.1. Annexin V (Endonexin II, Placental anticoagulant protein I, vascular anticoagulant-alpha, Lipocortin V, Placental protein 4; PP4, Anchorin CII) (Bohn H et al, 1979 ; Ernest Beutler et al, 2001 ;Reutelingsperger CP ,2001 ;Hendrikus H et al , 2005). Annexin V (Gene map locus 4q26-q28), a member of the Annexin family with an unknown function up to now , is a cellular protein which is known as a phosphatidylserine-binding protein (Ernest Beutler et al , 2001) . Annexin family has over 160 proteins. They all share the property of Ca 2+ dependent binding to negatively charged phospholipid surfaces (Gerke V et al, 2002). At late of 70s Annexin V for the first time was purified from an EDTA extract of human placenta (Bohn H et al, 1979; Hendrikus H et al, 2005). After ward, a protein (Fig. 2.1.1.) was discovered independently in blood vessels which was named vascular anticoagulant protein Alpha (VAC-Alpha) which inhibits blood coagulation (Reutelingsperger CP, 1985). The mechanism of anticoagulation is based on the high-affinity binding to Phospholipids (Fiedler K et al, 1995). This is the property which makes Annexin V quite effective in inhibiting the prothrombinase complex (Reutelingsperger CP, 1988; Homburg CH et al, 1995). In vitro, Annexin V is defined as a Phospholipid binding protein with a high affinity for PS (phosphatidylserine) (D’Arceuil H et al, 2000). One of the important functions of Annexin V is forming a shield around negatively-charged phospholipid molecules (Dumont EA et al, 2000; Dreier R et al, 1998). This formation of Annexin V shield would block the entry of phospholipid molecules into coagulation clotting reactions (Glaser M et al, 2003). An increased quantity of phospholipid molecules on cell membranes which speed up coagulation reactions is seen in the absence of the shield(Koopman G et al , 1994 ; Vermes I et al , 1995 ). Annexin V binds quite tightly to anionic phospholipids (Catherine .R et al, 1992). There are evidences suggesting Annexin V interacts with phospholipid vesicles (Hendrikus H et al, 2005). Annexin V can form a shield around phospholipid molecules which blocks their entry into coagulation (clotting) reactions (Ernest Beutler et al, 2001). The formation of this shield is disrupted by the abnormal antibodies in case of anti phospholipid antibody syndrome (Hayes MJ, 2004). In the absence of the shield, there is an increased quantity of phospholipid molecules on cell membranes (Gerke V et al, 2002). These molecules speed up coagulation reactions and cause the abnormal blood clotting which is characteristic of the anti phospholipid antibody syndrome (Ernest Beutler et al, 2001; Huber R et al , 1992).
Figure 2.1.1.X-ray analysis on Annexin V. Tertiary
Structure of Annexin V is revealed (Consists of 319
amino acids).Green spheres are indicating Ca 2+



2.2. Phosphatidylserine - C13H24NO10P (MW: 385.304)
Phosphatidylserine (Fig. 2.2.1.) is a phospholipid component essential to the functioning of all the cells of the body which is mainly known as a molecule which enables brain cells to metabolise glucose (Vermes I et al, 1995). It is mainly located entirely on the inner layer of the plasma membrane. Phosphatidylserine is mainly known as effective treatment for Alzheimer's disease . Phosphatidylserine is usually on the cytosolic side, of cell membranes (Catherine .R et al, 1992; Calderon F et al, 2008). By a flipase enzyme Phosphatidylserine become exposed on the surface when a cell undergoes apoptotic cell death (Boersma et al, 2003; Belhocine T et al, 2002). The normal distribution of Phosphatidylserine is altered during platelet activation and cellular apoptosis (Fadok VA et al, 1992) Phosphatidylserine modulates the activity of several enzymes involved in cellular signalling. Annexins (particularly Annexin V) bind to and polymerize through protein-protein interactions on membrane patches which are expressing phosphatidylserine. Annexin V specifically tends to bind to phosphatidylserine (Calderon F et al, 2008). As stated before Phosphatidylserine is almost exclusively located on the cytoplasmic side of the plasma membrane and after stimulation, Phosphatidylserine becomes exposed on the outer side of plasma membrane at a proportion depending on the type and the nature of the stimuli (Cohen Zoë et al , 2004 ;Kenis H et al ,2004).

Figure 2.2.1.Chemical structure
of Phosphatidylserine.
C13H24NO10P


2.3. Platelet Thrombocytes (Fig. 2.3.1.) are smallest (2-4µm in diameter) cytoplasmic bodies derived from megakaryocyte cytoplasm in bone marrow (White, GC, 1980). The development of megakaryocytes and consequently the production of platelets are unique processes in body (Dahlbäck B, 2000). Megakaryocyte maturation involves nuclear duplication without cell division (many sets of chromosomes in nucleus 8-64C) which resulting in giant cells. Over the process of cytoplasmic fragmentation of these giant cells, platelets are realised into blood stream .The production of platelet is controlled by thrombopoietin, IL-3, IL-6 and IL-11(Sixma JJ et al, 1977). Platelets have no nucleus. Platelets play a critical role in normal haemostasis .Their life span is between 7 to10 days with the normal count 1.5 - 4.0 x 105 ml -1 in blood (up to 1/3 of them are generally stored in spleen) (George.JG , 2000;Dahlbäck.B , 2000)
Figure 2.3.1. Platelets.
The main cells in the coagulation process.





There some types of granules in platelets which store different substances .Granules of platelets are including:
-α granules: fibrinogen, FV, FVIII, PDGF, PF4 VWF, b-thromboglobulin
-Dense bodies: Ca 2+, 5-HT, ADP
-Lysosomes: acid hydrolases

2.4. Activation of the platelets and activators
Platelets are normally circulating in blood in resting condition .They need to get activated to aggregate. They can become activated by some substances such as ADP and Thrombin. ADP (Adenosine diphosphate) and Thrombin can activate the platelets. Over the activation process their shape change. Platelets contract into a spheroid shape and throw out long protuberances “pseudopodia”. Platelets adhere to exposed structures in the sub endothelium .In this process, fibrinogen receptors (GPIIb/IIIa) on the platelet surface become activated and increase affinity for fibrinogen. Platelet cytoplasmic granules fuse with the plasma membrane and their contents are released into the blood (George.JG, 2000; Dahlbäck.B, 2000) .The platelets express more phosphatidylserine (PS) on the outer leaflet of the plasma membrane when they become activated (Cohen Zoë et al, 2004). Some substances could activate the platelets for instance ADP and Thrombin would be described.Thrombin needs to act on Protease-activated receptors (PRA) (a subfamily of related G protein-coupled receptors). Then it can activate the platelets. The mechanism is that after binding the receptors (PRA’s) become activated by cleavage of part of their extracellular domain. These receptors are presence on other cells such as endothelial cells, myocytes and neurons too. (Ayyanathan K et al, 1996) .Thrombin act on mainly PAR’s 1, 3 and 4 Thrombin cleaves the N-terminus of the receptor. The cleaved N-Terminus in turn would act as a tethered ligand. A part of the receptor itself acts as the agonist which can cause a physiological response , in the cleaved state (J Clin Invest , 2003) .ADP mainly binds to P2Y12.This receptors is mainly involved in platelet aggregation.P2Y12 is a potential target for the treatment of clotting disorders ( Murugappa S et al , 2006).
2.4.1. Activator ADP
Adenosine diphosphate is ester of pyrophosphoric acid with the nucleoside adenosine is a nucleotide. ADP (Fig. 2.4.1.1.) is more known as an energy transfer molecule. ADP is stored in dense granules of platelets (Born GV, 1962).ADP is know as activator of platelets activity. ADP induces platelet aggregation. It is by binding to specific receptor on the plasma membrane of platelet (Ernest Beutler et al, 2001). There is a molecule which inhibits this effect. It is Prostaglandin E1. Prostaglandin E1 interferes binding ADP to the receptors. The effect of ADP is enhanced by leptin (Nakata, M et al, 1999; Ozata M, et al, 2001). ADP is normally saved in dense granules of the platelets. ADP binds to GPIIb/GPIIIa (platelet membrane receptor) (Born GV et al, 1976).After initial activation the platelets undergo a change in shape. As stated before the disc shape cell would change into to a spherical form with protruding pseudopodia (Ernest Beutler et al, 2001). ADP stimulates the thromboxane A2 generation and release of arachidonic acid. ADP, inhibits adenylate cyclise activity, and therefore lowers cAMP activity. (Cohen Zoë et al, 2004). Inside the platelet When ADP binds to ADP leads to mobilization of intracellular calcium ions via activation of phospholipase C. This make a change in platelet shape, and consequently to platelet aggregation
Figure 2.4.1.1. Chemical structure of ADP



2.4.2. Activator Thrombin
Thrombin (Fig. 2.4.2.1.) with gene locus 11p11-q12 is a serine protease known as activated form of coagulation factor II. The main function of thrombin is converting soluble fibrininogen into insoluble strands of fibrin (Evans, I et al, 1992). It as well catalyses some other reactions in coagulation cascade. Thrombin is not known as a normal constituent of the circulating blood and would be generated by the catalytic cleavage of prootrombin(factor II) which is its plasma precursor (E.W.Davie et al , 2003).Thrombin is a glycoprotein formed by two peptides chains of 36 and 259 amino-acids linked by disulfure bonds (Francis CW et al , 1983).As already stated the earliest function of thrombin which has already been identified is the cleavage of fibrinogen into fibrin monomers and consequently the activation of the fibrin-stabilizing factor (factor XIII) and protein C (M.T. Stubbs et al , 1993).Therefore Thrombin would be more than a simple plasma enzyme because of its properties to stimulate platelets which cause them to expand aggregate and release components granules8alpha and dense) (Francis CW et al, 1983). LPS-induced liver injury is cause by thrombin as well which alters the synthesis, expression and release of proteins from endothelial cells (Liu CY et al, 1979). This process would results in increasing in production of factor VIII, tPA, PAI, platelet-derived growth factor (PDGF), factor XIII, platelet activating factor (PAF), and as well modifies the interactions between endothelial cells and the underlying matrix or even between endothelial cells and the expression of adhesion glycoproteins to the cell surface. That is a way to increase the binding of inflammatory cells to the endothelium (J.A. Huntington et al, 2005). Inducing chemotaxis in neutrophils and macrophages is performed by thrombin too (M.T. Stubbs et al, 1993) .The mechanism of activation of platelet by thrombin is based on receptor activation. Thrombin binds to its receptor then thrombin cleaves the amino-terminal extension to expose a new amino-acid sequence (Ernest Beutler et al, 2001). This new amino acid-sequence binds to the third extracellular loop of the receptor and causes activation. The similar mechanism exist on other ells as well (Francis CW et al, 1983).
Figure 2.4.2.1. Human Thrombin






2.5. Inhibition of the platelets and inhibitors
The inhibition process is the opposite of the activation. By definition inhibition the platelets means inhibiting clotting activity of platelets. Inhibitors of a platelets interfere with a chemical reaction, growth or other biological activity of the platelets where as an activator has the opposite effect (Siegle AM et al, 1982). For normal platelets keeping the equilibrium between activating and inhibiting processes is quite essential. It is important not only for platelets but also for vascular system as well .As it is linked with cardiovascular disease, Heparin and Aspirin are mow known as anti-platelet factors (Ernest Beutler et al, 2001). .The substances which eventually inhibit the platelets coagulation activity. Over the inhibiting the platelets, they tend to aggregate less (Joel S. Bennett, 2001). The molecules which were externalised over the activation would become less externalised. For instance some receptors such as GP IIb/IIIa and Glycoprotein IIb/IIIa (IIbß3). Prostacyclin (PG-I2), nitric oxide (NO) and Aspirin are some substances which can work as inhibit cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) are known, as two intracellular messengers, to mediate the effects of platelets inhibitors. Another instance is Aspirin which would be described (Gambaryan Stepan et al, 2004; Priyanka Dikshit et al, 2006).

2.5.1. Aspirin
Aspirin (acetyl salicylic acid) (Fig 2.5.1.1.) as a NSAID (non steroidal anti-inflammatory drug) inhibits platelet prostaglandin synthesis and the ADP- and collagen-induced platelet release reaction (Siegle AM et al, 1982). Although the whole mechanism of this irreversible inhibition of platelets is still unknown but may involve protein acetylation cyclo-oxygenase 1 (COX-1) which can permanently inactivates the enzyme activity Cyclooxygenase 1 enzyme catalyses the conversion of arachidonic acid to prostaglandin H2. (G J Roth et al, 1975; Barsom Aktas et al, 2005; Helgason, C.M, 2000). More importantly .Aspirin Induces Apoptosis through -Release of Cytochrome c from Mitochondria (Katja C Zimmermann et al , 2000) and -The inhibition of Proteasome Function (Priyanka Dikshit et al ,2006 ; Ernest Beutler et al, 2001).
Figure 2.5.1.1.
Chemical structure of Aspirin


2.6. Flowcytometry machine
Flowcytometry is a technique to counting, examine, and sort the microscopic particles which are suspended in a stream of fluid. Flowcytometry is one of the biggest advances in platelet function analysis (A.D Michelson et al, 1996; A.D. Michelson et al, 1999). The most commonly used routine tests of flowcytometry is the quantification of glycoprotein receptor density (R.J. Cohn et al , 1997; T.L. Lindahl et al , 1992) Only small quantities of blood are required for flowcytometry and platelets can be analysed in their circulating state. (A.D. Michelson et al ,1999 ; A.D. Michelson et al ,2002 .Platelet function in whole blood can be comprehensively evaluated by flowcytometry (Alice Longobardi Givan ,2001. Flowcytometry is a technique used for counting, examining, and sorting microscopic particles suspended in a fluid. Multiparametric analysis of the physical/chemical characteristics of single cell by an optical/electronic detection apparatus. Particles 0.2 to 150 micrometers. In this experiment flowcytometry is used to measure the phosphatidylserine exposed on the surface of platelets. They are bind with Annexin V which has been labelled with fluorescent substances (Florin). Light scatter and florescence channels were set at logarithmic gain (Alice Longobardi Givan, 2001). Forward scatter data are indicating fluorescence intensities were obtained from 10000 platelets and analysed. In this experiment flowcytometry is set up on FL2-H: which is set for 585/42 nm of light and yellow-green color. The result showing fluorescent intensity are express by a unit which is molecules of equivalent soluble fluorescein (MESF) (Alice Longobardi Givan ,2001).That indicates the number of molecules bind with Annexin V(in this experiment phosphatidylserine) revealed on the surface of platelet. In face in this experiment, phosphatidylserine is indirectly measured. Phosphatidylserine is bind with Annexin V and Annexin V was labelled with fluorescent. The fluorescent on the Annexin V is measured which indirectly is indicating the Annexin V and more importantly the phosphatidylserine express on the surface of the platelets (A.D Michelson et al , 1996 ; A.D. Michelson et al ,1999).
Figure 2.6.1. The mechanism of action of flowcytometry.