Noriko Tamura

Involvement of Glycoprotein VI in Platelet Thrombus Formation on Both Collagen and von Willebrand Factor Surfaces Under Flow Conditions

Background—We studied the role of glycoprotein (GP) VI in platelet adhesion and thrombus formation on the immobilized collagen and von Willebrand factor (vWF) surface under flow conditions. Methods and Results—Whole blood obtained from 2 patients with GP VI–deficient platelets and the effects of the Fab of anti–GP VI antibody (Fab/anti–GP VI) were tested. Blood containing platelets rendered fluorescent by mepacrine was perfused on immobilized type I collagen or vWF under controlled wall shear rate. Platelet adhesion and thrombus formation were detected by epifluorescent videomicroscopy. The percentage of surface coverage by the platelets was calculated. Fc receptor &ggr;-chain and spleen tyrosine kinase (Syk) were immunoprecipitated from the lysate of platelets stimulated by vWF plus ristocetin and then analyzed by antiphosphotyrosine immunoblotting. No platelet attachment was seen on the surface of collagen even after 9 minutes of perfusion of blood at relatively low (100 s−1) or high (1500 s−1) wall shear rate, either in the case of blood containing GP VI–deficient platelets or in the presence of Fab/anti–GP VI, whereas significant platelet thrombus formation was noted after control blood perfusion. Such interference with the actions of GP VI also reduced firm platelet adhesion on immobilized vWF. vWF-induced tyrosine phosphorylation of GP VI–associated Fc receptor &ggr;-chain followed by Syk activation occurred in normal platelets, but little activation of Syk occurred in GP VI–deficient platelets. Conclusions—GP VI plays crucial roles in platelet thrombus formation on the surface of collagen under flow conditions in humans and is also involved in the process of firm platelet adhesion on the surface of vWF.

Functional Significance of Adenosine 5′-Diphosphate Receptor (P2Y12) in Platelet Activation Initiated by Binding of von Willebrand Factor to Platelet GP Ibα Induced by Conditions of High Shear Rate

Background—The role of the adenosine 5′-diphosphate receptor P2Y12 in platelet activation initiated by the von Willebrand factor (VWF)–GP Ib&agr; interaction under high shear rate was investigated. Methods and Results—Blood samples were obtained from 11 donors. Shear-induced platelet aggregation was detected by optically modified cone-plate viscometer. Shear-induced VWF binding, P-selectin expression, and microparticle release were detected by flow cytometry. Platelet interaction with immobilized VWF was also investigated by parallel-plate flow chamber equipped with epifluorescent videomicroscopy. Effects of a selective P2Y12 antagonist AR-C69931 MX were tested. AR-C69931 MX inhibited shear-induced platelet aggregation in a dose-dependent manner, achieving the maximum inhibition at 100 nmol/L. The extent of aggregation after exposure to a shear rate of 10 800 s−1 for 6 minutes in the presence of 100 nmol/L AR-C69931 MX was 32.4±8.2% (mean±SD), which was significantly lower than the value in the controls of 69.7±9.6% (P <0.01). The inhibiting effects of AR-C69931 MX were reversed by exogenous addition of adenosine 5′-diphosphate. Shear-induced VWF binding and P-selectin surface translocation, which occurred in 4696±911 and 5964±784, respectively, of 10 000 measured platelets, was also inhibited by AR-C69931 MX (100 nmol/L) to 1948±528 and 2797±718, respectively (P =0.0018 and P =0.0009). Microparticle release was similarly inhibited. In a flow chamber experiment, firm platelet attachment on immobilized VWF was inhibited by AR-C69931 MX, whereas transient interaction was not influenced. All the above reactions were completely inhibited by blocking VWF–GP Ib&agr; interaction. Conclusions—We have demonstrated that the stimulation of P2Y12 is involved in platelet activation initiated by the binding of VWF to GP Ib&agr; induced by a high shear rate.

Different effects of various anti‐GPIIb‐IIIa agents on shear‐induced platelet activation and expression of procoagulant activity

Summary.  Inhibitors of the platelet glycoprotein (GP)IIb‐IIIa receptor (integrin αIIbβ3) reduce acute thrombotic events in patients with coronary artery disease. To characterize the mechanism of action of these drugs, we evaluated the effects of different GPIIb‐IIIa antagonists on shear‐induced platelet aggregation, activation, and the expression of procoagulant activity. Samples of platelet‐rich plasma from 16 volunteers were exposed to the shear rate of 10 800 s−1 for 6 min in an optically modified cone‐plate viscometer. Abciximab, tirofiban and eptifibatide inhibited aggregation to a similar extent (mean ± SD: 74.1 ± 8.5%, 69.5 ± 13.6%, 65.6 ± 17.0%, respectively), but only abciximab inhibited significantly microparticle release associated with shear‐induced platelet activation (64.4 ± 13.6%, P = 2.2 × 10−7; tirofiban = 20.0 ± 23.4%; eptifibatide = 23.9 ± 17.4%). P‐selectin platelet surface translocation was also strongly inhibited by abciximab, weakly by eptifibatide, but not by tirofiban. The addition of anti‐αvβ3 to tirofiban enhanced the inhibiting effects on shear‐induced P‐selectin translocation and microparticle release. Shearing of platelet‐rich plasma shortened the re‐calcification clotting time after addition of kaolin from 106.9 ± 14.3 to 94.2 ± 10.7 s (mean ± SD; P = 0.0013). This effect, which is mediated by the appearance of procoagulant phospholipids on the surface of sheared platelets and microparticles, was prevented by abciximab and by the combination of tirofiban and anti‐αvβ3, but not by tirofiban alone or eptifibatide. The ability to inhibit shear‐induced platelet activation, as evidenced by microparticle release and P‐selectin surface translocation as well as the expression of procoagulant activity, differentiates the effects of anti‐GPIIb‐IIIa agents, which may explain the distinct antithrombotic efficacy of the agents.

Lnk regulates integrin αIIbβ3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk-/- mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin alphaIIbbeta3-mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk-/- mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk-/- platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the beta3 integrin subunit, and reduced binding of Fyn to integrin alphaIIbbeta3. These results provide new insight into the mechanism of alphaIIbbeta3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events.

Platelets, after Exposure to a High Shear Stress, Induce IL-10-Producing, Mature Dendritic Cells In Vitro

There is evidence for immune system involvement in atherogenesis. In the present study the effect of platelets on dendritic cells (DC), an important immunologic regulator, was examined in vitro. Platelet-rich plasma, after exposure to shear stress, was added to human monocyte-derived immature DC, which were then examined for surface Ag expression, allogeneic T lymphocyte stimulatory activity, and cytokine production. After exposure, the number of anti-CD40 ligand (anti-CD40L) and anti-P-selectin IgG molecules bound per platelet was increased. These activated platelets induced DC maturation, as revealed by significant up-regulation of CD83, CD80, and CD86 Ags. The addition of platelets in the presence of IFN-γ plus LPS significantly enhanced IL-10 production from immature DC. After platelet addition, mature DC provoked a significant proliferation of allogeneic naive T lymphocytes. These activated T cells showed lower IFN-γ production than those stimulated by LPS- and IFN-γ-treated DC. CD40L on the platelet surface was not involved in maturation of DC, as mAb to CD40L failed to block maturation. The effect of platelets was observed even if platelets and DC were separated using large pore-sized membranes or when platelets were depleted from plasma by centrifugation. Furthermore, it was abrogated after the depletion of protein fraction. Thus, soluble protein factors excreted from activated platelets contribute to IL-10-producing DC maturation.

Overview of confinement and MHD stability in the Large Helical Device

The Large Helical Device is a heliotron device with L = 2 and M = 10 continuous helical coils with a major radius of 3.5–4.1 m, a minor radius of 0.6 m and a toroidal field of 0.5–3 T, which is a candidate among toroidal magnetic confinement systems for a steady state thermonuclear fusion reactor. There has been significant progress in extending the plasma operational regime in various plasma parameters by neutral beam injection with a power of 13 MW and electron cyclotron heating (ECH) with a power of 2 MW. The electron and ion temperatures have reached up to 10 keV in the collisionless regime, and the maximum electron density, the volume averaged beta value and stored energy are 2.4 × 1020 m−3, 4.1% and 1.3 MJ, respectively. In the last two years, intensive studies of the magnetohydrodynamics stability providing access to the high beta regime and of healing of the magnetic island in comparison with the neoclassical tearing mode in tokamaks have been conducted. Local island divertor experiments have also been performed to control the edge plasma aimed at confinement improvement. As for transport study, transient transport analysis was executed for a plasma with an internal transport barrier and a magnetic island. The high ion temperature plasma was obtained by adding impurities to the plasma to keep the power deposition to the ions reasonably high even at a very low density. By injecting 72 kW of ECH power, the plasma was sustained for 756 s without serious problems of impurities or recycling.

How is turbulence intensity determined by macroscopic variables in a toroidal plasma

We report observations of the dynamic response of micro-fluctuations and turbulent flux to a low-frequency heating power modulation in the Large Helical Device. The responses of heat flux and micro-fluctuation intensity differ from that of the change in temperature gradient. This result violates the local transport model, where turbulence is determined by the local temperature gradient. A new relationship between flux, gradient and turbulence is found. In addition to the temperature gradient, the heating rate is proposed as a new, direct controlling parameter of turbulence to explain the fast response of turbulence against periodic modulation of heating power.

Control of the radial electric field shear by modification of the magnetic field configuration in LHD

Control of the radial electric field, Er, is considered to be important in helical plasmas, because the radial electric field and its shear are expected to reduce neoclassical and anomalous transport, respectively. In general, the radial electric field can be controlled by changing the collisionality, and positive or negative electric fields have been obtained by decreasing or increasing the electron density, respectively. Although the sign of the radial electric field can be controlled by changing the collisionality, modification of the magnetic field is required to achieve further control of the radial electric field, especially to produce a strong radial electric field shear. In the Large Helical Device (LHD) the radial electric field profiles are shown to be controlled by the modification of the magnetic field by (1) changing the radial profile of the effective helical ripples, ?h, (2) creating a magnetic island with an external perturbation field coil and (3) changing the local island divertor coil current.

Towards an emerging understanding of non-locality phenomena and non-local transport

In this paper, recent progress on experimental analysis and theoretical models for non-local transport (non-Fickian fluxes in real space) is reviewed. The non-locality in the heat and momentum transport observed in the plasma, the departures from linear flux-gradient proportionality, and externally triggered non-local transport phenomena are described in both L-mode and improved-mode plasmas. Ongoing evaluation of ‘fast front’ and ‘intrinsically non-local’ models, and their success in comparisons with experimental data, are discussed

On impurity handling in high performance stellarator/heliotron plasmas

The Large Helical Device (LHD) and Wendelstein 7-X (W7-X, under construction) are experiments specially designed to demonstrate long-pulse (quasi steady state) operation, which is an intrinsic property of stellarators and heliotrons. Significant progress has been made in establishing high performance plasmas. A crucial point is the increasing impurity confinement at high density observed at several machines (TJ-II, W7-AS, LHD) which can lead to impurity accumulation and early pulse termination by radiation collapse. In addition, theoretical predictions for non-axisymmetric configurations predict the absence of impurity screening by ion temperature gradients in standard ion-root plasmas. Nevertheless, scenarios were found where impurity accumulation was successfully avoided in LHD and W7-AS due to the onset of friction forces in the (high density and low temperature) scrape-off-layer (SOL), the generation of magnetic islands at the plasma boundary and to a certain degree also by edge localized modes, flushing out impurities and reducing the net impurity influx into the core. In both the W7-AS high density H-mode regime and in the case of application of sufficient electron cyclotron radiation heating power a reduction in impurity core confinement was observed. The exploration of such purification mechanisms is a demanding task for successful steady-state operation. Impurity transport at the plasma edge/SOL was identified to play a major role for the global impurity behaviour in addition to the core confinement.