Page 201 - Haematologica May 2022

Letters to the Editor
Mitochondrial ATP generation in stimulated platelets is essential for granule secretion but dis- pensable for aggregation and procoagulant activity
Platelets, although anucleate, contain well-coupled and functional mitochondria1,2 and are capable of mitochondr- ial oxidative phosphorylation (OXPHOS).3 Simultaneous inhibition of glycolysis and OXPHOS abolishes agonist- induced aggregation and granule secretion.3 Recent stud- ies have shown that there is considerable plasticity in energy metabolism of platelets.1 Yet, we demonstrated a switch to aerobic glycolysis from OXPHOS in stimulated platelets.2 Small-molecules that divert the flux from aero- bic glycolysis towards OXPHOS prevent platelet activa- tion.2 In addition, several studies including our own have established that inhibitors of OXPHOS alone do not compromise platelet aggregation.1,2,4 These observations led us to question whether mitochondrial ATP generation is dispensable for platelet function.
We undertook a comprehensive study of the effect of mitochondrial inhibitors (antimycin, oligomycin) and an uncoupler (CCCP; carbonyl cyanide 3-chlorophenylhy- drazone) on platelet activity. We also established the probable mechanistic basis underlying their effects on platelet function. The underpinning modes of action for these molecules differ considerably. Antimycin acts by inhibiting complex III of the electron transport chain, thereby preventing both oxidation of energy substrates (Online Supplementary Figure S1A) as well as ATP synthe- sis. Oligomycin targets FoF1 ATPase (complex V) leading to a block in ATP generation although oxidation of fuels continues to the extent allowed by proton leak (Online Supplementary Figure S1A). CCCP uncouples mitochondr- ial respiration and OXPHOS by dissipating the proton gradient (Online Supplementary Figure S1B, C) thus permit- ting maximal oxidation of fuels (Online Supplementary Figure S1A) albeit compromising formation of ATP. Hence, it is reasonable to expect that the effects of these molecules will not be identical. Since only oligomycin specifically and uniquely inhibits mitochondrial ATP gen- eration, our inferences about the role of mitochondrial ATP in platelet function are based primarily on observa- tions with oligomycin.
Platelet aggregation induced by either thrombin (Figure 1A, B) or collagen (Online Supplementary Figure S1D, G) was profoundly impaired in the presence of CCCP while the presence of antimycin or oligomycin did not appear to have a significant effect, which was consistent with earlier reports.1,2,4 Furthermore, we found similar results with aspirinated platelets (Online Supplementary Figure S1E, I), suggesting that the effect of CCCP is likely inde- pendent of thromboxane A2 generation. Aggregation is mediated by fibrinogen which binds with high affinity to platelet surface integrin aIIbβ3 in an active conformation. In keeping with our observation on platelet aggregation, CCCP brought about a significant drop in thrombin- induced aIIbβ3 integrin activation while antimycin and oligomycin had no such influence (Figure 1D, G).
A fraction of stimulated platelets, characterized by expression of phosphatidylserine on the outer leaflet of the plasma membrane, provide a procoagulant surface for generation of a fibrin clot at the site of vessel injury.5 The extent of phosphatidylserine exposure (Figure 1E, H) upon thrombin challenge was significantly retarded by CCCP but unaffected by either antimycin or oligomycin. These observations led us to deduce that mitochondrial ATP generation is dispensable for platelet integrin activa- tion, aggregation and procoagulant activity. Nevertheless,
disruption of mitochondrial membrane potential by CCCP impeded these processes.
Platelet responses to agonist stimulation are energy- demanding.3,6 However, the energy requirements differ considerably, with shape change being the least energy- requiring process, followed by aggregation, dense/a granule release and acid hydrolase secretion, in increas- ing order.3,7 We reasoned that inhibition of mitochondrial ATP generation could affect greater energy-intensive processes, such as exocytosis of granule contents. Release of adenine nucleotides and surface expression of P-selectin are markers of dense and a granule secretion, respectively. ATP release from platelets stimulated with either thrombin (Figure 1A, C) or collagen (Online Supplementary Figure S1D, H), as well as P-selectin exter- nalization induced by thrombin (Figure 1F, I) were signif- icantly compromised in the presence of oligomycin and CCCP. Antimycin had no significant effect on either dense or a granule secretion (Figure 1A, C, F, I; Online Supplementary Figure S1D, H), which was indicative of greater compensation by glycolytic ATP generated in the presence of antimycin (Figure 3C). Glycolysis rate is enhanced in antimycin-treated platelets in order to com- pensate for significant inhibition in oxidation of fuels.4 Contrasting this, either residual oxidation allowed in mitochondria of oligomycin-treated platelets, or maximal oxidation provoked by CCCP, could restrict the compen- satory increase in glycolysis and ensuing ATP availability, thus failing to sustain platelet functions with higher ener- gy cost, such as granule secretion.
Expression of P-selectin on the surface of activated platelets triggers its interaction with the counter-ligand P- selectin glycoprotein ligand-1 (PSGL-1) present on leuko- cytes.8 Hence, we measured platelet-leukocyte aggregates in whole blood challenged with thrombin receptor-acti- vating peptide (TRAP)-6 by flow cytometry. Both oligomycin and CCCP inhibited TRAP-stimulated platelet-neutrophil interaction (Online Supplementary Figure S2A-E, K), which could be attributable to impaired P-selectin externalization in these platelets. However, only CCCP was able to prevent TRAP-induced platelet- monocyte interaction (Online Supplementary Figure S2F-J, L). Given that thrombin-induced phosphatidylserine exposure on platelets was unaffected by oligomycin, a phosphotidylserine-Tim4 interaction9 could be mediating the platelet-monocyte interaction in oligomycin-treated platelets despite compromised P-selectin-facilitated inter- actions. This hypothesis was supported by our observa- tion that oligomycin inhibited platelet-monocyte interac- tions (Online Supplementary Figure S1L) upon stimulation with ADP which, unlike TRAP, does not induce phos- phatidylserine exposure.
Several prothrombotic factors are released from platelet granules upon stimulation, including ADP and fibrinogen.10 ADP, in particular, plays a pivotal role in recruiting platelets to the ‘shell’ region of a growing thrombus.11 As we found that mitochondrial ATP sus- tained granule release, we hypothesized that it could be essential for the process of thrombosis. We studied platelet thrombus formation on immobilized collagen under arterial shear (1500 s-1). Washed human platelets were allowed to perfuse over the collagen-coated surface for 5 min. Total surface area covered by platelet thrombi was significantly diminished in the presence of oligomycin (Figure 2A, E) and CCCP (Figure 2A, F) but unaffected by antimycin (Figure 2A, D), which could be attributable to impaired ADP release consequent to oligomycin and CCCP treatment. This hypothesis was supported by the observation that, neither oligomycin
haematologica | 2022; 107(5)
1209