Platelet Function Tests

Hematology Lectures

1. Platelet Function Testing By: Ahmed Makboul Ahmed Assistant Lecturer of Clinical Pathology, SECI, Assiut University

2. Outlines: - Platelet structure. - Functions of Platelets. - Overview of normal platelet function. - Platelet function tests: Overview. Light transmission platelet aggregometry (LTA). Other platelet function tests.

3. INTRODUCTION: Stages of Hemostasis: 1. Primary Hemostasis: - Platelets and the injured vessels interact to form the primary hemostatic plug, which is a clump of platelets. - It only temporarily stops the bleeding and is very fragile. 2. Secondary Hemostasis: - The fibrin-platelet plug is then formed by a series of interactions involving the coagulation factors (coagulation cascade) . The fibrin stabilizes the clot covering the hole in the vessel. 3. Fibrinolysis: - The process of removing the clot once the wound has been healed.

4. PLATELET STRUCTURE: Normal human platelets are: Size: Small in size (0.5 x 3.0 μ m) Shape: - Discoid in shape have a mean volume of 7–11 fL. - Anucleated . Number: They circulate in relatively high numbers (between 150 and 400 x 10 9 /L). Life span: Their lifespan is approximately 10 days (9 – 12 days).

5. Platelet Ultrastructure 1 2 3 4

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7. 1. Peripheral Zone: Function: - Adhesion and aggregation. - Responsible for cell's negative charge. a). Glycocalyx  (exterior coat): It contains receptors : - GPIb -IX-V: It is the major platelet receptor for VWF. - GPIa /- Ia and GPVI: receptors for collagen. - GPIIb-IIIa : receptor for vWF . - Thromboxane A2 (TXA2) receptor . - ADP receptors: P2Y12 and P2Y1. b). Membrane: - Has a number of phospholipids and proteins embedded within it. - Responsible for the release of fatty acid derivatives: PGs, prostacyclins .

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9. 2. Structural Zone: - Organized network of protein filaments. - Maintain shape of resting platelet—discoid. - 3 principle types of filaments: a). Microtubules: - Located beneath the cell membrane of resting platelet. - Maintain discoid shape. b). Microfilaments: - Mediate contractile events. c). Intermediate filaments.

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11. 3. Membranous Zone: Function: - Structure and support . a). Open Canalicular System (OCS): - Responsible for the secretion of granule contents . - Provides a route for entry and secretion. b). Dense Tubular System (DTS): - Responsible for the storage of calcium . - Major site of prostaglandin and thromboxane synthesis

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13. 4. Organelle Zone: Function: - Secretion and storage. a). Mitochondria. b). Glycogen particles : support metabolic activities c). Granules: dispersed within the cytoplasm, which serve as storage sites for proteins and other substances necessary for platelet function: i . Dense granules: contain platelet agonists and signaling molecules that amplify platelet activation (approximately 3-8 dense granules per platelet): - Agonists: ADP. ATP. - Serotonin. - Calcium.

14. ii. Alpha granules: contain proteins that enhance adhesion , growth factors , and coagulation factors (approximately 80 alpha granules per platelet). - Adhesive proteins: Fibronectin. vWF . Thrombospondin. Vitronectin . - Growth modulators: PDGF. TGF- β . Platelet factor 4 (PF4).

15. - Coagulation factors: Factor V. High-molecular-weight kininogen (HMWK). C1 inhibitor. Fibrinogen. Factor XI. Protein S. Plasminogen activator inhibitor-1 (PAI-1). - Membranes of platelet alpha granules contain P-selectin.

16. FUNCTIONS OF PLATELETS: Role of Platelets in the Circulation 1. Surveillance of blood vessel continuity: Checks endothelial lining for gaps and breaks. Fill-in small gaps caused by separation of endothelial cells. 2. Formation of primary hemostatic plug. 3. Surface for coagulation factors to make secondary hemostatic plug. 4. Aid in healing injured tissue. Platelet Function 1. Adhesion. 2. Activation and shape change. 3. Secretion or release. 4. Aggregation.

17. OVERVIEW OF NORMAL PLATELET FUNCTION: Endothelial cells produce a number of potent antiplatelet substances (e.g., nitric oxide, prostacyclin, and CD39) that normally inhibit vessel wall–platelet interactions . Vessel wall damage exposes highly adhesive substrates (e.g., P selectin, von Willebrand factor (VWF), collagen, and many other extracellular matrix components), which overcome these inhibitory factors and result in a sequence of stepwise events resulting in the formation of a hemostatic plug:

18. 1. Platelet Adhesion: Platelets are marginated along the vessel wall where they are well-positioned to monitor the integrity of the endothelium. When vessel wall injury occurs and there is endothelial damage, the initial response of platelets is that of adhesion to collagen fibers in the exposed subendothelium . Initial binding of platelets is considered to occur via GPIa-IIa receptor , which allows for further binding to collagen via the GPVI receptor , initiating transmembrane and, subsequently, intracellular signalling . Adhesion of platelets to the exposed subendothelium is influenced by shear rates . At high shear , GPIa-IIa and GPVI are not sufficient to initiate binding to collagen, and binding of the GPIb -IX-V receptor to vWF that is immobilized on collagen, becomes essential in platelet adhesion.

19. 2. Platelet Activation and Granule Release: Platelet adhesion at the site of vessel wall damage initiates activation events that result in aggregation: 1. Adherent platelets undergo a dramatic shape change to an irregular sphere with multiple pseudopodia spreading on the subendothelium increasing their area of surface contact. 2. Adherent platelets also secrete or release the contents of their storage granules -- the alpha- and dense granules -- by an exocytic process . This provides a high local concentration of effector molecules essential for platelet plug formation at the site of vascular injury.

20. 2. Platelet Activation and Granule Release: 3. Platelet activation stimulates the formation of another aggregating agent, thromboxane A2 via the arachidonic acid cascade. 4. ADP, thromboxane A2 and thrombin bind to specific platelet membrane receptors and stimulate aggregation on and around the platelets adherent to the subendothelium via receptor-mediated signal transduction events.

21. 3. Platelet Aggregation: Aggregation is an active metabolic process: binding of any of the agonists to their respective membrane receptors initiates signalling pathways that ultimately convert GPIIb-IIIa - from a low affinity resting state to a high-affinity activated state for binding extracellular soluble ligands such as plasma fibrinogen and vWF . Divalent fibrinogen and multivalent von Willebrand factor function as bridges between GPIIb-IIIa receptors on adjacent activated platelets, thus allowing platelet aggregation to proceed to form a platelet plug that arrests blood loss from a vascular injury.

22. PLATELET FUNCTION TESTING: Overview: - Platelet function testing is indicated to determine if bleeding is due to defective platelet function: Adhesion Activation Granule release Aggregation - Defective platelet function suspected when platelet count is normal in setting of bleeding that is characteristic of disorder of primary hemostasis. - Light transmission platelet aggregometry (LTA) is considered gold standard for platelet function testing . - Platelet function is evaluated by observing response of platelets to agonists that bind to platelets via their receptors leading to activation and aggregation.

23. Light transmission platelet aggregometry (LTA): CLINICAL APPLICATIONS: 1. Diagnosis of Bleeding Disorder: - Pattern of response to agonists can help in determining if there is platelet function defect and can help categorize type of defect. 2. Monitoring Antiplatelet Therapy: - Effects of antiplatelet medications can be seen with LTA. - LTA used for monitoring antiplatelet therapy typically in research setting . - At present, clinical guidelines advise that there is no indication for routine laboratory monitoring of antiplatelet therapy.

24. PRINCIPLE: - PRP has high optical density and transmits light poorly because platelets prevent passage of light. PPP has been depleted of platelets and is able to transmit light well ( low optical density ). - When substance that promotes platelet activation and aggregation (agonist) is added to PRP, normal platelets are activated and aggregate. - Platelet aggregometer measures change in optical density (or light transmittance) over time of stirred PRP in cuvettes at 37°C after addition of agonists. Aggregation of platelets in PRP results in decrease in optical density (increased light transmittance) as aggregated platelets fall out of path of light.

25. METHOD: 1. SPECIMEN: 1.1. Collection: - Patients should not take medications known to affect platelet function for 10-14 days before specimen collection. - 3.2% sodium citrate: 1:9 citrate-to-whole blood ratio. 1.2. Transport: - After collection, samples should be transported to laboratory at room temperature . - Tubes should not be subjected to vibration, shaking, vortexing , continuous mixing, or agitation . - Samples should be tested between 30 min and no more than 4 hr from blood collection.

26. 2. PROCESSING:   2.1. Citrated PRP: - Prepared by centrifugation at 800 – 1000 rpm  for 15–20 minutes. - Platelet Count of PRP: Platelet count of PRP is performed and should be 200-400 x 10⁹/L. If platelet count is > 400 x 10⁹/L, concentration can be adjusted with autologous PPP. - PRP is added to a cuvette at 37C and preincubated for up to 5 minutes. - The PRP is stirred at a recommended speed (e.g., 1000–1200 rpm using a magnetic stir bar) to allow platelets to come in contact with each other.

27. 2.2. Instrument calibration: Aggregometers are calibrated for light transmission using autologous PPP and PRP: 100% transmission is set with autologous platelet-poor plasma (PPP) (prepared by centrifugation at 3000 rpm  for 10 minutes). 0% transmission is set with PRP and a stable baseline established before addition of agonist .

28. 2.3. Addition of agonist: Agonist is added at maximum of one-tenth volume of agonist to PRP to initiate aggregation (10% of total volume). Baseline panel of agonists includes: Agonist Dose Comment ADP  2.5–20 μ mol /L Collagen  1–5 μ g/mL Usually mediates a steep aggregation curve but after a characteristic lag phase. Epinephrine  3–30 μ mol /L Arachidonic acid (AA)  0.5–1.5 mmol /L Ristocetin  0.5–2 mg/mL Not strictly an activating agonist but stimulates platelet agglutination through binding of plasma vWF to GPIb .

29. INTERPRETATION OF RESULTS: - LTA measures change in optical density (or light transmittance) of stirred PRP that occurs after addition of agonists. - Aggregation tracing presents change in light transmission (y-axis) against time in minutes (x-axis). - Several parameters are evaluated for interpretation of these studies: 1. Lag phase (time between addition of agonist and start of aggregation). 2. Platelet shape change. 3. Primary aggregation slope. 4. Secondary aggregation. 5. Disaggregation. 6. Maximum amplitude. 7. Percent aggregation.

30. Normal LTA tracing with ADP, epinephrine, collagen, and ristocetin agonists

31. INTERPRETATION OF ABNORMAL LTA RESULTS: 1. Defective platelet adhesion: a). Abnormal aggregation response only to ristocetin agonist may be noted if there is: defect in GPIb receptor ( Bernard Soulier Syndrome ) decreased/abnormal vWF ( vWD ( . - Examination of peripheral blood smear can be helpful in distinguishing vWD and BSS: BSS is usually accompanied by macrothrombocytopenia (giant platelets). - Addition of cryoprecipitate (source of vWF ) corrects abnormal response to ristocetin that may be seen in vWD , but not in BSS. - vWF antigen and activity studies and factor VIII activity assay can be performed if vWD is suspected.

32. In vWD , there is increased response to low-dose ristocetin . This occurs in: Type 2B vWD Increased affinity of vWF for platelet GPIb -V-IX. Mutations causing type 2B vWD lead to gain-of-function mutations in GPIb binding site on vWF , leading to spontaneous binding of vWF to platelets. Spontaneous binding of vWF to platelets leads to formation of platelet aggregates. These aggregates are removed from circulation leading to thrombocytopenia. Platelet type (pseudo) vWD Caused by mutations in GPIba gene ( vWF receptor on platelets). This mutation results in abnormal GPIb receptor with increased binding (gain-of-function) affinity for normal vWF

33. These 2 disorders can be differentiated by: 1. Modification of low dose Ristocetin -induced platelet aggregation (RIPA) test: - Patient platelets mixed with normal plasma. - Patient plasma mixed with normal platelets. Increased low-dose RIPA with patient platelets mixed with normal plasma is consistent with platelet-type vWD Increased low-dose RIPA with patient plasma mixed with normal platelets is consistent with type 2B vWD 2. Genetic studies: can also be used to distinguish type 2B vWD from platelet-type vWD .

34. b). Abnormal aggregation response only to collagen agonist may be noted if there is: defect in collagen receptors ( GPIa-IIa or GPVI) . - Flow cytometry can be used to evaluate for low levels of GPIa-IIa or GPVI.

35. 2. Defective platelet activation and granule release: a). Secretion defects: - Defect in platelet-dense granules is due to either: Contents of granules (storage pool disorder). Inability to release granules (release defect). - Manifests as lack of secondary aggregation on LTA in response to ADP , epinephrine , collagen .

36. Additional studies can be performed to distinguish between storage pool disorder and release defect: 1. Electron microscopy can be used to examine contents of platelet-dense granules to investigate storage pool disorder. 2. Measurement of total platelet content of ADP and ATP and release of ATP in response to agonists can help to distinguish between storage pool disorder and release defect: Storage pool defects have decrease in amount of stored and released ADP with increased ratio of ATP to ADP . Release defects have normal stored ADP levels , normal ATP:ADP ratio , but decreased ADP release .

37. b). Thromboxane pathway defects: In these defects, there are: Absence/ severe reduction of aggregation in response to arachidonic acid (for cyclooxygenase defects , as in case of administration of aspirin), or absence of aggregation response to both arachidonic acid and thromboxane mimetics (for thromboxane receptor defects ) . c). P2Y12 inhibitors (e.g. clopidogrel ): Reduced or absent aggregation to ADP with notable deaggregation even at high concentrations of agonist.

38. 3. Defective platelet aggregation: - Lack of platelet aggregation in response to all agonists except for ristocetin indicates defect in platelet aggregation. This aggregation pattern can be seen in Glanzmann Thrombasthenia (GT) in which patients lack or have defect of GPIIb-IIIa receptor . - Similar aggregation pattern may be seen in severe hypofibrinogenemia or afibrinogenemia : Defects in aggregation can be remedied by addition of cryoprecipitate (source of fibrinogen) to PRP in the case of hypofibrinogenemia or afibrinogenemia . GT diagnosis can be confirmed by performing flow cytometry to examine number of GPIIb-IIIa receptors on platelets.

39. Disorders Aggregation Response Comment ADP Collagen AA Ristocetin Primary wave Secondary wave Defective adhesion: Bernard Soulier syndrome N N N N A - Platelet morphology. - vWD assay. vWD N N N N A Defective activation and secretion: Storage pool disease N A A N N - ADP/ATP levels Secretion defect N A A N N P2Y12 inhibitor ( Clopidogrel ) R/A R/A N N N COX deficiency N A/N R A N TX Synthase deficiency N A/N R A N Aspirin ingestion N A/N R A N Defective aggregation: Glanzmann Thrombasthenia A A A A N GPIIb / IIIa level by flow cytometry N: Normal, A: Absent, R: Reduced

40. From: Dacie and Lewis Practical Hematology, 12 th Edition, 2017.

41. Other Platelet Function Tests: 1. Whole-Blood (Impedance) Aggregometry: - Uses electric impedance to evaluate platelet aggregation in whole blood. - Citrated whole blood sample is stirred at 37°C. - Electric current is passed between 2 electrodes immersed in sample. Impedance is measured between electrodes. - Platelet aggregation occurs on surface of electrodes and increases impedance. - Addition of agonist to sample increases aggregation of platelets on surface of electrodes. This is detected as increase in impedance measured in ohms. - WBA uses same agonists as LTA: ADP. Epinephrine. Collagen. Ristocetin . AA. - WBA requires one-quarter of blood needed for LTA.

42. 2. Platelet Function Analyzer (PFA-100®): - PFA-100® uses whole blood to measure platelet function under high shear flow conditions. - Sample collected in 3.2% sodium citrate. - Whole blood passes through opening cut into coated membrane. Membrane coated with collagen and epinephrine or collagen and ADP as agonists. - These agonists promote platelet adhesion, activation, and aggregation leading to occlusion of aperture and cessation of blood flow. - Time to occlusion of opening by platelet plug is measured: Prolonged occlusion or “closure” times suggest impaired platelet function.

43. 3. Flow cytometry:   - Whole-blood flow cytometry offers a very attractive and reliable test for the diagnosis of various platelet receptor, granular, and other defects. - Platelet responses to various agonists can also be studied by measuring various activation markers. This is potentially useful for measuring platelet function in thrombocytopenia, as flow cytometry responses are independent of platelet count.

44. Applications of flow cytometry platelet function tests: i. Diagnosis of platelet function disorders: - Mainly used clinically for quantification of surface GP receptor density: Glanzmann thrombasthenia : Flow cytometry shows lack of surface GPIIb-IIIa . Bernard- Soulier syndrome: Flow cytometry shows lack of surface GPIb . ii. Platelet activation markers: - Degranulation markers: CD62p, CD63, and CD40L. iii. Measuring platelet production. iv. Accurate platelet counting: platelet to RBCs ratio. v. Blood bank tests: - Quality control of concentrates. - Leukocyte contamination. vi. Antiplatelet drugs monitoring.

45. 4. Measurement of Platelet Nucleotides: - ATP and ADP are released from platelet granules after platelet activation. - Evaluation of released adenine nucleotides (ATP and ADP) can be used as independent measure of granule release. Release of ATP from dense granules is quantitated using luciferin/luciferase system. ATP release occurs during secondary aggregation phase in LTA. Measurement of ATP release alone does not distinguish between storage pool and release defects. - Measurement of total platelet content of ADP and ATP and release of ATP in response to agonists can help to distinguish between a storage pool disorder and a release defect: Storage pool defects have decrease in amount of stored and released ADP with increased ATP-to-ADP ratio . Release defects have normal ADP levels , normal ATP:ADP ratio , but decreased ADP release .

46. 5. The VerifyNow ®:  The VerifyNow ®  device is a fully automated and near patient testing aggregation system, is available solely for the monitoring of the three major classes of antiplatelet drugs (e.g., GPIIb / IIIa inhibitors , aspirin , and P2Y12  receptor inhibitors/ antagonists ). 6. Plateletworks ®: Plateletworks ® is a standardized platelet counting ratio technique, based upon comparing platelet counts within a control EDTA tube and after aggregation with platelet agonists within citrated tubes, could theoretically also be applied to the diagnosis of platelet disorders, although experience with this assay is limited.