Marina Pereira Colella

Crizanlizumab for the Prevention of Pain Crises in Sickle Cell Disease

Background The up‐regulation of P‐selectin in endothelial cells and platelets contributes to the cell–cell interactions that are involved in the pathogenesis of vaso‐occlusion and sickle cell–related pain crises. The safety and efficacy of crizanlizumab, an antibody against the adhesion molecule P‐selectin, were evaluated in patients with sickle cell disease. Methods In this double‐blind, randomized, placebo‐controlled, phase 2 trial, we assigned patients to receive low‐dose crizanlizumab (2.5 mg per kilogram of body weight), high‐dose crizanlizumab (5.0 mg per kilogram), or placebo, administered intravenously 14 times over a period of 52 weeks. Patients who were receiving concomitant hydroxyurea as well as those not receiving hydroxyurea were included in the study. The primary end point was the annual rate of sickle cell–related pain crises with high‐dose crizanlizumab versus placebo. The annual rate of days hospitalized, the times to first and second crises, annual rates of uncomplicated crises (defined as crises other than the acute chest syndrome, hepatic sequestration, splenic sequestration, or priapism) and the acute chest syndrome, and patient‐reported outcomes were also assessed. Results A total of 198 patients underwent randomization at 60 sites. The median rate of crises per year was 1.63 with high‐dose crizanlizumab versus 2.98 with placebo (indicating a 45.3% lower rate with high‐dose crizanlizumab, P=0.01). The median time to the first crisis was significantly longer with high‐dose crizanlizumab than with placebo (4.07 vs. 1.38 months, P=0.001), as was the median time to the second crisis (10.32 vs. 5.09 months, P=0.02). The median rate of uncomplicated crises per year was 1.08 with high‐dose crizanlizumab, as compared with 2.91 with placebo (indicating a 62.9% lower rate with high‐dose crizanlizumab, P=0.02). Adverse events that occurred in 10% or more of the patients in either active‐treatment group and at a frequency that was at least twice as high as that in the placebo group were arthralgia, diarrhea, pruritus, vomiting, and chest pain. Conclusions In patients with sickle cell disease, crizanlizumab therapy resulted in a significantly lower rate of sickle cell–related pain crises than placebo and was associated with a low incidence of adverse events. (Funded by Selexys Pharmaceuticals and others; SUSTAIN ClinicalTrials.gov number, NCT01895361.)

Hydroxyurea is associated with reductions in hypercoagulability markers in sickle cell anemia

1 Mackman N. The many faces of tissue factor. J Thromb Haemost 2009; 7(Suppl 1): 136–9. 2 Mackman N. Tissue-specific hemostasis in mice. Arterioscler Thromb Vasc Biol 2005; 25: 2273–81. 3 Gregory SA, Morrissey JH, Edgington TS. Regulation of tissue factor gene expression in the monocyte procoagulant response to endotoxin. Mol Cell Biol 1989; 9: 2752–5. 4 Osterud B. Tissue factor expression in blood cells. Thromb Res 2010; 125(Suppl 1): S31–4. 5 Osterud B, Flaegstad T. Increased tissue thromboplastin activity in monocytes of patients with meningococcal infection: related to an unfavourable prognosis. Thromb Haemost 1983; 49: 5–7. 6 Mackman N, Brand K, Edgington TS. Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites. J Exp Med 1991; 174: 1517–26. 7 Mackman N. Regulation of the tissue factor gene. FASEB J 1995; 9: 883–9. 8 Hall AJ, Vos HL, Bertina RM. Lipopolysaccharide induction of tissue factor in THP-1 cells involves Jun protein phosphorylation and nuclear factor kappaB nuclear translocation. J Biol Chem 1999; 274: 376–83. 9 GuhaM, O Connell MA, Pawlinski R, Hollis A,McGovern P, Yan SF, Stern D, Mackman N. Lipopolysaccharide activation of the MEKERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression. Blood 2001; 98: 1429–39. 10 van den EijndenMM, Steenhauer SI, Reitsma PH, Bertina RM. Tissue factor expression duringmonocyte-macrophage differentiation.Thromb Haemost 1997; 77: 1129–36. 11 Ahamed J, Niessen F, Kurokawa T, Lee YK, Bhattacharjee G, Morrissey JH, Ruf W. Regulation of macrophage procoagulant responses by the tissue factor cytoplasmic domain in endotoxemia. Blood 2007; 109: 5251–9. 12 Pawlinski R,Wang JG, Owens AP 3rd,Williams J, Antoniak S, Tencati M, Luther T, Rowley JW, Low EN, Weyrich AS, Mackman N. Hematopoietic and nonhematopoietic cell tissue factor activates the coagulation cascade in endotoxemic mice. Blood 2010; 116: 806–14. 13 Del Conde I, Shrimpton CN, Thiagarajan P, Lopez JA. Tissue-factorbearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005; 106: 1604–11. 14 Wang JG, Williams JC, Davis BK, Jacobson K, Doerschuk CM, Ting JP, Mackman N. Monocytic microparticles activate endothelial cells in an IL-1beta-dependent manner. Blood 2011; 118: 2366–74. 15 Owens AP 3rd, Mackman N. Microparticles in hemostasis and thrombosis. Circ Res 2011; 108: 1284–97. 16 Owens AP 3rd, Passam FH, Antoniak S, Marshall SM, McDaniel AL, Rudel L, Williams JC, Hubbard BK, Dutton JA, Wang J, Tobias PS, Curtiss LK, Daugherty A, Kirchhofer D, Luyendyk JP, Moriarty PM, Nagarajan S, Furie BC, Furie B, Johns DG, et al. Monocyte tissue factor-dependent activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin. J Clin Invest 2012; 122: 558–68. 17 Furlan-Freguia C, Marchese P, Gruber A, Ruggeri ZM, Ruf W. P2X7 receptor signaling contributes to tissue factor-dependent thrombosis in mice. J Clin Invest 2011; 121: 2932–44. 18 Wang JG, Manly D, Kirchhofer D, Pawlinski R, Mackman N. Levels of microparticle tissue factor activity correlate with coagulation activation in endotoxemic mice. J Thromb Haemost 2009; 7: 1092–8. 19 Aras O, Shet A, Bach RR, Hysjulien JL, Slungaard A, Hebbel RP, Escolar G, Jilma B, Key NS. Induction of microparticleand cellassociated intravascular tissue factor in human endotoxemia. Blood 2004; 103: 4545–53. 20 Baroni M, Pizzirani C, Pinotti M, Ferrari D, Adinolfi E, Calzavarini S, Caruso P, Bernardi F, Di Virgilio F. Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles. FASEB J 2007; 21: 1926–33. 21 Rao LV, Kothari H, Pendurthi UR. Tissue factor encryption and decryption: facts and controversies. Thromb Res 2012; 129(Suppl 2): S13–7. 22 Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH, Griffiths RJ, Gabel CA. Altered cytokine production in mice lacking P2X(7) receptors. J Biol Chem 2001; 276: 125–32.

Hydroxyurea Is Associated With Reductions In Hypercoagulability Markers In Sickle Cell Anemia

1 Mackman N. The many faces of tissue factor. J Thromb Haemost 2009; 7(Suppl 1): 136–9. 2 Mackman N. Tissue-specific hemostasis in mice. Arterioscler Thromb Vasc Biol 2005; 25: 2273–81. 3 Gregory SA, Morrissey JH, Edgington TS. Regulation of tissue factor gene expression in the monocyte procoagulant response to endotoxin. Mol Cell Biol 1989; 9: 2752–5. 4 Osterud B. Tissue factor expression in blood cells. Thromb Res 2010; 125(Suppl 1): S31–4. 5 Osterud B, Flaegstad T. Increased tissue thromboplastin activity in monocytes of patients with meningococcal infection: related to an unfavourable prognosis. Thromb Haemost 1983; 49: 5–7. 6 Mackman N, Brand K, Edgington TS. Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites. J Exp Med 1991; 174: 1517–26. 7 Mackman N. Regulation of the tissue factor gene. FASEB J 1995; 9: 883–9. 8 Hall AJ, Vos HL, Bertina RM. Lipopolysaccharide induction of tissue factor in THP-1 cells involves Jun protein phosphorylation and nuclear factor kappaB nuclear translocation. J Biol Chem 1999; 274: 376–83. 9 GuhaM, O Connell MA, Pawlinski R, Hollis A,McGovern P, Yan SF, Stern D, Mackman N. Lipopolysaccharide activation of the MEKERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression. Blood 2001; 98: 1429–39. 10 van den EijndenMM, Steenhauer SI, Reitsma PH, Bertina RM. Tissue factor expression duringmonocyte-macrophage differentiation.Thromb Haemost 1997; 77: 1129–36. 11 Ahamed J, Niessen F, Kurokawa T, Lee YK, Bhattacharjee G, Morrissey JH, Ruf W. Regulation of macrophage procoagulant responses by the tissue factor cytoplasmic domain in endotoxemia. Blood 2007; 109: 5251–9. 12 Pawlinski R,Wang JG, Owens AP 3rd,Williams J, Antoniak S, Tencati M, Luther T, Rowley JW, Low EN, Weyrich AS, Mackman N. Hematopoietic and nonhematopoietic cell tissue factor activates the coagulation cascade in endotoxemic mice. Blood 2010; 116: 806–14. 13 Del Conde I, Shrimpton CN, Thiagarajan P, Lopez JA. Tissue-factorbearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005; 106: 1604–11. 14 Wang JG, Williams JC, Davis BK, Jacobson K, Doerschuk CM, Ting JP, Mackman N. Monocytic microparticles activate endothelial cells in an IL-1beta-dependent manner. Blood 2011; 118: 2366–74. 15 Owens AP 3rd, Mackman N. Microparticles in hemostasis and thrombosis. Circ Res 2011; 108: 1284–97. 16 Owens AP 3rd, Passam FH, Antoniak S, Marshall SM, McDaniel AL, Rudel L, Williams JC, Hubbard BK, Dutton JA, Wang J, Tobias PS, Curtiss LK, Daugherty A, Kirchhofer D, Luyendyk JP, Moriarty PM, Nagarajan S, Furie BC, Furie B, Johns DG, et al. Monocyte tissue factor-dependent activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin. J Clin Invest 2012; 122: 558–68. 17 Furlan-Freguia C, Marchese P, Gruber A, Ruggeri ZM, Ruf W. P2X7 receptor signaling contributes to tissue factor-dependent thrombosis in mice. J Clin Invest 2011; 121: 2932–44. 18 Wang JG, Manly D, Kirchhofer D, Pawlinski R, Mackman N. Levels of microparticle tissue factor activity correlate with coagulation activation in endotoxemic mice. J Thromb Haemost 2009; 7: 1092–8. 19 Aras O, Shet A, Bach RR, Hysjulien JL, Slungaard A, Hebbel RP, Escolar G, Jilma B, Key NS. Induction of microparticleand cellassociated intravascular tissue factor in human endotoxemia. Blood 2004; 103: 4545–53. 20 Baroni M, Pizzirani C, Pinotti M, Ferrari D, Adinolfi E, Calzavarini S, Caruso P, Bernardi F, Di Virgilio F. Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles. FASEB J 2007; 21: 1926–33. 21 Rao LV, Kothari H, Pendurthi UR. Tissue factor encryption and decryption: facts and controversies. Thromb Res 2012; 129(Suppl 2): S13–7. 22 Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH, Griffiths RJ, Gabel CA. Altered cytokine production in mice lacking P2X(7) receptors. J Biol Chem 2001; 276: 125–32.

Elevated plasma levels and platelet‐associated expression of the pro‐thrombotic and pro‐inflammatory protein, TNFSF14 (LIGHT), in sickle cell disease

Chronic vascular inflammation and endothelial activation may initiate vaso‐occlusion in sickle cell disease (SCD). TNFSF14 (CD258; LIGHT), a recently‐identified pro‐thrombotic and pro‐inflammatory tumour necrosis factor (TNF)‐superfamily cytokine, has a potent activating effect on endothelial cells. We evaluated whether TNFSF14 production is altered in SCD and whether platelets contribute to this production. TNFSF14 was measured in platelet‐free plasma from healthy‐control individuals (CON), steady‐state sickle cell anaemia (SCA), SCA on hydroxycarbamide therapy (SCAHC) and haemoglobin SC (HbSC) patients. Mean plasma TNFSF14 was significantly increased in SCA, SCAHC and HbSC, compared to CON individuals. In SCA/SCAHC patients, plasma TNFSF14, showed no correlation with haematological variables, but was significantly correlated with serum lactate dehydrogenase and inflammatory markers (CD40LG , IL8 and ICAM1). Platelet‐membrane TNFSF14 expression was significantly augmented on SCA platelets, and correlated with platelet activation; furthermore, measurement of platelet TNFSF14 release indicated that platelets may be a major source of circulating TNFSF14 in SCA. Interestingly, high plasma TNFSF14 was significantly associated with elevated tricuspid regurgitant velocity (≥2·5 m/s) in a population of SCA/SCAHC patients. The pro‐inflammatory and atherogenic cytokine, TNFSF14, could contribute to endothelial activation and inflammation in SCA; future investigations may confirm whether this protein contributes to major clinical complications of the disease, such as pulmonary hypertension, and represents a potential therapeutic target.

Elevated hypercoagulability markers in hemoglobin SC disease

Hemoglobin SC disease is a very prevalent hemoglobinopathy; however, very little is known about this condition specifically. There appears to be an increased risk of thromboembolic events in hemoglobin SC disease, but studies evaluating the hemostatic alterations are lacking. We describe the findings of a cross-sectional observational study evaluating coagulation activation markers in adult patients with hemoglobin SC, comparing them with those in sickle cell anemia patients and healthy controls. A total of 56 hemoglobin SC and 39 sickle cell anemia patients were included in the study, all in steady state, and 27 healthy controls. None of the patients was taking hydroxyurea. Hemoglobin SC patients had a significantly up-regulated relative expression of tissue factor, as well as elevations in thrombin-antithrombin complex and D-dimer, in comparison to controls (P<0.01). Hemoglobin SC patients had lower tissue factor expression, and thrombin-antithrombin complex and D-dimer levels when compared to sickle cell anemia patients (P<0.05). Markers of endothelial activation (soluble thrombomodulin and soluble vascular cell adhesion molecule-1) and inflammation (tumor necrosis factor-alpha) were both significantly elevated in hemoglobin SC patients when compared to controls, being as high as the levels seen in patients with sickle cell anemia. Overall, in hemoglobin SC patients, higher hemolytic activity and inflammation were associated with a more intense activation of coagulation, and hemostatic activation was associated with two very prevalent chronic complications seen in hemoglobin SC disease: retinopathy and osteonecrosis. In summary, our results demonstrate that hemoglobin SC patients have a hypercoagulable state, although this manifestation was not as intense as that seen in sickle cell anemia.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.

Role of innate immunity-triggered pathways in the pathogenesis of Sickle Cell Disease: a meta-analysis of gene expression studies.

Despite the detailed characterization of the inflammatory and endothelial changes observed in Sickle Cell Disease (SCD), the hierarchical relationship between elements involved in the pathogenesis of this complex disease is yet to be described. Meta-analyses of gene expression studies from public repositories represent a novel strategy, capable to identify key mediators in complex diseases. We performed several meta-analyses of gene expression studies involving SCD, including studies with patient samples, as well as in-vitro models of the disease. Meta-analyses were performed with the Inmex bioinformatics tool, based on the RankProd package, using raw gene expression data. Functional gene set analysis was performed using more than 60 gene-set libraries. Our results demonstrate that the well-characterized association between innate immunity, hemostasis, angiogenesis and heme metabolism with SCD is also consistently observed at the transcriptomic level, across independent studies. The enrichment of genes and pathways associated with innate immunity and damage repair-associated pathways supports the model of erythroid danger-associated molecular patterns (DAMPs) as key mediators of the pathogenesis of SCD. Our study also generated a novel database of candidate genes, pathways and transcription factors not previously associated with the pathogenesis of SCD that warrant further investigation in models and patients of SCD.

Haemostatic management of extreme challenges to haemostasis in acquired von Willebrand syndrome

Summary.  Acquired von Willebrand syndrome (AVWS) is a rare hemorrhagic condition for which very little information is available regarding the management of extreme challenges to Haemostasis. The AVWS is more common in the elderly, who are frequently exposed to invasive procedures and/or chemotherapy. Haematopoietic stem cell transplantation (HSCT) is a situation in which the haemostatic capacity is challenged by severe thrombocytopaenia, chemotherapy‐associated mucosal barrier breakdown and the need for invasive procedures. In our report, we present and discuss the haemostatic management of a patient with AVWS who was refractory to Von Willebrand factor concentrate replacement during the course of an autologous HSCT to treat multiple myeloma. Patients with AVWS are frequently exposed to high‐risk haemostatic challenges, and additional information about the haemostatic management of these situations is necessary.

Evaluation of the immature platelet fraction contribute to the differential diagnosis of hereditary, immune and other acquired thrombocytopenias

The differential diagnosis of immune (ITP) and hereditary macrothrombocytopenia (HM) is key to patient management. The immature platelet fraction (IPF) represents the subset of circulating platelets with higher RNA content, and has been shown to distinguish hypo- from hyperproliferative thrombocytopenias. Here we evaluated the diagnostic accuracy of IPF in the differential diagnosis between HM and other thrombocytopenias in a population of patients with post-chemotherapy thrombocytopenia (n = 56), bone marrow failure (n = 22), ITP (n = 105) and HM (n = 27). TPO levels were also measured in HM and ITP matched for platelet counts. Platelet counts were similar in all patient groups. Higher IPF values were observed in both ITP (12.3%; 2.4–65.6%) and HM (29.8%; 4.6–65.9%) compared to hypoproliferative thrombocytopenias. IPF values were also higher in HM compared to ITP, yielding a diagnostic accuracy of 0.80 (95%CI 0.70–0.90; P < 0.0001) to distinguish these two conditions. Intra- and inter-assays reproducibility of IPF in HM patients revealed that this is a stable parameter. In conclusion, IPF is increased in HM compared to both ITP and other thrombocytopenias and contributes to the differentiation between ITP and HM. Further studies are warranted to understand the biological rationale of these findings and to its incorporation in diagnostic algorithms of HM.

The impact of antibody profile in thrombosis associated with primary antiphospholipid syndrome

Triple positivity (TP) for antiphospholipid antibodies(aPL) may identify aPL carriers with poorer prognosis. The clinical impact of TP in primary antiphospholipid syndrome(PAPS) remains unclear and further clinical evidences are needed to validate TP as a marker of severity. The aim of this study was to evaluate the impact of TP on the clinical course of PAPS with thrombosis(t‐PAPS). We performed a retrospective analysis of a cohort of t‐PAPS patients, comparing groups of patients with TP and non‐TP profiles according to their demographic, clinical and laboratory features. We included 105 patients with t‐PAPS, the median follow‐up time of 3.7 years. Twenty‐two patients(21%) had TP; the demographic distribution, the presence of cardiovascular risk factors and the site of thrombosis were similar between TP and non‐TP patients. The frequency of thrombotic events did not differ between TP and non‐TP patients during the study period. Pregnancy morbidities were more frequent in women with t‐PAPS and TP than in those with non‐TP profile (80% vs. 52.8%, P = 0.05). Patients with t‐PAPS and TP presented, at diagnosis, higher dRVVT ratio (median R = 2.44 vs. 1.57, P < 0.0001), higher aCL titer (median = 50UI vs. 35 UI, P < 0.0001), lower C3 levels (median = 1.08 vs. 1.30 mg dL−1, P = 0.001), lower C4 levels (median = 0.22 vs. 0.25 mg dL−1, P = 0.05) and higher frequency of positive ANA test (50% vs. 20%, P = 0.008) than patients with t‐PAPS and non‐TP. Lower‐than‐normal levels of C3 was independently associated with TP (OR = 5.1, P = 0.02). The presence of TP in patients with t‐PAPS was associated with immune derangement, with no effect on the clinical course of the disease.