Edentulism and transplant-associated complications in patients with multiple myeloma undergoing autologous hematopoietic stem cell transplantation.

PURPOSE: Patients undergoing autologous hematopoietic stem cell transplantation (HSCT) are at risk for oral complications which may cause significant morbidity. The aim of this study was to compare the incidence of toxicities and complications in edentulous and dentate patients undergoing autologous HSCT for multiple myeloma. METHODS: We conducted a retrospective case-control study to analyze the incidence of bacteremia, fever, and oral mucositis, between edentulous and dentate patients. All patients underwent dental evaluation, received dental treatment if indicated, and were cleared before transplantation. The two groups were matched for age, gender, ethnicity, disease stage, time from diagnosis to transplant, performance status, and conditioning regimen. RESULTS: A total of 45 edentulous and 90 dentate patients were enrolled. All patients were male with a median age of 60 years and a mean performance status by Karnofsky score of 90 %. Two thirds had stage III MM with a median time from diagnosis to transplantation of 12 months, and all received melphalan as part of the conditioning regimen. The incidence of bacteremia (p = 0.553), fever (p = 0.245), severity of oral mucositis (p = 0.465), and other post-transplant toxicities were similar between both groups. CONCLUSIONS: There were no significant differences in the incidence of bacteremia, fever, severity of oral mucositis, or other complications between edentulous and dentate patients with multiple myeloma after autologous HSCT.

Paraneoplastic thrombocytosis in ovarian cancer.

BACKGROUND: The mechanisms of paraneoplastic thrombocytosis in ovarian cancer and the role that platelets play in abetting cancer growth are unclear. METHODS: We analyzed clinical data on 619 patients with epithelial ovarian cancer to test associations between platelet counts and disease outcome. Human samples and mouse models of epithelial ovarian cancer were used to explore the underlying mechanisms of paraneoplastic thrombocytosis. The effects of platelets on tumor growth and angiogenesis were ascertained. RESULTS: Thrombocytosis was significantly associated with advanced disease and shortened survival. Plasma levels of thrombopoietin and interleukin-6 were significantly elevated in patients who had thrombocytosis as compared with those who did not. In mouse models, increased hepatic thrombopoietin synthesis in response to tumor-derived interleukin-6 was an underlying mechanism of paraneoplastic thrombocytosis. Tumor-derived interleukin-6 and hepatic thrombopoietin were also linked to thrombocytosis in patients. Silencing thrombopoietin and interleukin-6 abrogated thrombocytosis in tumor-bearing mice. Anti-interleukin-6 antibody treatment significantly reduced platelet counts in tumor-bearing mice and in patients with epithelial ovarian cancer. In addition, neutralizing interleukin-6 significantly enhanced the therapeutic efficacy of paclitaxel in mouse models of epithelial ovarian cancer. The use of an antiplatelet antibody to halve platelet counts in tumor-bearing mice significantly reduced tumor growth and angiogenesis. CONCLUSIONS: These findings support the existence of a paracrine circuit wherein increased production of thrombopoietic cytokines in tumor and host tissue leads to paraneoplastic thrombocytosis, which fuels tumor growth. We speculate that countering paraneoplastic thrombocytosis either directly or indirectly by targeting these cytokines may have therapeutic potential. (Funded by the National Cancer Institute and others.).

Signal transducer and activator of transcription 3 (STAT3) regulates collagen-induced platelet aggregation independently of its transcription factor activity.

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 PLCγ2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLCγ2 was detected in HEK293 cells transfected with cDNAs for Syk and PLCγ2 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.

Polyethylene glycol modification of adenovirus reduces platelet activation, endothelial cell activation, and thrombocytopenia.

Thrombocytopenia is one of the complications for in vivo administration of adenovirus serotype 5 (Ad5) vectors after intravenous injection. In this paper, we investigated the mechanism of Ad5-induced thrombocytopenia and how these effects are attenuated by polyethylene glycol (PEG) modification of Ad5 (Ad-PEG). After intravenous injection, accelerated platelet loss was observed in Ad-injected mice but not in their Ad-PEG-injected counterparts. This platelet loss induced by Ad5 corresponded with increases in coagulation D-dimer levels, splenomegaly, and, later, production of megakaryocytes in the bone marrow. In contrast, these responses were blunted or ablated after injection of Ad-PEG. Ad5 activated both platelets and endothelial cells directly in vitro as evidenced by induction of P-selectin and the formation of von Willebrand factor-platelet strings and in vivo as evidenced by the induction of E-selectin messenger RNA. PEGylation blunted these observed activations. These data suggest that Ad5 may induce thrombocytopenia by direct activation of endothelial cells in addition to its direct effects on platelets. This link provides an important clue for the understanding of the mechanisms of thrombocytopenia associated with Ad5. Given that PEGylation blunted interactions of Ad with platelets and endothelial cells, reduced D-dimer formation, reduced thrombocytopenia, and reduced splenomegaly, these data suggest that this simple vector modification may have utility to improve the safety of Ad vectors for human gene therapy.

A recombinant fragment of von Willebrand factor reduces fibrin-rich microthrombi formation in mice with endotoxemia.

INTRODUCTION: Disseminated fibrin deposition in the microvasculature such as in disseminated intravascular coagulation (DIC) arises from uninhibited activated coagulation secondary to sustained systemic inflammation. Currently there is no treatment for DIC. Treating the underlying trigger and supportive care are the current recommendations to manage DIC. This study aims at using recombinant von Willebrand factor (VWF) A2 domain polypeptide to inhibit VWF-mediated platelet adhesion to fibrin and prevent DIC. MATERIALS AND METHODS: We use flow chamber assay to test the capacity of purified A2 protein to inhibit platelet adhesion to immobilized fibrin(ogen) and platelet-fibrin clot formation. We use a murine model of lipopolysaccharide-induced DIC to examine the effect of A2 protein on DIC. RESULTS: The A2 protein blocked flow-dependent platelet adhesion to fibrin, delayed fibrin polymerization, and inhibited platelet-fibrin clot formation in vitro. The infusion of the purified A2 protein to the endotoxin-treated mice prevented fibrin-rich microthrombi formation in brain, lung, kidney, and liver. It also attenuated levels of inflammatory mediators, and markedly reduced mortality rates at 96hours. CONCLUSIONS: The A2 protein inhibited platelet interaction with fibrin(ogen). Furthermore, A2 prevented disseminated fibrin-rich microthrombi and decrease mortality in a lipopolysaccharide-induced DIC murine model. A2 could provide a novel therapeutic approach in critically ill patients with uninhibited activated coagulation and disseminated fibrin deposition such as DIC.

Polymorphisms beta2-glycoprotein I: phospholipid binding and multimeric structure.

beta2-glycoprotein I is a phospholipid-binding protein of 326 amino acids and is found in plasma at a concentration of approximately 200 microg/ml. It has a sequence of positively charged amino acids located at the carboxy terminus that mediates anionic phospholipid binding. Two polymorphisms (306Cys-->Gly and 316Trp-->Ser) located at the phospholipid-binding site have been described. Homozygous state for either mutation and a compound heterozygous state show no phospholipid binding. Interestingly, heterozygotes for either 306Cys-->Gly or 316Trp-->Ser mutation have normal cardiolipin binding suggesting that beta2-glycoprotein I may circulate as a multimeric structure where wild-type subunits compensate the defective binding of the mutant ones. We investigated the effect of these mutations on quaternary structure of beta2-glycoprotein I and phospholipid binding. As previously reported, under native conditions, beta2-glycoprotein I shows an apparent molecular weight of approximately 320 kDa and it can be dissociated into subunits of lower molecular weight by boiling in 6 M urea. We show that the multimeric structure is not affected by the presence of mutations in the phospholipid-binding domain. beta2-glycoprotein I induces aggregation of anionic phospholipid vesicles suggesting again a multivalent interaction where at least two binding sites are required to bridge adjacent vesicles. beta2-glycoprotein I-induced aggregation does not cause vesicle fusion or damage as demonstrated by fluorescence resonance energy transfer (FRET) or encapsulated calcein release. In conclusion, the normal cardiolipin binding in heterozygous state for mutations at phospholipid-binding domain may be due to the multimeric structure of beta2-glycoprotein I.

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.

The catalytic subunit of protein phosphatase 1 gamma regulates thrombin-induced murine platelet alpha(IIb)beta(3) function.

BACKGROUND: Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin alpha(IIb)beta(3). 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 alpha(IIb)beta(3), the role of PP1c in platelet reactivity is unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using gamma isoform of PP1c deficient mice (PP1cgamma(-/-)), 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 PP1cgamma(-/-) platelets showed decreased alpha(IIb)beta(3) activation despite comparable levels of alpha(IIb)beta(3), PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin alpha(IIb)beta(3) signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cgamma(-/-) platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cgamma(-/-) mice. Phosphorylation of glycogen synthase kinase (GSK3)beta-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cgamma(-/-) platelets by an AKT independent mechanism. Inhibition of GSK3beta partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cgamma(-/-) platelets. CONCLUSIONS/SIGNIFICANCE: These studies illustrate a role for PP1cgamma in maintaining GSK3beta-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.

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

Integrin alpha(IIb)beta(3) 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 alpha(IIb)beta(3) signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin alpha(IIb)beta(3) in resting platelets and in human embryonal kidney 293 cells expressing alpha(IIb)beta(3). The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin alpha(IIb) is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to alpha(IIb)beta(3) during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Ac(alpha) in 293 cells decreased alpha(IIb)beta(3)-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Ac(alpha) expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated alpha(IIb)beta(3) adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac (alpha)-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2A(calpha) expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac (alpha) can negatively regulate integrin alpha(IIb)beta(3) signaling by suppressing the ERK1/2 signaling pathway.