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.

Abnormal platelet function in C3-deficient mice.

UNLABELLED: 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.

Calf compartment syndrome and lumbar plexopathy following topical bovine thrombin-induced coagulopathy.

We describe a patient with acquired thrombin inhibitor who developed tibial and peroneal neuropathies followed by lumbar plexopathy as a result of large calf and psoas muscle hematomas. Thrombin time, Prothombin time, and partial thromboplastin time were prolonged after repeated exposures to topical bovine thrombin in two orthopedic procedures Specific coagulation tests revealed that the coagulopathy was the result of an inhibitor to bovine thrombin that cross-reacted with human thrombin. We emphasize the risk of spontaneous hematomas that can compromise peripheral nerves as a result of an acquired coagulopathy following bovine thrombin exposure.

Primary antiphospholipid antibody syndrome with mutations in the phospholipid binding domain of beta(2)-glycoprotein I.

beta(2)-Glycoprotein I, an anionic phospholipid-binding 50-kDa plasma protein, circulates in the plasma at a concentration of 30-200 microg/ml. Its physiological role remains uncertain, but an important clue to this role is suggested by the finding that antibodies to this protein are frequently found in patients with antiphospholipid antibodies and thrombosis. beta(2)-Glycoprotein I belongs to the complement control protein (CCP) superfamily with five CCP domains. The fifth CCP domain of beta(2)-glycoprotein I has a unique structure and contains a stretch of positively charged amino acids that mediates the binding to phospholipids. This interaction may mediate the clearance of anionic phospholipid-containing surfaces from the circulation. Mutations in this domain affect its binding to phospholipids. We have identified a patient with primary antiphospholipid syndrome who is a compound heterozygous for two mutations in the fifth CCP. One mutation is located in exon 7 (codon 306), and the second mutation is in exon 8 (codon 316). The mutant beta(2)-glycoprotein I was present in normal quantities in his plasma but did not bind to cardiolipin. He had recurrent deep vein thrombosis and pulmonary embolism at age 28 and a thrombotic stroke at age 35, with no other identifiable risk factor for a hypercoagulable state. This report offers some insight into the mechanism of formation of antiphospholipid antibodies and suggests the possible role of the deficiency of beta(2)-glycoprotein I in the pathogenesis of thrombosis.

Peliosis hepatis after treatment with 2-chloro-3'-deoxyadenosine.

Peliosis hepatis is an unusual disorder associated with a variety of diseases and treatments. This is the first report of peliosis hepatis associated with administration of 2-chloro-3'-deoxyadenosine. The literature is reviewed.

Polymorphism of beta2-glycoprotein I at codons 306 and 316 in patients with systemic lupus erythematosus and antiphospholipid syndrome.

OBJECTIVE: To determine the frequency of mutations in the phospholipid binding domain of beta2-glycoprotein I (beta2GPI) in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS), and to analyze the clinical correlations of such mutations with thromboembolic complications. METHODS: Exons 7 and 8 of beta2GPI, which encode for its fifth domain, were amplified by polymerase chain reaction, and the presence of mutations was determined by restriction digestion and single-strand conformation polymorphism analysis. A clinical correlation with these mutations and the presence of antiphospholipid antibodies (aPL), lupus anticoagulant (LAC), anti-beta2GPI antibody, and the development of thromboembolic complications was performed using chi-square and Fisher's exact tests. RESULTS: From a total of 143 patients studied, we found that 5.6% were heterozygous for the mutation at exon 7 (codon 306), and 7.7% were heterozygous for the mutation at exon 8 (codon 316). No homozygous subjects were found for either mutation. No significant correlation between these mutations and the presence of aPL, LAC, or anti-beta2GPI antibodies was found. In patients with SLE (n = 95), 4 of 6 patients with exon 8 mutation had thrombosis, compared with 22 of 82 patients without the mutation (P = 0.043). CONCLUSION: The prevalence of mutations in the fifth domain of beta2GPI in these patients with SLE and/or APS were similar to those previously reported for the general population. Heterozygosity for either mutation does not influence the incidence of aPL, but in patients with SLE, the mutation at exon 8 may predispose to thrombosis as an independent factor.