Platelet Mitochondrial Fusion and Function in Vascular Integrity

Platelets are anucleate cells that serve as primary guardians of vascular integrity by initiating hemostasis and tissue repair in response to vascular injury¹. Derived from megakaryocytes, platelets are endowed with approximately 5–10 mitochondria, which are essential for launching such injury responses, and for maintaining their short lifespan of 7–10 days.² Despite being short lived, platelet protective responses are required to maintain mitochondrial integrity and function. In the absence of a nucleus that actively provide transcripts for mitochondria biogenesis, dynamic processes, such as mitochondrial fusion and fission, along with mitochondrial transcripts, are needed to maintain cellular bioenergetics and function. Mitochondrial fusion, conserved from yeast to humans, is a complex active protective process that merges 2 or more mitochondria (within minutes), and exchanges mtDNA, proteins, lipids and other molecules between mitochondria.³ An early study suggested that mitochondrial fusion can occur in vitro in a cell free state⁴. Whether mitochondrial fusion can occur in a platelet that is devoid of a nucleus, and thus whether platelet mitofusins (key to mitochondria fusion) play a role in determining platelet function that can impact severe vascular macro- and micro-thrombotic diseases, such as ischemic stroke and acute respiratory distress syndrome (ARDS)⁵, respectively, remain unexplored.

Mitofusin 1 (MFN1) and 2 (MFN2) are outer mitochondrial membrane-anchored dynamin family GTPases that mediate membrane fusion, a process involving sequential tethering, docking and extension of membrane contacts through homo- and heterooligomeric interactions.⁶ In Mfn2^−/− fibroblasts, mitochondria resemble swollen spheres, while those in Mfn1^−/− are fragmented in appearance.⁷ Global knockouts of either mitofusins cause early embryonic mortality in mice, whereas conditional cell type specific deletions of Mfn1 or Mfn2 have uncovered a protective role of mitochondrial fusion (e.g. neurodegeneration in cerebellum⁸) and additional functions of MFN2 in interactions between mitochondrial membranes and endoplasmic reticulum (ER).⁹ Mutations within Mfn2 gene cause the autosomal dominant disease Charcot-Marie-Tooth type 2A characterized by axonal dysfunction in peripheral motor and sensory neurons.¹⁰ A single nucleotide polymorphic variant rs1474868 within the MFN2 gene locus (alternative transcription start site), previously associated with mitochondrial DNA abundance, correlated with reduced platelet counts and reduced MFN2 RNA expression levels.¹¹ Mechanistic insights into MFN2 regulation of platelet numbers and function, particularly in severe vascular thrombotic diseases, have not been previously investigated.

In the current issue of Circulation Research, Jacob et al ¹² describe the generation and phenotype of conditional platelet/megakaryocyte lineage specific MFN2-knockout (Figure 1). The study demonstrates that MFN2 maintains platelet mitochondrial integrity, and is essential for maintaining platelet lifespan and hemostasis, particularly in the severe vascular thrombotic disease states in ischemic stroke and ARDS. Platelet counts were modestly lower in both male and female PF4-Cre Mfn2^−/− mice with platelet survival being significantly reduced in circulation and ex vivo. The Mfn2^−/− platelets were characterized by mitochondrial membrane depolarization, increased caspase-3 activation and increased platelet surface phosphatidylserine exposure supporting an apoptotic process because of impaired mitochondrial fusion. Importantly, there were greater fractions of immature or younger platelets in PF4Cre Mfn2^−/− mice with higher mean platelet volume as compared with controls. Mfn2^−/−platelets had lesser mitochondrial numbers and demonstrated greater membrane depolarization with reduced platelet basal, and maximal oxygen consumption rate and ATP-linked respiration, likely due to impaired assembly and activity of complex I. Deletion of MFN2 blunted platelet mitochondrial response to thrombin and disrupted calcium flux. However, there was no effect on glycolysis or ATP stores, suggesting that platelet mitochondria, even if depleted in numbers or function, have sufficient reserves to meet energy demands in the absence of major disease stressors.

Fig 1. Impact of platelet/megakaryocyte Mfn2 deletion on platelet biology and its functional consequences.

As demonstrated by Jacob et al in this issue, the impaired mitochondrial fusion in megakaryocyte caused by Mfn2 deletion (PF4-Cre) in mice results into fragmented mitochondria in megakaryocytes and stressed mature platelets leading to depolarized mitochondria, impaired activation, and shorter lifespan. The lack of platelet Mfn2 can cause low platelet counts, reduced cerebral infarcts in cerebral ischemia/reperfusion injury mice model and enhanced vascular permeability and bleeding in response to inflammatory insult. Effect of impaired mitochondrial fusion on other aspects of platelets (top right grey box) remain undefined. OCR – oxygen consumption rate. Mfn2 – Mitofusin 2 gene.

Restriction of platelet hyperreactivity can be protective against severe vascular thrombotic diseases as observed with the use of low dose aspirin in the secondary prevention of myocardial infarction but at the expense of major bleeding events¹³. Notably, arterial occlusion time in PF4-Cre Mfn2−/− mice (Mfn2−/− platelets) (FeCl₃ injury) did not significantly differ when compared with control mice. Yet similar to low dose aspirin and myocardial infarction, smaller cerebral infarcts were observed in the PF4Cre Mfn2−/− mice after cerebral ischemia-reperfusion and bleeding time was prolonged. Moreover, in a well-established model of ARDS (LPS induced lung injury), platelet Mfn2 deletion resulted in a more profound vascular permeability and hemorrhage in the lungs. This could mean that the absence of MFN2 in platelets probably do not disrupt immediate platelet response to vascular injury. The lack of difference in arterial occlusion time may also be due to greater involvement of younger platelets in primary plug formation during clotting, which are relatively resistant to Mfn2 deletion, as shown by authors in other assays. Further detailed studies are needed to determine the exact role of MFN2 in thrombosis. Combined, these mouse platelet Mfn2−/− studies demonstrate that the functional importance of MFN2 is largely dependent on the type, degree and dynamics (acute vs chronic) of the insult.

To corroborate the platelet Mfn2-knockouts with human relevance, subjects with the T/T allele (polymorphism rs1474868 in human Mfn2 gene) showed significantly reduced expression of platelet-specific isoform of Mfn2 RNA and protein, lower mitochondrial membrane potential and higher caspase-3 activity. Furthermore, megakaryocytes (from cultured human cord blood) of the T/T genotype displayed characteristically smaller fragmented mitochondria. These combined data suggest that conditional platelet-specific deletion of MFN2 in mice mimics the phenotype of humans with rs1474868 variant. Although humans with this polymorphic variant did not have increased risks of ARDS or thrombocytopenia, the T/T genotype was significantly associated with 28-day mortality in patients with ARDS. The Mfn2−/− platelets in their mice studies also formed lesser conjugates with neutrophils. Platelet-neutrophil conjugates are important in NETosis which is known to be involved in ARDS pathogenesis. Thus, in addition to thrombosis, such results may also be in part due to other roles played by platelets, including immunoregulation and repair.

These important results from Jacob et al opens the field of platelet and megakaryocyte mitochondrial dynamics to additional questions and studies. The observation of more profound effects of platelet MFN2 mutation or deletion in megakaryocytes than in platelets, especially young platelets, appears counterintuitive. The authors surmised that this could be due to different rates of Mfn2 RNA turnover. Alternatively, megakaryocytes donate mitochondria to proplatelets and thus may require mitochondrial fission, unopposed when MFN2 is lacking. Analyses of megakaryocyte mitochondrial turnover and contribution to proplatelets would definitively address the role played by fusion and fission. Another aspect that requires further investigation are the roles of MFN2 beyond mitochondrial fusion, such as regulation of mitophagy¹⁴ and mitochondrial interactions with ER. MFN2 appears to play a key role in mitochondrial-ER tethering and Ca²⁺ signaling⁹. Recently the role of the unfolded protein response pathway (involving ER stress) in platelet function and turnover has been described¹⁵. Studying the effect of MFN2 on mitochondrial-ER (platelet dense tubular system) connections and their significance in platelet function may inform the role of nuclear transcripts in this process and its contributions to diseases beyond vascular thrombotic diseases. Platelets, beyond their earliest known role in cardiovascular diseases, have also emerged to impact immune responses during infections, tumor development, sepsis, and other pathologies ^{16, 17}. Platelets are believed to work as sentinels for vasculature and immune cells. They can remotely regulate other cell or tissue types by releasing vesicular or naked mitochondria, soluble mediators, and microvesicles. The effects of impaired mitochondrial fusion can impact these functions beyond hemostasis and thrombosis. Such regulators of mitochondrial dynamics may be developed as therapeutic targets to modulate platelet function in diseases involving vascular integrity.

In summary, the present study by Jacob et al provides compelling evidence for a functional role of platelet MFN2 in the regulation of mitochondrial health in megakaryocytes and mature platelets. Deletion of MFN2 in mouse platelets sensitized mice to ARDS-associated loss of vascular integrity and reproduced, at least in part, the defects in mitochondrial fusion and platelet phenotype observed in humans with MFN2 T/T variant. These findings demonstrate the significance of platelet Mfn2 for platelet health and lifespan which is critical for hemostasis and vascular integrity. The precedence is now set to study platelet mitochondrial dynamics in other pathophysiological conditions and functional roles. The intricacies of platelet mitochondrial dynamics in regulating platelet survival and activation, and its impact on diverse pathophysiological phenomena are only beginning to be uncovered.