Characterization of a novel focal adhesion kinase inhibitor in human platelets.
Focal adhesion kinase (FAK) is activated in human platelets downstream of integrins, e.g. alpha(IIb)beta(3), and other adhesion receptors e.g. GPVI. Mice in which platelets lack FAK have been shown to exhibit extended bleeding times and their platelets have been shown to display decreased spreading on fibrinogen-coated surfaces. Recently, a novel FAK inhibitor (PF-573,228) has become available, its selectivity for FAK shown in vitro and in cell lines. We determined the effect of this inhibitor on platelet function and signaling pathways. Like murine platelets lacking FAK, we found that PF-573,228 was effective at blocking human platelet spreading on fibrinogen-coated surfaces but did not affect the initial adhesion. We also found a reduced spreading on CRP-coated surfaces. Further analysis of the morphology of platelets adhered to these surfaces showed the defect in spreading occurred at the transition from filopodia to lamellipodia. Similar to that seen with murine neutrophils lacking FAK, we also observed an unexpected defect in intracellular calcium release in human platelets pre-treated with PF-573,228 which correlated with impaired dense granule secretion and aggregation. The aggregation defect could be partially rescued by addition of ADP, normally secreted from dense granules, suggesting that PF-573,228 has effects on FAK downstream of alpha(IIb)beta(3) and elsewhere. Our data show that PF-573,228 is a useful tool for analysis of FAK function in cells and reveal that in human platelets FAK may regulate a rise in cell calcium and platelet spreading.
Jones
Matthew L
ML
Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
Shawe-Taylor
Amelia J
AJ
Williams
Christopher M
CM
Poole
Alastair W
AW
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors; D051379:Mice; D010974:Platelet Aggregation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors; D015363:Quinolones / Q000494:pharmacology*; D013450:Sulfones / Q000494:pharmacology*; D000072377:Syk Kinase; D054462:p21-Activated Kinases / Q000037:antagonists & inhibitors
C521108:6-(4-(3-(methylsulfonyl)benzylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-3,4-dihydroquinolin-2(1H)-one; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D015363:Quinolones; D013450:Sulfones; D000244:Adenosine Diphosphate; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D054462:p21-Activated Kinases; D002118:Calcium