Francisca C. Gushiken

N-acetylcysteine reduces the size and activity of von Willebrand factor in human plasma and mice

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by systemic microvascular thrombosis caused by adhesion of platelets to ultra-large vWF (ULVWF) multimers. These multimers accumulate because of a deficiency of the processing enzyme ADAMTS13. vWF protein forms long multimers from homodimers that first form through C-terminal disulfide bonds and then join through their N termini by further disulfide bonding. N-acetylcysteine (NAC) is an FDA-approved drug that has long been used to treat chronic obstructive lung disease and acetaminophen toxicity and is known to function in the former disorder by reducing mucin multimers. Here, we examined whether NAC could reduce vWF multimers, which polymerize in a manner similar to mucins. In vitro, NAC reduced soluble plasma-type vWF multimers in a concentration-dependent manner and rapidly degraded ULVWF multimer strings extruded from activated ECs. The effect was preceded by reduction of the intrachain disulfide bond encompassing the platelet-binding A1 domain. NAC also inhibited vWF-dependent platelet aggregation and collagen binding. Injection of NAC into ADAMTS13-deficient mice led to the rapid resolution of thrombi produced by ionophore treatment of the mesenteric venules and reduced plasma vWF multimers. These results suggest that NAC may be a rapid and effective treatment for patients with TTP.

Protein phosphatase 2A negatively regulates integrin alpha(IIb)beta(3) signaling.

Integrin αIIbβ3 activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin αIIbβ3 signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin αIIbβ3 in resting platelets and in human embryonal kidney 293 cells expressing αIIbβ3. The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin αIIb is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to αIIbβ3 during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Acα in 293 cells decreased αIIbβ3-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated αIIbβ3 adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac α-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2Acα expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac α can negatively regulate integrin αIIbβ3 signaling by suppressing the ERK1/2 signaling pathway.

Abnormal platelet function in C3-deficient mice

Summary.  Background and Objectives: The complement system is a biochemical cascade composed of several plasma proteins that can interact with endothelial cells and blood cells, including platelets. In order to investigate the effect of the complement system on platelets, we studied platelet function in C3‐deficient mice that lack complement activity. Method and Results: Tail‐cut bleeding time was prolonged and platelet aggregation in response to protease‐activated receptor‐4 (PAR4) peptide was decreased in C3‐deficient mice as compared with wild‐type littermates. Platelet aggregation in response to other agonists (ADP and collagen) was similar between C3‐deficient mice and their normal littermates. Isolated platelets from wild‐type mice aggregate less in C3‐deficient plasma than in normal plasma, and, conversely, addition of plasma from wild‐type mice or plasma‐purified C3 improved aggregation of C3‐deficient platelets. We also monitored the formation of murine arteriole or venule thrombi in an intravital microscopy thrombosis model. We found that C3‐deficient mice had a significantly delayed thrombotic response in arterioles as compared with their wild‐type littermates. Furthermore, thrombi in C3‐deficient mice were less stable and embolized more frequently than those in wild‐type mice. Conclusions: Platelets of C3‐deficient mice have subnormal function, resulting in a prolonged tail‐cut bleeding time and delayed thrombosis after vessel wall injury.

Protein phosphatase 2B inhibition promotes the secretion of von Willebrand factor from endothelial cells

Background: Secretion of Weibel–Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. Objective and method: In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. Results: We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell‐permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell‐stimulating agent. PP2B inhibitor‐induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA‐treated HUVECs, suggesting that this process may be involved in CsA‐mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA‐treated mice, and small interfering RNA‐mediated knockdown of the α and β isoforms of the PP2B‐A subunit in HUVECs enhanced VWF secretion. Conclusions: These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.

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 αIIbβ3. 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 αIIbβ3, the role of PP1c in platelet reactivity is unclear. Methodology/Principal Findings Using γ isoform of PP1c deficient mice (PP1cγ−/−), 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 PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets. Conclusions/Significance These studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.

The β isoform of the catalytic subunit of protein phosphatase 2B restrains platelet function by suppressing outside-in αII b β3 integrin signaling.

Calcium‐dependent signaling mechanisms play a critical role in platelet activation. Unlike calcium‐activated protease and kinase, the contribution of calcium‐activated protein serine/threonine phosphatase in platelet activation is poorly understood.

Signal Transducer and Activator of Transcription 3 (STAT3) Regulates Collagen-Induced Platelet Aggregation Independently of Its Transcription Factor ActivityClinical Perspective

Background— Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. Methods and Results— The signal transducer and activator of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk (spleen tyrosine kinase) and the substrate PLC&ggr;2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLC&ggr;2 was detected in HEK293 cells transfected with cDNAs for Syk and PLC&ggr;2 and stimulated with interleukin-6. Pharmacological inhibition of STAT3 blocked ≈50% of collagen- and a collagen-related peptide–induced but not thrombin receptor–activating peptide– or ADP-induced aggregation and ≈80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet-specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine interleukin-6 enhanced the collagen-induced STAT3 activation in human platelets. Conclusions— These data demonstrate a nontranscriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for the platelet hyperactivity found in conditions of inflammation.Background —Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. Methods and Results —The signal transducers and activators of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk and the substrate PLCγ2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLCγ2 was also detected in HEK293 cells transfected with cDNAs for Syk and PLCγ2, and stimulated with interleukin-6 (IL-6). Pharmacological inhibition of STAT3 blocked ~50% of collagen- and a collagen-related peptide-, but not TRAP- or ADP-induced aggregation and ~80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine IL-6 enhanced the collagen-induced STAT3 activation in human platelets. Conclusions —These data demonstrate a non-transcriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for platelet hyperactivity found in conditions of inflammation.