Spectrophotometric method for the assay of human blood coagulation factor VIII.

A spectrophotometric method for the assay of human blood coagulation factor VIII in plasma is presented. The chromogenic assay for factor VIII:C in plasma is performed in 3 steps: activation of factor VIII by thrombin; activation of factor X in a mixture of factor X, factor IXa, phospholipids/Ca2+ and plasma containing activated factor VIII, and determination of the rate of factor Xa formation with the chromogenic substrate S2337. Within-assay variation was between 5 and 6.

Assembly of the intrinsic factor X activating complex--interactions between factor IXa, factor VIIIa and phospholipid.

The activation of blood coagulation factor X by factor IXa is strongly stimulated by the non-enzymatic cofactors phospholipid, Ca2+ and activated factor VIII. In this paper we present a method by which we were able to determine binding affinities of factor IXa for phospholipids (either in the absence or presence of factor VIIIa) from kinetic measurements of factor X activation. It is shown that rates of factor X activation in the presence of phospholipids can be saturated with an excess factor VIIIa at limiting amounts of factor IXa and vice versa.

Impaired factor X and prothrombin activation associated with decreased phospholipid exposure in platelets from a patient with a bleeding disorder.

Platelets from a platelet factor 3-deficient patient, which was first described by Weiss et al (Am J Med 67:206, 1979), were found to be equally impaired in their ability to promote factor X and prothrombin activation. Compared to normal platelets, the patient's platelets showed upon stimulation with thrombin plus collagen a much slower generation and a considerably lower level of platelet prothrombin- and factor X-converting activities. Treatment of stimulated platelets with phospholipases revealed a decreased exposure of negatively charged phospholipid at the outer surface of the patient's platelets, relative to control's.

The role of activated human platelets in prothrombin and factor X activation.

The effect of activated human platelets in intrinsic factor X activation was compared with their effect in prothrombin activation. Compared with unstimulated platelets, platelets triggered by the combined action of collagen plus thrombin showed a tenfold activity increase in prothrombin activation, and a 20-fold rate enhancement in factor X activation. Treatment of collagen plus thrombin-stimulated platelets with N.

Purification and properties of an activating enzyme of blood clotting factor X from the venom of Cerastes cerastes.

An activator of blood coagulation factor X was found in the venom of the horned viper Cerastes cerastes, and was purified by gel filtration, ion-exchange chromatography and chromatofocussing. The activator is a protein composed of a heavy and a light polypeptide chain linked by disulfide bonds. Two subforms of the activator were found.