Intravenous thrombolysis has changed the way that acute ischemic stroke is treated. But there are downsides to recombinant tissue-plasminogen activator (rt-PA), including the potential for intracranial hemorrhage upon treatment, and there is only a narrow window of time after the symptoms occur to treat it. Therefore, the search is on for new and better treatments that not only deliver more efficacy but also do not worsen and become more common. In vitro methods are valuable for evaluating the properties of thrombolytics affected by structural modifications and serve as an effective platform for screening numerous drug variants in a cost-efficient and ethical way.

Ace Therapeutics offers a variety of in vitro tests to screen for effective thrombolytic drugs.

Related Services

• Thrombolytics Development for Stroke

Common Assays Used for In Vitro Testing of Thrombolytics

In vitro testing of thrombolytics generally follows the physiological fibrinolytic pathway, but assays vary in complexity, analysis time, reproducibility, and targeted properties. No single method is superior, so selecting the most relevant assays for specific purposes is essential. To gain a comprehensive understanding of the thrombolytics being studied, it may be necessary to employ a combination of different testing methods.

Fig.1. Overview of thrombolytic enzymatic activity assays. (Nikitin, et al., 2021)

Clot Lysis Assay

In this assay, macroscopic clots are prepared either by mixing thrombin with plasminogen and fibrinogen, or clotting human plasma or blood. Thrombolytics-induced lysis can be roughly monitored visually to report the complete clot lysis time or more accurately, by the change in clot-related light scattering. Additional measurement options include assessing the release of red blood cells from whole blood clots, changes in clot mass, viscoelastic properties using thromboelastography, and fluorescence changes upon dissolution of labeled clots.

This assay better mimics in vivo conditions compared to the chromogenic/fluorogenic substrate and fibrin plate assays, but its higher procedural complexity can reduce accuracy and reproducibility. Furthermore, the assay evaluates the overall effectiveness of thrombolytics rather than solely their enzymatic activity.

Fibrin Plate Assay

The fibrin plate assay utilizes the physiological fibrinolytic pathway to evaluate thrombolytics. Plates are prepared by combining melted agarose with plasminogen, thrombin, and fibrinogen. When thrombolytics are added, fibrin dissolution is measured by the size of the clear zone created.

This method is more natural than chromogenic or fluorogenic assays and yields robust, reproducible results. However, it is labor-intensive and time-consuming, and the size of the clear zone can be influenced by both fibrinolytic activity and the diffusibility of thrombolytics. To address these limitations, modified techniques such as the dyed fibrin plate assay and fibrin microplate assay have been developed.

Chromogenic/Fluorogenic Substrate Assay

The chromogenic/fluorogenic substrate assay checks if the thrombolytic enzymes are present, separating synthetic peptide substrates into a chromophore or fluorophore measured by spectroscopy. This method is fast, highly reproducible, and shows enzyme directly. However it lacks fibrin, a key component of thrombolytic function. For this small fibrin clots, fibrinogen, or fibrin fragments can be added to the reaction solution. This assay can either be cleaved directly by the activators of plasminogen, or in a couple whereby a thrombolytic drug switches on plasminogen to plasmin, which then cleaves the synthetic substrate.

Inhibition Resistance Assays

The assays are carried out by incubating thrombolytics with an inhibitor at a specific concentration for a set amount of time. Then the free and active thrombolytics level is calculated by enzyme-linked immunosorbent assay (ELISA) or the chromogenic/fluorogenic assay. These experiments yield valuable information regarding the kinetics and thermodynamics of the inhibition process.

Receptor Binding Assays

Receptor binding assays assess the affinity of thrombolytics to membrane receptors, similar to how they are evaluated for fibrin layers. One approach is the measurement of binding affinities and kinetics by surface plasmon resonance (SPR), or the concentration of thrombolytics attached to immobilized receptors can be determined by the use of specific antibodies as part of an ELISA technique or the chromogenic/fluorogenic wash. There are also reports of affinities for soluble receptor fragments, as measured by isothermal titration calorimetry.

Fibrin Immobilization Assay

The fibrin immobilization assay involves immobilizing fibrin layers or fragments on sensor chips to analyze the binding of thrombolytics using surface plasmon resonance (SPR). This method provides insights into both the affinity and kinetics of the drug-fibrin interaction. Alternatively, fibrin can be immobilized on microplate wells, where it is incubated with thrombolytics, and the amount of bound molecules is measured using ELISA or the chromogenic/fluorogenic assay.

Clot Binding Assay

This assay tests whether thrombolytics are binding to macroscopic fibrin clots by adding fibrinogen to the agents and triggering clotting with thrombin. Centrifuged to separate the clot and the supernatant is analyzed for unbound thrombolytics with ELISA, fibrin plate, or chromogenic/fluorogenic assay. While this assay requires careful optimization for reproducibility, it effectively utilizes macroscopic clots without the need for complex instrumentation.

1. Nikitin, D., et al. (2021). Development and testing of thrombolytics in stroke.Journal of Stroke, 23(1), 12-36.