Kinetics of the tissue factor-dependent activation of coagulation Factors IX and X in a bovine plasma system.

The kinetics of the tissue factor-dependent activation of Factors IX and X have been studied in a bovine plasma system. Heat-defibrinated plasma, which lacks Factors VIII, V, and fibrinogen, was depleted of Factors IX and X by using specific antibodies coupled to Sepharose. Activation initiated by the addition of bovine brain thromboplastin was studied by adding back to the plasma radiolabeled Factor IX or X at varying levels and measuring the generation of labeled activation peptides as previously described. In the presence or absence of Factor IX, the activation of Factor X is characterized by a 15-s lag before the attainment of maximum rate. This lag is shortened, but not abolished, by pretreatment of the plasma with Factor X,. In contrast, the activation of Factor IX in the absence of Factor X shows a lag of about 5 min before maximum rate is attained. This is shortened to 20 s by the addition of plasma levels of Factor X or by pretreatment of the plasma with low levels of Factor X,. Pretreatment with thrombin has no effect. The ability of pretreatment of plasma with Factor X, reproducibly to shorten the lag phase of activation permitted the determination of the kinetic parameters of the activation of each factor. The apparent K,,, for Factor X is 430 nM (24 pg/ml) and the maximum velocity, V,,, is 800 pmol/min/unit of thromboplastin. The apparent K,,, for Factor IX activation is 73 11~ (4 pg/ml), and V,,,, is 115 pmol/min/unit of thromboplastin. These figures refer to activation of each factor in the absence of the other, but, as substrates for the same enzyme, each competitively inhibits the activation of the other. The apparent Ki for the inhibition of Factor X activation by Factor IX is 220 w (12 pg/ml), and the apparent Ki for inhibition of Factor IX activation by Factor X is 64 m (3.6 pg/ml). From these results it is predicted that, at plasma levels of Factors IX and X, the rate of activation of Factor X by Factor VII, is 168 pmol/min/unit of thromboplastin, and the rate of Factor IX activation, similarly calculated, is 25 pmol/min/ unit of thromboplastin.