| Popis: |
The utility of immobilized enzymes in blood coagulation studies is readily apparent. An enzymatic transformation can be stopped by centrifugation and removal of the enzyme beads. This could leave an “activated” factor free of the contaminating “activator”. The further degradation which prolonged exposure to the enzyme often produces can thus easily be stopped. The action of immobilized thrombin on factor VIII warranted testing, since we have observed that hirudin added to a factor VIII solution that has been maximally activated with thrombin prevents the subsequent destruction of “activated” factor VIII. Thrombin was covalently bound to agarose and shown to be active in hydrolyzing a synthetic tripeptide (S-2160) with a pH optimum of 8.5. Tested on human or bovine plasma, immobilized thrombin had no effect on factor VIII activity over 24 hours. Incubated with purified bovine factor VIII (100 units/ml) and tested at various concentrations, no activation of factor VIII activity could be observed. Slow destruction of activity was noted, so that in 16 hours only 20% of factor VIII activity remained compared with glycine-agarose beads as control.It was assumed that a complex of factor VIII and thrombin was necessary for the activated state. To test this, thrombin was labeled with 125I. This iodinated thrombin maintained full factor VIII activating activity. A time dependent binding of factor VIII and thrombin was observed following chromatography on Bio-Gel A 1.5M. This observation, plus the absence of activation of factor VIII by solid phase thrombin, suggests that a factor VIII-thrombin complex rather than limited proteolysis is the mechanism of factor VIII activity enhancement by thrombin.Supported by the National Hemophilia Foundation. |
