1. by Abdelbaset Elzagallaai, Sergio D. Rosé, and José-Marı́a Trifaró
Platelet secretion induced by phorbol esters stimulation is mediated through phosphorylation of MARCKS: a MARCKS-derived peptide blocks MARCKS phosphorylation and serotonin release without affecting pleckstrin phosphorylation
by Abdelbaset Elzagallaai, Sergio D. Rosé, and José-Marı́a Trifaró
Blood
Volume 95(3):
February 1, 2000
©2000 by American Society of Hematology

2. Effect of permeabilization on [3H]5-HT output from platelets
Effect of permeabilization on [3H]5-HT output from platelets.(A) Time course of [3H]5-HT output from intact and permeabilized cells in the presence or absence of PMA. [3H]5-HT–labeled platelets were used and were permeabilized for 4 different periods of tim...
Effect of permeabilization on [3H]5-HT output from platelets.(A) Time course of [3H]5-HT output from intact and permeabilized cells in the presence or absence of PMA. [3H]5-HT–labeled platelets were used and were permeabilized for 4 different periods of time with 15 μM of digitonin in K+-glutamate buffer. When indicated, PMA (100 nM) was present during the entire incubation period. After incubation, platelets were recovered by centrifugation, and the content of [3H]5-HT was measured in the medium and in the platelets. [3H]5-HT outputs were expressed as a percentage of total content. Values represent the mean ± SEM of at least 8 different preparations. (B) Effect of permeabilization is shown for different periods of time on the responses to a short period of stimulation with PMA. [3H]5-HT–labeled platelets were permeabilized as indicated in A, and they were stimulated for 45 seconds with 100 nM of PMA. Platelets were recovered by stimulation, and [3H]5-HT output was measured. Bars represent the mean ± SEM of 4 different preparations.
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology

3. Leakage of proteins from digitonin-permeabilized platelets
Leakage of proteins from digitonin-permeabilized platelets.Platelets were incubated with 15 μM of digitonin in K+-glutamate buffer for different periods of times.
Leakage of proteins from digitonin-permeabilized platelets.Platelets were incubated with 15 μM of digitonin in K+-glutamate buffer for different periods of times. Platelets were then recovered by centrifugation, and pellets (P) were resuspended in K+-glutamate buffer to the same volumes of the corresponding supernatants (S; incubation media). Equal aliquots (1:1) of P and S were run on SDS-PAGE, followed by immunoblotting with antibodies against PKC, pleckstrin, and MARCKS. Under these conditions, MARCKS was not detected in the medium (S). Therefore, S was concentrated 30 times; aliquots were run again and tested for the presence of MARCKS (1:30). In this case, a strong band was observed, and this is shown at the bottom of the figure. The degree of platelet permeabilization in this experiment was determined by incubating fixed platelets for 15 minutes with rhodamine-phalloidin. Results obtained for intact (Int.) and permeabilized (Per.) platelets after 5 and 20 minutes of digitonin treatment are shown at the top of the figure. Phase contrast (PC) and fluoresence (F) fields of the preparations are shown at left and right, respectively.
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology

4. Immunostaining of platelets with MARCKS and CD41a antibodies
Immunostaining of platelets with MARCKS and CD41a antibodies.Intact platelets were fixed, permeabilized, and immunostained with either human MARCKS mouse monoclonal antibody (C and D) or human CD41a (fibrinogen receptor) mouse monoclonal antibody (E and F).
Immunostaining of platelets with MARCKS and CD41a antibodies.Intact platelets were fixed, permeabilized, and immunostained with either human MARCKS mouse monoclonal antibody (C and D) or human CD41a (fibrinogen receptor) mouse monoclonal antibody (E and F). The second antibody used in both cases was affinity-purifed Cy3-conjugated donkey Fab2 fragment raised against mouse IgG (dilution 1:200). Panels A and B show control platelets incubated only with the second antibody.
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology

5. Detection of MARCKS in platelet extracts and its phosphorylation during PKC activation.Platelets labeled with [32P]Pi were permeabilized with 15 μM of digitonin in K+-glutamate buffer for 5 minutes.
Detection of MARCKS in platelet extracts and its phosphorylation during PKC activation.Platelets labeled with [32P]Pi were permeabilized with 15 μM of digitonin in K+-glutamate buffer for 5 minutes. During the last 3 minutes of permeabilization, 100 nM of PMA in 0.05% DMSO (final concentration) or 0.05% DMSO (control) were present in the incubation medium. Reactions were stopped with RIPA buffer containing protease and phosphatase inhibitors. SDS-PAGE was performed on boiled extracts of these preparations to separate heat-stable proteins. Proteins were electrotransferred to nitrocellulose membranes, and autoradiography was first performed. This was followed by Western blotting with a mouse monoclonal antibody (4 μg/mL) raised against the C-terminal domain of human MARCKS. Two bands of 83 and 85 kd were detected by the antibody, which corresponded to 2 phosphorylated protein bands found in the autoradiography. Although PMA increased the phosphorylation of both MARCKS bands, the upper band was heavily phosphorylated, increasing the amount of slow-moving MARCKS (85 kd), as indicated by a much heavier immunoreactivity of this band in the PMA-treated preparations.
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology

6. Effects of peptides MPSD and Ala-MPSD on PKC-induced [3H]5-HT release from permeabilized platelets.[3H]5-HT–labeled platelets were permeabilized for 5 minutes with 15 μM of digitonin in K+-glutamate buffer.
Effects of peptides MPSD and Ala-MPSD on PKC-induced [3H]5-HT release from permeabilized platelets.[3H]5-HT–labeled platelets were permeabilized for 5 minutes with 15 μM of digitonin in K+-glutamate buffer. Permeabilization was done in the absence or presence of 10 μM of either peptide MPSD or peptide Ala-MPSD. MPSD has a 25–amino acid sequence corresponding to the phosphorylation site domain of MARCKS (A). This is the site for the binding of F-actin and calmodulin (A). In Ala-MPSD, the 4 serine residues of MPSD have been substituted by alanines (A). When indicated, PMA (100 nM) was present in the permeabilization medium during the last 3 minutes. Platelets were recovered by centrifugation and resuspended in the same medium for 45 seconds. At the end of this stimulation period, [3H]5-HT content was measured in the medium and in platelets. [3H]5-HT outputs were expressed as a percentage of total content after subtraction of spontaneous release (B). Each bar represents the mean ± SEM of results obtained from 3 different experiments (bottom). A minimum of 24 samples per condition were measured.
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology

7. Concentration-dependent inhibition by MPSD of [3H]5-HT output in response to PKC activation.Platelets were labeled with [3H]5-HT, permeabilized with digitonin, and incubated with 100 nM of PMA in the absence or presence of either MPSD or Ala-MPSD, as indica...
Concentration-dependent inhibition by MPSD of [3H]5-HT output in response to PKC activation.Platelets were labeled with [3H]5-HT, permeabilized with digitonin, and incubated with 100 nM of PMA in the absence or presence of either MPSD or Ala-MPSD, as indicated in the legend to Figure 5. Inhibition of PMA-induced [3H]5-HT output by different concentrations of MPSD was expressed as a percentage of the [3H]5-HT output in the presence of PMA. Ala-MPSD at the higher concentration tested (10 μM) did not modify the secretory response induced by PMA. Values represent mean ± SEM of 8 different platelet preparations.
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology

8. Effect of calmodulin and PIP2 on the inhibition by MPSD of [3H]5-HT output in response to PKC activation.Platelets were labeled with [3H]5-HT, permeabilized with digitonin, and incubated with 100 nM of PMA in the absence or presence of 10 μM of MPSD. When i...
Effect of calmodulin and PIP2 on the inhibition by MPSD of [3H]5-HT output in response to PKC activation.Platelets were labeled with [3H]5-HT, permeabilized with digitonin, and incubated with 100 nM of PMA in the absence or presence of 10 μM of MPSD. When indicated, 10 μM of either calmodulin or PIP2 were present in the incubation medium. [3H]5-HT outputs were expressed as a percentage of total content. Values represent mean ± SEM of at least 8 different platelet preparations.
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology

9. Effects of MPSD and Ala-MPSD on the phosphorylation of MARCKS induced by PKC activation.Platelets were labeled with [32P]Pi, permeabilized with digitonin, incubated with 100 nM PMA in the absence or presence of 10 μM of either MPSD or Ala-MPSD, and subseque...
Effects of MPSD and Ala-MPSD on the phosphorylation of MARCKS induced by PKC activation.Platelets were labeled with [32P]Pi, permeabilized with digitonin, incubated with 100 nM PMA in the absence or presence of 10 μM of either MPSD or Ala-MPSD, and subsequently stimulated as described in the legend to Figure 5. At the end of the stimulation periods, heat-stable platelet extracts were prepared, and their proteins were separated by SDS-PAGE as indicated in “Materials and methods.” Proteins were then electrotransferred to nitrocellulose membranes; these were exposed to hyperfilm, and the autoradiographies thus obtained were scanned as indicated in “Materials and methods.” Panel A shows the autoradiography of an experiment carried out to test the effect of MPSD on protein phosphorylation, and panel B shows a similar experiment performed with Ala-MPSD. Double arrows in A and B show the position of MARCKS, and single arrowheads indicate the position of myosin light chain (MLC). The open triangles in A indicate the position of proteins p25, p31, p50, and p66. The asterisk in A indicates the position of peptide MPSD, which was less phosphorylated in the absence (basal PKC activity) than in the presence of PMA (basal + stimulated PKC activity). Note the absence of a phosphorylated band when the experiment was performed with Ala-MPSD (B). On the right side in both panels, scannings of the autoradiographies are shown. The numbers beside the MARCKS and MLC peaks are arbitrary units obtained from computer integration of peak areas. Similar results were obtained in 3 other experiments (see Figure9B).
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology

10. Effects of MPSD and Ala-MPSD on the phosphorylation of pleckstrin (p47) and myosin light chain (p20) induced by PKC activation.(A) An experiment similar to that described in the legend to Figure 8was performed on [32P]PI–labeled platelets except that, here,...
Effects of MPSD and Ala-MPSD on the phosphorylation of pleckstrin (p47) and myosin light chain (p20) induced by PKC activation.(A) An experiment similar to that described in the legend to Figure 8was performed on [32P]PI–labeled platelets except that, here, total platelet extracts (heat-stable plus heat-sensitive proteins) were prepared. SDS-PAGE, electrotransfers, autoradiography, scanning, and integration of peak areas (arbitrary units) were performed as indicated in the legend to Figure 8 and in “Materials and methods.” The arrow and the arrowhead indicate the position of pleckstrin and myosin light chain (MLC), respectively. The asterisk shows the position of phosphorylated MPSD, which in this gel system migrated with another unknown phosphoprotein present in all lines. Pleckstrin and MLC phosphorylations were not modified by either MPSD or Ala-MPSD. (B) Cumulative data on protein phosphorylation obtained from experiments carried out on different platelet preparations are shown. Experiments were performed as described above in A for pleckstrin and MLC and as indicated in legend to Figure 8 for MARCKS. Bars represent mean ± SEM of [32P]Pi incorporation, expressed as a percentage of control (absence of PMA), obtained from 3 to 4 different experiments for each condition tested.
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology

11. PMA–concentration-dependent responses
PMA–concentration-dependent responses.Platelets were labeled with either [32P]Pi or [3H]5-HT, permeabilized with digitonin, and stimulated with increasing concentrations (1-500 nM) of PMA. [3H]5-HT release and protein phosphorylation were measured.
PMA–concentration-dependent responses.Platelets were labeled with either [32P]Pi or [3H]5-HT, permeabilized with digitonin, and stimulated with increasing concentrations (1-500 nM) of PMA. [3H]5-HT release and protein phosphorylation were measured. (A) Autoradiography of SDS-PAGE gels of heat-stable platelet extracts (MARCKS) and whole platelet extracts (pleckstrin) is shown. The concentrations of PMA used are indicated at the bottom of the figure. Arrowheads indicate the smallest concentrations of PMA producing a significant increase in the phosphorylation of each protein. (B) PMA–concentration-dependent curves for [3H]5-HT release and pleckstrin and MARCKS phosphorylation are shown. Values represent the mean ± SEM of 6 different platelet preparations.
Abdelbaset Elzagallaai et al. Blood 2000;95:
©2000 by American Society of Hematology