Flow cytometry detection of platelet procoagulation activity and microparticles in patients with unstable angina treated by percutaneous coronary angioplasty and stent implantation

Platelet activation is known to participate to the pathogenesis of acute coronary syndromes. Aminophospholipid exposure and microparticles shedding are hallmarks of full platelet activation and may account for the dissemination of prothrombotic seats. Using flow cytometry analysis of annexin V binding to externalized aminophospholipids, we followed platelet procoagulant activity (PPA) and platelet microparticles (PMP) shedding in venous and coronary whole blood samples from 30 patients with unstable angina before and after percutaneous coronary angioplasty (PTCA) and stent implantation. Baseline values of PPA and PMP were significantly more elevated in patients than in control subjects (p < 0.005). PMP percentage was significantly higher in coronary than in venous blood, and in coronary blood of patients with proximal instead of mid/distal lesions of coronary arteries. No enhancement of platelet reactivity to TRAP and collagen was induced by procedure. Whereas activated GpIIb-IIIa and P-selectin expression decreased 24 h and 48 h after procedure, PPA and PMP remained as elevated as before. Thus, flow cytometry is a reliable method for detection of fully activated platelets in whole blood samples. Annexin V binding analysis demonstrates the persistance of in vivo platelet activation, despite the use of antiaggregating agents.     

Expression of the transcription factor Evi-1 in human erythroleukemia cell lines and in leukemias

The Evi-1 proto-oncogene is a zinc finger DNA binding protein. Although activation of the Evi-1 gene has been associated with chromosomal rearrangements of the 3q25-q28 region, ectopic expression of Evi-1 could also be observed in acute myelogenous leukemias and myelodysplastic syndromes without cytogenetic abnormalities of the 3q26 locus. In this study, human erythroleukemic cell lines were screened for the expression of Evi-1 mRNA by northern blotting. Evi-1 was expressed in all the erythroid cell lines, whether undifferentiated (K 562, HEL, LAMA 84) or exhibiting spontaneous terminal erythroid differentiation (KU 812, JK-1). Evi-1 mRNA levels were constant or elevated in hemoglobin-synthesizing KU 812 or K 562 cells in response to erythropoietin or hemin treatment, respectively. In human acute myeloblastic leukemias (AML), 11/30 expressed Evi-1 by RT-PCR. Among these cases, 4/6 erythroleukemias without abnormalities of the 3q25-q28 region were found positive. The presence of acidophilic erythroblasts (15–47% of bone marrow cells) accounted for the existence of a terminal erythroid differentiation in all Evi-1-positive AML M6, whereas one negative case was poorly differentiated and referred to as AML M6 variant. These results suggest that Evi-1 mRNA expression can coexist with erythroid differentiation.     

Increased proliferation of bone marrow-derived fibroblasts in primitive hypertrophic osteoarthropathy with severe myelofibrosis

Pachydermoperiostosis or primary hypertrophic osteoarthropathy (HOA) is a rare congenital growth disorder of connective tissue. We report a case of severe myelofibrosis in a patient with HOA. When cultured in vitro, patient bone marrow-derived fibroblasts displayed a high proliferative potential with a shortened doubling time (24 hours v 36 to 48 hours for normal fibroblasts). The role of platelet-derived growth factor (PDGF), previously implicated in the pathogenesis of secondary acquired myelofibrosis, was studied. HOA fibroblasts expressed an increased number of PDGF-BB binding sites (300,000 sites/cell v 200,000 sites/cell for normal fibroblasts) without any modification of affinity. The increased expression of PDGF-R beta appeared to result from an accelerated rate of PDGF-R beta resynthesis with normal kinetics of endocytosis. As a consequence, a several-fold increase of PDGF-R beta tyrosine kinase activity was observed. No autocrine mechanism of growth was suspected as neither spontaneous PDGF- R beta autophosphorylation nor mitogenic activity in HOA fibroblast- conditioned medium was detected. Patient serum and platelet lysate were less potent than controls in inducing [3H]thymidine incorporation into HOA fibroblasts. This was inconsistent with a paracrine mechanism of growth. In vitro, human serum or PDGF-BB were not more mitogenic for HOA than normal fibroblasts. High levels of cyclin D1, a putative oncogene, were detected in serum-deprived HOA fibroblasts. Cyclin D1 overexpression could be implicated in the accelerated growth of these cells. Our results suggest that the mechanism of fibroblastic proliferation observed in this case of myelofibrosis might differ from those reported in other acquired myeloproliferative syndromes and could be associated with an intrinsic abnormality of HOA fibroblast growth.     

Expression of two type 2N von Willebrand disease mutations identified in exon 18 of von Willebrand factor gene

Type 2N von Willebrand disease (VWD) is characterized by a markedly decreased affinity of von Willebrand factor (VWF) for factor VIII (FVIII). The FVIII binding site has been localized within the first 272 amino acid residues of mature VWF, encoded by exons 18-23. Two substitutions in exon 18 of VWF gene, inducing candidate mutations Y795C and C804F were identified in the heterozygous state in two French patients who also displayed the frequent R854Q mutation in exon 20. Expression studies in Cos-7 cells showed that these abnormalities, which implicate cysteine residues, induced secretion, multimerization and FVIII binding defects of corresponding recombinant VWF. Results from transfection experiments with R854Q, performed to reproduce the hybrid VWF present in patient plasma, were in agreement with those obtained for patient's plasma VWF. These findings confirm the importance of the VWF D' domain in FVIII binding. In addition, this work shows that exon 18 should preferentially be sequenced in type 2N VWD patients when the frequent R854Q mutation in exon 20 has been excluded or detected in the heterozygous state.     

Activity of the caspase-3/CPP32 enzyme is increased in "early stage" myelodysplastic syndromes with excessive apoptosis, but caspase inhibition does not enhance colony formation in vitro

OBJECTIVE: Excessive apoptosis may have a role in the ineffective hematopoiesis and cytopenias observed in myelodysplastic syndromes. The goals of this study were 1) to quantify apoptosis in patients with "early stage" myelodysplasia [including patients with refractory anemia (RA), RA with ringed sideroblasts (RARS), RA with excess blasts and with less than 10% blasts (RAEB(<10))], and in patients with "late stage" myelodysplasia [including RAEB with more than 10% blasts (RAEB(>10)), RAEB in transformation (RAEB-t), and acute myeloid leukemia secondary to myelodysplasia (LAM2)]; 2) to study the activation of the caspase-3/CPP32 enzyme, a major "effector" caspase in hematopoiesis, in patients with "early stage" myelodysplasia, and 3) to evaluate the effect of caspase inhibition on the apoptotic phenotype and clonogenicity of hematopoietic progenitors in vitro in these patients. MATERIALS AND METHODS: Patients: Fifty-four patients with myelodysplastic syndromes, including 30 with "early stage" myelodysplasia and 24 with "late stage" myelodysplasia were studied. Study of apoptosis: TUNEL assay performed on bone marrow smears and/or quantification of annexin V positive bone marrow mononuclear cells by flow cytometric analysis. Caspacse-3/CPP32 activity: Quantitative measurement of caspase-3/CPP32 activity on total bone marrow mononuclear cells using a fluorogenic substrate. Effect of the caspase-inhibitor Z-VAD-FMK: 1) on the apoptotic phenotype of total bone marrow mononuclear cells and 2) on the clonogenicity of hematopoietic progenitor cells. RESULTS: The group of 30 patients with "early stage" myelodysplasia had statistically increased apoptosis compared to the group of 24 patients with "late stage" myelodysplasia (44.1% +/- 4.8 vs 21.8% +/- 3.6; p = 0.02) using the TDT-mediated dUTP nick-end labeling (TUNEL) assay. In the group of patients with RAEB, those with MDS(RAEB<10) had excessive apoptosis compared to those with MDS(RAEB>10) (44.0% +/- 3.5% vs 29.5% +/- 3.6%;p = 0.042) The median caspase-3 activity in 20 "early stage" myelodysplasia patients was 19,000 U (range 3,460-41,000) and significantly increased compared to normal individuals (4,256 U, range 3,200-5,200; p = 0.032) Bone marrow mononuclear cells from 12 "early stage" MDS patients (including 11 from the 20 studied for caspase-3 activity) were incubated with or without the broad-spectrum caspase inhibitor Z-VAD-FMK. In 4 of 9 evaluable patients (44.4%) with excessive apoptosis, the number of annexin V positive cells decreased in a dose-dependent manner in the presence of Z-VAD-FMK. However, in none of these patients was caspase inhibition with Z-VAD-FMK able to enhance colony formation in vitro. CONCLUSION: These results confirm that a major characteristic of patients with "early stage" myelodysplasia is increased apoptosis. The results also indicate that excessive apoptosis in these patients is accompanied by increased caspase-3/CPP32 activity. However, caspase inhibition with the broad-spectrum inhibitor Z-VAD-FMK cannot improve hematopoiesis in this group of patients, even when apoptosis is attenuated.     

Ineffective erythropoiesis in myelodysplastic syndromes: correlation with Fas expression but not with lack of erythropoietin receptor signal transduction

Ineffective erythropoiesis in myelodysplasia is characterized by a defect in erythroid progenitor growth and by abnormal erythroid differentiation. Increased apoptosis of erythroid, granulocytic and megakaryocytic lineages is thought to account for cytopenias. Erythropoietin (Epo)-induced BFU-E and CFU-E growth was studied in 25 myelodysplastic syndrome (MDS) marrow specimens and found to be drastically diminished. To investigate the functionality of Epo-R in MDS marrow, we focused on Epo-induced STAT5 activation. Epo was able to stimulate STAT5 DNA binding activity in all normal and 12/24 MDS marrows tested, with no correlation between the level of STAT5 activation and the development of erythroid colonies in response to Epo. In contrast, impaired proliferation of erythroid progenitors was related to an increased expression of the transmembrane mediator of apoptotic cell death Fas/CD95 on the glycophorin A+ subpopulation. Therefore we conclude that the stimulation of pro-apoptotic signals rather than the defect of anti-apoptotic pathways resulting from Epo-stimulated Jak2-STAT5 pathway, predominantly accounts for ineffective erythropoiesis in myelodysplasia.     

In vitro proliferation and differentiation of erythroid progenitors from patients with myelodysplastic syndromes: evidence for Fas-dependent apoptosis

Erythropoiesis results from the proliferation and differentiation of pluripotent stem cells into immature erythroid progenitors (ie, erythroid burst-forming units (BFU-Es), whose growth, survival, and terminal differentiation depends on erythropoietin (Epo). Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). We used a 2-step liquid-culture procedure to study erythropoiesis in MDS. CD34(+) cells from the marrow of patients with MDS were cultured for 10 days in serum-containing medium with Epo, stem cell factor, insulin-like growth factor 1, and steroid hormones until they reached the proerythroblast stage. The cells were then placed in medium containing Epo and insulin for terminal erythroid differentiation. Numbers of both MDS and normal control cells increased 10(3) fold by day 15. However, in semisolid culture, cells from patients with refractory anemia (RA) with ringed sideroblasts and RA or RA with excess of blasts produced significantly fewer BFU-Es than cells from controls. Fluorescence in situ hybridization analysis of interphase nuclei from patients with chromosomal defects indicated that abnormal clones were expanded in vitro. Epo-signaling pathways (STAT5, Akt, and ERK 1/2) were normally activated in MDS erythroid progenitors. In contrast, apoptosis was significantly increased in MDS cells once they differentiated, whereas it remained low in normal cells. Fas was overexpressed on freshly isolated MDS CD34(+) cells and on MDS erythroid cells throughout the culture. Apoptosis coincided with overproduction of Fas ligand during the differentiation stage and was inhibited by Fas-Fc chimeric protein. Thus, MDS CD34(+)-derived erythroid progenitors proliferated normally in our 2-step liquid culture with Epo but underwent abnormal Fas-dependent apoptosis during differentiation that could be responsible for the impaired erythropoiesis.     

Megakaryocyte growth and development factor-induced proliferation and differentiation are regulated by the mitogen-activated protein kinase pathway in primitive cord blood hematopoietic progenitors.

In several erythroleukemia cell lines, activation of mitogen-activated protein kinases (MAPK) by phorbol esters or megakaryocyte growth and development factor (MGDF) is required for induction of megakaryocytic phenotype and growth arrest. To support this model, we have examined the effect of a specific inhibitor of this pathway (PD98059) on human CD34(+) hematopoietic progenitors isolated from cord blood (CB), induced to differentiate along the megakaryocytic lineage in liquid cultures supplemented with rhuMGDF. RhuMGDF induced a sustained activation of MAPK in megakaryocytes and this activation was completely inhibited in the presence of low concentrations of PD98059 (6 to 10 micromol/L). At this concentration, PD98059 induced an increase in cell proliferation, resulting in accumulation of viable cells and a prolongation of the life time of the cultures. This increase correlated with an increase in DNA synthesis rather than with a reduction in apoptosis. This effect was combined with developmental changes indicative of delayed megakaryocytic differentiation: (1) PD98059-treated cells tended to retain markers of immature progenitors as shown by the increased proportion of both CD34(+) and CD41(+)CD34(+) cells. (2) PD98059-treated cultures were greatly enriched in immature blasts cells. (3) PD98059 increased megakaryocytic progenitors able to form colonies in semisolid assays. Thus, the MAPK pathway, although not required for megakaryocyte formation, seems to be involved in the transition from proliferation to maturation in megakaryocytes. Inhibition of MAPK activation also led to an increase in the number and size of erythroid colonies without affecting granulocyte/macrophage progenitor numbers suggesting that, in addition to the megakaryocytic lineage, the MAPK pathway could play a role in erythroid lineage differentiation.     

Critical role for PI 3-kinase in the control of erythropoietin-induced erythroid progenitor proliferation

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate-2 (IRS2), Src homology 2 domain-containing inositol 5'-phosphatase (SHIP), Grb2-associated binder-1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2), which, in turn, down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.