Platelet Structure and Function Flashcards
1
Q
What is the division of megakaryocytes called ?
A
- 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
2
Q
What is the most immature form of
Megakaryocytes and what are characteristic features ?
A
- 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
3
Q
When megakaryocytes express this surface marker
they become less responsive to which cytokine ?
A
- GPIb/IX/V [vWF receptor]
- become less responsive to thrombopoietin
- undergo fewer endomitotic cycles
4
Q
What surface proteins are expressed on Megakaryocytes
when they become committed to the lineage ?
A
- 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
5
Q
What factors when expressed by Megakaryocytes
can lead to bone marrow fibrosis development ?
A
- Transforming growth factor beta 1 (TGFB1)
- Platelet-derived growth factor (PDGF)
6
Q
What is the specific mechanism of the megakaryocytic
pathology seen in Wiskott Aldridge Syndrome ?
A
- 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
7
Q
What are some of the receptors that are found on
normal platelets ?
A
- 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
8
Q
What is the role of the vW receptor GPIb/IX/V ?
A
- 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
9
Q
What are the two collagen receptors on platelets ?
A
- 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
10
Q
What are the two ADP platelet receptors ?
A
- 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
11
Q
What is the adrenergic receptor on platelets and
how does it function ?
A
- 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
12
Q
What are the thrombin receptors on platelets
and what is their function ?
A
- 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
13
Q
What are the thromboxane and prostaglandin receptors
on platelets and how do they function ?
A
- 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
14
Q
What is the platelet receptor for Fibrinogen ?
A
- 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
15
Q
What is the CD36 receptor and its ligands ?
A
- 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
16
Q
What is CD31 and how does it function on platelets ?
A
- 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
17
Q
What organelles are found within the platelet cytoplasm ?
A
- mitochondira
- lysosomes
- glycogen
- alpha granules
- dense granules
18
Q
What is stored in alpha granules ?
A
- 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
19
Q
What is stored in dense granules ?
A
- 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
20
Q
How do platelets promote hemostasis ?
A
- 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
21
Q
What are the platelet inhibiting factors produced by
the endothelium to prevent not necessary adhesion ?
A
- ecto-ADPase
- Nitrous oxide
- Prostaglandin I2
22
Q
What is the initial step in binding/platelet adhesion to the endothelium?
A
- 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
23
Q
What is the next step in platelet adhesion
after vWF transiently binds ?
A
- 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
24
Q
After platelet adhesion, what are essential steps
to further platelet activation ?
A
- production of thrombin during coagulation
- release of ADP from dense granules
- and platlet production of Thromboxane A2
25
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
26
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
27
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
28
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
29
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
30
Other than ADP, what other molecules can
initiate platelet aggregation ?
* epinephrine
* thrombin
* collagen
* platelet activating factor
31
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
32
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.
33
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
34
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
35
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
36
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
37
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
38
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
39
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
40
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
41
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
42
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
43
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
44
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
45
What technique is used to evaluate the
ultrastructural components of platelets ?
* EM
* used for evaluation of cytoplasmic granules mostly
46
What is Glanzmann thrombasthenia ?
* congenital deficiency or dysfunction of GP IIb/IIIa
* receptor for fibrinogen
* autosomal recessive disorder
* manifests as lifelong mucocutaneous bleeding
47
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
48
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
49
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
50
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
51
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
52
What situations can mimic platelet storage
pool disorders due to ongoing platelet activation ?
* cardiopulmonary bypass
* DIC
* TTP/HUS
53
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
54
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.
55
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
56
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
57
Where does thrombopoietin originate ?
* liver
