Alexander Tsygankov | Temple University School of Medicine (original) (raw)

Papers by Alexander Tsygankov

International Journal of Molecular Sciences

The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key ... more The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of...

International Journal of Molecular Sciences

The two members of the UBASH3/TULA/STS-protein family have been shown to critically regulate cell... more The two members of the UBASH3/TULA/STS-protein family have been shown to critically regulate cellular processes in multiple biological systems. The regulatory function of TULA-2 (also known as UBASH3B or STS-1) in platelets is one of the best examples of the involvement of UBASH3/TULA/STS proteins in cellular regulation. TULA-2 negatively regulates platelet signaling mediated by ITAM- and hemITAM-containing membrane receptors that are dependent on the protein tyrosine kinase Syk, which currently represents the best-known dephosphorylation target of TULA-2. The biological responses of platelets to collagen and other physiological agonists are significantly downregulated as a result. The protein structure, enzymatic activity and regulatory functions of UBASH3/TULA/STS proteins in the context of platelet responses and their regulation are discussed in this review.

Journal of Biological Chemistry, 1996

Fyn is a Src family protein-tyrosine kinase functionally associated with the T-cell antigen recep... more Fyn is a Src family protein-tyrosine kinase functionally associated with the T-cell antigen receptor (TcR)/ CD3 receptor complex. We have demonstrated earlier that the TcR/CD3-induced activation of Fyn results in tyrosine phosphorylation of several Fyn-associated proteins, including a protein of 116 kDa. In this report, we identify the Fyn-associated 116-kDa phosphoprotein (p116) as c-Cbl. The identity of p116 has been demonstrated by its specific reactivity with anti-Cbl and similarity of phosphopeptides generated by V8 proteolysis of phospho-Cbl and p116. We demonstrate here that the association of Fyn and c-Cbl is direct and does not require the presence of other proteins. We also demonstrate that Fyn is the Src family kinase that preferentially interacts with c-Cbl in T cells. The fraction of c-Cbl capable of coprecipitating with Fyn is increased by TcR/ CD3 ligation. This increase is likely due to the involvement of Fyn SH2 in the interactions between Fyn and tyrosine-phosphorylated c-Cbl.

The human immunodeficiency virus type-1 (HIV-1) is the major etiological agent of acquired immuno... more The human immunodeficiency virus type-1 (HIV-1) is the major etiological agent of acquired immunodeficiency syndrome (AIDS), the cause of over 30 million deaths worldwide. Highly active antiretroviral therapy (HAART) has demonstrated great efficacy at suppressing viral load and is therefore the standard therapeutic treatment for HIV-1 infection. Noncompliance due to severe HAART-associated side effects significantly undermines therapeutic efficacy. Dronabinol, the synthetic form of the cannabinoid THC found in marijuana, is FDA-approved for countering some of these side effects. Studies have reported that cannabinoids restrict HIV-1 replication, although no mechanism has yet been proposed. Thus the purpose of this study was to characterize the effects of cannabinoids on HIV-1 infection and to determine the molecular basis of cannabinoid-induced viral suppression. By transcriptomic sequencing of T cells treated with cannabinoids, we have found that the expression of BAG6, a protein u...

The TULA-family consists of two proteins implicated in cellular regulation. TULA-1 is expressed i... more The TULA-family consists of two proteins implicated in cellular regulation. TULA-1 is expressed in T-cells and is involved in apoptosis. TULA-2 is a ubiquitously expressed phosphatase that suppresses receptor-mediated signaling. T cells from mice lacking TULA-1 and 2 (double knockout, or dKO) are hypersensitive to TCR stimulation. This may be due to these proteins having a similar function working synergistically or dissimilar functions having a convergent effect. To understand functional interaction of these proteins we have characterized TULA-family knockout mice without and during an immune challenge. We show that CD4+ T cells of dKO mice have a characteristic CD45RB distribution, and that within the CD45RBlow subset effector/memory T cells are expanded only in dKO, but not in single knockouts (sKO) of either TULA-1 or TULA-2. However, CD4+ T cells of sKO and wild-type (WT) mice respond differently to TCR stimulation as seen using signaling and responses in vitro. To evaluate con...

Angiogenesis is an important process in maintaining normal physiology as well as in the pathology... more Angiogenesis is an important process in maintaining normal physiology as well as in the pathology of many diseases. Angiogenesis based therapies have the potential to have a phenomenal impact on a diseases affecting more than one billion people worldwide, including all cancers, cardiovascular disease, blindness, arthritis, complications of AIDS, diabetes, Alzheimer's disease, and more than 70 other major health conditions affecting children and adults, in developed and developing nations. In this study, we investigate the role of Interleukin-19 (IL-19) and Allograft inflammatory factor-1 (AIF-1) in endothelial cells (EC) proliferation, migration, activation and angiogenic potential. IL-19 is a recently described member of the IL-10 family of anti-inflammatory cytokines. Nothing has been reported on the expression or mechanism(s) of IL-19 effects in endothelial cells. We have found that IL-19 is expressed in aortic endothelium, and can be induced in cultured EC by serum and infla...

Inflammatory Bowel Diseases, 2007

The Journal of biological chemistry, Jan 8, 2016

Protein tyrosine phosphatase TULA-2 has been shown to regulate receptor signaling in several cell... more Protein tyrosine phosphatase TULA-2 has been shown to regulate receptor signaling in several cell types, including platelets. Platelets are critical for maintaining vascular integrity; this function is mediated by platelet aggregation in response to recognition of the exposed basement membrane collagen by the GPVI receptor, which is non-covalently associated with the signal-transducing FcRγ polypeptide chain. Our previous studies suggested that TULA-2 plays an important role in negatively regulating signaling through GPVI/FcRγ and indicated that the tyrosine protein kinase Syk is a key target of TULA-2's regulatory action in platelets. However, the molecular basis of the down-regulatory effect of TULA-2 on Syk activation via FcRγ remained unclear. In this study we demonstrate that suppression of Syk activation by TULA-2 is mediated, to a substantial degree, by dephosphorylation of pY346, a regulatory site of Syk, which becomes phosphorylated soon after receptor ligation and play...

Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in ... more Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in the heart and subsequently leads to myocardial remodeling, deteriorated cardiac function and heart failure. However, the etiology of the cardiac disease is unknown. Therefore, we assessed the gene expression in the left ventricle of diabetic and non-diabetic mice using Affymetrix microarray analysis. Allograft inflammatory factor-1 (AIF-1), one of the top downregulated B cell inflammatory genes, is associated with B cell functions in inflammatory responses. Real-time reverse transcriptase-polymerase chain reaction confirmed the Affymetrix data. The expression of CD19 and AIF-1 were downregulated in diabetic hearts as compared to control hearts. Using in vitro migration assay, we showed for the first time that AIF-1 is responsible for B cell migration as B cells migrated to GFP-AIF-1-transfected H9C2 cells compared to empty vector-transfected cells. Interestingly, overexpression of AIF-1 ...

Journal of Biological Chemistry

Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans ... more Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans and an inner layer of β-glucans and chitin. The fungal cell wall is the primary target for antifungals and is recognized by host immune cells. Environmental conditions such as carbon sources, pH, temperature, and oxygen tension can modulate the fungal cell wall architecture. Cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways, are responsible for sensing environmental cues and mediating cell wall alterations. While iron has recently been shown to affect β-1,3-glucan exposure on the cell wall, we report here that iron changes the composition of all major C. albicans cell wall components. Specifically, high iron decreased the levels of mannans (including phosphomannans) and chitin and increased β-1,3-glucan levels. These changes increased the resistance of C. albicans to cell wall-perturbing antifungals. Moreover, high iron cells exhibited adequate m...

Blood

FcγRIIA-mediated platelet activation is important in the pathogenesis of heparin-induced thromboc... more FcγRIIA-mediated platelet activation is important in the pathogenesis of heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis disorders. FcgRIIa is also the major transmembrane signaling adapter for the human platelet fibrinogen receptor, integrin aIIbb3. In HIT, antibody to PF4/heparin is necessary but not sufficient for the disease. There is considerable inter-individual variation in platelet FcγRIIA activation; the reasons remain unclear. To identify potential regulators in the FcγRIIA pathway, we determined the platelet RNA expression profile in 154 healthy human donors who were phenotyped for platelet aggregation via FcgRIIA. 87 mRNAs and 4 DE miRNAs were identified as differentially expressed (DE) between hyper- and hypo-responsive donors by microarray and Nanostring analysis, respectively. Among them, hyper-responders show significantly lower T-cell ubiquitin ligand-2 (TULA-2) mRNA level and higher miR-148a-3p and miR-25-3p levels t...

Blood

Platelet FcγRIIA is central to the pathophysiology of immune-mediated thrombocytopenia and thromb... more Platelet FcγRIIA is central to the pathophysiology of immune-mediated thrombocytopenia and thrombosis syndromes, such as heparin-induced thrombocytopenia (HIT). FcγRIIA is also the major transmembrane signaling adapter for αIIbβ3 outside-in signaling. In HIT, antibody to heparin/PF4 is necessary but not sufficient for disease to occur. Inter-individual variation in platelet activation via FcγRIIA contributes to HIT risk, but the molecular basis for the variation is incompletely understood. In our PRAX1 study of platelet reactivity and RNA expression (Edelstein, Nature Med 2013; Simon, Blood 2014), we identified differentially expressed mRNAs from healthy donors with different platelet reactivity to FcγRIIA stimulation. We observed significant differential expression of molecules involved in ubiquitination processes in relation to platelet reactivity to FcγRIIA stimulation. Syk is a protein tyrosine kinase and the major signaling node downstream of platelet receptors that use immunot...

Blood

Thienopyridines are a class of anti-platelet drugs that are metabolized in the liver to several m... more Thienopyridines are a class of anti-platelet drugs that are metabolized in the liver to several metabolites of which only one active metabolite can irreversibly antagonize the platelet P2Y12 receptor. Possible effects of these drugs and the role of activated platelets in inflammatory responses have previously been investigated in a variety of animal models, demonstrating that thienopyridines could alter inflammation. Interestingly, P2Y12 may also be expressed in other cells of the immune system, indicating that the effects of the drug could directly target other cells rather than platelets. Furthermore, neutrophil functions could be inhibited by the in vitro generated metabolites of a thienopyridine such as prasugrel, although these cells did not express P2Y12 receptor. Hence, it is not clear whether thienopyridine effects are caused only by the P2Y12antagonism or whether also off-target effects of other metabolites intervene. To address this question, we investigated P2Y12 deficien...

Blood

Heparin-induced thrombocytopenia (HIT) is a life-threatening disease in which IgG antibodies agai... more Heparin-induced thrombocytopenia (HIT) is a life-threatening disease in which IgG antibodies against the heparin-PF4 complex activate platelets via FcγRIIA. We previously reported that TULA-2 serves as a negative regulator of FcγRIIA pathway by dephosphorylating Syk in HEL cells. To further investigate the effect of TULA-2 on the FcγRIIA pathway and HIT, we crossed TULA-2-/- with FcγRIIA+/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of Syk, LAT, and PLCγ2 in platelets after FcγRIIA activation. Integrin activation, calcium mobilization, and P-selectin exposure were also enhanced in TULA-2-/- murine platelets compared to TULA-2+/+. Further, anti-GPIX antibody-induced HIT-like thrombocytopenia and thrombin generation were also augmented in TULA-2-/- mice (Figure A). We also found that decreased TULA-2 level shortened tail-bleeding time in mice (Figure B), suggesting a role of TULA-2 in physiological hemostasis. Additionally, TULA-2 KO and WT platelets did not show signif...

TH Open

Platelet activation is essential for hemostasis. Central to platelet activation are the signals t... more Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors such as glycoprotein VI, the protease-activated receptors, and C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a HemITAM (hem-immunoreceptor tyrosine activation motif)-bearing receptor that binds podoplanin and signals through spleen tyrosine kinase (Syk). T-cell ubiquitin ligand-2 (TULA-2) is a protein tyrosine phosphatase that is highly expressed in platelets and targets phosphorylated Y352 of Syk. We wanted to determine whether TULA-2 regulates Syk phosphorylation and activity downstream of CLEC-2. To that end, we used TULA-2 knockout mice and wild-type (WT) littermate controls. We found that TULA-2 deficiency enhances the aggregation and secretion response following stimulation with an excitatory CLEC-2 antibody or the CLEC-2 agonist rhodocytin. Consistently, Syk phosphorylation of Y346 is enhanced, as well as phosphorylation of the downstream s...

Journal of Cellular Physiology

Frontiers in cardiovascular medicine, 2018

Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in ... more Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in the heart and subsequently leads to myocardial remodeling, deteriorated cardiac function and heart failure. However, the etiology of the cardiac disease is unknown. Therefore, we assessed the gene expression in the left ventricle of diabetic and non-diabetic mice using Affymetrix microarray analysis. Allograft inflammatory factor-1 (AIF-1), one of the top downregulated B cell inflammatory genes, is associated with B cell functions in inflammatory responses. Real-time reverse transcriptase-polymerase chain reaction confirmed the Affymetrix data. The expression of CD19 and AIF-1 were downregulated in diabetic hearts as compared to control hearts. Using migration assay, we showed for the first time that AIF-1 is responsible for B cell migration as B cells migrated to GFP-AIF-1-transfected H9C2 cells compared to empty vector-transfected cells. Interestingly, overexpression of AIF-1 in diabet...

Arteriosclerosis, thrombosis, and vascular biology, 2016

The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in t... more The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2(-)(/-) mice with transgenic FcγRIIA (+/+) mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2(-)(/-) murine platelets. Compared...

International Journal of Molecular Sciences

The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key ... more The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of...

International Journal of Molecular Sciences

The two members of the UBASH3/TULA/STS-protein family have been shown to critically regulate cell... more The two members of the UBASH3/TULA/STS-protein family have been shown to critically regulate cellular processes in multiple biological systems. The regulatory function of TULA-2 (also known as UBASH3B or STS-1) in platelets is one of the best examples of the involvement of UBASH3/TULA/STS proteins in cellular regulation. TULA-2 negatively regulates platelet signaling mediated by ITAM- and hemITAM-containing membrane receptors that are dependent on the protein tyrosine kinase Syk, which currently represents the best-known dephosphorylation target of TULA-2. The biological responses of platelets to collagen and other physiological agonists are significantly downregulated as a result. The protein structure, enzymatic activity and regulatory functions of UBASH3/TULA/STS proteins in the context of platelet responses and their regulation are discussed in this review.

Journal of Biological Chemistry, 1996

Fyn is a Src family protein-tyrosine kinase functionally associated with the T-cell antigen recep... more Fyn is a Src family protein-tyrosine kinase functionally associated with the T-cell antigen receptor (TcR)/ CD3 receptor complex. We have demonstrated earlier that the TcR/CD3-induced activation of Fyn results in tyrosine phosphorylation of several Fyn-associated proteins, including a protein of 116 kDa. In this report, we identify the Fyn-associated 116-kDa phosphoprotein (p116) as c-Cbl. The identity of p116 has been demonstrated by its specific reactivity with anti-Cbl and similarity of phosphopeptides generated by V8 proteolysis of phospho-Cbl and p116. We demonstrate here that the association of Fyn and c-Cbl is direct and does not require the presence of other proteins. We also demonstrate that Fyn is the Src family kinase that preferentially interacts with c-Cbl in T cells. The fraction of c-Cbl capable of coprecipitating with Fyn is increased by TcR/ CD3 ligation. This increase is likely due to the involvement of Fyn SH2 in the interactions between Fyn and tyrosine-phosphorylated c-Cbl.

The human immunodeficiency virus type-1 (HIV-1) is the major etiological agent of acquired immuno... more The human immunodeficiency virus type-1 (HIV-1) is the major etiological agent of acquired immunodeficiency syndrome (AIDS), the cause of over 30 million deaths worldwide. Highly active antiretroviral therapy (HAART) has demonstrated great efficacy at suppressing viral load and is therefore the standard therapeutic treatment for HIV-1 infection. Noncompliance due to severe HAART-associated side effects significantly undermines therapeutic efficacy. Dronabinol, the synthetic form of the cannabinoid THC found in marijuana, is FDA-approved for countering some of these side effects. Studies have reported that cannabinoids restrict HIV-1 replication, although no mechanism has yet been proposed. Thus the purpose of this study was to characterize the effects of cannabinoids on HIV-1 infection and to determine the molecular basis of cannabinoid-induced viral suppression. By transcriptomic sequencing of T cells treated with cannabinoids, we have found that the expression of BAG6, a protein u...

The TULA-family consists of two proteins implicated in cellular regulation. TULA-1 is expressed i... more The TULA-family consists of two proteins implicated in cellular regulation. TULA-1 is expressed in T-cells and is involved in apoptosis. TULA-2 is a ubiquitously expressed phosphatase that suppresses receptor-mediated signaling. T cells from mice lacking TULA-1 and 2 (double knockout, or dKO) are hypersensitive to TCR stimulation. This may be due to these proteins having a similar function working synergistically or dissimilar functions having a convergent effect. To understand functional interaction of these proteins we have characterized TULA-family knockout mice without and during an immune challenge. We show that CD4+ T cells of dKO mice have a characteristic CD45RB distribution, and that within the CD45RBlow subset effector/memory T cells are expanded only in dKO, but not in single knockouts (sKO) of either TULA-1 or TULA-2. However, CD4+ T cells of sKO and wild-type (WT) mice respond differently to TCR stimulation as seen using signaling and responses in vitro. To evaluate con...

Angiogenesis is an important process in maintaining normal physiology as well as in the pathology... more Angiogenesis is an important process in maintaining normal physiology as well as in the pathology of many diseases. Angiogenesis based therapies have the potential to have a phenomenal impact on a diseases affecting more than one billion people worldwide, including all cancers, cardiovascular disease, blindness, arthritis, complications of AIDS, diabetes, Alzheimer's disease, and more than 70 other major health conditions affecting children and adults, in developed and developing nations. In this study, we investigate the role of Interleukin-19 (IL-19) and Allograft inflammatory factor-1 (AIF-1) in endothelial cells (EC) proliferation, migration, activation and angiogenic potential. IL-19 is a recently described member of the IL-10 family of anti-inflammatory cytokines. Nothing has been reported on the expression or mechanism(s) of IL-19 effects in endothelial cells. We have found that IL-19 is expressed in aortic endothelium, and can be induced in cultured EC by serum and infla...

Inflammatory Bowel Diseases, 2007

The Journal of biological chemistry, Jan 8, 2016

Protein tyrosine phosphatase TULA-2 has been shown to regulate receptor signaling in several cell... more Protein tyrosine phosphatase TULA-2 has been shown to regulate receptor signaling in several cell types, including platelets. Platelets are critical for maintaining vascular integrity; this function is mediated by platelet aggregation in response to recognition of the exposed basement membrane collagen by the GPVI receptor, which is non-covalently associated with the signal-transducing FcRγ polypeptide chain. Our previous studies suggested that TULA-2 plays an important role in negatively regulating signaling through GPVI/FcRγ and indicated that the tyrosine protein kinase Syk is a key target of TULA-2's regulatory action in platelets. However, the molecular basis of the down-regulatory effect of TULA-2 on Syk activation via FcRγ remained unclear. In this study we demonstrate that suppression of Syk activation by TULA-2 is mediated, to a substantial degree, by dephosphorylation of pY346, a regulatory site of Syk, which becomes phosphorylated soon after receptor ligation and play...

Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in ... more Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in the heart and subsequently leads to myocardial remodeling, deteriorated cardiac function and heart failure. However, the etiology of the cardiac disease is unknown. Therefore, we assessed the gene expression in the left ventricle of diabetic and non-diabetic mice using Affymetrix microarray analysis. Allograft inflammatory factor-1 (AIF-1), one of the top downregulated B cell inflammatory genes, is associated with B cell functions in inflammatory responses. Real-time reverse transcriptase-polymerase chain reaction confirmed the Affymetrix data. The expression of CD19 and AIF-1 were downregulated in diabetic hearts as compared to control hearts. Using in vitro migration assay, we showed for the first time that AIF-1 is responsible for B cell migration as B cells migrated to GFP-AIF-1-transfected H9C2 cells compared to empty vector-transfected cells. Interestingly, overexpression of AIF-1 ...

Journal of Biological Chemistry

Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans ... more Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans and an inner layer of β-glucans and chitin. The fungal cell wall is the primary target for antifungals and is recognized by host immune cells. Environmental conditions such as carbon sources, pH, temperature, and oxygen tension can modulate the fungal cell wall architecture. Cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways, are responsible for sensing environmental cues and mediating cell wall alterations. While iron has recently been shown to affect β-1,3-glucan exposure on the cell wall, we report here that iron changes the composition of all major C. albicans cell wall components. Specifically, high iron decreased the levels of mannans (including phosphomannans) and chitin and increased β-1,3-glucan levels. These changes increased the resistance of C. albicans to cell wall-perturbing antifungals. Moreover, high iron cells exhibited adequate m...

Blood

FcγRIIA-mediated platelet activation is important in the pathogenesis of heparin-induced thromboc... more FcγRIIA-mediated platelet activation is important in the pathogenesis of heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis disorders. FcgRIIa is also the major transmembrane signaling adapter for the human platelet fibrinogen receptor, integrin aIIbb3. In HIT, antibody to PF4/heparin is necessary but not sufficient for the disease. There is considerable inter-individual variation in platelet FcγRIIA activation; the reasons remain unclear. To identify potential regulators in the FcγRIIA pathway, we determined the platelet RNA expression profile in 154 healthy human donors who were phenotyped for platelet aggregation via FcgRIIA. 87 mRNAs and 4 DE miRNAs were identified as differentially expressed (DE) between hyper- and hypo-responsive donors by microarray and Nanostring analysis, respectively. Among them, hyper-responders show significantly lower T-cell ubiquitin ligand-2 (TULA-2) mRNA level and higher miR-148a-3p and miR-25-3p levels t...

Blood

Platelet FcγRIIA is central to the pathophysiology of immune-mediated thrombocytopenia and thromb... more Platelet FcγRIIA is central to the pathophysiology of immune-mediated thrombocytopenia and thrombosis syndromes, such as heparin-induced thrombocytopenia (HIT). FcγRIIA is also the major transmembrane signaling adapter for αIIbβ3 outside-in signaling. In HIT, antibody to heparin/PF4 is necessary but not sufficient for disease to occur. Inter-individual variation in platelet activation via FcγRIIA contributes to HIT risk, but the molecular basis for the variation is incompletely understood. In our PRAX1 study of platelet reactivity and RNA expression (Edelstein, Nature Med 2013; Simon, Blood 2014), we identified differentially expressed mRNAs from healthy donors with different platelet reactivity to FcγRIIA stimulation. We observed significant differential expression of molecules involved in ubiquitination processes in relation to platelet reactivity to FcγRIIA stimulation. Syk is a protein tyrosine kinase and the major signaling node downstream of platelet receptors that use immunot...

Blood

Thienopyridines are a class of anti-platelet drugs that are metabolized in the liver to several m... more Thienopyridines are a class of anti-platelet drugs that are metabolized in the liver to several metabolites of which only one active metabolite can irreversibly antagonize the platelet P2Y12 receptor. Possible effects of these drugs and the role of activated platelets in inflammatory responses have previously been investigated in a variety of animal models, demonstrating that thienopyridines could alter inflammation. Interestingly, P2Y12 may also be expressed in other cells of the immune system, indicating that the effects of the drug could directly target other cells rather than platelets. Furthermore, neutrophil functions could be inhibited by the in vitro generated metabolites of a thienopyridine such as prasugrel, although these cells did not express P2Y12 receptor. Hence, it is not clear whether thienopyridine effects are caused only by the P2Y12antagonism or whether also off-target effects of other metabolites intervene. To address this question, we investigated P2Y12 deficien...

Blood

Heparin-induced thrombocytopenia (HIT) is a life-threatening disease in which IgG antibodies agai... more Heparin-induced thrombocytopenia (HIT) is a life-threatening disease in which IgG antibodies against the heparin-PF4 complex activate platelets via FcγRIIA. We previously reported that TULA-2 serves as a negative regulator of FcγRIIA pathway by dephosphorylating Syk in HEL cells. To further investigate the effect of TULA-2 on the FcγRIIA pathway and HIT, we crossed TULA-2-/- with FcγRIIA+/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of Syk, LAT, and PLCγ2 in platelets after FcγRIIA activation. Integrin activation, calcium mobilization, and P-selectin exposure were also enhanced in TULA-2-/- murine platelets compared to TULA-2+/+. Further, anti-GPIX antibody-induced HIT-like thrombocytopenia and thrombin generation were also augmented in TULA-2-/- mice (Figure A). We also found that decreased TULA-2 level shortened tail-bleeding time in mice (Figure B), suggesting a role of TULA-2 in physiological hemostasis. Additionally, TULA-2 KO and WT platelets did not show signif...

TH Open

Platelet activation is essential for hemostasis. Central to platelet activation are the signals t... more Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors such as glycoprotein VI, the protease-activated receptors, and C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a HemITAM (hem-immunoreceptor tyrosine activation motif)-bearing receptor that binds podoplanin and signals through spleen tyrosine kinase (Syk). T-cell ubiquitin ligand-2 (TULA-2) is a protein tyrosine phosphatase that is highly expressed in platelets and targets phosphorylated Y352 of Syk. We wanted to determine whether TULA-2 regulates Syk phosphorylation and activity downstream of CLEC-2. To that end, we used TULA-2 knockout mice and wild-type (WT) littermate controls. We found that TULA-2 deficiency enhances the aggregation and secretion response following stimulation with an excitatory CLEC-2 antibody or the CLEC-2 agonist rhodocytin. Consistently, Syk phosphorylation of Y346 is enhanced, as well as phosphorylation of the downstream s...

Journal of Cellular Physiology

Frontiers in cardiovascular medicine, 2018

Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in ... more Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in the heart and subsequently leads to myocardial remodeling, deteriorated cardiac function and heart failure. However, the etiology of the cardiac disease is unknown. Therefore, we assessed the gene expression in the left ventricle of diabetic and non-diabetic mice using Affymetrix microarray analysis. Allograft inflammatory factor-1 (AIF-1), one of the top downregulated B cell inflammatory genes, is associated with B cell functions in inflammatory responses. Real-time reverse transcriptase-polymerase chain reaction confirmed the Affymetrix data. The expression of CD19 and AIF-1 were downregulated in diabetic hearts as compared to control hearts. Using migration assay, we showed for the first time that AIF-1 is responsible for B cell migration as B cells migrated to GFP-AIF-1-transfected H9C2 cells compared to empty vector-transfected cells. Interestingly, overexpression of AIF-1 in diabet...

Arteriosclerosis, thrombosis, and vascular biology, 2016

The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in t... more The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2(-)(/-) mice with transgenic FcγRIIA (+/+) mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2(-)(/-) murine platelets. Compared...