A practical method to study functional impairment of proteins by glycation and effects of inhibitors using current coagulation/fibrinolysis reagent kits

Objectives We undertook the present work to device a simple method to study the effects of inhibitors on functional impairment of proteins by the action of glycating agents. Design and methods For that purpose, we first tested the feasibility and optimized the conditions to employ glycation of human plasma coupled with AT III and plasminogen activity measurement, using coagulation test kits available in most clinical laboratories. Results Using D-BUT-CHT-lys-pNA as a plasmin-specific substrate, we show that incubation of plasma with fructose, glyceraldehyde or MG but not glucose decreases plasminogen activity reaching more than 40% in 16 h. A parallel dose-dependent decrease in heparin activation of AT III by up to a 50% was demonstrated using SAR-PRO-ARG-pNA as a specific thrombin substrate. We studied the effects of aminoguanidine, carnosine, quercetin aglycone, alpha tocopherol and ascorbic acid. Conclusion The methods afforded good discrimination between the known different reactivities of glycating sugars as well as the action of known antiglycation agents. They provide a practical system for monitoring the action of putative antiglycation agents.