Development and validation of a high throughput whole blood thrombolysis plate assay

T. Bonnard, L. S. Law, Zachary Tennant, C. E. Hagemeyer

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The objective of this work was to develop a high throughput assay for testing in vitro the thrombolytic activity using citrated whole blood samples, and to overcome the limitations of currently available techniques. We successfully developed a method that involves forming halo shaped, tissue factor induced, whole blood clots in 96 well plates, and then precisely measuring the thrombolysis process with a spectrophotometer plate reader. We here describe the implementation of this novel method, which we refer to as halo assay, and its validation with plasmin, urokinase and tissue plasminogen activator at different doses. The resulting data is a highly detailed thrombolysis profile, allowing comparison of different fibrinolytic agents. The time point analysis allows kinetic data to be collected and calculated to determine key parameters such as the activation time and the rate of fibrinolysis. We also assessed the capacity of the model to study the effect of clot maturation time on the fibrinolytic rate, an aspect of thrombosis rather unexplored with currently available methods, but of increasing importance in drug development. This novel thrombolysis assay could be an extremely useful research tool; to study the complex process of thrombolysis, and a valuable translational clinical tool; as a screening device to rapidly identify hypo-or hyper-fibrinolysis.

Original languageEnglish
Article number2346
Number of pages10
JournalScientific Reports
Volume7
Issue number1
DOIs
Publication statusPublished - 1 Dec 2017

Cite this

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title = "Development and validation of a high throughput whole blood thrombolysis plate assay",
abstract = "The objective of this work was to develop a high throughput assay for testing in vitro the thrombolytic activity using citrated whole blood samples, and to overcome the limitations of currently available techniques. We successfully developed a method that involves forming halo shaped, tissue factor induced, whole blood clots in 96 well plates, and then precisely measuring the thrombolysis process with a spectrophotometer plate reader. We here describe the implementation of this novel method, which we refer to as halo assay, and its validation with plasmin, urokinase and tissue plasminogen activator at different doses. The resulting data is a highly detailed thrombolysis profile, allowing comparison of different fibrinolytic agents. The time point analysis allows kinetic data to be collected and calculated to determine key parameters such as the activation time and the rate of fibrinolysis. We also assessed the capacity of the model to study the effect of clot maturation time on the fibrinolytic rate, an aspect of thrombosis rather unexplored with currently available methods, but of increasing importance in drug development. This novel thrombolysis assay could be an extremely useful research tool; to study the complex process of thrombolysis, and a valuable translational clinical tool; as a screening device to rapidly identify hypo-or hyper-fibrinolysis.",
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Development and validation of a high throughput whole blood thrombolysis plate assay. / Bonnard, T.; Law, L. S.; Tennant, Zachary; Hagemeyer, C. E.

In: Scientific Reports, Vol. 7, No. 1, 2346, 01.12.2017.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Law, L. S.

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