Platelet Production In Mice Research Articles

Luteolin inhibits GPVI-mediated platelet activation, oxidative stress, and thrombosis.

Introduction: Luteolin inhibits platelet activation and thrombus formation, but the mechanisms are unclear. This study investigated the effects of luteolin on GPVI-mediated platelet activation in vitro and explored the effect of luteolin on thrombosis, coagulation, and platelet production in vivo. Methods: Washed human platelets were used for aggregation, membrane protein expression, ATP, Ca2+, and LDH release, platelet adhesion/spreading, and clot retraction experiments. Washed human platelets were used to detect collagen and convulxin-induced reactive oxygen species production and endogenous antioxidant effects. C57BL/6 male mice were used for ferric chloride-induced mesenteric thrombosis, collagen-epinephrine induced acute pulmonary embolism, tail bleeding, coagulation function, and luteolin toxicity experiments. The interaction between luteolin and GPVI was analyzed using solid phase binding assay and surface plasmon resonance (SPR). Results: Luteolin inhibited collagen- and convulxin-mediated platelet aggregation, adhesion, and release. Luteolin inhibited collagen- and convulxin-induced platelet ROS production and increased platelet endogenous antioxidant capacity. Luteolin reduced convulxin-induced activation of ITAM and MAPK signaling molecules. Molecular docking simulation showed that luteolin forms hydrogen bonds with GPVI. The solid phase binding assay showed that luteolin inhibited the interaction between collagen and GPVI. Surface plasmon resonance showed that luteolin bonded GPVI. Luteolin inhibited integrin αIIbβ3-mediated platelet activation. Luteolin inhibited mesenteric artery thrombosis and collagen- adrenergic-induced pulmonary thrombosis in mice. Luteolin decreased oxidative stress in vivo. Luteolin did not affect coagulation, hemostasis, or platelet production in mice. Discussion: Luteolin may be an effective and safe antiplatelet agent target for GPVI. A new mechanism (decreased oxidative stress) for the anti-platelet activity of luteolin has been identified.

Inflammatory platelet production stimulated by tyrosyl-tRNA synthetase mimicking viral infection

Platelets play a role not only in hemostasis and thrombosis, but also in inflammation and innate immunity. We previously reported that an activated form of tyrosyl-tRNA synthetase (YRSACT) has an extratranslational activity that enhances megakaryopoiesis and platelet production in mice. Here, we report that YRSACT mimics inflammatory stress inducing a unique megakaryocyte (MK) population with stem cell (Sca1) and myeloid (F4/80) markers through a mechanism dependent on Toll-like receptor (TLR) activation and type I interferon (IFN-I) signaling. This mimicry of inflammatory stress by YRSACT was studied in mice infected by lymphocytic choriomeningitis virus (LCMV). Using Sca1/EGFP transgenic mice, we demonstrated that IFN-I induced by YRSACT or LCMV infection suppressed normal hematopoiesis while activating an alternative pathway of thrombopoiesis. Platelets of inflammatory origin (Sca1/EGFP+) were a relevant proportion of those circulating during recovery from thrombocytopenia. Analysis of these "inflammatory" MKs and platelets suggested their origin in myeloid/MK-biased hematopoietic stem cells (HSCs) that bypassed the classical MK-erythroid progenitor (MEP) pathway to replenish platelets and promote recovery from thrombocytopenia. Notably, inflammatory platelets displayed enhanced agonist-induced activation and procoagulant activities. Moreover, myeloid/MK-biased progenitors and MKs were mobilized from the bone marrow, as evidenced by their presence in the lung microvasculature within fibrin-containing microthrombi. Our results define the function of YRSACT in platelet generation and contribute to elucidate platelet alterations in number and function during viral infection.

Rational Design of a Bifunctional AND-Gate Ligand To Modulate Cell-Cell Interactions.

Protein "AND-gate" systems, in which a ligand acts only on cells with two different receptors, direct signaling activity to a particular cell type and avoid action on other cells. In a bifunctional AND-gate protein, the molecular geometry of the protein domains is crucial. Here we constructed a tissue-targeted erythropoietin (EPO) that stimulates red blood cell (RBC) production without triggering thrombosis. The EPO was directed to RBC precursors and mature RBCs by fusion to an anti-glycophorin A antibody V region. Many such constructs activated EPO receptors in vitro and stimulated RBC and not platelet production in mice but nonetheless enhanced thrombosis in mice and caused adhesion between RBCs and EPO-receptor-bearing cells. On the basis of a protein-structural model of the RBC surface, we rationally designed an anti-glycophorin-EPO fusion that does not induce cell adhesion in vitro or enhance thrombosis in vivo. Thus, mesoscale geometry can inform the design of synthetic-biological systems.

Pre-clinical development of a cryopreservable megakaryocytic cell product capable of sustained platelet production in mice.

Platelet (PLT) transfusions are the most effective treatments for patients with thrombocytopenia. The growing demand for PLT transfusion products is compounded by a limited supply due to dependency on volunteer donors, a short shelf-life, risk of contaminating pathogens, and alloimmunization. This study provides preclinical evidence that a third-party, cryopreservable source of PLT-generating cells has the potential to complement presently available PLT transfusion products. CD34+ hematopoietic stem/progenitor cells derived from umbilical cord blood (UCB) units were used in a simple and efficient culture system to generate a cell product consisting of megakaryocytes (MKs) at different stages of development. The cultures thus generated were evaluated ex vivo and in vivo before and after cryopreservation. We generated a megakaryocytic cell product that can be cryopreserved without altering its phenotypical and functional capabilities. The infusion of such a product, either fresh or cryopreserved, into immune-deficient mice led to production of functional human PLTs which were observed within a week after infusion and persisted for 8 weeks, orders of magnitude longer than that observed after the infusion of traditional PLT transfusion products. The sustained human PLT engraftment was accompanied by a robust presence of human cells in the bone marrow (BM), spleen, and lungs of recipient mice. This is a proof-of-principle study demonstrating the creation of a cryopreservable megakaryocytic cell product which releases functional PLTs in vivo. Clinical development of such a product is currently being pursued for the treatment of thrombocytopenia in patients with hematological malignancies.

Platelet Biogenesis in the Lung Circulation

Platelets are indispensable in hemostasis, thrombosis, and immune responses. In humans, billions of platelets are produced each day from megakaryocytes, however the mechanisms of mature platelet production are incompletely understood. Megakaryocytes are produced in the bone marrow and have been visualized to communicate with the bone marrow sinusoids to release proplatelet fragments. Megakaryocytes have also been found in other tissues, including the lung, but the function of megakaryocytes in these locations is unclear. Historical data indicate that the lung may be a site of platelet biogenesis. The concentration of megakaryocytes in the blood exiting the lung is much lower than the blood entering the lung (implying filtering) and conversely, platelet counts are higher in blood draining from the lungs. Additionally, when the lung circulation in entirely bypassed, megakaryocytes accumulate in the blood and there is a high incidence of thrombocytopenia. However, direct proof of platelet biogenesis in the lung is lacking. We used lung intravital microscopy combined with fluorescently labeled mouse strains and directly visualized intravascular megakaryocytes releasing platelets in the lung circulation. We also visualized megakaryocytes in the bone marrow and spleen releasing proplatelet fragments, and megakaryocyte migration in toto from the bone marrow, which are presumably the source material for lung platelet production. The megakaryocyte-releasing events in the lung were quantified and represent at least half of the total platelet production in mice, which can be increased by the application of thrombopoietin. We also observed a much larger extravascular pool of megakaryocytes in the lung that were not platelet generating as observed by lung intravital imaging. The function of these lung-resident megakaryocytes is unknown, but RNA-Seq data points to a potential role in lung immunity. Orthotopic, single-lung transplantation experiments into thrombocytopenic and hematopoietic progenitor-deficient animals (c-mpl-/-) revealed that peripheral blood platelet counts and bone marrow hematopoietic progenitors could be fully reconstituted by the lung transplant procedure implying the presence of hematopoietic progenitors in the mouse lung. Indeed, these progenitors were directly detected in the extravascular lung and purified populations of hematopoietic progenitors in the lung could correct thrombocytopenia in c-mpl-/-animals. Finally, the lung transplant procedure produced donor-derived chimerism of other hematopoietic lineages such as neutrophils and lymphocytes. We conclude that the lung has significant hematopoietic potential including being a major site of platelet biogenesis. DisclosuresNo relevant conflicts of interest to declare.

Detection of abundant megakaryocytes in pulmonary artery blood in lung cancer patients using a microfluidic platform

ObjectivesThe lung was recently re-discovered as a hematopoietic organ for platelet production in mice. However, evidence for the role of the lung in thrombopoiesis in humans is still limited. In this study, we examined megakaryocytes in the pulmonary and systemic circulation, specifically in pulmonary arterial blood (PAB), venous blood (PVB) and peripheral blood using a newly developed microfluidic platform for rare cell isolation. Materials and methodsWe analyzed 23 lung cancer patients who underwent surgery in our institute. PAB and PVB were obtained from the resected lung immediately after surgery. Blood samples were size-selected using a filtration-based microfluidic device and enriched rare cells on glass slide specimens were stained with Papanicolaou (Pap), immunocytochemistry (ICC), and immunofluorescence (IF). Lung tissues were also analyzed by immunohistochemistry. ResultsPap/ICC/IF showed the presence of abundant CD61+/cytokeratin- giant cells with a megakaryocyte lineage in PAB, but only a few in PVB. These megakaryocytes were found to consist of CD61+/CD41+ immature megakaryocytes and CD61+/CD41- mature megakaryocytes with the potential to produce platelets. These findings were confirmed by the conventional hematological analysis of blood smears stained with Giemsa. In analysis of lung cancer, CD61+ megakaryocytes were observed exclusively in the capillaries of non-cancerous tissue, whereas platelets were selectively observed in the tumor blood vessels of cancerous tissue. ConclusionsThese results indicate that numerous megakaryocytes migrate from systemic bone marrows to accumulate in PAs and arrest of mature megakaryocytes in the capillaries of normal lung, suggesting the possibility that the lung plays a physiological role in the systemic thrombopoiesis in lung cancer patients.

Extended Platelet In Vivo Survival Results in Exhausted Platelets

Platelet lifespan is limited to 10 days in humans and 5 days in mice. The intrinsic apoptosis pathway regulates the survival of platelets, where the pro-survival protein Bcl-xLrestrains the essential death mediators Bak and Bax (Mason et al., Cell 2007). Hence, platelet lifespan and platelet counts in mice are increased in the absence of Bak and Bak/Bax. While platelet production in mice is normal in the absence of intrinsic apoptosis (Josefsson et al., J Exp Med 2011), the function of these long-lived platelets has not been investigated. In the current study we examined the functional outcome of extended platelet survival.We found that washed platelets from mice with a constitutive deletion of Bak and a platelet-specific deletion of Bax (Bak-/-BaxPf4Δ/Pf4Δ) were fully resistant to apoptosis induced by the BH3-mimetic ABT-737, as demonstrated by lack of phosphatidylserine exposure (binding of AnnexinV) and unaltered mitochondrial membrane potential. Tail bleeding times into 37°C saline, were extended in the absence of either Bak alone or both Bak and Bax. Furthermore, the electrolytic thrombosis model showed that despite normal time to arterial occlusion, the thrombi formed in Bak-/- BaxPf4Δ/Pf4Δ mice were unstable, a trend also observed in Bak-/-Baxfl/fl mice.The formation of stable thrombi is dependent on the release of secondary agonists, such as ADP and Thromboxane, from activated platelets. To investigate potential defects in platelet signaling pathways in the absence of Bax and/or Bak, we performed in vitro platelet activation assays. Flow cytometric measurements revealed that activation of the PAR4 receptor (by PAR4-AP) or GPVI (by convulxin) led to reduced integrin activation (JON/A) and degranulation (P-selectin exposure) in the absence of Bak and Bak/Bax, while loss of Bax alone had no effect. In contrast, the response to activation with ADP, which does not induce granule release, was similar in platelets from all genotypes. Similarly, platelet aggregation in response to intermediate concentrations of PAR4-AP was severely reduced in the absence of Bak and Bak/Bax, but normal in response to ADP. We next investigated if abnormal degranulation in response to agonists could explain the aggregation defect. Platelet aggregation was performed with PAR4-AP and the platelet supernatants were collected after centrifugation. Dense granule release (ATP and serotonin) and alpha granule release (PF4) were significantly reduced from platelets deficient in Bak, Bak/Bax, but not Bax alone. Untreated resting platelets of all genotypes contained similar amount of granular proteins (ATP, serotonin and PF4). Hence, altered granule content was not the reason behind the abnormality.We next explored if platelet age was a factor behind the observed functional differences. To be able to directly compare platelet function in Bak/Bax deficient mice and wild-type controls, we synchronized platelet age to ~3 days in all genotypes. Platelets were depleted in vivo by injection of anti-platelet serum (APS). Newly generated platelets were collected at 72 h post injection, a time-point were platelet counts had returned to normal. Remarkably, synchronized platelet age normalized PAR4-AP and convulxin dependent integrin activation (JON/A) and degranulation (P-selectin exposure) in the absence of Bak and Bak/Bax to control levels. Similarly, the platelet aggregation and release defects were rescued.Lastly we investigated if synchronizing platelet age would revert the hemostatic defect of Bak/Bax mice in vivo. We determined tail bleeding times using mice, which were either untreated or depleted of platelets 72 h prior to the experiment. Strikingly, synchronization of platelet age to 3 days rescued the hemostatic defect in Bak-/-BaxPf4Δ/Pf4Δ mice. We conclude that extended platelet survival leads to platelet exhaustion, with reduced ability to mobilize granular release. Our studies suggest that, in the context of blood bank storage, extending platelet survival times by pharmacologically inhibiting apoptosis may result in a hemostatically compromised product. DisclosuresNo relevant conflicts of interest to declare.

Mutations in Tropomyosin 4 Cause Macrothrombocytopenia in Mice and Humans

A genome-wide scan in nearly 70,000 individuals showed that the common SNP rs8109288 in the first intron of the human TPM4 gene exerts an effect on the volume and count of platelets (Gieger et al. Nature 2011). We isolated a mouse line with an ENU-induced missense mutation in Tpm4. Mice carrying this mutation exhibited dose-dependent macrothrombocytopenia, while other blood cell counts were normal. Bone marrow transplant experiments demonstrated that the phenotype is intrinsic to hematopoietic cells. Notably, Tpm4 insufficiency did not affect the life span or in vitro function of mutant platelets, and there was no evidence of an increased propensity to bleeding. Megakaryocyte numbers in the bone marrow were increased, although maturation as measured by ploidy appeared normal. Mutant megakaryocytes displayed altered morphology indicating fragmentation, and markedly decreased proplatelet formation in vitro.Based on Gieger et al., we examined the functional requirement for TPM4 in human megakaryocytes. We found that the localisation of TPM4 in proplatelet-forming megakaryocytes was extremely similar to the localisation in their mouse counterparts, suggesting an identical role. Furthermore, knock down of TPM4by shRNA in human megakaryocytes did not affect maturation as measured by CD41 and CD42 expression, but significantly reduced the number of proplatelet-forming cells. The occasional megakaryocyte that did form proplatelets did not exhibit the typical “beads-on-a-string” phenotype. Typically, one large bulb at the end of a protrusion or a string with no clearly distinguishable beads was observed.We therefore performed a look-up in the BRIDGE consortium database, which enrolled 542 cases with inherited bleeding and platelet disorders of unknown aetiology in the NIHR BioResource for exome sequencing. Calling of variants revealed single nucleotide variants with consequences in TPM4which were absent from ~30,000 control haplotypes, in three BRIDGE cases. Two cases with a stop codon at residue 105 and R91H variant presented with macrothrombocytopenia and mild bleeding symptoms with platelet counts of 103 and 128 x10e9/L and volumes of 15.10 and 14.00 fl, respectively. The remaining case with variant D20N (not conserved, genomic evolutionary rate profiling score of 2.68 compared to 4.62 for the other variants) had a count in the normal range (232 x10e9/L) and a reduced platelet volume of 7.20 fl.Together, these results provide compelling evidence that Tropomyosin 4 is a crucial regulator of platelet production in mice and humans, being specifically required for the terminal stages of platelet formation. Our studies demonstrate that the common intronic variant exerts a subtle effect, whilst two extremely rare variants have a more robust effect on platelet formation leading to counts and volumes at the tails of the population distribution. The lack of concordance between mice and humans with regard to bleeding may be explained by strong modifiers at loci other than TPM4. DisclosuresNo relevant conflicts of interest to declare.

PKCα negatively regulates in vitro proplatelet formation and in vivo platelet production in mice

Proplatelet formation is a part of the intricate process by which platelets are generated by their precursor cell, the megakaryocyte. The processes that drive megakaryocyte maturation and platelet production are however still not well understood. The protein kinase C (PKC) family of serine/threonine kinases has been demonstrated as an important regulator of megakaryocyte maturation and proplatelet formation, but little investigation has been made on the individual isoforms. We have previously shown, in mouse models, that PKCα plays a vital role in regulating platelet function, so in this study we aimed to investigate the role of PKCα in megakaryocyte function using the same Prkca−/− mice. We assessed the role of global PKC and specifically PKCα in proplatelet formation in vitro, analyzed polyploidy in Prkca−/−-derived megakaryocytes and followed platelet recovery in platelet-depleted Prkca−/− mice. We show reduced proplatelet formation in the presence of global PKC blockade. However, in the presence of a selective classical PKC isoform inhibitor, Go6976, proplatelet formation was conversely enhanced. PKCα null megakaryocytes also showed enhanced proplatelet formation, as well as a shift to greater polyploidy. In vivo, platelet production was enhanced in response to experimentally induced immune thrombocytopenia. In conclusion, our data indicate that classical PKC isoforms, and more specifically PKCα, are negative regulators of proplatelet formation. PKCα appears to negatively regulate endomitosis, with the enhanced polyploidy observed in Prkca−/−-derived megakaryocytes. In vivo, these observations may culminate in the observed ability of Prkca−/− mice to recover more rapidly from a thrombocytopenic insult.

Ponatinib Induced Thrombocytopenia Might Inhibit Proplatelet Formation of Megakaryocytes Via the Pathways Including Rho/Rock and Rac.

Abstract 2186 Background:Ponatinib (AP24534) is identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML (Chronic Myeloid Leukemia). Ponatinib potently inhibited in vitro proliferation of Ba/F3 cells expressing BCR-ABL T315I mutation (IC 50; 11 nM). In PACE (Ponatinib Ph+ ALL and CML Evaluation) clinical trial indicated that 57% had CCyR (complete cytogenetical response), and 47% had MMR (major molecular response) in CML-chronic phase with T315I mutation. Ponatinib has substantial activity in heavily pretreated patients and those with refractory T315I mutation. However, approximately one third of ponatinib-treated patients had moderate to severe thrombocytopenia (J.E. Cortes et al. 2011 ASH Annual Meeting). The mechanism of ponatinib-induced thrombocytopenia remains unknown. In this study, we evaluated the effects of ponatinib on megakaryocytic progenitor cells, megakaryocytopoiesis, megakaryocyte and platelet production in mice. Method:All animal procedures were approved by the Institutional Animal Care and Use Committee of Iwate Medical University. Male ddY mice at 8 weeks of age were used in all experiments. We studied in vitro culture of megakaryocytic colonies (CFU-Meg), megakaryocyte ploidy analyses in vitro culture and proplatelet formation (PPF) assay in vitro. Murine bone marrow cells were cultured in methylcellulose with mIL-3, mIL-6 and TPO at 37°C in 5% CO2 and 20% O2 for 7 days in the presence of ponatinib (0.01, 0.1, 1, 10, 100 nM). PPF: Murine megakarocytes were partially purified from bone marrow cells using BSA gradient. They were plated in 96 micro-well culture plates (300 megakaryocyte/well) and cultured in IMDM, supplemented with 1% ITS-G (serum-free medium) in the presence of ponatinib (0.01, 0.1, 1, 10, 100 nM), at 37°C in 5% CO2and 20% O2. After 24 hr incubation, the megakaryocytes with proplatelets in each well were counted. Activated Rho and activated Rac in murine platelets were measured by the Western blot using Rhotekin-binding domain (RBD) beads and PAK-PBD Affinity beads respectively. The phosphorylation of Lyn (Src family kinase) in murine platelets was also evaluated by the Western blots. Results:CFU-Megs did not decrease significantly at 0.01, 0.1, 1, 10 and 100 nM (31.8 +/− 1.4 to 42.3 +/− 2.4 cells) and decreased significantly (17.0 +/− 1.6 cells p<0.01) at 1000 nM of ponatinib. PPF were decreased significantly at 0.1, 1, 10, 100 nM ponatinib (0 nM: 26.4 ± 0.8 %, 0.1 nM: 19.2 ± 1.7% p<0.05, 1 nM: 19.4 ± 2.1 % p<0.05, 10 nM: 17.9 ± 1.1% p<0.01, 100 nM: 12.5 ± 1.1 % p<0.001, 1000 nM: 11.6 ± 0.9 % p<0.001). The decreases in PPF were cancelled significantly by the addition of Y27632, Rho-associate kinase ROCK inhibitor (ponatinib 10 nM; 17.9 ± 1.1%, ponatinib 10 nM + Y27632 10μM; 29.8 ± 1.7% p<0.001, ponatinib 100 nM; 12.5 ± 1.1%, ponatinib 100 nM + Y27632 10μM; 19.3 ± 1.4% p<0.001). There was no difference in DNA ploidy of cultured megakaryocytes in the presence of ponatinib (0.01 to 100 nM). Next we have tried to understand the precise role of Rho/Rock and Rac pathways in platelets. Our data showed that Rho was upregulated and that activated Rac was downregulated at 50 nM of ponatinib. Ponatinib reduced the levels of phosphorylated Lyn (p-Lyn). Discussion and Conclusion:The Rho/ROCK pathway was reported to be negative regulators (Blood 2007 109; 4229) and positive regulators (Blood 2007 110; 3637) in PPF. Ponatinib induced thrombocytopenia might not be due to the inhibition of megakaryocyte colony formations but the inhibition of PPF of megakaryocytes via pathways including Rho/Rock and Rac. Disclosures:No relevant conflicts of interest to declare.

VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice

AbstractIt has previously been reported that VEGF-A stimulates megakaryocyte (MK) maturation in vitro. Here we show that treatment of mice with the isoform VEGF-A165 resulted in a significant increase in circulating numbers of platelets. Using specific VEGFR1 and VEGFR2 blocking mAbs and selective VEGFR1 and 2 agonists, PlGF-2 and VEGF-E, respectively, we show directly that stimulation of VEGFR1, but not VEGFR2, increases circulating platelet numbers in vivo. Using flow cytometric analysis of harvested MKs, we show that while PlGF does not change the absolute numbers of MKs present in the bone marrow and the spleen, it increases both their maturation and cell-surface expression of CXCR4 in the bone marrow. Histology of the bone marrow revealed a redistribution of MKs from the endosteal to the vascular niche in response to both VEGF-A165 and PlGF-2 treatment in vivo. Antagonism of CXCR4 suppressed both the VEGFR1-stimulated redistribution of megakyocytes within the bone marrow compartment and the VEGF-A165–induced thrombocytosis. In conclusion, we define a novel proinflammatory VEGFR1-mediated pathway that stimulates the maturation and up-regulation of CXCR4 on megakaryocytes, leading to their redistribution within the bone marrow environment, thereby enhancing platelet production in vivo.

Bortezomib Induced Thrombocytopenia Might Be Due to the Inhibition of Proplatelet Formation of Megakaryocyte.

AbstractAbstract 3696 Background:Bortezomib is potent and reversible proteasome inhibitor that has been extensively used for multiple myeloma. Several clinical studies demonstrated that overall response rates using bortezomib alone to relapsed or refractory patients with multiple myeloma were 33 to 50%. The most common grade 3 adverse event was a cyclic thrombocytopenia, which was reported in 20–30% of patients in several clinical studies. The mechanism by which bortezomib causes thrombocytopenia remains unknown. In this study, we evaluated the effect of bortezomib on megakaryocytic progenitor cells, megakaryocytopoiesis, megakaryocyte and platelet production in mice. Method:All animal procedures were approved by the Institutional Animal Care and Use Committee in Iwate Medical University. Male ddY at 8 weeks of age mice were used in all experiments. In vivo experiments: (a) The mice received 2.5 mg/kg bortezomib via tail-vein injection. Blood was obtained and the following experiments were carried out at day 2, 4, 6, 8, 10 after intravenous injection (n=9, each group). Complete blood counts were measured. Reticulated platelet (RP) was analyzed by flow cytometry using thiazole orange (TO) to evaluate platelet kinetics. Plasma TPO level were measured by ELISA. Bone marrow megakaryocyte's number and morphology from femur in bortezomib- and control-treated mice were observed by microscopy. Femur was fixed in 10% buffered formalin, decalcificated, embedded in paraffin and stained for Hematoxylin-Eosin (H-E). (b) Bortezomib (2.5 mg/kg) was administrated via tail-vein to mice. After 24hr, bone marrow cells were cultured in MegaCult®-C at 5% CO2 and 20% O2for 7 days. The megakaryocytic colonies (CFU-Megs-in vivo) were counted. In vitro experiments: (c) Bone marrow cells, obtained from non-treated mice, were cultured at 37°C in 5% CO2and 20% O2 for 7 days with bortezomib (0.01, 0.1, 1, 10, 100 ng/ml). CFU-Megs were counted (CFU-Megs-in vitro). (d) Proplatelet formation: Murine megakarocytes were partially purified from bone marrow using BSA gradient. They were plated in 96 micro-well culture plates (300 megakaryocyte)well) and cultured in IMDM in duplicates, supplemented with 1 × ITS-G (Life technologies) and each concentration of bortezomib (0.01, 0.1, 1, 10, 100 ng/ml), at 37°C in 5% CO2and 20% O2. After 24 hr incubation, the megakaryocytes with proplatelets in each well were counted. Results:(a) Control mice did not have any significant change in platelet counts, % reticulated platelets and plasma TPO levels at days 0, 2, 4, 6, 8, 10. While, bortezomib treated mice (2.5mg/kg) had a significant reduction in platelet counts at day 2 (470 ± 210 × 109/L. P<0.001), at day 4 (667 ± 118 × 109/L, P<0.001). The platelet counts returned to normal value at day 6 (903 ± 548 × 109/L) and day 10 (1122 ± 187 × 109/L). RP (%) began to increase at day 6 (8.8 ± 4.0 %). Plasma TPO levels tend to increase at day 4. Means megakaryocytes's number in one field of femur was similar in between bortezomib non-treated and –treated mice. The megakaryocytes were similar in morphology at each day, too. (b) CFU-Megs-in vivo were similar in number between bortezomib non-treated and –treated mice (38.0 ± 6.1 vs 34.5 ± 5.6 per 1 × 105 bone marrow cells respectively). (c) CFU-Megs-in vitro were not decreased significantly at 0.001 to 1 ng/ml and decreased significantly (p<0.01) at 10 and 100 ng/ml of bortezomib. (d) Proplatelet formation (PPF) were decreased significantly at 0.01, 0.1, 1, 10, 100 ng/ml bortezomib (0 mg)ml: 25.2 ± 4.8%, 0.01ng/ml: 23.8 ± 4.9%, 0.1 ng/ml: 18.4 ± 3.1% p<0.01, 1 ng/ml: 13.2 ± 3.8% p<0.001, 10 ng/ml: 13.3 ± 2.1% p<0.001, 100ng/ml: 5.9 ± 1.4 % p<0.001). Discussion & Conclusion:Bortezomib did not adversely affect on megakaryocytic prognitors nor megakaryocytes. It did inhibit PPF, that is, the step of platelet production, even when bortezomib plasma concentration levels have gone down. Plasma TPO level showed an inverse relationship against circulating platelet counts. Based on the evidence in which Cmax of plasma bortezomib concentration was under 100 ng/ml in bortezomib-injected mice (2.5mg/kg), bortezomib induced thrombocytopenia might be due to the inhibition of proplatelet formation of megakaryocyte. Disclosures:No relevant conflicts of interest to declare.

Proteomic analysis of platelet alpha-granules using mass spectrometry.

Platelets have three major types of secretory organelles: lysosomes, dense granules, and alpha-granules. alpha-Granules contain several adhesive proteins involved in hemostasis, as well as glycoproteins involved in inflammation, wound healing, and cell-matrix interactions. This article represents the first effort to define the platelet alpha-granule proteome using mass spectrometry (MS). We prepared a subcellular fraction enriched in intact alpha-granules from human platelets using sucrose gradient ultracentrifugation. alpha-Granule proteins were separated and identified using sodium dodecylsulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem MS. In the sucrose fraction enriched in alpha-granules, we identified 284 non-redundant proteins, 44 of which appear to be new alpha-granule proteins, on the basis of a literature review. Immunoelectron microscopy confirmed the presence of Scamp2, APLP2, ESAM and LAMA5 in platelet alpha-granules for the first time. We identified 65% of the same proteins that were detected in the platelet releasate (J. A. Coppinger et al. [Blood 2004;103: 2096-104]) as well as additional soluble and membrane proteins. Our method provides a suitable tool for analyzing the granule proteome of patients with storage pool deficiencies.

A Single Intravenous Dose of Murine Megakaryocyte Growth and Development Factor Potently Stimulates Platelet Production, Challenging the Necessity for Daily Administration

AbstractThe thrombopoietic efficacy of recombinant forms of c-mplligand is being actively investigated in preclinical studies using daily dosing schedules. However, a comprehensive kinetic study of the thrombopoietic response to a single injection of recombinant c-mpl ligand has not been performed. Here, we present the results of a detailed kinetic analysis of the platelet response to a single intravenous administration of pegylated recombinant murine megakaryocyte growth and development factor (PEG-rmMGDF) in mice. In addition, we compare the efficacy of single versus daily dosing in stimulating platelet production. A single intravenous injection of PEG-rmMGDF produced a marked and dose-dependent elevation in platelet number and a moderate increase in mean platelet volume (MPV). After administration of 25 or 250 μg/kg of PEG-rmMGDF, platelet number was first increased on day 3 and peaked at 2.7-fold (25 μg/kg) and 5.7-fold of normal (250 μg/kg) on day 5. Thereafter, platelet number declined and returned to baseline by days 9 and 14, with the 25 and 250 μg/kg doses, respectively. MPV began to increase on day 2 after PEG-rmMGDF, reaching maximum values of 1.2-fold (25 μg/kg) and 1.5-fold of normal (250 μg/kg) on day 4. Subsequently, MPV declined and was downregulated on days 6 to 7 (25 μg/kg) and day 8 (250 μg/kg). Based on these results, we evaluated the platelet response to PEG-rmMGDF administered intravenously as a single dose versus daily for 5 days. A single administration of 100 μg/kg produced a higher platelet number on day 5 than daily administration of 100 or 20 μg/kg for 5 days. However, the thrombocytosis was less sustained after single versus daily dosing. The smaller platelet number increase on day 5 after daily dosing reflected the production of larger platelets, rather than suppression of thrombopoiesis. Our results indicate that PEG-rmMGDF administered as a single intravenous dose potently stimulates platelet production in mice, challenging the need for its daily administration. Adoption of an intermittent administration schedule of this cytokine could be more efficacious and is merited in future clinical trials.

A Single Intravenous Dose of Murine Megakaryocyte Growth and Development Factor Potently Stimulates Platelet Production, Challenging the Necessity for Daily Administration

The thrombopoietic efficacy of recombinant forms of c-mplligand is being actively investigated in preclinical studies using daily dosing schedules. However, a comprehensive kinetic study of the thrombopoietic response to a single injection of recombinant c-mpl ligand has not been performed. Here, we present the results of a detailed kinetic analysis of the platelet response to a single intravenous administration of pegylated recombinant murine megakaryocyte growth and development factor (PEG-rmMGDF) in mice. In addition, we compare the efficacy of single versus daily dosing in stimulating platelet production. A single intravenous injection of PEG-rmMGDF produced a marked and dose-dependent elevation in platelet number and a moderate increase in mean platelet volume (MPV). After administration of 25 or 250 μg/kg of PEG-rmMGDF, platelet number was first increased on day 3 and peaked at 2.7-fold (25 μg/kg) and 5.7-fold of normal (250 μg/kg) on day 5. Thereafter, platelet number declined and returned to baseline by days 9 and 14, with the 25 and 250 μg/kg doses, respectively. MPV began to increase on day 2 after PEG-rmMGDF, reaching maximum values of 1.2-fold (25 μg/kg) and 1.5-fold of normal (250 μg/kg) on day 4. Subsequently, MPV declined and was downregulated on days 6 to 7 (25 μg/kg) and day 8 (250 μg/kg). Based on these results, we evaluated the platelet response to PEG-rmMGDF administered intravenously as a single dose versus daily for 5 days. A single administration of 100 μg/kg produced a higher platelet number on day 5 than daily administration of 100 or 20 μg/kg for 5 days. However, the thrombocytosis was less sustained after single versus daily dosing. The smaller platelet number increase on day 5 after daily dosing reflected the production of larger platelets, rather than suppression of thrombopoiesis. Our results indicate that PEG-rmMGDF administered as a single intravenous dose potently stimulates platelet production in mice, challenging the need for its daily administration. Adoption of an intermittent administration schedule of this cytokine could be more efficacious and is merited in future clinical trials.

Purification and characterization of thrombopoietin.

A thrombopoietic factor, termed thrombopoietin (TPO), was highly purified directly from the plasma of sublethally irradiated 1,100 rats by measuring the production of megakaryocytes from a highly enriched population of rat megakaryocyte progenitor cells (CFU-MK). The rat plasma TPO is a glycoprotein and strongly hydrophobic. The total activity and purification yields obtained were about 29% and 1.49 x 10(8), respectively. The amino acid sequences of the two peptide fragments prepared from the purified 19 kDa TPO were analyzed, and used for the cloning of rat and human TPO cDNAs. It was found that the 19 kDa TPO was truncated but comprised at least 163 amino acids. The sequence of human TPO cDNA revealed that the TPO was identical to the c-Mpl ligand. Both rat and human TPOs expressed in COS-1 cells exhibited significant activity toward the CFU-MK in vitro, and were active in stimulating platelet production in mice. These results indicate that a thrombopoietic factor originally found in the irradiated rat plasma is a ligand for the rat c-Mpl.

Thrombopoietin and the humoral regulation of thrombocytopoiesis.

In 1994 four biotechnology research groups reported the isolation and cloning of the ligand for the cytokine receptor c-Mpl and showed it to be the long-sought regulator of platelet production, thrombopoietin. Thrombopoietin is a hematopoietic growth factor of 332 amino acids composed of an amino terminal domain homologous to erythropoietin and a highly glycosylated carboxyl domain. The erythropoietin-like domain is the functional domain, whereas the glycosylated domain appears to stabilize circulating thrombopoietin. Thrombopoietin stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet-producing megakaryocytes. Thrombopoietin induces dramatic increases in megakaryocyte number and platelet production in mice, indicating that it regulates both thrombopoiesis and megakaryocytopoiesis. Thrombopoietin also accelerates the recovery of platelets in myelosuppressed animals, suggesting that it will be clinically useful for the treatment of thrombocytopenia.

Stimulation of thrombopoiesis in mice by human recombinant interleukin 6.

To date, testing of various cytokines for the stimulation of blood cell production has not demonstrated a consistent effect on peripheral platelet levels. In this report, we provide evidence that human recombinant IL-6 increased platelet production in mice, as measured by both peripheral platelet levels and [75Se]selenomethionine (75SeM) incorporation into newly forming platelets. Peripheral white blood cell counts also were increased, but only to a modest extent, and hematocrit values were unchanged. A dose-response relationship between the amount of IL-6 administered and platelet count, 75SeM incorporation, and white blood cell count was demonstrated. Detectable megakaryocyte and granulocyte-macrophage colony-forming cells in mice that had received IL-6 also were increased in both bone marrow and spleen. These results demonstrate the ability of a purified, recombinant protein to stimulate platelet production in vivo.

The Effect of Partial Splenectomy on Platelet Production in Mice

The effect of total and partial splenectomy on the number and production of circulating platelets was studied in mice. Five days after total and partial splenectomy the number of the peripheral blood platelets increased by 87% and 60%, respectively and the incorporation of 75selenium methionine (75Se-Met) into platelets was enhanced indicating that the thrombocytosis was due to increased platelet production. The results obtained by the two operative procedures were compared. Since previous work from our laboratory has shown that a factor produced by splenic lymphocytes affects the platelet number in mice, it is suggested that the differences in the number of circulating platelets observed in animals after total and partial splenectomy may reflect a difference in the number of spleen lymphocytes removed.

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

AbstractThe 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.