MEGAKARYOPOIESIS, PLATELET STRUCTURE AND FUNCTION

Question 1

What is Megakaryopoiesis/ Thrombopoiesis

Answer 1

Megakaryopoiesis is the process of platelet (thrombocyte) formation.

Platelets are formed by ligations of the cytoplasm of megakaryocytes (precursor cells for platelets).

◼ A single megakaryocyte can give rise to thousands of thrombocytes (platelets).
◼ The term “thrombocytopoiesis” is sometimes used to emphasize the cellular nature.

Question 2

What is the unique maturation process of megakaryocytes?

Answer 2

Megakaryocytes, which are the precursor cells for platelets are derived from the hematopoietic stem cell through successive lineage commitment steps, and they undergo a unique maturation process that includes:

▪ polyploidization, (having more than usual number of complete sets of chromosomes in a single cell)
▪ development of an extensive internal demarcation membrane system and finally
▪ formation of proplatelet processes.

Question 3

What is Thrombopoietin?
What are its functions?

Answer 3

Thrombopoietin ( which is the platelet growth factor) is majorly produced in the liver and it stimulates megakaryopoiesis, the process of megakaryocyte maturation and differentiation.

Thrombopoietin, upon release, binds to its receptor, c-mpl, found on megakaryocyte progenitor cells.

Following binding, intracellular signaling leads to:
i. megakaryocyte growth,
ii. maturation,
iii. membrane stability,
iv. platelet granule formation
v. the demarcation of the cytoplasm into regions destined to fragment into mature platelets.

Question 4

Thrombopoietin doesn’t act on the last step of megakaryopoiesis (proplatelet processes) T/F

Answer 4

True

This last step of proplatelet process and platelet formation, in vitro , has been shown to be independent of thrombopoietin

Question 5

Discuss the formation of proplatelet processes

Answer 5

These “proplatelet processes” further fragment into platelets.

◼ This last step of proplatelet process and platelet formation, in vitro , has been shown to be independent of thrombopoietin

◼ Platelets are shed from these processes into vascular sinusoids within the bone marrow.

Question 6

How is Megakaryopoiesis regulated? What is its clinal significance?

Answer 6

◼ Megakaryocyte differentiation is regulated both positively and negatively by transcription factors and cytokine signaling.

◼ Clinically, acquired and inherited mutations affecting megakaryocytic transcription factors and thrombopoietin signaling have been identified in disorders of thrombocytopenia and thrombocytosis.

Question 7

What is the most important hematopoietic cytokine for platelet production?

Answer 7

Thrombopoietin

Question 8

Process of thrombopoiesis

Question 9

Discuss Megakaryocyte maturation?
How long do they take to mature?

Answer 9

Megakaryocyte mature in about 10 days with:

1. Development of granules [alpha granules and Dense bodies]
2. Development of membrane glycoproteins which are essential for its function.
3. Progressive folding and invagination of its membrane.

Question 10

How are platelets formed from the megakaryocytes?

Answer 10

Platelet fragments are released as proplatelets from the megakaryocyte

Further fragmention occurs in the lungs
Spleen : 30% pooled

Question 11

What is the platelet lifespan?
Where are they destroyed?

Answer 11

◼ Lifespan: 8-12 days
◼ Destroyed: by Spleen and Liver macrophages

Question 12

Describe the structure of platelets
Discuss the functions of glycoproteins

Answer 12

Platelets are extremely small and discoid, 3.0 × 0.5 μm in diameter.

◼ The glycoproteins of the surface coat are particularly important in the platelet reactions of adhesion and aggregation.

◼ Adhesion to collagen is facilitated by glycoprotein Ia

◼ Glycoproteins Ib (defective in Bernard–Soulier syndrome) and IIb/IIIa (also called αIIb and β3, defective in Glanzmann’s thrombasthenia) are important in the attachment of platelets to von Willebrand factor (VWF) and hence to vascular subendothelium.

◼ The binding site for IIb/IIIa is also the receptor for fibrinogen which, like VWF, is important in platelet–platelet aggregation.

The plasma membrane invaginates into the platelet interior to form an open membrane (canalicular) system which provides a large reactive surface to which the plasma coagulation proteins may be selectively absorbed.

◼ The membrane phospholipids (previously known as platelet factor 3) are of particular importance in the conversion of coagulation factor X to Xa and prothrombin (factor II) to thrombin (factor IIa).

Question 13

What are the types of storage granules of platelets?
What is used to activate them during vessel damage?

Answer 13

The platelet contains three types of storage granules:
▪ dense,
▪ α and
▪ lysosomes

Platelets are also rich in signalling and cytoskeletal proteins, which support the rapid switch from quiescence to activation that follows vessel damage.

During the release reaction, the contents of the granules are discharged into the open canalicular system.

Question 14

What is the constituent of α granules ?

Answer 14

The more frequent specific α granules contain
▪ clotting factors, (factor 5, fibronectin & fibrinogen)
* Heparin antagonist PF4
▪ VWF,
▪ platelet‐derived growth factor (PDGF) and other proteins.

Question 15

What is the constituent of dense granules?

Answer 15

Dense granules are less common and contain
▪ adenosine diphosphate (ADP),
▪ adenosine triphosphate (ATP),
▪ serotonin and
▪ Ca2+

Question 16

What is the constituent of Lysosomes?

Answer 16

Lysosomes contain hydrolytic enzymes.

Question 17

Discuss platelet function (including the 3 major platelet functions)

Answer 17

The main function of platelets is the formation of mechanical plugs during the haemostatic response to vascular injury. In the absence of platelets, spontaneous leakage of blood through small vessels may occur.

There are three major platelet functions:
i) adhesion, ii) aggregation iii) release reactions and amplification.

The immobilization of platelets at the sites of vascular injury requires specific platelet–vessel wall (adhesion to exposed collagen and platelet–platelet (aggregation) interactions, both partly mediated through VWF

Question 18

Discuss the VWF in platelet adhesion
Where is it stored?
When is it released?

Answer 18

• Von Willebrand factor (VWF) VWF is involved in shear‐dependent platelet adhesion to the vessel wall and to other platelets (aggregation)
• It also carries factor VIII
• VWF is synthesized both in endothelial cells and megakaryocytes, and
  stored in Weibel–Palade bodies and platelet α granules, respectively.
• Plasma VWF is almost entirely derived from endothelial cells, with two distinct pathways of secretion. The majority is continuously secreted and a minority is stored in Weibel– Palade bodies.
• The stored VWF can raise the plasma levels when released under the influence of several stimulants, such as stress, exercise, adrenaline and infusion of desmopressin (1‐diamino‐8‐D‐arginine vasopressin; DDAVP).
• The VWF released from Weibel–Palade bodies is in the form of large and ultra‐large multimers, the most adhesive and reactive form of VWF.
• They are in turn cleaved in plasma to smaller multimers and monomeric VWF by the specific plasma metalloprotease, ADAMTS 13

Question 19

How are Platelets activated, what are their agonists?

Answer 19

Platelet activation :
following adhesion, platelets are activated by a number of agonists
such as ADP, and collagen present at the site of injury, by binding to specific receptors on the platelet surface

Question 20

Discuss Platelet aggregation

Answer 20

Platelet aggregation

• Upon activation, platelets become spherical and extend pseudopodia to
  enable them to attach to other platelets and to the vessel wall.
• Adhesion applies also to recruitment of circulating platelets into the thrombus.
• vWF, also plays a fundamental role in this process
• Adhesion to the growing thrombus is supported by binding of fibrinogen to the integrin αIIbβ3, a process that is more correctly termed aggregation.

Question 21

Discuss platelet adhesion

Answer 21

Platelet adhesion to the blood vessel wall and to each other (aggregation). The binding of glycoprotein (GP)Ib to vonWillebrand factor leads to platelet adhesion to the subendothelium. It also exposes the GPIIb/IIIa binding sites.

The GPIa site permits direct adhesion of platelets to collagen and also further exposes the GPIIb/IIIa binding site. This leads to further binding to von Willebrand factor and so results in further platelet adhesion to the subendothelium.

Platelets also attach to each other, platelet aggregation. The GPIb and IIb/IIIa are involved via von Willebrand factor. Fibrinogen is also directly involved via receptors for it on GPIIb/IIIa

Question 22

What is the function of platelets in would healing?

Answer 22

PDGF found in the α granules of platelets stimulates vascular smooth muscle cells to multiply and this may hasten vascular healing following injury

Question 23

Discuss the Platelet release reaction and amplification

Answer 23

Primary activation by various agonists induces intracellular signalling, leading to the release of α granule contents.

These have an important role in platelet aggregate formation and stabilization and, in addition, the ADP released from dense granules has a major positive feedback role in promoting platelet activation.

Thromboxane A2 (TXA2) is important in secondary amplification of platelet activation to form a stable platelet aggregate.

Question 24

After platelet aggregation and release, the exposed membrane phospholipid (platelet factor 3) is available for two reactions in the coagulation cascade:

Answer 24

• Both phospholipid‐mediated reactions are calcium‐ion dependent. The first (tenase) involves factors IXa, VIIIa and X in the formation of factor Xa.
• The second (prothrombinase) results in the formation of thrombin from the interaction of factors Xa, Va and prothrombin (II).
• The phospholipid surface forms an ideal template for the crucial concentration and orientation of these proteins.

Question 25

What are the natural inhibitors of platelet function?

Answer 25

1. Nitric oxide (NO) is constitutively released from endothelial cells and also from macrophages and platelets. It inhibits platelet activation and promotes vasodilatation.
2. Prostacyclin synthesized by endothelial cells also inhibits platelet function and causes vasodilatation by raising cyclic guanosine monophosphate (GMP) levels.
3. An ectonucleotidase (CD39) acts as an ADPase and helps prevent platelet aggregation in the intact vessel wall.