Timothy J. Rink

Agonists stimulate divalent cation channels in the plasma membrane of human platelets.

Agonists such as thrombin, PAF (platelet‐activating factor) and ADP are known to cause a larger elevation in [Ca2+]i, in quin2‐loaded platelets in the presence of extracellular Ca2+ than in its absence. The simplest interpretation of these observations is that in the presence of extracellular calcium there is an influx component across the cell surface. In the presence of Mn2+, a divalent cation which is known to avidly bind to quin2 and to quench its fluorescence, the agonists produce a small initial rise in quin2 fluorescence followed by a decrease in fluorescence to well below the resting level. The result indicates entry of Mn2+, presumably through some form of receptor‐operated Ca2+ channel.

Miniaturized FRET assays and microfluidics: key components for ultra-high-throughput screening.

Assay miniaturization applicable across a wide range of target classes, along with automation and process integration, are well-recognized goals for ultra-high-throughput screening on an industrial scale. This report summarizes the implementation of fluorescence resonance energy transfer (FRET)-based biochemical and cell-based assays in 3456-well NanoWelltrade mark assay plates using key components of Auroras ultra-high-throughput screening system.

Stimulation of Ca2+ efflux from fura-2-loaded platelets activated by thrombin or phorbol myristate acetate

We investigated the restoration of [Ca2+]i in fura‐2‐loaded human platelets following discharge of internal Ca2+ stores in the absence of external Ca2+. After stimulation by thrombin [Ca2+]i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca2+]i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca2+]i and restore resting levels within 5 min, an effect that was mimicked by phorbol‐12‐myristate‐13‐acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca2+ efflux, perhaps via protein kinase C actions on a plasma membrane Ca2+ pump.

Elevation of pHi is not an essential step in calcium mobilisation in fura-2-loaded human platelets

Human platelets were co‐loaded with the fluorescent indicators BCECF and fura‐2 to mesure pHi and [Ca2+]i and incubated with aspirin to block cyclooxygenase. Either pHi and shape change and aggregation or pHi and [Ca2+]i were measured simultaneously in the same stirred cuvette, at 37°C. In Hepes‐buffered saline containing 1 mM Ca2+, mean resting pHi was 6.98±0.01 (SE, n = 59). Changes of pHi up to ±0.35 units, imposed by additions of NH4Cl, CO2 or nigericin, produced no shape change or aggregation and only insignificant changes in [Ca2+]i. Sufficient thrombin to raise [Ca2+]i over 1 μM and cause rapid shape change and aggregation increased pHi by no more than 0.05 units, and the increase in pHi lagged behind the elevation of [Ca2+]i. We conclude that changes in pHi do not form a necessary or sufficient component of the pathways leading to receptor‐mediated Ca2+ mobilisation or the stimulation of shape change or aggregation.

Effects of ionic substitution on [Ca2+]i rises evoked by thrombin and PAF in human platelets

We have investigated the effects of substituting extracellular Na+ by choline or K+ on responses of quin2- and fura-2-loaded human platelets to thrombin and platelet-activating factor (PAF). Na+ substitution by choline did not affect the extent of the rise in [Ca2+]i evoked by either agonist. The response to thrombin, but not PAF, was slightly slowed. High K+ did not activate the cells, but the rises in [Ca2+]i evoked by both agonists were slowed and reduced. Shape change evoked by both agonists was little affected by either substitution. Aggregation evoked by PAF was reduced in high K+ but unaffected in choline. Thrombin-induced aggregation was unaffected by either substitution, even when the rise in [Ca2+]i was markedly reduced. The results suggest that the mechanism which generates Ca2+ fluxes in platelets is not voltage-dependent; but high K+ appears to interfere with the influx mechanism.

The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function

1 The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2 Siguazodan selectively inhibited the major cyclic AMP‐hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP‐inhibited PDE or PDE‐III. 3 In platelet‐rich plasma (PRP), siguazodan inhibited U46619‐induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4 In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium ([Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5 Siguazodan has anti‐platelet actions over the same concentration range that it is an inotrope and vasodilator.

Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2-loaded human platelets

The adenylate cyclase stimulator forskolin was used to study the inhibitory effects of elevated cAMP on the activation of washed human platelets loaded with the fluorescent Ca2+ indicator quinl. In the presence of 10 μM isobutylmethylxanthine forskolin inhibited rises in [Ca2+]i evoked by thrombin and platelet‐activating factor (PAF) due to both Ca2+ influx and release from internal stores with similar potency. Aggregation evoked by thrombin and PAF was suppressed whilst partial shape‐change persisted, even in the absence of a measurable rise in [Ca2+]i. Forskolin did not affect the rise in [Ca2+ i evoked by Ca2+ ionophore; aggregation was suppressed but shape‐change persisted.

Free Ca2+ requirements of agonist-induced thromboxane A2 synthesis in human platelets

In quin2-loaded human platelets ionomycin raised cytosolic free calcium to greater than 1 microM and generated less than 1 ng thromboxane. Collagen alone or in the presence of EPO92 generated up to 32 and 16 ng thromboxane respectively; in the latter case at calcium levels around 120 nM. Thrombin maximally raised calcium to greater than 1 microM and generated up to 27 ng thromboxane, although in the presence of 1 mM EGTA these calcium and thromboxane levels were reduced to 200 nM and 5 ng respectively.

Agonist Stimulated Changes in Human Endothelial Cell Cytosolic Calcium

A considerable amount of evidence exists to suggest that a rise in cytoplasmic free calcium concentration, [Ca2+]i, evokes morphological, metabolic and secretory responses in endothelial cells in response to stimulation by many inflammatory mediators. One of the most potent agents for stimulating the release of the vasodilators endothelial-derived relaxing factor (EDRF) and prostacyclin (PGI2) is the calcium ionophore A23187. However, the hypothesis that elevated [Ca2+]i is the physiological messenger is held with some caution since A23187-evoked rises in [Ca2+]i sufficient to evoke these responses can conceivably be far larger than those physiologically attained on stimulation with inflammatory mediators. With the development of the fluorescent [Ca2+] indicator dyes quin2, fura-2 and indo-1, increases in [Ca2+]. have been observed in endothelial cells from bovine or porcine aortae or from human umbilical vein in response to histamine, bradykinin, thrombin, adenine nucleotides and PAF. Furthermore, recent work has shown that these mediator-evoked rises in [Ca2+]i are both sufficient and necessary for the production and release of PGI2 (Hallam et al., 1988c). Our attention has now focussed on the mechanisms of Ca2+ movement and the sources of trigger Ca2+.