Characterization of t-PA sensitivity in a healthy population: an in vitro study

Introduction: In stroke cases, early treatment with alteplase, a tissue plasminogen activator, is recommended to activate plasminogen and localize clot breakdown. However, up to 5% of patients experience severe fibrinogen depletion [1], suggesting individual variability in response to alteplase. This study aims to develop a biological test to detect and analyze this sensitivity, as well as tools to better understand the mechanisms behind this hypersensitivity.

Method: To examine alteplase’s in vitro effect on plasma fibrinogen, alteplase was added to plasma from 50 healthy donors (final concentration 3 μg/mL) and measured fibrinogen, alpha-2-antiplasmin, and plasminogen activity after a 10-minute incubation at 37°C, using ACLTOP 750®​ (Werfen, Spain). Results were expressed as the percentage decrease in activity before and after alteplase addition. Assays for alpha-2-macroglobulin, tPA, PAI-1, and TAFI (Asserachrom®​ Stago, France), as well as plasmin generation (Fluoroskan Ascent, Thrombinoscope), were also conducted. Statistical analysis used the Mann-Whitney test for group comparisons and Spearman’s test for correlations, performed on GraphPad V8. A P-value<0.05 was considered statistically significant.

Results: Our study revealed a variable response to tPA in control plasmas, allowing us to classify any control showing a fibrinogen decrease of over 20% as a tPA hypersensitive control. Among the controls, 9 plasmas were deemed hypersensitive, while 41 were non-hypersensitive ([Fig. 1]). No correlation was observed between the percentage decrease in fibrinogen and the initial levels of plasminogen or alpha-2-antiplasmin. However, hypersensitive controls exhibited significantly higher consumption of plasminogen and alpha-2-antiplasmin, with a greater percentage decrease compared to non-hypersensitive controls (p<0.0001, Mann-Whitney test), attributed to a significantly higher endogenous plasmin potential in the hypersensitive group (p<0.05, Mann-Whitney test). No significant difference in basal levels of t-PA, PAI-1, TAFI and alpha-2-macroglobulin was found between non-hypersensitive and hypersensitive controls (p>0.05).

Fig. 1  % decrease in fibrinogen between hypersensitive and non hypersensitive controls.

Conclusion: The increased sensitivity to alteplase appears to involve excessive plasminogen activation, resulting in greater plasmin production and higher alpha-2-antiplasmin consumption. The key contribution of this study is its description of a simple and quick test (fibrinogen assay before and after t-PA spiking) that may predict hemorrhagic risk following thrombolysis. However, further evaluation is needed to confirm its predictive accuracy and correlation with bleeding risk in ischemic stroke patients. If validated, this test could prompt neurologists to adjust therapeutic management or t-PA dosage while ensuring that lower doses of t-PA don’t, conversely, lead to ineffective thrombolysis and increased morbi-mortality. To investigate these aspects, a clinical study will be conducted at our institution, which performs around 400 thrombolysis procedures annually.

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2025

• References

• 1 Matrat A, 1 De Mazancourt P, 1 Derex L, 1 Nighoghossian N, 1 Ffrench P, 1 Rousson R. 1 et al. Characterization of a severe hypofibrinogenemia induced by alteplase in two patients thrombolysed for stroke. Thromb Res. janv 2013; 131 (01) e45-48