In Vitro Clot Lysis as a Potential Indicator of Thrombus Resistance to Fibrinolysis – Study in Healthy Subjects and Correlation with Blood Fibrinolytic Parameters

Summary

Using an in vitro model of clot lysis, the individual response to a pharmacological concentration of recombinant tissue plasminogen activator (rt-PA) and the influence on this response of the physiological variations of blood parameters known to interfere with the fibrinolytic/thrombolytic process were investigated in 103 healthy donors. ¹²⁵I-fibrin labelled blood clots were submersed in autologous plasma, supplemented with 500 ng/ml of rt-PA or solvent, and the degree of lysis was determined after 3 h of incubation at 37° C. Baseline plasma levels of t-PA, plasminogen activator inhibitor 1 (PAI-1), plasminogen, α₂-anti-plasmin, fibrinogen, lipoprotein (a), thrombomodulin and von Willebrand factor as well as platelet and leukocyte count and clot retraction were also determined in each donor. rt-PA-induced clot lysis varied over a wide range (28-75%) and was significantly related to endogenous t-PA, PAI-1, plasminogen (p <0.001) and age (p <0.01). Multivariate analysis indicated that both PAI-1 antigen and plasminogen independently predicted low response to rt-PA. Surprisingly, however, not only PAI-1 but also plasminogen was negatively correlated with rt-PA-ginduced clot lysis. The observation that neutralization of PAI-1 by specific antibodies, both in plasma and within the clot, did not potentiate clot lysis indicates that the inhibitor, including the platelet-derived form, is insufficient to attenuate the thrombolytic activity of a pharmacological concentration of rt-PA and that its elevation, similarly to the elevation of plasminogen, is not the cause of clot resistance but rather a coincident finding. It is concluded that the in vitro response of blood clots to rt-PA is poorly influenced by the physiological variations of the examined parameters and that factors other than those evaluated in this study interfere with clot dissolution by rt-PA. In vitro clot lysis test might help to identify patients who may be resistant to thrombolytic therapy.

• References

• 1 Verstraete M. Advances in thrombolytic therapy. Cardiovasc Drug Therapy 1992; 06: 111-124
• 2 Collen D. Towards improved thrombolytic therapy. Lancet 1993; 342: 34-36
• 3 Musselman DR, Tate DA, Oberhardt BJ, Abruzzini AF, Blauwet MB, Koch G, Dehmer GJ. Differences in clot lysis among patients demonstrated in vitro with three thrombolytic agents (tissue-type plasminogen activator, streptokinase and urokinase). Am J Cardiol 1994; 73: 544-549
• 4 Colucci M, Cavallo LG, Agnelli G, Mele A, Burgi R, Heim J, Semeraro N. Properties of chimeric (tissue-type/urokinase-type) plasminogen activators obtained by fusion at the plasmin cleavage site. Thromb Haemost 1993; 69: 466-472
• 5 Kluft C. Constitutive synthesis of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1): Conditions and therapeutic targets. Fibrinolysis 1994; 08 (Suppl. 02) 1-7
• 6 Rånby M, Bergsdorf N, Nilsson T, Mellbring G, Winblad B, Bucht G. Age dependence of tissue plasminogen activator concentrations in plasma, as studied by an improved enzyme-linked immunosorbent assay. Clin Chem 1986; 32: 2160-2165
• 7 Erickson LA, Ginsburg MH, Loskutoff DJ. Detection and partial characterization of an inhibitor of plasminogen activator in human platelets. J Clin Invest 1984; 74: 1465-1472
• 8 Onundarson PT, Francis CW, Marder VJ. Depletion of plasminogen in vitro or during thrombolytic therapy limits fibrinolytic potential. J Lab Clin Med 1992; 120: 120-128
• 9 Juhan-Vague I, Alessi MC, Fossat C, Declerck PJ, Kruithof EKO. Plasma determination of plasminogen activator inhibitor 1 antigen must be performed in blood collected on antiplatelet/anticoagulant mixture. Thromb Haemost 1987; 58: 1096
• 10 Eriksson E, Tengborn L, Risberg B. The effect of various anticoagulant/antiplatelet mixtures on determination of plasminogen activator inhibitor, platelet proteins and hemostasis parameters. Thromb Haemost 1989; 61: 511-516
• 11 Coller H. Platelets and thrombolytic therapy. N Engl J Med 1990; 322: 33-42
• 12 Stringer HAR, van Swieten P, Heijnen HFG, Sixma JJ, Pannekoek H. Plasminogen activator inhibitor-1 released from activated platelets plays a key role in thrombolysis resistance. Arterioscler Thromb 1994; 14: 1452-1458
• 13 Robbie LA, Bennet B, Croll AM, Brown PAJ, Booth NA. Proteins of the fibrinolytic system in human thrombi. Thromb Haemost 1996; 75: 127-133
• 14 Sane DC, Stump DC, Topol EJ, Sigmon KN, Kereiakes DJ, George BS, Mantell SJ, Macy E, Collen D, Califf RM. Correlation between baseline plasminogen activator inhibitor levels and clinical outcome during therapy with tissue plasminogen activator for acute myocardial infarction. Thromb Haemost 1991; 65: 275-279
• 15 Barbash JI, Hod H, Roth A, Miller HI, Rath S, Zahav YH, Modan M, Zivelin A, Laniado S, Seligsohn U. Correlation of baseline plasminogen activator inhibitor activity with patency of infarct artery after thrombolysis in acute myocardial infarction. Am J Cardiol 1989; 64: 1231-1235
• 16 Kurnik PB. Circadian variation in the efficacy of tissue-type plasminogen activator. Circulation 1995; 91: 1341-1346
• 17 Blann AD. A reliable marker of endothelial cell dysfuntion: does it exist? BrJ Haematol. 1995; 90: 244-248
• 18 Seigneur M, Dufourcq P, Conri C, Constans J, Mercie P, Pruvost A, Bois-seau M. Plasma thrombomodulin: new approach of endothelium damage. IntAngiol 1993; 12: 355-359
• 19 Colucci M, Gelato AV, Scopece SM, Altomare DF, Rinaldi M, Semeraro N. Resistance to fibrinolysis in patients with obstructive jaundice. Fibrinolysis 1996; 10 (Suppl. 03) (Abstract). 49