D-Phe-Pro-Arg-Chloromethylketone: Its Potential Use in Inhibiting the Formation of In Vitro Artifacts in Blood Collected During Tissue-Type Plasminogen Activator Thrombolytic Therapy

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Abstract
In vitro artifacts due to proteolysis may occur in blood samples containing recombinant tissue-type plasminogen activator (rt-PA) due to continued activation of plasminogen to plasmin by rt-PA. The aim of this study was to identify a rapid inhibitor of rt-PA that would not interfere in assays designed to monitor thrombolytic events. When rt-PA was added at 5 μg/ml to whole blood and incubated at 25° C, fibrinogen decreased 50 percent, plasminogen levels decreased 90 percent and α2-antiplasmin decreased below detectable levels. If D-Phe-Pro-Arg-chloromethylketone (PPACK) or aprotinin were added before the addition of rt-PA there was no significant loss of fibrinogen. Only PPACK completely inhibited changes in fibrin degradation products, plasminogen and α2-antiplasmin. PPACK was also found to inhibit the binding of rt-PA to plasma protease inhibitors in vitro. Rhesus monkeys were infused with rt-PA and blood samples were taken with either PPACK or aprotinin in the collection syringe. There was a significant increase in the recovery of immunoreactive rt-PA and consistent measures of fibrinogen, FDPs, plasminogen, and α2-antiplasmin in the PPACK group as compared to the aprotinin group which indicates that PPACK will prevent the in vitro formation of artifacts due to the presence of active rt-PA