Platelet Glutamate and Aspartate Uptake in Huntington's Disease

Abstract A possible involvement of amino acid uptake mechanisms in the etiology of the human neurodegenerative disease, Huntington's disease (HD), was investigated. Measurement of glutamate (Glu) and aspartate (Asp) uptake was performed in blood platelets, which have previously been shown to constitute a peripheral model system for central amino acid uptake processes. Analyses of Glu and Asp accumulation at 10⁻⁷ M and kinetic examination of the high affinity site for Glu indicate no significant differences between control and HD platelets. A genetically determined defect in amino acid uptake therefore does not seem to underlie the nerve cell loss observed in HD patients.     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at ~14°C, whereas, freeze-dried platelets exhibited a main phase transition ~12°C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42°C, whereas proteins in the rehydrated platelets denatured at 48°C.     

On the degradation of heparin by human blood platelets.

Human blood platelets are able to degrade heparin from different tissues and species. The main degradation product is an oligosaccharide. Low molecular weight components such as inorganic sulfate or monosaccharides, i.e. products released by exoenzymes are not detected. The in vitro degradation of heparin by the crude enzyme is observed at pHs below 6.5 with an optimum temperature around 37 degrees C. The presence of sulfate in the substrate structure is shown to be essential for the enzyme activity. Since the oligosaccharides formed have only 10 per cent of the anticoagulant activity of the heparins tested, it is conceivable that the platelet enzyme may play an important role in the inactivation of some of the biological properties of heparin.     

Antimalarial drugs impact chemical messenger secretion by blood platelets

BackgroundAdvances in antimalarial drug development are important for combating malaria. Among the currently identified antimalarial drugs, it is suggested that some interact directly with the malarial parasites while others interact indirectly with the parasites. While this approach leads to parasite elimination, little is known about how these antimalarial drugs impact immune cells that are also critical in malarial response.MethodsHerein, the effects of two common antimalarial drugs, chloroquine and quinine, on platelets were explored at both the bulk level, using high performance liquid chromatography, and the single cell level, using carbon-fiber microelectrode amperometry, to characterize any changes in chemical messenger secretion.ResultsThe data reveal that both drugs cause platelet activation and reduce the number of platelet exocytosis events as well as delay fusion pore opening and closing.ConclusionsThis work demonstrates how chloroquine and quinine quantitatively and qualitatively impact in vitro platelet function.General significanceOverall, the goal of this work is to promote understanding about how antimalarial drugs impact platelets as this may affect antimalarial drug development as well as therapeutic approaches to treat malarial infection.     

A Mechanism for Localized Dynamics-driven Affinity Regulation of the Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

Binding of the A1 domain of von Willebrand factor (vWF) to glycoprotein Ibα (GPIbα) results in platelet adhesion, activation, and aggregation that initiates primary hemostasis. Both the elevated shear stress and the mutations associated with type 2B von Willebrand disease enhance the interaction between A1 and GPIbα. Through molecular dynamics simulations for wild-type vWF-A1 and its eight gain of function mutants (R543Q, I546V, ΔSS, etc.), we found that the gain of function mutations destabilize the N-terminal arm, increase a clock pendulum-like movement of the α2-helix, and turn a closed A1 conformation into a partially open one favoring binding to GPIbα. The residue Arg⁵⁷⁸ at the α2-helix behaves as a pivot in the destabilization of the N-terminal arm and a consequent dynamic change of the α2-helix. These results suggest a localized dynamics-driven affinity regulation mechanism for vWF-GPIbα interaction. Allosteric drugs controlling this intrinsic protein dynamics may be effective in blocking the GPIb-vWF interaction.     

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.     

Wiskott-Aldrich Syndrome Protein (WASp) Controls the Delivery of Platelet Transforming Growth Factor-β1

Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp^−/−) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10–15% released after 30–60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp^−/− platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp^−/− platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly.     

cGMP-independent inhibition of integrin alphaIIbbeta3-mediated platelet adhesion and outside-in signalling by nitric oxide

We examined the influence of S-nitrosoglutathione (GSNO) on alpha(IIb)beta(3) integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin beta(3) phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of beta(3) on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin beta(3) phosphorylation.     

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3^?/?) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1β (IL-1β), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3^?/? mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.