GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6-/- platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5\u27-diphosphate stimulation in GRK6-/- platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation

Binding of CIB1 to the αIIb tail of αIIbβ3 is required for FAK recruitment and activation in platelets.

It is believed that activation of c-Src bound to the integrin β3 subunit initiates outside-in signaling. The involvement of αIIb in outside-in signaling is poorly understood.We have previously shown that CIB1 specifically interacts with the cytoplasmic domain of αIIb and is required for αIIbβ3 outside-in signaling. Here we evaluated the role of CIB1 in regulating outside-in signaling in the absence of inside-out signaling.We used αIIb cytoplasmic domain peptide and CIB1-function blocking antibody to inhibit interaction of CIB1 with αIIb subunit as well as Cib1-/- platelets to evaluate the consequence of CIB1 interaction with αIIb on outside-in signaling.Fibrinogen binding to αIIbβ3 results in calcium-dependent interaction of CIB1 with αIIb, which is not required for filopodia formation. Dynamic rearrangement of cytoskeleton results in CIB1-dependent recruitment of FAK to the αIIb complex and its activation. Disruption of the association of CIB1 and αIIb by incorporation of αIIb peptide or anti-CIB1 inhibited both FAK association and activation. Furthermore, FAK recruitment to the integrin complex was required for c-Src activation. Inhibition of c-Src had no effect on CIB1 accumulation with the integrin at the filopodia, suggesting that c-Src activity is not required for the formation of CIB1-αIIb-FAK complex.Our results suggest that interaction of CIB1 with αIIb is one of the early events occurring during outside-in signaling. Furthermore, CIB1 recruits FAK to the αIIbβ3 complex at the filopodia where FAK is activated, which in turn activates c-Src, resulting in propagation of outside-in signaling leading to platelet spreading

Schematic representation of the initial steps of outside-in signaling through α_IIbβ₃.

<p>Inactive α_IIbβ₃ in resting platelets complexed with JAM-A (left). Initial steps of outside-in signaling initiated by Fg binding to α_IIbβ₃ prior to the association of CIB1 leading to filopodia formation (middle). Signaling complex recruited by CIB1 to the α_IIb tail leading to Src activation and platelet spreading (right).</p

Association of c-Src with CIB1-FAK complex augments its activity.

<p>(A) Washed platelets were exposed to BSA or adhered to Fg for 45 min. The pre-cleared detergent lysates of these platelets were immunoprecipitated with anti-CIB1 or cIgG. Autoradiogram (AR) of MBP phosphorylation (upper panel). Western blot of immunoprecipitates probed with anti-Src (upper middle), and with anti-FAK (lower middle). The membrane was reprobed with anti-CIB1 to ensure equal amount of protein in the precipitates (lower panel). (B) Densitometric analysis of co-immunoprecipitated c-Src (upper middle blot in (A) (**<i>P < 0</i>.<i>01</i>). Error bars indicate mean ± SEM of at least three independent experiments. (C) Src kinase activity in CIB1 immunoprecipitates from platelets attached to Fg compared to BSA (***<i>P<0</i>.<i>001</i>).</p

Interaction of CIB1 with α_IIbβ₃ requires intracellular calcium rise.

<p>(A) DIC images of human platelets treated as indicated. (B) Confocal images of platelets in suspension pre-treated with or without BAPTA-AM (50 μM) and then allowed to spread on immobilized Fg-coated coverglass for 45 min. Accumulation of CIB1 at the filopodia and the membrane periphery are indicated by arrows; original magnification, X 1600. (C) Western blot analysis of cIgG or anti-CIB1 immunoprecipitates of lysates of platelets treated as in B. Input represents sample of platelet lysate before immunoprecipitation. α_IIb bands are shown in upper blot. Same blot was reprobed with anti-CIB1 to ensure an equal amount of protein in the immunoprecipitates (lower blot). (D) Densitometric analysis of coimmunoprecipitated α_IIb bands normalized to corresponding CIB1 band (**<i>P<0</i>.<i>01</i>). Error bars indicate mean ± SEM of at least three independent experiments.</p

Dynamic cytoskeletal rearrangement is required for activation of FAK.

<p>(A) Confocal images of vehicle treated (DMSO) platelets, of platelets pre-treated with cytochalasin D (10 μM) for 15 min (Pre-CD) and then allowed to spread on Fg for 30 min, of platelets allowed to spread for 30 min prior to treatment with cytochalasin D for additional 15 min (Post-CD). Arrow indicates CIB1 staining at the tip of the filopodia. Arrowhead indicates filopodia lacking CIB1 staining; original magnification, X 1600. (B) Western blot analysis of anti-CIB1 immunoprecipitates of lysates from platelets treated as in A. Top blot shows phosphorylated Y³⁹⁷FAK. The blot was reprobed for total FAK co-immunoprecipitated with CIB1 (upper middle), and for CIB1 to ensure equal amount of protein in the immunoprecipitates (bottom). Samples from B were also blotted for α_IIb (lower middle). Shown are the representative blots from three separate experiments performed independently.</p

Association of CIB1 with α_IIb is needed for platelet spreading but not filopodia formation.

<p>(A) Western blot analysis of anti-CIB1 immunoprecipitates of lysates from platelets incorporated with either α_IIb peptide (2.5 μM) or anti-CIB1 antibody (0.1 mg/ml) as indicated. DMSO, scrambled α_IIb peptide (2.5 μM) and irrelevant isotype specific antibody (cIgG, 0.1 mg/ml) were used as control. Upper blot shows the co-immunoprecipitation of α_IIb. Same blot was reprobed with anti-CIB1 to ensure equal amount of protein in the immunoprecipitates (lower blot). Input represents sample of platelet lysate prior to immunoprecipitation. (B) Quantitation of α_IIb bands from (A) normalized to corresponding CIB1 band. (<i>*P<0</i>.<i>05)</i> (C) Confocal images of platelets incorporated with α_IIb peptide, anti-CIB1 or corresponding controls DMSO, scrambled α_IIb peptide, or cIgG and were allowed to spread on Fg for 45 min and stained for F-actin; boxed area is enlarged to visualize colocalization (arrows); non-specific red speckles are shown by arrowheads; original magnification, X 1600. (D) Quantitation of platelets from C showing CIB1 staining at the filopodia (***<i>P<0</i>.<i>001</i>). Error bars indicate mean ± SEM of at least three independent experiments.</p