A NOVEL ASSAY TO QUANTIFY FIBRINOLYTIC POTENTIAL OF PLATELETS UPON STIMULATION

Abstract

Platelets forms a blood clot to minimize blood loss at sites of injury. These clots also need to be removed to restore blood flow through a process called fibrinolysis, where the role of platelets remain unclear. This is of interest for diagnosis and management of patients with platelet dysfunction, who often bleed. However, there are no clinical assays that can investigate the fibrinolytic properties of platelets. Therefore, the primary aim of this project is to develop an assay that can quantify the fibrinolytic potential of platelets that is expressed when stimulated by agonists. To achieve this, a modified clot lysis assay was developed. By uncoupling the platelet activation step (using thrombin, ADP, or convulxin) from clotting (using batroxobin, thrombin-like enzyme that does not stimulate platelets), clot lysis times were obtained from both resting and activated platelets, and their difference was considered as the fibrinolytic potential. The fibrinolytic potential increased in a platelet concentration-dependent manner, with thrombin having the greatest effect followed by convulxin. ADP did not affect fibrinolysis, suggesting that different signalling pathways have differential fibrinolytic functional consequences. In addition, fibrinolytic potential increase was driven by TAFIa. Though attempts were made to test the assay using platelets from patients with platelet dysfunction, due to technical difficulties, the experiments were inconclusive and require further research. The assay was further modified to be sensitive to mouse platelets. Although our preliminary experiments showed increased net fibrinolytic potential in a platelet concentration-dependent manner, further optimization of the protocol is needed to increase reproducibility and consistency with mice samples. Taken together, we have developed a platelet fibrinolytic function assay. Its ability to measure resting platelet activity represents the ability to provide individual baseline data. Therefore, this assay could represent the beginning of individualized care for patients with platelet dysfunction or bleeding disorders.