Immature megakaryocytes undergo apoptosis in the absence of thrombopoietin

OBJECTIVES: We prospectively performed a two-step risk assessment in patients in the early phase after acute myocardial infarction (MI). BACKGROUND: Noninvasive methods like Holter electrocardiographic monitoring (HM) and determination of the left ventricular ejection fraction (EF) as well as the invasive technique of programmed ventricular stimulation (PVS) have been used to identify patients in the late phase after MI as candidates for prophylactic implantation of a cardioverter/defibrillator. However, it is unclear whether these results can be transferred to patients following acute MI. METHODS: A series of 657 patients with acute MI (< or = 75 years) underwent HM and EF. If one of the two methods yielded abnormal findings (HM > or = 20 ventricular ectopic beats/h/> or =10 ventricular pairs/day/ventricular tachycardia; EF < or = 40%), PVS was done (abnormal PVS: induction of monomorphic ventricular tachycardia, duration >10 s, cycle length > or = 230 ms). RESULTS: Of 657 patients, 304 (46%) had either an abnormal HM or EF. The PVS performed in 146 of 304 patients was abnormal in 22. During a mean follow-up of 37 months, there were 106 (16%) deaths, being sudden in 24 (3.6%), nonsudden cardiac in 45 (6.8%). The incidence of arrhythmic events (sudden cardiac death, symptomatic ventricular tachycardia, cardiac arrest) was 18% (4/22) with an abnormal PVS and only 4% (5/124) with a normal PVS (odds ratio 4.0, p=0.032). CONCLUSIONS: The rate of arrhythmic events is low in post-MI patients in the 1990s. Nevertheless, a two-step risk stratification is helpful in selecting candidates for a defibrillator trial aiming at primary prevention of sudden cardiac death after MI.     

Mpl ligand (thrombopoietin) and platelet regulation

After 35 years of research, the physiological regulator of platelet production has been isolated and its gene cloned. This discovery originates from studies performed with the myeloproliferative leukemia virus (MPLV), a murine retrovirus which induces an acute myeloproliferative syndrome in adult mice. MPLV carries in its genome the v-mpl oncogene which corresponds to a truncated form the c-mpl proto-oncogene. c-mpl encodes a cytokine receptor (Mpl-R) belonging to the hematopoietin receptor superfamily. Among the hematopoietic cell lineages, Mpl-R is preferentially expressed on late megakaryocyte progenitors, megakaryocytes and platelets. The ligand for Mpl-R, called Mpl-L or TPO or MGDF or megapoietin, is a glycosylated hormone of 352 amino acids in human which comprises two domains: the N-terminus domain shares 50% similarity with erythropoietin and is responsible for the biological activity; the C-terminus part is required for secretion. Notwithstanding its major action on megakaryocytopoiesis and thrombocytopoiesis, Mpl-L also potentiates the action of other cytokines on several hematopoietic lineages. Mpl-L/TPO/MGDF, the homeostatic regulator of platelet production, might be a useful therapeutical cytokine to treat thrombocytopenia induced in patients by chemotherapy.     

Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin

Thrombopoietin (TPO) has recently been cloned and shown to regulate megakaryocyte and platelet production by activating the cytokine receptor c-mpl. To determine whether TPO is the only ligand for c-mpl and the major regulator of megakaryocytopoiesis, TPO deficient mice were generated by gene targeting. TPO-/- mice have a >80% decrease in their platelets and megakaryocytes but have normal levels of all the other hematopoietic cell types. A gene dosage effect observed in heterozygous mice suggests that the TPO gene is constitutively expressed and that the circulating TPO level is directly regulated by the platelet mass. Bone marrow from TPO-/- mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in the megakaryocytes that are present. These results demonstrate that TPO alone is the major physiological regulator of both proliferation and differentiation of hematopoietic progenitor cells into mature megakaryocytes but that TPO is not critical to the final step of platelet production.     

Endogenous thrombopoietin levels and effect of recombinant human thrombopoietin on megakaryocyte precursors in term and preterm babies

We have isolated genomic DNA containing the human tissue inhibitor of metalloproteinases-4 gene (TIMP4) and determined the structure of the exons comprising the gene. Like other members of the TIMP family, the TIMP-4 protein is encoded by five exons. These span 6 kb of genomic DNA, so that TIMP4 is similar in size to Timp1 but considerably smaller than TIMP2 and TIMP3. The exon-intron boundaries of TIMP4 are at locations very similar to those of the other TIMP genes, demonstrating the high degree of conservation of gene structure in this family. The human and mouse TIMP-4 genes map to comparable locations in the respective genomes, localizing to human chromosome 3p25 and mouse chromosome 6.     

Characterization of adhesive interactions between human endothelial cells and megakaryocytes

Cell-cell adhesion is essential for many immunological functions and is believed to be important in the regulation of hematopoiesis. Adhesive interactions between human endothelial cells and megakaryocytes were characterized in vitro using the CMK megakaryocytic cell line as well as marrow megakaryocytes. Although there was no adhesion between unactivated human umbilical vein endothelial cells (HUVEC) and megakaryocytes, treatment of HUVEC with inflammatory cytokines such as IL-1 beta, tumor necrosis factor alpha, INF-gamma, or the phorbol ester phorbol myristate acetate (PMA) resulted in a time- and dose-dependent increase in adhesion. Stimulation of marrow megakaryocytes or CMK cells with the cytokines IL-1 beta, GM-CSF, IL-6, IL-3, or PMA augmented their adhesion to endothelium. Monoclonal antibodies against the LFA-1 subunit of the leukocyte adherence complex CD18 inhibited the binding of marrow megakaryocytes or CMK cells to HUVEC. Adhesion blocking experiments also demonstrated that the VLA-4/VCAM-1 pathway was important for megakaryocyte attachment to HUVEC. Adhesion promoted maturation of megakaryocytic cells as measured by increased expression of glycoproteins GpIb and GpIIb/IIIa and by increased DNA content. These observations suggest that alterations in megakaryocyte adhesion may occur during inflammatory conditions, mediated by certain cytokines, resulting in augmented megakaryocyte maturation.     

Binding characteristics of aromatase inhibitors and phytoestrogens to human aromatase

We have evaluated the binding characteristics of three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione (7alpha-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)], and two flavone phytoestrogens (chrysin, and 7,8-dihydroxyflavone) to aromatase through a combination of computer modeling and inhibitory profile studies on the wild-type and six aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y). We have generated two aromatase models based on the x-ray structures of cytochrome P450-cam and cytochrome P450bm3, respectively. A major difference between the cytochrome P450cam-based and cytochrome P450bm3-based models is in the predicted lengths of helices F and G. In the cytochrome P450cam-based model, helices F and G lie antiparallel and extend across the active-site face of the molecule from one edge to the center, so that the carboxyl-terminal residues of helix F and the N-terminal residues of helix G make a major contribution to the structure of the active site. In the cytochrome P450bm3-based model, both helices are longer and so extend almost all the way across the active-site face of the molecule. Considering the size of the androgen substrate, we evaluated our results mainly based on the cytochrome P450cam model. The mutations involved in this study are thought to be at or near the proposed active site pocket. The inhibitory profile analysis has produced very interesting results and provided a molecular basis as to how seven aromatase inhibitors with different structures bind to the active site of aromatase. Furthermore, the investigation reveals that phytoestrogens bind to the active site of aromatase in a different orientation from that in the estrogen receptor.     

Binding to human extracellular matrix by Neisseria meningitidis

Adhesion of Neisseria meningitidis strains to extracellular matrix (ECM) and purified matrix components was examined. Most strains bound to subendothelial ECM as well as to immobilized fibronectin and types I, III, and V collagen. Strains from healthy carriers adhered significantly better than isolates from patients. The binding site was localized to the central 75-kDa cell-binding domain of the fibronectin molecule. This domain has not been described previously to interact with bacterial structures.     

Binding of collagen alpha1 chains to human platelets

We previously reported that purified alpha1 chains of type 1 chick skin collagen induce platelet aggregation. We now describe immunological and biochemical evidence that the peptide binds to intact platelets as an early event in the induction of platelet aggregation and the release reaction. Antibody against alpha1 (I) was obtained by immunizing rabbits with complete Freund's adjuvant mixed with purified alpha1. Immunofluorescence studies showed that alpha1(I)-treated platelets exhibited strong immunofluorescence. The intensity of fluorescence was markedly decreased by the pretreatment of platelets with alpha1-CB5 and glucosylgalactosylhydroxylysine. Dose-response curves of platelet aggregation induced by alpha1 and the binding of alpha1 by washed intact platelets are correlated. The biochemical studies showed that the binding of the alpha1 chain to washed intact platelets was platelet concentration and temperature dependent, and that it reached a maximum in 10 min. The process was reversible and specific, with an association constant of 1.7 muM. The inhibitor of alpha1-induced platelet aggregation, glucosylgalactosyl hydroxylysine, inhibited the alpha1 binding. These results suggest that alpha1(I) chains bind to specific receptor site(s) on platelet membranes to trigger aggregation and the release reaction.     

Giant intranuclear granules in human megakaryocytes: an ultrastructural study

The appearance of "giant perichromatin granules" to date has been considered pathognomonic of nasopharyngeal angiofibroma. We present the finding of giant intranuclear granules in human megakaryocytes. The patient was a 34-year-old woman with a diagnosis of Sebastian platelet syndrome. The bone marrow aspirate showed an increased number of megakaryocytes. Ultrastructural study revealed giant perichromatin-like granules in the nuclei of 80% megakaryocytes. These granules were round or oval and size ranged 230 to 540 nm. Further studies are needed in our patient to determine the significance of these granules.     

Binding of human serum amyloid P component (hSAP) to human neutrophils

Human serum amyloid P component (hSAP) and human C-reactive protein (hCRP) are normal serum constituents related to the pentraxin family of plasma proteins. hSAP has morphological and immunochemical identity and extensive sequence similarity to the amyloid P (AP) component found in normal tissues and particularly in amyloid deposits. hCRP and its proteolytic products have been previously shown to bind and to interact with various types of human leukocytes. Binding-displacement experiments with 125I-labeled hSAP and hCRP show that both proteins have specific high-affinity binding sites on normal human polymorphonuclear leukocytes (PMN) and each can compete efficiently with the binding of the other. Scatchard analysis of hSAP-displacement curves reveals a heterogeneous population of hSAP-binding sites existing on the PMN cells, among them about 300,000 low-affinity binding sites with Kd < or = 5 x 10(-6) M and about 30,000 high-affinity binding sites with Kd < or = 5 x 10(-8) M. hAP was found to be degraded by enzymes from human neutrophils to yield a mixture of low-molecular-mass peptides, similarly to the case of CRP reported previously. The binding of hSAP can be efficiently inhibited by this peptide mixture. The results suggest that both hCRP and hSAP, together with related peptides, may participate in vivo in an unknown mechanism of regulation of human neutrophils.     

Binding of enterostatin to the human neuroepithelioma cell line SK-N-MC

SK-N-MC cells were found to possess binding sites for enterostatin, a peptide with central effects on appetite and sympathetic activation of brown adipose tissue during high-fat feeding. Scatchard analyses of the binding indicated one high-affinity binding (Kd = 0.5-1.5 nM) and one low-affinity binding (Kd = 15-30 nM) for 3H-enterostatin (APGPR). 125I-YGGAPGPR showed similar binding parameters as for the low affinity binding of 3H-APGPR. Met-enkephalin and beta3-casomorphin1-5 were found to displace the binding of 3H-APGPR to the SK-N-MC cells. Affinity purification of solubilized cells revealed an APGPR-binding protein estimated to 53 kDa which may represent a distinct enterostatin receptor. Cross-linking of 125I-YGGAPGPR to intact cells labeled one major protein with the same molecular size. There was no binding of enterostatin to four other human neuroblastoma/neuroepithelioma cell lines, named IMR-92, LAN#5, NB-1 #14 and SH5-SY.     

Binding of Cryptococcus neoformans to heterologously expressed human complement receptors

Previously, we demonstrated that monoclonal antibodies (MAb) directed against any of the three defined complement receptors (CR) for the third component of complement (CR1, CR3, and CR4) profoundly inhibited the binding of serum-opsonized Cryptococcus neoformans to monocyte-derived macrophages. These studies suggested either that a synergistic interaction between multiple CR was required for optimal binding of C. neoformans or that the MAb were exerting nonspecific effects (such as receptor coassociation). In the present studies, we took a novel approach to dissecting out the contributions of individual receptors to binding of a microbial pathogen. Chinese hamster ovary (CHO) cells stably transfected with human CR1, CR3, or CR4 were challenged with serum-opsonized C. neoformans. We found that CHO cells transfected with any of the three receptors bound C. neoformans, with the avidity of binding to CR3 being the greatest followed in decreasing order by CR1 and CR4. Following binding of C. neoformans to transfected CHO cells, most organisms remained surface attached only, although for each receptor a significant percentage (18.5 to 27.3%) of C. neoformans was internalized. Both C. neoformans and sheep erythrocytes that were selectively opsonized with the fragments of the third component of complement, C3b and iC3b, were bound preferentially by CHO cells transfected with CR1 and CR3, respectively. These data establish CR1, CR3, and CR4 as receptors independently capable of binding C. neoformans opsonized with fragments of C3. Moreover, our study demonstrates the usefulness of transfected cell lines as a powerful tool for identifying the contribution of individual receptors to the binding of a microbial pathogen.     

Binding of amyloid beta-protein to intracellular brain proteins in rat and human

Amyloid beta-protein (Abeta), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of Abeta to intracellular proteins has not been studied. We have developed an overlay assay to study Abeta binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to Abeta 1-40 and Abeta 1-42. No major difference was observed in the Abeta binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of Abeta to brain proteins resides between 12-28 amino acid sequence of Abeta. The presence of several intracellular Abeta-binding (AbetaB) proteins suggests that these proteins may either protect Abeta from its fibrillization or alternatively promote Abeta polymerization. Identification of these proteins and their binding affinities for Abeta are needed to assess their potential role in the pathogenesis of Alzheimer's disease.     

Binding of codeine, morphine, and methadone to human serum proteins

The binding properties of codeine, morphine (as representative opium alkaloids), and methadone (a synthetic pharmacologically similar compound) were studied with selected human serum proteins. The methodology involved equilibrium and dynamic dialysis using 3H-and/or 14C-labeled compounds. For estimation of the percent binding with equilibrium dialysis, concentrations of the ligand used were approximately therapeutic blood levels and another concentration 30-60 times higher. The percent binding to whole human serum ranged from about 20% for morphine to almost 60% for methadone. Of the human serum proteins investigated, the highest percent binding was found with albumin, except for methadone for which it was beta-globulin III. The affinity for other serum proteins varied with the ligand. In studies with albumin using dynamic dialysis, the plots of nubar divided by free concentration versus nubar were similar for all three ligands studied and had positive slopes, unlike those reported for acidic compounds for which the slope is always negative. In studies of binding of one ligand in the presence of another, significant competition was demonstrated, suggesting that the same binding sites were involved.     

Suppression of the voltage-gated K+ current of human megakaryocytes by thrombin and prostacyclin

We examined the effects of platelet activators and inhibitors of platelet function on the voltage-gated delayed rectifier K+ current of human megakaryocytes. We found that both the activators such as thrombin, the thrombin receptor peptide (TRP42-47) and ADP and the inhibitors such as prostacyclin suppressed the delayed rectifier current through two different mechanisms. The cAMP dependent protein kinase (A-kinase) inhibitor IP20 blocked the suppression of the delayed rectifier current by prostacyclin and failed to block the suppression by thrombin, TRP42-47 and ADP. The effects of IP20 suggest that the action of prostacyclin is mediated by A-kinase and the action of the three activators is not mediated by A-kinase. Pertussis toxin (PTX) an inhibitor of the inhibitory GTP-binding proteins (Gi) blocked the suppression of the delayed rectifier current by thrombin, TRP42-47 and ADP and failed to block the suppression by prostacyclin. The effects of PTX suggests that the action of the three activators is mediated by Gi or some other PTX-sensitive GTP-binding protein. We speculate that thrombin and other platelet activators that activate Gi may be suppressing the delayed rectifier current via a direct interaction of Gi or a subunit of it with the delayed rectifier potassium channel itself.     

Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand

The site and mechanism of platelet production by bone marrow megakaryocytes (MKs) has been the subject of extensive studies, but is still a matter of controversy. However, the recent discovery of the Mpl ligand (Mpl-l), also called megakaryocyte growth and development factor (MGDF) or thrombopoietin, has resulted in considerable progress in the understanding of the maturation of the MK lineage. To better understand the mechanism of platelet production, we examined the late stage of MK maturation by electron microscopy in cells cultured in the presence of Mpl-l. Human bone marrow CD34+ CD38+ cells, which contain late MK progenitors, were purified by flow cytometry and cultured in a serum-free liquid medium containing recombinant human Mpl-l (MGDF 10 ng/mL) for 7 days. In this system, the majority of cultured cells were large MKs with lobulated polyploid nuclei. The MKs displayed a smooth surface with harmonious cytoplasmic maturation and abundant, regularly distributed demarcation membranes and alpha-granules, and even some dense granules. Interestingly, approximately 30% of the MKs observed displayed morphologic evidence of platelet production: at optical microscopy, MKs formed long filamentous cytoplasmic extensions (proplatelets) that fragmented into platelet-sized particles. Moreover, flow cytometric analysis of this cultured cell population showed GPIIb-positive particles of the size of platelets. Electron microscopic observation showed that MKs producing platelets displayed thin pseudopods on the surface, and that the channels of the demarcation membrane system were dilated, allowing long strands of cytoplasm to extend from the cell periphery. These cytoplasmic strands displayed beading with constrictions separating platelet-sized segments; the more distal to the cell core, the smaller the fragments were. They eventually detached from the cell core into the culture medium either occasionally still elongated or, more often, separated into individual platelets. Parallel longitudinal and perpendicular microtubules were visualized in the constricted regions of these cytoplasmic strips; immunogold study of tubulin localization confirmed this subcellular distribution. On both sides of the constricted areas, vacuoles were noted, the fusion of which might have led to the detachment of individual platelets. Finally, in close proximity to the platelet-forming MKs, numerous microparticles were shed. Although some of these particles might correspond to transverse sections of pseudopods, this did not seem to be the case, since they were rarely seen around thrombin-stimulated MKs with surfaces bristled by numerous pseudopods. Flow cytometry showed that apart from shed cytoplasmic fragments of platelet size, numerous smaller particles strongly labeled for CD41 were also released by mature MKs. In conclusion, this study describes the ultrastructure of human platelet production in cultured MKs, involving the formation of proplatelets and the shedding of microparticles.     

Integrins involved in the adhesion of megakaryocytes to fibronectin and fibrinogen

We studied integrins involved in the adhesion of resting and activated megakaryocytes (MK) to fibronectin (FN) and fibrinogen (FGN). Guinea pig MK were isolated and in some experiments were activated by thrombin. MK adhering to FN or FGN coated on coverslips were quantitated by a computerized image analysis program. The binding of soluble human FN to MK was detected by Western blotting. Anti-integrin antibodies, disintegrins, and cyclic RGD peptides were used to identify integrins involved in the adhesion of MK to FN or FGN. Resting MK adhered to coverslips with immobilized FN. The adhesion of MK to FN was primarily inhibited by an anti-alpha5 antibody and EMF-10, a distintegrin highly specific for alpha5 beta1. However, the adhesion of MK to FN was not blocked by agents that inhibit alphaIIb beta3, alphav beta3 or alpha4 beta1. A beta1 activating antibody increased the number of MK bound to FN due to the activation of alpha5 beta1. The binding of soluble FN was also primarily inhibited by agents that block alpha5 beta1. Resting MK did not adhere to FGN. However, MK activated by thrombin did adhere to FGN. This binding was mediated by alphaIIb beta3, because binding was inhibited by bitistatin, a disintegrin, and a cyclic RGD peptide that are known to block this integrin. The binding of thrombin-activated MK to FN was mediated by both alpha5 beta1 and alphaIIb beta3 based on the additive effect of agents that inhibit these integrins. The study indicates that resting MK bind to FN but not to FGN and that alpha5 beta1 is the major integrin involved in the binding of MK to FN. Activated MK bind to FGN primarily by alphaIIb beta3. However, the binding of activated MK to FN is due to both alpha5 beta1 and alphaIIb beta3. The demonstration that alpha5 beta1 and that alphaIIb beta3 are involved in MK adhesion indicates that these integrins may have a role in MK maturation and platelet production.