Genesis and Characterization of Human eNOS-Based Platelet Subpopulations. Implications for Haemostasis and Thrombosis

Background: Myocardial infarction (MI) and ischemic stroke are responsible for nearly half of all cardiovascular deaths in Canada. Platelets play a critical role in MI and ischemic stroke by forming thrombi that occlude coronary and cerebral arteries. In spite of abundant pharmacological agents (acetylsalicylic acid, clopidogrel, prasugrel, ticagrelor, abciximab, eptibifatide) used to prevent platelet thrombus formation, the incidence of arterial thrombosis remains high. One of the reasons might be incomplete understanding of platelet biology and their function. Anucleate platelets derived from bone marrow megakaryocytes (MKs) have long been considered simple cell fragments that act as vascular system “band-aids”. Hence, investigations into biochemically distinct subpopulations with differential functional roles in haemostasis and thrombosis have been limited. In 1990 Radomski et al. proposed that platelets have endothelial nitric oxide synthase (eNOS)-signalling pathway present and when activated generate nitric oxide (NO). This endogenous negative-feedback mechanism provides a balance for platelet pro-aggregatory properties by inhibiting their adhesion and aggregation. However, in recent years existence of this pathway within platelets has been questioned. To explain some discrepancies in findings, we propose that differences in platelet eNOS levels might account for a part of the divergent results. Therefore, the over-arching goal of this study was to explore if two platelet subpopulations exist based on the presence or absence of eNOS-signalling, and thereby represent functionally distinct platelet subpopulations with differential roles in adhesion and aggregation. Furthermore, inflammation that contributes to atherosclerosis and thrombosis may modulate the bone marrow microenvironment and promote formation of MKs that generate platelets with enhanced reactivity, therefore increasing risk of acute ischemic events. In this thesis, I have addressed three hypotheses: 1) Subpopulations of eNOS-negative (eNOSneg) and eNOS-positive (eNOSpos) platelets exist, and due to their biochemical differences perform differential roles in haemostasis and thrombosis. 2) Subpopulations of eNOS-negative (eNOSneg) and eNOS-positive (eNOSpos) megakaryocytes/blasts exist and give rise to their respective eNOS-based platelet subpopulations. 3) Pro- and anti-inflammatory cytokines interferon-γ (IFN-γ) and interleukin-10 (IL-10) counter-regulate formation of eNOS-based platelet subpopulations via their effects on megakaryocyte/blast eNOS expression, and IFN-γ promotes differentiation of megakaryocytes/blasts lacking eNOS that give rise to more reactive eNOSneg platelets. Methods: Prostacyclin-washed platelets were isolated from healthy humans. Human megakaryoblastic cell line (Meg-01) was used as a surrogate of human bone marrow MKs and to generate Meg-01-derived platelets. In addition, eNOS-GFP mouse platelets and bone marrow samples were used for experiments. Production of NO was assessed by fluorescent NO indicator - DAF-FM diacetate. Detection of eNOS was performed by flow cytometry, western blot, and confocal microscopy. Results: Our results show existence of mouse and human platelet subpopulations based on the presence (eNOSpos) or absence (eNOSneg) of functional intracellular eNOS and NO generation. In contrast to majority of human platelets that have eNOS and generate NO, most of mouse platelets lack eNOS and generate no or low amounts of NO. Our study also demonstrates that less abundant eNOSneg platelets, due to the absence of NO generation, are more reactive and initiate aggregate formation, while eNOSpos platelets limit aggregate size by generating NO. In addition, down-regulated sGC-PKG-signalling within eNOSneg platelets facilitates their refractoriness to endothelial-derived NO and increases activation of platelet fibrinogen receptor - integrin αIIbβ3. Consequently, eNOSneg platelets more readily bind to collagen compared to more abundant eNOSpos platelets that attach to collagen at later time points and form the bulk of an aggregate, ultimately limiting its size by NO generation. Similar to human platelets, the majority of Meg-01 cells are eNOSpos and NO-producers. The majority of mouse platelets and mouse MKs are eNOSneg. Pro-inflammatory IFN-γ in a concentration-dependent manner decreases total amount of MK eNOS, which reduces number of eNOSpos Meg-01 cells and Meg-01-derived eNOSpos platelets. Anti-inflammatory IL-10 in a concentration-dependent manner partially restores MK total eNOS levels and number of Meg-01-derived eNOSpos platelets. Conclusions: These findings demonstrate novel characteristics and complexity of platelets and their regulation of adhesion and aggregation. The identification of eNOS-based platelet subpopulations has potentially important consequences to human disease as impaired platelet NO-signalling has been identified in patients with coronary artery disease. Our study also provides potential insight on how chronic inflammatory states can contribute to thrombosis by promoting formation of eNOSneg MKs that generate more reactive eNOSneg platelets.