Redistribution of glycoprotein Ib within platelets in response to protease-activated receptors 1 and 4: roles of cytoskeleton and calcium

Summary.  Thrombin activates human platelets by hydrolyzing the protease-activated receptors PAR-1 and PAR-4, exposing new N-terminal sequences which act as tethered ligands, and binding to glycoprotein (GP) Ib, whose surface accessibility transiently decreases when platelets are stimulated by the enzyme. In an attempt to better understand this latter process, we used the peptides SFLLRNPNDKYEPF (PAR-1-AP or TRAP) and AYPGKF (PAR-4-AP) to study whether hydrolysis of both PAR receptors leads to GPIb redistribution. Both peptides induced surface clearance of GPIb with a maximum at 2 min and 5 min for PAR-1-AP and PAR-4-AP, respectively, followed by a slow return to the surface with levels normalizing between 30 and 60 min. Translocation was associated with the formation of clusters of GPIb as revealed by fluorescence microscopy. This transient redistribution of GPIb was blocked by cytochalasin D and in large part by the membrane permeable Ca²⁺ chelator, BAPTA. The inhibitor of phosphatidylinositol 3-kinase and myosin light chain kinase, wortmannin, did not significantly modify internalization of GPIb, although its return to the surface was delayed for PAR-1-AP. PAR receptor–mediated association of GPIb to the insoluble cytoskeleton was blocked by cytochalasin D, while BAPTA alone increased and stabilized the presence of GPIb. Globally, immunoprecipitation experiments and analysis of the cytoskeleton confirmed that GPIb translocation is powered by a contractile mechanism involving Ca²⁺ mobilization, actin polymerization, and myosin incorporation into the cytoskeleton and that both PAR-1 and PAR-4 can activate this process.     

ITAM receptor‐mediated generation of reactive oxygen species in human platelets occurs via Syk‐dependent and Syk‐independent pathways

Background: Ligation of the platelet‐specific collagen receptor, GPVI/FcRγ, causes rapid, transient disulfide‐dependent homodimerization, and the production of intracellular reactive oxygen species (ROS) generated by the NADPH oxidase, linked to GPVI via TRAF4. Objectives: The aim of this study was to evaluate the role of early signaling events in ROS generation following engagement of either GPVI/FcRγ or a second immunoreceptor tyrosine‐based activation motif (ITAM)‐containing receptor on platelets, FcγRIIa. Methods and Results: Using an H₂DCF‐DA‐based flow cytometric assay to measure intracellular ROS, we show that treatment of platelets with either the GPVI agonists, collagen‐related peptide (CRP) or convulxin (Cvx), or the FcγRIIa agonist 14A2, increased intraplatelet ROS; other platelet agonists such as ADP and TRAP did not. Basal ROS in platelet‐rich plasma from 14 healthy donors displayed little inter‐individual variability. CRP, Cvx or 14A2 induced an initial burst of ROS within 2 min followed by additional ROS reaching a plateau after 15–20 min. The Syk inhibitor BAY61‐3606, which blocks ITAM‐dependent signaling, had no effect on the initial ROS burst, but completely inhibited the second phase. Conclusions: Together, these results show for the first time that ROS generation downstream of GPVI or FcγRIIa consists of two distinct phases: an initial Syk‐independent burst followed by additional Syk‐dependent generation.     

Effect of multimer size and a natural dimorphism on the binding of convulxin to platelet glycoprotein (GP)VI

BACKGROUND: Convulxin (CVX), a C-type lectin from the venom of Crotalus durissus terrificus, is a potent activator of human platelets, binding predominantly to glycoprotein (GP)VI. Native CVX is an octamer composed of four alphabeta-heterodimers [(alphabeta)(4)]. Two different native sequences have been reported, one bearing lysine (K), the other glutamic acid (E), at beta chain residue 89, but the physiological relevance of this difference is unknown. OBJECTIVE: We used the Drosophila S2 system to express recombinant CVX (rCVX) heterodimers (alphabeta) and site-directed mutagenesis to evaluate the influence of multimer size and the substitution betaK89E on CVX function. METHODS: By flow cytometry, native CVX and both recombinant forms bind to human platelets in whole blood. By surface plasmon resonance (BIAcore, Piscataway, NJ, USA), the calculated equilibrium dissociation constants (K(D)) were: rCVX alphabeta89K, 11.3 x 10(-8) m; rCVX alphabeta89E, 9 x 10(-8) m; and native CVX, 2.8 x 10(-8) m. RESULTS: Thus, the affinities of the two rCVX forms for human, recombinant GPVI are essentially the same, but the relative affinity of native CVX is about 3-fold higher. The minimum concentration of native CVX that induces maximal human platelet aggregation (70 pm) is roughly 400-fold lower than that of either rCVX (29 nm). CONCLUSIONS: These results are consistent with the hypothesis that the ability of the native CVX octamer to cluster mobile GPVI molecules within the platelet membrane may be the single most important factor that contributes to the efficiency with which CVX is able to induce platelet activation.     

PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets

To cite this article: Harper MT, Poole AW. PKC inhibition markedly enhances Ca²⁺ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets. J Thromb Haemost 2011; 9 : 1599–607. Summary. Background: Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca²⁺ signaling must be tightly controlled. Thrombin‐induced Ca²⁺ signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca²⁺signal, although the mechanisms by which this occurs and its physiological relevance are still unclear. Objectives: To investigate the mechanisms by which PKC inhibitors enhance thrombin‐induced Ca²⁺ signaling, and to determine the importance of this pathway in platelet activation. Methods: Cytosolic Ca²⁺ signaling was monitored in fura‐2‐loaded human platelets. Phosphatidylserine (PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry. Results: PKC inhibition by bisindolylmaleimide‐I (BIM‐I) enhanced α‐thrombin‐induced Ca²⁺ signaling in a concentration‐dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or γ‐thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM‐I had little effect on α‐thrombin‐induced signaling following treatment with the PAR1 antagonist, SCH‐79797. BIM‐I enhanced Ca²⁺ release from intracellular stores and Ca²⁺ entry, as assessed by Mn²⁺ quench. However, the plasma membrane Ca²⁺ ATPase inhibitor, 5(6)‐carboxyeosin, did not prevent the effect of BIM‐I. PKC inhibition strongly enhanced α‐thrombin‐induced PS exposure, which was reversed by blockade of PAR1. Conclusions: Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca²⁺ release and Ca²⁺ entry, which leads to reduced platelet PS exposure.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

The −589C>T polymorphism in the interleukin‐4 gene (IL‐4) is associated with a reduced risk of myocardial infarction in young individuals

Summary. Background: Inflammatory reactions contribute to the development of arterial disease. We investigated the role of interleukin‐4 (IL‐4) in the development of myocardial infarction (MI) by genotyping patients with MI and control subjects for the ?589C>T (rs2243250) single nucleotide polymorphism (SNP), which tags a functional haplotype of IL‐4. Methods and results: Study of Myocardial Infarctions Leiden (SMILE) included 560 men with a first MI and 646 control subjects. The Valencia study included 305 patients with MI at ≤52 years (men and women) and 310 control subjects. In SMILE no clear overall association with the ?589C>T genotype was found [odds ratio (OR) 0.84; 95% CI 0.37–1.95 for ?589TT and 0.82; 95% CI 0.62–1.07 for ?589CT compared with ?589CC]. In patients younger than 50 years, carriership of one or two ?589T alleles was associated with a reduced risk of MI (OR 0.57: 95% CI 0.34–0.95). This result was replicated in the Valencia study, where carriers of one or two ?589T alleles had a reduced risk of MI (OR 0.67: 95% CI 0.47–0.95), with a strong protective effect of the ?598T allele in homozygous ?589T (OR 0.33: 95% CI 0.10–1.05). In the control subjects of the Valencia study, the ?589T allele was associated with reduced levels of F1+2. Conclusion: Our data indicate that the IL‐4 haplotype tagged by the ?589T allele reduces the risk of MI in young individuals.     

Design and biological functionality of a novel hybrid Ti‐6Al‐4V/hydrogel system for reconstruction of bone defects

We have designed a unique injectable bioactive hydrogel comprising of alginate, gelatin, and nanocrystalline hydroxyapatite and loaded with osteoblasts, with the ability to infiltrate into three‐dimensional Ti‐6Al‐4V scaffolds with interconnected porous architecture, fabricated by electron beam melting. A two‐step crosslinking process using the EDC/NHS and CaCl₂ was adopted and found to be effective in the fabrication of cell‐loaded hydrogel/Ti‐6Al‐4V scaffold system. This hybrid Ti‐6Al‐4V scaffold/hydrogel system was designed for the reconstruction of bone defects, which are difficult to heal in the absence of suitable support materials. The hybrid Ti‐6Al‐4V/hydrogel system favourably modulated the biological functions, namely, adhesion, proliferation, cell‐to‐cell, and cell‐material communication because of the presence of extracellular matrix‐like hydrogel in the interconnected porous structure of 3D printed Ti‐6Al‐4V scaffold. The hydrogel was cytocompatible, which was proven through live/dead assay, the expression level of prominent proteins for cell adhesion and cytoskeleton, including 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay. Furthermore, the high bone formation ability of the hydrogel was confirmed using alkaline phosphatase assay. A high equilibrium water content (~97%) in the hydrogel enables the delivery of cells and bioactive molecules, necessary for bone tissue growth. Although not studied, the presence of hydrogel in the pores of the scaffold can provide the space for the cell migration as well as vascularization through it, required for the effective exchange of nutrients. In conclusion, we underscore that the 3D‐printed Ti‐6Al‐4V scaffold‐loaded with bioactive hydrogel to treat the bone defects significantly impacted cellular functions and cell‐material interaction.     

Phorbol ester‐evoked Ca2+signaling in human platelets is via autocrine activation of P2X1 receptors,not a novel non‐capacitative Ca2+entry

Summary. Background: Platelets are reported to possess a protein kinase C (PKC)‐dependent non‐capacitative Ca²⁺entry (NCCE) pathway. The phorbol ester, phorbol, 12‐myristate, 13‐acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store‐operated Ca²⁺entry in human platelets for Na⁺/Ca²⁺ exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA‐evoked Ca²⁺entry. Objectives: To investigate the roles of NCXs and dense granule secretion in PMA‐evoked Ca²⁺signaling in human platelets. Methods: Fura‐2‐ or sodium‐binding benzofuran isophthalate (SBFI)‐loaded platelets were used to monitor cytosolic Ca²⁺or Na⁺ concentrations. Dense granule secretion was monitored as ATP release using luciferin–luciferase. Results: The NCX inhibitors KB‐R7943 or SN‐6, and removal of extracellular Na⁺, significantly reduced PMA‐evoked Ca²⁺entry. PMA‐evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro‐31‐8220 and significantly slowed by KB‐R7943. The P_2X1 antagonists Ro‐0437626 or MRS‐2159, or desensitization of P_2X1 receptors by prior treatment with α,β‐Methylene‐ATP or omitting apyrase from the medium, reduced PMA‐evoked Ca²⁺entry. Ro‐0437626 or chelation of extracellular Ca²⁺ slowed but did not abolish PMA‐evoked ATP release, indicating that PMA‐evoked dense granule secretion does not require P_2X1 receptor activation but is accelerated by P_2X1‐mediated Ca²⁺entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. Conclusion: PMA‐evoked Ca²⁺entry results from an NCX3‐dependent dense granule secretion and subsequent P_2X1 receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.     

Regulatory mechanisms of C4b‐binding protein (C4BP)α and β expression in rat hepatocytes by lipopolysaccharide and interleukin‐6

Summary. Background: C4b‐binding protein (C4BP), a multimeric protein structurally composed of α chains (C4BPα) and a β chain (C4BPβ), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)‐6. However, it is not fully understood how lipopolysaccharide (LPS) and IL‐6 affect the plasma C4BP antigen level and C4BPα and C4BPβ expression in hepatocytes. Objectives: To assess the effect of LPS and IL‐6 on plasma C4BP, PS–C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. Results: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg^?1) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL‐6 (10 μg kg^?1) injection, and then gradually decreased up to 24 h after IL‐6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPα and C4BPβ in rat hepatocytes, and this effect was inhibited by NFκB and MEK/ERK inhibitors. IL‐6 mediated increase in C4BPβ expression in rat hepatocytes, which leads to increased plasma PS–C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT‐3. Conclusion: LPS decreases both C4BPα and C4BPβ expression via the NFκB and MEK/ERK pathways, whereas IL‐6 specifically increases C4BPβ expression via the STAT‐3 pathway, causing an increase in plasma PS–C4BP complex, and thus decreasing the anticoagulant activity of PS.     

Knockout of a P‐glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua

P‐glycoprotein [P‐gp or the ATP‐binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P‐gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR‐associated 9 (CRISPR/Cas9) gene‐editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P‐gp protein (SeP‐gp) predicted to display structural characteristics common to P‐gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP‐gp ORF was established using the CRISPR/Cas9 gene‐editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP‐gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta‐cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP‐gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP‐gp may contribute to abamectin and EB resistance in S. exigua.     

Granulocyte colony‐stimulating factor produces a decrease in IFNγ and increase in IL‐4 when administrated to healthy donors

Hematopoietic stem cells transplantation (HSCT) is the leading curative therapy for a variety of hematological and hereditary diseases; however, graft versus host disease (GVHD), an immunologic phenomenon that is favored by Th1 cytokines and cytotoxic cells from donors, is present frequently and is one of the most important causes of transplant related mortality. Peripheral blood HSCT is the preferred source of stem cells in almost 100% of the cases of autologous HSCT and in 70% of allogeneic transplants. The best mobilizing agent to get the stem cells out from the bone marrow is the Granulocyte‐Colony Stimulating Factor (G‐CSF). In this work, our main objective was to study a possible correlation between the graft cell dose and the patient's clinical outcome. We evaluated the immunologic changes produced by G‐CSF in the lymphocyte and cytokine profiles in allogeneic HSC donors. HSC from twelve donors were mobilized with G‐CSF at 16 μg/kg/day, for 5 days. Basal Peripheral Blood (BPB), Mobilized Peripheral Blood (MPB), and aphaeresis mononuclear cells (G‐MNC) samples were taken from all donors. Using flow cytometry, we quantified CD19⁺, CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, NK, NKT, DC1, and DC2 cells. Cytokines were determined by ELISA in culture supernatants. CD19⁺ (p = 0.001), DC1 (p < 0.002) and DC2 (p < 0.001) cells were increased in MPB with respect to BPB. An increase in Th2 cytokines such as (IL‐4) and a decrease in Th1 cytokines (IFNγ, IL‐2) were also found in MPB samples. In conclusion, Th1 and Th2 cytokines are relevant in predicting the clinical outcome after allogeneic peripheral blood HSCT. J. Clin. Apheresis 25:181–187, 2010. © 2010 Wiley‐Liss, Inc.