The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin alpha 2 beta 1.
BACKGROUND
Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets.


OBJECTIVE
We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line.


METHODS
An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells.


RESULTS
We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1.


CONCLUSIONS
Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.
School of Animal and Microbial Sciences, University of Reading, Reading, UK.
J M
JM
Stevens
P A
PA
Jordan
T
T
Sage
J M
JM
Gibbins
D000730:Androstadienes / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D047468:Immunoprecipitation; D038982:Integrin alpha2beta1 / Q000096:biosynthesis*; D008533:Megakaryocytes / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics*; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D000077191:Wortmannin; D000099200:Scaffold Protein ILK
D000730:Androstadienes; D003094:Collagen; D004791:Enzyme Inhibitors; D038982:Integrin alpha2beta1; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D013917:Thrombin; D000077191:Wortmannin; D000099200:Scaffold Protein ILK