Protein-tyrosine phosphatases: a new frontier in platelet signal transduction.

Platelet activation must be tightly controlled in order to allow platelets to respond rapidly to vascular injury and prevent thrombosis from occurring. Protein-tyrosine phosphorylation is one of the main ways in which activation signals are transmitted in platelets. Although much is known about the protein-tyrosine kinases (PTKs) that initiate and propagate activation signals, relatively little is known about the protein-tyrosine phosphatases (PTPs) that modulate these signals in platelets. PTPs are a family of enzymes that dephosphorylate tyrosine residues in proteins and regulate signals transmitted within cells. PTPs have been implicated in a variety of pathological conditions, including cancer, diabetes and autoimmunity, but their functions in hemostasis and thrombosis remain largely undefined. Exciting new findings from a number of groups have revealed that PTPs are in fact critical regulators of platelet activation and thrombosis. The primary aim of this review is to highlight the unique and important functions of PTPs in regulating platelet activity. Establishing the functions of PTPs in platelets is essential to better understand the molecular basis of thrombosis and may lead to the development of improved antithrombotic therapies.

Distinct and overlapping functional roles of Src family kinases in mouse platelets.

BACKGROUND AND OBJECTIVES: Src family kinases (SFKs) play a critical role in initiating and propagating signals in platelets. The aims of this study were to quantitate SFK members present in platelets and to analyze their contribution to platelet regulation using glycoprotein VI (GPVI) and intregrin αIIbß3, and in vivo. METHODS AND RESULTS: Mouse platelets express four SFKs, Fgr, Fyn, Lyn and Src, with Lyn expressed at a considerably higher level than the others. Using mutant mouse models, we demonstrate that platelet activation by collagen-related peptide (CRP) is delayed and then potentiated in the absence of Lyn, but only marginally reduced in the absence of Fyn or Fgr, and unaltered in the absence of Src. Compound deletions of Lyn/Src or Fyn/Lyn, but not of Fyn/Src or Fgr/Lyn, exhibit a greater delay in activation relative to Lyn-deficient platelets. Fibrinogen-adherent platelets show reduced spreading in the absence of Src, potentiation in the absence of Lyn, but no change in the absence of Fyn or Fgr. In mice double-deficient in Lyn/Src or Fgr/Lyn, the inhibitory role of Lyn on spreading on fibrinogen is lost. Lyn is the major SFK-mediating platelet aggregation on collagen at arterial shear and its absence leads to a reduction in thrombus size in a laser injury model. CONCLUSION: These results demonstrate that SFKs share individual and overlapping roles in regulating platelet activation, with Lyn having a dual role in regulating GPVI signaling and an inhibitory role downstream of αIIbß3, which requires prior signaling through Src.

Src family kinases are essential for primary aggregation by G(i) -coupled receptors.

INTRODUCTION AND BACKGROUND: Adrenaline stimulates biphasic aggregation in plasma through the G(i) -coupled α(2A) -adrenoreceptor. In the present study, we demonstrate that both primary and secondary wave aggregation induced by adrenaline in plasma is blocked by two structurally distinct inhibitors of Src family kinases, dasatinib and PD0173952. METHODS AND RESULTS: In contrast, primary aggregation is partially inhibited or unaffected in the presence of inhibitors of cyclo-oxygenase, phosphoinositide (PI) 3-kinases, and P2Y(1) and P2Y(12) ADP receptors, although secondary aggregation is abolished. The ability of adrenaline to inhibit adenylyl cyclase and to synergize with platelet agonists in mediating platelet activation in plasma is retained in the presence of Src family kinase inhibition. Moreover, adrenaline does not activate Src family kinases, as determined by western blotting of their regulatory tyrosines, suggesting that constitutive signaling from Src family kinases may underlie their role in activation. Adrenaline is widely used in clinical laboratories for investigation of patients with suspected bleeding disorders. In a group of 90 unrelated patients with a clinically diagnosed platelet bleeding disorder, we identified four who did not exhibit primary wave aggregation in response to adrenaline, although the catecholamine potentiated the response to other agonists, and five who failed to undergo secondary wave aggregation. In contrast, adrenaline stimulated biphasic aggregation in 60 controls. All of the patients with a defective response to adrenaline had impaired ADP-induced platelet activation. CONCLUSIONS: The present results indicate a previously unappreciated role for Src family kinases in mediating G(i) signaling in plasma, and demonstrate heterogeneity in response to adrenaline in patients with a clinically diagnosed platelet disorder.

CD148 enhances platelet responsiveness to collagen by maintaining a pool of active Src family kinases.

SUMMARY BACKGROUND: We have previously shown that the receptor-like protein tyrosine phosphatase (PTP) CD148 is essential for initiating glycoprotein VI (GPVI) signaling in platelets. We proposed that CD148 does so by dephosphorylating the C-terminal inhibitory tyrosine of Src family kinases (SFKs). However, this mechanism is complicated by CD148-deficient mouse platelets having a concomitant reduction in GPVI expression. OBJECTIVES: To investigate the effect of CD148 on GPVI signaling independent of the decrease in GPVI expression and to further establish the molecular basis of the activatory effect of CD148 and downregulation of GPVI. METHODS: CD148-deficient mouse platelets were investigated for functional and biochemical defects. The DT40/NFAT-lucifierase reporter assay was used to analyze the effect of CD148 on GPVI signaling. CD148-SFK interactions and dephosphorylation were quantified using biochemical assays. RESULTS: CD148-deficient mouse platelets exhibited reduced collagen-mediated aggregation, secretion and spreading in association with reduced expression of GPVI and FcR gamma-chain and reduced tyrosine phosphorylation. The phosphorylation status of SFKs suggested a global reduction in SFK activity in resting CD148-deficient platelets. Studies in a cell model confirmed that CD148 inhibits GPVI signaling independent of a change in receptor expression and through a mechanism dependent on tyrosine dephosphorylation. Recombinant CD148 dephosphorylated the inhibitory tyrosines of Fyn, Lyn and Src in vitro, although paradoxically it also dephosphorylated the activation loop of SFKs. CONCLUSIONS: CD148 plays a critical role in regulating GPVI/FcR gamma-chain expression and maintains a pool of active SFKs in platelets by directly dephosphorylating the C-terminal inhibitory tyrosines of SFKs that is essential for platelet activation.

Proteomic analysis of integrin alphaIIbbeta3 outside-in signaling reveals Src-kinase-independent phosphorylation of Dok-1 and Dok-3 leading to SHIP-1 interactions.

BACKGROUND AND OBJECTIVES: Outside-in integrin alphaIIbbeta3 signaling involves a series of tyrosine kinase reactions that culminate in platelet spreading on fibrinogen. The aim of this study was to identify novel tyrosine phosphorylated signaling proteins downstream of alphaIIbbeta3, and explore their role in platelet signaling. METHODS AND RESULTS: Utilizing proteomics to search for novel platelet proteins that contribute to outside-in signaling by the integrin alphaIIbbeta3, we identified 27 proteins, 17 of which were not previously shown to be part of a tyrosine phosphorylation-based signaling complex downstream of alphaIIbbeta3. The proteins identified include the novel immunoreceptors G6f and G6b-B, and two members of the Dok family of adapters, Dok-1 and Dok-3, which underwent increased tyrosine phosphorylation following platelet spreading on fibrinogen. Dok-3 was also inducibly phosphorylated in response to the GPVI-specific agonist collagen-related peptide (CRP) and the PAR-1 and -4 agonist thrombin, independently of the integrin alphaIIbbeta3. Tyrosine phosphorylation of Dok-1 and Dok-3 was primarily Src kinase-independent downstream of the integrin, whereas it was Src kinase-dependent downstream of GPVI. Moreover, both proteins inducibly interacted with Grb-2 and SHIP-1 in fibrinogen-spread platelets. CONCLUSIONS: This study provides new insights into the molecular mechanism regulating alphaIIbbeta3-mediated platelet spreading on fibrinogen. The novel platelet adapter Dok-3 and the structurally related Dok-1 are tyrosine phosphorylated in an Src kinase-independent manner downstream of alphaIIbbeta3 in human platelets, leading to an interaction with Grb2 and SHIP-1.

Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation.

Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.

Aortic endothelial cell von Willebrand factor content, and circulating plasminogen activator inhibitor-1 are increased, but expression of endothelial leukocyte adhesion molecules is unchanged in insulin-dependent diabetic BB rats.

Endothelial cell injury has been implicated in the increased incidence of vascular disease associated with diabetes mellitus. In diabetic humans, elevated plasma von Willebrand Factor (vWF) has been interpreted as an indication of endothelial damage. In contrast, in an animal model of inherited insulin-dependent diabetes, the bio-breeding (BB) rat, plasma vWF levels did not differ from those in age-matched control rats during the first 7 months of diabetes although morphological evidence of mild aortic endothelial alteration or injury was observed. In the present study efforts have been made to define the endothelial alterations in BB diabetic rats compared to controls more precisely over this time period. Thus, adhesion molecules: intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) were evaluated by in situ immunohistochemistry, vWF content was determined by biochemical analysis of aortic extracts and by quantitative immunohistochemistry, plasma vWF levels were measured by ELISA and vWF mRNA by RNAse protection assay. Neither age nor diabetic state significantly affected either the expression of adhesion molecules, or the levels of circulating vWF. Endothelial vWF content was significantly increased in the diabetic vessels, as observed by both approaches but the vWF mRNA content was not different from that in control vessels. Plasma plasminogen activator inhibitor (PAI-1) activity was significantly increased in diabetic animals. In conclusion, endothelial alterations in BB rats associated with diabetes, together with the raised plasma PAI-1 levels, promote the thrombogenic potential of the vessel wall, and are consistent with an increased risk for vascular disease.

Changes in the pattern of distribution of von Willebrand factor in rat aortic endothelial cells following thrombin generation in vivo.

The pattern of distribution of von Willebrand factor (VWF) in relatively large sheets of rat aortic endothelial cells (EC) obtained by the Häutchen technique were analysed by immunocytochemistry and light microscopy. EC were examined pre and post administration of a procoagulant mixture of factor Xa (F.Xa) and phosphotidylcholine/phosphotidylserine (PCPS) vesicles which was demonstrated to result in the selective loss of high molecular weight multimers (HMWM) of plasma VWF in the rat. In placebo animals the pattern was heterogenous both in overall distribution and in individual cells which showed both a diffuse and granular pattern. Groups of intensely stained EC were oriented parallel to the longitudinal axis of the aorta and staining was particularly prominent around the orifices of the intercostal arteries, implicating shear-stress as a possible factor in VWF expression by EC. Changes in the pattern of distribution of staining were observed at various time points post-infusion of F.Xa/PCPS, suggesting the immediate release of VWF from EC stores followed by the recruitment of EC to synthesize and store VWF. These changes are consistent with the decrease in EC Weibel-Palade Body (WPB) content observed by EM in previously reported studies using this model.

Cell layer-specific expression of cyclic nucleotide phosphodiesterases in rat arteries: molecular cloning using isolated cell layers from paraformaldehyde-fixed tissues.

We describe a method for the isolation of small quantities of large poly (A)+ mRNA from blood vessels of the rat as well as from distinct cell layers of the rat aorta. The poly (A)+ mRNA isolated by this method is suitable for use in reverse transcription polymerase chain reaction (RT-PCR) amplification of low abundance messages. In this method, anesthetized rats are perfused with ice-cold phosphate-buffered paraformaldehyde to allow for the in situ fixation of many of the main arteries of the rat. Following the in situ fixation of the rat vasculature, selected blood vessels can be removed, cleaned, and poly (A)+ mRNA purified. In addition, the distinct cell layers of the paraformaldehyde-fixed aorta can be mechanically separated and poly (A)+ mRNA purified selectively from each. The application of this method to the study of enzymes involved in cyclic nucleotide-mediated cell-signaling is illustrated by the cloning of two cyclic AMP phosphodiesterases from rat arteries, and from the selective amplification of message for these enzymes from different cell layers isolated from the rat aorta. This method should be applicable to determine if selected mRNAs are present in selected blood vessels of the rat, or within distinct cell layers of particular large blood vessels.