Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation.
Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin alphaIIbbeta3) is activated by agonist-mediated G(q) stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADP- and U46619-induced aggregation and anti-alphaIIbbeta3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially inhibited SFLLRN-induced aggregation, and was accompanied by diminished dense-granule secretion. When either PKC activation or intracellular calcium release was abrogated, aggregation and fibrinogen receptor activation with U46619 or SFLLRN was partially restored by additional selective activation of the G(i) signalling pathway. In contrast, when both PKC activity and intracellular calcium increase were simultaneously inhibited, the complete inhibition of aggregation that occurred in response to either U46619 or SFLLRN could not be restored with concomitant G(i) signalling. We conclude that, while the PKC- and calcium-regulated signalling pathways are capable of inducing activating fibrinogen receptor independently and that each can synergize with G(i) signalling to cause irreversible fibrinogen receptor activation, both pathways act synergistically to effect irreversible fibrinogen receptor activation.
Quinton
Todd M
TM
Department of Physiology, Temple University Medical School, 3420 N. Broad Street, Philadelphia, PA 19140, U.S.A.
Kim
Soochong
S
Dangelmaier
Carol
C
Dorsam
Robert T
RT
Jin
Jianguo
J
Daniel
James L
JL
Kunapuli
Satya P
SP
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D054641:Dual Specificity Phosphatase 2; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D039222:Integrin alpha1beta1 / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000187:drug effects / Q000378:metabolism; D039341:Receptors, Fibrinogen / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction; D013928:Thromboxane A2 / Q000378:metabolism
D004791:Enzyme Inhibitors; D007211:Indoles; D039222:Integrin alpha1beta1; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D039341:Receptors, Fibrinogen; C082835:thrombin receptor peptide (42-47); D004533:Egtazic Acid; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D011493:Protein Kinase C; D054648:Protein Phosphatase 2; C517032:DUSP2 protein, human; D054641:Dual Specificity Phosphatase 2; D017027:Protein Tyrosine Phosphatases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; C064758:Ro 31-8220