The ß isoform of the catalytic subunit of protein phosphatase 2B restrains platelet function by suppressing outside-in αII b ß3 integrin signaling.

BACKGROUND: 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. OBJECTIVE: To assess the role of catalytic subunit of protein phosphatase 2B (PP2B) or calcineurin in platelet function. RESULTS: Here, we showed that an increase in PP2B activity was associated with agonist-induced activation of human and murine platelets. Pharmacological inhibitors of the catalytic subunit of protein phosphatase 2B (PP2B-A) such as cyclosporine A or tacrolimus (FK506) potentiated aggregation of human platelets. Murine platelets lacking the ß isoform of PP2B-A (PP2B-Aß(-/-) ) displayed increased aggregation with low doses of agonist concentrations. Loss of PP2B-Aß did not affect agonist-induced integrin αII b ß3 inside-out signaling, but increased basal Src activation and outside-in αII b ß3 signaling to p38 mitogen-activated protein kinase (MAPK), with a concomitant enhancement in platelet spreading on immobilized fibrinogen and greater fibrin clot retraction. Fibrinogen-induced increased p38 activation in PP2B-Aß(-/-) platelets were blocked by Src inhibitor. Both PP2B-Aß(-/-) platelets and PP2B-Aß-depleted human embryonal kidney 293 αII b ß3 cells displayed increased adhesion to immobilized fibrinogen. Filamin A, an actin crosslinking phosphoprotein that is known to associate with ß3 , was dephosphorylated on Ser(2152) in fibrinogen-adhered wild-type but not in PP2B-Aß(-/-) platelets. In a FeCl3 injury thrombosis model, PP2B-Aß(-/-) mice showed decreased time to occlusion in the carotid artery. CONCLUSION: These observations indicate that PP2B-Aß by suppressing outside-in αII b ß3 integrin signaling limits platelet response to vascular 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 alpha and beta 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.

Human platelets contain a glycosylated estrogen receptor beta.

Platelets play an important role in the coronary thrombus formation that leads to myocardial ischemia and infarction. Gender differences in the development of coronary heart disease and its outcomes are partly regulated by estrogen and its receptors, but the roles of the latter in thrombogenicity are less well-defined. We previously demonstrated the presence of estrogen receptor (ER) beta in cells of the megakaryocytic lineage. In this study, we characterize human platelet ERbeta and its expression using biochemical and molecular biological techniques. Western immunoblotting showed that platelet ERbeta migrated with an apparent molecular mass approximately 3.7 kDa larger than ERbeta in a variety of cell lines (including those of prostate and breast origin). A rigorous investigation of platelet ERbeta mRNA by reverse transcriptase-polymerase chain reaction revealed normal transcripts and a single alternately spliced mRNA. However, this variant form was smaller, lacking exon 2, and could not account for the larger protein size seen in platelets. Treatment of ERbeta with N-glycosidase F, which removes core carbohydrate residues, caused a more rapid migration through polyacrylamide gels but had no effect on ERbeta from human cell lines. We conclude that the larger form of ERbeta in human platelets is not attributable to alternate mRNA splicing but primarily to tissue-specific glycosylation.

The Pl(A2) polymorphism of integrin beta(3) enhances outside-in signaling and adhesive functions.

Genetic factors are believed to influence the development of arterial thromboses. Because integrin alpha(IIb)beta(3) plays a crucial role in thrombus formation, we analyzed receptor adhesive properties using Chinese hamster ovary and human kidney embryonal 293 cells overexpressing the Pl(A1) or Pl(A2) polymorphic forms of alpha(IIb)beta(3). Soluble fibrinogen binding was no different between Pl(A1) and Pl(A2) cells, either in a resting state or when alpha(IIb)beta(3) was activated with anti-LIBS6. Pl(A1) and Pl(A2) cells bound equivalently to immobilized fibronectin. In contrast, significantly more Pl(A2) cells bound to immobilized fibrinogen in an alpha(IIb)beta(3)-dependent manner than did Pl(A1) cells. Disruption of the actin cytoskeleton by cytochalasin D abolished the increased binding of Pl(A2) cells. Compared with Pl(A1) cells, Pl(A2) cells exhibited a greater extent of polymerized actin and cell spreading, enhanced tyrosine phosphorylation of pp125(FAK), and greater fibrin clot retraction. These adhesion differences appear to depend on a signaling mechanism sensitive to receptor occupancy. Thus, the Pl(A2) polymorphism altered integrin-mediated functions of adhesion, spreading, actin cytoskeleton rearrangement, and clot retraction.

Human megakaryocytes and platelets contain the estrogen receptor beta and androgen receptor (AR): testosterone regulates AR expression.

Gender differences in vascular thromboses are well known, and there is evidence that platelets may be involved in these differences and that sex hormones affect platelet function. We characterized the expression of the estrogen receptor alpha (ER alpha), estrogen receptor beta (ER beta), progesterone receptor (PR), and androgen receptor (AR) in the megakaryocyte lineage. Megakaryocytes generated ex vivo from normal human CD34(+) stem cells contained RNA for ER beta and AR, which increased with cell differentiation. Platelets and human erythroleukemia (HEL) cells also contained ER beta and AR transcripts. No ER alpha or PR messenger RNA or protein was detected in the megakaryocyte lineage. Immunofluorescence microscopy showed that ER beta protein was present in glycoprotein (GP) IIb(+) megakaryocytes and the HEL megakaryocytic cell line in a predominantly cytoplasmic location. AR showed a cytoplasmic and nuclear distribution in GPIIb(+) and GPIIb(-) cells derived from CD34(+) cells and in HEL cells. Western immunoblotting confirmed the presence of ER beta and AR in platelets. Megakaryocyte and HEL AR expression was up-regulated by 1, 5, and 10 nmol/L testosterone, but down-regulated by 100 nmol/L testosterone. These findings indicate a regulated ability of megakaryocytes to respond to testosterone and suggest a potential mechanism through which sex hormones may mediate gender differences in platelet function and thrombotic diseases.

Chronic myeloid leukemic granulocytes exhibit reduced and altered binding to P-selectin: modification in the CD15 antigens and sialylation.

Granulocytes from patients with chronic myeloid leukemia (CML) egress from the bone marrow before they are functionally mature and exhibit delayed emigration to sites of infection. To understand these defects, we have compared the binding of normal and CML granulocytes to the 293 cell line transfected with cDNA for P-selectin. The CML granulocytes show significantly reduced binding to P-selectin relative to normal cells. The binding of normal granulocytes to P-selectin was significantly reduced when the cells were treated with anti CD15, polyclonal anti-P-selectin, monoclonal anti-P-selectin (G1) antibodies or neuraminidase. On average, only 8% of the CML granulocyte population bound to P-selectin. The antibodies and neuraminidase were ineffective in inhibiting the binding of this population of leukemic cells. These data show that the morphologically mature CML granulocytes consist of a heterogeneous population of cells, some of which do not bind to P-selectin and others which adhere to the molecule via modified sugars.