6. platelet function_tests

Platelet function tests

1. PLATELET FUNCTION TESTS Presenter : DR. G Santhi priya Moderator : DR. Divyasree B N 20/08/2018 SEMINAR

2. CONTENTS Introduction Platelet formation Platelet structure Platelet functions Platelet function tests 20/08/2018 SEMINAR

3. INTRODUCTION Platelets are 2 nd major component of the hemostatic system There role is first established by Bizzozero in 1892 20/08/2018 SEMINAR

4. PLATELET FORMATION From megakaryocytes in the bone marrow Endomitosis : involves nuclear replication without cellular division To generate 2000 – 3000 platelets per magakaryocyte 20/08/2018 SEMINAR

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6. PLATELET STRUCTURE Platelets are small, anucleate cells Discoid in shape Diameter : 1.5-3.5 µm Normal count : 150 – 440 x 10 9 /L Storage granules : α , dense, lysosomes Primary function : rapid formation of a vascular plug following vessel injury 20/08/2018 SEMINAR

7. Peripheral zone Structural zone Organelle zone Membrane systems 20/08/2018 SEMINAR

8. α Granules Group 1- hemostatic proteins Fibrinogen Factor v Von willebrand factor Plasminogen Plasminogen activator inhibitor(PAI-1) α 2antiplasmin Group II- non hemostatic proteins Platelet specific Β thromboglobulin Platelet factor 4 Platelet derived growth factor Non platelet specific Albumin Thrombospondin Fibronectin 20/08/2018 SEMINAR

9. PLATELET FUNCTIONS Surveillance of blood vessel continuity Formation of primary haemostatic plug Surface for coagulation factor to make secondary haemostatic plug Aid in healing of injured tissue 1. Adhesion 2. Shape change 3. Aggregation 4. Secretion 20/08/2018 SEMINAR

10. ADHESION The initiating event following vascular damage to exposed subendothelial matrix proteins The platelet glycoprotein (GP) receptors which mediate adhesion are dependent on the rate of shear Intermediate to high shear (arterioles) -strictly dependent on von Willebrand factor ( vWF ) and its receptor, the GPIb –IX, V complex 20/08/2018 SEMINAR

11. lower rates of shear (venous circulation) and in the static conditions (experimental purposes) Adhesion - subendothelial matrix proteins such as collagen and fibrinogen Adhesion is strengthened –activation of platelet surface integrins , which leads to an increase in affinity for their adhesive ligands 20/08/2018 SEMINAR

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14. SHAPE CHANGE Upon activation, platelets become spherical and extend pseudopodia This enables to attach to other platelets and to the vessel wall Shape change is mediated by phosphorylation of myosin light chains As a consequence of elevation in intracellular Ca2+ ions, which activate myosin light chain kinase 20/08/2018 SEMINAR

15. Inhibition of myosin light chain phosphatase As the platelet flattens and spreads, granules and organelles are squeezed into the centre of the cell, resulting in a characteristic fried egg appearance Granules are secreted from the centre of the cell directly into the surface open canalicular system 20/08/2018 SEMINAR

16. Dense granules - adenine nucleotides ADP and ATP, the amine 5-HT and Ca2+ Rapid release of ADP - plays a major positive feedback role in promoting platelet activation α - granules contain the adhesive proteins fibrinogen and vWF α-Granule release of vWF is critical for normal thrombus formation at intermediate and high rates of flow 20/08/2018 SEMINAR

17. AGGREGATION 20/08/2018 SEMINAR

18. PLATELET FUNCTION TESTS ADHESION TESTS Retention in a glass-bead column Baumgartner’s technique PFA-100 AGGREGATION TESTS Turbidometric technique using ADP Collagen Ristocetin Adrenaline (epinephrine) Thrombin Arachidonic acid Endoperoxide analogues Calcium ionophore INVESTIGATION OF GRANULE CONTENT AND RELEASE Dense bodies Electron microscopy ADP and ATP content (bioluminescence) Serotonin release GRANULES b- thromboglobulin Platelet factor 4 VWF Fluorescence by flow cytometry 20/08/2018 SEMINAR

19. PROSTAGLANDIN PATHWAYS TXB2 radioimmunoassay PLATELET COAGULANT ACTIVITY Prothrombin consumption index FLOW CYTOMETRY Glycoprotein surface expression Activation P selectin (CD62) surface expression FIBRINOGEN BINDING Annexin binding (to phosphatidyl serine) Conformational changes in Gp IIbIIIa Platelet granule fluorescence 20/08/2018 SEMINAR

20. PFA-100 [Platelet Function Analyser ] 20/08/2018 SEMINAR

21. New generation of rapid whole-blood analyzers Designed to screen for platelet function defect Whole blood is passed through a hole in a membrane coated with collagen and either ADP or epinephrine Combined influence of high shear rates and VWF, platelets encountering this membrane spontaneously adhere to collagen And aggregate because of stimulation by ADP or epinephrine Measures closure time (CT), the amount of time required for a platelet plug to occlude the aperture and stop flow 20/08/2018 SEMINAR

22. CT is prolonged in - severe congenital and acquired platelet dysfunction Defects in platelet glycoprotein receptors GPIIb / IIIa ( Glanzmann thrombasthenia ) and GPIb (Bernard- Soulier syndrome) Monitoring of patients taking aspirin – aspirin resistance 20/08/2018 SEMINAR

23. Hematologic variables - influence PFA-100 CT independent of platelet function are Severe thrombocytopenia Anemia CT seems to be sensitive to VWF levels, as indicated by prolonged CT in patients who have von Willebrand disease 20/08/2018 SEMINAR

24. ADVANTAGES Whole blood test High shear Small blood volumes Simple Rapid DISADVANTAGES Inflexible vWf dependent Insensitive to clopidogrel 20/08/2018 SEMINAR

25. OTHER ADHESION TESTS : Retention in a glass bead column Baumgartners technique 20/08/2018 SEMINAR

26. Retention in a glass-bead column 20/08/2018 SEMINAR

27. AGGREGATION TESTS Measures the ability of various agonists to platelets to induce in vitro activation and platelet-to-platelet activation Change in their shape from discoid to spiny spheres Associated with a transient increase in optical density 20/08/2018 SEMINAR

28. STRONG AGONISTS Directly induce platelet aggregation, TxA2 synthesis and platelet granule secretion Eg : Collagen Thrombin TxA2 WEAK AGONISTS Induce platelet aggregation without inducing secretion Eg : ADP Epinephrine 20/08/2018 SEMINAR

29. 20/08/2018 SEMINAR Mechanism Platelets are non- thrombogenic in their resting state The expose receptor on their surface on activation by agonists and becomes sticky Activated platelets adhere on the surfaces of the Sensor and aggregates form a insulation layer The impedance between the electrodes rises The increase of impedance is transformed into aggregation units and plotted against time

30. Normal aggregation curve 12/23/2017 30 Seminar

31. Platelet aggregation curves in von Willebrand disease and Bernard- Soulier syndrome (absent aggregation with ristocetin , normal aggregation with ADP, epinephrine, and arachidonic acid) 12/23/2017 31 Seminar

32. Platelet aggregation curves in storage pool defect (absent second wave of aggregation with ADP and epinephrine, absent or greatly diminished aggregation with collagen, and normal ristocetin aggregation) 12/23/2017 32 Seminar

33. Platelet aggregation curves in Glanzmann’s thrombasthenia (absent aggregation with all agonists except ristocetin ) 12/23/2017 33 Seminar

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35. SECRETION ASSAYS Measured by 2 different ways Measuring ATP secretion from platelets 14 C-labelled serotonin 20/08/2018 SEMINAR

36. ATP secretion from platelets PRP or whole blood is incubated with firefly luciferase and its substrate luciferin Addition of platelet agonists causes release of ATP, a cofactor in a light-producing luciferin-luciferase assay Resulting light can be measured with a photodetector system Often performed simultaneously with a platelet aggregation assay 14 C-labelled serotonin : measured by allowing platelets to take up 14 C-labelled serotonin Its release is then measured in response to agonists 20/08/2018 SEMINAR

37. FLOW CYTOMETRY Method used to measure multiple properties of single cells in suspension Using specific monoclonal antibodies, it can be used to gauge the expression of proteins in these cells Antibodies are conjugated with fluorescent tags that are activated as the cells pass in a single-file stream through the flow cytometer 20/08/2018 SEMINAR

38. Light emitted from each fluorochrome can be individually detected and its intensity measured for each cell Diagnosis of inherited defects in platelet surface glycoproteins Decreased expression or absence of GPIb - Bernard- Soulier syndrome GPIIb / IIIa - Glanzmann thrombasthenia Multiple antibodies against different GPIIb / IIIa epitopes - acquired Glanzmann thrombasthenia 20/08/2018 SEMINAR

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41. REFERENCES 1.Hoffbrand A V, Catovsky D, Tuddenham E G D, editors. Postgraduate Hematology.5 th ed. Massachusetts:Blackwell Publishing Ltd; 2005.P.808-24. 2. Seegmiller A, Sarode R. Laboratory Evaluation of Platelet Function. Hematol Oncol Clin N Am 2007;21:731–42. 3. Quinn M, Fitzgerald D, Platelet function, assesment, diagnosis and treatment. New Jersy:Humana press;2005:P.202-328. 4. Platelet Function Testing: Light Transmission Aggregometry [LTA], 29-4-2012, Available from http://www.platelet-research.org/3/aggregometry.htm. 5.Laffan M A, Manning R , Investigation of hemostasis , Bain B.J, Bates I, Laffan M.A, Lewis S.M, Dacie and Lewis Practical Haematology . 11 th ed. London: 2012. P.424-39. 20/08/2018 SEMINAR

42. Thank you!!! 20/08/2018 SEMINAR