Platelet Structure and Function Flashcards
What is the division of megakaryocytes called ?
- Endomitosis
- it is an increase in the cellular DNA content without proliferation
- polyploid DNA content may reach 32 to 64 times that of a normal diploid cell
What is the most immature form of
Megakaryocytes and what are characteristic features ?
- Megakaryoblasts
- present in low numbers
- small cells with high N:C ratios often with cytoplasmic blebs
- Express:
- CD34, CD41, thrombopoietin receptor (c-MPL) and the alpha chemokine receptor CXCR4
When megakaryocytes express this surface marker
they become less responsive to which cytokine ?
- GPIb/IX/V [vWF receptor]
- become less responsive to thrombopoietin
- undergo fewer endomitotic cycles
What surface proteins are expressed on Megakaryocytes
when they become committed to the lineage ?
- CD61, CD41
- GPIIb/IIIa
- c-Mpl (thrombopoietin receptor)
- GPIb/IX/V
- this comes at an even later stage and causes the cells to be less responsive to TPO
Note: the alpha and dense granules form when the megakaryocytic cytoplasm becomes filled with platelet specific organelles and proteins
What factors when expressed by Megakaryocytes
can lead to bone marrow fibrosis development ?
- Transforming growth factor beta 1 (TGFB1)
- Platelet-derived growth factor (PDGF)
What is the specific mechanism of the megakaryocytic
pathology seen in Wiskott Aldridge Syndrome ?
- megakaryocytes lose their ability to extend protoplatelets into the blood stream
- platelets are abnormally released into the bone marrow
- leads to thrombocytopenia and bleeding diathesis
What are some of the receptors that are found on
normal platelets ?
- glycoprotein surface receptors:
- GPIIb/IIIa -Fibrinogen receptor
- GPIa/IIa and GPVI- Collagen receptor
- GPIV- Thrombospondin
- GPIb/IX/V- vWF receptor
- GPIc/IIa- Fibronectin
- ADP receptors: P2Y1, P2Y12, P2X1
- Thrombin receptors: PAR-1 and PAR-4
- Thromboxane and aderenergic receptors
What is the role of the vW receptor GPIb/IX/V ?
- secreted by endothelial cells into the extracellular matrix
- facilitates platelet adhesion to the endothelial surface ***
- binding of vWF to GPI/IX/V initiated by the conformational change brought about by shear stress or simulated by Ristocetin
- exposes GPIb binding epitope
- IMP: also serves as one of the thrombin receptors on the platelet surface
What are the two collagen receptors on platelets ?
- GPVI and GPIa/IIa
- collagen helps platelets bind during initial platelet plug formation, but also collagen is a potent platelet activator
- Polymorphisms if GPIa/IIa exist
- lead to altered binding to collagen
What are the two ADP platelet receptors ?
- P2Y1
- classic 7 transmembrane G-coupled protein, only 150 copies per platelet
- distributed in many other tissues
- involved in the initial platelet shape change with mobilization of intracytoplasmic Ca++
- but not able to support the full platelet aggregation response
- classic 7 transmembrane G-coupled protein, only 150 copies per platelet
- P2Y12
- activates PI3K pathway, completes platelet aggregation
- potentiates platlet secretion and clot stabilization
- leads to inhibition of adenylate cyclase
- IMP: only found on platelets, no other tissues
- P2X1 receptor
- ATP-gated cation channel that raises cytoplasmic calcium levels
- can also bind to collagen under shear stress
What is the adrenergic receptor on platelets and
how does it function ?
- platelets can take up and store epinephrine in their dense granules
- alpha2A-adrenergic receptor on platelets
- expressed in low numbers
- epinephrine is a weak activator of platelets
- leads to platelet secretion and aggregation
What are the thrombin receptors on platelets
and what is their function ?
- PAR-1 and PAR-4
- protease activated receptors
- Because thrombin enzymatically cleaves these receptors they are not easily inactivated
- down regulation occurs by internalization of the receptors
What are the thromboxane and prostaglandin receptors
on platelets and how do they function ?
- Thromboxane A2
- metabolic product of cyclooxygenase
- released from platelets and interacts with TxA2 receptors on adjacent platelets
- stimulates platelet activation via calcium mobilization
- Prostaglandin D2 receptors
- lead to decreased platelet activation
- release of cAMP
What is the platelet receptor for Fibrinogen ?
- GPIIb/IIIa
- most abundant surface protein on platelets
- its expression is limited to megakaryocytes and platelets
- it is an integrin that requires Ca++ and Mg++ for use
- in resting platelets it is in an inactivated conformation and does not interact with fibrinogen
- becomes activated when vWF binds GPIb/IX/V or some other platelet agonist
What is the CD36 receptor and its ligands ?
- CD36 is also known as GPIV
- broad ligand specificity and one of the most abundant surface proteins on platelets
- receptor for Thrombospondin 1 (TSP-1)
- released from alpha granules
- reinforces the molecular bridge formed between platelets and fibrinogen
- Also a scavenger receptor for oxidized lipids and apoptotic cells on macrophages
What is CD31 and how does it function on platelets ?
- also known as Platelet endothelial cell adhesion molecule-1 (PECAM-1)
- abundantly expressed protein on the platelets surface
- note: platelets deficient in PECAM-1 are capable of activation and aggregation
- CD31 reduces activation of platelets when it is stimulated by Thrombin and ADP
- CD31 enhances platelet spreading on fibrinogen and clot retraction
What organelles are found within the platelet cytoplasm ?
- mitochondira
- lysosomes
- glycogen
- alpha granules
- dense granules
What is stored in alpha granules ?
- granules are abundant in platelets, carry proteins
- contain 2 morphologically distinct compartments
- Contain MANY things including:
- Platelet factor 4 (PF4)
- Fibronectin
- vWF
- Coagulation factors: Fibrinogen, V, VII, XI, XIII, protein S, plasminogen
- Cellulalar mitogens: PDGF, TGFB, VEGF
- Protease inhibitors: alpha 2 antitrypsin, PAI-1, TFPI, TAFI
- Immunoglobulins, Albumin, amyloid precursor protein
What is stored in dense granules ?
- small granules, few in number (<10 per platelet)
- fuse with plasma membrane for release
- Contents include:
- ADP, ATP
- Serotonin, Histamine
- Calcium, Magnesium
- Polyphosphates, glutamate, sphingosine, I-phosphate, epinephrine
How do platelets promote hemostasis ?
- adhere to sites of vascular injury
- undergo an activation process leading to the release of compounds from their granules
- aggregate to form a hemostatic platelet plug
- provide a procoagulant surface for activated coagulation protein complexes on their phospholipid membrane
What are the platelet inhibiting factors produced by
the endothelium to prevent not necessary adhesion ?
- ecto-ADPase
- Nitrous oxide
- Prostaglandin I2
What is the initial step in binding/platelet adhesion to the endothelium?
- vWF becomes exposed from the subendothelial matrix
- either by injury, shear stress, collagen exposure or ristocetin
- binds platelets in the shear stream
- allos platelets to roll and transiently bind
- transiently is bound to platelet GPIb/IX/V receptor
- binding activates intracytoplasmic calcium release
- leads to activation of GPIIb/IIIA receptor
What is the next step in platelet adhesion
after vWF transiently binds ?
- extracellular matrix collagen binds to the platelet receptors GPVI and GPIa/IIa
- a stronger bond to collagen and weaker bond to vWF allow the platelet to roll along the damaged endothelium
Note: with longer adhesion times the actin cytoskeleton restructures itself to extend filopodia where eventually the adherent platelets forma a pancake like layer
After platelet adhesion, what are essential steps
to further platelet activation ?
- production of thrombin during coagulation
- release of ADP from dense granules
- and platlet production of Thromboxane A2
What happens as a consequence of platelets binding to
the subendothelium ?
- the platelets then get stimulated by thrombin and ADP
- ADP binds to P2Y1 and P2Y12
- allows calcium release
- thrombin binds to receptors PAR-1 and PAR-4 which leads to activation of receptors and ultimately increases in cytoplasmic calcium
- this results in re-organization of GPIIb/IIIa
- allows binding of Fibrinogen to the receptor and causes platelet aggregation
Which pathway in platelet activation is thought to be
essential in amplification of platelet activation and aggregation?
- Cyclooxygenase pathway –> production of Thromboxane A2 from Arachadonic acid
- TXA2 binds to it’s receptors on adjacent platelets and induces activation and aggregation
- this process works in parallel to the others
What is the process for platelet granule release ?
- increase in cytoplasmic calcium leads to platelet granule release
- it is a processes of exocytosis
- alpha granules
- degranulate directly into the channels
- dense granules
- merge with the plasma membrane
What are platelet microparticles and why are they important ?
- they are tiny, membrane bound particles shed by platelets
- they are rich in surface glycoproteins, GPIIb/IIIa receptors, and phospholipids
- further amplify thrombotic process
- Fibrinogen binds to GPIIb/IIIa
- Coagulation cascade gets activated with phospholipids
- represent 70-90% of all microparticles
What is essential for platelet aggregation ?
- Fibrinogen must bind to the GPIIb/IIIa receptor on platelets/between platelets and forms a bridge
- conformation change of the receptor is induced by increase in cytoplasmic calcium and release/binding of ADP to receptors
Other than ADP, what other molecules can
initiate platelet aggregation ?
- epinephrine
- thrombin
- collagen
- platelet activating factor
What molecular found in alpha granules and expressed
on activated platelets binds monocytes ?
- P-selectin
- serves to localize monocytes to areas of tissue damage
- alos helps with tissue factor expression and inflammatory changes
What is a frequent cause of
pseudothrombocytopenia ?
- it is a lab phenomenon, caused by EDTA
- often due to cold reactive platelet agglutinins or platelets binding neutrophils (platelet satellitism)
- agglutinins are often seen in patients with high immunoglobulins or infections
- IMP: usually only bind platelets when calcium is chelated (EDTA)
- Note: Abciximab (anti-GP IIb/IIIa) pseudothrombocytopenia has been reported
- better to collect in citrate or heparin tubes
- the mean platelet volume would be increased size of the platelets because the platelets are stuck together as they go through the automated cell counter
Note: peripheral platelet destruction or a primary marrow process can also lead to large platelet production.
What is true about the PFA-100 ?
- it has a high negative predictive value
- normal results support normally functioning platelets
Exceptions:
- platelet secretion defects
- platelet storage pool disorders
- mild type I vWD
What is the gold standard for
evaluating platelet disorders?
- light transmission platelet aggregometry
- specimen must be run within 4 hours of collection
- uses platelet rich plasma
- very labor intensive and time consuming
- Intereference from:
- hyperlipidemia
- hemolysis
- thombocytopenia
- anticoagulation
What are the usual agonists used
in platelet aggregometry ?
- ADP, Epinephrine and collagen
- AA
- can be used
- helps exclude the effect of aspirin and or NSAIDS
What does the mean platelet volume (MPV)
tell you ?
- it is an indication of the platelet size
- can be an indication of platelet turnover
- newly released platelets from the bone marrow are larger and tend to decrease in size with age in circutlation
Note: platelets live for 7-10 days before being cleared
average platelet loss per day is 7000
What are the specimens that can be used for
platelet aggregation studies ?
- whole blood using impedence
- platelet rich plasma using turbidimetric techniques
- measured by spectrophotometry
How is platelet aggregometry interpreted ?
- optimal aggregomtry:
- biphasic pattern for ADP and Epinephrine
- initial increase is due to primary aggregation via GP IIb/IIIa
- second wave is due to platelet degranulation
- AA, Thrombin, and collagen
- usually single wave agonist
- biphasic pattern for ADP and Epinephrine
How is ristocetin used in evaluating
platelet aggregation ?
- ristocetin is an antibiotic
- facilitates binding of vWF to the glycoprotein IB/IX/V
- use various concentrations of ristocetin
- for a normal result the patient must have functional vWF and normal GP IB/IX/V
- allows detection of Bernard Soulier syndrome as well as vWD
What is the basis of the of the PFA-100
test of platelet function ?
- device that measures platelet-related primary hemostasis within a citrated whole blood specimen
- membrane with a central aperature
- uses collagen and epinephrine as well as collagen and ADP
- the instrument measures closure time
In the PFA-100, what is the primary
screening cartridge and what do the results mean?
- collagen-epinephrine cartridge
- detects platelet dysfunction induced by intrinsic platelet defects, vWD, or platelet inhibiting agents
- abnormal results with:
- intrinsic platelet defects
- vWD
- platelet inhibiting agents
When would you expect to see an abnormal
result on PFA-100 with collagen-ADP ?
- abnormal result with:
- platelet disorders
- vWD
- normal results with
- aspirin like drugs
- high concentration of ADP overcomes them
What conditions produce abnormal results
with both collagen-epinephrine and collagen-ADP
cartridges ?
- vWD
- intrinsic platelet dysfunction
- non-aspirin drugs
Note: sample must be run within 5 hours,
- affected by:
- low platelet counts
- low hematocrit
- BUT not affected by heparin
How is the reticulated platelet count performed ?
- flow cytometry essentially
- measures platelets based on increased RNA content
- thiazole orange dye binds RNA and DNA
- helps evaluate whether thrombocytopenia is due to decreased platelet destruction or increased platelet production
- platelets newly released from bone marrow usually have more RNA content
What technique is used to evaluate the
ultrastructural components of platelets ?
- EM
- used for evaluation of cytoplasmic granules mostly
What is Glanzmann thrombasthenia ?
- congenital deficiency or dysfunction of GP IIb/IIIa
- receptor for fibrinogen
- autosomal recessive disorder
- manifests as lifelong mucocutaneous bleeding
What is the pattern of abnormality seen with
platelet testing for Glanzmann thrombasthenia ?
- PFA- abnormal
- LTA
- no aggregation with ADP, collagen, epinephrine or AA
- ristocetin aggregation will be normal
Which disorder can present similarly
to Glanzmann thrombasthenia ?
- Afibrinogeniemia
- rare deficiency of fibrinogen
- can present with similar aggregation results, but the results should correct with the addition of fibrinogen to the specimen
What is Bernard Soulier disease ?
- congenital deficiency of the platelet glycoprotein GP Ib/IX/V
- receptor for vWF mediated aggregation in primary hemostasis
- incomplete autosomal recessive
- presents with severe bleeding
- moderate to severe thrombocytopenia and large platelets
What are the platelet study findings in
Bernard Soulier disease ?
- normal aggregation on LTA to
- ADP, collagen, epinephrine and AA
- abnormal aggregation
- ristocetin
Note: the abnormality can be confirmed with flow cytometry
What is the pattern seen on LTA
for dense granule disease ?
- decreased aggregation response to ADP, epinephrine and collagen
- normal aggregation to AA and Ristocetin
Note: acquired platelet disorders can be seen with myeloproliferative disorders
What situations can mimic platelet storage
pool disorders due to ongoing platelet activation ?
- cardiopulmonary bypass
- DIC
- TTP/HUS
What is Scott Syndrome ?
- defect in a phospholipid flipase
- patients will have normal platelet aggregation studies
- abnormal platelet procoagulant activity
- phosphilipid is needed for clotting cascade to effectively work
What finding is usually seen on platelet
aggregation for a myeloproliferative disorder ?
- epinephrine aggregation is reduced or absent altogether
- possibly due to down regulation of alpha 2 adrenergic receptors
see pg. 142 for additional findings.
What is Wiskott Aldrich Syndrome ?
- condition with thrombocytopenia and small platelets
- X-linked recessive
- characterized by recurrent infections, eczema, and thrombocytopenia
- also a T lymphocyte function
- MPV is often low and lymphocytes are often low in CD43
- platelet dysfunction is severe
- platelets cannot aggregate and looks like a storage pool disorder
When would you expect to see post
transfusion pupura ?
- 5-12 days after transfusion
- it is an immune mediated destruction of platelets (both native and transfused)
- patients often have abnormal platelet antigens
Where does thrombopoietin originate ?
- liver
