Platelet Structure and Function

Question 1

What is the division of megakaryocytes called ?

Answer 1

• 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

Question 2

What is the most immature form of

Megakaryocytes and what are characteristic features ?

Answer 2

• 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

Question 3

When megakaryocytes express this surface marker

they become less responsive to which cytokine ?

Answer 3

• GPIb/IX/V [vWF receptor]
• become less responsive to thrombopoietin
  
  • undergo fewer endomitotic cycles

Question 4

What surface proteins are expressed on Megakaryocytes

when they become committed to the lineage ?

Answer 4

• 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

Question 5

What factors when expressed by Megakaryocytes

can lead to bone marrow fibrosis development ?

Answer 5

• Transforming growth factor beta 1 (TGFB1)
• Platelet-derived growth factor (PDGF)

Question 6

What is the specific mechanism of the megakaryocytic

pathology seen in Wiskott Aldridge Syndrome ?

Answer 6

• 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

Question 7

What are some of the receptors that are found on

normal platelets ?

Answer 7

• 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

Question 8

What is the role of the vW receptor GPIb/IX/V ?

Answer 8

• 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

Question 9

What are the two collagen receptors on platelets ?

Answer 9

• 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

Question 10

What are the two ADP platelet receptors ?

Answer 10

• 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
• 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

Question 11

What is the adrenergic receptor on platelets and

how does it function ?

Answer 11

• 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

Question 12

What are the thrombin receptors on platelets

and what is their function ?

Answer 12

• 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

Question 13

What are the thromboxane and prostaglandin receptors

on platelets and how do they function ?

Answer 13

• 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

Question 14

What is the platelet receptor for Fibrinogen ?

Answer 14

• 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

Question 15

What is the CD36 receptor and its ligands ?

Answer 15

• 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

Question 16

What is CD31 and how does it function on platelets ?

Answer 16

• 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

Question 17

What organelles are found within the platelet cytoplasm ?

Answer 17

• mitochondira
• lysosomes
• glycogen
• alpha granules
• dense granules

Question 18

What is stored in alpha granules ?

Answer 18

• 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

Question 19

What is stored in dense granules ?

Answer 19

• 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

Question 20

How do platelets promote hemostasis ?

Answer 20

• 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

Question 21

What are the platelet inhibiting factors produced by

the endothelium to prevent not necessary adhesion ?

Answer 21

• ecto-ADPase
• Nitrous oxide
• Prostaglandin I2

Question 22

What is the initial step in binding/platelet adhesion to the endothelium?

Answer 22

• 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

Question 23

What is the next step in platelet adhesion

after vWF transiently binds ?

Answer 23

• 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

Question 24

After platelet adhesion, what are essential steps

to further platelet activation ?

Answer 24

• production of thrombin during coagulation
• release of ADP from dense granules
• and platlet production of Thromboxane A2

Question 25

What happens as a consequence of platelets binding to the subendothelium ?

Answer 25

* 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

Question 26

Which pathway in platelet activation is thought to be essential in amplification of platelet activation and aggregation?

Answer 26

* 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

Question 27

What is the process for platelet granule release ?

Answer 27

* 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

Question 28

What are platelet microparticles and why are they important ?

Answer 28

* 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

Question 29

What is essential for platelet aggregation ?

Answer 29

* 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

Question 30

Other than ADP, what other molecules can initiate platelet aggregation ?

Answer 30

* epinephrine * thrombin * collagen * platelet activating factor

Question 31

What molecular found in alpha granules and expressed on activated platelets binds monocytes ?

Answer 31

* P-selectin * serves to localize monocytes to areas of tissue damage * alos helps with tissue factor expression and inflammatory changes

Question 32

What is a frequent cause of pseudothrombocytopenia ?

Answer 32

* 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.

Question 33

What is true about the PFA-100 ?

Answer 33

* 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

Question 34

What is the gold standard for evaluating platelet disorders?

Answer 34

* 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

Question 35

What are the usual agonists used in platelet aggregometry ?

Answer 35

* ADP, Epinephrine and collagen * AA * can be used * helps exclude the effect of aspirin and or NSAIDS

Question 36

What does the mean platelet volume (MPV) tell you ?

Answer 36

* 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

Question 37

What are the specimens that can be used for platelet aggregation studies ?

Answer 37

* whole blood using impedence * platelet rich plasma using turbidimetric techniques * measured by spectrophotometry

Question 38

How is platelet aggregometry interpreted ?

Answer 38

* 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

Question 39

How is ristocetin used in evaluating platelet aggregation ?

Answer 39

* 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

Question 40

What is the basis of the of the PFA-100 test of platelet function ?

Answer 40

* 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

Question 41

In the PFA-100, what is the primary screening cartridge and what do the results mean?

Answer 41

* 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

Question 42

When would you expect to see an abnormal result on PFA-100 with collagen-ADP ?

Answer 42

* abnormal result with: * platelet disorders * vWD * normal results with * aspirin like drugs * high concentration of ADP overcomes them

Question 43

What conditions produce abnormal results with both collagen-epinephrine and collagen-ADP cartridges ?

Answer 43

* 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

Question 44

How is the reticulated platelet count performed ?

Answer 44

* 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

Question 45

What technique is used to evaluate the ultrastructural components of platelets ?

Answer 45

* EM * used for evaluation of cytoplasmic granules mostly

Question 46

What is Glanzmann thrombasthenia ?

Answer 46

* congenital deficiency or dysfunction of GP IIb/IIIa * receptor for fibrinogen * autosomal recessive disorder * manifests as lifelong mucocutaneous bleeding

Question 47

What is the pattern of abnormality seen with platelet testing for Glanzmann thrombasthenia ?

Answer 47

* PFA- abnormal * LTA * no aggregation with ADP, collagen, epinephrine or AA * ristocetin aggregation will be normal

Question 48

Which disorder can present similarly to Glanzmann thrombasthenia ?

Answer 48

* Afibrinogeniemia * rare deficiency of fibrinogen * can present with similar aggregation results, but the results should correct with the addition of fibrinogen to the specimen

Question 49

What is Bernard Soulier disease ?

Answer 49

* 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

Question 50

What are the platelet study findings in Bernard Soulier disease ?

Answer 50

* normal aggregation on LTA to * ADP, collagen, epinephrine and AA * abnormal aggregation * ristocetin Note: the abnormality can be confirmed with flow cytometry

Question 51

What is the pattern seen on LTA for dense granule disease ?

Answer 51

* decreased aggregation response to ADP, epinephrine and collagen * normal aggregation to AA and Ristocetin Note: acquired platelet disorders can be seen with myeloproliferative disorders

Question 52

What situations can mimic platelet storage pool disorders due to ongoing platelet activation ?

Answer 52

* cardiopulmonary bypass * DIC * TTP/HUS

Question 53

What is Scott Syndrome ?

Answer 53

* 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

Question 54

What finding is usually seen on platelet aggregation for a myeloproliferative disorder ?

Answer 54

* epinephrine aggregation is reduced or absent altogether * possibly due to down regulation of alpha 2 adrenergic receptors see pg. 142 for additional findings.

Question 55

What is Wiskott Aldrich Syndrome ?

Answer 55

* 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

Question 56

When would you expect to see post transfusion pupura ?

Answer 56

* 5-12 days after transfusion * it is an immune mediated destruction of platelets (both native and transfused) * patients often have abnormal platelet antigens

Question 57

Where does thrombopoietin originate ?

Answer 57

* liver