Shirley A. Steward

The roles of ADP and TXA2 in botrocetin/VWF‐induced aggregation of washed platelets

Summary.  Background: Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib‐IX‐V complex initiates a cascade of events leading to αIIbβ3 activation and platelet aggregation. The roles of ADP and thromboxane A2 (TXA2) in agglutination‐induced GPIbα‐mediated platelet activation have not been fully described. Methods: Botrocetin and human VWF were used to stimulate washed mouse platelets. Platelets deficient in TXA2 receptors, Gαq, or αIIbβ3, and inhibitors and chelating agents were used to investigate the roles of TXA2, ADP, αIIbβ3 and Ca2+ in botrocetin/VWF‐induced signaling. Results: Our data demonstrate that botrocetin/VWF/GPIbα‐mediated agglutination results in calcium‐independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPIbα‐elicited TXA2 production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA2‐induced αIIbβ3 activation and ADP signaling. TXA2 or ADP can activate αIIbβ3, but both are required for α‐granule secretion and aggregation. Botrocetin/VWF‐induced dense granule secretion is Gαq‐dependent. α‐Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an αIIbβ3‐dependent manner. Conclusions: In contrast to adhesion or shear stress‐induced GPIb‐elicited signaling, agglutination‐elicited GPIb signaling that activates αIIbβ3 requires TXA2. Agglutination‐elicited TXA2 production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. Therefore, our results demonstrate that agglutination‐elicited GPIb signaling causes αIIbβ3 activation by a mechanism that is distinct from those used by adhesion, or shear stress‐induced GPIb signaling.

Identification of the Src Family Kinases, Lck and Fgr in Platelets Their Tyrosine Phosphorylation Status and Subcellular Distribution Compared With Other Src Family Members

We have identified the Src family members, Lck and Fgr in resting human and rodent platelets and compared their subcellular distributions and tyrosine phosphorylation status to those of the other Src family kinases to gain insights into the signal transduction pathways active in maintaining platelets in the circulation. Like Fyn, Lyn, and Yes, most of Fgr and Lck was detergent-insoluble in human and rat platelets. In comparison, Src showed higher detergent solubility than the Src-related kinases. Most all human platelet Src was detergent-soluble, while that of rodent platelets was present in all detergent fractions. We also compared the tyrosine-phosphorylation status of Lck and Fgr to other Src family members in resting platelets using immunoprecipitation and immunoblotting. All of these Src family members except Fgr exhibited substantial phosphotyrosine antibody labeling. The partitioning of these kinases, with the exception of Src, with the detergent-insoluble fraction, their tyrosine-phosphorylation status, and co-localization with endocytotic vesicles lead us to hypothesize that the Src family kinases are involved in signaling events that drive cytoskeletal reorganization and active endocytosis of plasma proteins by circulating platelets.

Epigenetic and molecular profiles of erythroid cells after hydroxyurea treatment in sickle cell anemia

Hydroxyurea has been shown to be efficacious for the treatment of sickle cell anemia (SCA), primarily through the induction of fetal hemoglobin (HbF). However, the exact mechanisms by which hydroxyurea can induce HbF remain incompletely defined, although direct transcriptional effects and altered cell cycle kinetics have been proposed. In this study, we investigated potential epigenetic and alternative molecular mechanisms of hydroxyurea-mediated HbF induction by examining methylation patterns within the (G)γ-globin promoter and miRNA expression within primary CD71(+) erythrocytes of patients with SCA, both at baseline before beginning hydroxyurea therapy and after reaching maximum tolerated dose (MTD). Using both cross-sectional analysis and paired-sample analysis, we found that the highly methylated (G)γ-globin promoter was inversely correlated to baseline HbF levels, but only slightly altered by hydroxyurea treatment. Conversely, expression of several specific miRNAs was significantly increased after hydroxyurea treatment, and expression of miR-26b and miR-151-3p were both associated with HbF levels at MTD. The significant associations identified in these studies suggest that methylation may be important for regulation of baseline HbF, but not after hydroxyurea treatment, whereas changes in miRNA expression may be associated with hydroxyurea-mediated HbF induction. This study was registered at ClinicalTrials.gov (NCT00305175).

Hydroxycarbamide alters erythroid gene expression in children with sickle cell anaemia

Sickle cell anaemia (SCA) is a severe debilitating haematological disorder associated with a high degree of morbidity and mortality. The level of fetal haemoglobin (HbF) is well‐recognized as a critical laboratory parameter: lower HbF is associated with a higher risk of vaso‐occlusive complications, organ damage, and early death. Hydroxycarbamide treatment can induce HbF, improve laboratory parameters, and ameliorate clinical complications of SCA but its mechanisms of action remain incompletely defined and the HbF response is highly variable. To identify pathways of hydroxycarbamide activity, we performed microarray expression analyses of early reticulocyte RNA obtained from children with SCA enrolled in the HydroxyUrea Study of Long‐term Effects (NCT00305175) and examined the effects of hydroxycarbamide exposure in vivo. Hydroxycarbamide affected a large number of erythroid genes, with significant decreases in the expression of genes involved in translation, ribosome assembly and chromosome organization, presumably reflecting the daily cytotoxic pulses of hydroxycarbamide. Hydroxycarbamide also affected expression of numerous genes associated with HbF including BCL11A, a key regulator of baseline HbF levels. Together, these data indicate that hydroxycarbamide treatment for SCA leads to substantial changes in erythroid gene expression, including BCL11A and other potential signalling pathways associated with HbF induction.

AlphaIIbbeta3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause alpha-granule secretion by platelets.

Summary.  The peptide LSARLAF (LSA) causes αIIbβ3‐dependent platelet activation that results in α‐granule secretion and aggregation. LSARLAF‐induced, αIIbβ3‐mediated outside‐in signaling causing α‐granule secretion and platelet aggregation was studied using washed mouse platelets. ADP receptor antagonists, enzyme inhibitors, normal platelets and platelets from mice that lack either Gαq or thromboxane (Tx) A2 receptors were used for this investigation. The results demonstrate that LSA‐induced αIIbβ3‐mediated signaling producing aggregation of washed platelets is mediated through the release of ADP and thromboxane, which cause α‐granule release by mediating their effects though Gαq and/or Gi depending on the level of LSA used to activate the platelets. Specifically, αIIbβ3 elicited aggregation of washed platelets in response to a low level of LSA requires signaling through the ADP receptor P2Y1 and Gαq, and the ADP receptor P2Y12 and Gi as well as TxA2 receptors. However, this aggregation is independent of Gαq and TxA2 signaling in response to high LSA concentrations, but is dependent on ADP signaling through its receptor P2Y12, and therefore presumably Gi, regardless of the level of LSA used to activate the platelets. PKC function is required for ADP secretion and the subsequent signaling through P2Y12 regardless of the level of LSA used to activate the platelets. The end point of the LSA‐induced αIIbβ3‐mediated signaling characterized in this study is α‐granule secretion, which provides the fibrinogen required for aggregation of washed platelets.

Microarray analysis of liver gene expression in iron overloaded patients with sickle cell anemia and beta-thalassemia

Chronic transfusion therapy is used clinically to supply healthy erythrocytes for patients with sickle cell anemia (SCA) or beta‐thalassemia major (TM). Despite the benefits of red blood cell transfusions, chronic transfusions lead to iron accumulation in key tissues such as the heart, liver, and endocrine glands. Transfusion‐acquired iron overload is recognized as a cause of morbidity and mortality among patients receiving chronic transfusions. At present, there is little understanding of molecular events that occur during transfusional iron loading and the reasons for the large inter‐individual variation observed clinically in transfusion‐acquired iron accumulation. To address these issues, we examined whether any liver‐expressed genes in SCA or TM patients with transfusional iron overload were associated with the degree of iron accumulation. Specifically, we performed microarray analysis on liver biopsy specimens comparing SCA patients with mild or severe iron overload and also compared SCA with TM patients. Fifteen candidate genes were identified with significantly differential expression between the high and low liver iron concentrations. SCA patients and 20 candidate genes were detected between the SCA and TM patient comparison. Subsequent quantitative PCR experiments validated 12 candidate genes; with GSTM1, eIF5a, SULF2, NTS, and HO‐1 being particularly good prospects as genes that might affect the degree of iron accumulation. Future work will determine the baseline expression of these genes prior to transfusional iron overload and elucidate the full impact of these genes on the inter‐individual variation observed clinically in transfusion‐acquired iron accumulation. Am. J. Hematol. 2009.

Inverse relationship between megakaryocyte buoyant density and maturity

Summary. We examined the relationship between rat megakaryocyte buoyant density and maturation stage in continuous Percoll density gradients. An average of 88% of megakaryocytes had buoyant densities <1–054 g/ml. There was an inverse relationship between megakaryocyte buoyant density and maturation. Morphologically mature forms comprised 90% of the megakaryocytes with buoyant densities of 1.030–1.033 g/ml. In contrast, immature morphology was present in three‐quarters of megakaryocytes with buoyant densities of 1.042–1.046 g/ml. These morphological findings were confirmed by [3H]thymidine labelling studies. Cell viability assessed by trypan blue exclusion was highest among more dense megakaryocytes of which the majority were immature. The lowest trypan blue exclusion was found in the less dense, predominantly mature megakaryocytes indicating that these cells are more susceptible to membrane damage during marrow suspension. Megakaryocyte DNA content distributions and platelet antigen levels, determined by two‐colour flow cytometry, were also related to megakaryocyte density; the more dense megakaryocytes showed an approximately two‐fold higher proportion of 8N cells and less platelet antibody binding than did less dense megakaryocytes. These studies suggest that megakaryocytes can be fractionated according to their buoyant densities into immature and mature populations suitable for molecular studies of differentiation.