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Platelet function and dysfunction
- 1. Platelet Function and Dysfunction Paul Basciano MD October 24, 2013
- 2. Outline • • • • • • • • Thrombopoiesis Platelet Structure Platelet Function Platelet Activation and Aggregation Biology Assessment of Platelet Function Inherited Disorders of Platelet Function Acquired Disorders of Platelet Function Treatment of Platelet Dysfunction
- 4. PLATELET PRODUCTION, STRUCTURE, AND FUNCTION
- 5. Thrombopoiesis • Humans produce 1x 10^11 platelets per day • Can increase 10-20 fold • Each megakaryocyte can produce 1000 platelets • The lifespan of a platelet is approximately 10 days • Platelet production from MK progenitors takes 4-7days • Most of this time is for the maturation of the megakaryocyte • Endomitosis • Cytoplasmic maturation
- 6. Thrombopoietin • TPO is one of the key factors for platelet production • Produced constitutively by the liver (and kidney) • Circulating levels are determined by the megakaryocyte and platelet biomass • Low platelets more circulating TPO increased platelet production • ITP is an exception to this • Normal platelets-> low TPO->low platelet production
- 8. Platelet Structure • Alpha granules (many): PF4, vWF, fibronectin, thromboglobulin, FV, FXI, PS, TGFB, PDGF • Dense granules (3-8/cell): ADP, ATP, serotonin, pyrophosphate, calcium • Open canalicular system • Dense tubular system
- 9. Platelet Functions • Primary hemostasis—the “platelet plug” • Secondary hemostasis—the coagulation cascade • Vasculature maintenance • Reservoir for soluble factors
- 10. Collagen (especially Type I and III) Fibronectin Thrombospondin Laminin Bound-vWF
- 11. Endothelial Damage Basement Membrane Exposures Platelet Adhesion Platelet Activation Phospholipid Exposure Coagulation Cascade (Secondary Hemostasis) Release of Soluble Factors Shape Change Integrin activation Activation of Other Platelets Platelet Aggregation
- 12. Adhesion Phase: vWF GP Ib/IX/V Complex • Major receptor modulating interaction with vWF • GP Ibα is essential for arterial thrombus formation (Fab development) • No inherent signaling ability (no coupling to G proteins, no TK activity) • Found in lipid rafts in association with other GPs • Defects define Bernard Soulier Syndrome • Macrothrombocytopenia • No response to ristocetin on aggregometry
- 13. AdhesionPhase: Collagen GP VI • Low-affinity but high impact for binding to collagen • Signals through Src kinases, as well as association with FcRg dimer which signals through ITAMs • Ab in development blocks thrombosis but does not prolong bleeding time GP Ia/IIa No specific human disease has been identified with a defect in either
- 15. Platelet Activation: Granule Secretion and Integrin Activation • Initial binding leads to the release of agonists from the first-responding platelets which in turn leads to recruitment and activation of other near-by platelets • ADP • TxA2 • Epinephrine • Thrombin • All lead to the activation of GP IIb/IIIa (αIIb/β3 integrin) via inside-out signaling • Also lead to more granule secretion->feed forward mechanism • Activation of GPIIb/IIIa allows it to bind to fibrinogen (and vWF), to cross link platelets and allow the thrombus to form
- 16. Platelet Activation: Outside-In and Inside-Out signaling
- 17. Granule Secretion/GPCRs: ADP and P2Y1 and P2Y12 Receptors • Released from platelet dense granules and from RBCs • Interacts with the GPCRs • Leads to Ca elevation, TxA2 synthesis, protein phosphorylation, and shape change, granule secretion, activation of GPIIb/IIIa and aggregation • P2Y12 is more important • Few patients identified with dysfunction and bleeding diathesis • Major target of thienopyridine drugs • P2Y1 is necessary for full signaling and is a potential drug target
- 18. GPCRs: Thromboxane A2 and Tpa receptor • The product of COX and TxA2 synthase enzymes using arachadonic acid (AA) as a precursor • Freely diffuses across plasma membrane • High activity during aggregation when platelets are closely apposed • Causes shape change, phospholipid hydrolysis, Ca mobilization, secretion, and aggregation via the TPa receptor • Aspirin inhibits COX-1 irreversibly
- 19. GPCRs: Epinephrine and α2A Receptor • Weak agonist—cannot induce shape change or activate PLC • Reduces levels of cAMP within the platelet
- 20. GPCRs: Thrombin and PAR receptors • Likely the most potent platelet activator • Acts via PAR-1 and PAR-4 (protease activated receptors) • The ligand is tethered to the receptor but hidden until it is cleaved Nature 407, 258-264(14 September 2000) • Also Ib/IX/V complex • Impaired thrombin responsiveness in BSS—Ib/IX possibly localizes thrombin to the PARs • PAR-1 is likely the primary mediator, but PAR4 is necessary for full activation • No clinical examples of PAR deficiencies have been described
- 21. Adhesion Integrin Activation and Platelet Aggregation Secretion GPCRs activation Ca++, PKC Fibrinogen GP IIb/IIIa (αIIBβIII)
- 23. Evaluation of Platelet Function • • • • • Peripheral Blood Smear Bleeding Time PFA-100 Platelet aggregometry and secretion Thromboelastography
- 24. Peripheral Blood Smear • Keys to look for: – Number of platelets – Size of platelets – Granulations within platelets – WBC inclusions
- 25. Bleeding Time • Controversial, and mainly abandoned • Invasive, difficult to standardize • Requires dedicated technician to perform test over a relatively long period of time on one patient
- 26. PFA-100 Epi or ADP collagen collagen • Rapid, automated, general assessment of platelet and overall hemostatic function – Replacing bleeding time • Citrated whole blood passed through aperture and time to closure with platelet plug is measured – Collagen with either epinephrine or ADP • High negative predictive value; low specificity
- 27. PFA-100 • Influenced by many factors: – Hematocrit – Platelet count – Blood group – Timing and processing
- 29. Platelet Aggregometry • Usually performed on platelet-rich plasma • Measures transmission of light through the solution full of platelets – As platelets aggregate, light transmission increases • Uses a panel of agonists to determine specific defect • Not well-standardized
- 31. Aggregometry • Weak agonists: – ADP and epinephrine: biphasic platelet aggregation • Strong agonists – Collagen, TRAP (thrombin), arachadonic acid TXA2 • (Ristocetin: Agglutination) • Can also measure secretion of ATP and ADP
- 34. Bernard-Soulier Syndrome • • • • • • Reduced/lack of GP Ib/IX Rare, autosomal recessive Moderate thrombocytopenia Large platelets Prolonged bleeding time Lack of response to ristocetin on platelet aggregometry (agglutination) – All other agonists are normal • ITP vs BSS • Can diagnose via flow cytometry
- 35. Glanzmann Thombasthenia • Qualitative and/or quantitative abnormality of GP IIb/IIIa • More severe mucocutaneous bleeding than other platelet disorders • Rare, autosomal recessive • No aggregation response to any agonists except ristocetin • Normal secretion to thrombin, but reduced to weak agonists • Can diagnose via flow cytometry
- 36. Storage Pool Disorders: Dense Granules • 3-8 dense granules per platelet; will not see problems on light microscopy • Contain ADP, ATP, serotonin, Ca, pyrophosphate • Moderate bleeding diathesis • Second wave of aggregation to ADP and Epi is lost • Reduced collagen response • Seen in association with Hermansky-Pudlak, ChediakHigashi, and Wiskott Aldrich, TAR, and Griscelli syndromes
- 37. Hermansky-Pudlak • Rare autosomal disorder • Largest concentration in Puerto Rico • Occulocutaneous albinism, congenital nystagmus, decreased visual acuity • Also granulomatous colitis and pulmonary fibrosis
- 38. Chediak-Higashi • Occulocutaneous albinism • Immune deficiency • Cytotoxic T and NK cell dysfunction • Neurologic symptoms • Cytoplasmic inclusions • LYST gene on chromosome 1 ASH Image Bank
- 39. Storage Pools Disorders: Alpha Granules • Gray platelet syndrome – – – – – Mild thrombocytopenia Macrothrombocytopenia Pale platelets on light microscopy Variable inheritance Variable aggregation patterns • • Response to ADP and epi normal Collage, thrombin, and ADP impaired – Splenomegaly, fibrosis • Quebec platelet disorder – Autosomal dominant – Delayed bleeding – Increased proteolysis of alpha granule proteins (elevated platelet urokinase type plasminogen activator) – Thrombocytopenia – Reduced aggregation with epinephrine
- 40. Signal Transduction and Activation Abnormalities • Abnormalities for specific agonist receptors or abnormalities of the signal transduction cascade – G proteins, phospholipase C, calcium mobilization, pleckstrin phosphorylation, phospholipase C, PKD – Thromboxane synthesis
- 41. MYH-9 abnormalities (May-Hegglin) • MYH-9 mutations (myosin heavy chain) • Macrothrombocytopenia • Most commonly described as MayHegglin; – Fechtner, Epstein, Sebastian Syndromes are all part of the same spectrum • Neutrophil inclusions (Dohle bodies) • Abnormalities in kidneys, ears, heart
- 42. Wiskott-Aldrich • Characterized by moderate to severe thrombocytopenia, with small platelet volumes • Wide clinical variability: – susceptibility to infections associated with adaptive and innate immune deficiency – eczema – isolated thrombocytopenia – X-linked neutropenia (XLN) • Mutation is WASP—actin remodeling protein • Platelet volumes are usually 3.5-5fL (normal=710fL)
- 43. Scott Syndrome • Defect in platelet pro-coagulant activity • Failure to move phosphatidylserine to the outer membrane • Normal bleeding time and normal platelet aggregation
- 44. Bleeding disorder Low Platelet Count Small Platelets Large Platelets Normal Platelet Count, Normal Size Very Abnormal Aggregometry Normal Aggregometry Glanzmann Wiskott-Aldrich No Color Issues Bernard Soulier Scott Quebec Color Issues Pale Platelets Pale People Gray Platelet Normal Neutrophils Kind-of adapted from Williams Hematology, 7th Ed. Neutrophil Inclusions Hermansky-Pudlack Chediak-Higashi
- 46. Aspirin • Irreversibly inhibits COX-1 and leads to inhibition of thromboxane A2 production • The most common antithrombotic in use • Inhibition with low-dose is complete (50100mg) – Essential thrombocythemia • Major bleeding risk is approximately 1% per year • Other NSAIDs block COX-1 to a lesser degree
- 47. Thienopyridines • Clopidogrel, ticlopidine, and prasagurel • Work (mainly? in part?) by blocking the ADP receptor • Multiple methods to asses effect: aggregometry, PFA-100 (ADP), Verify-Now – All have significant variability in detection of ‘resistance’ and questionable clinical utility
- 48. GP IIb/IIIa inhibitors • Abciximab, eptifibatide, tirofiban • Complete thrombasthenic state (Glanzman’s thrombasthenia) • High incidence of bleeding complications • May also cause acute thrombocytopenia – – – – Rapid onset, 30min to one day Variable recovery May be severe May also induce platelet clumping/ pseudothrombocytopenia
- 49. Other medications • Penicillins (especially high dose) – Onset 2-3d, lasts 3-10d after discontinuation • Cephalosporins • SSRIs
- 51. Aggregometry in Platelet Disorders Disorder Ristocetin ADP Epinephrine Arachadonic Acid Collagen Bernard Soulier --- +++ +++ +++ +++ Glanzmann +++ --- --- --- --- Storage Pool/Release Defect + Primary only Primary only ASA Primary only Clopidogrel --- Primary only ----- ---
- 52. Treatment • DDAVP for mild bleeding diathesis, uremic • Platelet transfusions – Allo-immunization against absent GP receptors may occur • • • • Estrogens Amicar Novo7 (Esp. Glanzmann) TPO agents (Bernard Soulier, MYH-9, ?others)