Early processing of prothrombin and factor X by the vitamin K-dependent carboxylase.
Binding interactions between the membrane-associated vitamin K-dependent carboxylase and its prothrombin and factor X substrates have been investigated in liver microsomes. Both substrates are firmly attached to microsomal membrane fragments which also harbor the carboxylase. In vitro 14CO2 gamma-carboxylation of these substrates, triggered by reduced vitamin K1H2, resulted in release of 14C-labeled prothrombin precursors from the membrane fragments, but no release of 14C-labeled factor X precursors could be demonstrated, which suggested a difference in early processing of these substrates by the carboxylase. Warfarin treatment of rats resulted in a 3-fold increase in the membrane concentration of factor X antigens and a 20-fold increase in 14C gamma-carboxylation of the membrane pool of factor X carboxylase substrates. There was a dose-response relationship between the amount of drug administered to the rats and 14C labeling of the membrane pool of factor X carboxylase substrates. On the other hand, the membrane concentration of prothrombin antigens did not increase in response to the drug, and 14CO2 gamma-carboxylation of the membrane pool of prothrombin carboxylase substrates was the same in warfarin and saline-treated rats. The results demonstrate significant differences in the interaction between the carboxylase and its prothrombin and factor X substrates. It appears that the different interactions result from binding of the prothrombin and the factor X precursors to separate microsomal membrane proteins that are involved in the gamma-carboxylation reaction. Warfarin appears to induce the factor X precursor-specific but not the prothrombin precursor-specific binding proteins, which suggests a new mechanism for the action of warfarin. These binding proteins may be under different genetic control. Treatment of the prothrombin and the factor X carboxylase substrates with endonuclease H showed that the rat prothrombin and the human factor X carboxylase substrates are high mannose glycoproteins. The human prothrombin and the rat factor X carboxylase substrates did not, on the other hand, change their migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after endonuclease H treatment. The data demonstrate differences in the glycoprotein nature of the rat and the human carboxylase substrates.
Wallin
R
R
Department of Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Martin
L F
LF
D000818:Animals; D002021:Buffers; D019735:Carbon-Carbon Ligases; D002793:Cholic Acids; D004591:Electrophoresis, Polyacrylamide Gel; D005170:Factor X / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007093:Imidazoles; D007118:Immunoassay; D007163:Immunosorbent Techniques; D008025:Ligases / Q000378:metabolism*; D008297:Male; D008358:Mannose / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism*; D051381:Rats; D014859:Warfarin / Q000494:pharmacology
D002021:Buffers; D002793:Cholic Acids; D006023:Glycoproteins; D007093:Imidazoles; D010446:Peptide Fragments; D011498:Protein Precursors; D014859:Warfarin; C029899:imidazole; D011516:Prothrombin; D005170:Factor X; D006596:Hexosaminidases; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase; D008358:Mannose; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate