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.

Usharani, P; Warn-Cramer, Bonnie J.; Kasper, Carol K.; Bajaj, S. Paul

doi:10.1172/jci111700

Cited by 30 publications

(19 citation statements)

References 41 publications

(34 reference statements)

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“…Neutralization of an Anti-factor IX mAb by the Mutant Proteins-The ability of the IX mutant proteins to bind to a mAb (12), which interferes with the interaction of IXa and VIIIa, was studied using a coagulant based assay as described previously (40). For mutants that possessed Յ8% coagulant activity, a competition-based assay in which the mutant protein competed with the normal IX in binding to the mAb was used (40).…”

Section: Resultsmentioning

confidence: 99%

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Protease and EGF1 Domains of Factor IXa Play Distinct Roles in Binding to Factor VIIIa

Mathur¹,

Bajaj

1999

Journal of Biological Chemistry

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Previous studies revealed that cleavage at Arg-318 -Ser-319 in the protease domain autolysis loop of factor IXa results in its diminished binding to factor VIIIa. Now, we have investigated the importance of adjacent surface-exposed helix 330 -338 (162-170 in chymotrypsin numbering) of IXa in its interaction with VIIIa. IX WT , eight point mutants mostly based on hemophilia B patients, and a replacement mutant (IX helixVII in which helix 330 -338 is replaced by that of factor VII) were expressed, purified, and characterized. Each mutant was activated normally by VIIa-tissue factor-Ca 2؉ or XIa-Ca 2؉ . However, in both the presence and absence of phospholipid, interaction of each activated mutant with VIIIa was impaired. The role of IXa EGF1 domain in binding to VIIIa was also examined. Two mutants (IX Q50P and IX PCEGF1 , in which EGF1 domain is replaced by that of protein C) were used. Strikingly, interactions of the activated EGF1 mutants with VIIIa were impaired only in the presence of phospholipid. We conclude that helix 330 in IXa provides a critical binding site for VIIIa and that the EGF1 domain in this context primarily serves to correctly position the protease domain above the phospholipid surface for optimal interaction with VIIIa.

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Section: Resultsmentioning

confidence: 99%

“…For mutants that possessed Յ8% coagulant activity, a competition-based assay in which the mutant protein competed with the normal IX in binding to the mAb was used (40). Each mutant protein bound to the mAb with an equal affinity…”

Section: Resultsmentioning

confidence: 99%

Protease and EGF1 Domains of Factor IXa Play Distinct Roles in Binding to Factor VIIIa

Mathur¹,

Bajaj

1999

Journal of Biological Chemistry

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“…The fIX zymogen (Hematologic Technologies) was depleted of residual fVIl and fX by sequential monoclonal antibody affinity chromatography prior to reductive tritiation (16). The resulting 3H-flX preparation had a specific activity of 2 x 108 dpm/mg and retained 97% of the original clotting activity measured in fIX-deficient plasma (George King Biomedical).…”

Section: Methodsmentioning

confidence: 99%

Anticoagulant repertoire of the hookworm Ancylostoma caninum.

Stassens¹,

Bergum²,

Gansemans³

et al. 1996

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

239

191

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Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substance(s) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor Vlla and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXadependent inhibition of fVIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.

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“…However, a subsequent study by the same group (Spitzer et al [13]) showed that factors IX Los Angeles and Long Beach had identical sequences (Ile 397 + Thr) and suggested the difference between them in the bovine brain PT was due to factor 1X antigen level. A reexamination of the data of Usharani et al [12] by Spitzer et al [ 131 revealed a correlation between the level of factor IX antigen and degree of prolongation of the bovine brain PT.…”

Section: Introductionmentioning

confidence: 94%

Comparison of the behavior of normal factor IX and the factor IX BM variant hilo in the prothrombin time test using tissue factors from bovine, human, and rabbit sources

et al. 1993

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A subset of hemophilia B patients have a prolonged bovine-brain prothrombin time. These CRM+ patients are classified as having hemophilia Bm. The prolongation of the prothrombin time has been reported only with bovine brain (referred to as ox brain in some literature) as the source of thromboplastin; prothrombin times determined with thromboplastin from rabbit brain or human brain are not reported to be prolonged. Factor IX from a hemophilia Bm patient (factor IX Hilo) was isolated. The activity of factor IX Hilo was compared to that of normal factor IX in prothrombin time assays when the thromboplastin source was of bovine, rabbit, or human origin. Factor IX, either normal or Hilo, prolonged a prothrombin time regardless of the tissue factor source. However, unless thromboplastin was from a bovine source, this prolongation required high concentrations of factor IX. Further, factor IX normal was as effective as factor IX Hilo in prolonging the prothrombin time when rabbit or human thromboplastin was used. With bovine thromboplastin, factor IX Hilo was significantly better than factor IX normal at prolonging the prothrombin time. The amount of prolongation was dependent on the amount of factor IX Hilo added. In addition, the prolongation was dependent on the concentration of factor X present in the sample. The prothrombin time changed as much as 20 seconds when the factor X concentration was varied from 50% to 150% to normal (fixed concentration of factor IX Hilo). These results demonstrate the difficulty of classifying the severity of a hemophilia Bm patient based on the bovine brain prothrombin time unless both the factor IX and factor X concentrations are known.

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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.

Cited by 30 publications

References 41 publications

Protease and EGF1 Domains of Factor IXa Play Distinct Roles in Binding to Factor VIIIa

Protease and EGF1 Domains of Factor IXa Play Distinct Roles in Binding to Factor VIIIa

Anticoagulant repertoire of the hookworm Ancylostoma caninum.

Comparison of the behavior of normal factor IX and the factor IX BM variant hilo in the prothrombin time test using tissue factors from bovine, human, and rabbit sources

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