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Prothrombin biosynthesis: characterization of processing events in rat liver microsomes
Abstract:
Plasma and hepatic microsomal forms of rat prothrombin have been compared by sodium dodecyl sulfate-polyacrylamide electrophoresis and isoelectric focusing. The major prothrombin species that accumulated in the microsomes of rats treated with warfarin had a molecular weight of 78 500 and a pI in 8 M urea of 6.3-6.5. Plasma prothrombin had a molecular weight of 83 500 and a pI of 5.3-5.7. Microsomes from normal rat liver contain a second pool of precursor with a molecular weight of 83 500, and digestion with the glycosidase Endo H indicated that this form has been processed to contain complex carbohydrates, while the Mr 78 500 form is a high mannose form and is the substrate for the vitamin K dependent carboxylase. Treatment of rats with tunicamycin revealed that glycosylation was not essential for carboxylation or secretion from the liver. Comparison of the aglyco forms of prothrombin and its precursors suggests that the intracellular forms contain a basic, Mr approximately 1500 peptide that is missing from the plasma form of prothrombin.
Insights
Warfarin treatment alters rat prothrombin, creating a smaller microsomal form (78,500 MW) that serves as a substrate for vitamin K-dependent carboxylation. This contrasts with plasma prothrombin (83,500 MW).
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Prothrombin is a key protein in blood coagulation.
- Vitamin K-dependent carboxylation is essential for prothrombin function.
- Warfarin is an anticoagulant that inhibits vitamin K metabolism.
Purpose of the Study:
- To compare plasma and hepatic microsomal forms of rat prothrombin.
- To investigate the characteristics of prothrombin precursors in rat liver microsomes.
- To elucidate the role of glycosylation in prothrombin processing and function.
Main Methods:
- Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) for molecular weight determination.
- Isoelectric focusing (IEF) for charge characterization.
- Endo H digestion to assess glycosylation status.
- Tunicamycin treatment to study the effect of glycosylation inhibition.
Main Results:
- Warfarin-treated rats showed a major microsomal prothrombin species (78,500 MW, pI 6.3-6.5) distinct from plasma prothrombin (83,500 MW, pI 5.3-5.7).
- Intracellular precursors (83,500 MW) in normal rat liver microsomes contain complex carbohydrates, while the 78,500 MW form is a high mannose precursor and substrate for vitamin K-dependent carboxylase.
- Glycosylation is not essential for carboxylation or hepatic secretion of prothrombin, as shown by tunicamycin treatment.
- Aglyco forms suggest intracellular prothrombin precursors possess a basic peptide (approx. 1500 MW) absent in plasma prothrombin.
Conclusions:
- Warfarin induces the accumulation of a specific, partially processed intracellular prothrombin form.
- The 78,500 MW microsomal prothrombin is a direct substrate for vitamin K-dependent carboxylation.
- Intracellular prothrombin processing involves modifications, including the potential addition and removal of a basic peptide, and differential glycosylation, independent of carboxylation and secretion.