Effect of reaction condition on microstructure and properties of (NiCuZn)Fe2O4 nanoparticles synthesized via co-precipitation with ultrasonic irradiation.

Nano-spinel ferrites synthesized via chemical co-precipitation method are small in size and have serious agglomeration phenomenon, which makes separation difficult in the subsequent process. Ni0.4Cu0.2Zn0.4Fe2O4 ferrites nanoparticles were synthesized via co-precipitation assisted with ultrasonic irradiation produced by ultrasonic cleaner with 20 kHz frequency using chlorinated salts and KOH as initial materials. The effects of ultrasonic power (0, 40 W, 60 W, 80 W) and reaction temperature on the microstructure and magnetic properties of ferrite nanoparticles were investigated. The structure analyses via XRD revealed the successful formation of pure (NiCuZn)Fe2O4 ferrites nanospinel without any impurity. The crystallites sizes were less than 40 nm and the lattice constant was near 8.39 Å. The TEM showed ferrite particle polygonal. M-H analyses performed the saturation magnetization and coercivity of ferrite nanoparticles obtained at the reaction temperature of 25℃ were higher than at 50℃ with same power. The samples exhibited the highest values of Ms 55.67 emu/g at 25℃ and 47.77 emu/g at 50℃ for 60 W and the lowest values of Hc 71.23 Oe at 25℃ for 40 W and 52.85 Oe at 50℃ for 60 W. The squareness ratio (SQR) were found to be lower than 0.5, which revealed the single magnetic domain nature (NiCuZn)Fe2O4 nanoparticles. All the outcomes show the ultrasonic irradiation has positive effects on improving the microstructure and increasing magnetic properties.

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.

Inhibition of Hb Binding to GP1bα Abrogates Hb-Mediated Thrombus Formation on Immobilized VWF and Collagen under Physiological Shear Stress.

BACKGROUND: Reports including our own describe that intravascular hemolysis increases the risk of thrombosis in hemolytic disorders. Our recent study shows that plasma Hb concentrations correlate directly with platelet activation in patients with paroxysmal nocturnal hemoglobinuria (PNH). The binding of Hb to glycoprotein1bα (GP1bα) increases platelet activation. A peptide AA1-50, designed from N-terminal amino acid sequence of GP1bα significantly inhibits the Hb binding to GP1bα as well as Hb-induced platelet activation. This study further examined if the Hb-mediated platelet activation plays any significant role in thrombus formation on subendothelium matrix under physiological flow shear stresses and the inhibition of Hb-platelet interaction can abrogate the above effects of Hb. METHODS AND RESULTS: Study performed thrombus formation assay in vitro by perfusing whole blood over immobilized VWF or collagen type I in presence of Hb under shear stresses simulating arterial or venous flow. The Hb concentrations ranging from 5 to 10 µM, commonly observed level in plasma of the hemolytic patients including PNH, dose-dependently increased thrombus formation on immobilized VWF under higher shear stress of 25 dyne/cm2, but not at 5 dyne/cm2. The above Hb concentrations also increased thrombus formation on immobilized collagen under both shear stresses of 5 and 25 dyne/cm2. The peptide AA1-50 abrogated invariably the above effects of Hb on thrombus formation. CONCLUSIONS AND SIGNIFICANCE: This study therefore indicates that the Hb-induced platelet activation plays a crucial role in thrombus formation on immobilized VWF or collagen under physiological flow shear stresses. Thus suggesting a probable role of this mechanism in facilitating thrombosis under hemolytic conditions.

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.

Platelet glycoprotein Ib beta/IX mediates glycoprotein Ib alpha localization to membrane lipid domain critical for von Willebrand factor interaction at high shear.

The localization of the platelet glycoprotein GP Ib-IX complex (GP Ibα, GP Ibß, and GP IX) to membrane lipid domain, also known as glycosphingolipid-enriched membranes (GEMs or raft) lipid domain, is essential for the GP Ib-IX complex mediated platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation. To date, the mechanism for the complex association with the GEMs remains unclear. Although the palmitate modifications of GP Ibß and GP IX were thought to be critical for the complex presence in the GEMs, we found that the removal of the putative palmitoylation sites of GP Ibß and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ibα disulfide linkage with GP Ibß markedly decreased the amount of the GEM-associated GP Ibα without altering the GEM association of GP Ibß and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ibα interaction with vWf at high shear instead of in static condition or under low shear stress. Thus, for the first time, we demonstrated that GP Ibß/GP IX mediates the disulfide-linked GP Ibα localization to the GEMs, which is critical for vWf interaction at high shear.

Protein phosphatase 2A negatively regulates integrin alpha(IIb)beta(3) signaling.

Integrin alpha(IIb)beta(3) activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin alpha(IIb)beta(3) signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin alpha(IIb)beta(3) in resting platelets and in human embryonal kidney 293 cells expressing alpha(IIb)beta(3). The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin alpha(IIb) is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to alpha(IIb)beta(3) during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Ac(alpha) in 293 cells decreased alpha(IIb)beta(3)-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Ac(alpha) expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated alpha(IIb)beta(3) adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac (alpha)-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2A(calpha) expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac (alpha) can negatively regulate integrin alpha(IIb)beta(3) signaling by suppressing the ERK1/2 signaling pathway.

Recombinant CUB-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions.

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.

Gain of von Willebrand factor-binding function by mutagenesis of a species-conserved residue within the leucine-rich repeat region of platelet glycoprotein Ibalpha.

Glycoprotein (GP) Ibalpha, a member of the leucine-rich repeat (LRR) protein family, mediates platelet adhesion to immobilized von Willebrand factor (VWF). We investigated the role in VWF binding of charged residues in the LRR region of GP Ibalpha that are conserved in human, canine, and murine proteins. Substitution of His86 with either Ala or Glu resulted in a gain of VWF-binding function as judged by increased VWF binding in the presence of the modulators ristocetin and botrocetin and by enhanced adhesion of Chinese hamster ovary (CHO) cells expressing the mutant GP Ibalpha to immobilized VWF under conditions of flow. This is the first report of a gain-of-function phenotype resulting from mutations in the LRR region of GP Ibalpha. Because His86 is 2 nm away from the region of GP Ibalpha with the largest surface of contact with VWF, the data suggest that the LRRs regulate GP Ibalpha affinity for VWF allosterically.

The alpha1 helix-beta13 strand spanning Leu214 to Val229 of platelet glycoprotein Ibalpha facilitates the interaction with von Willebrand factor: evidence from characterization of the epitope of monoclonal antibody AP1.

The glycoprotein Ib-IX-V (GP Ib-IX-V) complex mediates platelet binding to von Willebrand factor (VWF) through its largest polypeptide, GP Ibalpha. Of the many GP Ibalpha monoclonal antibodies described, AP1 is of particular interest because it blocks static VWF binding induced by 2 modulators, ristocetin and botrocetin, and platelet adhesion to VWF surfaces under flow. We mapped the AP1 binding site to a region encompassing Arg218 to Tyr228, comprising the alpha1 helix and beta13 strand defined by the GP Ibalpha crystal structure. AP1 binding absolutely required Arg218, Asp222, and Glu225. We evaluated the ability of cells expressing mutants of this region to bind VWF under static conditions in the presence of modulators, and to attach to and roll on a VWF matrix under flow. These data indicate that 2 regions within the sequence Arg218 to Tyr228 have important roles in VWF binding: the alpha1 helix has a regulatory role and the beta turn and beta13 strand bind VWF directly. Despite this, the only effect of a synthetic peptide corresponding to Leu214 to Val229 was to slightly increase the rolling velocity of GP Ibalpha-expressing Chinese hamster ovary (CHO) cells on VWF. This region thus appears to be more important for maintaining the regional conformation of GP Ibalpha, thereby facilitating the interaction with VWF.