Protein tyrosine phosphatase PTPN22 negatively modulates platelet function and thrombus formation.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a protein tyrosine phosphatase that negatively regulates T-cell signaling. However, whether it is expressed and functions in platelets remains unknown. Here we investigated the expression and role of PTPN22 in platelet function. We reported PTPN22 expression in both human and mouse platelets. Using PTPN22-/- mice, we showed that PTPN22 deficiency significantly shortened tail-bleeding time and accelerated arterial thrombus formation without affecting venous thrombosis and the coagulation factors VIII and IX. Consistently, PTPN22-deficient platelets exhibited enhanced platelet aggregation, granule secretion, calcium mobilization, lamellipodia formation, spreading, and clot retraction. Quantitative phosphoproteomic analysis revealed the significant difference of phosphodiesterase 5A (PDE5A) phosphorylation in PTPN22-deficient platelets compared with wild-type platelets after collagen-related peptide stimulation, which was confirmed by increased PDE5A phosphorylation (Ser92) in collagen-related peptide-treated PTPN22-deficient platelets, concomitant with reduced level and vasodilator-stimulated phosphoprotein phosphorylation (Ser157/239). In addition, PTPN22 interacted with phosphorylated PDE5A (Ser92) and dephosphorylated it in activated platelets. Moreover, purified PTPN22 but not the mutant form (C227S) possesses intrinsic serine phosphatase activity. Furthermore, inhibition of PTPN22 enhanced human platelet aggregation, spreading, clot retraction, and increased PDE5A phosphorylation (Ser92). In conclusion, our study shows a novel role of PTPN22 in platelet function and arterial thrombosis, identifying new potential targets for future prevention of thrombotic or cardiovascular diseases.

Dimethyl fumarate inhibits antibody-induced platelet destruction in immune thrombocytopenia mouse.

BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune disease characterized as a low platelet count resulting from immune-mediated platelet destruction. Dimethyl fumarate (DMF) is widely applied for the treatment of several autoimmune diseases with immunosuppressive effect. However, whether it ameliorates ITP is unclear. This study aims to evaluate whether DMF has a preventive effect on ITP in mice. METHODS: DMF (30, 60 or 90 mg/kg body weight) was intraperitoneally injected into mice followed by injection of rat anti-mouse integrin GPIIb/CD41antibody to induce ITP. Peripheral blood was isolated to measure platelet count and spleen mononuclear cells were extracted to measure Th1 and Treg cells along with detecting the levels of IFN-γ, and TGFß-1 in plasma and CD68 expression in spleen by immuohistochemical staining. Additionally, macrophage cell line RAW264.7 was cultured and treated with DMF followed by analysis of cell apoptosis and cycle, and the expression of FcγRI, FcγRIIb and FcγRIV mRNA. RESULTS: DMF significantly inhibited antiplatelet antibody-induced platelet destruction, decreased Th1 cells and the expression of T-bet and IFN-γ, upregulated Treg cells and the expression of Foxp3 and TGF-ß1 as well as reduced CD68 expression in the spleen of ITP mouse. DMF-treated RAW264.7 cells showed S-phase arrest, increased apoptosis and downregulated expression of FcγRI and FcγRIV. Meanwhile, in vitro treatment of DMF also decreased the expression of cyclin D1 and E2, reduced Bcl-2 level and increased Bax expression and caspase-3 activation. CONCLUSIONS: In conclusion, DMF prevents antibody-mediated platelet destruction in ITP mice possibly through promoting apoptosis, indicating that it might be used as a new approach for the treatment of ITP.

Tacrolimus ameliorates thrombocytopenia in an ITP mouse model.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by lower platelet count resulting from immune cells-mediated platelet clearance. Tacrolimus is an immunosuppressive agent which selectively inhibits T cell activation. Whether tacrolimus plays a role in ITP remains unclear. This study aimed to investigate the effect of tacrolimus on ITP in mice. An ITP mouse model was established by injection of rat anti-mouse integrin GPIIb/CD41 immunoglobulin and treated with tacrolimus followed by isolation of peripheral blood mononuclear cells and plasma. The mRNA expression of T-bet, GATA3, and Foxp3 was measured by RT-PCR, and level of IFN-γ, IL-12p70, IL-4, IL-13, and TGF-ß in plasma was measured by ELISA. Tacrolimus inhibited antiplatelet antibody-mediated platelet clearance in ITP mouse model. Meanwhile, tacrolimus-treated ITP mice displayed a significant decrease in the mRNA expression of T-bet and plasma level of IFN-γ and IL-12p70 compared with ITP mice but without differences when compared with normal mice. Furthermore, the expression of GATA3, Foxp3, and plasma level of IL-4 and TGF-ß were upregulated in tacrolimus-treated ITP mice without significant differences to normal mice (except TGF-ß). Tacrolimus prevents antiplatelet antibody-mediated thrombocytopenia in ITP mice possibly through regulating T cell differentiations, suggesting it might be a novel approach for preventing ITP.

Salidroside inhibits platelet function and thrombus formation through AKT/GSK3ß signaling pathway.

Salidroside is the main bioactive component in Rhodiola rosea and possesses multiple biological and pharmacological properties. However, whether salidroside affects platelet function remains unclear. Our study aims to investigate salidroside's effect on platelet function. Human or mouse platelets were treated with salidroside (0-20 µM) for 1 hour at 37°C. Platelet aggregation, granule secretion, and receptors expression were measured together with detection of platelet spreading and clot retraction. In addition, salidroside (20 mg/kg) was intraperitoneally injected into mice followed by measuring tail bleeding time, arterial and venous thrombosis. Salidroside inhibited thrombin- or CRP-induced platelet aggregation and ATP release and did not affect the expression of P-selectin, glycoprotein (GP) Ibα, GPVI and αIIbß3. Salidroside-treated platelets presented decreased spreading on fibrinogen or collagen and reduced clot retraction with decreased phosphorylation of c-Src, Syk and PLCγ2. Additionally, salidroside significantly impaired hemostasis, arterial and venous thrombus formation in mice. Moreover, in thrombin-stimulated platelets, salidroside inhibited phosphorylation of AKT (T308/S473) and GSK3ß (Ser9). Further, addition of GSK3ß inhibitor reversed the inhibitory effect of salidroside on platelet aggregation and clot retraction. In conclusion, salidroside inhibits platelet function and thrombosis via AKT/GSK3ß signaling, suggesting that salidroside may be a novel therapeutic drug for treating thrombotic or cardiovascular diseases.

p47phox deficiency impairs platelet function and protects mice against arterial and venous thrombosis.

NADPH oxidase-derived reactive oxygen species (ROS) regulates platelet function and thrombosis. It remains controversial regarding NOX2's role in platelet function. As a regulatory subunit for NOX2, whether p47phox regulates platelet function remains unclear. Our study intends to evaluate p47phox's role in platelet function. Platelets were isolated from wild-type or p47phox-/- mice followed by analysis of platelet aggregation, granule secretion, surface receptors expression, spreading, clot retraction and ROS generation. Additionally, in vivo hemostasis, arterial and venous thrombosis was assessed. Moreover, human platelets were treated with PR-39 to inhibit p47phox activity followed by analysis of platelet function. p47phox deficiency significantly prolonged tail-bleeding time, delayed arterial and venous thrombus formation in vivo as well as reduced platelet aggregation, ATP release and αIIbß3 activation. In addition, p47phox-/- platelets presented impaired spreading on fibrinogen or collagen and defective clot retraction concomitant with decreased phosphorylation of Syk and PLCγ2. Moreover, CRP or thrombin-stimulated p47phox-/- platelets displayed reduced intracellular ROS generation which was further decreased after inhibition of NOX1. Meanwhile, p47phox deficiency increased VASP phosphorylation and decreased phosphorylation of ERK1/2, p38, ERK5 and JNK without affecting AKT and c-PLA2 phosphorylation. Furthermore, p47phox translocates to membrane to interact with both NOX1 and NOX2 after stimulation with CRP or thrombin. Finally, inhibition of p47phox activity by PR-39 reduced ROS generation, platelet aggregation and clot retraction in human platelets. In conclusion, p47phox regulates platelet function, arterial and venous thrombus formation and ROS generation, indicating that p47phox might be a novel therapeutic target for treating thrombotic or cardiovascular diseases.

Thalidomide prevents antibody-mediated immune thrombocytopenia in mice.

Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disorder characterized by immune-mediated platelet destruction, leading to lower platelet count. Thalidomide is considered as a novel immunomodulatory drug for treating several autoimmune diseases. Whether thalidomide can ameliorate ITP remains unclear. This study aims to evaluate the effect of thalidomide on ITP mouse model. ITP mouse model was established through intraperitoneal injection of rat anti-mouse integrin GPIIb/CD41 immunoglobulin. Thalidomide (10, 20 or 50 mg/kg body weight) was intraperitoneally injected into mice followed by antibody injection. Then, peripheral blood and plasma was isolated for analysis of platelet count and the level of IFN-γ and IL-17 in plasma. Meanwhile, spleen was extracted to measure the expression of CD68, a macrophage marker. In addition, macrophage cell line RAW264.7 was cultured and treated with thalidomide followed by analysis of cell viability, apoptosis as well as cell cycle. Thalidomide prevented antiplatelet antibody-mediated platelet destruction in ITP mouse model. Compared with vehicle (phosphate-buffered saline), thalidomide significantly inhibited the secretion of IFN-γ and IL-17 in ITP mouse and reduced the expression of CD68 in spleen. After thalidomide treatment, the cell viability of RAW264.7 cell was significantly reduced and the cell number in S phase was also significantly decreased. In addition, the expression of cyclin E2 was significantly reduced. In conclusion, thalidomide prevents antiplatelet antibody-mediated platelet destruction in ITP mouse possibly through reducing the number of macrophages, suggesting that it might be a novel approach for treating ITP.

All-Trans Retinoic Acid Impairs Platelet Function and Thrombus Formation and Inhibits Protein Kinase CßI/δ Phosphorylation.

All-trans retinoic acid (ATRA) is widely used for induction of complete remission in patients with acute promyelocytic leukemia (APL). ATRA also regulates protein kinase C (PKC) activity. Therapeutic use of ATRA reportedly interferes with hemostatic function in APL patients, including effects on coagulation or other vascular cells, although effects of ATRA on platelets remain unclear. This study aims to investigate the effect of therapeutic-relevant doses of ATRA on platelet function. Human platelets were preincubated with ATRA (0-20 µM) for 1 hour at 37°C, followed by analysis of aggregation, granule secretion, receptor expression by flow cytometry, platelet spreading, or clot retraction. Additionally, ATRA (10 mg/kg) was injected intraperitoneally into mice and tail bleeding time and arterial thrombus formation were evaluated. ATRA inhibited platelet aggregation and adenosine triphosphate release induced by collagen (5 µg/mL) or thrombin (0.05 U/mL) in a dose-dependent manner without affecting P-selectin expression or surface levels of glycoprotein (GP) Ibα, GPVI, or αIIbß3. ATRA-treated platelets demonstrated reduced spreading on immobilized fibrinogen or collagen and reduced thrombin-induced clot retraction together with reduced phosphorylation of Syk and PLCγ2. In addition, ATRA-treated mice displayed significantly impaired hemostasis and arterial thrombus formation in vivo. Further, in platelets stimulated with either collagen-related peptide or thrombin, ATRA selectively inhibited phosphorylation of PKCßI (Ser661) and PKCδ (Thr505), but not PKCα or PKCßII phosphorylation (Thr638/641). In conclusion, ATRA inhibits platelet function and thrombus formation, possibly involving direct or indirect inhibition of PKCßI/δ, indicating that ATRA might be beneficial for the treatment of thrombotic or cardiovascular diseases.

Increased GPIbα shedding from platelets treated with immune thrombocytopenia plasma.

Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disease, characterized by accelerated platelet destruction/clearance or decreased platelet production. ADAM17-mediated platelet receptor GPIbα extracellular domain shedding has been shown to be involved in platelet clearance. Whether GPIbα shedding participates in the pathogenesis of ITP remains poorly understood. This study aims to investigate the role of GPIbα shedding in the development of ITP via incubating normal platelets with ITP plasma to mimic ITP in vivo environment. Plasma was isolated from ITP patients or healthy control and incubated with platelets in vitro followed by measuring GPIbα expression by flow cytometry and western blot, ADAM17 expression by western blot, ROS generation and platelet activation by flow cytometry. Compared with control plasma, ITP plasma-treated platelet displayed significantly reduced GPIbα surface expression, increased ADAM17 expression and ROS generation. However, metalloproteinase inhibitor GM6001 blocked the ITP-plasma-induced decrease in GPIbα surface expression, increase in ADAM17 expression and platelet activation. In addition, inhibitors of NADPH oxidase or mitochondria respiration significantly inhibited ROS generation from ITP plasma-treated platelets. Moreover, ROS inhibition or blocking FcγRIIa attenuated the decrease in GPIbα surface expression, platelet activation and ROS generation (for blocking FcγRIIa) in ITP plasma-treated platelets. In conclusion, ITP plasma induces platelet receptor GPIbα extracellular domain shedding, suggesting that it might participate in the pathogenesis of ITP and targeting it might be a novel approach for treating ITP.