Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site.
Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and component heavy and light chains isolated, the heavy chains were no longer a substrate for proteolysis by activated protein C. However, when factor VIII heavy chains were recombined with light chain, the reconstituted factor VIII activity was inactivated by activated protein C. The rate of factor VIII inactivation catalyzed by activated protein C was reduced by the presence of free light chain. The extent of this inhibition was dependent upon the concentration of light chain. Control experiments indicated that this protective effect of free light chain was not the result of inhibition of the activated protein C - lipid interaction. Fluorescence analysis demonstrated binding between the factor VIII light chain, chemically modified with eosin maleimide, and activated protein C, modified at its active site by dansyl-Glu-Gly-Arg chloromethyl ketone. Similar to proteolysis of factor VIII by activated protein C, this binding was dependent upon a lipid surface. Based upon the degree of fluorescence quenching, a spatial distance of 26 A was calculated separating the two fluorophores. These results demonstrate direct binding of activated protein C to the factor VIII light chain and suggest that this binding is an obligate step for activated protein C-catalyzed inactivation of factor VIII.
Fay
P J
PJ
University of Rochester School of Medicine, NY 14642.
Walker
F J
FJ
D001665:Binding Sites; D004789:Enzyme Activation; D005169:Factor VIII / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000648:ultrastructure; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D017293:Protein S; D013057:Spectrum Analysis
D006023:Glycoproteins; D046911:Macromolecular Substances; D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D005169:Factor VIII