Thrombocytes and Thrombopoiesisand Thrombopoiesis

thrombocytes and Thrombopoiesis

1. Thrombocytes and
Thrombopoiesis

DR. AMANY M. ELSHAMY
LECTURER OF BIOCHEMISTRY AND MOLECULAR DIAGNOSTICS
PH.D OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

MLS, AUC, CAIRO

2. Outlines

3. Mukipotential hematopoietic
stem cell
(Hemocytoblast)

Common lymphoid progenitor

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Myeloblast (Large granular lymphocyte)

Common myeloid progenitor

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Ut | Plasma cell
Thrombocytes

Macrophage

4. Hematopoiesis

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6. Maturation

» In healthy intact bone marrow tissue, megakaryocytes, under the
influence of an array of stromal cell cytokines, cluster with
hematopoietic stem cells in vascular niches adjacent to venous
sinusoid endothelial cells.

* Responding to the growth factor thrombopoietin (TPO),
megakaryocyte progenitors are recruited from common myeloid
progenitors and subsequently differentiate through several
maturation stages.

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Megakaryoblast Promegakaryocyte Granular megakaryocyte Mature megakaryocyte
(Stage I) (Stage Il) (Stage III) (Stage IV)

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Megakaryoblast Promegakaryocyte Granular megakaryocyte — Mature megakaryocyte
(Stage 1) (Stage 1) (Stage Ill) (Stage IV)

* The development process of
megakaryocytes and platelets in bone
marrow is known as megakaryopoiesis.

+ It is divided into four stages.

+ Mature megakaryocytes extend long and
slender cytoplasmic processes
(proplatelets) between endothelial cells
of sinusoids in the bone marrow and
platelets are released from
fragmentation of these processes.

9. Megakaryocyte Megakaryocyte
differentiation maturation Platelet release
CO CXCL12
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— 1

Osteoblastic
niche

10. + Each megakaryocyte produces 1000 to 5000
platelets, leaving behind a ‘bare’ nucleus
which is removed by macrophages.

+» A unique feature of thrombocytopoiesis is
endomitosis. This refers to nuclear division
with cytoplasmic maturation but without

Megakaryopoiesis cell division.

+ As the cell matures from megakaryoblast to
the megakaryocyte, there is a gradual increase
in cell size, number of nuclear lobes, and
red-pink granules and gradual decrease in
cytoplasmic basophilia.

11. Megakaryocyte

+ Upon complete maturation, megakaryocytes
extend pseudopods through the walls of the
marrow sinusoids and individual platelets
break off into the peripheral circulation.

+ There is evidence that some of the
megakaryocytes are carried to the lungs where
platelets are released.

+ Ahumoral factor, thrombopoietin, controls

the maturation of megakaryocytes.

12. Megakaryocyte

Megakaryocytes, the most
abundant cells of the platelet
series in the marrow, are large
and contain numerous nuclear
lobes with dense nuclear
chromatin, and small
aggregates of granules in the
cytoplasm.

13. Megakaryocyte

+ Megakaryocyte is 30 to 50 mm in
diameter with a multilobulated
nucleus and abundant granular
cytoplasm.

+ Megakaryocytes account for less than than
0.5% of all bone marrow cells, and
on a normal Wright-stained bone r 4
marrow aspirate smear the Megakaryocytes are the largest cells in the bone marrow
microscopist may identify two to four
megakaryocytes per 10x low-power
field.

14. Thrombocytes

Thrombocytes= Mature

Platelets (PLTs)

+ Origin: Myeloid progenitor cell
in response to growth factor
Thrombopoietin, gives rise to
Megakaryocytes.

« Mature platelets have no
nucleus.

« Reference range (SI units) is
150,000 - 450,000 pL.

15. Size: 2-4 um

Appearing as purple blue

Leu cells in stained blood film.

Life span: 8-12 days.

16. Platelets

» Platelets

» Platelets are derived from cytoplasmic fragmentation of bone
marrow cells called megakaryocytes.

+ They measure 2 to 3 p in diameter and do not contain a nucleus.

+ It is round or oval, anucleate (for this reason some hematologists

prefer to call platelets “cell fragments”), and slightly granular.

17. Platelets

+ Platelets remain viable in circulation for approximately 10 days.
+ About one-third of the total platelets in the body are in the spleen and

the remainder in peripheral blood.

+ Uncontrolled platelet and hemostatic activation are responsible for deep
vein thrombosis, pulmonary emboli, acute myocardial infarctions (heart
attacks), cerebrovascular accidents (strokes), peripheral artery disease.
and repeated spontaneous abortions (miscarriages).

» Elevated platelet counts, called thrombocytosis, signal inflammation or

trauma but convey modest intrinsic significance.

18. Hite ste wi

Platelets

19. Ultrastructure of Platelets

Open Saraloyeg system Glycogen

Alpha granule

+— Cell membrane

Dense tubular

system Microtubule

Dense granule

Mitochondrion

20. Ultrastructure of Platelets

21. Ultrastructure of Platelets

+ Ultrastructurally, the following three zones can be distinguished:

+» (1) Peripheral zone: exterior coat (glycocalyx), cell membrane, open
canalicular system.

» (2) Sol-gel zone: microfilaments, circumferential microtubules, dense
tubular system.

+ (3) Organelle zone: alpha granules, dense granules, mitochondria,

lysosomes.

23. Glycogen Glycocalyx

Electron dense granule:
nucleotides (ADP),
Ca?*, serotonin

Lysosome

Plasma membrane

Platelet
phospholipid

== Open canalicular
system

Specific a-granule:
fibrinogen, factor V,

VWF, fibronectin,

heparin antagonist (PF 4), «
PDGF, other proteins
Submembranous filaments
(platelet contractile protein)

Mitochondrion Dense tubular system

Ultrastructure of Platelets

24. Platelets

+ The central cytoplasm
is dominated by three
platelet granules:

+ the 5 (dense) granules
+ a granules

+ lysosomal granules.

Tarima
potes ol

Acid proteases je

Acid hydrolases

Membrane proteins / / ~

agranule o. o

Adhesion molecules

Onokines/chemokines o

Angiogenic factors |, ©

6 granule

Nucleotides

4 Cations

\\ Bioactive amines
GTP-ases

) Membrane proteins

Growth factors

Coagulation factors

Immune mediators

25. T granule
PDI
TLR-9

Lysosome
Acid proteases
Acid hydrolases

Membrane proteins

6 granule
Nucleotides
a granule Cations
Adhesion molecules Bioactive amines
Cytokines/Chemokines GTP-ases
Angiogenic factors Membrane proteins
Growth factors

Coagulation factors
Immune mediators

26. Platelet Membrane

27. Platelet Membrane Glycoproteins

* The cell membrane contains integral membrane glycoproteins (Gp),
which play an important role in hemostasis.

* Important platelet membrane glycoproteins and their functions are as
follows:

e Gp Ib-IX-V: This is a constitutively active receptor that mediates

vWF-dependent adhesion of platelets to subendothelial collagen.

28. Platelet Membrane Glycoproteins

Important platelet membrane glycoproteins and their functions are as follows:

e Gp Ib-IX-V: This is a constitutively active receptor that mediates vWF-
dependent adhesion of platelets to subendothelial collagen.

+ Note: Von Willebrand factor (vWF) is a glycoprotein crucial to primary
hemostasis through platelet and subendothelial collagen adhesion, and

the intrinsic coagulation cascade, through factor VIII stabilization.

29. Platelet Membrane Glycoproteins

Gp Ilb/Illa: On activation,
serves to bind fibrinogen

and thus mediates
aggregation. Also receptor
for vWF, fibronectin, and
thrombospondin.

Gp la-Ila: Constitutively
active receptor for
collagen and mediates
platelet adhesion
independent of vWF.

30. GP1b/IX/V or (CD42b/CD42a/V)
VW and Mac-1 receptor

GPlba

GPla, GPIIb/Illa
{alibB3 or CD41/CD61)
Fibrinogen receptor

GPVI and
FcRy-chain

LAMP2 (CD107b) _
LAMP3 (CD63)

P-Selectin
PAR-1 and PAR-4 (CD62)

Thrombin receptor

31. Platelet
Antigens

32. Platelet Antigens

» Platelets possess HLA antigens and platelet-specific antigens.
+ HLA class I antigens induce alloimmunization (immune response

to nonself antigens ) and cause refractoriness to platelet

transfusions when platelets are obtained from random donors.

33. Platelet Antigens

* The platelet-specific antigen systems are now known as human
platelet antigen (HPA) systems.

» Platelet-specific antigens play an important role in neonatal

alloimmune thrombocytopaenic purpura (NATP) and in post

transfusion purpura.

34. GPilla naz

Only one half of the GPIb-IX-V complex is shown. The HPA polymorphisms and hereditary defects are schematised according to the legend:

Glanzmann s thrombasthenia: ih, Bemard-Soulier syndrome: V7
von Willebrand s diseace-piatelet form: db HPApotymomtism: MIN

35. Platelet functions:

«Maintenance of
Thrombocytes Vascular Integrity.
functions «Formation of the Primary
Hemostatic Plug.
« Accelerate thrombin
formation.

36. Thrombocytes
functions

Maintenance of Vascular

Integrity.

¢ Platelets, or thrombocytes, are true
blood cells that maintain blood vessel
integrity by initiating vessel wall repairs.

« Platelets rapidly adhere to the surfaces
of damaged blood vessels, form
aggregates with neighboring platelets
to plug the vessels, and secrete
proteins and small molecules that
trigger thrombosis, or clot formation.

37. Primary Hemostatic Plug.

¢ Platelets are the major cells
Thrombocytes that control hemosfasis, a
functions series of cellular and plasma-
based mechanisms that seal
wounds, repair vessel walls,
and maintain vascular
patency (unimpeded blood
flow).

38. Role of Platelets in
normal Hemostasis

39. Role of Platelets in Haemostasis

» Activation of platelets refers to adhesion, aggregation, and release

reaction of platelets which occurs after platelet stimulation (i.e.

after vascular damage).

40. Role of Platelets in Haemostasis

* Platelets do not normally adhere to each other or to the
vessel wall but can form a plug that aids in cessation of
bleeding when vascular disruption occurs.

« Injury to the intimal layer in the vascular wall exposes
subendothelial collagen to which platelets adhere. This
process requires von Willebrand factor (vWF), a
protein in the subendothelium that is lacking in patients
with von Wille brand disease.

+ VWF binds to glycoprotein (GP) I/IX/V on the platelet
membrane.

41. Role of Platelets in Haemostasis

+ Following adhesion, platelets initiate a release reaction that
recruits other platelets from the circulating blood to seal the
disrupted vessel. Up to this point, this process is known as
primary hemostasis.

+ Platelet aggregation is reversible and is not associated with
secretion. Additionally, heparin does not interfere with this
reaction and thus hemostasis can occur in the heparinized
patient. Adenosine diphosphate (ADP) and serotonin
are the principal mediators in platelet aggregation.

42. Role of Platelets in Haemostasis

+ Adhesion: This means binding of
platelets to nonendothelial surfaces,
particularly subendothelium which is
uncovered following vascular injury.

+ von Willebrand factor (vWF)

mediates the adhesion of platelets to

subendothelium via GpIb on the surface

of platelets.

Platelet

-— Gplb receptor

von Willebrand factor

vA

Endothelial cells
Cave) (BV SNS)

Subendothelial
collagen

43. Role of Platelets in Haemostasis

e Release reaction (secretion):

Immediately after adhesion and shape

change, process of release reaction or

secretion begins. In this process, E)

contents of platelet_organelles are HT : mm 77,

P mmm me cf ts

released to the exterior.

44. integrin activation

“5 coagulation
+
i 1 fe

CA ADP | secretion of coagulation factors

resting thrombin

Mi GPCRs ( GPVI Sy resting
© granules x ECM VA activated

45. Role of Platelets in Haemostasis

e Aggregation: This may be defined
as binding of platelets to each
other. ADP released from platelets

a
or from damaged cells binds to ”

specific receptors on platelet

surface.

46. Aggregation

Gpllb/Illa receptors

EN
fil

Fibrinogen

47. Role of Platelets in Haemostasis

e Platelet procoagulant activity: When platelets are activated,
negatively charged phospholipids (phosphatidylserine and
phosphatidylinositol) located in the inner half of the lipid bilayer
become exposed on the outer surface to critical binding site for
several procoagulant proteins. These phospholipids play an active
role in coagulation by providing surface for the interaction of some

coagulation factors.

48. Cytoskeletal changes

ADP
> serotonin
o

o
© 5 granules

ollagen

49. PLTs activation

+ TxA2 serves as a positive-feedback mediator during platelet
activation.

+ TxA2 acts to activate adjacent platelets, generate more TxA2, and
amplify the action of other, more potent, platelet agonists. When
TxA2 binds to its cell surface receptor TP, platelet activation leads
to platelet-shape change, activation of phospholipase A2, platelet
degranulation of dense granules and alpha granules, and platelet
aggregation.

« TxA2 also induces vasoconstriction of smooth muscle

50. Membrane phospholipids

Inhibition by
steroids Phospholipases

Arachidonic acid

INES Or ee

thromboxane NSAIDs
A2 Prostaglandin G2

Prostaglandin H2

| Thromboxane synthetase

Thromboxane A2

|

Promotes platelet aggregation

51. Platelet procoagulant activity. Platelets provide surface for some
important coagulation reactions

Platelet surface Platelet surface

IX-VIII-Ca** V-Xa-Ca**
2 Xa Prothrombin —— a — Thrombin

In addition platelets also secrete calcium, FV, fibrinogen, and FXII and
contribute to the coagulation system.

52. To sum up

53. Primary platelet adhesion Platelet aggregation

> Unactivated platelet = Contact and
aggregation
—> En Activated
= Rolling Granule ieee 7 Seco
contact | [Aghesion| pareve —_— en
=> Spreading =
Collagen
Endothelial vWf deposited
cell on subendothelium
Platelet receptors engaged GPIb ar Quels es
a ae Collagen, Wi
nding sites Fibrinogen
Fibronectin
Gamer Boogy

Vitronectin

54. Blood flow
—
Tacens = MaA2 Cal'aDP, VWEfibrinogen,
K Senn Derseganales agite
agan
al amen al
collagen We

Er?

Recruitment — Activation — (Calciom) sienaling

® range (4)
Fibrin formation

| ‘amplified Clot retraction

BEE

Boundaries of the clot
Bleeding stopped

55. Injured blood vessel

Protein Wave First Wave Second Wave
Hemostasis Hemostasis Hemostasis
Lumen
Plasma fibronectin Platelet adhesion Coagulation
deposition & aggregation
Blood
flow

q?

Subendolhelium

> Resting Platelet WW Activated Platelet

Thrombin

X Gpvi

Be GPIbXV AS) 028 or a IIbB3 integrin

© Platelet Granule
coco Fibrinogen (Fa) Fibrin Plasma fibronectin (pFn) —— Collagen

es Von Wilebrand Factor (VWF)

56. [EEES
Eu — E]
\ Z
==

57. + Thrombocytopenia may be associated with:

* A, Postsplenectomy

* B. Hypersplenism

* C. Acute blood loss

+ D. Increased prolifera

+ tion of pluripotential stem cells

+ Platelets

+ A release ADP after activation

» B are also called megakaryocytes

+ C can only be activated via their glycoprotein receptors
+ D have dense vesicles containing histamine

58. + During primary homeostasis
«A fibrinogen is converted to fibrin

«B the initial vasoconstriction does not require platelet
activation

*C thromboxane A2 causes platelet adhesion
«D activation of lipoxygenase is a vital step

59. «Which ofthe following blood cells play an im portant
role in blood clotting?

*(a) Throm bocytes
+ (b) Neutrophils
*(c) leucocytes

» (d) Erythrocytes

60. + Serum differs from blood as it lacks

+ (a) antibodies

+ (b) clotting factors

+ (c)albumins

+ (d) globulins

Which ofthe following is correct?

*(a) Serum contains blood and fibrinogen

*(b) Plasma is blood without lym phocytes

*(c) Blood comprises plasma, RBC, WBC and platelets
+ (d) Lymph is plasma with RBC and WBC

61. Platelets are formed from what type of cell?
a) Melanocytes

b) Macrophages

c) Astrocytes

d) Megakaryocytes

Clum ping of cells is known as

a) clotting

b) mutation

c) agglutination

d) glutathione

62. + Which of the following is correct?
a) Lymph = Plasma + WBC’ + RBC’
b) Plasma = Blood — Lymphocytes
c) Neuron = Cyton + Dendron + Axon + Synapse
d) Blood = Plasma + RBC’ + WBC% + Platelets

+ Which of the following plasma protein is involved in coagulation
of blood?
a) Albumin
b) Globulin
c) Fibrinogen
d) Amylase

63. Platelets

A release ADP after activation

B are also called megakaryocytes

C can only be activated via their glycoprotein
receptors

D have dense vesicles containing histamine

64. During primary homeostasis

A fibrinogen is converted to fibrin

B the initial vasoconstriction does not require platelet
activation

C thromboxane A2 causes platelet adhesion

D activation of lipoxygenase is a vital step

65. Activation of thrombin

A is triggered by thrombomodulin

B always requires the presence of activated
platelets

C is Ca” independent

D depends on Factor X

66. Which of the following is NOT one of the four major
physiologic events of hemostasis 2

A. Fibrinolysis
B. Vasodilatation
C. Platelet plug formation

D. Fibrin production

67. Which is required for platelet adherence to injured
endothelium?

A. Thromboxane A,

B. Glycoprotein (GP) Ilb/Illa

C. Adenosine diphosphate (ADP)
D. Von Willebrand factor (vWF)