Spring 2010 Hematology Section (50 points total). Please answer the questions below in your own words.

Handwritten answers are okay as long as they are legible. If I cant read it, I cant grade it! You should be able to answer these questions based on the lecture materials and supplemental readings that I gave you. Due: Wednesday, April 28th at 5:00 PM. 1. Describe how plasma concentrations of erythropoietin (Epo) and thrombopoietin (Tpo) are regulated by the body. Consider what would happen after you donate blood and try to represent this graphically by plotting relative plasma concentrations of Epo and Tpo versus time. (10 points) Erythropoietin (Epo) is produced mainly in the renal cortex. Epo is regulated by a feed back mechanism relying on blood oxygen levels. When oxygen concentrations are low hypoxia inducible factors (HIFs) are activated, once activated they mediate the activation of the Epo gene and increase the plasma concentration of Epo. The increased Epo levels can then stimulate the colony forming unit erythrocyte (CFU-E) to give rise to more red blood cells. On the other hand thrombopoietin (Tpo), which is constitutively produced mainly by the liver is regulated differently than Epo. Tpo plasma concentrations are regulated by the circulating platelets, which contain receptors for Tpo. When platelet counts are normal or elevated they bind most of the circulating Tpo and less is left to stimulate the megakaryocytes. When platelet counts drop more Tpo becomes available to bind to the megakaryocytes to stimulate the platelet supply. After you donating blood you would see arise in Epo concentrations, to replace the red blood cells that were lost. However, you would not see a rise in Epo right away because it does need to be synthesized. Tpo is constitutively produced so you would not see a rise in Tpo plasma levels after donating blood.

2. Epo How and

Tpo

Hypoxia inducible factor-1 (HIF) is the bodys master oxygen sensor. is HIF regulated under normoxic hypoxic conditions? What are

Synthesis

Time

some therapeutic approaches that target HIF regulation? Are there any potential risks associated with these approaches? (15 points) Under normoxic conditions, the HIF - isoform is degraded by proline hydroxylation pathways, once hydroxylated the von Hippel Lindau can bind and signal for the ubiquitin-proteasome pathway. So under normoxic conditions HIF is degraded, initiated by the proline hydroxylation. On the other hand, under hypoxic conditions, the HIF - protein is stabilized and can dimerize with the HIF- isoform and translocate into the nucleus. Once inside the nucleus the heterodimerized factor can bind to hypoxia response elements (HRE) motifs and initiate a variety of events such as increase levels of Epo and vascular endothelial growth factors (VEGF). Some therapeutic approaches that target HIF regulation, is in the treatment of chronic kidney disease and stimulate erythropoiesis. If one could inhibit the hydroxylation of proline even under normoxic conditions, you could potentially stimulate the production of Epo and eventually the generation of more red blood cells. The potential risk associated with this approach is potentially inhibiting proiine hydroxylation on a global scale, you would only want to locally inhibit the hydroxylation just in the kidney and allow it to occur else where in the body.

3.

Eltrombopag and romiplostim are currently FDA-approved for the treatment of idiopathic thrombocytopenic purpura (ITP). Briefly describe the mechanism of action for each of these drugs. What are some possible risks associated with this general approach? Are there any potential advantages to using one of these drugs versus the other? (15 points) Eltrombopag is a non-peptide drug that is a thrombopoietin receptor agonist. Once bound to the Tpo receptor, eltrombopag interacts with specific amino acids in or near the transmembrane domain within the receptor. So once eltrombopag binds to the receptor it acts similar to endogenous Tpo, binding activates the JAK-STAT pathway and causing a cascade of events eventually leading to a change in gene expression to induce differentiation and proliferation of megakaryocytes. Romiplostim is a peptide fusion protein; it contains Fc portions of IgG, which is bound to two Tpo peptides via a glycine bridge. The peptide component of romiplostim stimulates the TPO receptor and activates intracellular transcriptional pathways leading to increased platelet production. According to the clinical study done by the manufacturers of Eltrombopag, the adverse effects were no different from what was seen in placebo. Studies have only been conducted for 156 weeks for romiplostim and 151 days for eltrombopag and adverse effects such as headache, ecchymoses and epistaxix have been reported. Since these drugs may be used for long term, there are

issues that have been raised such as reduced platelet activation threshold, rebound thrombocytopenia to name a few. Using eltrombopag versus the recombinant romiplostim is that patients on eltrombopag could benefit from the platelets being produced by the megakaryocytes in response to using eltrombopag plus its effect on mature platelet such as stimulating platelet Tpo receptors.

4.

What occurs during the production of the neutrophils in the bone marrow? How does the kinetics of neutrophil production differ from that of the other blood cells? (10 points) To produce neutrophils the pluripotent hematopoietic stem cells is stimulated by IL-1, IL-3, AND IL6 plus stem cell factors into the myloid stem cell. Granulocytemacrophage colony- stimulating factor (GM-CSF) plus IL-3 stimulated the uncommitted myloid stem cell to commit into developing into a colony forming unit granulocyte-macrophage (CFU-GM). To convert the CFU-GM into a neutrophil, the CFU-GM is stimulated by colony forming unit granulocyte colony stimulating factor (G-CSF) plus IL-3. Circulating levels of neutrophils are much higher when compared to other leukocytes but much lower then when compared to red blood cells. On the other hand, neutrophils are produced in abundance by the bone marrow and kept on reserve. If there is an infection the body can recruit the reserve neutrophils into the battlefield. More neutrophils can be produced by colony stimulating factors released by activated macrophages.