Assignment 1

I have handed in these two assignments as the start of the project .
Assignment one:
Cytometric measurement of Annexin V by flow cytometry
Supervised by: Dr James Vickers
Saman Yazdani

Annexin V is defined as a cellular protein with an unknown function, Ernest Beutler (2000). Therefore recognition of some functions of Annx5 could be the main object. The quantitative measurement of Annx5 is another way to get more information about this protein.Annx5 is called with other names as well like: Alternative titles; symbols Annexin V; ANX5 , endonexin II; ENX2, placental Anticoagulant protein I , Vascular anticoagulant-alpha lipocortin V placental protein 4; PP4 , anchorin CII and it gen map locus is: 4q26-q28 , Tait et al (1991). AnxA5 is a member of the Annexin family. This a multiprotein family of over 160 protein that share the property of Ca2+ dependent binding to negatively charged phospholipid surface Gerke V, et al (2002) . It is also indicated that Annexin V forms the voltage-dependent Ca (2+) channels in phospholipids bilayers and was the first ion channel to be structurally and functionally characterized. Demange et al. (1994) . At the end of the 1970s, anxA5 was first isolated from human placenta, Bohn H (1979). A few years later was discovered independently in blood vessels and named vascular anticoagulant protein alpha (VAC-alpha) because of its property to inhibit blood coagulation, Reutelingsperger CP (1985). The anticoagulant mechanism is based on the high-affinity binding to PS. This property makes Anx5 very effective in inhibiting the prothrombinase complex; Reutelingsperger CP et al (1988) .Elucidation of its primary structure revealed that VAC- alpha (vascular anticoagulant protein alpha) was a member of the Annexin family. It was therefore termed Annexin V. According to this system, human Annexin V was renamed anxA5. After its discovery, anxA5 was produced by the expression of its complementary DNA in the bacterium Escherichia coli, Maurer-Fogy I et al (1988), Iwasaki A et al (1987).Anx5 has an important role in Apoptosis as well, Ernest Beutler (2000) .Apoptosis, which is the major form of PCD, Leist M (2001), plays an important role in the development, homeostasis, and disease of an organism. In pathology, 2 opposing situations can arise concerning apoptosis: 1 situation in which there is abundant apoptosis, as in cases of transplanted organ rejections, AIDS, septic shock, and cardiovascular and neurodegenerative diseases, and 1 situation in which there is insufficient apoptosis, as in cases of cancer , Blankenberg FG et al (2001). Fadok et al (1992) revealed that PS is expressed on the cell surface of apoptotic cells, Fadok V et al (1992). Viable cells retain PS Predominantly located in the inner leaflet of the cell membrane, Zwaal RF et al (1997). This notion led, Koopman et al (1994) and others to design an apoptosis detection assay on the basis of fluorescence (fluorescin isothiocyanate)-labelled anxA5, Koopman (1994), Martin SJ (1995), Homburg (1995). To be able to discern between apoptotic cells and necrotic cells, which have compromised plasma membrane integrity, propidium iodide (PI) was added. By this means, viable, apoptotic, and necrotic cells can be discriminated by either fluorescence microscopy or flow cytometry. PCD is a rapidly changing topic. The current state of the art concerning apoptosis is described in several recent reviews, Leist M (2001), Kroemer G et al (2005), and Zimmermann KC et al (2001). A5 in involves in the following Biological Process: anti-apoptosis , Signal transduction , Negative regulation of coagulation , Ernest Beutler (2000) . A5 has a high affinity to bind to phosphatidylserine. Phosphatidylserine is translocated from the inner side of the membrane to the outer layer, if cell undergoes death by apoptosis (or necrosis) and serves as one of the several signals by which cell destined for death and also is recognized by phagocytes, Van England et al (1998). That is what is focused on in this experiment. The high affinity of anx5 to phosphatidylserine.The base of this experiment is ( Design Experiment) : First the sample is taken from the donors ( up to 5 ) there are centrifuged and washed and platelets are separated and then as a base line the amount of Anx5 on the surface of the platelets is measured by flow cytometry , conference abstract . Then in separate times some substances are added to the platelets.
ADP, A23187 ( Calcimycin, Calcium Ionophore, Antibiotic A23187 ,Calcium Ionophore A23187)and PGE1 (prostaglandin E1) .ADP is an activator of the platelets and A23187 & PGE2 will decrease the platelets activity, Carlo patrono et al 2001.
Prostaglandin E1 stimulated the formation of platelet radioactive 3′:5′-cyclic AMP in a dose-dependent manner, G. Ball et al (1970) .Which eventually causes inhibiting activity of the platelets. There are two hypotheses in this regard:
-Activation of the platelets will increase A5 binding to the platelets surface
Or
-Activation of the platelets has no effect on the Anx5 binding to the platelets surface.

The explanation of hypothesis is: Phosphatidylserine, which is one of the “eat me” signals at the surface of the apoptotic cell .A5, would bind to this molecule.
--In the case of increasing activity of the platelets: ADP will cause phosthatydil serine inside the platelets appears more on the surface of the platelets, E. E. Nishazawa (2008). As stated before Anx5 is bound to phosphatydil serine and therefore the appearance of Anx5 will be increased (it is assumed).
--In the case of inhibiting activity of the platelets:
A23187 and PGE1 will be added to the platelets separately .A23187 and PGE1 are inhibitor for platelet activates (conference abstract), the manifestation of phosphatydil serine will be reduces on the surface of the platelets consequently the Anx5 binding will decrease (it is assumed).
When the phsphatadyil serine appears on the surface of platelets ad stated before, Anx5 is bound and then by flowcytometry the level of Anx5 is measured.
, Alice Longobardi Givan (2001).The final part is getting conclusion .As stated above this experiment is designed to either prove or disapprove the two indicated hypothesis. The final conclusion will help to get a basic understating of the role of ADP, A23187 and PGE1 on the appearing of Anx5 on the surface of the platelets which might be helpful for future investigations.


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