The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

BACKGROUND:Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin alpha(IIb)beta(3). Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with alpha(IIb)beta(3), the role of PP1c in platelet reactivity is unclear. METHODOLOGY/PRINCIPAL FINDINGS:Using gamma isoform of PP1c deficient mice (PP1cgamma(-/-)), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cgamma(-/-) platelets showed decreased alpha(IIb)beta(3) activation despite comparable levels of alpha(IIb)beta(3), PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin alpha(IIb)beta(3) signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cgamma(-/-) platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cgamma(-/-) mice. Phosphorylation of glycogen synthase kinase (GSK3)beta-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cgamma(-/-) platelets by an AKT independent mechanism. Inhibition of GSK3beta partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cgamma(-/-) platelets. CONCLUSIONS/SIGNIFICANCE:These studies illustrate a role for PP1cgamma in maintaining GSK3beta-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation

Expression of thrombin receptors, α_IIbβ₃ and P-selectin in PP1cγ^−/− platelets.

<p>(<b>A</b>) Lysate from resting washed platelets was evaluated by SDS-PAGE and immunoblotted with anti-PAR3 antibody (∼43 kDa). The blot was stripped and reprobed with anti- PAR4 (∼38 kDa) and then with anti-actin (loading) antibodies. Blots are representative of two experiments. Washed platelets were incubated with anti-JON/A PE antibody (<b>B</b>) or anti-α_IIb FITC antibody (<b>C</b>) or anti-P-selectin antibody (<b>D</b>) and subjected to flow cytometry. Results are expressed as ±SEM of 5–6 experiments. Compared to thrombin stimulated WT platelets, the decreased binding of JON/A antibody to PP1cγ^−/− platelets was significant at *p = 0.003.</p

Thrombin specific impairment in soluble fibrinogen binding and aggregation in PP1cγ^−/− platelets.

<p>Washed platelets from WT or PP1cγ^−/− mice were stimulated with ADP, collagen or CRP (<b>A</b>), thrombin (<b>B</b>) or PAR4-AP (<b>C</b>) in the presence of Alexa 488 fibrinogen and bound fibrinogen analyzed by flow cytometry as mean fluorescence intensity (MFI). Data are ±SEM of 9–11 experiments for ADP, thrombin and collagen and 13–14 experiments for PAR4-AP. As compared to the WT platelets, the decreased fibrinogen binding in PP1cγ^−/− platelets were significant at *p = 0.02 (for 0.01 U/ml thrombin); #p = 0.01 (for 0.02 U/ml thrombin); *p = 0.05 (for 200 µM PAR4-AP). In other experiments (n = 3), platelets were pretreated with 0.5 mM EDTA or 2 mM apyrase (ADP scavenger) prior to stimulation with 0.02 U/ml thrombin. (D) Percentage aggregation for WT and PP1cγ^−/− platelets in response to the indicated doses of thrombin, ADP and collagen. Studies are indicated as ±SEM of 5–6 experiments. The decreased aggregation in 0.02 U/ml thrombin stimulated PP1cγ^−/− platelets compared to WT platelets was significant at *p = 0.04.</p

Decreased phosphorylation of GSK3β-Ser9 in thrombin stimulated PP1cγ^−/− platelets.

<p>(<b>A</b>). Platelet lysate from resting (0) and thrombin activated (0.005, 0.01 and 0.02 U/ml) was separated on an SDS-PAGE and immunoblotted with antibodies to AKT-Ser473, GSK3β- Ser9 and AKT (total). (<b>B and C</b>) Densitometric quantification of phosphorylation levels for AKT-Ser473 and GSK3β-Ser9 in WT and PP1cγ^−/− stimulated platelets. The decreased phosphorylation of GSK3β-Ser9 in thrombin-stimulated PP1cγ^−/− platelets was significant at ♦p = 0.05 (for 0.01 U/ml thrombin); *p = 0.02 (for 0.02 U/ml thrombin). Data are expressed as ±SEM of 5 experiments. (<b>D</b>) Effect of GSK3β inhibitor VIII on thrombin-induced fibrinogen binding on WT and PP1cγ^−/− platelets. Compared with WT platelets, PP1cγ^−/− platelets displayed ∼40% decreased fibrinogen binding in response to 0.02 U/ml thrombin in the presence of solvent control (DMSO) (*P = 0.048) and GSK3 inhibitor VIII (GSK3β) partially reduced the difference to 23%. Data are expressed as ±SEM of 3–4 experiments.</p

Role of PP1cγ in thrombosis and hemostasis injury models.

<p>(<b>A</b>). Light/dye -induced microvascular thrombosis was studied in the venules of the cremaster muscles of WT and PP1cγ^−/− mice by intra vital microscopy. The onset of thrombosis (onset) and the cessation of blood flow following the injury were monitored. Results are expressed as ±SEM of 13 experiments. Compared to the WT, the delayed time for flow cessation in PP1cγ^−/− mice was significant at *p = 0.03. (<b>B</b>). Tail vein bleeding times from 13 WT and PP1cγ^−/− mice are shown.</p

Outside-in integrin α_IIbβ₃ signaling functions in PP1cγ^−/− platelets.

<p>(<b>A</b>). Washed platelets were incubated with immobilized fibrinogen-coated micro titer wells for the indicated periods of time and adhesion quantified. The data are expressed as ±SEM of 3 experiments. (<b>B</b>) Fibrin clot retraction was examined following the addition of thrombin to platelet-rich plasma. Volume of the clot is expressed as ±SEM of 3 experiments.</p

PP1c isoforms in platelets.

<p>(<b>A–C</b>) Platelet lysate from wild type (WT) or PP1cγ^−/− mice were evaluated by SDS-PAGE and Western blotting using isoform specific antibodies to α, β and γ (recognizes γ1 and γ2 splice variants). These membranes were stripped and reprobed with actin to demonstrate equal protein loading (lower panels). Blots are representative of three independent experiments.</p