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 Ibβ/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.

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 Ibβ 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 (Hα_SSMα^−/−). 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 (Hα_SSMα^−/−ApoE^−/− and Hα_WTMα^−/−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.

We have previously reported that the structural elements of the GP Ib-IX complex required for its localization to GEMs reside in the Ibβ and IX subunits. In order to identify them, we generated a series of cell lines expressing mutant GP Ibβ and GP IX where 1) the cytoplasmic tails (CTs) of either or both GP Ibβ and IX are truncated, and 2) the transmembrane domains (TMDs) of GP Ibβ and GP IX were swapped with the TMD of a non-GEMs associating molecule, human transferrin receptor (Trf). Sucrose density fractionation analysis showed that the removal of either or both of the CTs from GP Ibβ and GP IX does not alter GP Ibα-GEMs association when compared to the wild type. In contrast, swapping of the TMDs of either GP Ibβ or GP IX with that of Trf 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%) when compared to 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 (vWf)-immobilized surface to a much lesser extent than the previously reported disulfide linkage dysfunctional GP Ibα-expressing cells. Thus, our data demonstrates that the bundle of GP Ibβ and GP IX TMDs instead of their individual CTs is the structural element that mediates the β/IX complex localization to the membrane GEMs, which through the α/β disulfide linkage brings GP Ibα into the GEMs.

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 The Authors IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 68(10):823–829, 2016

We have shown that the platelet counter-receptor for integrin Mac-1 (α_Mβ₂, CD11b/CD18) is glycoprotein (GP) Ibα, the largest component of the GP Ib-IX-V complex, better known for its role in mediating platelet attachment to sites of vessel wall injury. The GP Ibα-Mac-1 interaction mediates the firm adhesion of leukocytes on platelet thrombi, enabling their subsequent transmigration into the vessel wall. Our previous studies showed that the I-domain of Mac-1 binds the C-terminal flanking sequences (Phe201–Gly268) of GP Ibα, as demonstrated by the fact that binding is almost completely inhibited by the I domain ligands fibrinogen and heparin, the I-domain monoclonal antibody LPM19c, and the GP Ibα-specific monoclonal antibody AP1. The latter antibody is of interest because it also inhibits the binding of von Willebrand factor and thrombin. We have mapped the AP1 epitope to a 10-amino acid sequence spanning Arg218 to Tyr228. In the current investigation, we constructed a series of cell lines expressing mutants of human GP Ibα, either by replacement of the human sequence with the corresponding dog sequence (dog GP Ibα does not bind human Mac-1) or by targeted mutagenesis, and tested their ability to bind the recombinant Mac-1 I domain. The GP Ibα region Phe201-Asn223 was crucial for Mac-1 binding, with residues Arg218, Asp222 and Asn223 playing vital roles. In addition, a peptide containing the AP1 epitope (Leu214-Val229) bound Mac-1 I-domain specifically and saturably. Peptide binding was blocked by LPM19c and soluble GP Ibα, and by the M2 peptide, which corresponds to the GP Ibα-binding site in the Mac-1 I domain (Phe201-Lys217). Peptide binding was also blocked by an antibody against the M2 sequence. The AP1 peptide inhibited the attachment of GP Ib-IX-V complex-expressing CHO cells to immobilized Mac-1 I domain, and the adhesion of THP-1 cells—a monocytoid line expressing Mac-1—to immobilized GP Ibα. This peptide did not inhibit attachment of the cells VWF surfaces, or thrombin-induced platelet aggregation. In summary, we have defined the GP Ibα sequence Arg214 to Val229 as a critical binding site for Mac-1. Because a peptide corresponding to this region inhibits GP Ibα binding to Mac-1 but blocks neither platelet adhesion to immobilized VWF nor thrombin-induced platelet aggregation, it has potential to guide the development of agents that will specifically inhibit leukocyte-platelet complexes that promote vascular inflammation.