Assignment 2

I have handed in these two assignments as the start of the project .

Cytometric measurement of Annexin 5 by flowcytometry
Supervised by : Dr J Vickers.
Saman Yazdani

1- Describe the hypothesis to be tested in your project
This experiment is aimed for a basic understanding of the system. It is a quantitative measurement of Annexin5 (a family of calcium-binding proteins).
There are two hypotheses on the table:
-Activation of platelets increases A5 binding to platelets.
Or
-Activation of platelets has no effect on A5binding to platelets surface. (Conference abstract)

First, the amount of A5 on the surface of platelets is measured by flow cytometry (as a base line).A5 is bound to phospholipids inside the platelets include: phosphatidyl inositol 1, phostatidyl serine and ethanol amine (conference abstract).
This measurement is kept as a base line to be compared. Then some substances include: ADP and A23187( Calcimycin, Calcium Ionophore, Antibiotic A23187 ,Calcium Ionophore A23187), (both as activator) and PGE1 (prostaglandin E1-as inhibitor)
Are separately added to the platelets (conference abstract).
If the indicated substances (ADP, A23187 and PGE1) had any effect on the presence and exhibition of those phospholipids, then it can be displayed (it is assumed). The fact is the indicated phospholipids would bind to A5 and then they expo and present Anx5 to the surface of the platelets (it is expected). In flowcytometry a material (A5 binding) would bind to A5 out side the platelets and then A5 binding is measured, it does indirectly shows the amount of A5 out side the platelets.
As stated above, the hypothesis is base on the fact that, the ADP, A23187 & PGE1 (as activator and inhibitors) could cause a change in the manifestation the Anx5 on the surface of the platelets.

2. Briefly outline the type of experimental design you intend to employ, include appropriate control strategies.

Experimental Design.
Materials & Method:
Blood samples (up to 5) .ADP and A23187 (activator) PGE1 (inhibitor), Flow cytometry machine) either 2 or 4 laser channels) and general haematology laboratory equipments.
First of all, the blood is taken from donors (not necessarily patient).It would be centrifuged and the plasma is separated and then platelet is reached from the Buffy coat layer. Then the sample will be washed to have more pure platelets.
In order to have a base line for experiment, before doing the rest of process, the level of A5 is measured by flowcytometry .In this experiment the effect of some substances is measured in order to realise the activation or inhibition of platelets.
As mentioned before this experiment is aimed to either prove or disapprove the indicated hypothesis. Further explanations are about the assumptions coming part5.However it is essential to know that why this experiment is done and what should be focused in all part of the experiment.
It is required to remember that the Anx5 cellular protein which binds with some types of phospholipids (mainly phospholipids serine) and then appears on the surface side of the platelets. (Conference abstract).
Like every other experiment, the first time is a negative control, means measuring with out adding any substance and just measuring the Anx5 as a base line.

3. What type of statistical analysis will be used to analyse your data?
Using SPSS software or simple excel spread sheets could be used to evaluate and analyse the data. The main output of this experiment is some figures by flowcytometry (and graphs) which indicate the amount of A5 in different cases include before and after adding those activator or inhibitors.
A schematic diagram like follow is expected.
Blue: A5 on the surface of platelet before adding ADP
Red: A5 on the surface of platelet before adding A23187
Yellow: A5 on the surface of platelet before adding PGE1
This could be extend with different substances and materials as well .Up to know what ever is reached is this fact that those indicated substances have effect( either increase or decrease) on the level of A5 presenting on the surface of platelets (conference abstract). Although more specific and details are always expected over any experiment.
As stated above, by changing the amount of those indicated substances their effect will be changed as well, hence in t2 the more indicated substances would be added, t3 ant t4 and t5 as well.

4- Outline the number and type of experimental observations that are required to carry out this type of analysis.
As the out put results of flow cytometry is an average of a lot of measurements of doing the test, Longobardi Givan (2001); therefore even if in each of those conditions, there are measured once, should not cause technical errors.
Up to 10 donors blood could give satisfactory data to be analysed.


5-In the majority of experimental designs it is necessary to make a number of assumptions. Outline some of the assumptions you have made when designing experiments for your project. Give reasons for these decisions

ADP-induced platelet aggregation when added in vitro and increase aggregation, Carlo patrono et al 2001, means increasing the activity of platelets. In the opposite side PGE1 and A23187 decrease the activity of platelets (conference abstract).Another fact which has already been mention is the affinity of phosphatydil serine to bind with Anx5 and presenting on the surface of the platelets. Consequently the main assumption could be finding a logical link between the activation of platelets (or inhibition) and level of presenting of Anx5 on the surface of the platelets.
One of the assumptions could be null and void, means there is not any relation ship between these parameters.
Another assumption is, the more the platelets are activated, the more Anx5 they would expose. The third assumption is vice verse the second one, the more the platelets are activated the less Anx5 they would expose.
It is assumed that the assumption would be rejected, because lots of other researchers earlier have been proved that the phosphatydil molecules inside the platelet surface would be undergone due to the activity of the platelets, E.E Nishizawa et el (2008).If so, the level of manifesting the anx5 must logically change on the surface of the platelets (either increase or decrease). Base on those three indicated assumptions the experiment and following the hypothesis would be carried on.
There are other physiological activators which have similar effect on the platelets: thrombin, collagen, thromboxane A2, adrenaline, platelet activating factor, 5-HT, vasopressin, George, JG. (2000) and Dahlbäck, B (2000). Blood coagulation. It is assumed to use other substances over the experiment as well.



References:
-Alice Longobardi Givan , Flow Cytometry: First Principles, Second Edition.
Copyright 2001 by Wiley-Liss, Inc.
ISBNs 0-471-38224-8 (Paper); 0-471-22394-8 (Electronic)

- Blankenberg FG, Strauss HW. Will imaging of apoptosis play a role in clinical
care? A tale of mice and men. Apoptosis. 2001;6:117–123

- Bohn H, Kraus W. Isolation and characterization of a new placenta specific
protein (PP10) . Arch Gynecol. 1979; 227:125–134.

-Carlo Patrono, Barry Coller, James E. Dalen, Garret A. FitzGerald, Valentin Fuster, Michael Gent, Jack Hirsh and Gerald Roth . Platelet-Active Drugs: The Relationships among Dose, Effectiveness, and Side Effects .On line version of chest journal 2001.

Dahlbäck, B. Blood coagulation. The Lancet 2000; 355: 1627-1632

-E. E. Nishizawa 1 J. F. Mustard 1 .The Effect of Synthetic Phosphatidyl Serines on Platelet Aggregation, Blood Coagulation and Haemostasis. British Journal of Hematology 2008 ; Volume 20 Issue 1, Pages 45 – 54.

-Ernest Beutler, Marshall A.Lichtman, Barry S.Coller, Thomas J.Kipps, Uri Seligsohn. Anti phospholipid syndrome.Williams Haematology 2000;(pp. 1715-1728).

-Fadok V, Voelker DR, Campbell PA, et al. Exposure of phosphatidylserine on the
surface of apoptotic lymphocytes triggers specific recognition and removal by
macrophages. J Immunol. 1992;148:2207–2216

-G. Ball, G. G. Brereton, Mary Fulwood, D. M. Ireland and Patricia Yates ,(1970) ,
Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3´:5´-cyclic monophosphate,Biochem. Journal. (1970) 120 (709–718)

-Gerke V, Moss SE. Annexins: from structure to function. Physiol Rev. 2002;82:
331–371

-George, JG. Platelets. The Lancet 2000; 355: 1531-1539

-Hendrikus H. Boersma, Bas L.J.H. Kietselaer,Leo M.L. Stolk, PharmD,
Abdelkader Bennaghmouch, Leonard Hofstra,Jagat Narula, . Past, Present, and Future of Annexin A5: From Protein Discovery to Clinical Applications. The Journal of Nuclear medicine. 2005; Vol. 46. No. 12

-Homburg CH, de Haas M, von dem Borne AE, et al. Human neutrophils lose their
Surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in
Vitro; Blood. 1995;85:532–540.

-Iwasaki A, Suda M, Nakao H, et al. Structure and expression of cDNA for an
inhibitor of blood coagulation isolated from human placenta: a new lipocortinlike
protein. J Biochem (Tokyo). 1987;102:1261–1273

-Koopman G, Reutelingsperger CP, Kuijten GA, et al. Annexin V for flow
cytometric detection of phosphatidylserine expression on B cells undergoing
apoptosis. Blood 1994;84:1415–1420

-Kroemer G, Martin SJ. Caspase-independent cell death. Nat Med. 2005;11:725–
730.

-Leist M, Jaattela M. Four deaths and a funeral: from caspases to alternative
mechanisms. Nat Rev Mol Cell Biol. 2001;2:589 –598
1985;151:625– 629

-Martin SJ, Reutelingsperger CP, McGahon AJ, et al. Early redistribution of
plasma membrane phosphatidylserine is a general feature of apoptosis regardless
of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp
Med. 1995;182:1545–1556.

-Maurer-Fogy I, Reutelingsperger CP, Pieters J, et al. Cloning and expression of
cDNA for human vascular anticoagulant, a Ca2_-dependent phospholipid-binding
protein. Eur J Biochem 1988;174:585–592.

-Reutelingsperger CP, Kop JM, Hornstra G, Hemker HC. Purification and characterization of a novel protein from bovine aorta that inhibits coagulation:
inhibition of the phospholipid-dependent factor-Xa-catalyzed prothrombin activation, through a high-affinity binding of the anticoagulant to the phospholipids. Eur J Biochem. 1988;173:171–178

-Tait JF, Smith C, Blankenberg FG. Structural requirements for in vivo detection
of cell death with 99mTc-annexin V. J Nucl Med. 2005;46:807– 815

-Trotter,PJ Orchard, MA and Walker,J H . Relocation of annexin V to platelet membranes is a phosphorylation-dependent process. Biochem Journal 1997; 1 December , pp. 447–452. [Pubmed]


-Zimmermann KC, Green DR. How cells die: apoptosis pathways. J Allergy Clin
Immunol. 2001;108(suppl):S99 –S103

-Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid
asymmetry in blood cells. Blood. 1997;89:1121–1132.

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.


References:
-Alice Longobardi Givan , Flow Cytometry: First Principles, Second Edition.
Copyright 2001 by Wiley-Liss, Inc.ISBNs 0-471-38224-8 (Paper); 0-471-22394-8 (Electronic)

- Blankenberg FG, Strauss HW. Will imaging of apoptosis play a role in clinical
care? A tale of mice and men. Apoptosis. 2001;6:117–123

- Bohn H, Kraus W. Isolation and characterization of a new placenta specific
protein (PP10) . Arch Gynecol. 1979; 227:125–134.

-Carlo Patrono, Barry Coller, James E. Dalen, Garret A. FitzGerald, Valentin Fuster, Michael Gent, Jack Hirsh and Gerald Roth . Platelet-Active Drugs: The Relationships among Dose, Effectiveness, and Side Effects .On line version of chest journal 2001.

-Dahlbäck, B. Blood coagulation. The Lancet 2000; 355: 1627-1632

-E. E. Nishizawa 1 J. F. Mustard 1 .The Effect of Synthetic Phosphatidyl Serines on Platelet Aggregation, Blood Coagulation and Haemostasis. British Journal of Hematology 2008 ; Volume 20 Issue 1, Pages 45 – 54.

-Ernest Beutler, Marshall A.Lichtman, Barry S.Coller, Thomas J.Kipps, Uri Seligsohn. Anti phospholipid syndrome.Williams Haematology 2000;(pp. 1715-1728).

-Fadok V, Voelker DR, Campbell PA, et al. Exposure of phosphatidylserine on the
surface of apoptotic lymphocytes triggers specific recognition and removal by
macrophages. J Immunol. 1992;148:2207–2216

-G. Ball, G. G. Brereton, Mary Fulwood, D. M. Ireland and Patricia Yates ,(1970) ,
Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3´:5´-cyclic monophosphate.Biochem. Journal. (1970) 120 (709–718)

- Gerke V, Moss SE. Annexins: from structure to function. Physiol Rev. 2002;82:
331–371

-George, JG. Platelets. The Lancet 2000; 355: 1531-1539

-Hendrikus H. Boersma, Bas L.J.H. Kietselaer,Leo M.L. Stolk, PharmD,
Abdelkader Bennaghmouch, Leonard Hofstra,Jagat Narula, . Past, Present, and Future of Annexin A5: From Protein Discovery to Clinical Applications. The Journal of Nuclear medicine. 2005; Vol. 46. No. 12

-Homburg CH, de Haas M, von dem Borne AE, et al. Human neutrophils lose their
Surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in
Vitro; Blood. 1995;85:532–540.

-Iwasaki A, Suda M, Nakao H, et al. Structure and expression of cDNA for an
inhibitor of blood coagulation isolated from human placenta: a new lipocortinlike
protein. J Biochem (Tokyo). 1987;102:1261–1273

-Koopman G, Reutelingsperger CP, Kuijten GA, et al. Annexin V for flow
cytometric detection of phosphatidylserine expression on B cells undergoing
apoptosis. Blood 1994;84:1415–1420

-Kroemer G, Martin SJ. Caspase-independent cell death. Nat Med. 2005;11:725–
730.

-Leist M, Jaattela M. Four deaths and a funeral: from caspases to alternative
mechanisms. Nat Rev Mol Cell Biol. 2001;2:589 –598
1985;151:625– 629

-Martin SJ, Reutelingsperger CP, McGahon AJ, et al. Early redistribution of
plasma membrane phosphatidylserine is a general feature of apoptosis regardless
of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp
Med. 1995;182:1545–1556.

-Maurer-Fogy I, Reutelingsperger CP, Pieters J, et al. Cloning and expression of
cDNA for human vascular anticoagulant, a Ca2_-dependent phospholipid-binding
protein. Eur J Biochem 1988;174:585–592.

- Reutelingsperger CP, Kop JM, Hornstra G, Hemker HC. Purification and characterization of a novel protein from bovine aorta that inhibits coagulation:
inhibition of the phospholipid-dependent factor-Xa-catalyzed prothrombin activation, through a high-affinity binding of the anticoagulant to the phospholipids. Eur J Biochem. 1988;173:171–178

-Tait JF, Smith C, Blankenberg FG. Structural requirements for in vivo detection
of cell death with 99mTc-annexin V. J Nucl Med. 2005;46:807– 815

-Trotter,PJ Orchard, MA and Walker,J H . Relocation of annexin V to platelet membranes is a phosphorylation-dependent process. Biochem Journal 1997; 1 December , pp. 447–452. [Pubmed]

-Zimmermann KC, Green DR. How cells die: apoptosis pathways. J Allergy Clin
Immunol. 2001;108(suppl):S99 –S103

-Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid
asymmetry in blood cells. Blood. 1997;89:1121–1132.