Mitochondrial Uncoupling Prevents Procoagulant Platelet Formation Resulting in Decreased Thrombus Stability
Authors: Kholmukhamedov Andaleb , Sakurai Yumiko , Hardy Elaissa L. , Lam Wilbur A , Jobe Shawn M. 
Published: October 2019

Abstract
Secondary hemostasis is the cascade of coagulation reactions that has two pathways, both of which cultivate in the formation of insoluble fibrin to stabilize the primary platelet clot. Although some biochemical reactions of secondary hemostasis can occur in solution their amplification, to the extent capable of supporting active hemostasis, occurs only in the presence of the membranes provided by cells and particles of blood and vasculature. Procoagulant platelets, a subpopulation of activated platelets, are believed to be the key contributor of the phosphatidylserine surface for the membrane dependent reactions. This subpopulation of activated platelets is generated upon the onset of mitochondrial permeability transition (MPT) triggered by mitochondrial calcium overload or intramitochondrial reactive oxygen species (ROS) formation. MPT leads to phosphatidyl serine (PSer) exposure by yet unknown mechanisms. Prevention of MPT however, completely disrupts transition of platelets to a procoagulant phenotype. It has been debated for more than a decade whether procoagulant platelets get formed during thrombogenesis in vivo. Numerous recent studies suggest that higher procoagulant platelet formation potential correlates with pathologic thromboses. However, the role of procoagulant platelet phenotype in hemostasis has been poorly investigated. Here we demonstrate that procoagulant platelets are essential in secondary hemostasis.
Washed human platelets were stimulated with thrombin (THR), convulxin (CVX) or both. PSer exposure, granule release and integrin activation were measured by annexin V (AnnV), P-selectin and PAC1, respectively. Aggregation was done by standard aggregometry. Thrombin generation was measured using tissue factor initiated thrombinoscopy. Hemostasis experiments were performed as described previously (Sakurai Y, Hardy ET, Ahn B, et al. A microengineered vascularized bleeding model that integrates the principal components of hemostasis. Nature communications. 2018;9(1):509). Isolated platelets were treated with vehicle (DMSO) or DNP, washed, resuspended in plasma, mixed with red and white blood cell mass (so called "red layer") at a ratio of 1 to 1. Whole blood then was perfused over the bleeding channels at a shear rate of 500 s-1.
Mitochondrial transmembrane potential is the primary driving force for mitochondrial calcium uptake, as well as the generation of ROS upon platelet stimulation. Mitochondrial uncouplers dissipate the proton gradient across the inner mitochondrial membrane leading to mitochondrial depolarization. Dissipation of membrane potential, by uncouplers carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone and 2,4-dinitrophenol (FCCP and DNP, respectively), decreased formation of procoagulant platelets in a dose dependent manner as measured by AnnV binding. DNP decreased CVX stimulated THR generation to the baseline. Although mitochondrial uncoupling alleviated the spatio-temporal transition of proaggregatory platelets to procoagulant phenotype, it did not affect THR nor CVX stimulated platelet aggregation. Mitochondrial uncoupling did not affect granule release, whereas it did increase platelet accretion by ~10-fold. On hemostasis model there was no statistical difference between experimental groups in platelet accumulation as measured by CD41 fluorescence, whereas fibrin (59D8 fluorescence) in DMSO group was ~2-fold higher than in DNP. More striking finding is that bleeding time after DMSO treatment averaged 10.5 minutes, whereas hemostasis in DNP treatment group was never achieved.
Together, these results identify a key role of procoagulant platelets in the formation of a stable clot; hence procoagulant platelets are essential in secondary hemostasis. Manipulations targeted to affect percentage of procoagulant subpopulation may provide a novel pharmacologic strategy for the treatment of procoagulant related hematopathologies.

Disclosures
Jobe: CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.