Functional Relationship Between Cyclic AMP-dependent Protein Phosphorylation and Platelet Inhibition

Overview

Journal Biochem J

Specialty Biochemistry

Date 1990 Nov 1

PMID 1700902

Citations 9

Authors

W Siess

E G Lapetina

Affiliations

Soon will be listed here.

Abstract

Exposure of human platelets to prostacyclin (PGI2), iloprost or prostaglandin E1 (PGE1) elicits the cyclic AMP-dependent phosphorylation of proteins of 22, 24, 30, 39, 50, 60 and 250 kDa (P22, P24 etc.). P22 was recently identified as rap 1B, a ras-like protein, and P24 was shown to be the beta-chain of glycoprotein Ib. We found that cyclic AMP-dependent phosphorylation of all proteins except P22 was maximal 1 min after exposure of platelets to PGI2, iloprost or PGE1; maximal phosphorylation of P22 occurred after 45 min of incubation. Inhibition of thrombin-induced platelet activation required only a 30 s incubation with PGI2 or iloprost; at this time phosphorylation of P22 was only slightly increased. Although at maximal concentrations PGI2 was more potent than PGE1 in inhibiting thrombin-induced platelet activation, no difference in the degree and the kinetics of cyclic AMP-dependent protein phosphorylation was found. Platelets that had been preincubated and washed in the presence of PGE1 and later resuspended in the absence of PGE1 responded fully to activation by thrombin despite maximal phosphorylation of P22 and P24. Furthermore, addition of PGI2 to PGE1-washed platelets prevented thrombin-induced platelet activation, but did not evoke further phosphorylation of P22 or P24. Phosphorylation of P39 and P50 correlated better with PGI2-induced inhibition of platelet activation. In experiments in which PGE1-induced inhibition of platelet activation was overcome by the addition of thrombin, no dephosphorylation of proteins phosphorylated by cyclic AMP-dependent kinases was observed. These experiments indicate that: (a) phosphorylation of rap 1B and glycoprotein Ib is not related to platelet inhibition by cyclic AMP; (b) phosphorylation of other proteins such as P39 and P50 probably plays a role in mediating cyclic AMP-dependent platelet inhibition; (c) reactions other than cyclic AMP-dependent protein phosphorylation may participate in platelet inhibition by cyclic AMP.

Citing Articles

Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS.

Swieringa F, Solari F, Pagel O, Beck F, Huang J, Feijge M Sci Rep. 2020; 10(1):11389.

PMID: 32647264 PMC: 7347634. DOI: 10.1038/s41598-020-68379-3.

New Concepts and Mechanisms of Platelet Activation Signaling.

Estevez B, Du X Physiology (Bethesda). 2017; 32(2):162-177.

PMID: 28228483 PMC: 5337829. DOI: 10.1152/physiol.00020.2016.

Activated platelets enhance ovarian cancer cell invasion in a cellular model of metastasis.

Holmes C, Levis J, Ornstein D Clin Exp Metastasis. 2009; 26(7):653-61.

PMID: 19444623 DOI: 10.1007/s10585-009-9264-9.

Interaction of antiplatelet drugs in vitro: aspirin, iloprost, and the nitric oxide donors SIN-1 and sodium nitroprusside.

Negrescu E, Grunberg B, Kratzer M, Lorenz R, Siess W Cardiovasc Drugs Ther. 1995; 9(4):619-29.

PMID: 8547213 DOI: 10.1007/BF00878095.

Magnier C, Bredoux R, Kovacs T, Quarck R, Papp B, Corvazier E Biochem J. 1994; 297 ( Pt 2):343-50.

PMID: 8297341 PMC: 1137835. DOI: 10.1042/bj2970343.

References

Lapetina E, Lacal J, REEP B, Molina y Vedia L . A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets. Proc Natl Acad Sci U S A. 1989; 86(9):3131-4. PMC: 287079. DOI: 10.1073/pnas.86.9.3131. View

Siess W, Lapetina E . Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases. Biochem J. 1989; 258(1):57-65. PMC: 1138323. DOI: 10.1042/bj2580057. View

Kawata M, Kikuchi A, Hoshijima M, Yamamoto K, Hashimoto E, Yamamura H . Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets. J Biol Chem. 1989; 264(26):15688-95. View

Siess W . Molecular mechanisms of platelet activation. Physiol Rev. 1989; 69(1):58-178. DOI: 10.1152/physrev.1989.69.1.58. View

Hoshijima M, Kikuchi A, Kawata M, Ohmori T, Hashimoto E, Yamamura H . Phosphorylation by cyclic AMP-dependent protein kinase of a human platelet Mr 22,000 GTP-binding protein (smg p21) having the same putative effector domain as the ras gene products. Biochem Biophys Res Commun. 1988; 157(3):851-60. DOI: 10.1016/s0006-291x(88)80953-7. View

Edwards R, MacDermot J, Wilkins A . Prostacyclin analogues reduce ADP-ribosylation of the alpha-subunit of the regulatory Gs-protein and diminish adenosine (A2) responsiveness of platelets. Br J Pharmacol. 1987; 90(3):501-10. PMC: 1917175. DOI: 10.1111/j.1476-5381.1987.tb11199.x. View

Molina y Vedia L, REEP B, Lapetina E . Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein. Proc Natl Acad Sci U S A. 1988; 85(16):5899-902. PMC: 281872. DOI: 10.1073/pnas.85.16.5899. View

Pizon V, Chardin P, Lerosey I, Olofsson B, Tavitian A . Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region. Oncogene. 1988; 3(2):201-4. View

Yoshida K, Nachmias V . Phorbol ester stimulates calcium sequestration in saponized human platelets. J Biol Chem. 1987; 262(33):16048-54. View

10.

Taylor S . cAMP-dependent protein kinase. Model for an enzyme family. J Biol Chem. 1989; 264(15):8443-6. View

11.

Tsai A, Hsu M, Vijjeswarapu H, Wu K . Solubilization of prostacyclin membrane receptors from human platelets. J Biol Chem. 1989; 264(1):61-7. View

12.

Halbrugge M, Walter U . Purification of a vasodilator-regulated phosphoprotein from human platelets. Eur J Biochem. 1989; 185(1):41-50. DOI: 10.1111/j.1432-1033.1989.tb15079.x. View

13.

ORourke F, Zavoico G, Feinstein M . Release of Ca2+ by inositol 1,4,5-trisphosphate in platelet membrane vesicles is not dependent on cyclic AMP-dependent protein kinase. Biochem J. 1989; 257(3):715-21. PMC: 1135647. DOI: 10.1042/bj2570715. View

14.

Siess W, Winegar D, Lapetina E . Rap1-B is phosphorylated by protein kinase A in intact human platelets. Biochem Biophys Res Commun. 1990; 170(2):944-50. DOI: 10.1016/0006-291x(90)92182-y. View

15.

WHITE T, Lacal J, Reep B, Fischer T, Lapetina E, White 2nd G . Thrombolamban, the 22-kDa platelet substrate of cyclic AMP-dependent protein kinase, is immunologically homologous with the Ras family of GTP-binding proteins. Proc Natl Acad Sci U S A. 1990; 87(2):758-62. PMC: 53345. DOI: 10.1073/pnas.87.2.758. View

16.

Halbrugge M, Friedrich C, Eigenthaler M, Schanzenbacher P, Walter U . Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets in response to cGMP- and cAMP-elevating vasodilators. J Biol Chem. 1990; 265(6):3088-93. View

17.

McKay D, Steitz T . Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA. Nature. 1981; 290(5809):744-9. DOI: 10.1038/290744a0. View

18.

Knight D, Scrutton M . Cyclic nucleotides control a system which regulates Ca2+ sensitivity of platelet secretion. Nature. 1984; 309(5963):66-8. DOI: 10.1038/309066a0. View

19.

KASER-GLANZMANN R, Gerber E, LUSCHER E . Regulation of the intracellular calcium level in human blood platelets: cyclic adenosine 3',5'-monophosphate dependent phosphorylation of a 22,000 dalton component in isolated Ca2+-accumulating vesicles. Biochim Biophys Acta. 1979; 558(3):344-7. DOI: 10.1016/0005-2736(79)90271-2. View

20.

Haslam R, Lynham J, Fox J . Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets. Biochem J. 1979; 178(2):397-406. PMC: 1186528. DOI: 10.1042/bj1780397. View