Background: Clot retraction, the ultimate phase of platelet thrombus formation, is critical for clot stabilization. It requires functional αIIbβ3 receptors (Glanzmann thrombasthenia patients have diminished or absent clot retraction), fibrin, and the integrated actions of the platelet actin-myosin contractile and cytoskeletal systems. Disturbances in the mechanical properties of a clot have clinical implications as they are associated with both bleeding and thrombosis.

Aims: We recently demonstrated that platelets treated with the αIIbβ3 antagonist peptide RGDW, which eliminates fibrinogen-mediated platelet aggregation, are still able to retract clots. We have exploited this observation to develop a robust high-throughput screening assay to identify chemical compounds that inhibit clot retraction.

Methods: We miniaturized and standardized a clot retraction assay in a 384 well microtiter plate format. 1. Wash fresh platelets and treat with RGDW to prevent platelet-fibrinogen interaction. 2. Add test compounds to the wells of polystyrene 384-well microtiter plates using a pin tool. 3. Add CaCl2 and thrombin to the wells. 4. Add platelet/RGDW mixture, 5. After 60 min, image the plate using Image Xpress (Figure). Negative controls consist of wells containing platelets without compounds. Positive controls are wells containing platelets that are not stimulated with thrombin or platelet treated with mAb 7E3, an anti-αIIbβ3 mAb known to inhibit clot retraction.

Results: To date, we have screened more than 400,000 compounds with a ‘hit’ rate of 0.34%. The Z’ factor was 0.5-1.0, indicating that the assay has a high degree of reproducibility. Confirmation and curation of the hits is underway, and several classes of compounds have been identified, including kinase inhibitors with known antiplatelet effects as well as many compounds that have not previously been reported to have antiplatelet activity

Conclusion(s): Our novel high-throughput screen has identified compounds that inhibit clot retraction. Ongoing studies will identify their mechanism(s) of action.

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Figure. Miniaturized clot-retraction assay in a 384-well plate format. The retracted clots in the negative control lane -green outline- without compound appear as small, dense dots, whereas the clots in the positive control wells -red outline-, which do not contain thrombin, and the wells with compounds that inhibit clot retraction -yellow outline- appear uniformly dark.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/high-throughput-screening-to-identify-novel-small-molecule-inhibitors-of-clot-retraction/