[Inhibition of protein kinase A leads to cleavage of platelet GP I balpha and downregulation of GP I b-dependent platelet aggregation].
OBJECTIVE
To explore the regulatory role of protein kinase A (PKA) in platelet surface glycoprotein (GP) I balpha expression.


METHODS
Washed platelets from healthy volunteers were incubated with PKA inhibitor. The N-terminal fragment of GP I balpha (glycocalicin, GC) in the supernatant of platelet suspensions was detected by Western blot and GP I balpha surface expression by flow cytometry. Calpain activity was determined by cytoskeletal proteins proteolysis and calpain surface expression by flow cytometry. The effect of PKA inhibitor on ristocetin-induced platelet aggregation was measured by platelet aggregometer.


RESULTS
After PKA was inhibited in washed platelets, GP I balpha was cleaved and released to the supernatant, which significantly decreased the surface expression of GP I balpha (P < 0.05). The event was suppressed by pre-treatment with various calpain inhibitors, indicating that PKA inhibitor-mediated shedding was calpain dependent. The actin-binding protein (ABP) and talin proteolysis demonstrated that calpain was activated by PKA inhibitor and expressed on the platelet membrane. Ristocetin-induced aggregation was inhibited by PKA inhibitor.


CONCLUSIONS
PKA inhibition results in calpain-dependent GP I balpha shedding, which thus reduces GP I balpha surface expression and GP I balpha-dependent platelet aggregation. These results might provide a view to develop new drugs for thrombotic diseases.
Yan
Rong
R
School of Biological Science and Medical Engineering, Beijing University of Aeronautics & Astronautics, Beijing 100191, China.
Dai
Ke-Sheng
KS
D001792:Blood Platelets / Q000187:drug effects*; D002154:Calpain / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D005434:Flow Cytometry; D006801:Humans; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis
D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D017868:Cyclic AMP-Dependent Protein Kinases; D002154:Calpain