Vicinal thiols are required for activation of the αIIbβ3 platelet integrin

Summary. Background: Closely spaced thiols in proteins that interconvert between the dithiol form and disulfide bonds are called vicinal thiols. These thiols provide a mechanism to regulate protein function. We previously found that thiols in both αIIb and β3 of the αIIbβ3 fibrinogen receptor were required for platelet aggregation. Methods and Results: Using p‐chloromercuribenzene sulfonate (pCMBS) we provide evidence that surface thiols in αIIbβ3 are exposed during platelet activation. Phenylarsine oxide (PAO), a reagent that binds vicinal thiols, inhibits platelet aggregation and labeling of sulfhydryls in both αIIb and β3. For the aggregation and labeling studies, binding of PAO to vicinal thiols was confirmed by reversal of PAO binding with the dithiol reagent 2,3‐Dimercapto‐1‐propanesulfonic acid (DMPS). In contrast, the monothiol β‐mercaptoethanol did not reverse the effects of PAO. Additionally, PAO did not inhibit sulfhydryl labeling of the monothiol protein albumin, confirming the specificity of PAO for vicinal thiols in αIIbβ3. As vicinal thiols represent redox sensitive sites that can be regulated by reducing equivalents from the extracellular or cytoplasmic environment, they are likely to be important in regulating activation of αIIbβ3. Additionally, when the labeled integrin was passed though a lectin column containing wheat germ agglutinin and lentil lectin a substantial amount of non‐labeled αIIbβ3 eluted separately from the labeled receptor. This suggests that two populations of integrin exist on platelets that can be distinguished by thiol labeling. Conclusion: A vicinal thiol‐containing population of αIIbβ3 provides redox sensitive sites for regulation of αIIbβ3.     

A mutation in the β3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIbβ3 to an active state

Summary. Background: The cytoplasmic tails of α_IIb and β₃ regulate essential α_IIbβ₃ functions. We previously described a variant Glanzmann thrombasthenia mutation in the β₃ cytoplasmic tail, IVS14: ?3C>G, which causes a frameshift with an extension of β₃ by 40 residues. Objectives: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of α_IIbβ₃ from a resting state to an active state. Methods: We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild‐type (WT) α_IIb as follows: β₃‐742stop, a truncated mutant to evaluate the effect of deleted residues; β₃‐749stop, a truncated mutant that preserves the NPLY conserved sequence; and β₃‐749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. Results and conclusions: Surface expression of α_IIbβ₃ of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti‐ligand‐induced‐binding‐site 6 [antiLIBS6] or PT25‐2). Activation of the α_IIbβ₃ mutants was only achieved when both PT25‐2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the β₃ tail is important for maintaining α_IIbβ₃ in a resting conformation.     

A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts

α_Vβ₃ Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near‐infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α_Vβ₃ integrin‐overexpressing tumors. DA364's binding affinity for α_Vβ₃ integrin was first evaluated in vitro. Human α_Vβ₃ integrin‐positive, U‐87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α_Vβ₃ integrin on U‐87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic‐RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α_Vβ₃ integrin‐overexpressing tumors. Copyright © 2011 John Wiley & Sons, Ltd.     

Characteristics of recombinant W501S mutated human γ‐glutamyl carboxylase

Summary. A mutation (W501S) in the vitamin K‐dependent γ‐glutamyl carboxylase (VKC) that leads to a congenital bleeding disorder was recently discovered in two patients. To characterize the enzyme defect, recombinant VKC‐W501S was expressed in and purified from insect cells. The major effect of the mutation appears to be to decrease the affinity of the carboxylase for the propeptide of its substrates. This observation agrees with recent data that place part of the propeptide binding site within residues 495–513 of VKC. Additionally, we demonstrate that the affinity between descarboxy osteocalcin (d‐OC) and VKC remains unaffected by the W501S mutation. This confirms earlier data that the high‐affinity site for d‐OC is not located on the propeptide binding domain of VKC. Two properties of the enzyme suggest an explanation for the observation that vitamin K supplementation ameliorates the effects of the mutation: (i) since full carboxylation requires the propeptide to remain bound to the enzyme sufficiently long for full carboxylation, a reduced affinity can cause its premature release before carboxylation is complete; (ii) propeptide binding results in a decrease of the K_M for vitamin K hydroquinone in wild‐type, but not in mutant carboxylase, resulting in increased vitamin K requirement of affected subjects.     

Functional association of phosphoinositide‐3‐kinase with platelet glycoprotein Ibα, the major ligand‐binding subunit of the glycoprotein Ib–IX–V complex

Summary. Background: The adhesion receptor glycoprotein (GP)Ib–IX–V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet‐mediated tumor metastasis. The cytoplasmic C‐terminal domain of the ligand‐binding GPIbα subunit contains binding sites for filamin (residues 561–572, critically Phe568/Trp570), 14‐3‐3ζ (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide‐3‐kinase (PI3‐kinase) regulatory subunit, p85. Objectives: We previously showed that, as compared with wild‐type receptor, deleting the contiguous sequence 580–590 or 591–610, but not upstream sequences, of GPIbα expressed as a GPIb–IX complex in Chinese hamster ovary cells inhibited VWF‐dependent Akt phosphorylation, which is used as a read‐out for PI3‐kinase activity. Pulldown experiments using glutathione‐S‐transferase (GST)–p85 or GST–14‐3‐3ζ constructs, and competitive inhibitors of 14‐3‐3ζ binding, suggested an independent association of 14‐3‐3ζ and PI3‐kinase with GPIbα. The objective of this study was to analyze a further panel of GPIbα deletion mutations within residues 580–610. Results: We identified a novel deletion mutant, Δ591–595, that uniquely disrupts 14‐3‐3ζ binding but retains the functional p85/PI3‐kinase association. Deletion of other sequences within the 580–610 region were less discriminatory, and either partially affected p85/PI3‐kinase and 14‐3‐3ζ binding (Δ580–585, Δ586–590, Δ596–600, Δ601–605), or strongly inhibited binding of both proteins (Δ606–610). Conclusions: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14‐3‐3ζ in GPIb‐dependent signaling or platelet functional studies involving truncation of the C‐terminal residues in cell‐based assays and mouse models. The Δ591–595 mutation provides another strategy for determining the function of GPIbα‐associated 14‐3‐3ζ by selective disruption of 14‐3‐3ζ but not p85/PI3‐kinase binding.     

RGD‐ligand mimetic antagonists of integrin αIIbβ3 paradoxically enhance GPVI‐induced human platelet activation

Summary. Background: The integrin α_IIbβ₃ is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of α_IIbβ₃, for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD‐ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. Objectives: To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. Methods: We assessed the effect of lotrafiban, and other α_IIbβ₃ antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro‐coagulant activity) and dense granule release. Results: In all cases, no effect of α_IIbβ₃ antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)‐associated FcR γ‐chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y₁₂ antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. Conclusions: These data provide a mechanism for enhancement of platelet activity by α_IIbβ₃ inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.     

TGFβ3 secretion by three‐dimensional cultures of human dental apical papilla mesenchymal stem cells

Mesenchymal stem cells (MSCs) can be isolated from dental tissues, such as pulp and periodontal ligament; the dental apical papilla (DAP) is a less‐studied MSC source. These dental‐derived MSCs are of great interest because of their potential as an accessible source for cell‐based therapies and tissue‐engineering (TE) approaches. Much of the interest regarding MSCs relies on the trophic‐mediated repair and regenerative effects observed when they are implanted. TGFβ3 is a key growth factor involved in tissue regeneration and scarless tissue repair. We hypothesized that human DAP‐derived MSCs (hSCAPs) can produce and secrete TGFβ3 in response to micro‐environmental cues. For this, we encapsulated hSCAPs in different types of matrix and evaluated TGFβ3 secretion. We found that dynamic changes of cell–matrix interactions and mechanical stress that cells sense during the transition from a monolayer culture (two‐dimensional, 2D) towards a three‐dimensional (3D) culture condition, rather than the different chemical composition of the scaffolds, may trigger the TGFβ3 secretion, while monolayer cultures showed almost 10‐fold less secretion of TGFβ3. The study of these interactions is provided as a cornerstone in designing future strategies in TE and cell therapy that are more efficient and effective for repair/regeneration of damaged tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

Human target validation of phosphoinositide 3‐kinase (PI3K)β: effects on platelets and insulin sensitivity,using AZD6482 a novel PI3Kβ inhibitor

Nylander S, Kull B, Björkman JA, Ulvinge JC, Oakes N, Emanuelsson BM, Andersson M, Skärby T, Inghardt T, Fjellström O, Gustafsson D. Human target validation of phosphoinositide 3‐kinase (PI3K)β:effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kβ inhibitor. J Thromb Haemost 2012; 10: 2127–36. See also Jackson SP, Schoenwaelder SM. Antithrombotic phosphoinositide 3‐kinase β inhibitors in humans – a ‘shear’ delight! This issue, pp 2123–6. Summary. Background: Based on in vitro and animal data, PI3Kβ is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear. Objective: To strengthen the PI3Kβ target validation using the novel, short‐acting inhibitor AZD6482. Methods and results: AZD6482 is a potent, selective and ATP competitive PI3Kβ inhibitor (IC₅₀ 0.01 μm ). A maximal anti‐platelet effect was achieved at 1 μm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti‐thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3‐h infusion. The ex vivo anti‐platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin‐induced human adipocyte glucose uptake in vitro (IC₅₀ of 4.4 μm ). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 μm but reduced by about 60% at a plasma exposure of 27 μm . In man, the homeostasis model analysis (HOMA) index increased by about 10–20% at the highest plasma concentration of 5.3 μm . Conclusions: This is the first human target validation for PI3Kβ inhibition as anti‐platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at ‘supratherapeutic’ plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.     

Regulation of drug transporter mRNA expression by interferon‐γ in primary human hepatocytes

Interferon (IFN)‐γ is known to downregulate expression of drug detoxifying proteins such as cytochromes P‐450 (CYPs) in human hepatocytes. The present study was designed to determine whether IFN‐γ may also impair expression of influx and efflux drug transporters, which constitute important determinants of the liver detoxification pathway. Exposure of primary human hepatocytes to 10 ng/mL IFN‐γ was found to downregulate mRNA levels of sinusoidal influx transporters such as sodium‐taurocholate cotransporting polypeptide, organic anion transporting polypeptide (OATP) 2B1, OATP1B1, and OATP1B3. IFN‐γ concomitantly reduced mRNA expression of drug efflux pumps such as multidrug resistance gene 1, multidrug resistance protein (MRP) 2, MRP3, breast cancer resistance protein and bile salt export pump. Such IFN‐γ‐mediated repression of major hepatic drug transporters may contribute to impaired liver clearance of drugs administrated to patients suffering from inflammation or viral infections associated with increased secretion of IFN‐γ.     

Improvements in endotoxemic syndromes using a disintegrin,rhodostomin, through integrin αvβ3‐dependent pathway

Summary. Background and objectives: Septic shock is a major cause of morbidity and mortality in intensive care units, but there is still no effective therapy for the patients. We evaluated the effects of rhodostomin (Rn), an Arg‐Gly‐Asp‐containing snake venom disintegrin, on lipopolysaccharide (LPS)‐activated phagocytes in vitro and LPS‐induced endotoxemia in vivo. Methods and results: Rn inhibited adhesion, migration, cytokine production and mitogen‐activated protein kinase (MAPK) activation of macrophage induced by LPS. Flow cytometric analysis revealed that Rn specifically blocked anti‐αv mAb binding to RAW264.7. Besides inhibiting MAPK activation of THP‐1, Rn bound to LPS‐activated THP‐1 and specifically blocked anti‐αvβ3 mAb binding to THP‐1. Binding assays proved that integrin αvβ3 was the binding site for rhodostomin on phagocytes. Rn reversed the enhancement of fibronectin and vitronectin on LPS‐induced monocyte adhesion and cytokine release. Transfection of integrin αv siRNA also inhibited LPS‐induced activation of monocyte, and Rn exerted no further inhibitory effect. Furthermore, Rn significantly decreased the production of tumor necrosis factor‐α (TNF‐a), interleukin (IL)‐6, ‐1β and ‐10 and attenuated cardiovascular dysfunction, including blood pressure and heart pulse, and thrombocytopenia in LPS‐induced endotoxemic mice. Rn also protected against tissue inflammation as evidenced by histological examination. Conclusions: Rn may interact with αvβ3 integrin of monocytes/macrophages leading to interfere with the activation of phagocytes triggered by LPS. These results suggest that the protective function of Rn in LPS‐induced endotoxemia may be attributed to its anti‐inflammation activities in vivo.     

Noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for γ-melanocyte-stimulating hormone (γ-msh) and measurement of immunoreactive γ-msh in plasma of healthy subjects

A noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for _γ-melanocyte-stimulating hormone (_γ-MSH) was developed. _γ-MSH (1–12) was biotiny-lated, trapped onto an anti-_γ-MSH (1–12) IgG-coated polystyrene bead, eluted at pH 1 after washing to eliminate other biotiny-lated substances, and measured using two streptavidin-coated polystyrene beads and affinity-purified anti-_γ-MSH (1–12) Fab'-per-oxidase conjugate. The detection limit of _γ-MSH (1–12) was 10–30 amol (16–48 fg)/assay and 130–400 fmol (210–630 pg)/L of plasma. There was little or only slight cross reaction with α-MSH, b?-MSH, and -MSH. By this immunoassay, the concentration and molecular size of immunoreactive _γ-MSH in plasma of healthy subjects were examined, and the results were compared with those by competitive enzyme immunoassay. Immunoreactive _γ-MSH measured by competitive enzyme immunoassay was a mixture of substances with high molecular weights (100–500 kDa), and its concentration was calculated to be 50–60 pmol/L using _γ-MSH (1–12) as standard. Immunoreactive _γ-MSH detected by the noncompetitive enzyme immunoassay after removal of high molecular weight substances was not homogeneous and smaller than _γ-MSH (1–12), and its concentration was ～ 1 pmol/L. The exact nature of these immunoreactive _γ-MSHs remains to be elucidated. _γ-MSH (1–12) added to plasma was degraded rapidly, and the concentration of _γ-MSH (1–12) was very low, if any, in plasma of healthy subjects.     

Possible involvement of β-PKC rather than that of α-PKC in differentiation of 3T3–L1 cells to adipocytes

Abstract. The change of subspecies of protein kinase C (PKC) was studied in 3T3—L1 cells in terms of their differentiation to adipocytes. 3T3—L1 cells feasible to differentiate to adipocytes by exposure to 3–isobutyl-1–methylxanthine (IBMX) and dexamethasone had both α- and β-PKC. However, 3T3–Ll cells unfurnished with such feasibility had only α-PKC. α-PKC, therefore, seems to be more deeply involved in differentiation of 3T3–LI cells to adipocytes than α-PKC.     

Platelet hyperreactivity and a prothrombotic phenotype in mice with a gain‐of‐function mutation in phospholipase Cγ2

Summary. Background: Agonist‐induced platelet activation involves different signaling pathways leading to the activation of phospholipase C (PLC) β or PLCγ2. Activated PLC produces inositol 1,4,5‐trisphosphate and diacylglycerol, which trigger Ca²⁺ mobilization and the activation of protein kinase C, respectively. PLCβ is activated downstream of Gq‐coupled receptors for soluble agonists with only short interaction times in flowing blood. In contrast, PLCγ2 becomes activated downstream of receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI or activated integrins. Objective and methods: We speculated that PLCγ2 activity might be optimized for sustained but submaximal signaling to control relatively slow platelet responses. To test this hypothesis, we analyzed platelets from mice heterozygous for a gain‐of‐function mutation in the Plcg2 gene (Plcg2^Ali5/+). Results: Plcg2^Ali5/+ platelets showed enhanced Ca²⁺ mobilization, integrin activation, granule secretion and phosphatidylserine exposure upon GPVI or C‐type lectin‐like receptor‐2 stimulation. Furthermore, integrin α_IIbβ₃ outside‐in signaling was markedly enhanced in the mutant platelets, as shown by accelerated spreading on different matrices and faster clot retraction. These defects translated into virtually unlimited thrombus formation on collagen under flow in vitro and a prothrombotic phenotype in vivo. Conclusions: These results demonstrate that the enzymatic activity of PLCγ2 is tightly regulated to ensure efficient but limited platelet activation at sites of vascular injury.     

The glycoprotein Ibα–von Willebrand factor interaction induces platelet apoptosis

Summary. Background: The interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbα–VWF interaction. Objectives: To investigate whether the GPIbα–VWF interaction induces platelet apoptosis and the role of 14‐3‐3ζ in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild‐type (1b9) or mutant GPIb–IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin‐induced GPIbα–VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear‐induced apoptosis was eliminated by the anti‐GPIbα antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb–IX with GPIbα truncated at residue 551 or a serine‐to‐alanine mutation at the 14‐3‐3ζ‐binding site in GPIbα. Conclusions: This study demonstrates that the GPIbα–VWF interaction induces apoptotic events in platelets, and that the association of 14‐3‐3ζ with the cytoplasmic domain of GPIbα is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.     

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary. Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin α_IIbβ₃ is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to α_IIbβ₃ was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with α_IIbβ₃ is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with α_IIbβ₃ to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.