Basciano platelets fellows 2013


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Basciano platelets fellows 2013

  1. 1. Platelet Function and Dysfunction Paul Basciano MD January 17, 2013
  2. 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. 4. 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
  5. 5. 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
  6. 6. Thrombopoietin in Disease
  7. 7. 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
  8. 8. Platelet Functions• Primary hemostasis—the “platelet plug”• Secondary hemostasis—the coagulation cascade• Vasculature maintenance• Reservoir for soluble factors
  9. 9. Collagen (especially Type I and III)FibronectinThrombospondinLamininBound-vWF
  10. 10. BasementEndothelial Membrane Platelet AdhesionDamage Exposures Platelet Activation Shape Change Phospholipid Exposure Release of Soluble Factors Integrin activation Activation of OtherCoagulation Cascade Platelets(Secondary Hemostasis) Platelet Aggregation
  11. 11. Initiation Phase: vWFGP Ib/IX/V Complex• Major receptor modulating interaction with vWF• GP Iba 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
  12. 12. Initiation Phase: CollagenGP 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 timeGP Ia/IIaNo specific human disease has been identified with adefect in either
  13. 13. Platelet Activation: 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• Activation of GPIIb/IIIa allows it to bind to fibrinogen (and vWF), to cross link platelets and allow the thrombus to form
  14. 14. Platelet Activation: Outside-In and Inside-Out signaling
  15. 15. ADP: P2Y1 and P2Y12• Released from platelet dense granules and from RBCs• Interacts with the GPCRs • Leads to Ca elevation, TxA2 synthesis, protein phophorylation, 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
  16. 16. Thromboxane A2: Tpa receptor• The product of COX and TxA2 synthase enzymes• 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
  17. 17. Thrombin: PAR receptors• Likely the most potent platelet activator• Acts via PAR-1 and PAR-4 (protease activated receptors) 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
  18. 18. Epinephrine• Weak agonist—cannot induce shape change or activate PLC• Reduces levels of cAMP within the platelet
  20. 20. Evaluation of Platelet Function• Peripheral Blood Smear• Bleeding Time• PFA-100• Platelet aggregometry and secretion• Thromboelastography
  21. 21. Peripheral Blood Smear• Keys to look for: – Number of platelets – Size of platelets – Granulations within platelets – WBC inclusions
  22. 22. 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
  23. 23. PFA-100 • Rapid, automated, general assessment of platelet and overall Epi or ADP hemostatic functioncollagen collagen – 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
  24. 24. PFA-100• Influenced by many factors: – Hematocrit – Platelet count – Blood group – Timing and processing
  25. 25. 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
  26. 26. 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
  28. 28. Inherited Platelet Dysfunction
  29. 29. 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)• ITP vs BSS• Can diagnose via flow cytometry
  30. 30. Glanzmann Thombasthenia• More severe mucocutaneous bleeding than other platelet disorders• Qualitative and/or quantitative abnormality of GP IIb/IIIa• Rare, autosomal recessive• No aggregation response to any agonists except ristocetin• Normal secretion to thrombin, but reduced to weak agonists• Can diagnose via flow cytometery
  31. 31. 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, Chediak- Higashi, and Wiskott Aldrich, TAR, and Griscelli syndromes
  32. 32. Hermansky-Pudlak • Rare autosomal disorder • Largest concentration in Puerto Rico • Occulocutaneous albinism, congenital nystagmus, decreased visual acuity • Also granulomatous colitis and pulmonary fibrosis
  33. 33. Chediak-Higashi • Oculocutaneous albinism • Immune deficiency • Cytotoxic T and NK cell dysfunction • Neurologic symptoms • Cytoplasmic inclusions • LYST gene on chromosome 1ASH Image Bank
  34. 34. 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
  35. 35. 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
  36. 36. MYH-9 abnormalities (May-Hegglin) • MYH-9 mutations (myosin heavy chain) • Macrothrombocytopenia • Most commonly described as May- Hegglin; – Fechtner, Epstein, Sebastian Syndromes are all part of the same spectrum • Neutrophil inclusions (Dohle bodies) • Abnormalities in kidneys, ears, heart
  37. 37. 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=7- 10fL)
  38. 38. Scott Syndrome• Defect in platelet pro-coagulant activity• Failure to move phosphatidylserine to the outer membrane• Normal bleeding time and normal platelet aggregation
  39. 39. Acquired Platelet Defects
  40. 40. 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 (50- 100mg)• Major bleeding risk is approximately 1% per year• Other NSAIDs block COX-1 to a lesser degree
  41. 41. 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
  42. 42. 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
  43. 43. Other medications• Penicillins (especially high dose) – Onset 2-3d, lasts 3-10d after discontinuation• Cephalosporins• SSRIs
  44. 44. ADP Epi Collagen Ristocetan Bernard Soulier Glanzmann Storage Pool/Secretion
  45. 45. Aggregometry in Platelet Disorders Disorder Ristocetin ADP Epinephrine Arachadonic Collagen AcidBernard --- +++ +++ +++ +++SoulierGlanzmann +++ --- --- --- ---Storage + Primary only Primary only Primary onlyPool/ReleaseDefectASA Primary only ---Clopidogrel --- --- ---
  46. 46. 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)