ALBERT

Description: Abstract 1015 Poster Board I-37 Background: Early mortality, mostly from hemorrhagic complications, occurs in less than 10% of patients currently treated in clinical trials for acute promyelocytic leukemia (APL). However, data about the proportion of patients developing such complications prior to clinical trial enrollment are scarce in the literature (Sanz M, et al Blood 2009). Approximately 5% of newly diagnosed patients with APL have been reported not to be eligible for participation in clinical trials due to very poor clinical condition, and their outcome has never been reported. However, enrollment on clinical trials may be difficult in specific clinical situations, such as after hours/weekend admissions and/or emergent requirement for therapy. This study reports the incidence, time of occurrence and clinical features of APL patients with a focus on early mortality. Methods: 150 consecutive APL patients treated at Stanford University between 8/1986 and 7/2009 were identified. Thirteen patients were excluded for lack of appropriate clinical information. Clinical features of patients with APL were analyzed for factors that might be relate to prognosis, including age, gender, white blood cell count, platelet count, fibrinogen, PTT, and INR. Continuous variables were compared with the t-test and categorical variables by Fisher's exact test or X-square statistic. The Kaplan–Meier method was applied to assess overall survival time. Results: Of the 137 patients included in this analysis, the median age at diagnosis was 45 (1-93) years and 78 (57%) were females. Using the PETHEMA criteria, there were 37, 46 and 20 patients with high-, intermediate- and low-risk disease (34 patients could not be classified based on partial/missing data). With a median follow-up time of 748 (0-6,235) days for the entire cohort, 52 (38%) have died. 19 (14%) and 11 (8%) of these patients died within 7 and 3 days of presentation, respectively. Patients with high-risk features had a 13% and 24% chance of dying with 3 and 7 days of presentation, respectively, with significantly inferior outcomes (p=0.045) when compared to those with intermediate-risk patients (6% and 13%) and low-risk disease (5% and 5%). Patients with unknown risk category faired similarly to low-risk patients. The most common cause of early mortality in these 19 patients was intracranial hemorrhage (n=11). Patients with early death (ED) (either

Description: This study describes the magnitude of risk of therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML) in 578 individuals diagnosed with Ewing sarcoma and enrolled on Children's Oncology Group therapeutic protocol, INT-0091. Between 1988 and 1992, patients with or without metastatic disease were randomized to receive doxorubicin, vincristine, cyclophosphamide, and dactinomycin (regimen A) or these 4 drugs alternating with etoposide and ifosfamide (regimen B). Between 1992 and 1994, patients with metastatic disease were nonrandomly assigned to receive high-intensity therapy (regimen C: regimen B therapy with higher doses of doxorubicin, cyclophosphamide, and ifosfamide). Median age at diagnosis of Ewing sarcoma was 12 years, and median length of follow-up, 8 years. Eleven patients developed t-MDS/AML, resulting in a cumulative incidence of 2% at 5 years. While patients treated on regimens A and B were at a low risk for development of t-MDS/AML (cumulative incidence: 0.4% and 0.9% at 5 years, respectively), patients treated on regimen C were at a 16-fold increased risk of developing t-MDS/AML (cumulative incidence: 11% at 5 years), when compared with those treated on regimen A. Increasing exposure to ifosfamide from 90 to 140 g/m2, cyclophosphamide from 9.6 to 17.6 g/m2, and doxorubicin from 375 to 450 mg/m2 increased the risk of t-MDS/AML significantly.

Description: The del(5q) is the most commonly reported deletion in de novo MDS and is found in 10–15% of all patients. Our group demonstrated haploinsufficiency for the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region in patients with the 5q- syndrome (Boultwood et al, Br J Haematol2007, 139:578–89). Haploinsufficiency of RPS14 has been shown to be the mechanism underlying the erythroid defect in this disorder (Ebert et al, Nature2008, 451:335–9). We have recently shown that haploinsufficiency of RPS14 in patients with the 5q- syndrome is associated with deregulated expression of ribosomal- and translation-related genes, suggesting that the 5q- syndrome represents a disorder of aberrant ribosome biogenesis (Pellagatti et al, Br J Haematol2008, 142:57–64). The del(5q) in the 5q-syndrome is cytogenetically indistinguishable from the del(5q) found in other MDS and in the vast majority of these patients the CDR of the 5q- syndrome will be deleted (and therefore one allele of RPS14 will be lost). We are investigating the hypothesis that haploinsufficiency of RPS14 and consequent deregulated ribosome biogenesis may also play a role in the pathogenesis of non-5q- syndrome MDS patients with del(5q). Using Affymetrix U133 Plus2.0 arrays, we have studied the expression profiles of a group of 579 ribosomal- and translation-related genes in the CD34+ cells of 21 non-5q- syndrome MDS patients with del(5q) and 95 MDS patients without del(5q). 168 of 579 ribosomal-and translation-related probe sets were found to be significantly differentially expressed between these two groups, with approximately 90% of these showing lower expression levels in patients with del(5q). Hierarchical clustering using this set of 168 genes gave a good separation between patients with and without the del(5q). RPS14 was one of the most significant differentially expressed genes, with lower expression levels in patients with del(5q) confirming its haploinsufficient status in these patients. Other significant differentially expressed genes include the ribosomal protein RPL22L1, and the translation initiation factors EIF4EBP3 and EIF4B. Interestingly, when samples from 16 patients with 5q- syndrome were included in the analysis, hierarchical clustering using significantly differentially expressed ribosomal- and translation-related genes showed that most patients with 5q- syndrome and most patients with del(5q) clustered together. We are currently using polysome profile analysis on bone marrow cells to examine the levels of the 40S ribosomal subunit in patients with del(5q) and without del(5q). Our results support the hypothesis that haploinsufficiency of RPS14 and deregulation of ribosomal- and translation-related genes contribute to disease pathogenesis in MDS patients with del(5q). An exciting possibility is that other MDS with the del(5q) and the 5q- syndrome share a related molecular basis in that they are all disorders of defective ribosomal biogenesis.

Description: Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.

Description: Activated protein C (APC) plays a critical anticoagulant role by inactivating factor Va (FVa) and factor VIIIa (FVIIIa) and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor (EPCR) can initiate PAR-1 mediated cytoprotective signalling. Although protein S constitutes a critical cofactor for APC anticoagulant function, the molecular basis through which protein S interacts with APC is not fully understood. In this study, we employed a site-directed mutagenesis strategy to characterise the effects of four single amino acid substitutions (D35T, D36A, L38D and A39V) within a region of the APC Gla domain important for protein S cofactor enhancement. To maintain Gla domain structural integrity, each residue was substituted with the corresponding residue of the human prothrombin Gla domain. Protein C variants were expressed in HEK 293 cells and purified by ion-exchange chromatography. Upon activation, the amidolytic activity of each recombinant APC variant was identical to that of wild type APC. The anticoagulant function of recombinant wild type and variant APC was compared in a tissue factor-initiated thrombin generation assay using protein C-deficient plasma. Wild type APC diminished thrombin generation in a concentration-dependent manner as expected. Variants APC-D35T, APC-D36A and APC-A39V exhibited only mildly impaired (

Description: Protamine sulphate is a positively-charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, protamine also possesses intrinsic anticoagulant properties. Furthermore, administration of excess protamine in the neutralization of UFH is associated with increased bleeding, particularly following cardiothoracic surgery. In this study we have investigated the molecular mechanisms underlying the anticoagulant properties of protamine. In pooled normal plasma, we observed a dose-dependent prolongation of both PT and APTT assays with increasing protamine concentrations (0–30μg/ml). The anticoagulant effects of protamine in normal plasma were also examined using a tissue factor-initiated thrombin generation assay. 30μg/ml protamine resulted in a two-fold prolongation of lag-time, a two-fold reduction in peak thrombin generation, and a 41±17% (p=0.047) decrease in endogenous thrombin potential (ETP). In heparinised plasma (0.3U/ml), addition of increasing protamine concentration initially reversed the anticoagulant effect of heparin, resulting in a progressive increase in ETP to that of normal plasma. However, further increases in protamine resulted in a dose-dependent reduction in ETP to a minimum of 61±16%. Recent studies have shown that platelet factor 4 (PF4), another cationic protein used to reverse heparin, can bind to the anionic Gla domain of protein C, thereby enhancing APC generation (up to 25-fold). Consequently, we investigated potential interaction(s) between protamine and the protein C anticoagulant pathway. As expected, in normal plasma, APC (0–20nM) caused a concentration-dependent prolongation in APTT to 180±8%, and a parallel reduction in ETP. However in the presence of 30μg/ml protamine, the effects of APC on both the APTT and ETP were markedly enhanced compared to the effect of either substance alone. As APC down-regulates thrombin generation by inactivating FVa and FVIIIa, we used a phospholipid-dependent FVa proteolysis assay to elucidate the mechanism responsible for the synergistic interaction between APC and protamine. The ability of APC to reduce FVa cofactor activity in this assay (in the presence or absence of protein S) was not significantly affected by the presence of protamine. However, a potent synergistic anticoagulant interaction between APC and protamine was also observed in plasma from patients with homozygous FV Leiden, suggesting protamine enhances APC cleavage of FVa at position Arg-306. To determine whether protamine influences the rate of FVIIIa proteolysis by APC, we expressed and purified an APC-resistant FVIII variant (R336Q/R562Q). FVIII-deficient plasma was spiked with physiological concentrations of wild type or variant FVIII, and the anticoagulant effects of protamine ± APC studied using plasma thrombin generation assays. Similar synergistic anticoagulant effects of APC in combination with protamine were observed for both wildtype and variant FVIII. To assess whether protamine also influences procoagulant processes, the rate of factor V activation by thrombin was analysed by SDS-PAGE. In the presence of protamine (30μg/ml), FVa generation was significantly reduced. In addition to inhibiting the rate of FVa generation, we also observed that protamine significantly impaired the functional activity of the prothrombinase complex in a concentration dependent manner. In contrast, cationic polybrene had no significant effect on either rate of FVa generation or prothrombinase complex activity. In conclusion, we have shown a novel and profound anticoagulant synergy between protamine sulphate and APC. Moreover, we demonstrate that this synergistic effect is mediated by independent effects on FVa generation and proteolysis respectively. These novel findings provide further insights into the molecular mechanism underlying the anticoagulant effect of excess protamine in human plasma.

Description: ABO blood group is an important determinant of plasma von Willebrand factor antigen (VWF:Ag) levels, with lower levels in group O. Previous reports have suggested that ABO(H) sugars affect the susceptibility of VWF to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 repeats-13) cleavage. To further test this hypothesis, we collected plasma from individuals with the rare Bombay blood group. VWF:Ag levels were significantly lower in Bombay patients (median, 0.69 IU/mL) than in groups AB, A, or B (P 〈 .05) and lower than in group O individuals (median, 0.82 IU/mL). Susceptibility of purified VWF fractions to recombinant ADAMTS13 cleavage, assessed using VWF collagen-binding assay (VWF:CB), was increased in Bombays compared with either group O or AB. Increasing urea concentration (0.5 to 2 M) increased the cleavage rate for each blood group but eliminated the differences between groups. We conclude that reduction in the number of terminal sugars on N-linked glycan increases susceptibility of globular VWF to ADAMTS13 proteolysis and is associated with reduced plasma VWF:Ag and VWF:CB levels.

Description: Antithymocyte/antilymphocyte globulins are polyclonal antihuman T-cell antibodies used clinically to treat acute transplant rejection. These reagents deplete T cells, but a rabbit antihuman thymocyte globulin has also been shown to induce regulatory T cells in vitro. To examine whether antithymocyte globulin–induced regulatory cells might be functional in vivo, we generated a corresponding rabbit antimurine thymocyte globulin (mATG) and tested its ability to induce regulatory cells in vitro and whether those cells can inhibit acute graft-versus-host disease (GVHD) in vivo upon adoptive transfer. In vitro, mATG induces a population of CD4+CD25+ T cells that express several cell surface molecules representative of regulatory T cells. These cells do not express Foxp3 at either the protein or mRNA level, but do show suppressive function both in vitro and in vivo when adoptively transferred into a model of GVHD. These results demonstrate that in a murine system, antithymocyte globulin induces cells with suppressive activity that also function in vivo to protect against acute GVHD. Thus, in both murine and human systems, antithymocyte globulins not only deplete T cells, but also appear to generate regulatory cells. The in vitro generation of regulatory cells by anti-thymocyte globulins could provide ad-ditional therapeutic modalities for immune-mediated disease.

Description: The multimeric size and the function of circulating von Willebrand factor are modulated via its proteolytic cleavage by the plasma metalloproteinase, ADAMTS13. It is unclear how ADAMTS13 activity is regulated within the vascular system. In the absence of a regulatory mechanism, ADAMTS13 activity might compromise platelet adhesion at sites of vascular injury. We hypothesized that at sites of vascular injury, ADAMTS13 activity could be regulated locally by coagulation proteinases. Initiation of coagulation in human plasma resulted in the disappearance of added full-length recombinant ADAMTS13. This loss was inhibited by hirudin. Using purified proteins, we showed that ADAMTS13 is proteolyzed at several cleavage sites by thrombin in a time- and concentration-dependent manner. Furthermore, this proteolysis ablated ADAMTS13 activity against purified von Willebrand factor. Preincubation of thrombin with soluble thrombomodulin, but not heparin, inhibited the proteolysis of ADAMTS13, suggesting the involvement of thrombin exosite I (and not exosite II) in ADAMTS13 recognition. Plasmin also cleaved ADAMTS13 into similar fragments, resulting in the loss of ADAMTS13 activity. This study demonstrates the susceptibility of ADAMTS13 to proteolytic inactivation and suggests possible roles for thrombin and plasmin at sites of vascular injury.

Description: Refractory Anemia with Ringed Sideroblasts (RARS) is characterized by severe ineffective erythropoesis, cytochrome c release, and mitochondrial iron overload. (Tehranchi, 2003). Granulocyte-CSF inhibit erythroid apoptosis in vitro as well as in vivo (Tehranchi 2005) The molecular mechanisms underlying the erythroid apoptosis in RARS and the effects of G-CSF were studied by gene expression profiling of erythroblasts from 8 healthy controls and 6 RARS patients. CD34+ selected marrow cells were cultured for 7 days in Iscove’s medium with 15% BIT9500. At day 7 an aliquot of RARS cells were treated with G-CSF (100ng/ml) for 4 hours. The gene expression profiles were determined using Affymetrix, U133 Plus2.0 chips (Pellagatti, 2006). Statistical analysis showed that 1426 probe-sets were significantly differentially expressed (P