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Protein kinase Cε and protein kinase Cθ double-deficient mice have a bleeding diathesis
A J Unsworth1, B A Finney, L Navarro-Nunez
1Department of Biochemistry, University of Oxford, UK.
Background:
In comparison to the classical isoforms of protein kinase C (PKC), the novel isoforms are thought to play minor or inhibitory roles in the regulation of platelet activation and thrombosis.
Objectives:
To measure the levels of PKCθ and PKCε and to investigate the phenotype of mice deficient in both novel PKC isoforms.
Methods:
Tail bleeding and platelet activation assays were monitored in mice and platelets from mice deficient in both PKCθ and PKCε.
Results:
PKCε plays a minor role in supporting aggregation and secretion following stimulation of the collagen receptor GPVI in mouse platelets but has no apparent role in spreading on fibrinogen. PKCθ, in contrast, plays a minor role in supporting adhesion and filopodial generation on fibrinogen but has no apparent role in aggregation and secretion induced by GPVI despite being expressed at over 10 times the level of PKCε. Platelets deficient in both novel isoforms have a similar pattern of aggregation downstream of GPVI and spreading on fibrinogen as the single null mutants. Strikingly, a marked reduction in aggregation on collagen under arteriolar shear conditions is observed in blood from the double but not single-deficient mice along with a significant increase in tail bleeding.
Conclusions:
These results reveal a greater than additive role for PKCθ and PKCε in supporting platelet activation under shear conditions and demonstrate that, in combination, the two novel PKCs support platelet activation.
Insights
Novel protein kinase C (PKC) isoforms, PKCθ and PKCε, play a greater than additive role in supporting platelet activation under shear conditions. Combined deficiency significantly increases tail bleeding and reduces platelet aggregation.
Area of Science:
- Hematology
- Molecular Biology
- Biochemistry
Background:
- Classical protein kinase C (PKC) isoforms are well-studied in platelet regulation.
- Novel PKC isoforms (PKCθ and PKCε) were previously thought to have minor or inhibitory roles in platelet activation and thrombosis.
Purpose of the Study:
- To quantify PKCθ and PKCε levels in platelets.
- To investigate the functional consequences of combined PKCθ and PKCε deficiency in mice.
Main Methods:
- Assessed tail bleeding times in wild-type and knockout mice.
- Performed platelet aggregation and activation assays.
- Studied platelet adhesion and spreading on fibrinogen.
Main Results:
- PKCε minimally supports GPVI-stimulated aggregation and secretion but not spreading.
- PKCθ minimally supports fibrinogen adhesion and filopodial generation but not GPVI-induced aggregation.
- Combined PKCθ and PKCε deficiency markedly reduces collagen-induced platelet aggregation under shear stress.
- Double-deficient mice exhibit significantly increased tail bleeding times.
Conclusions:
- PKCθ and PKCε have a synergistic role in platelet activation under shear conditions.
- The combined absence of PKCθ and PKCε significantly impairs platelet function and hemostasis.
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