Platet expression (Research Paper Sample)

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 SEQ Figure \* ARABIC 1- platelet when disc-shaped
(Source: Dixon et al., 2006)
(Source: Machlus and Italiano, 2013)
Figure SEQ Figure \* ARABIC 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 SEQ Table \* ARABIC 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 SEQ Table \* ARABIC 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 SEQ Table \* ARABIC 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 thro...