Biochemical characterization of spleen tyrosine kinase (SYK) isoforms in platelets.

Alternate splicing is among the regulatory mechanisms imparting functional diversity in proteins. Studying protein isoforms generated through alternative splicing is therefore critical for understanding protein functions in many biological systems. Spleen tyrosine kinase (Syk) plays an essential role in ITAM/hemITAM signaling in many cell types, including platelets. However, the spectrum of Syk isoforms expressed in platelets has not been characterized. Syk has been shown to have a full-length long isoform SykL and a shorter SykS lacking 23 amino acid residues within its interdomain B. Furthermore, putative isoforms lacking another 23 amino acid-long sequence or a combination of the two deletions have been postulated to exist. In this report, we demonstrate that mouse platelets express full-length SykL and the previously described shorter isoform SykS, but lack other shorter isoforms, whereas human platelets express predominantly SykL. These results both indicate a possible role of alternative Syk splicing in the regulation of receptor signaling in mouse platelets and a difference between signaling regulation in mouse and human platelets.

Platelets express two sizes of the Syk molecule with possible alternate functions in the cell. We need to understand how these two differ in their structure so that further studies can be developed by selectively deleting one of them to evaluate their function in platelets. This study shows that platelet Syk molecules differ in their structure with and without a linker region in the molecule.

Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets.

Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor-based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses.

Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice.

BACKGROUND AND OBJECTIVE: CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c-terminal catalytic peptide in platelets. Therefore, we investigated the functional role of this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region. METHODS AND RESULTS: We used an antibody specific to the c-terminus of CD45 to confirm the presence of a truncated CD45 isoform in platelets. We also examined ex vivo and in vivo platelet function using CD45 knockout (KO) mice. Aggregation and secretion mediated by the glycoprotein VI (GPVI) receptor was impaired in CD45 KO platelets. Consequently, CD45 KO mice had impaired hemostasis indicated by increased tail bleeding times. Also, using a model of pulmonary embolism we showed that CD45 KO mice had defective in vivo thrombus formation. Next, we investigated whether or not the truncated isoform of CD45 had a role in GPVI signaling. The full-length isoform of CD45 is known to regulate Src family kinase (SFK) activation in lymphocytes. We find a similar role for the truncated isoform of CD45 in platelets. SFK activation was impaired downstream of the GPVI receptor in the CD45 KO murine platelets. Consequently, Syk, PLCγ2, and pleckstrin phosphorylations were also impaired in CD45 KO murine platelets. CONCLUSION: We conclude that the truncated CD45 isoform regulates GPVI-mediated signaling and platelet functional responses by regulating SFK activation.

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice.

OBJECTIVE: 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). APPROACH AND RESULTS: 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 with wild-type mice, TULA-2-/- mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2-/- and TULA-2+/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2+/- mice, whose platelets contained 50% as much protein as the TULA-2+/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. CONCLUSIONS: Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

TULA-2 Protein Phosphatase Suppresses Activation of Syk through the GPVI Platelet Receptor for Collagen by Dephosphorylating Tyr(P)346, a Regulatory Site of Syk.

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 the regulatory action of TULA-2 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 Tyr(P)346, a regulatory site of Syk, which becomes phosphorylated soon after receptor ligation and plays a critical role in initiating the process that yields fully activated Syk. TULA-2 is capable of dephosphorylating Tyr(P)346 with high efficiency, thus controlling the overall activation of Syk, but is less efficient in dephosphorylating other regulatory sites of this kinase. Therefore, dephosphorylation of Tyr(P)346 may be considered an important "checkpoint" in the regulation of Syk activation process. Putative biological functions of TULA-2-mediated dephosphorylation of Tyr(P)346 may include deactivation of receptor-activated Syk or suppression of Syk activation by suboptimal stimulation.

Anti-miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice.

Fc receptor for IgG IIA (FcγRIIA)-mediated platelet activation is essential in heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis disorders. There is considerable interindividual variation in platelet FcγRIIA activation, the reasons for which remain unclear. We hypothesized that genetic variations between FcγRIIA hyper- and hyporesponders regulate FcγRIIA-mediated platelet reactivity and influence HIT susceptibility. Using unbiased genome-wide expression profiling, we observed that human hyporesponders to FcγRIIA activation showed higher platelet T-cell ubiquitin ligand-2 (TULA-2) mRNA expression than hyperresponders. Silent interfering RNA-mediated knockdown of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase following FcγRIIA activation in HEL cells. Significantly, we found miR-148a-3p targeted and inhibited both human and mouse TULA-2 mRNA. Inhibition of miR-148a in FcγRIIA transgenic mice upregulated the TULA-2 level and reduced FcγRIIA- and glycoprotein VI-mediated platelet αIIbß3 activation and calcium mobilization. Anti-miR-148a also reduced thrombus formation following intravascular platelet activation via FcγRIIA. These results show that TULA-2 is a target of miR-148a-3p, and TULA-2 serves as a negative regulator of FcγRIIA-mediated platelet activation. This is also the first study to show the effects of in vivo miRNA inhibition on platelet reactivity. Our work suggests that modulating miR-148a expression is a potential therapeutic approach for thrombosis.

C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets.

C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-ß-cyclodextrin (MßCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MßCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling.

Glyoxylate lowers metabolic ATP in human platelets without altering adenylate energy charge or aggregation.

Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced.

A novel histidine tyrosine phosphatase, TULA-2, associates with Syk and negatively regulates GPVI signaling in platelets.

T-cell ubiquitin ligand-2 (TULA-2) is a recently discovered histidine tyrosine phosphatase thought to be ubiquitously expressed. In this work, we have investigated whether TULA-2 has a key role in platelet glycoprotein VI (GPVI) signaling. This study indicates that TULA-2 is expressed in human and murine platelets and is able to associate with Syk and dephosphorylate it. Ablation of TULA-2 resulted in hyperphosphorylation of Syk and its downstream effector phospholipase C-γ2 as well as enhanced GPVI-mediated platelet functional responses. In addition, shorter bleeding times and a prothrombotic phenotype were observed in mice lacking TULA-2. We therefore propose that TULA-2 is the primary tyrosine phosphatase mediating the dephosphorylation of Syk and thus functions as a negative regulator of GPVI signaling in platelets.

Cbl-b is a novel physiologic regulator of glycoprotein VI-dependent platelet activation.

Cbl-b, a member of the Cbl family of E3 ubiquitin ligases, plays an important role in the activation of lymphocytes. However, its function in platelets remains unknown. We show that Cbl-b is expressed in human platelets along with c-Cbl, but in contrast to c-Cbl, it is not tyrosine-phosphorylated upon glycoprotein VI (GPVI) stimulation. Cbl-b, unlike c-Cbl, is not required for Syk ubiquitylation downstream of GPVI activation. Phospholipase Cgamma2 (PLCgamma2) and Bruton's tyrosine kinase (BTK) are constituently associated with Cbl-b. Cbl-b-deficient (Cbl-b(-/-)) platelets display an inhibition in the concentration-response curve for GPVI-specific agonist-induced aggregation, secretion, and Ca(2+) mobilization. A parallel inhibition is found for activation of PLCgamma2 and BTK. However, Syk activation is not affected by the absence of Cbl-b, indicating that Cbl-b acts downstream of Syk but upstream of BTK and PLCgamma2. When Cbl-b(-/-) mice were tested in the ferric chloride thrombosis model, occlusion time was increased and clot stability was reduced compared with wild type controls. These data indicate that Cbl-b plays a positive modulatory role in GPVI-dependent platelet signaling, which translates to an important regulatory role in hemostasis and thrombosis in vivo.

Curcumin inhibits GPVI-mediated platelet activation by interfering with the kinase activity of Syk and the subsequent activation of PLCgamma2.

Turmeric (Curcuma longa), a herbal remedy and culinary spice, has been used in traditional Indian culture for millennia. An active ingredient found in turmeric is curcumin (diferuloylmethane). In the current study, we investigated the antiplatelet properties of this naturally occurring compound. Curcumin inhibited human platelet aggregation and dense granule secretion induced by GPVI agonist convulxin in a concentration-dependent manner. At 50 microM, it effectively inhibited the maximal extent of aggregation and dense granule secretion to as much as 75%. It also dramatically inhibited the activation-dependent tyrosine phosphorylation of Y753 and Y759 on PLCgamma2, but did not affect the phosphorylation of Y145 residue on the cytosolic adaptor protein SLP-76. Interestingly, curcumin had no significant effect on the phosphorylation of Y525/Y526 present on the activation loop of Syk (spleen tyrosine kinase), but had a significant inhibitory effect on in vitro Syk kinase activity. Moreover, the inhibitory action of curcumin is not due to an inhibition of thromboxane generation because all our studies were performed using aspirin-treated platelets. We conclude that curcumin inhibits platelet activation induced by GPVI agonists through interfering with the kinase activity of Syk and the subsequent activation of PLCgamma2.

Rapid ubiquitination of Syk following GPVI activation in platelets.

Spleen tyrosine kinase (Syk) activation is a key intermediate step in the activation of platelets by the physiologic agonist collagen. We have found that Syk is rapidly ubiquitinated upon activation of platelets by collagen, collagen-related peptide (CRP), and convulxin. The Src family kinase inhibitors prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases. The ubiquitination of Syk did not cause degradation of the protein as evidenced by the lack of effect of proteasomal and lysosomal inhibitors. We separated ubiquitinated Syk from its nonubiquitinated counterpart and used an in vitro kinase assay to compare their activities. We found that the ubiquitinated Syk appeared to be about 5-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that most (60%-75%) of the active Syk is in the ubiquitinated fraction. This result explains the apparent high specific activity of ubiquitinated Syk. In c-Cbl-deficient mice, Syk is not ubiquitinated, implicating c-Cbl as the E3 ligase involved in Syk ubiquitination. Furthermore, Syk is not dephosphorylated in these mice. We propose that c-Cbl plays a regulatory role in glycoprotein VI (GPVI)/Fc receptor gamma (FcRgamma)-chain-dependent platelet activation through its interaction with Syk.