et al. 1981

J. Clin. Invest.

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A B S T R A C T Factor IXchapel Hill (Factor IXCH), an abnormal Factor IX molecule isolated from the plasma of a patient with mild hemophilia B, has previously been shown to exhibit delayed activation by Factor XIa and calcium. In this study, we have found that Factor IXCH is cleaved upon incubation with human Factor XIa and calcium; however, cleavage of this protein is not observed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, the rate of disappearance of the zymogen parallels both the appearance of the heavy chain and the generation of clotting activity. In addition, a protein band that migrates with an apparent molecular weight of 45,000 also increases in parallel with clotting activity. Factor IXCH and normal Factor IX (Factor IXN), after incubation with Factor XIa and calcium, were subjected to amino terminal sequence analysis. Activated Factor IXN is cleaved at an arginine-alanine (Arg-Ala) bond and an arginine-valine (Arg-Val) bond as demonstrated by formation of the three amino terminal sequences corresponding to the amino terminal of the light chain, heavy chain, and activation peptide. However, activated Factor IXCH has only two amino terminal sequences, corresponding to the original amino terminal sequence and the heavy chain (formed by cleavage at the Arg-Val bond). It is concluded that the major defect in Factor IXCH is, the inability of Factor XIa to cleave the Arg-Ala bond at a significant rate. The rate of formation ofclotting activity of Factor IXCH is -60% ofthe rate of formation of clotting activity of Factor IXN. The specific clotting activity of activated Factor IXCH is between 20 and 33% of activated Factor IXN.

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“…This protein is deficient or functionally defective in patients with hemophilia B (3)(4)(5). The vitamin K-dependent blood coagulation proteins, including factor IX, constitute a class of calcium-binding proteins that contain 'Y carboxyglutamic acid residues, which confer their metalbinding properties (6,7).…”

mentioning

confidence: 99%

Immunoaffinity purification of factor IX (Christmas factor) by using conformation-specific antibodies directed against the factor IX-metal complex.

Liebman¹,

Limentani²,

Furie³

et al. 1985

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

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Factor TX is a vitamin K-dependent blood dotting zymogen that is functionally defective or absent in patients with hemophilia B. A method of imnmunoaffinity chromatogiaphy has been developed for a one-step high yield purification of factor IX directly front plasma. The technique utilizes conformation-specific antibodies that bind solely to the metal-stabilized factor TX conformer, but not to the conformer of factor IX found in the absence of metal ions. Anti-factor IXCa(II) antibodies were immobilized on an agarose matrix. Human plasma in the presence of 7.5 mM MgCI2 was applied to the antibody-agarose column. The factor TX that binds to these antibodies was specifically eluted by metal chelation with EDTA. This immunopurification resulted in a 10,000-fold onestep purification of the fully functional zymogen. Purified factor TX yielded a single band upon gel electrophoresis in NaDodSO4 and had a specific activity of 120-150 units/mg. The purified factor IX was separated from other vitamin K-dependent blood clotting proteins and hepatitis virus; no activated factor TX was detected. This method has application for the large scale purification of factor TX for the treatment of hemophilia B.Factor IX, a vitamin K-dependent plasma protein with a mo- (9), and immunochemical methods using conformation-specific rabbit polyclonal (10) and murine monoclonal antibodies (11) that bind solely to the metal-stabilized conformation.Although we have used these conformation-specific antibodies to develop specific immunoassays for prothrombin species with various contents of y-carboxyglutamic acid (12, 13), we have recently recognized the advantages of conformation-specific antibodies for the immunoaffinity chromatographic purification of proteins. Unlike standard methods for immunopurification, in which the harsh conditions required for the dissociation of the antibody-antigen complex can lead to denaturation and loss of function of the protein (14), this method involves mild and specific elution conditions. Antibodies that only recognize the metal-stabilized conformer of the protein are used to transiently immobilize the protein to a solid support. The antibody-protein complex is disrupted by the removal of bound metal ions from the protein with a metal chelator. The general technique, applied to the purification of factor IX (Christmas factor), has potential application for the large scale purification of factor IX for specific replacement therapy in hemophilia B.

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Purification and characterization of an abnormal factor IX (Christmas factor) molecule. Factor IX Chapel Hill.

Cited by 33 publications

References 40 publications

Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

Characterization of the defect in activation of factor IX Chapel Hill by human factor XIa.

Immunoaffinity purification of factor IX (Christmas factor) by using conformation-specific antibodies directed against the factor IX-metal complex.

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