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

Platelet-derived Toll-like receptor 4 (Tlr-4) is sufficient to promote microvascular thrombosis in endotoxemia.

Endotoxin (lipopolysaccharide, LPS) produced by gram-negative bacteria initiates a host of pro-inflammatory effects through Toll-like receptor 4 (TLR-4). We reported previously that LPS enhances microvascular thrombosis in cremaster venules of wild-type mice, but had no effect in mice deficient in TLR-4. Since TLR-4 is expressed on various cell types, the cellular origin of TLR-4 responsible for the LPS-enhanced thrombosis remains undetermined. Platelets are known to express functional TLR-4. Platelet-derived TLR-4 has been suggested to mediate various inflammatory responses in endotoxemia, including production of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β), two cytokines reported to enhance microvascular thrombosis. We determined whether platelet-derived TLR-4 was sufficient to mediate the enhanced thrombosis induced by endotoxin and whether these responses were accompanied by systemic increases in TNF-α and IL-1β. We isolated platelets from wild-type mice and transfused them into either of two strains of TLR-4-deficient mice (C57BL/10ScN and B6.B10ScN-TLR-4(lps-del)/Jth). The mice were then injected with LPS or saline, and the kinetics of thrombosis were studied 4 hours later. Transfusion of wild-type platelets restored responsiveness to LPS in TLR-4-deficient mice with regards to microvascular thrombosis but not to plasma levels of TNF-α or IL-1β. The accelerated rates of microvascular thrombosis induced by platelet transfusions were specific to TLR-4, since isolation and transfusion of platelets derived from TLR-4-deficient donors did not restore responsiveness to LPS. These studies demonstrate that platelet-derived TLR-4 is sufficient to promote microvascular thrombosis in endotoxemia, independent of systemic increases in TNF-α or IL-1β

Differential effects of l-NAME on rat venular hydraulic conductivity

The role of nitric oxide (NO) in microvascular permeability remains unclear because both increases and decreases in permeability by NO synthase (NOS) inhibitors have been reported. We sought to determine whether blood-borne constituents modify venular permeability responses to the NOS inhibitorNG-nitro-l-arginine methyl ester (l-NAME). We assessed hydraulic conductivity (Lp) of pipette-perfused rat mesenteric venules before and after exposure to 10-4 M l-NAME. In the absence of blood-borne constituents, l-NAME reducedLp by nearly 50% (from a median of 2.4 × 10-7cm · s-1 · cmH2O-1,n = 17, P \u3c 0.001). The reduction inLp by l-NAME was inhibited by a 10-fold molar excess of l-arginine but notd-arginine (n = 6). In a separate group of venules, blood flow was allowed to resume during exposure tol-NAME. In vessels perfused by blood duringl-NAME exposure, Lp increased by 78% (from 1.4 × 10-7cm · s-1 · cmH2O-1,n = 10, P \u3c 0.01).NG-nitro-d-arginine methyl ester did not affect Lp in either of the two groups. These data imply that NO has direct vascular effects on permeability that are opposed by secondary changes in permeability mediated by blood-borne constituents

Mitochondria Are Fast Ca 2؉ Sinks in Rat Extraocular Muscles: A Novel Regulatory Influence on Contractile Function and Metabolism

] i and force significantly more in extraocular muscles. Mitochondrial volume density and capillary density were three times greater, and citrate synthase and cytochrome c oxidase were only ϳ2-fold higher in extraocular muscle. Calcineurin A␣, calcineurin B, and peroxisome proliferator activated receptor (PPAR)␥ were more abundant in extraocular muscle. CONCLUSIONS. These data support the hypothesis that mitochondria serve as Ca 2ϩ sinks in extraocular muscles. The high mitochondrial content of these muscles may partly reflect this additional function. It is likely that mitochondrial Ca 2ϩ influx increases the dynamic response range of the extraocular muscles and matches metabolic demand to supply. (Invest Ophthalmol Vis Sci. 2005;46:4541-4547

Dissociation between Corneal and Cardiometabolic Changes in Response to a Time-Restricted Feeding of a High Fat Diet

Mice fed a high fat diet (HFD) ab libitum show corneal dysregulation, as evidenced by decreased sensitivity and impaired wound healing. Time-restricted (TR) feeding can effectively mitigate the cardiometabolic effects of an HFD. To determine if TR feeding attenuates HFD-induced corneal dysregulation, this study evaluated 6-week-old C57BL/6 mice fed an ad libitum normal diet (ND), an ad libitum HFD, or a time-restricted (TR) HFD for 10 days. Corneal sensitivity was measured using a Cochet-Bonnet aesthesiometer. A corneal epithelial abrasion wound was created, and wound closure was monitored for 30 h. Neutrophil and platelet recruitment were assessed by immunofluorescence microscopy. TR HFD fed mice gained less weight (p < 0.0001), had less visceral fat (p = 0.015), and had reduced numbers of adipose tissue macrophages and T cells (p < 0.05) compared to ad libitum HFD fed mice. Corneal sensitivity was reduced in ad libitum HFD and TR HFD fed mice compared to ad libitum ND fed mice (p < 0.0001). Following epithelial abrasion, corneal wound closure was delayed (~6 h), and neutrophil and platelet recruitment was dysregulated similarly in ad libitum and TR HFD fed mice. TR HFD feeding appears to mitigate adipose tissue inflammation and adiposity, while the cornea remains sensitive to the pathologic effects of HFD feeding

A Low-Fat/Sucrose Diet Rich in Complex Carbohydrates Reverses High-Fat/Sucrose Diet-Induced Corneal Dysregulation

High-fat/sucrose diet feeding in mice causes loss of corneal nerve function and impairs corneal wound healing. While changing to a diet with a low fat/sugar composition and enrichments in complex carbohydrates mitigates the reduction in nerve function, it remains to be determined if it has an effect on corneal wound healing. In this study, 6-week-old C57BL/6 male mice were fed either a normal diet or a high-fat/sucrose diet for 20 weeks. A third group (diet reversal) was placed on a high-fat/sucrose diet for 10 weeks followed by a normal diet for an additional 10 weeks. A central corneal epithelial abrasion wound was created, and wound closure was monitored. Neutrophil and platelet recruitment was assessed by immunofluorescence microscopy. Mice fed the high-fat/sucrose diet-only had greater adiposity (p p ≤ 0.01) and, at 30 h post-wounding, fewer neutrophils reached the wound center and fewer extravascular platelets were present at the limbus (p < 0.05). Diet restored normal wound closure and neutrophil and platelet influx in the injured cornea. These data suggest compositional changes to the diet may be an effective diet-based therapeutic strategy for maintaining or restoring corneal health

Platelets from wild-type mice do not restore LPS-enhanced cytokine release in TLR-4-deficient mice.

<p>Plasma cytokine levels of TNF-α (A) and IL-1β (B) were measured by ELISA in response to LPS in wild-type mice, TLR-4-deficient mice, and TLR-4-deficient mice transfused with platelets from wild-type mice. All TLR-4-deficient mice were of a C57BL/6 background. Data shown as mean ± SE, n = 4 per group. Analysis was done with one-way ANOVA with Bonferroni correction.</p

Platelets from wild-type mice restore LPS-enhanced thrombosis in TLR-4-deficient mice in a BL/10 background.

<p>TLR-4-deficient mice in a C57BL/10 background were transfused with platelets from either wild-type (A) or TLR-4-deficient (B) mice and exposed to saline or LPS. Microvascular thrombosis kinetics were assessed by a light-dye injury model using intravital microscopy. Initial platelet adhesion to the microvascular wall (Onset) and vessel occlusion for 60 seconds (Flow Cessation) were recorded in minutes. Data shown as mean ± SE, n = 12 per group, *:p<0.05 (for the comparison between saline and LPS by unpaired t-tests).</p

Platelet aggregation responses are comparable in wild-type and TLR-4-deficient mice.

<p>Maximal aggregation of platelet-rich plasma from TLR-4-deficient and wild-type mice in the presence of platelet agonist ADP (A) or collagen (B) across dose ranges. All TLR-4-deficient mice were of a C57BL/6 background. Data shown as mean ± SE, n = 4 to 8 per group. Analysis was done with one-way ANOVA with Bonferroni correction.</p