platelet function, disorders and its assesment

platelet, platelet functional assays, aggregation tests, platelet disorders, transfusion medicine, hematology

1. Platelet; Functions, Disorders and its Assessment

2. Platelet function Responsible for primary hemostasis via 5 mechanisms- Adhesion Secretion Aggregation Coagulation (provide coagulant surface) Clot retraction Physiologically contribute to hemostasis and pathologically leads to thrombotic occlusion of vessel with obstruction to blood flow and subsequent tissue damage

3. Additional role of platelets Inflammation Antimicrobial host defense Mitogenesis Wound healing

4. Primary hemostasis Adhesion Upon damage to vessel vWF will bind to the exposed subendothelial collagen via its A3 domain, after which it adopts its platelet-binding conformation. VWF binds to platelets via GPIb –V–IX complex The interaction of GPVI with collagen will result in further platelet activation

5. Platelet activation Once platelets have adhered they degranulate, releasing stored secondary agents such as ADP, ATP, and synthesize thromboxane A2 ADP activates P2Y1 and P2Y12 which further degranulates platelets

6. Platelet Aggregation Agonists ADP Collagen Epinephrine AA Thrombin Serotonin

7. Platelet function Platelets: procoagulant surface for activation of coagulation cascade as explained by cell model of coagulation

8. Platelet function

9. Platelet disorders Common bleeding disorders: congenital or acquired. Any abnormality in platelet count and function or both can result in bleeding Platelet disorders Quantitative disorders Qualitative disorders Inherited Acquired Thrombocytosis Thrombocytopenia Platelet count > 4 lac /µ L Platelet count <1.5 lac /µ L Rare Common

10. Quantitative disorders: THROMBOCYTOSIS ( > 4 50000/ cumm ) Reactive thrombocytosis Iron deficiency Inflammatory disorders Tissue injury/trauma, Sepsis or infection Malignancy Post splenectomy Rebound thrombocytosis following hemorrhage Primary thrombocytosis Bone marrow disorders Essential thrombocytosis Polycythemia vera Chronic myelogenous leukemia Myeloid metaplasia

11. Increased destruction Decreased production Sequestration Dilutional Congenital B12 and folate deficiency Medications ( valproic acid, chemotherapy) Radiation Toxins (alcohol) Infections (parvovirus, CMV, ehrlichiosis) Liver disease (TPO deficiency) Primary marrow disorders myelodysplasia, myelofibrosis, Acute & chronic leukemias, lymphoproliferative disorders Marrow replaced by solid tumors Granulomatous diseases of the marrow Non immune Immune ITP Infections (HIV, HCV, H.pylori ) Lymphoproliferative disorders Medications (e.g. quinine, quinidine, gold, heparin, glycoprotein IIb/IIIa inhibitors) Posttransfusion thrombocytopenia DIC Thrombotic microangiopathies (TTP, HUS, eclampsia, HELLP syndrome) Cardiopulmonary bypass, intra-aortic balloon pump, ventricular assist devices Abnormal vascular surfaces (aneurysms, heart valves, Merritt- Kasabach syndrome) Hemophagocytosis splenomegaly Massive transfusion Crystalloid infusion Quantitative disorders: Thrombocytopenia ( <150000/ cumm )

12. Platelet Function Disorders An abnormality in platelet function can result in bleeding even when the platelet count is normal Most platelet function disorders are associated with a relatively mild bleeding tendency Can be aquired which is more common Can be inherited/congenital which is more rare

13. Inherited Disorders of Platelet Function

14. Inherited Disorders of Platelet Function

15. Inherited causes of Platelet Function Disorders

16. Characteristics of Congenital Platelet Function Disorders

17. Platelet function disorders: Acquired Uraemia Dysproteinemias Extracorporeal perfusion Acquired von Willebrand disease Acquired storage pool deficiency Antiplatelet antibodies Liver disease Drugs and other agents Acute leukemias & myelodysplastic syndromes Myeloproliferative disorders Essential thrombocythemia Polycythemia vera Chronic myeloid leukemia Primary myelofibrosis

18. Acquired platelet function disorders: Site of platelet dysfunction Systemic illness Potential mechanisms Platelet aggregation Studies Treatment Intrinsic disorders of platelet function Chronic Myeloproli . Disorders Defect at level of c. megakaryocyte : 1) Abnormal lipid peroxidation & responses to Tx A2 2) Abnormal expression of Fc γ R. 3) Combined defect in memb.expression & activation of GPIIb / IIIa complexes 4) Acquired storage pool disorder Defective aggregation Treatment of underlying disorders

19. Site of platelet dysfunction Systemic illness Potential mechanisms Platelet aggregation Studies Treatment Intrinsic disorders of platelet function MDS/ Leukemias Defective megakaryopoiesis : Dilated canalicular system and abnormal microtubule formation 2) Reduced or giant granules may form by fusion of several single granules 3) Acquired membrane defect with abnormal Gp expression Multiple aggre . defects Treatment of underlying disorders Acquired platelet function disorders:

20. Site of platelet dysfunction Systemic illness Potential mechanisms Platelet aggregation Studies Treatment Extrinsic disorders of platelet function Uremia platelet GPIb /IX receptor number and function ↓ 2) ↑proteolysis by ADAMTS13 enz . 3) Defective activation of GPIIb / IIIa for binding fibrinogen and VWF 4) Defective platelet secretion of ADP ↓ Aggregation with collagen, ADP and epinephrine Dialysis Correction of anemia DDAVP Platelet transfusion rFVIIa Cryo . Acquired platelet function disorders:

21. Site of platelet dysfunction Systemic illness Potential mechanism Platelet aggregation Studies Treatment Extrinsic disorders of platelet function Liver dysfunction Altered platelet mem . palmate and stearate meta. Defective aggregation Correction of underlying ds . DDAVP Platelet Tx . Paraprotenemia Nonspecific binding of Igs to platelet surface Defective aggregation Plasmaphresis Tt of underlying ds Platelet tx only during life threatening bleeding Hypothermia ↓ plasma soluble P- selectin expression 2) ↓ levels of TX-A2 ↓ Platelet activation Correction of hypothermia Acquired platelet function disorders:

22. Site of platelet dysfunction Systemic illness Potential mechanism Platelet aggregation Studies Treatment Extrinsic disorders of platelet function CABG 1) Plt activation & fragmentation due to hypothermia, contact with synthetic surfaces & blood/air interface, damage caused by blood suctioning & exposure to traces of thrombin, plasmin , ADP, or complement 2) Drugs ( heparin, protamine , & Aspirin) and production of FDPs expected to impair plt . function Defective aggregation DDAVP Platelet Tx Aprotonin Antifibrinolytic rFVIIa Acquired platelet function disorders:

23. Drug induced: Platelet function disorders CYCLOOXYGENASE 1 INHIBITORS Aspirin- irreversible inhibitor Other- reversible inhibitor- indomethacin, ibuprofen, naproxen, sulfinpyrazone, sulindac , meclofenamic acid, diflunisal , piroxicam , tolmetin , zomepirac , phenylbutazone Inhibits endoperoxidase and TxA2 synthesis hence preventing aggregation and secretions ADP RECEPTOR ANTAGONISTS Ticlopidine , clopidogrel , prasugrel , ticagrelor GPIIb-IIIa ANTAGONISTS Abciximab , tirofiban , eptifibatide

24. Drug induced: Platelet function disorders DRUGS THAT INCREASE PLATELET CYCLIC AMP OR CYCLIC GMP Inhibiting the cyclic nucleotide phosphodiesterases , or increasing adenyl cyclase activity Prostaglandin I 2 and analogues Results in inhibition of platelet responses- adhesion, aggregation and secretion Phosphodiesterase inhibitors (dipyridamole, cilostazol , caffeine, theophylline, aminophylline) Nitric oxide and nitric oxide donors

25. Acquired causes of platelet function disorders ANTIMICROBIALS Penicillins , Cephalosporins , Nitrofurantoin, Hydroxychloroquine, Miconazole Inhibit platelet aggregation & adherence to subendothelial structures Effect is dose-dependent, taking approx. 2-3 days to manifest and 3-10 days to abate ANTICOAGULANT S Heparin THROMBOLYTIC AGENTS Streptokinase, tissue plasminogen activator, urokinase

26. Acquired causes of platelet function disorders CARDIOVASCULAR DRUGS β- blockers (propranolol), Vasodilators (nitroprusside, nitroglycerin ), Diuretics (furosemide), CCB, Quinidine, ACE inhibitors Act through inhibition of aggregation or modification of platelet surface PSYCHOTROPICS AND ANESTHETICS TCA ( e.g imipramine) inhibit platelet aggregation and secretion responses to ADP, epinephrine, and collagen Phenothiazines ( e.g chlorpromazine, promethazine), SSRI ( e.g fluoxetine )- reversible serotonin deficiency in dense- granules General anesthesia (halothane)

27. Acquired causes of platelet function disorders CHEMOTHERAPEUTIC AGENTS Mithramycin , BCNU (1,3-bis(2-chloroethyl)-1-nitrosurea), Daunorubicin , Dasatinib MISCELLANEOUS AGENTS Dextrans and hydroxyethyl starch Lipid-lowering agents- clofibrate , halofenate e- Aminocaproic acid, Antihistamines, Ethanol, Vitamin E Radiographic contrast agents Food items and food supplements - omega-3 fatty acids, vitamin E, onions, garlic, ginger, cumin, turmeric, clove, black tree fungus, Ginkgo

28. Assessment of suspected platelet disorder

29. History & Examination Petechiae Ecchymosis Bleeding on cut and easy bruising Clinical assessment Ecchymosis Petechiae

30. Platelet count, size & morphology Hematology analyzer Peripheral smear (morphology & no.) Lab investigations

31. Ivy method (N: 3-10 min) BP cuff is placed on upper arm & inflated to 40 mmHg. A lancet or scalpel blade: shallow incision on volar aspect of forearm Standard-sized incision: 10 mm long & 1 mm deep Time measured: when the incision is made until all bleeding has stopped. Every 30 seconds, filter paper or a paper to wipe off the blood. Duke method (N: 2-5 min) Patient is pricked with a lancet to 3-4mm deep at earlobe or fingertip and bleeding is wiped every 15s then bleeding time is noted once bleeding ceases Bleeding time

32. Platelet function analyzer PFA 100/200 Rapid, simple, & reproducible Measures primary platelet-related hemostasis under conditions of high shear, with platelet plug formation Asses adhesion function (dependent on VWF rather than fibrinogen) Very sensitive to vWF levels but test results can also be prolonged in cases with thrombocytopenia & anemia itself. Screening for suspected platelet disorder

33. Different platelet activating agents are used Aperture closure time is noted Uses- VWD and its treatment monitoring; identification of inherited and acquired platelet defects; monitoring of antiplatelet therapy Platelet function analyzer PFA

34. Lab evaluation of aggregation Gold standard for platelet function test It by passes the test of adhesion Light transmission aggregometry (LTA): most common method Platelet aggregation studies

35. Platelet Aggregation: LTA Light focused on sample cuvette contained PRP PRP stirred and recorder identified baseline – 0% transmittance Agonist added Transmitted light changes in response to degree of PLT shape changes Change in light transmission continuously monitored and recorded As PLT aggregates form, recorder moves towards 100% transmittance Abnormalities Decreased or absent aggregation Platelet-rich plasma in an optical aggregometer. Platelet count is approximately 200 × 10 9 /L, and platelets are maintained in suspension by a magnetic stir bar turning at 1000 rpm.

36. PLT Aggregation: WB Parallel electrodes (DC) immersed in saline-diluted whole blood Add agonist PLTs aggregate on electrodes, reducing current Change is current directly proportional to level of PLT aggregation The aggregating platelets form a layer on the electrodes, and current is impeded by the platelet layer. Resistance (Ω) is proportional to aggregation, providing a tracing that resembles optical aggregometry.

37. Platelet Aggregation Curve Primary wave Reversible Measures ability of platelets to respond to an external agonist Without enough stimulus or without an intact prostaglandin pathway (to TxA 2 ), platelets disaggregate Secondary wave Irreversible Results in complete release of primarily dense granules contents, most importantly ADP, after stimulation.

38. Common aggregation agonists ADP (P2Ya/P2Y12) Biphasic curve: 1 o & 2 o waves (requires intact PG pathway) No biphasic response: if ADP is added at too low/too high conc. Epinephrine (a 2 -andrenergic receptor) Biphasic curve; requires intact PG pathway Collagen ( GPIa / IIa /GPVI) Lag phase followed by 2 o wave only Thrombin (PAR1/PAR4/ GPIba /GPV) Independent of PG or ADP pathways Irreversible aggregation only Arachadonic acid ( TPa / TPb ) 2 o wave only; assesses COX pathway Ristocetin ( GPIb /V/IX/ vWF ) Binds to vWF/GPIb/IX complex & results in agglutination Evaluates adhesion reaction 41

39. Lumiaggregometry: luminescence technology Measure platelet aggregation and secretion of ATP. This modification is sensitive to ATP release & marker of platelet activation. ATP is measured by its reaction with firefly luciferin to give chemiluminescence Resulting light emission is detected, amplified, & recorded by instrument. Performed by using whole blood or PRP. USES Quantitative analysis of Dense Granule Release Secretion Study and Aggregation performed simultaneously in same sample Diagnose Storage Pool Disease and Secretion Defect

40. Platelet Aggregation Curve- Verify Now Cartridge containing fibrinogen-coated microparticles using Arachidonic Acid as agonist. Whole blood Rapid, easy to perform More specific than optical aggregometry Qualitative – Can measure Aggregation time interval expressed as aspirin reaction units (ARUs) Studies GPIIb-IIIa antagonists function.

41. This two-step -uses a cell counter to measure total platelet count in a whole blood sample and then to re-determine the platelet count on a second sample that has been exposed to a known platelet agonist The Plateletworks tubes can be run on any hematology analyzer utilizing impedance methodology Platelet Aggregation Curve- plateletworks

42. lumi-aggregometer Secretion of radio-labelled serotonin used to study dense granules α-granule secretion most commonly assayed by flow cytometry- studying P-selectin expression on platelet surface after activation ELISA and Multiplex assay are also employed- to study PF4 and β- TG Assessment of secretion of platelet granule contents

43. Flow cytometry F luorochrome tagged monoclonal antibodies( Mabs ) against a platelet-specific antigen Most common platelet activation markers assessed P-selectin, glycoproteins are studied Can be used to study platelet anomaly of number and function of platelet receptors Assessment of suspected platelet disorder:

44. Transmission electron microscopy For the quantitation of dense granules Ultrastructural evaluation Thromboxane A2 formation Investigation of patients with suspected abnormalities of arachidonic acid metabolism. Released by activated platelets and rapidly metabolised Studied by immunoassay or mass spectrometry Assessment of suspected platelet disorder:

45. Molecular genetic testing genetic testing can aid in accurate diagnosis of the inherited disorders Next generation sequencing (NGS) panels, now available, offer an efficient approach to molecular diagnosis Can help in genetic counselling and prenatal diagnosis Assessment of suspected platelet disorder:

46. Other tests available Platelet adhesion Impact; Cone and Plate(let) Analyzer Global thrombosis test GTT Viscoelastic test- TEG/ROTEM/platelet Assessment of suspected platelet disorder:

47. Assessment of suspected platelet disorder