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

Summary

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

Key words

Tissue-type plasminogen activator - Tripeptide chloromethyl-ketone inhibitor - Thrombolytic and coagulation assays

• References

• 1 Pennica D, Holmes WE, Kohr WS, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 1983; 301: 214
• 2 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
• 3 Bergmann S, Fox KA A, Ter-Pogossian MM, Sobel BE, Collen D. Clot selective coronary thrombolysis with tissue-type plasminogen activator. Science 1983; 220: 1181-1183
• 4 Walker JE, Ogston D. The inhibition of tissue activator and urokinase by human plasma. Thromb Haemostas 1982; 47: 265-268
• 5 Chmielewska J, Ranby M, Wiman B. Evidence for a rapid inhibitor to tissue plasminogen activator in plasma. Thromb Res 1983; 31: 427-436
• 6 Emeis J, van Hinsbergh V, Verheijen J, Wijngaards G. Inhibition of tissue plasminogen activator by conditioned medium from cultured human and porcine vascular endothelial cells. Biochem Biophys Res Comm 1983; 110: 392-398
• 7 Haggroth L, Mattsson C, Friberg J. Inhibition of the human tissue plasminogen activator in plasma from different species. Thromb Res 1984; 33: 583-584
• 8 Kettner C, Shaw E. D-Phe-Pro-Arg-CH2-Cl - A selective affinity label for thrombin. Thromb Res 1979; 14: 969-973
• 9 Korninger C, Stassen JM, Collen D. Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb Haemostas 1981; 46: 658-661
• 10 Rampling MW, Gaffney PJ. The sulphite precipitation method for fibrinogen measurement: its use on small sample in the presence of fibrinogen degradation products. Clin Chim Acta 1976; 67: 43
• 11 Vermylen C, DeVreker RA, Verstraete M. A rapid enzymatic method for assay of fibrinogen fibrin polymerization time (FPT test). Clin Chim Acta 1963; 8: 418-424
• 12 Von Clauss A. Gerinnungsphysiologische Schnellmethode zur Be-stimmung des Fibrinogens. Acta haemat 1957; 17: 237-246
• 13 Friberger P, Claeson G, Knoes M, Aurell L, Arielly S, Simonsson R. Activity of plasminogen activators on tripeptide chromogenic substrates. In: Progress in Chemical Fibrinolysis and Thrombolysis. Davidson JF. (eds.) Churchill Livingstone; London: 1979: 149-153
• 14 Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem 1982; 257: 2920-2925
• 15 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 16 Kudryk B. et al A monoclonal antibody with the ability to distinguish between fragments derived from fibrinogen and fibrin. Mol Immunol 1983; 20: 1191-120
• 17 Friberger P, Knos M, Gustavsson S, Aurell L, Claeson G. Methods for determination of plasmin, antiplasmin and plasminogen by means of substrate S-2251. Haemostasis 1978; 7: 138
• 18 Friberger P, Knos M. Plasminogen determination in human plasma. In Chromogenic Peptide Substrates. Scully MS, Kakkar VV. (eds.) Curchill Livingstone; Edinburgh, Scotland: 1979: 128-140
• 19 Alkjaersig N. The purification and properties of human plasminogen. Biochem J 1964; 93: 171-182
• 20 Edy J, De Cock F, Collen D. Inhibition of plasmin by normal and antiplasmin-depleted plasma. Thromb Res 1976; 8: 513-515
• 21 Steichele DF, Herschlein HJ. Zur antifibrinolytischen Wirkung des Trypsin-Kallikrein-Inactivators. Med Welt 1961; 42: 2170
• 22 Loskutoff DJ. Plasminogen activator inhibitor (PAI): Biochemical and functional aspects. Thromb Haemostas 1985; 54: 118
• 23 Juhan-Vague I, Moerman B, DeCock Aillaud MF, Collen D. Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathological conditions. Thromb Res 1984; 33: 523-530
• 24 Wiman B, Csemiczky G, Marsk L, Robbe H. The fast acting inhibitor of tissue plasminogen activator in plasma during pregnancy. Thromb Haemostas 1984; 52: 124-126