Factor IX antigen by radioimmunoassay. Abnormal factor IX protein in patients on warfarin therapy and with hemophilia B.

A B S T R A C T Human Factor X was isolated from Cohn fraction III and characterized by polyacrylamide gel electrophoresis, amino acid composition, and isoelectric focusing. Two molecular forms with biological activity were observed at isoelectric points of 4.8 and 5.0. Antisera generated to Factor X was monospecific and used to establish an equilibrium competitive inhibition radioimmunoassay. This assay was specific for human Factor X and did not cross-react with human prothrombin or bovine Factor X within the sensitivity range of 6-300 ng Factor X antigen/ml. The mean concentration of Factor X based on the antigen was 11.9 ,ug0ml, whereas concentration values based on coagulant activity was 7.8 ,ug/ml. This 30% difference in measurement appears to result from the presence of a subpopulation of Factor X molecules devoid of coagulant activity. The radioimmunoassay was used to qualitatively and quantitatively compare purified Factor X to plasmic Factor X obtained from normal, warfarintreated, acquired Factor X-deficient, and congenitaldeficient patients. In all but one case, the Factor X present in these plasmas was immunochemically identical to the purified Factor X and permitted precise quantitation of these abnormal Factor X molecules. Factor X procoagulant activity was analyzed relative to Factor X antigen and the specific activities were used to characterize normal and abnormal Factor X molecules. Reduced Factor X activity in plasmas from warfarin-treated and acquired Factor Xdeficient patients was attributed to both decreases in Factor X antigen and decreased function of the Factor X molecules. Congenitally deficient patients, in general, showed a reduction in Factor X antigen in parallel with Factor X procoagulant activities This is publication

Section: Discussionmentioning

confidence: 99%

Combined Functional and Immunochemical Analysis of Normal and Abnormal Human Factor X

Fair¹,

Plow²,

Edgington³

1979

“…Affinity and ion-exchange chromatography were performed similarly to the method of Baugh and Travis (12), except soybean trypsin inhibitor was used as the affinity ligand instead of aprotinin. Soybean trypsin inhibitor-agarose was prepared by CNBr activation (6) of 50 ml Sepharose 4B, followed by stirring overnight with 1 g soybean trypsin inhibitor at 4°C. Columns (2.5 X 4 cm) of resin were equilibrated with Tris-buffered saline and the sample of lysed granules from 12 U of buffy coat applied.…”

Section: Methodsmentioning

confidence: 99%

“…Factor IX clotting activity and inhibition were determined by the one-stage partial thromboplastin time with kaolin and deficient plasma as previously described (6). For Factor IXa coagulant activity, kaolin was omitted and substrate plasma, phospholipid, and sample were warmed for 15 s at 370C before recalcification; tipping in siliconized glass tubes and reagents were as otherwise described for the assay with contact activation (6).…”

Section: Methodsmentioning

confidence: 99%

“…For Factor IXa coagulant activity, kaolin was omitted and substrate plasma, phospholipid, and sample were warmed for 15 s at 370C before recalcification; tipping in siliconized glass tubes and reagents were as otherwise described for the assay with contact activation (6). In testing for inhibition of Factor IX coagulant activity, elastase-degraded Factor IX was added to normal and Factor IX-deficient plasmas before the kaolin activated, one-stage assay.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Cleavage and inactivation of Factor IX by granulocyte elastase.

Takaki¹,

Enfield²,

Thompson³

1983

Self Cite

A B S T R A C T Radioiodinated Factor IX was cleaved by a crude sonicate from leukocytes. In the absence of calcium, fragments of <15,000 mol wt were seen from reduced samples on gel electrophoresis. After digestion in 2 mM calcium, however, electrophoresis of reduced samples showed, in addition to low molecular weight fragments, protein bands corresponding in size to heavy and light chains of Factor XIa-activated Factor IX. The cleaving activity in leukocyte sonicates was inhibited by soybean trypsin inhibitor, but only to a small extent by aprotinin.Granulocyte elastase was isolated from purified polymorphonuclear leukocyte granules by affinity chromatography on soybean trypsin inhibitor-agarose and further chromatography on carboxymethyl cellulose. The purified fraction contained two isozymes on acidic gels which cleaved both an ester sensitive to elastase and radiolabeled Factor IX. These two activities were inhibited by elastase-specific chloromethyl ketone. The isolated protease fraction rapidly inactivated apparent Factor IX activity in a coagulant assay system. The degree of inactivation correlated with the amount of intact, radiolabeled Factor IX cleaved. As with the crude sonicate, generation of the larger heavy and light chain-sized fragments was dependent upon calcium.To assess directly the effect of elastase on Factor IX, an immunospecific, active site-directed assay was developed. In this assay, the sample was incubated with solid-phase antibody to Factor IX and the amount of activated product was detected as that which had com-

“…It is present as a zymogen with a molecular weight of 57,500 at a concentration of 2.6-5 gg/ml in human plasma (2)(3)(4). Problems that arise in the quantitative analysis of Factor IX activation include the lack ofspecificity ofcoagulation assays and the lack ofavailability of chromogenic substrates specifically cleaved by Factor IXa.…”

Section: Introductionmentioning

confidence: 99%

Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX.

Walsh¹,

Bradford²,

Sinha³

et al. 1984

Abstract. The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX by a modification of a method described for bovine Factor IX activation by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707). Initial rates of trichloroacetic acid-soluble 3H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl2 (5 mM) and with pure (>98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of 3H preceded the appearance of Factor IXa activity, and the percentage of 3H released remained constant when the mole fraction of 3H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r > 0.98, P < 0.001) was observed between initial rates of 3H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include a Km (0.49 tiM) of about five-to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (kt) (7.7/s) is -20-50 times higher than that reported by