Lecture 5- do not Flashcards
what are platelets
Complex, Anucleate cell fragments that are 2-4 um
-in a wrights stain they appear Circular to irregular in shape
‒ Cytoplasm light blue to colourless
‒ ‘Granular’ looking
■ Granules are abundant
■ Red to violet in colour
■ Only slightly visible using light microscopy
-flat or discoid shape or bi convex
Platelet Morphology
Complex, granular structure
-have alpha granules, mitochondrion , open canicular system
‒ Involved in clotting of blood and tissue repair
-Platelets circulate in an inactivated or resting state
-become activated by tissue injury
-Activation includes shape change, adhesion, aggregation & secretion (granule release)
Platelet precursors arise
from which stem cells?
MHCs - common myeloid progenitor
BM megakaryocytopoiesis - sit right beside because the venous sinusoid
-cytokines help to go from one stage to next
Megakaryocytic Progenitors
Proliferation Phase
● BFU-Meg clones hundreds of daughter cells (mitosis) (least mature) - job is proliferation
● CFU-Meg clones a dozen daughter cells (mitosis) - more intermediate forms
● LD-CFU-Meg undergoes the first stage of Endomitosis (most mature) most ready to produce the precursors for the BM
-need flow cytometry to test cant tell by just looking
a Megakaryocytic erythrocyte Progenitors with CFU GEMM acts on BFU Meg using TPO, Meg - CSF and IL 3
Endomitosis
DNA is duplicated without cell division (replicates nucleaus cytoplasm) (do not see separation of parents from daughters causing larger cells) no cytokinesis
● Unique to Megakaryocyte linage
● MKs become ‘polyploid’
‒ Contain multiple copies of DNA within a single cell
‒ Most have a ploidy of 16N(range 4N - 64N)
● MKs are the largest normally occurring cells in the marrow (20-90µm)
‒ More DNA= larger cell or more cytoplasm synthesized= more
PLTs
-how does the cytoplasm become more abundant - the higher the N the more ptls being produced
what is Terminal Differentiation of Megakaryocytic Progenitors
Series of stages where MK differentiation becomes recognizable in BM using LD CFU MEG
● Broken into three morphological stages:
1. MK-I or Megakaryoblast- round nucleus, , Endomitosis only , basophilic cytoplasm with granules
- MK-II or Promegakaryocyte- indented nucleus, condensed chromatin,, Endomitosis ENDS, cytoplasm is abundant, basophilic and granular
- MK-III or Megakaryocyte -LARGEST (undergoes proplatelet process and platelet shedding)- multilobed nucleus, condensed chromatin, NO MITOSIS OR ENDOMITOSIS, azurophilic and granular cytoplasm
● MK-I can look like any blast
● MK-II & -III identified in BM
how are platelets formed
-Massive amounts of DNA produce equally massive amounts of cytoplasm and proteins
-plasma membrane ‘invades’ the cytoplasm in a series of channels –
this is the Demarcation System or DMS - divide the megakaryocyte cytoplasm
-Form basis for fragmentation into single PLTs
- After the cytoplasm is released as platelets, the MK nucleus that
remains is reabsorbed by Macrophages in the bone marrow
-PLATELET SHEDDING
-MKs sit adjacent to BM venous sinusoids
-DMS dilates & tubules called ‘proplatelet processes’ (pseudopodia) develop
-Across processes constrictions form, ready to fragment
-These pierce through or squeeze between endothelial cells and break off or shed into the central vein of BM and then into peripheral circulation
Control of Thrombopoiesis
Thrombopoietin or TPO
Glycoprotein hormone produced by kidney and liver, that circulates in plasma
‒ Also produced by smooth muscle cells, and stromal cells
Binds to megakaryocytes and circulating platelets via MPL membrane receptor
-PLTs and MKs degrade TPO, taking it out of circulation or removing stimulus for PLT production (primary platelet count control mechanism)
● decrease PLTs = INCREASE circulating TPO
‒ stimulates platelet production
TPO Induces:
● Stem cells to differentiate into MK progenitors
● MK progenitors to differentiate into MK
● Proliferation and maturation of MK
● Platelet release (PLT shedding) from MK
Thrombopoietin INDUCES
-Stem cells to differentiate into MK progenitors
● MK progenitors to differentiate into MK
● Proliferation and maturation of MK
● Platelet release (PLT shedding) from MK
acts In synergy with various cytokines (e.g., Interleukins, IL-3, IL-6 & IL-11)
increases platelet production
WHAT IS THE Platelet Plasma Membrane LIKE
●Phospholipid bilayer
● Receptors on the membranesurface
● Thick surface or glycocalyx - also absorbs plasma proteins
● The outer membrane also extends inside – similar to the
DMS of MK
● Surface is negatively charged ‒ Repels other PLTs so they
don’t stick together spontaneously
what are the Platelet Plasma Membrane: Receptors like
▪ Promotes PLT adhesion and aggregation
what are some Platelet Plasma Inner Organelles
Surface-Connected Canalicular System (SCCS)
‒ Inner extension of plasma membrane that binds
coagulation factors and provides a route for secretion of α-granule
contents
● Dense Tubular System (DTS)
‒ Supports PLT activation, provides cytoskeleton
● Microfilaments & Microtubules
‒ Maintain PLT (discoid) shape
‒ Allow for shape change (via membrane contraction and pseudopod extension)
● Granules
‒ α- and dense granules –
‒ Also contain few lysosomes (similar content to Neutrophils)
what are α-Granules all about
●50-80 per PLT
● Some proteins absorbed from plasma
● Some made in MK
● Released through SCCS during PLT activation
‒ α-granule membrane fuses with SCCS and contents are released
‒ E.g., Coagulation factors
(Fibrinogen, Factor V and VWF)
what are Dense Granules
2 – 7 per PLT (endocytosed)
● Migrate to PLT plasma membrane to release contents directly into the plasma when PLT is activated
-contains ADP - non metabolic and support platelet aggregation by binding to P2Y and ADP receptors
-Serotonin - Vasoconstrictor
CA and MG - divalent cations support platelet activation and coagulation
Platelet Function - first line of defense during blood lose -
PLTs circulate in a resting or inactivated state
● PLTs attach to injured vessel
● Upon activation, PLTs:
‒ Adhere, Aggregate & Secrete
● Part of PRIMARY HEMOSTASIS or Blood Clotting
● Platelet ‘Plug’ formation is the start of clot formation
Resting to Activated Platelet how does that happen in 3 steps
Initially discoid (resting)
● Change shape
‒ When activated become- Round with long, finger-like projections (pseudopods) and lamelipodia
‒ Sticky and adhere to one another and to blood vessel surfaces to form a PLT plug
Adhesion
‒ Receptors to Collagen or VWF
● Aggregation
‒ PLT to PLT receptors
● Secretion
‒ From granules through SCCS or directly to membrane
what happens when Platelet Activated - secondary hemostasis
Release molecules that cause fibrinogen to be converted to fibrin
* Fibrin molecules combine:
‒ Form a long, sticky fibrous mesh
‒ Traps other platelets, RBCs & WBCs
* Summon more platelets to the damaged vessel
* Activate other clotting factors
Fibrin clot (or red clot) formation
‒ Series of plasma protein interactions on PLT surface
‒ PLT phospholipid surface is essential to solid clot formation
what is Primary Hemostasis
Injury or trauma to blood vessel endothelium
● Blood vessels contract to seal wound & reduce blood flow in
general area
● Collagen exposed
● Damaged endothelial cells release adhesion molecules
& VWF
● Platelets adhere to Collagen &/or VWF
what is Platelet Adhesion
all occurs simultaneously
Occurs via receptors specific to Collagen or VWF
● GP Ib/IX/V –
‒ Binds VWF & Thrombin
-vwf acts as bridge
● GP Ia/IIa & GP VI –
‒ Bind Collagen
● Firm but Reversible binding
● PLT loses disc shape and spreads
extending of the pseudopodia
what is Platelet Aggregation
PLT to PLT binding to build up the plug
● PLT microenvironment supports binding via activation of GPIIb/IIIa
receptor
● Fibrinogen with Ca2+ and VWF bind GPIIb/IIIa of adjacent PLTs
● PLT shape continues to spread and form pseudopods
‒ Forms large clump(s) that cover greater surface area
● Irreversible – shape
some granules contain the plt proteins and in the system and also in the endothelial cells
what is Platelet Secretion
Secretion of α- & dense granules
‒ Via SCCS or directly to PLT plasma
membrane, respectively
● Occurs simultaneously with adhesion and aggregation
● Amplification of aggregation by
releasing more Fibrinogen & Ca2+
alpha granules are also coag proteins
what is primary and secondary hemostasis
●Primary – Platelet plug
(or white clot) is insufficient to repair most damaged vessels
-plts and vWf
● A solid clot requires Secondary
Hemostasis – Fibrin clot (or red clot) formation
‒ Series of plasma protein interactions on PLT surface
‒ PLT phospholipid surface is essential to solid clot formation
how many PLT are in PB
● R.I. 150 – 450 x 10^9 /L decreases as you get older
● Live an average of 8-12 days
● 2/3 of total platelet numbers circulate in PB
● 1/3 are sequestered in splenic pool ‒ Released when sudden depletion of PLTs occurs
-acts as a reserve for after surgery or injury
-if the spleen is inflamed there will be low plt due to increased sequestration
-megakaryocytic precursors made in the red pulp MPV corresponds to larger platelet diameter
Thrombocytopenia
● < 150 x 10^9 /L
● Caused by:
‒ decreased Production
■ E.g., Acute Leukemias, BM failure like aplastic anemia (can
manifests as bruising in patient) or Myelodysplasia
‒ Splenic sequestration
‒ increase Peripheral destruction
■ Antibodies to PLTs
■ , MAHA, hemolytic anemias- DIC & TTP)
Thrombocytosis
● > 450 X 10^9/L
● Causes include:
‒ Reactive
■ E.g., Injury, surgery, stress, pregnancy, or immune response
‒ Pathological
■ E.g., Chronic Leukemia or Chronic bleeding
Abnormal PLT Morphology
‘Giant’ platelets
Agranular platelets
Elongated platelets
● ‘Giant’ platelets- same size or larger then RBC
‒ Great variation in size – 20um
‒ Result of abnormal megakaryopoiesis
● Agranular platelets
‒ Appear light gray due to lack of protein within granules
● Elongated platelets - like a dumbbell two platelets at each end
‒ Reticulated PLTs – released early – larger proplatelet processes
(cylindrical and beaded seen in citrated whole blood)
Most common cause of
PLT clumping in PBF:
● Poor venipuncture technique with CBC sample
‒ Slow or difficult draws
‒ Improper or delayed mixing
● We can also see similar
results with EDTA induced PLT clumping
● Platelets start to aggregate or ‘clump’
‒ Leads to falsely decreased PLT count on CBC
● ‘Clumps’ can also be counted as WBC by analyzers
‒ Falsely high WBC count on CBC
‘Platelet Satellitism’
● In vitro phenomenon known as
‘Platelet Satellitism’
‒ Rare and only seen in certain patients
● Caused by patient’s PLT response to EDTA in ETS tube
‒ EDTA causes PLTs to adhere to Neutrophil surface
● Find out after examining CBC & PBF
‒ Pseudothrombocytopenia on CBC
‒ PLTs surrounding Neuts on PBS
How to resolve?
Poor venipuncture
EDTA-induced PLT clumping or PLT Satellitism
● Poor venipuncture
‒ Reject sample and recollect in new lavender-top EDTA tube
‒ Troubleshoot technique
● EDTA-induced PLT clumping or PLT Satellitism
‒ Reject sample and recollect using Sodium Citrate anticoagulant
■ Draw a light blue-top tube (liquid anticoagulant)
■ Adjust the CBC results for the dilution factor
real pr thrmbon pseudopedia
ttur thrmbo - pt with low plt
pseudo thyr - lab error
check with a smear if you dont see large clumps look for platelet clumps in the smear
