Increased Phosphorylation of Specific Blood Platelet Proteins in Association with the Release Reaction

Abstract

Calcium ions and cyclic nucleotides appear to be the intracellular second messengers most intimately involved in determining the responses of blood platelets to extracellular stimuli andmay, at least in part, regulate platelet function by activation of protein kinases (Haslam, 1975). In an attempt to relate protein phosphorylation to platelet function, we have measured the changes in phosphorylation of individual proteins in intact platelet that have been labelled with [3zP]Pi before addition of aggregation and release-inducing stimuli. While this work was in progress, Lyons et al. (1975), using a similar approach, reported that both thrombin and phytohaemagglutinin increase phosphorylation of two platelet proteins. Suspensions of washed human platelets were prepared by a modification of the method of Mustard et al. (1972). The platelets were initially washed with phosphate-free Tyrode's solution containing 0.35 % bovine serum albumin, then incubated for 60min at a platelet count of 1 x 109-2x 109/ml with 0.2mCi/ml of carrier-free 32Pi and O.~.UM-[G-~H]~hydroxytryptamine (1 8Ci/mmol), then washed once more and finally resuspended in Tyrode's solution without albumin (4x 10'-6x 10' platelets/ml). Samples (1 ml) were stirred at 37°C with aggregating agents and/or inhibitors of aggregation for various times, during which platelet aggregation was recorded turbidometrically, and after which the samples were either centrifuged for determination of the release of [G-3H]5-hydroxytryptamine (Haslam et al., 1975) or acidified with O.~M-HCIO, to precipitate platelet protein, which was washed with O.~M-HCIO, and dissolved in 0.1 M-sodium phosphate buffer, pH7.0, containing 3 % (w/v) sodium dodecylsulphate and 0.1 M-dithiothreitol. The dissolved protein was electrophoresed on cylindrical sodium dodecyl sulphate/ polyacrylamide gels, and stained and destained as described by Fairbanks et al. (1971). Gels were scanned at 550nm by using a Gilford spectrophotometer and linear transport, and divided into 1 mm slices with a Bio-Rad gel slicer. Slices were placed in polythene vials containing 0.01 % (w/v) 4-methylumbelliferone in water and counted for &renkov radiation (efficiency 40 %). Suspensions of collagen fibres were prepared as described elsewhere (Haslam et al., 1975). The divalent cation ionophore A23 187 (Eli Lilly and Co., Indianapolis, IN, U.S.A.) was dissolved in dimethyl sulphoxide, which had no effect on the parameters studied at the final concentration used (0.2%, v/v). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of platelet protein gave a reproducible pattern of about 28 stained polypeptide chains (Fig. la) , which included a major peak identified as platelet actin (mol.wt. 42000). Prior aggregation of platelets