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The role of protein kinases in glycoprotein GMP-140 expression on activated human platelets
International Congress Series 1223 (2001) 117 – 121
The role of protein kinases in glycoprotein GMP-140 expression on activated human platelets Masaru Shojia,*, Ryoko Kudob, Moe Wakuia, Mari Nakanob, Shoji Tsutayab, Junko Saitob, Hideetsu Takamatsub, Minoru Yasujimaa a
Department of Laboratory Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan b Department of Clinical Laboratory, Hirosaki University Hospital, Japan
Abstract The expression of glycoprotein GMP-140 (P-selectin, CD-62P) is stimulated in human platelets by vasopressin via G protein-coupled receptors. In order to clarify the subsequent intracellular signal pathway, we examined the role of protein kinases in vasopressin action on thrombocytic CD62P expression in normal subjects. Staurosporine, a protein kinase (PK) C inhibitor, inhibited platelet aggregation and CD62P expression, whereas phorbol ester (PMA), a PKC stimulator, provoked them. On the other hand, 8-bromo-adenosine-30,50-monophosphate-cyclic AMP (8-Br-cAMP), a membrane permeant cAMP analogue, had no effect on platelet activation. PMI, a PKA inhibitor, had a little effect on them. From these results, it is suggested that PKC plays a dominant role in AVP action on platelet aggregation and CD62P expression. D 2001 Elsevier Science B.V. All rights reserved. Keywords: Platelet; CD62P; P-selectin; Vasopressin; Protein kinase C; Protein kinase A; 8-Br-cAMP; Staurosporine
1. Introduction Vasopressin is well known as an antidiuretic hormone secreted from the posterior pituitary. Vasopressin is also a vasoactive substance [1] and has been reported to stimulate platelet aggregation via its G protein-coupled membrane receptors. Both V1a and V2 *
Corresponding author. Tel.: +81-172-39-5124; fax: +81-172-39-5124. E-mail address: [email protected] (M. Shoji).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 4 2 6 - 5
118
M. Shoji et al. / International Congress Series 1223 (2001) 117–121
receptors are responsible for platelet activation [2 –4]. Vasopressin V1a action on the cardiovascular system is mediated via protein kinase C stimulation, whereas renomedullary V2 action is via protein kinase A [1,5]. However, downstream signal transductions within the platelets are uncertain [6,7]. Recently, we have found that vasopressin stimulates glycoprotein GMP-140 (CD62P) expression on human platelets in a dose-dependent manner [8]. CD62P is one of the surface markers and plays a role in platelet aggregation [9,10]. In order to clarify the downstream event of the stimulation, we identified the effects of inhibitors and stimulators for protein kinases on vasopressin-induced CD62P expression in normal subjects.
Fig. 1. The effects of PMA (A) and staurosporine (B) on vasopressin-induced CD62P expression.
M. Shoji et al. / International Congress Series 1223 (2001) 117–121
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2. Methods Twenty-seven normal healthy subjects were enrolled in the study. Blood samples were taken from individual antecubital veins, and whole peripheral blood was diluted with HEPES buffer. Selection of activated platelets by the expression of CD62P was made according to the simultaneity of the stainings with FITC-labeled anti-CD62P and PE-labeled anti-CD42b antibodies, as well as by the use of precise gating on the flow cytometer (Becton-Dickinson,
Fig. 2. The effects of 8-Br-cAMP (A) and PMI (B) on vasopressin-induced CD62P expression.
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M. Shoji et al. / International Congress Series 1223 (2001) 117–121
FACSCalibur) [11,12]. This expression of the surface markers was stimulated by the application of 10 5 M vasopressin. Inhibitors and stimulators for protein kinase A (PKA) and protein kinase C (PKC) used in this study were as follows: 10 5 M of 8-bromoadenosine-30,50-monophosphate-cyclic AMP (8-Br-cAMP), 10 5 M of protein kinase A inhibitor peptide, 10 5 M of phorbol 12-myristate 13-acetate (PMA), and 10 5 M of staurosporine. Data are shown as means and standard errors. Statistical significance between the groups was determined by ANOVA and subsequent Newman –Keul’s test. P values less than 0.05 were considered statistically significant.
3. Results Serial doses of AVP-provoked platelet aggregation and CD62P expression in a dosedependent manner within the range of 10 9 – 10 7 nM (data not shown). Phorbol ester (PMA), a PKC stimulator, provoked basal platelet CD62P expression ( p < 0.01) (Fig. 1A). It also strengthened AVP-induced platelet activation ( p < 0.01). Staurosporine, a PKC inhibitor, inhibited this ( p < 0.01) (Fig. 1B). On the other hand, 8-Br-cAMP, a membrane permeant cAMP analogue, had no effect on platelet activation (Fig. 2A). PMI, a PKA inhibitor, had little effect on them (Fig. 2B).
4. Discussion In the present study, PKC blocker showed significant inhibition of platelet CD62P expression induced by vasopressin. PMA itself provoked platelet activation. However, neither PKA stimulator nor inhibitor had a significant effect. The data are consistent in that PKC plays a dominant role in vasopressin-triggered platelet activation. Although vasopressin has been reported to stimulate platelet aggregation via its G protein-coupled membrane receptors, there has been controversy regarding the receptors involved. From pharmacological studies, Thibonnier et al. [3] concluded that V1a receptor agonists and antagonists appeared to have much more affinity. In contrast, Wun et al. [4] reported that desmopressin, a V2 receptor agonist, has a stimulatory effect on platelets. There is general agreement that vasopressin V1a action on cardiovascular system is mediated via protein kinase C stimulation, whereas reno-medullary V2 action is via protein kinase A [1,5]. However, downstream signal transductions within the platelets are uncertain. Therefore, the present findings support the conclusion that V1a receptors are of importance for mediating vasopressin action in platelets. Accumulated evidence suggests that PKC plays a significant role in activating platelets. PKC agonists and stimulants cause platelet aggregation. Platelet aggregation by thrombin has also been reported to be PKC-dependent [13]. CD62P is an adhesion molecule that plays a role in platelet aggregation and platelet –leukocyte interaction [10]. Activated CD62P expression is also sensitive to the PKC pathway [14]. Therefore, it is plausible that vasopressin shares a common pathway with other G protein-coupled membrane receptor stimulants.
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In conclusion, it is suggested that PKC plays a dominant role in the AVP stimulation of CD62P expression in human platelets.
Acknowledgements The authors are grateful to Miss Akiko Inaba for her expert secretarial assistance. The current study was supported in part by a Grant-in-Aid for Scientific Research (Nos. 09672347 and 12672236) from the Culture Ministry of Japan, the Karoji Medical Fund for Medical Research at Hirosaki University, Kurozumi Medical Foundation, Research Foundation for Community Medicine ‘‘Research Meeting on Hypertension and Arteriosclerosis’’, and the Charitable Trust Clinical Pathology Research Foundation of Japan.
References [1] L. Share, Role of vasopressin in cardiovascular regulation, Physiol. Rev. 68 (1988) 1248 – 1284. [2] M. Thibonnier, J.M. Roberts, Characterization of human platelet vasopressin receptors, J. Clin. Invest. 76 (1985) 1857 – 1864. [3] M. Thibonnier, A.L. Bayer, Z. Leng, Cytoplasmic and nuclear signaling pathways of V1-vascular vasopressin receptors, Regul. Pept. 45 (1993) 79 – 84. [4] T. Wun, T.G. Paglieroni, N.A. Lachant, Desmopressin stimulates the expression of P-selectin on human platelets in vitro, J. Lab. Clin. Med. 126 (1995) 401 – 409. [5] K. Ota, T. Kimura, M. Inoue, T. Funyu, M. Shoji, K. Sato, M. Ohta, T. Yamamoto, K. Abe, Effects of V1and V2-vasopressin (AVP) antagonists on the pressor, AVP and atrial natriuretic peptide responses to a hypertonic saline infusion in conscious anephric rats, Eur. J. Endocrinol. 133 (1995) 127 – 132. [6] D. Vittet, M.N. Mathieu, B. Cantau, C. Chevillard, Vasopressin inhibition of human platelet adenylate cyclase: variable responsiveness between donors and involvement of a G-protein different from Gi, Eur. J. Pharmacol. 150 (1988) 367 – 372. [7] W.K. Pollock, D.E. MacIntyre, Desensitization and antagonism of vasopressin-induced phosphoinositide metabolism and elevation of cytosolic free calcium concentration in human platelets, Biochem. J. 234 (1986) 67 – 73. [8] M. Shoji, M. Nakano, S. Tsutaya, H. Takamatsu, M. Yasujima, Vasopressin stimulates the membrane expression of P-selectin and GPIIb-IIIa on human platelets, 1999 World Congress on Neurohypophysial Hormones (Edinburgh, Scotland), 1999, pp. 106 (Abstracts). [9] T.F. Tedder, D.A. Steeber, A. Chen, P. Engel, The selectins: vascular adhesion molecules, FASEB J. 9 (1995) 866 – 873. [10] J. Yang, B.C. Furie, B. Furie, The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte – endothelial and leukocyte – platelet interaction, Thromb. Haemostasis 81 (1999) 1 – 7. [11] L. Corash, Measurement of platelet activation by fluorescence-activated flow cytometry, Blood Cells 16 (1990) 97 – 106. [12] W. Jy, L.L. Horstman, H. Park, W.W. Mao, P. Valant, Y.S. Ahn, Platelet aggregates as markers of platelet activation: characterization of flow cytometric method suitable for clinical applications, Am. J. Hematol. 57 (1998) 33 – 42. [13] M. Geanacopoulos, J. Turner, K.E. Bowling, S.R. Vandenberg, A.R. Gear, The role of protein kinase C in the initial events of platelet activation by thrombin assessed with a selective inhibitor, Thromb. Res. 69 (1993) 113 – 124. [14] T. Fujimoto, R.P. McEver, The cytoplasmic domain of P-selectin is phosphorylated on serine and threonine residues, Blood 82 (1993) 1758 – 1766.
The role of protein kinases in glycoprotein GMP-140 expression on activated human platelets Masaru Shojia,*, Ryoko Kudob, Moe Wakuia, Mari Nakanob, Shoji Tsutayab, Junko Saitob, Hideetsu Takamatsub, Minoru Yasujimaa a
Department of Laboratory Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan b Department of Clinical Laboratory, Hirosaki University Hospital, Japan
Abstract The expression of glycoprotein GMP-140 (P-selectin, CD-62P) is stimulated in human platelets by vasopressin via G protein-coupled receptors. In order to clarify the subsequent intracellular signal pathway, we examined the role of protein kinases in vasopressin action on thrombocytic CD62P expression in normal subjects. Staurosporine, a protein kinase (PK) C inhibitor, inhibited platelet aggregation and CD62P expression, whereas phorbol ester (PMA), a PKC stimulator, provoked them. On the other hand, 8-bromo-adenosine-30,50-monophosphate-cyclic AMP (8-Br-cAMP), a membrane permeant cAMP analogue, had no effect on platelet activation. PMI, a PKA inhibitor, had a little effect on them. From these results, it is suggested that PKC plays a dominant role in AVP action on platelet aggregation and CD62P expression. D 2001 Elsevier Science B.V. All rights reserved. Keywords: Platelet; CD62P; P-selectin; Vasopressin; Protein kinase C; Protein kinase A; 8-Br-cAMP; Staurosporine
1. Introduction Vasopressin is well known as an antidiuretic hormone secreted from the posterior pituitary. Vasopressin is also a vasoactive substance [1] and has been reported to stimulate platelet aggregation via its G protein-coupled membrane receptors. Both V1a and V2 *
Corresponding author. Tel.: +81-172-39-5124; fax: +81-172-39-5124. E-mail address: [email protected] (M. Shoji).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 4 2 6 - 5
118
M. Shoji et al. / International Congress Series 1223 (2001) 117–121
receptors are responsible for platelet activation [2 –4]. Vasopressin V1a action on the cardiovascular system is mediated via protein kinase C stimulation, whereas renomedullary V2 action is via protein kinase A [1,5]. However, downstream signal transductions within the platelets are uncertain [6,7]. Recently, we have found that vasopressin stimulates glycoprotein GMP-140 (CD62P) expression on human platelets in a dose-dependent manner [8]. CD62P is one of the surface markers and plays a role in platelet aggregation [9,10]. In order to clarify the downstream event of the stimulation, we identified the effects of inhibitors and stimulators for protein kinases on vasopressin-induced CD62P expression in normal subjects.
Fig. 1. The effects of PMA (A) and staurosporine (B) on vasopressin-induced CD62P expression.
M. Shoji et al. / International Congress Series 1223 (2001) 117–121
119
2. Methods Twenty-seven normal healthy subjects were enrolled in the study. Blood samples were taken from individual antecubital veins, and whole peripheral blood was diluted with HEPES buffer. Selection of activated platelets by the expression of CD62P was made according to the simultaneity of the stainings with FITC-labeled anti-CD62P and PE-labeled anti-CD42b antibodies, as well as by the use of precise gating on the flow cytometer (Becton-Dickinson,
Fig. 2. The effects of 8-Br-cAMP (A) and PMI (B) on vasopressin-induced CD62P expression.
120
M. Shoji et al. / International Congress Series 1223 (2001) 117–121
FACSCalibur) [11,12]. This expression of the surface markers was stimulated by the application of 10 5 M vasopressin. Inhibitors and stimulators for protein kinase A (PKA) and protein kinase C (PKC) used in this study were as follows: 10 5 M of 8-bromoadenosine-30,50-monophosphate-cyclic AMP (8-Br-cAMP), 10 5 M of protein kinase A inhibitor peptide, 10 5 M of phorbol 12-myristate 13-acetate (PMA), and 10 5 M of staurosporine. Data are shown as means and standard errors. Statistical significance between the groups was determined by ANOVA and subsequent Newman –Keul’s test. P values less than 0.05 were considered statistically significant.
3. Results Serial doses of AVP-provoked platelet aggregation and CD62P expression in a dosedependent manner within the range of 10 9 – 10 7 nM (data not shown). Phorbol ester (PMA), a PKC stimulator, provoked basal platelet CD62P expression ( p < 0.01) (Fig. 1A). It also strengthened AVP-induced platelet activation ( p < 0.01). Staurosporine, a PKC inhibitor, inhibited this ( p < 0.01) (Fig. 1B). On the other hand, 8-Br-cAMP, a membrane permeant cAMP analogue, had no effect on platelet activation (Fig. 2A). PMI, a PKA inhibitor, had little effect on them (Fig. 2B).
4. Discussion In the present study, PKC blocker showed significant inhibition of platelet CD62P expression induced by vasopressin. PMA itself provoked platelet activation. However, neither PKA stimulator nor inhibitor had a significant effect. The data are consistent in that PKC plays a dominant role in vasopressin-triggered platelet activation. Although vasopressin has been reported to stimulate platelet aggregation via its G protein-coupled membrane receptors, there has been controversy regarding the receptors involved. From pharmacological studies, Thibonnier et al. [3] concluded that V1a receptor agonists and antagonists appeared to have much more affinity. In contrast, Wun et al. [4] reported that desmopressin, a V2 receptor agonist, has a stimulatory effect on platelets. There is general agreement that vasopressin V1a action on cardiovascular system is mediated via protein kinase C stimulation, whereas reno-medullary V2 action is via protein kinase A [1,5]. However, downstream signal transductions within the platelets are uncertain. Therefore, the present findings support the conclusion that V1a receptors are of importance for mediating vasopressin action in platelets. Accumulated evidence suggests that PKC plays a significant role in activating platelets. PKC agonists and stimulants cause platelet aggregation. Platelet aggregation by thrombin has also been reported to be PKC-dependent [13]. CD62P is an adhesion molecule that plays a role in platelet aggregation and platelet –leukocyte interaction [10]. Activated CD62P expression is also sensitive to the PKC pathway [14]. Therefore, it is plausible that vasopressin shares a common pathway with other G protein-coupled membrane receptor stimulants.
M. Shoji et al. / International Congress Series 1223 (2001) 117–121
121
In conclusion, it is suggested that PKC plays a dominant role in the AVP stimulation of CD62P expression in human platelets.
Acknowledgements The authors are grateful to Miss Akiko Inaba for her expert secretarial assistance. The current study was supported in part by a Grant-in-Aid for Scientific Research (Nos. 09672347 and 12672236) from the Culture Ministry of Japan, the Karoji Medical Fund for Medical Research at Hirosaki University, Kurozumi Medical Foundation, Research Foundation for Community Medicine ‘‘Research Meeting on Hypertension and Arteriosclerosis’’, and the Charitable Trust Clinical Pathology Research Foundation of Japan.
References [1] L. Share, Role of vasopressin in cardiovascular regulation, Physiol. Rev. 68 (1988) 1248 – 1284. [2] M. Thibonnier, J.M. Roberts, Characterization of human platelet vasopressin receptors, J. Clin. Invest. 76 (1985) 1857 – 1864. [3] M. Thibonnier, A.L. Bayer, Z. Leng, Cytoplasmic and nuclear signaling pathways of V1-vascular vasopressin receptors, Regul. Pept. 45 (1993) 79 – 84. [4] T. Wun, T.G. Paglieroni, N.A. Lachant, Desmopressin stimulates the expression of P-selectin on human platelets in vitro, J. Lab. Clin. Med. 126 (1995) 401 – 409. [5] K. Ota, T. Kimura, M. Inoue, T. Funyu, M. Shoji, K. Sato, M. Ohta, T. Yamamoto, K. Abe, Effects of V1and V2-vasopressin (AVP) antagonists on the pressor, AVP and atrial natriuretic peptide responses to a hypertonic saline infusion in conscious anephric rats, Eur. J. Endocrinol. 133 (1995) 127 – 132. [6] D. Vittet, M.N. Mathieu, B. Cantau, C. Chevillard, Vasopressin inhibition of human platelet adenylate cyclase: variable responsiveness between donors and involvement of a G-protein different from Gi, Eur. J. Pharmacol. 150 (1988) 367 – 372. [7] W.K. Pollock, D.E. MacIntyre, Desensitization and antagonism of vasopressin-induced phosphoinositide metabolism and elevation of cytosolic free calcium concentration in human platelets, Biochem. J. 234 (1986) 67 – 73. [8] M. Shoji, M. Nakano, S. Tsutaya, H. Takamatsu, M. Yasujima, Vasopressin stimulates the membrane expression of P-selectin and GPIIb-IIIa on human platelets, 1999 World Congress on Neurohypophysial Hormones (Edinburgh, Scotland), 1999, pp. 106 (Abstracts). [9] T.F. Tedder, D.A. Steeber, A. Chen, P. Engel, The selectins: vascular adhesion molecules, FASEB J. 9 (1995) 866 – 873. [10] J. Yang, B.C. Furie, B. Furie, The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte – endothelial and leukocyte – platelet interaction, Thromb. Haemostasis 81 (1999) 1 – 7. [11] L. Corash, Measurement of platelet activation by fluorescence-activated flow cytometry, Blood Cells 16 (1990) 97 – 106. [12] W. Jy, L.L. Horstman, H. Park, W.W. Mao, P. Valant, Y.S. Ahn, Platelet aggregates as markers of platelet activation: characterization of flow cytometric method suitable for clinical applications, Am. J. Hematol. 57 (1998) 33 – 42. [13] M. Geanacopoulos, J. Turner, K.E. Bowling, S.R. Vandenberg, A.R. Gear, The role of protein kinase C in the initial events of platelet activation by thrombin assessed with a selective inhibitor, Thromb. Res. 69 (1993) 113 – 124. [14] T. Fujimoto, R.P. McEver, The cytoplasmic domain of P-selectin is phosphorylated on serine and threonine residues, Blood 82 (1993) 1758 – 1766.