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Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader
Published on: May 24, 2024
Protein kinase C activates non-capacitative calcium entry in human platelets
1Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
The Journal of Physiology
|November 18, 2000
Summary
Human platelets possess a novel non-capacitative calcium (Ca2+) entry pathway, distinct from store-mediated mechanisms. This pathway is activated independently of intracellular calcium store depletion, offering new insights into platelet calcium regulation.
Area of Science:
- Cell Biology
- Physiology
- Biochemistry
Background:
- Calcium (Ca2+) influx in non-excitable cells is primarily governed by intracellular Ca2+ store filling.
- Store-mediated (capacitative) Ca2+ entry is thought to involve endoplasmic reticulum-plasma membrane coupling.
- The existence of alternative Ca2+ entry pathways in human platelets remains an area of investigation.
Purpose of the Study:
- To investigate the presence and characteristics of a non-capacitative calcium (Ca2+) entry mechanism in human platelets.
- To differentiate this novel pathway from established store-mediated Ca2+ entry.
- To elucidate the signaling pathways regulating this additional Ca2+ influx.
Main Methods:
- Measurement of cytosolic Ca2+ and Sr2+ using the fluorescent indicator fura-2 in human platelets.
- Stimulation of Ca2+ stores using thapsigargin and ionomycin.
- Activation of platelets with thrombin and protein kinase C (PKC) activators (phorbol-12-myristate-13-acetate, 1-oleoyl-2-acetyl-sn-glycerol).
- Inhibition studies using Ro-31-8220 (PKC inhibitor) and xestospongin C (IP3 receptor antagonist).
Main Results:
- Thrombin stimulation induced further divalent cation entry after store depletion, indicating a distinct pathway.
- Direct activation of protein kinase C (PKC) stimulated cation entry independently of Ca2+ store release.
- This non-capacitative entry was not inhibited by jasplakinolide and occurred even when store-mediated entry was blocked.
Conclusions:
- Human platelets exhibit a non-capacitative divalent cation entry pathway regulated independently of store-mediated entry.
- This pathway does not necessitate endoplasmic reticulum-plasma membrane coupling.
- Receptor-evoked Ca2+ entry in platelets can occur independently of Ca2+ store depletion, potentially via G-protein-coupled receptors and PKC activation.
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