Solid phase immunoradiometric assay of activated human coagulation factor IX

Bom, Vjj; Reinalda-Poot, Hanneke H; Poort, S R; Cupers, R; Bertina, Rogier M.

doi:10.1016/0049-3848(87)90328-8

Cited by 3 publications

(4 citation statements)

References 16 publications

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“…Carrier-free Na125I was purchased from the Radiochemical Centre, Amersham, UK. Antithrombin Hl-agarose was prepared as described previously (22). Gelatin (bactogelatin) was purchased from Difco Laboratories, Detroit, MI.…”

Section: Reagentsmentioning

confidence: 99%

“…This assay is based on binding of factor IXa to antithrombin IIIagarose in the first stage and to radiolabeled antibodies against factor IX in the second stage of the assay and has been described in detail previously (22). The factor IXa samples were diluted at least 5-fold in ice-cold assay buffer containing 0.05 M-Tris (pH 7.2), 0.15 M-NaCl, 0.01 M-EDTA, 0.001 M-benzamidine, 0.5 ml/1 of Tween-20, 30 g/1 bovine serum albu min, 1 g/1 of gelatin and 0.5 g/1 of azide and could be stored at -20° C for at least 1 month without affecting the assay results.…”

Section: Immunoradiometric Assay O F Factor Ixamentioning

confidence: 99%

“…In a recent study (21) we performed a detailed kinetic analysis of the activation of factor X by factor V ila using affinity-purified tissue factor apoprotein reconstituted with known amounts of phospholipids (an equimolar mixture of phosphatidylserine and phosphatidylcholine) in the presence of calcium. In the present study the same experimental system of purified factors and a recently developed sensitive and specific immunoradiometric assay of factor IXa (22) have been used to determine the kinetic parameters for the extrinsic factor IX activation. These have been compared with those obtained for the extrinsic activation of factor X.…”

Section: Introductionmentioning

confidence: 99%

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Extrinsic Activation of Human Blood Coagulation Factors IX and X

Bom¹,

Reinalda-Poot²,

Cupers³

et al. 1990

Thromb Haemost

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SummaryWe studied activation of human coagulation factors IX and X by factor VIIa in the presence of calcium ions, phospholipid (phosphatidylserine/phosphatidylcholine, 50/50, mol/mol) and purified tissue factor apoprotein. Activation of factor IX and factor X was found to occur without a measurable lag-phase and hence initial rates of factor IXa and factor Xa formation could be determined. Like previously observed for the activation of factor X, the activation of factor IX was saturable with respect to factor VIIa, tissue factor apoprotein and phospholipid. The results suggested that in the presence of a Ca2+ ions the same ternary complex of factor VIIa-tissue factor apoprolein-phospholipid is responsible for the activation of factor IX and factor X. Roth the apparent Km of 22 nM-factor IX and the apparent Kcat of 28 min−1 were about 3-fold lower than the coiicsponding parameters of factor X activation by this complex. Hence, the catalytic efficiency (Kcat/Km) of factor IX and factor X activation was about equal. However, the two substrates inhibited the activation of each other by competition for the same catalytic sites. The apparent Kinh of factor IX for inhibition of extrinsic factor X activation is 30 nM. The apparent Kinh of factor X for inhibition of extrinsic factor IX activation is 116 nM. From these kinetic data it was calculated that at plasma concentration of factors IX and X, the rate of extrinsic factor IX activation would be half the rate of factor X activation. These relative rates of extrinsic factor IX and factor X activation in combination with previously reported kinetic data on the activation of factor X by factor IXa in the presence of factor VIIIa provide support for the concept that at low levels of tissue factor, factor IXa formation might play an important role in the extiinsic pathway of coagulation in vivo.

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Section: Reagentsmentioning

confidence: 99%

Section: Immunoradiometric Assay O F Factor Ixamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Extrinsic Activation of Human Blood Coagulation Factors IX and X

Bom¹,

Reinalda-Poot²,

Cupers³

et al. 1990

Thromb Haemost

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“…In short, samples of 0.03 ml were added to mixtures composed of 0. Factor IXa concentration was determined by the immunoradiometric assay of factor IXa described previously (44). In this "sandwich" assay, factor IXa binds to antithrombin III coupled to agarose in the first stage and is detected by radioiodinated antibodies against factor IX in the second stage.…”

Section: Assay Of Factor Xa and Factor Ixamentioning

confidence: 99%

Extrinsic Activation of Human Coagulation Factors IX and X on the Endothelial Surface

Bom¹,

Hinsbergh²,

Reinalda-Poot³

et al. 1991

Thromb Haemost

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SummaryIn previous kinetic studies, the catalytic efficiency of the activation of human coagulation factors IX and X by factor VIIa in the presence of purified tissue factor apoprotein was found to be essentially equal. These activation reactions were now studied on the surface of human umbilical vein endothelial cells. The cells were stimulated with endotoxin to express tissue factor. This tissue factor activity was saturable with factor VIIa and could be inhibited by rabbit antibodies against human tissue factor apoprotein. Only stimulated cells supported factor VIIa activity. No difference in the reactivity of factor VII and VIIa was observed in the presence of factor X, due to rapid feedback activation of factor VII by factor Xa. However, the activation of factor IX by factor VII shows a 10 min lag-phase, which reflects that the activation of factor VII by factor IXa is a less efficient process. The kinetic parameters for the factor VIIa dependent activation of factor IX and factor X on the endothelial surface were: Km 0.09 εM, Vmax 0.13 pmol/min, and Km 0.071 εM, Vmax 0.41 pmol/min, respectively. The same ratio between the Vmax for factor X and factor IX activation was observed as in a cell free system. However, the Km of factor IX was 4-fold higher on the endothelial surface than in the cell free system. Together, these kinetic parameters will favour factor X activation 5-fold over factor IX activation at physiological concentrations of these proteins.The activation of factor X by factor VIIa on the endothelial surface was characterized by a short lag-phase, which was absent in factor IX activation. Further, both the activation of factor X and factor IX were down regulated by factor Xa.

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