GLA-CONTAINING PROTEINS FROM CALCIFIED HUMAN ATHEROSCLEROTIC PLAQUES

Vitamin K-dependent carboxylase activity has been detected in human andbovine vessel wall. Studies comparingthe carboxylases from liver and vessel wall revealed that the enzyme systems may be regarded as isoenzymes withwidely different substrate specificities. The carboxylated product of vessel wall carboxylase has not yet been identified, but it seems plausible that it will be found amongst the Gla-containing proteins which are abundantly present in calcified atherosclerotic plaques (Gla= gammacarboxyglutamicacid, the abnormal amino acid formed by vitamin K-dependent carboxylase). Therefore we have started to characterize the protein constituents of hardened atherosclerotic plaques.The calcified areas from human aortae were solubilized in EDTA and the proteins extracted were partly purified by batch-wise adsorption onto QAE and elution with high salt. The crudeplaque-extract did not contain prothrombin, factor X or protein C. This excludes the possibility that Gla-containing coagulation factors are bound non-specifically from blood. Osteocalcin accounted for 20% of the total amount of protein-bound Gla-residues.Another Gla-containing protein waspurified from the crude plaque-extract by employing high performance liquid chromatography (HPLC). Gel filtration yielded a Gla-rich protein with anapparent Mr of 25 kD. In vitro boththe crude plaque-extract and the purified Gla-containing protein strongly inhibited the precipitation of calcium phosphate and calcium carbonate. A similar effect was not found with humanserum albumin nor with a thermallydecarboxylated plaque-extract. If also in vivo the Gla-containing proteinsproduced by vessel wall carboxylase prevent the precipitation of calcium salts remains to be investigated.