Regulation of calcium mobilization and entry in human platelets by endothelium-derived factors

1994 ◽  
Vol 267 (1) ◽  
pp. C236-C244 ◽  
Author(s):  
J. Geiger ◽  
C. Nolte ◽  
U. Walter
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Human Platelets ◽  
Dependent Protein Kinase ◽  
Rapid Phase ◽  
No Donor ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
Camp And Cgmp ◽  
Stimulation Of

Stimulation of Ca2+ mobilization and entry by agonists such as ADP, thrombin, and thromboxane is an early step of platelet activation. Here, we compared the effects of adenosine 3',5'-cyclic monophosphate (cAMP)-elevating prostaglandins, guanosine 3',5'-cyclic monophosphate (cGMP)-elevating nitrovasodilators, membrane-permeant selective activators of cAMP- or cGMP-dependent protein kinases, and physiological endothelium-derived factors on the agonist-evoked Ca2+ mobilization and entry in human platelets. Prostaglandin E1, the prostacyclin analogue Iloprost, the nitric oxide (NO) donor 3-morpholinosydnonimine hydrochloride, and selective activators of cGMP- or cAMP-dependent protein kinase strongly inhibited the agonist-evoked Ca2+ mobilization from intracellular stores and associated late Ca2+ entry but had little effects on the rapid (1st) phase of ADP-evoked Ca2+ entry. During coincubation of platelets with endothelial cells, endothelium-derived factors that were released strongly inhibited platelet agonist-evoked Ca2+ mobilization and only moderately affected the rapid phase of ADP-evoked Ca2+ entry. These effects were partially prevented when endothelial cells were preincubated with cyclooxygenase and/or NO synthase inhibitors. Endothelial cells therefore produce sufficient quantities of labile platelet inhibitors whose effects on the platelet Ca2+ response resemble those observed with selective cAMP- and cGMP-dependent protein kinase activators.

Platelet Activating Factor (PAF) Stimulates Protein Kinase (PK) Activity In Human Platelets

1981 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer ◽  
L M Cagen ◽  
E E Muirhead
Keyword(s):  
Platelet Aggregation ◽  
Protein Kinase ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Alkyl Ether ◽  
Dependent Protein Kinase ◽  
Proteolytic Activation ◽  
Dependent Protein ◽  
Stimulation Of

Kishimoto et al.(J.B.C.255:2273,1980) have demonstrated a Ca2+ and phospholipid-dependent protein kinase (PK) from various mammalian tissues which is markedly stimulated by the addition of unsaturated diacylglycerol. PAF, an alkyl ether analogue of phosphatidyl choline induces platelet aggregation and secretion. We investigated the ability of the C18:0 analogue (l-alkyl-2acetyl-sn-glycero-3-phosphocholine) to stimulate Ca2+ dependent PK activity in homogenates of human platelets. Two hundred ml whole blood was collected in EDTA(5mM) and EGTA(5mM). PGl2(534nM) was included to prevent platelet activation during preparation. Platelets were washed twice in 0.05M Tris containing EDTA, EGTA,and PGl2, pH 7.5. On the 3rd wash PGI2 was omitted. The platelet pellet was then resuspended in the same buffer now containing leu- peptin(0.2mM) to inhibit proteolytic activation of PK. The suspension was sonicated and centrifuged at 100,000xg for 1 hr. PK activity was assayed in the supernatant and pellet suspension by measuring the incorporation of 32P into HI histone from [γ32P]ATP. The standard reaction mixture (0.3 ml) contained 250yl supernatant or pellet suspension, HI histone(60μg), [γ32P]ATP(3nmoles),magnesium acetate (13.3mM) diolein(500ng) Ca2+ and PAF for 3 min. at 30°.Basal PK activity was 14.6pmol/min/mg protein. PAF(0.8μg) which is just saturating dose for in vitro platelet aggregation, stimulate PK activity by 70% in the supernatant but was without effect on the pellet suspension. In the absence of Ca2+ and/or diolein there was no stimulation of PK by PAF. Phosphatidyl serine(PS)(5μg) also stimulated protein kinase by 100%.Stimulation of PK by both PAF and PS occurred at endogenous platelet Ca2+ concentrations (i.e. sufficient Ca2+ added to titrate EDTA and EGTA) and at higher Ca2+ concentration (by 0.2mM.)Supernatants from platelets prepared in the absence of PGI2 were not stimulated by PAF These data show that PAF activates a Ca2+ dependent protein kinase which may mediate its effects on human platelets.

scholarly journals A study of 3′:5′-cyclic mononucleotide-dependent protein kinase from canine prostate glands

1974 ◽  
Vol 144 (2) ◽  
pp. 377-383 ◽  
Author(s):  
B K Tsang ◽  
R L Singhal
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Metal Ion ◽  
Maximum Velocity ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Diethylaminoethyl Cellulose ◽  
Dependent Protein ◽  
Stimulation Of

1. An adenosine 3′:5′-cyclic monophosphate (cyclic AMP)-dependent protein kinase, located predominantly in the cytosol, was studied in canine prostate. 2. The enzyme exhibited cyclic AMP-binding activity, and could be isolated by chromatography on diethylaminoethyl cellulose. 3. The enzyme was maximally stimulated (fourfold) by 1μm-cyclic AMP, and half-maximal activation of the enzyme was observed in presence of 50nm-cyclic AMP. 4. Equilibrium studies at pH5.0 indicated the presence of one major class of binding site for cyclic AMP, with an association constant of approx. 108m-1. 5. Stimulation of the enzyme was also observed with the 3′:5′-cyclic monophosphate derivatives of cytidine, inosine, guanosine and uridine as well as with dibutyryl cyclic AMP, but higher concentrations of these cyclic nucleotides were required to provide the same degree of activation as that seen with cyclic AMP. 6. Comparing α-casein, protamine and different histone subfractions as substrates, highest cyclic AMP stimulation was demonstrated with histones. 7. Although maximum velocity of the enzyme was enhanced approximately fivefold in presence of cyclic AMP, kinetic studies indicated that the apparent Km for histone (0.5mg/ml) remained the same whether determined in the presence or absence of the cyclic nucleotide. 8. In addition, cyclic AMP did not significantly change the apparent Km for ATP (1.2×10-5m). 9. The purified enzyme showed an absolute requirement for bivalent metal ion. Substitution of Mn2+for Mg2+decreased basal protein kinase activity as well as the stimulation noted with cyclic AMP. Similarly, the basal activity was lowered when Mg2+was replaced by Ca2+and cyclic AMP produced only little stimulation of the prostatic enzyme.

Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cells

1995 ◽  
Vol 268 (1) ◽  
pp. C171-C180 ◽  
Author(s):  
K. S. Murthy ◽  
G. M. Makhlouf
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Muscle Cells ◽  
Rabbit Muscle ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
Camp And Cgmp

The signaling pathways mediating relaxation by vasoactive intestinal peptide (VIP), peptide histidine-isoleucine amide (PHI), isoproterenol (ISO), and sodium nitroprusside (SNP) were examined in dispersed rabbit and guinea pig gastric muscle cells. In rabbit muscle cells, SNP stimulated only guanosine 3',5'-cyclic monophosphate (cGMP) and cGMP-dependent protein kinase (cG-kinase) activity; VIP stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP, and both cG-kinase and cAMP-dependent protein kinase (cA-kinase) activities; PHI and ISO stimulated only cAMP and cA-kinase activity, and at higher concentrations, cross-activated cG-kinase. All four agents elicited concentration-dependent relaxation. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1 microM) selectively inhibited cA-kinase activity and abolished relaxation when only cA-kinase was elevated. 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c,d,e)- trinden-1-one (KT-5823; 1 microM) selectively inhibited cG-kinase activity and abolished relaxation when only cG-kinase was elevated. When both kinases were elevated, H-89 and KT-5823 partially inhibited relaxation and abolished relaxation in combination. In permeabilized guinea pig and rabbit muscle cells, all agents elicited relaxation and inhibited inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Both functions were inhibited in parallel fashion by protein kinase inhibitor PKI(6-22) and by KT-5823. We conclude that cA-kinase and cG-kinase act separately and in concert to inhibit IP3-dependent Ca2+ release and induce relaxation.

A predominant role for cAMP-dependent protein kinase in the cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein and platelet inhibition in humans

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4423-4429 ◽  
Author(s):  
Zhenyu Li ◽  
Jasna Ajdic ◽  
Martin Eigenthaler ◽  
Xiaoping Du
Keyword(s):  
Protein Kinase ◽  
Intracellular Signaling ◽  
Platelet Inhibition ◽  
Human Platelets ◽  
Predominant Role ◽  
Dependent Protein Kinase ◽  
No Donor ◽  
Vasp Phosphorylation ◽  
Dependent Protein

Abstract The vasodilator-stimulated phosphoprotein (VASP) plays an important role in cGMP-induced platelet inhibition. Since VASP is an in vitro substrate for cGMP-dependent protein kinase (PKG), it has been presumed that VASP phosphorylation induced by cGMP is mediated by PKG. Here we show that, in human platelets, phosphorylation of VASP at Ser239 induced by either cGMP analogs or nitric oxide (NO) donor glyco-SNAP1 is inhibited by PKA inhibitors KT5720, PKI, Rp-Br-cAMPS, and H89, but not by PKG inhibitors KT5823 or Rp-pCPT-cGMPS. Unlike human platelets, cGMP analog–induced phosphorylation of VASP in mouse platelets is inhibited by both PKG and PKA inhibitors. Ineffectiveness of PKG inhibitors in inhibiting VASP phosphorylation in human platelets is not due to an inability to inhibit PKG, as these PKG inhibitors but not PKA inhibitors inhibit a different cGMP-induced intracellular signaling event: phosphorylation of extracellular signal–responsive kinase. Furthermore, PKA inhibitors reverse cGMP-induced inhibition of thrombin-induced platelet aggregation, whereas PKG inhibitors further enhance the inhibitory effect of cGMP analogs. Thus, PKA plays a predominant role in the cGMP-induced phosphorylation of VASP and platelet inhibition in human platelets.

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