ADP causes partial degranulation of platelets in the absence of aggregation

Summary. Whole blood flow cytometry has revealed that platelets undergo partial degranulation in response to ADP, in the absence of aggregation, as evidenced by the expression of the P-selectin and CD63 antigens of the α-granule and lysosomal membranes respectively. With maximum ADP (10^-5 m ) fibrinogen bound to 76·1 ± 7·2% of platelets but P-selectin and CD63 antigen were expressed on 26·9±9·8% and 8·6±3·5% of platelets respectively. Maximum fibrinogen binding, P-selectin and CD63 expression induced by α-thrombin were 96·1±1·4%, 92·8±2·3% and 77·6±9·7% respectively. β-thromboglobulin release from the ADP-stimulated platelets correlated closely with the expression of P-selectin and CD63 ( r =0·98·0±02 for both antigens). No platelet aggregates were seen by flow cytometry and the absence of aggregation was confirmed by single cell counting. Addition of the GPIIb–IIIa antagonist echistatin. at concentrations that totally blocked fibrinogen binding to ADP-stimulated platelets, had no effect on the expression of the granule membrane antigens. The partial degranulation of normal platelets was independent of thrombin generation since it was not inhibited by hirudin (5 units/ml). In conclusion, ADP is capable of causing partial degranulation of platelets independently of aggregation, fibrinogen binding or thrombin generation. Thus release of potent procoagulant, vasoactive and mitogenic substances from the platelets could continue in the presence of thrombin inhibitors and GPIIb-IIIa antagonists.     

Two patterns of thrombopoietin signaling suggest no coupling between platelet production and thrombopoietin reactivity in thrombocytopenia-absent radii syndrome

Background

Thrombocytopenia with absent radii syndrome is defined by bilateral radius aplasia and thrombocytopenia. Due to impaired thrombopoietin signaling there are only few bone marrow megakaryocytes and these are immature; the resulting platelet production defect improves somewhat over time. A microdeletion on chromosome 1q21 is present in all patients but is not sufficient to form thrombocytopenia with absent radii syndrome. We aimed to refine the signaling defect in this syndrome.

Design and Methods

We report an extended study of 23 pediatric and adult patients suffering from thrombocytopenia with absent radii syndrome in order to scrutinize thrombopoietin signal transduction by immunoblotting and gel electrophoretic shift assays. In addition, platelet immunotyping and reactivity were analyzed by flow cytometry. Results were correlated with clinical data including age and platelet counts.

Results

Two distinct signaling patterns were identified. Juvenile patients showed abrogated thrombopoietin signaling (pattern #1), which is restored in adults (pattern #2). Phosphorylated Jak2 was indicative of activation of STAT1, 3 and 5, Tyk2, ERK, and Akt, showing its pivotal role in distinct thrombopoietin-dependent pathways. Jak2 cDNA was not mutated and the thrombopoietin receptor was present on platelets. All platelets of patients expressed normal levels of CD41/61, CD49b, and CD49f receptors, while CD42a/b and CD29 were slightly reduced and the fibronectin receptor CD49e markedly reduced. Lysosomal granule release in response to thrombin receptor activating peptide was diminished.

Conclusions

We show a combined defect of platelet production and function in thrombocytopenia with absent radii syndrome. The rise in platelets that most patients have during the first years of life preceded the restored thrombopoietin signaling detected at a much later age, implying that these events are uncoupled and that an unknown factor mediates the improvement of platelet production.     

Role of the kidneys in thrombopoietin production.

Extracts of plasma from normal rats, rats injected with rabbit anti-rat platelet serum (RARPS), nephrectomized rats and nephrectomized rats injected with RARPS were tested in thrombocytotic mice for the presence of thrombopoietin. Only fractions of plasma from unoperated rats that had been made thrombocytopenic by injection of RARPS gave positive results for thrombopoietin. The results suggest that the kidneys play a role in thrombopoietin production.     

Effects of thrombopoietin and erythropoietin on platelet production in rebound-thrombocytotic and normal mice.

The effects of thrombopoietin-rich (TSF-rich) kidney cell culture medium and partially purified preparations of human urinary and step I sheep plasma erythropoietin (Ep) on platelet production in mice were determined. After TSF and Ep preparations had been injected into mice in rebound thrombocytosis and into normal mice, the rate of incorporation of Na₂ ³⁵SO₄ into platelets was measured as an index of platelet production. TSF injections caused significant increases in platelet production of all the mice; however, greater effects were observed in rebound-thrombocytotic mice than in normal mice. In rebound-thrombocytotic mice, low dosages of Ep (0.25–1.0 units per mouse) significantly depressed percentage ³⁵S incorporation into platelets. The same low dosage of Ep did not alter platelet production rates in normal mice, but large doses (2.5–5.0 units per mouse) stimulated ³⁵S incorporation into platelets. Thus, these results show that Ep caused different responses in rebound-thrombocytotic and normal mice. Endotoxin, injected in dosages previously shown to be present in Ep, gave responses similar to that of the low dosages of Ep, ie, depressed thrombocytopoiesis in rebound-thrombocytotic mice but had no effect in normal mice. When two foreign proteins were injected in doses similar to the previously tested Ep protein content, platelet production was not stimulated. We conclude, therefore, that the thrombocytopoietic effects of Ep cannot be due to contamination with known humoral factors or foreign proteins.     

Effect of thrombopoietin on the development of megakaryocytes and platelets: an ultrastructural analysis

Megakaryocytopoiesis and platelet production can be assessed with reasonable accuracy by quantitative and functional analyses of circulating platelets. The evaluation of megakaryocytopoiesis in culture has remained unsatisfactory, particularly because platelet production is rarely observed. In mouse culture systems, megakaryocytes have been identified almost entirely by measurements of acetyl cholinesterase, size, and ploidy without concomitant assessment of maturation based on such criteria as the formation of granules, demarcation membranes, and cytoplasmic fragmentation. The availability of various thrombopoietic cytokines, in particular thrombopoietin (TPO), and their imminent clinical use has made a more detailed understanding of their effect on differentiation and maturation of the MK lineage more urgent. Therefore, ultrastructural analyses were performed on megakaryocyte-depleted serum-free mouse bone marrow cultures in the presence of TPO alone, TPO plus other cytokines, or under conditions in which TPO and/or other cytokines were blocked with neutralizing agents. These studies show that, while cytokines that use the gp130 receptor subunit may function synergistically with TPO, in the absence of TPO, such cultures do not yield morphologically recognizable MK. On the other hand, TPO alone is able to drive MK to full maturation as evidenced by the generation of granules, demarcation membranes, and cytoplasmic fragmentation into platelets.     

Assay for thrombopoietin: a comparison of time of isotope incorporation into platelets and the effects of different strains and sexes of mice.

Several workers have used mice for thrombocytopoiesis-stimulating factor (TSF) assays, but the methods have differed. In quest of the optimum TSF assay conditions, we investigated the effects of interval between isotope injection and measurement of 35S incorporation into platelets, different mouse strains and sexes of mice. The 35S incorporation into platelets of mice increased with increase of the interval after isotope injection. However, the greatest difference in radioactivity between control and TSF-injected mice occurred at 16-40 h for normal mice and 24 h for rebound-thrombocytotic mice. When C3H, BALB/c or B6D2F1 mice were injected with platelet specific antisera, similar degrees of thrombocytopenia and rebound-thrombocytosis occurred. After injection of a standard dose of TSF, C3H and B6D2F1 mice showed greater isotopic incorporation levels, compared to suitable controls, than did BALB/c mice. Female and male mice exhibited essentially the same response to a standard dose of TSF. The data show that the mouse strain and isotopic incorporation time influence the sensitivity of the TSF assay but sex of test animals does not.     

A mutation in Rab27a causes the vesicle transport defects observed in ashen mice

The dilute (d), leaden (ln), and ashen (ash) mutations provide a unique model system for studying vesicle transport in mammals. All three mutations produce a lightened coat color because of defects in pigment granule transport. In addition, all three mutations are suppressed by the semidominant dilute-suppressor (dsu), providing genetic evidence that these mutations function in the same or overlapping transport pathways. Previous studies showed that d encodes a major vesicle transport motor, myosin-VA, which is mutated in Griscelli syndrome patients. Here, using positional cloning and bacterial artificial chromosome rescue, we show that ash encodes Rab27a. Rab GTPases represent the largest branch of the p21 Ras superfamily and are recognized as key players in vesicular transport and organelle dynamics in eukaryotic cells. We also show that ash mice have platelet defects resulting in increased bleeding times and a reduction in the number of platelet dense granules. These defects have not been reported for d and ln mice. Collectively, our studies identify Rab27a as a critical gene for organelle-specific protein trafficking in melanocytes and platelets and suggest that Rab27a functions in both MyoVa dependent and independent pathways.     

Simultaneous absence of dopamine D1 and D2 receptor-mediated signaling is lethal in mice

Dopamine (DA) controls a wide variety of physiological functions in the central nervous system as well as in the neuroendocrine and gastrointestinal systems. DA signaling is mediated by five cloned receptors named D1-D5. Knockout mouse models for the five receptors have been generated, and, albeit impaired for some important DA-mediated functions, they are viable and can reproduce. D1 and D2 receptors are the most abundant and widely expressed DA receptors. Cooperative/synergistic effects mediated by these receptors have been suggested, in particular, in the control of motor behaviors. To analyze the extent of such interrelationship, we have generated double D1/D2 receptor mutants. Interestingly, in contrast to single knockouts, we found that concurrent ablation of the D1 and D2 receptors is lethal during the second or third week after birth. This dramatic phenotype is likely to be related to altered feeding behavior and dysfunction of the gastrointestinal system, especially because major anatomical changes were not identified in the brain. Similarly, in the absence of functional D1, heterozygous D2 mutants (D1r(-/-);D2r(+/-)) showed severe growth retardation and did not survive their postweaning period. The analysis of motor behavior in D1r/D2r compound mutants showed that loss of D2-mediated functions reduces motor abilities, whereas the effect of D1r ablation on locomotion strongly depends on the experimental paradigms used. These studies highlight the interrelationship between D1 and D2 receptor-mediated control of motor activity, food intake, and gastrointestinal functions, which has been elusive in the single-gene ablation studies.     

Lung dysfunction causes systemic hypoxia in estrogen receptor beta knockout (ERbeta-/-) mice

Estrogen receptor beta (ERbeta) is highly expressed in both type I and II pneumocytes as well as bronchiolar epithelial cells. ERalpha is not detectable in the adult lung. Lungs of adult female ERbeta knockout (ERbeta-/-) mice have already been reported to have fewer alveoli and reduced elastic recoil. In this article, we report that, by 5 months of age, there are large areas of unexpanded alveoli in lungs of both male and female ERbeta-/- mice. There is increased staining for collagen and, by EM, abnormal clusters of collagen fibers are seen in the alveolar septa of ERbeta-/- mice. Immunohistochemical analysis and Western blotting with lung membrane fractions of ERbeta-/- mice revealed down-regulation of caveolin-1, increased expression of membrane type-1 metalloproteinase, matrix metalloproteinase 2 (active form), and tissue inhibitors of metalloproteinases 2. Hypoxia, measured by immunohistochemical analysis for hypoxia-inducible factor 1alpha and chemical adducts (with Hypoxyprobe), was evident in the heart, ventral prostate, periovarian sac, kidney, liver, and brain of ERbeta-/- mice under resting conditions. Furthermore, both male and female adult ERbeta-/- mice were reluctant to run on a treadmill and tissue hypoxia became very pronounced after exercise. We conclude that ERbeta is necessary for the maintenance of the extracellular matrix composition in the lung and loss of ERbeta leads to abnormal lung structure and systemic hypoxia. Systemic hypoxia may be responsible for the reported left and right heart ventricular hypertrophy and systemic hypertension in ERbeta-/- mice.     

The role of the liver in the production of thrombopoietin compared with erythropoietin

The liver plays an important role in the production of haemopoietic hormones. It acts as the primary site of synthesis of erythropoietin (EPO) in the fetal stage, and it is the predominant thrombopoietin (TPO)-producing organ for life. In contrast to that of EPO and other liver proteins, the hepatic synthesis of TPO is influenced little by external signals. Hepatocytes express the TPO gene in a constitutive way, i.e. irrespective of the level of platelets in blood. Megakaryocytes and platelets remove the hormone from blood by means of their high-affinity TPO receptors. Normally, the plasma level of TPO is relatively low ( approximately 10(-12) mol/l). However, in thrombocytopenic states due to marrow failure or bleeding, the concentration of circulating TPO may increase greatly. The simple feedback regulation by TPO and its target cells is efficient in maintaining constant platelet numbers in healthy people. Persisting thrombocytopenia develops only in severe liver or marrow failure. On the other hand, an increase in circulating TPO and interleukin 6 (IL-6) may cause reactive thrombocytosis in inflammatory diseases, including cancer. The indications for recombinant human thrombopoietin (rHuTPO) therapy and its impact on transfusion medicine are still under investigation.     

Thrombocytopenic c-mpl(-/-) mice can produce a normal level of platelets after administration of 5-fluorouracil: the effect of age on the response

Administration of 5-fluorouracil (5-FU) to mice results in a marked increase in the level of circulating platelets in 10 days. Mice lacking Mpl, the receptor for thrombopoietin (TPO), are thrombocytopenic. To gain insight into the mechanism by which 5-FU produces such a substantial stimulation of platelet production, this study investigated whether 5-FU (150 mg/kg) produced thrombocytosis in c-mpl(-/-) mice, thus establishing whether TPO was required for this response. A 5- to 6-fold increase in platelet levels in c-mpl(-/-) mice (to approximately 1000 x 10(9)/L) was observed on days 20 and 25 after 5-FU injection. Thus, at the peak of the response, c-mpl(-/-) mice had platelet levels comparable to those in normal mice. Administration of 5-FU also produced thrombocytosis in previously splenectomized c-mpl(-/-) mice. Comparison of the platelet response to 5-FU in young (6-12 weeks) and old (33-46 weeks) c-mpl(-/-) mice found that older mice produced a much more marked response than younger mice, with a mean maximum platelet level of approximately 1700 x 10(9)/L. To determine whether this increase in circulating platelets was preceded by an increase in hematopoietic progenitors, serial cultures of bone marrow and spleen were evaluated. A considerable increase in all colony types studied was observed on days 15 and 20 in spleens of c-mpl(-/-) mice, but no similar elevations were detected in bone marrow. These results indicate that c-mpl(-/-) mice can achieve a normal level of platelets after 5-FU injection, by means of a TPO-independent mechanism, and that they respond to 5-FU myelosuppression by producing large numbers of megakaryocytic, myeloid, and erythroid progenitors. (Blood. 2001;98:1019-1027)     

Effect of thrombopoietin receptor agonists on the apoptotic profile of platelets in patients with chronic immune thrombocytopenia

Platelet survival depends upon mediators of apoptosis e.g., Bcl‐x_L, Bax, and Bak, which are regulated by thrombopoietin (TPO)‐mediated AKT signaling. Thrombopoietin receptor (TPO‐R) signaling might decrease platelet and/or megakaryocyte apoptosis and increase the platelet count. This study therefore explored anti‐apoptotic effects of TPO‐R‐agonists in vivo on platelets of patients with immune thrombocytopenia. Patients received eltrombopag or romiplostim for two weeks. Total, immature, and large platelet counts were assessed as were Bcl‐x_L inhibitor assay; Bcl‐x_L Western blot; and flow cytometric (FACS) analysis of the AKT‐signaling pathway. Eight/ten patients had platelet responses to eltrombopag and all three to romiplostim. Platelet sensitivity to apoptosis by Bcl‐xL inhibition was greater in pretreatment patients than controls. This sensitivity normalized after one week of therapy, but surprisingly returned to pretreatment levels at week two. FACS analysis revealed increased AKT‐pathway signaling after one week, followed by a decrease at week two. Platelet counts correlated with the Bcl‐x_L/Bak ratio. Platelet survival may be enhanced by TPO‐R‐agonists as a transient decrease in platelet sensitivity to apoptosis was accompanied by transient activation of AKT. However, this mechanism has only a short‐lived effect. Megakaryocytes and platelets already present at the start of TPO‐R‐agonist treatment appear to respond differently than those generated de novo. Am. J. Hematol. 89:E228–E234, 2014. © 2014 Wiley Periodicals, Inc.     

Suppressor of cytokine signaling gene expression in human pancreatic islets: modulation by cytokines

OBJECTIVE: Suppressor of cytokine signaling (SOCS) proteins negatively regulate signal transduction of several cytokines. Since cytokines participate in the pancreatic islet damage in type 1 diabetes, the aim of our study was to investigate the expression of SOCS-1, -2 and -3 in isolated human islets, in basal conditions and after exposure, in vitro, to a combination of interferon (IFN)-gamma, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha cytokines and in control and in type 1 diabetic human pancreata, to establish (i) whether SOCS molecules are constitutively expressed in human pancreatic islets and (ii) whether their expression can be modulated in vitro by proinflammatory cytokines or ex vivo by an islet inflammatory process. METHODS: Gene expression of SOCS-1, -2 and -3 was evaluated by RT-PCR in untreated and cytokine-treated isolated human pancreatic islets and their protein expression by immunohistochemistry in control and in type 1 diabetic human pancreata paraffin-embedded sections. RESULTS: We found that SOCS-1, -2 and -3 mRNA is constitutively, although weakly, expressed in human pancreatic islets, similar to the expression observed in control pancreata by immunohistochemistry. SOCS-1, -2 and -3 mRNA expression was strongly increased in human islets after exposure, in vitro, to IFN-gamma, IL-1beta and TNF-alpha. Accordingly, an intense and islet-specific immunohistochemical staining for all three SOCS was detected in pancreata from type 1 diabetic patients. CONCLUSION: SOCS-1, -2 and -3 genes are constitutively expressed in human pancreatic islets; their expression increases after exposure to proinflammatory cytokines and during an autoimmune inflammatory process, raising the possibility that these molecules act as key regulators of cytokine signaling in pancreatic islets.