Comprehensive Analysis of Procoagulant Platelets Exhibiting Features of Necrosis, Apoptosis and Platelet Activation

Our research focuses on understanding platelet metabolism and signaling during prothrombotic states, such as antiphospholipid syndrome and cancer, with the intent of identifying novel biomarkers and therapeutic targets. Our group has been at the forefront of a recent surge in interest about how platelet metabolism contributes to platelet activation and thrombosis. Accurate characterization of the platelet activation state in the circulation of patients to determine the risk of thrombosis as well as the therapeutic efficacy of antiplatelet agents remains a significant challenge.

Aerobic glycolysis, pentose phosphate pathway, and beta oxidation of fatty acids have been unraveled as the hallmark energy metabolism pathways in platelets and their potential has been established for therapeutic targeting. This protocol serves to aid in the identification and accurate functional characterization of procoagulant platelets, distinct from proaggregatory platelets, as well as platelets undergoing cell death by apoptosis or necrosis. To begin, supplement washed human platelets with 2.5 millimolar calcium by adding 0.5 microliters of 0.5 molar stock calcium chloride solution to 100 microliters of platelet suspension.

Keep one fraction of platelets unstimulated and stimulate the remaining fraction with a combination of thrombin and convulsant for 15 minutes at room temperature. After stimulation, add one microliter each of an annexin V, pack one, and anti-CD62P antibody to 100 microliters of stimulated platelet suspensions. Incubate the platelets in the dark at room temperature for 30 minutes.

After 30 minutes, fix the platelets by adding an equal volume of 2%formalin before analyzing the samples by flow cytometry to quantify procoagulant platelets. For the preparation of platelets, dilute washed human platelets to a concentration of one million platelets per milliliter and supplement with 2.5 millimolar calcium. Label the platelets with five micromolar ROD2 for mitochondrial calcium detection and incubate for 30 minutes in the dark.

For the preparation of platelets for caspase activation assay, after supplementing the platelets with calcium and stimulating a fraction, add active caspase labeling die to the stimulated platelet suspensions. Incubate the platelets for 30 minutes in the dark. After 30 minutes, fix the platelets by adding an equal volume of 2%formalin before analyzing the platelets by flow cytometry.

The results show that thrombin induced a dose dependent increase in the proportion of platelets expressing both phosphatidylserine and P-selectin. Thrombin stimulation also caused a time-dependent increase in mitochondrial calcium levels in platelets. However, thrombin stimulation resulted in a reduction of mitochondrial membrane potential in platelets.

Thrombin stimulation activated caspase-8 but not caspase-3 or 7 in platelets.