The catalytic subunit of protein phosphatase 1 gamma regulates thrombin-induced murine platelet alpha(IIb)beta(3) function

Abstract

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.

Figure 1. PP1c isoforms in platelets.

(A–C) 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.

Figure 2. Thrombin specific impairment in soluble fibrinogen binding and aggregation in PP1cγ^−/− platelets.

Washed platelets from WT or PP1cγ^−/− mice were stimulated with ADP, collagen or CRP (A), thrombin (B) or PAR4-AP (C) 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.

Figure 3. Expression of thrombin receptors, α_IIbβ₃ and P-selectin in PP1cγ^−/− platelets.

(A) 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) or anti-α_IIb FITC antibody (C) or anti-P-selectin antibody (D) 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.

Figure 4. Outside-in integrin α_IIbβ₃ signaling functions in PP1cγ^−/− platelets.

(A). 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) 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.

Figure 5. Role of PP1cγ in thrombosis and hemostasis injury models.

(A). 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). Tail vein bleeding times from 13 WT and PP1cγ^−/− mice are shown.

Figure 6. Decreased phosphorylation of GSK3β-Ser9 in thrombin stimulated PP1cγ^−/− platelets.

(A). 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 and C) 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. (D) 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.