Integrin-Dependent Transient Density Increase in Detergent-Resistant Membrane Rafts in Platelets Activated by Thrombin.

Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction via sucrose density gradient centrifugation. The platelet detergent-resistant membrane shifted to a higher density on the sucrose density gradient upon thrombin stimulation. The shift peaked at 1 min and returned to the control level at 60 min. During this time, platelets underwent clot retraction and spreading on a fibronectin-coated glass strip. Thrombin induced the transient tyrosine phosphorylation of several proteins in the detergent-resistant membrane raft fraction and the transient translocation of fibrin and myosin to the detergent-resistant membrane raft fraction. The level of phosphatidylserine (36:1) was increased and the level of phosphatidylserine (38:4) was decreased in the detergent-resistant membrane raft fraction via the thrombin stimulation. Furthermore, Glanzmann's thrombasthenia integrin αIIbß3-deficient platelets underwent no detergent-resistant membrane shift to a higher density upon thrombin stimulation. As the phosphorylation of the myosin regulatory light chain on Ser19 was at a high level in Glanzmann's thrombasthenia resting platelets, thrombin caused no further phosphorylation of the myosin regulatory light chain on Ser19 or clot retraction. These observations suggest that the fibrin-integrin αIIbß3-myosin axis and compositional change of phosphatidylserine species may be required for the platelet detergent-resistant membrane shift to a higher density upon stimulation with thrombin.

Phosphacan acts as a repulsive cue in murine and rat cerebellar granule cells in a TAG-1/GD3 rafts-dependent manner.

Phosphacan, a chondroitin sulfate proteoglycan, is a repulsive cue of cerebellar granule cells. This study aims to explore the molecular mechanism. The glycosylphosphatidylinositol-anchored neural adhesion molecule TAG-1 is a binding partner of phosphacan, suggesting that the repulsive effect of phosphacan is possibly because of its interaction with TAG-1. The repulsive effect was greatly reduced on primary cerebellar granule cells of TAG-1-deficient mice. Surface plasmon resonance analysis confirmed the direct interaction of TAG-1 with chondroitin sulfate C. On postnatal days 1, 4, 7, 11, 15, and 20 and in adulthood, phosphacan was present in the molecular layer and internal granular layer, but not in the external granular layer. In contrast, transient TAG-1 expression was observed exclusively within the premigratory zone of the external granular layer on postnatal days 1, 4, 7, and 11. Boyden chamber cell migration assay demonstrated that phosphacan exerted its repulsive effect on the spontaneous and brain-derived neurotrophic factor (BDNF)-induced migration of cerebellar granule cells. The BDNF-induced migration was inhibited by MK-2206, an Akt inhibitor. The pre-treatment with a raft-disrupting agent, methyl-ß-cyclodextrin, also inhibited the BDNF-induced migration, suggesting that lipid rafts are involved in the migration of cerebellar granule cells. In primary cerebellar granule cells obtained on postnatal day 7 and cultured for 7 days, the ganglioside GD3 and TAG-1 preferentially localized in the cell body, whereas the ganglioside GD1b and NB-3 localized in not only the cell body but also neurites. Pre-treatment with the anti-GD3 antibody R24, but not the anti-GD1b antibody GGR12, inhibited the spontaneous and BDNF-induced migration, and attenuated BDNF-induced Akt activation. These findings suggest that phosphacan is responsible for the repulsion of TAG-1-expressing cerebellar granule cells via GD3 rafts to attenuate BDNF-induced migration signaling.

Amyloid precursor protein 770 is specifically expressed and released from platelets.

Platelets not only play an essential role in hemostasis after vascular injury but are also involved in the development of coronary artery disease (CAD) and cerebrovascular lesions. Patients with CAD and cerebral ischemia are recommended to undergo antiplatelet therapy, but they have an increased incidence of major bleeding complications. Both assessment of the platelet activation status and response to antiplatelet therapy in each patient are highly desired. ß-Amyloid precursor protein (APP) 770 is expressed in vascular endothelial cells, and its extracellular region, a soluble form of APP770 (sAPP770, also called nexin-2), is proteolytically cleaved for shedding. Abundant sAPP770 is also released from activated platelets. In this study, we used peripheral blood samples from patients with CAD and control subjects and evaluated sAPP770 as a specific biomarker for platelet activation. First, the plasma levels of sAPP770 correlated well with those of the soluble form CD40 ligand (CD40L), an established biomarker for platelet activation. Additionally, flow cytometry analysis using peripheral blood cells showed that CD40L expression is up-regulated in activated T cells, whereas APP770 expression is negligible in all blood cell types except platelets. Following stimulation with collagen or ADP, aggregating platelets immediately released sAPP770. Finally, patients with dual antiplatelet therapy showed significantly lower levels of plasma sAPP770 than those with no therapy. Taken together, our data show that plasma sAPP770 could be a promising biomarker for platelet activation.

Human monoclonal antibodies neutralizing influenza virus A/H1N1pdm09 and seasonal A/H1N1 strains - Distinct Ig gene repertoires with a similar action mechanism.

Influenza virus causes acute respiratory infection in humans, and is a major public health concern globally. Antibodies play a central role in host protection against influenza virus. We isolated human monoclonal antibodies (hMAb) 206-2-4 and 201-6-8 by a human hybridoma protocol that neutralized various but distinct influenza virus (IFV) A/H1N1 strains, including 2009 pandemic strains. The half-inhibitory concentration of 206-2-4 and 201-6-8 against A/H1N1pdm09 strains was 2-100ng/mL and 5-20µg/mL, respectively. Prophylactic and therapeutic potencies of 206-2-4 were demonstrated in a mouse model of IFV infection at i.p. dosages of 0.25 and 2.5mg/kg, respectively, suggesting that 206-2-4 is one of the most potent hnMAbs against IFV reported thus far. The Ig genes of 206-2-4 and 201-6-8 were originated from distinct germ line repertoires, and accompanied by 63 and 23 somatic hypermutations, respectively. The hemagglutination inhibitory activity indicated that the mechanism of neutralization was to interfere the virus-receptor interaction. The binding epitope of the two antibodies was mapped to hemagglutinin 1 (HA1) amino acid residues 111-120. Additional interaction between the antibody and the HA1 globular head was necessary for neutralization. Such hnMAbs bearing distinct binding epitope have been rarely reported. The potency is likely due to the coverage of a wide surface area of HA protein by these hnMABs. IFV is a highly variable. Our knowledge on the mechanisms by which these cross-reactive hnMAbs function should help design a novel immunogen for the development of a vaccine effective against broader spectrum of IFV strains.

SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3ß at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-ß-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gß and 4.0% of PI3Kß, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbß3-myosin axis in platelet sphingomyelin-rich membrane rafts.

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-ß-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbß3-myosin complex is formed as a primary axis to promote platelet contraction.

Soluble amyloid precursor protein 770 is a novel biomarker candidate for acute coronary syndrome.

Most Alzheimer disease patients show deposition of amyloid ß (Aß) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid ß precursor protein (APP) 770, a different APP isoform from neuronal APP695, and that they produce amyloid ß peptide. We analyzed the glycosylation of APP770 and found that O-glycosylated sAPP770 is preferentially processed by proteases for Aß production. Because the soluble APP cleavage product sAPP is considered to be a possible marker for Alzheimer disease diagnosis, sAPP, consisting of a mixture of these variants, has been widely measured. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable us to discriminate between endothelial and neurological dysfunctions. Our recent findings, showing that the level of plasma sAPP770 is significantly higher in patients with acute coronary syndrome, raise the possibility that sAPP770 could be an indicator of endothelial dysfunction. In this review, we first describe the expression, glycosylation, and processing of APP770, and then discuss sAPP770 as a novel biomarker candidate of acute coronary syndrome.

An anti-sulfatide antibody O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the G protein α subunit in rat primary immature oligodendrocytes.

The association of sulfatide with specific proteins in oligodendrocytes was examined by co-immunoprecipitation with an anti-sulfatide antibody. Protein kinase activity was detected in precipitates with a monoclonal antibody to sulfatide (O4) from the rat primary immature oligodendrocytes. We conducted in vitro kinase assay of tyrosine phosphorylated proteins of 80, 59, 56, 53 and 40 kDa by gel electrophoresis. Of these proteins, the proteins of 59 kDa and 53/56 kDa were identified as the Src family tyrosine kinases Fyn and Lyn on the basis of their sequential immunoprecipitation with anti-Fyn and anti-Lyn antibodies, respectively. The 40 kDa protein was identified as the α subunit of the heterotrimeric G protein. These observations suggest that O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the α subunit of the heterotrimeric G protein.

Novel Bernard-Soulier syndrome variants caused by compound heterozygous mutations (case I) or a cytoplasmic tail truncation (case II) of GPIbα.

A defective platelet glycoprotein (GP) Ib/IX/V complex [von Willebrand factor (VWF) receptor] results in Bernard-Soulier syndrome (BSS), which is characterized by macrothrombocytopenia and impaired ristocetin- and thrombin-induced platelet aggregation. We found 2 independent BSS-variant families: Case I [compound heterozygous mutations, p.Glu331X and a frame shift by a deletion at c.1444delA of GPIbα (GP1BA) terminating at a premature stop codon (p.Thr452ProfsX58)], and case II [homozygous nonsense mutation at c.1723C>T, p.Gln545X]. Case I platelets expressed no GPIbα, resulting in absence of ristocetin-induced platelet aggregation (RIPA) and 50% reduction in thrombin-induced aggregation with no shape change. The mother's platelets had 50% the expression level of A-type GPIbα (4-repeated VNTR: variable number of tandem repeats, p.[Thr145Met; Ser399_Pro411[4]]); the father's platelets had the same expression level of C-type GPIbα (2-repeated VNTR, p.Ser399_Pro411dup) as the mother's platelets. The mother's RIPA was significantly higher than the father's. Thrombin-induced aggregation was normal in both parents. Case II platelets expressed a GPIbα with an abnormal cytoplasmic tail, p.Gln545X-truncated GPIbα, which complexed with GPIX and GPV on the cell surface; its expression level of the complex was normal. Case II platelets had reversible RIPA, with no ATP release, and weak thrombin-induced aggregation without shape change. These results suggest that a signaling process through the GPIbα cytoplasmic tail required for full platelet activation is defective in BSS variant case II and a length polymorphism of GPIbα is associated with a modified level of RIPA heterozygous BSS case I.

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones.

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C-terminal src kinase)-binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent-resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti-ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over-expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314-phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314-phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP-43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.

Soluble amyloid precursor protein 770 is released from inflamed endothelial cells and activated platelets: a novel biomarker for acute coronary syndrome.

BACKGROUND: Separate monitoring of the cleavage products of different amyloid ß precursor protein (APP) variants may provide useful information. RESULTS: We found that soluble APP770 (sAPP770) is released from inflamed endothelial cells and activated platelets as judged by ELISA. CONCLUSION: sAPP770 is an indicator for endothelial and platelet dysfunctions. SIGNIFICANCE: How sAPP770 is released in vivo has been shown. Most Alzheimer disease (AD) patients show deposition of amyloid ß (Aß) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid ß precursor protein (APP) 770, a different APP isoform from neuronal APP695, and produce Aß. Since the soluble APP cleavage product, sAPP, is considered to be a possible marker for AD diagnosis, sAPP has been widely measured as a mixture of these variants. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable discrimination between endothelial and neurological dysfunctions. Using our newly developed ELISA system for sAPP770, we observed that inflammatory cytokines significantly enhanced sAPP770 secretion by endothelial cells. Furthermore, we unexpectedly found that sAPP770 was rapidly released from activated platelets. We also found that cerebrospinal fluid mainly contained sAPP695, while serum mostly contained sAPP770. Finally, to test our hypothesis that sAPP770 could be an indicator for endothelial dysfunction, we applied our APP770 ELISA to patients with acute coronary syndrome (ACS), in which endothelial injury and platelet activation lead to fibrous plaque disruption and thrombus formation. Development of a biomarker is essential to facilitate ACS diagnosis in clinical practice. The results revealed that ACS patients had significantly higher plasma sAPP770 levels. Furthermore, in myocardial infarction model rats, an increase in plasma sAPP preceded the release of cardiac enzymes, currently used markers for acute myocardial infarction. These findings raise the possibility that sAPP770 can be a useful biomarker for ACS.

Ganglioside GD3 monoclonal antibody-induced paxillin tyrosine phosphorylation and filamentous actin assembly in cerebellar growth cones.

We have demonstrated that antibody to ganglioside GD3 (R24) immunoprecipitates src-family tyrosine kinase Lyn from primary cerebellar granule cells and R24 treatment of the intact cells induces Lyn activation and rapid tyrosine phosphorylation of several substrates, suggesting the functional association of ganglioside GD3 with Lyn. In this study, R24 treatment of primary cerebellar granule cells enhances phosphorylation of paxillin at tyrosine residue 118 and induces filamentous actin assembly and neurite outgrowth. R24 treatment of cerebellar growth cone membrane fraction induces prominent tyrosine phosphorylation of 68 kDa protein which comigrates with phosphopaxillin at tyrosine residue 118. Tyrosine phosphorylation of paxillin is known to regulate actin cytoskeleton-dependent changes in cell morphology. Signal transduction by ganglioside GD3 is involved in growth cone morphology via tyrosine phosphorylation of paxillin.

Impaired clot retraction in factor XIII A subunit-deficient mice.

Factor XIII (FXIII) is a plasma transglutaminase that cross-links fibrin monomers, alpha(2)-plasmin inhibitor, and so forth. Congenital FXIII deficiency causes lifelong bleeding symptoms. To understand the molecular pathology of FXIII deficiency in vivo, its knockout mice have been functionally analyzed. Because prolonged bleeding times, a sign of defective/abnormal primary hemostasis, were commonly observed in 2 separate lines of FXIII A subunit (FXIII-A) knockout mice, a possible role or roles of FXIII in platelet-related function was investigated in the present study. Although platelet aggregation induced by adenosine diphosphate or collagen was normal, clot retraction (CR) was lost in the platelet-rich plasma (PRP) of FXIII-A knockout mice. In contrast, there was no CR impairment in the PRP of tissue transglutaminase-knockout mice compared with that of wild-type mice. Furthermore, a transglutaminase inhibitor, cystamine, halted CR in the PRP of wild-type mice. These results indicate that the enzymatic activity of FXIII is necessary for CR, at least in mice.

Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors.

Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.