Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition

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.

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Possible Roles for Protein Phosphorylation in Platelet Responses

10.1055/s-0039-1687474 ◽

1979 ◽

Author(s):

R.J. Haslam ◽

J.E.B. Fox ◽

S.E. Salama ◽

J.A. Lynham

Keyword(s):

Cyclic Amp ◽

Protein Phosphorylation ◽

Protein Kinases ◽

Platelet Function ◽

Subcellular Fractionation ◽

Human Platelets ◽

Type I ◽

Sds Page ◽

Dependent Protein ◽

32P Incorporation

The relationships between the phosphorylation of specific platelet polypeptides and platelet function were studied using washed human platelets labelled by preincubation with [32p] Pi. Platelet polypeptides were separated by SDS-PAGE and 32P incorporation into them determined by autoradiography. Whereas induction of platelet aggregation alone did not affect protein phosphorylation, induction of the release reaction increased 3P incorporation into several polypeptides (P75,P47,P40,P27,P20,P19), including the P-light chain of platelet myosin (P20). These changes were inhibited by drugs that blocked Ca2 movements and may be due to activation of Ca2+-dependent protein kinases. Compounds that inhibited platelet function by increasing cyclic AMP (e.g. PCE1) also suppressed these reactions but, in addition, increased phosphorylation of other polypeptides (P50,P49,P36,P24,P22). Type I and Type II cyclic AMP-dependent protein kinases were present in platelets and may mediate Che latter effects of cyclic AMP. Subcellular fractionation of 32p-labelled platelets that had been exposed to PCE1 showed that P24 was present in membranes that could take up Ca2+ by an ATP-dependent mechanism. Membranes from PCE1-treated platelets took up Ca2+ more rapidly than control membranes. Thus, the cyclic AMP-dependent phosphorylation of P24 may stimulate the removal of Ca2+ from platelet cytosol and suppress Ca2+-dependent phosphorylation reactions necessary for release of granule constituents.

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Functional Relationship between Cyclic AMP-Dependent Protein Рhosphorylation and Platelet Inhibition

Advances in Experimental Medicine and Biology - Mechanisms of Platelet Activation and Control ◽

10.1007/978-1-4615-2994-1_18 ◽

1993 ◽

pp. 229-235 ◽

Cited By ~ 11

Author(s):

Wolfgang Siess ◽

Bernd Grünberg ◽

Karin Luber

Keyword(s):

Cyclic Amp ◽

Functional Relationship ◽

Platelet Inhibition ◽

Dependent Protein

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Defective nitrovasodilator-stimulated protein phosphorylation and calcium regulation in cGMP-dependent protein kinase-deficient human platelets of chronic myelocytic leukemia

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)38681-8 ◽

1993 ◽

Vol 268 (18) ◽

pp. 13526-13531

Author(s):

M. Eigenthaler ◽

H. Ullrich ◽

J. Geiger ◽

K. Horstrup ◽

P. Hönig-Liedl ◽

...

Keyword(s):

Protein Kinase ◽

Protein Phosphorylation ◽

Calcium Regulation ◽

Human Platelets ◽

Chronic Myelocytic Leukemia ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Myelocytic Leukemia ◽

Dependent Protein

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Ca2+-induced insulin secretion from electrically permeabilized islets. Loss of the Ca2+-induced secretory response is accompanied by loss of Ca2+-induced protein phosphorylation

Biochemical Journal ◽

10.1042/bj2850973 ◽

1992 ◽

Vol 285 (3) ◽

pp. 973-978 ◽

Cited By ~ 24

Author(s):

P M Jones ◽

S J Persaud ◽

S L Howell

Keyword(s):

Protein Kinase C ◽

Insulin Secretion ◽

Protein Kinase ◽

Cyclic Amp ◽

Protein Phosphorylation ◽

Prolonged Exposure ◽

Kinase C ◽

Dependent Protein ◽

32P Incorporation ◽

Rat Islets Of Langerhans

Increasing the cytosolic Ca2+ concentration of electrically permeabilized rat islets of Langerhans caused rapid increases in insulin secretion and in 32P incorporation into islet proteins. However, the secretory responsiveness of permeabilized islets was relatively transient, with insulin secretion approaching basal levels within 20-30 min despite the continued presence of stimulatory concentrations of Ca2+. The loss of Ca2(+)-induced insulin secretion was accompanied by a marked reduction in Ca2(+)-dependent protein phosphorylation, but not in cyclic AMP-dependent protein phosphorylation. Similarly, permeabilized islets which were no longer responsive to Ca2+ were able to mount appropriate secretory responses to cyclic AMP and to a protein kinase C-activating phorbol ester. These results suggest that prolonged exposure to elevated cytosolic Ca2+ concentrations results in a specific desensitization of the secretory mechanism to Ca2+, perhaps as a result of a decrease in Ca2(+)-dependent kinase activity. Furthermore, these studies suggest that secretory responses of B-cells to cyclic AMP and activators of protein kinase C are not dependent upon the responsiveness of the cells to changes in cytosolic Ca2+.

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Glycoprotein V is not the thrombin activation receptor on human blood platelets

Blood ◽

10.1182/blood.v68.3.720.bloodjournal683720 ◽

1986 ◽

Vol 68 (3) ◽

pp. 720-725 ◽

Cited By ~ 1

Author(s):

D Bienz ◽

W Schnippering ◽

KJ Clemetson

Keyword(s):

Platelet Activation ◽

Polyclonal Antibodies ◽

Blood Platelets ◽

Polyacrylamide Gel Electrophoresis ◽

Human Platelets ◽

Exchange Chromatography ◽

Thrombin Activation ◽

Strong Candidate ◽

Triton X ◽

Kinetics Of

Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.

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