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
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
14.
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
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
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
28.
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
30.
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
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
