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

Abstract

SummaryUsing 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.

Plasminogen Activator Inhibitor-1 Is a Major Determinant of Arterial Thrombolysis Resistance

“…4,5,22 However, the majority of platelet PAI-1 is inactive, 3,22 and several groups have concluded that PAI-1 does not contribute substantially to platelet-dependent inhibition of clot lysis in vitro when tPA is present in pharmacological concentrations. 7,8,10 Therefore, our studies, which strongly support an important role of PAI-1 in arterial thrombolysis resistance, underscore the importance of in vivo experiments in defining protein function. We hypothesize that the discordant effects of PAI-1 on clot lysis in vitro and in vivo are explained primarily by 2 factors.…”

“…7,8 Furthermore, the very high plasma concentrations of tPA attained during thrombolytic therapy have been predicted to overcome the inhibitory effects of plasma and thrombus-associated PAI-1. 9,10 The true role of PAI-1 in regulating the clearance of platelet-rich thrombi can only be assessed by in vivo experiments. Mice deficient in PAI-1 have been generated by gene targeting.…”

Multifunctional Nanoagent for Thrombus-Targeted Fibrinolytic Therapy

“…Final concentrations of tPA or nanoagent (0 – 30 nM) were calculated based on their total protein amount and a molecular weight of 68 kDa. The degree of lysis was measured at 1 h, 2 h and 3 h of incubation by γ-counting of supernatant (100 μl) and the fractional fibrinolysis was determined as described [24, 25]. …”

Minocycline and Tissue-Type Plasminogen Activator for Stroke

“…A wide range of clinically relevant concentrations of minocycline (0–30 μg/ml) were added to the supernatant, and clot lysis was immediately initiated by t-PA 2nM (Activase; Genentech). The degree of fibrinolysis was measured at various times of incubation by counting the percent soluble 125 I-fibrin degradation products [15, 16] . The kinetic parameters of amidolysis were measured in the presence or absence of minocycline with H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide dihydrochloride (S-2288; Chromogenic) as substrate.…”