The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing impor-

The sarcoplasmic reticulum Ca-ATPase inhibitor thapsigargin (Tg; 0.4-100 nM) produced concentration-related, strong and sustained contractions of the mouse-isolated anococcygeus muscle; these contractions were dependent on extracellular calcium but were only partially reduced (by about 50%) in the presence of verapamil (10 and 100 microM). The verapamil-resistant component of the Tg-induced contraction was relaxed by the general calcium entry blockers SKF96365 (0.4-40 microM) and cadmium (50-300 microM), and by the tyrosine kinase inhibitor genistein (10-180 microM). In single smooth muscle cells loaded with Fura-2, addition of Tg (100 nM) to calcium-free medium produced a small, transient increase in fluorescence; subsequent addition of calcium (2.5 mM) produced a larger and sustained increase which was abolished on return to calcium-free conditions, but was only partially reduced by verapamil (10 microM; by about 30%). Manganese quenching of Fura-2 was enhanced in cells treated with Tg. The verapamil-resistant calcium influx was reduced by SKF96365 (20 microM) and to a lesser extent by genistein (40 microM); cadmium (200 microM) produced an initial decrease in fluorescence followed by a marked increase. These results demonstrate that, in the mouse anococcygeus, Tg can cause sustained contractions and elevations of calcium influx in the presence of verapamil; the time-course, calcium dependence and, although to a lesser extent, pharmacology of these effects generally support the proposal that excitation-contraction coupling in this tonic smooth muscle involves sustained capacitative calcium influx.

1 The aim of this work was to determine whether Rho-kinase-mediated calcium sensitisation contributes to contractions of the mouse anococcygeus smooth muscle and, if so, whether the process was activated by receptor-dependent or receptor-independent mechanisms. 2 The Rho-kinase inhibitor Y27632 produced concentration-dependent decreases in tone raised by either the muscarinic receptor agonist carbachol (CCh), or the sarco-endoplasmic reticulum calcium ATPase inhibitor thapsigargin (Tg) (EC 50 values against CCh and Tg of 8.473.3 (n ¼ 6) and 6.172.1 (n ¼ 7) mm, respectively). Pretreatment of tissues with Y27632 also inhibited contractions produced by 65 mm external potassium (6977% (n ¼ 4) inhibition using 10 mm Y27632). Y27632 had no effect on contractions produced by the inhibitor of smooth muscle myosin light-chain phosphatase, calyculin-A.3 In b-escin-permeabilised preparations, both CCh and Tg produced significant increases in tone over-and-above that produced by a combination of calcium (1 mm) and GTP (100 mm). These responses to CCh and Tg were inhibited by Y27632 (10 mm). 4 Western blot analysis of fractionated tissue samples probed for RhoA immunoreactivity, indicated that both CCh and Tg were able to induce translocation of RhoA from the cytosol to the membrane. 5 These findings suggest that Rho-kinase-mediated calcium sensitisation is activated by both receptor-dependent and receptor-independent mechanisms in the mouse anococcygeus.

1 The object of this study was to determine whether inhibition of capacitative calcium entry is essential for relaxation of the mouse anococcygeus via the NO/cyclic GMP signalling pathway. 2 In intact muscles, thapsigargin (Tg; 100 nM)-induced tone was relaxed by NO, sodium nitroprusside (SNP), 8-Br-cyclic GMP, and nitrergic ®eld stimulation. The relaxations were similar in magnitude to those observed against carbachol (50 mM) tone and, with the exception of those to 8-Br-cyclic GMP, were reduced by the soluble guanylyl cyclase inhibitor 1H- [1,2,4] 4 In b-escin skinned preparations, NO had no e ect on tone induced by calcium (1 mM in the presence of 100 mM GTP). Carbachol and Tg produced further increases in calcium/GTP-induced tone and, in both cases, this additional tone was relaxed by NO and 8-Br-cyclic GMP. 5 The results support the hypothesis that the NO/cyclic GMP pathway inhibits capacitative calcium entry by re®lling the internal stores, since reduction in [Ca 2+ ] i was not observed in the presence of Tg. However, as muscle relaxation was still observed, impairment of capacitative calcium entry cannot be considered obligatory for relaxation. Results from skinned tissues suggest that inhibition of calcium sensitization processes, perhaps associated with store-depletion, may be an important mechanism of NO/cyclic GMP-induced relaxation.