Prolonged inhibition of protein kinase A results in metalloproteinase-dependent platelet GPIbalpha shedding.
BACKGROUND
The interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbalpha ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbalpha shedding. Here we show that prolonged inhibition of protein kinase A (PKA) results in metalloproteinase-dependent GPIbalpha shedding.


RESULTS
GPIbalpha was shed from platelets prolongedly incubated with PKA inhibitors in a dose-dependent manner. In platelets treated with PKA inhibitor H89, the level of GPIbalpha shedding was significantly higher than that in calcium ionophore or alpha-thrombin treated platelets, however, P-selectin surface expression was significantly lower. PKA inhibition mediated GPIbalpha shedding was reversed by PKA activator forskolin and partially inhibited by membrane-permeable calpain inhibitors. Furthermore, the metalloproteinase inhibitor GM6001 or EDTA completely inhibited H89 induced GPIbalpha shedding, indicating that it was metalloproteinase-dependent. Time course experiments revealed that the maximum GPIbalpha shedding occurred at 30 minutes after treatment with PKA inhibitor. Platelets prolongedly treated with PKA inhibitor exhibited significant decrease in botrocetin-induced aggregation and shear-induced adhesion on VWF.


CONCLUSIONS
These data show that prolonged inhibition of PKA results in metalloproteinase-dependent platelet GPIbalpha ectodomain shedding. This finding has physiological implications for hemostasis and limiting thrombus infinite formation after platelet activation, and it also suggests a novel strategy to develop new drugs for thrombotic diseases.
Dai
Kesheng
K
Department of Biological Science and Technology, Beihang University, Haidian district, Beijing, China. kdai@buaa.edu.cn
Yan
Rong
R
Li
Suping
S
Fan
Yubo
Y
Zhuang
Fengyuan
F
Ruan
Changgeng
C
D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003435:Crotalid Venoms / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex; D011480:Protease Inhibitors / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D014841:von Willebrand Factor / Q000378:metabolism
D002614:Chelating Agents; D003435:Crotalid Venoms; D004151:Dipeptides; D047908:Intracellular Signaling Peptides and Proteins; D007476:Ionophores; D007546:Isoquinolines; D008562:Membrane Glycoproteins; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D019007:P-Selectin; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D011480:Protease Inhibitors; D013449:Sulfonamides; C094990:adhesion receptor; C094363:protein kinase inhibitor (6-22)-amide; D014841:von Willebrand Factor; D005576:Colforsin; C030961:botrocetin; D004492:Edetic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D045726:Metalloproteases; D013917:Thrombin; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium