Platelets Expression of COX1, IL1 and IL10 - Research Proposal Example

PLATELET EXPRESSION OF COX1, IL1 and IL10 Platelets (thrombocytes) are one of the components of the blood; the others being erythrocytes, leukocytes and plasma. Platelets are anucleated and are fragments of the cytoplasm of the megakaryocytes. Platelets are specialized cells that minimize vessel injury and bleeding in the human body. They are produced by megakaryocytes; which are huge progenitor cells. The megakaryocytes release the platelets through a series of complex processes. The megakaryocyte mature and become polyploid, accumulating massive protein and membrane. The megakaryocyte then extends into sinusoidal blood vessels where they release the platelets by fission. The project aims to understand under which conditions, platelets express COX1, IL1 and IL10. Background Platelets are approximately 20% of the diameter of the red blood cells. They have proteins on their surfaces that enable them to stick to break in blood vessels. Their count is 150,000-350,000 per every microliter of blood (Andre, 2014). Each megakaryocyte produces an average of 1000-3000 platelets in a lifetime. An un-activated platelet is biconvex and disc-shaped, about 1-3 micrometers. In human beings, the average lifespan of a platelet is 7-10 days; however, the lifespan of an individual platelet is determined by the internal apoptotic regulating pathway (Machlus and Italiano, 2013). Platelets are formed from the cytoplasm of the megakaryocytes which are found in the bone-marrow. The megakaryocytes are approximately 75 micrometers in diameter. The megakaryocytes become polyploidy by endomitosis to assemble and release the platelets. They then mature, where the majority of the cytoplasm is packaged into proplatelets and the nucleus extruded. The platelets form at the tip of the proplatelets (Machlus and Italiano, 2013). The platelet formation is divided into two phases; one where the megakaryocyte mature ad when the megakaryocyte generate the platelets. The whole process takes place approximately in 5 days. During the first phase, the megakaryocyte matures and develops and it requires specific megakaryocyte growth factors. The megakaryocyte’s cytoplasm nuclear proliferates and enlarges as the megakaryocyte is filled with cytoskeletal proteins, platelet-specific granules and a sufficient membrane that completes the platelet assembly. The second phase is short and the megakaryocyte generates platelets by remodeling their cytoplasm into pro-platelets and later into pre-platelets that undergo fission and generate discoid platelets. The second phase may just take place in hours. Figure 1- platelet when disc-shaped (Source: Dixon et al., 2006) (Source: Machlus and Italiano, 2013) Figure 2-Schematic of platelet production Cytokines are antibody proteins that are mediators between the cells. Most of the cytokines are produced the cells themselves, rather than the immune cells because they have effects on non-immune cells. Cytokines include monikines (produced by monuclear phagocytic cells), lymphokines (activated lymphocytes) and interleukins (act as mediators between the leukocytes). Interleukins include the interleukin 1, Interleukin 10, Interleukin 12 and Interleukin 2 (Pathmicro.med.sc.edu, 2014). IL-1 is an inflammatory cytokine that is produced by activated macrophages. Cytokine Cell source Cell target The primary effects IL1 Monocytes Fibroblasts Epithelial cells Astrocytes Macrophages T-cells and B-cells Hypothalamus The liver Endothelial cells Costimulatory molecule Fever Inflammation/activation Acute phase reactants IL10 T-cells Macrophages T-cells It inhibits APC activity It inhibits cytokine production Table 1-characteristics of IL1 and IL10 IL-10 is also produced by activated macrophages and the Th2 cells. It is an inhibitory cytokine, having effect mainly on the production of IFN-by the Th2 cells. The IL-10 inhibits the production of cytokine by activating macrophages and the expressing class II MHC; hence resulting in a dampening of the immune response (Pathmicro.med.sc.edu, 2014). Arachinodic acid, which is released from the cellular phopholids by phospholipase, has to be biosynthesized from the fatty acid in dietary sources. The un-esterified intercellular arachinodic acid is converted by either cyclooxygenase or lipoxygenase. Cyclooxygenases are glycosylated membrane-bound enzymes that are ubiquitous in the animal cell. They are in two forms COX-1 and COX-2. The breakdown of arachinodic acid by cyclooxygenase forms prostaglandins, prostacyclin and thromboxane (GOLAN 2008, page 748,749). Expression mRNA cDNA Tissue location Cellular localization Role Inhibition Substrate selectivity constitutive 2.8kb Chromosome 9; 22kb Ubiquitous expression Endoplasmic reticulum Protection and maintenance of functions Pharmacologic: NSAIDS Increased 2- to 4-fold by inflammatory stimuli Arachinodic acid Eicosapentoic acids Table 2- Characteristics of COX1 The existence of COX1 was observed when the glucocorticoid dexamethasone inhibits the increase of COX1 activity with no effect on prostaglandins. There is little difference between COX1 and COX2, with COX2 being more inducible while COX1 is constitutively expressed. In COX1, the translational start sites and signal peptide are located in exons1 and 2. Studies on the tissue localization, under basal physiological conditions, show that COX1 can be expressed in all tissues, unlike COX2 that is restricted to the brain, testicles, kidney and the tracheal epithelial cells (Brooks et al., 1999). The expression and location of COX1 suggest that it controls the production of prostaglandins which are critical to the autocrine response to circulating hormones. The use of NSAIDS inhibits COX1; the side effects may include GI irritation, bronchospasm and platelet dysfunction. Coagulation Coagulation can be summarized as the process of blood clotting. Coagulation involves a controlled series of proteolytic initiation of a series of zymogens to achieve a timely and effective hemostasis (C ADAMS and J BIRD, 2009). Coagulation cascade is a succession of enzymatic alterations. A stepwise activation for inactive zymogens is critical for the formation of fibrin. The cascade model is illustrated below: Table 3-Cascade process (Source: C ADAMS and J BIRD, 2009) The coagulation phase can be divided into three phases: the initiation, amplification and propagation. The initiation phase is presaged by exposing the tissue factor to blood by damage or activation of the endothelium. The tissue factor is a 47kDa cell-bound trans-membrane glycoprotein and members of the class II cytokine and functions as a receptor and a cofactor of factors. Cytokines usually have influence on the tissue factor expression. IL1 leads to the union of monocytes and also transforms the growth factor (van der Poll et al., 2006). During the amplification phase, the FIXa and FVIIIa form an intrinsic tenase factor complex in the existence of calcium. Tenase is essential for the intensification of the clotting procedure that is required for a sustained haemostasis. The activated factors simulate a positive reaction which generates thrombin in large amounts for a stable clot. Thrombin then acts on the platelets using the platelet receptor Gplb; hence enhances platelet aggregation and provides a negatively charged phospholipid surface. The propagation phase depends on the use of activated platelets at the injury site and provides an appropriate localization of the components of thrombin, prothrombinase complex, a phospholipid layer and calcium. Thrombin burst leads to a development of fibrin and produces a fibrin clot. Aim: The project aims to determine under which conditions, platelets express COX-1, IL-1 and IL10. Research plan Human platelets- I will collect from a blood sample from a healthy subject using venipuncture, approximately 45 ml. The subject shall be free from any bleeding disorders and have abstained from alcohol 48hr prior to the donation. The donor will be informed of the consent in accordance with the Helsinki Declaration (Li and Diamond, 2013). Mouse platelets: I will collect the blood sample from the mice in accordance to the Institutional Animal Care and Committee guidelines. Blood will be collected in tubes that contain sodium citrate and then poured into centrifuge tubes. The tubes will then be centrifuged at 500g for approximately 20mins at 20oC. I will then transfer to the upper layer of the centrifuge into a silicone Pasteur pipette. The platelets will remain at temperatures slightly above room temperature (25oC) (Born and Cross, 1964). To remove any residual blood cell, the mixture will be re-suspended in the presence of anti-Ter 119. The project will be in two studies; for COX1 and for IL (1 and 10) STUDY ONE: COX1 I will divide the platelets into two sets; one with aspirin and the other without aspirin. Arachinodic acid is then added to the platelet set that does not have an aspirin and the Thromboxane measured. For the set that contains aspirin, LPS is added to stimulate the platelets. After approximately 5hours, arachinodic acid is added to the set and the level of Thromboxane measured. STUDY 2: IL1 AND IL10 The platelet is kept in citrated platelet rich plasma. I will then create a platelet plug by aggregating with thrombin, spin the plug gently and then incubate and rest it for approximately 7 days. The solution is then stimulated with LPS and the level of IL1 and IL10 measured at intervals of 24hours Limitation of the methods The total cost of the project is high, factoring in the costs of materials, the setup process and the storage costs. The success depends on the ability to choose a healthy donor (both human and mice). Any defect in the donor cells may affect the results of the experiment. Leukocytes bypass the leukocyte depletion step. The timing of the project cannot be fixed. The time needed for platelet preparation can be extended. Timeline Cell culture- two weeks Thromboxane- two weeks Timeline for COX1-4 weeks Timeline for IL1 and IL10- 8 weeks The total time needed- 12 weeks Cost Mouse platelet gratis TxA2 kit Cell culture- 150 monthly IL1 kit IL10 kit Lps sigma PMA sigma Ethical consideration The following approvals will be obtained from the NHS: Human Tissue Authority license (HTA). Research Ethics Committee (REC). Outcome At the end of the project, the following will be found out: The condition in which COX1 is expressed. Whether the platelet plug expresses IL1 or IL10. References Andreas viklund.com/, C. (2014). Platelets. [Online] Ouhsc.edu. Available at: http://www.ouhsc.edu/platelets/platelets/platelets%20intro.html [Accessed 10 Aug. 2014]. Born, G. And Cross, M. (1964). Effects of inorganic ions and of plasma proteins on the aggregation of blood platelets by adenosine diphosphate. The Journal of physiology, 170(2), pp.178-195. Brooks, P., Emery, P., Evans, J., Fenner, H., Hawkey, C., Patrono, C., Smolen, J., Breedveld, F., Day, R., Dougados, M. and others, (1999). Interpreting the clinical significance of the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2. Rheumatology, 38(8), pp.779--788. C ADAMS, R. and J BIRD, R. (2009). nep_1228 462..470 Review Article Review article: Coagulation cascade and therapeutics update: Relevance to nephrology. Part 1: Overview of coagulation, thrombophilias and history of anticoagulants. nephrology, 15(5), pp.1-4. Dixon, D., Tolley, N., Bemis-Standoli, K., Martinez, M., Weyrich, A., Morrow, J., Prescott, S. and Zimmerman, G. (2006). Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling. The Journal of Clinical Investigation, 116(10), p. GOLAN, D. E. (2008). Principles of pharmacology: the pathophysiologic basis of drug therapy. Philadelphia, Pa., [etc.], Lippincott Williams & Wilkins. Health Research Authority, (2014). Applying for approvals - Health Research Authority. [online] Available at: http://www.hra.nhs.uk/research-community/applying-for-approvals/ [Accessed 11 Aug. 2014]. Li, R. and Diamond, S. (2013). Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay. Thrombosis Research 133 (2014) 203–210, p.2. Machlus, K. and Italiano, J. (2013). The incredible journey: From megakaryocyte development to platelet formation. The Journal of cell biology, 201(6), pp.785--796. Pathmicro.med.sc.edu, (2014). CYTOKINES AND IMMUNOREGULATION. [online] Available at: http://pathmicro.med.sc.edu/mobile/m.immuno-13.htm [Accessed 10 Aug. 2014]. van der Poll, T., de Jonge,, E., ten Cate, H. and Levi, M. (2006). Cytokines as Regulators of Coagulation and Fibrinolysis. Molecular Mechanisms of Disseminated Intravascular Coagulation, 8(1), pp.1-4. Read More