MicroRNAs 10a and 10b Regulate the Expression of Human Platelet Glycoprotein Ibα for Normal Megakaryopoiesis.

MicroRNAs are a class of small non-coding RNAs that bind to the three prime untranslated region (3'-UTR) of target mRNAs. They cause a cleavage or an inhibition of the translation of target mRNAs, thus regulating gene expression. Here, we employed three prediction tools to search for potential miRNA target sites in the 3'-UTR of the human platelet glycoprotein (GP) 1BA gene. A luciferase reporter assay shows that miR-10a and -10b sites are functional. When miR-10a or -10b mimics were transfected into the GP Ibß/GP IX-expressing cells, along with a DNA construct harboring both the coding and 3'-UTR sequences of the human GP1BA gene, we found that they inhibit the transient expression of GP Ibα on the cell surface. When the miR-10a or -10b mimics were introduced into murine progenitor cells, upon megakaryocyte differentiation, we found that GP Ibα mRNA expression was markedly reduced, suggesting that a miRNA-induced mRNA degradation is at work. Thus, our study identifies GP Ibα as a novel target of miR-10a and -10b, suggesting that a drastic reduction in the levels of miR-10a and -10b in the late stage of megakaryopoiesis is required to allow the expression of human GP Ibα and the formation of the GP Ib-IX-V complex.

Membrane skeleton orchestrates the platelet glycoprotein (GP) Ib-IX complex clustering and signaling.

Platelet glycoprotein Ib-IX complex is affixed to the membrane skeleton through interaction with actin binding protein 280 (ABP-280). We find that removal of the ABP-280 binding sites in GP Ibα cytoplasmic tail has little impact on the complex clustering induced by antibody crosslinking. However, large truncation of the GP Ibα cytoplasmic tail allows the formation of larger patches of the complex, suggesting that an ABP-280 independent force may exist. Besides, we observe that the signaling upon GP Ib-IX clustering is elicited in both membrane lipid domain dependent and independent manner, a choice that relies on how the membrane skeleton interacts with the complex. Our findings suggest a more complex mechanism for how the membrane skeleton regulates the GP Ib-IX function. © 2016 IUBMB Life, 68(10):823-829, 2016.

Defective Association of the Platelet Glycoprotein Ib-IX Complex with the Glycosphingolipid-Enriched Membrane Domain Inhibits Murine Thrombus and Atheroma Formation.

Localization of the platelet glycoprotein Ib-IX complex to the membrane lipid domain is essential for platelet adhesion to von Willebrand factor and subsequent platelet activation in vitro. Yet, the in vivo importance of this localization has never been addressed. We recently found that the disulfide linkage between Ibα and Ibß is critical for the association of Ibα with the glycosphingolipid-enriched membrane domain; in this study, we established a transgenic mouse model expressing this mutant human Ibα that is also devoid of endogenous Ibα (HαSSMα(-/-)). Characterization of this model demonstrated a similar dissociation of Ibα from murine platelet glycosphingolipid-enriched membrane to that expressed in Chinese hamster ovary cells, which correlates well with the impaired adhesion of the transgenic platelets to von Willebrand factor ex vivo and in vivo. Furthermore, we bred our transgenic mice into an atherosclerosis-prone background (HαSSMα(-/-)ApoE(-/-) and HαWTMα(-/-)ApoE(-/-)). We observed that atheroma formation was significantly inhibited in mutant mice where fewer platelet-bound CD11c(+) leukocytes were circulating (CD45(+)/CD11c(+)/CD41(+)) and residing in atherosclerotic lesions (CD45(+)/CD11c(+)), suggesting that platelet-mediated adhesion and infiltration of CD11c(+) leukocytes may be one of the mechanisms. To our knowledge, these observations provide the first in vivo evidence showing that the membrane GEM is physiologically and pathophysiologically critical in the function of the glycoprotein Ib-IX complex.

The Transmembrane Domains of ß and IX Subunits Mediate the Localization of the Platelet Glycoprotein Ib-IX Complex to the Glycosphingolipid-enriched Membrane Domain.

We have previously reported that the structural elements of the GP Ib-IX complex required for its localization to glycosphingolipid-enriched membranes (GEMs) reside in the Ibß and IX subunits. To identify them, we generated a series of cell lines expressing mutant GP Ibß and GP IX where 1) the cytoplasmic tails (CTs) of either or both GP Ibß and IX are truncated, and 2) the transmembrane domains (TMDs) of GP Ibß and GP IX were swapped with the TMD of a non-GEMs associating molecule, human transferrin receptor. Sucrose density fractionation analysis showed that the removal of either or both of the CTs from GP Ibß and GP IX does not alter GP Ibα-GEMs association when compared with the wild type. In contrast, swapping of the TMDs of either GP Ibß or GP IX with that of transferrin receptor results in a significant loss (∼ 50%) of GP Ibα from the low density GEMs fractions, with the largest effect seen in the dual TMD-replaced cells (> 80% loss) when compared with the wild type cells (100% of GP Ibα present in the GEMs fractions). Under high shear flow, the TMD-swapped cells adhere poorly to a von Willebrand factor-immobilized surface to a much lesser extent than the previously reported disulfide linkage dysfunctional GP Ibα-expressing cells. Thus, our data demonstrate that the bundle of GP Ibß and GP IX TMDs instead of their individual CTs is the structural element that mediates the ß/IX complex localization to the membrane GEMs, which through the α/ß disulfide linkage brings GP Ibα into the GEMs.

Programmed cell death protein 5 interacts with the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) to regulate ß-tubulin folding.

Programmed cell death protein 5 (PDCD5) has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for binding partners of phosducin-like protein, a co-chaperone for the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT), revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and ß-tubulin, a key CCT-folding substrate, and specifically inhibited ß-tubulin folding. Cryo-electron microscopy studies of the PDCD5·CCT complex suggested a possible mechanism of inhibition of ß-tubulin folding. PDCD5 bound the apical domain of the CCTß subunit, projecting above the folding cavity without entering it. Like PDCD5, ß-tubulin also interacts with the CCTß apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and ß-tubulin suggest that PDCD5 sterically interferes with ß-tubulin binding to the CCTß apical domain and inhibits ß-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of ß-tubulin folding.