**7. ABCA1 and Microparticles**

Microparticle (MP) release is a means for cell communication and cell-cell interaction, in addition to direct interaction and release of signaling molecules. MPs are small vesicles released from activated and/or apoptotic cells with substantial heterogeneity in size (50–250 nm). MPs include intracellular components involved in cell signaling and have membrane proteins characteristic of the original parent cell. It has been stablished that MPs are both biomarkers and cell signaling effectors that contribute to maintain and/or initiate cell dysfunction [203]. In a wide variety of thrombotic disorders, platelet and endothelialderived MP levels are increased, with an interesting association between MP levels and pathophysiology, activity or progression of the disease [204]. MPs have procoagulant activity in several diseases including myocardial infarction [205,206], and may play a role in mediating inflammation-induced vascular calcification [207].

ABCA1 has a main role in facilitating outward bending or bulging of the plasma membrane [208]. It is currently known that the C-terminal of ABCA1 separately regulates its cholesterol floppase activity and cholesterol efflux activity [209]. Membrane dynamics are a prerequisite for HDL biogenesis and may also be required to release MPs to the medium [210]. ABCA1 and ApoA-I contribute to MP formation, mediating the production of MPs containing cholesterol. The addition of ApoA-I to human monocyte-derived macrophages markedly increased MP release, while ABCA1 inhibition with probucol and methyl-β cyclodextrin-induced membrane cholesterol depletion markedly reduced MP release and nascent HDL formation. MPs do not contain ApoA-I, but contain the plasma membrane marker flotilin-2, and CD63, an exosome marker. ABCA1 promotes cholesterol efflux, reduces cellular cholesterol accumulation and regulates anti-inflammatory activities in an ApoAI or annexin A1 (ANXA1)-dependent manner. ABCA1 anti-inflammatory activity seems to occur by mediating the efflux of ANXA1, which plays a critical role in anti-inflammatory effects, cholesterol transport, exosome and microparticle secretion and apoptotic cell clearance [211].

Although many studies have shown the importance of *ABCA1* gene variation in serum HDL-C levels, very few studies have reported the effect of gene variants on MP formation and their possible clinical consequences. It is known that ABCA1 participates in infectious and/or thrombotic disorders involving vesiculation [212], and in vitro studies and animal models indicate that ABCA1 also plays an important role in MP formation [21,208]. In Hamster kidney cells and mouse macrophages, ABCA1 was found not only to promote cholesterol efflux towards ApoA-I forming nascent HDL, but it also promoted the formation of ApoA-I-free MPs. This study also demonstrated that the *ABCA1* A937V mutation altered the formation of HDLs and concurrently reduced the release of MPs [208]. Moreover, in an experimental mouse model of cerebral malaria, Combes et al. evaluated the pathogenic implications of MP using *Abca1* deficient mice. Upon infection by *Plasmodium berghei ANKA*, these mice showed complete resistance to cerebral malaria, and MPs purified from infected animals were able to reduce normal plasma clotting time and to significantly enhance tumor necrosis factor release from naive macrophages [213,214]. *ABCA1* promoter variants associated with increased atherosclerotic burden [73] were found to be associated with decreased MP levels and were more prevalent in patients with uncomplicated malaria, suggesting that these polymorphisms have a protective effect against severe malaria in humans [215].

Calcium-dependent cytoskeleton proteolysis causes an eventual transient phospholipid density imbalance between the two plasma membrane leaflets driven by swift phosphatidylserine (PS) egress and lower reverse transport of phosphatidylcholine and sphingomyelin. This imbalance causes local instability of the plasma membrane and MP release upon raft clustering. The calcium-dependent channel TMEM16F plays a crucial role in calcium-induced phospholipid scrambling in the release of MPs exposing PS. TMEM16F mutations cause Scott Syndrome, a rare bleeding disorder characterized by defective platelet PS membrane exposure and MP shedding [216–218]. Because ABCA1 is known to have a role in exofacial PS translocation, Albrecht et al. analyzed the role of this protein in the pathophysiology of a Scott Syndrome patient who carried an *ABCA1* mutation (R1925Q). In vitro expression studies revealed that the 1925Q variant showed impaired trafficking to the plasma membrane, while wild-type ABCA1 overexpression in Scott Syndrome lymphocytes complemented the calcium-dependent PS exposure at the cell surface. Thus, this *ABCA1* mutation contributed to the defective PS translocation phenotype [219].

*Abca1*-deficient mice show alterations in PS exposure and significant reductions in circulating levels of MPs [212,220]. Moreover, silencing of *ABCA1* in human umbilical cord endothelial cell (HUVEC) cultures significantly reduced the release of MPs when subjected to frictional forces [221]. In this study, atheroprone shear stress conditions stimulated the formation and release of endothelial-derived MPs and hemodynamic forces were identified as an important determinant of MP plasma levels in healthy subjects. Sustained exposure to atheroprone low shear stress conditions increased both endothelial apoptosis and the release of MPs in the medium, when compared with physiological high shear stress conditions. Moreover, downregulation of ABCA1 expression by endogenously released nitric oxide (NO) contributed to limit the release of endothelial-derived MPs in HUVECs exposed to high shear stress [221].
