Platelets Flashcards
hemostasis
physiologic process by which the body stops bleeding while maintaining blood in the fluid state within the blood vessels
3 properties:
- keeps blood circulating in fluid state
- produces controlled, localized clot when injured
- dissolves clot once wound has healed
formation of the platelet plug
primary hemostasis
stabilization of the platelet plug through the activation of coagulation factors to form fibrin
secondary hemostasis
hemostasis players
interactions between
- blood vessels
- platelets
- coagulation proteins
- coagulation inhibitors
- fibrinolytic system
thrombopoitein analogs
- romiplostim
- eltrombopag
platelet production
cytokine influencers
- thrombopoietin
- IL-3 (early)
- IL-6, IL-11 (later)
molecular influencers
- GATA1 (early differentiation)
- RUNX1 (drives switch from mitosis to endomitosis (meg maturation)
sometimes the earliest PLT progenitor detectable by morphology
megakaryoblast
acute megakaryoblastic leukemia
- intermediate size
- granular basophilic cytoplasm with pseudopods
- most common form of AML seen in Down Syndrome before age 3
> associated with mutated GATA1
maturation from the megakaryoblast to mature megakaryocyte involves two main steps:
- endomitosis
- cytoplasmic maturation
this is followed by shedding of PLTs in a process called thrombocytopoiesis
how is the abundant cytoplasm state of mature megakaryocytes reached?
through repeated rounds of endomitosis
what is endomitosis?
- mitosis without telophase and cytokinesis
- increased DNA content and cytoplasm without cell separation into daughter cells
cytoplasmic maturation of megakaryocytes
- increased DNA content allows increased synthesis of cytoplasmic contents
> granules
> demarcation membrane system (DMS): membrane repository support increase in membrane SA needed with PLT formation
> microtubules and other organelles - PLTs form from the mature meg cytoplasm
thrombocytopoesis
- megs locate near BM sinusoids and shed PLTs into the bloodstream
- megs form cytoplasmic extensions - proplatelet processes
- processes pierce through or between sinusoid epithelial cells into venous blood
- organelles move along tubules to the end of the processes
- processes extend and branch
> until cytoplasm is a mass of protoplatelets
> protoplatelets released from meg body - protoplatelets fragment further into PLTs
- naked megakaryocte nucleus undergoes apoptosis
PLT morphology
- ~3um diameter
- 8-10 fL in volume
- no nucleus in mammals
- visible granular on peripheral blood film
- biconvex in circulation (in resting state) round on blood film
reticulated PLTs
- young PLTs containing high amounts of RNA
- larger than mature platelets
- released compensation for thrombocytopenia (if marrow is working)
> differentiate between thrombocytopenia due to PLT destruction vs. bone marrow failure
> signify marrow regeneration after chemotherapy or transplant
PLT ultrastructure
- peripheral zone
- receptor/transmitter region - sol-gel zone
- cytoskeleton - organelle zone
- metabolic machinery
peripheral zone
- glycocalyx
> PLT membrane surface
> thick and adhesive - ready for PLT function
> absorbs plasma proteins, transport to storage organelles via endocytosis - plasma membrane = invades the PLT interior, forming the Surface Connected Canalicular System
> functions as a pathway: extracellular Ca2+ uptake, secretion of intracellular material such as granule contents in platelet activation
peripheral zone plasma membrane contains:
- phospholipid bilayer
> inner phospholipids flip to the outer layer with activation
> phospholipids act as a surface for coagulation factors - glycoprotein receptors
> involved in PLT adhesion and aggregation
> including VWF (GPIIb/IX/V), fibrinogen (GPIIb/IIIa)
> collagen (GPVI)
Sol-Gel Zone
- circumferential microtubules: > located just within PLT membrane > encase the PLT; maintain discoid shape - microfilaments > located within PLT cytoplasm > composed of actin and myosin > becomes contractile with activation shape change granule content release
organelle zone
dense tubular system (DTS) - derived from endoplasmic reticulum - sequestration of calcium > controls PLT contraction & activation via Ca++ release - site of eicosanoid synthesis
eicosanoid synthesis
- AKA prostaglandin, cyclooxygenase or thromboxane pathway
> prostaglandins and thromboxanes = eicosanoid subtypes
> cyclooxygenase = enzyme involved in eicosanoid synthesis - PLT activation pathway
- starts in membrane and finishes in the dense tubular system
- aspirin inhibits cyclooxygenase function
alpha granules
- 50 to 80 granules/plt contents = - coagulation proteins = fibrinogen, factor V, VIII - vWF - PLT factor 4 - PLT derived growth factor - proteins!
dense granules
- 2--7 granules/plt contents = - ADP - ATP - Calcium - Mg - serotonin - small molecules!
primary hemostasis
platelet plug formation
secondary hemostasis
procoagulant function
PLT function
- initial adhesion to site of injury
- PLT activation with: shape change, secretion of granule contents, aggregation of platelets to form platelet plug
- providing procoagulant surface and scaffold for fibrin clot formation
platelet receptor to its agonist
GP-VI = collagen
PAR1 & PAR4 = thrombin
P2Y1 & P2Y12 = ADP
thromboxane receptor = thromboxane A2 (TXA2)
shape change of PLTs
- microtubules rearrange w activation
- from discoid shape at rest to spherical with projections
shape change of PLTs
- microtubules rearrange w activation
- from discoid shape at rest to spherical with projections
initial formation of platelet plug via adhesion, activaton, aggregation
primary hemotasis
defect in primary hemostasis results in
mucosal bleeding > nose bleeds > menorrhagia early post-op bleeding petechiae ex: VWD, PLT disorders
activation of a series of plasma proteins to form a fibrin clot
Secondary hemostasis; “Coagulation Cascade”
defects in secondary hemostasis
- joint bleeds
- delaye bleeding post-op
- deep hematomas
- example: hemaphilia A ( Congenital Factor VIII deficiency)
at around what count does bleeding normally occur
at 20 x 10^9 or even less
when are manual PLT counts used
for samples with many giant platelets
types of automated PLT counts
- electrical impedence counting
- fluorescent plt count
- flow cytometry
disadvantages of electrical impedance counting
- overlap between cells of extreme size may result in erroneous counts
- falsely high PLT counts
> extremely mcrocytic RBCs
> RBC fragments - falsely low PLt counts
> giant PLTs
> PLT clumping
> PLT satellitism
flow cytometry for PLT counting
- immunological PLT counting
- PLT antigens labeled (CD41 (GPIIb), CD61 (GPIIIa))
- antigensm ust be present in normal amounts
- expensive (not routine)
quantitative platelet abnormalities
- thrombocytosis = primary, secondary (reactive)
- thrombocytopenia
primary thrombocytosis
clonal disordes; MPNs
- essential thrombocythemia
- polycythemia vera
- primary myelofibrosis
- chronic myeloid leukemia
secondary (reactive) thrombocytosis
influence of proinflammatory cytokines (IL-6) and TPO
- infection/inflammation
- post-opeerative state
- iron deficiency/blood loss
- post-splenectomy
thrombocytopenia can arise due to
- decreased production
- splenic sequestraation
- dilutional thrombocytopenia
- increased destruction
decreased PLT production (congenital)
- macrothromboytopenias (eg. May-Hegglin anomaly)
- megakaryocyte hypoplasia (Fanconi anemia, thrombocytopenia with absent radii)
decreased PLT production (acquired)
- toixns: alcohol, drugs
- viral infection
- ineffective thrombopoiesis (B12/folate def; myelodysplastic syndrome)
May-Hegglin anomaly
- large PLTs + Dohle neutrophil inclusions
- thrombocytopenia in 30-50%
- MYH9 mutation
> nonmuscle myosin heavy chain => cytoskeletal protein
> autosomal dominant - other MYH9 disorders may also be associated with sensorineural hearing loss and/or renal disease
thrombocytopenia with absent radius
- severe thrbocytopenia at birth + congenital absence/hypoplasia of radial bones
- PLTs 10-30 x 10^9/L as infant but increase to normal levels by 1 y/o
- impaired DNA repair
- autosomal recessive, rare
splenic sequestration
- ” distributional thrombocytopenia”
- normally 1/3 PLTs in spleen; increasing spleen size = increased proportion of PLTs pooling in spleen
severe portal hypertension
up to 9% of PLTs may be sequestered in the spleen
- total PLT mass normal and clinical bleeding is uncommon
massive hemorrhage w RBC and fluid support but without PLT transfusion
dilutional thrombocytopenia
increased destruction of PLTs
IMMUNE
- ITP (immune thrombocytopenia purpura) = autoimmune
- FNAIT (fetal/neonatal alloimmune thrombocytopenia)
- drugs
= viral infections
NON-IMMUNE
- microangiopathic hemolytic anemia
- cardiopulmonary bypass
assessing PLT function
bleeding time PFA-100 PLT aggrgation studies thromboelasstography morphologic assessment & EM flow cytometry
bleeding time disadvanatges
poorly reproducible
not a good predictor of surgical bleeding
time consming
invasive
advantages of bleeding time
studies natural hemostasis
no expensive equipment
no anticoagulation artifacts
bleeding time may be prolonged in …
- thrombocytopenia or PLT function disorders (but not specific for PLT abnormalities)
- decreased or defective fibrinogen
- vWD
- vascular abnormalities (Ehlers-Danlos syndrome)
agglutination
PLT adhesion but not activation
> induced by Ristocetin
aggregation
adhesion and activation
primary wave
reversible aggregation by exogenous agonist only
secondary wave
irreversible aggregation resulting from release of endogenous ADP
Ristocetin
induces agglutination of PLTs by plasma VWF binding to PLT GPIb-IX
list of agonists
- ADP
- arachidonic acid
- epinephrine
- collagen
- thrombin receptor activating peptide
provides global assessment of the dynamics of clot development, stabilization, and dissolution
thromboelastography
> uses whole blood?
anti-CD42b
GPIb
what is the impact of Aspirin on the Eicosanoid pathway?
(ASA)
inhibition of cyclooygenase andd reduced thromboxanse A2 synthesis ; blocks platelet aggregation
- aggregation with arachidonic acid impaired, but normal with a direct agonist of the thromboane A2 receptor (U46619)
acquired platelet function disorders
- drugs (ASA, NSAIDs, etc.)
- liver disease
- uremia (chronic kidney disease)
- cardiopulmonary bypass
- hematologic neoplasms (MDS, MPNs, Multiple Myeloma - dysproteinemia)
congenital platelet function disorders
- Benard-Soulier syndrome (disorder of adhesion)
- Storage Pool Disease (diorders of granules)
- Glanzman’s Thrombasthemia (disorder of aggregation)
Bernard-Soulier syndrome
- defects in GPIb
> autosomal recessive
> VWF binding - thrombocytopenia with giant PLTs
- aggregation studies:
> normal aggregation to ADP, epi, collagen, AA
> reduced aggregation to thrombin and agglutination to ristocetin
T or F. PLt count for storage pol diseases are typically normal
T! non-specific abnormalities on aggregation studies
Storage Pool Diseases
- alpha granule def = Gray PLT syndrome; rare; PLTs grey due to def of alpa grans
- dene granule def = more common; reduce # of dense granules visible by whole mount PLT EM
Glanzmann’s Thrombasthenia
- autosomall recessive def of GPIIb/IIIa
- abnormal response to all activating agonists (ADP, collagen, AA, thrombin, epi)
- normal agglutination to Ristocetin
heparin-induced thrombocytopenia
- HIT; conundrum!
- drug rxn to heparin anticogulation
- thrombocytopenia
- but potential severe clots with rapid limps loss or death
- history key for diagnosis as is detection of anti-PF4/heparin Ab
- therapy is prompt anticoagulation with non-heparin anticoagulant
> no prophylactic PLT transfusions
when blood vessels and PLTs respond to vascular injury
primary hemostasis - blood vessels contract (vasoconstriction) - PLTs become activated > adhere to site of injury > secrete granule contents > aggregate to form PLT plug
the vascular system is supported by internal elastic lamina and connective tissue
- collagen
- fibroblasts
- smooth muscle cells (in arteries/arterioles)
endothelial and subendothelial structures and substances in the blood vessels have hemostatic properties: (3)
- anticoagulant (prevent clotting)
- procoagulant (promote clotting)
- fibrinolysis (promote clot lysis)
anticoagulant properties of the vascular system
- intact endothelium prevents thrombosis
- repels PLTs when intact
- non-reactive to coagulation factors
- thromboresistance achieved by substances and/or released by the endothelium
- enhances fibrinolysis
- barrier between blood and collagen/tissue factor
Procoagulant Properties
- vasoconstriction: decreease blood flow to site of injury
- exposure of subendothelial collagen and secretion of vWF by endothelial cells: allows for PLT adhesion (primary hemostasis)
- endothelial release of tissue factor (TF): activates the extrinsic coagulation pathway
- exposure of collagen: initiates activation of the intrinsic coagulation pathway
endothelial substances that promote clotting
- collagen: activates PLTs, PLT adhesion
- vWF: required for PLT adhesion
- tissue factor: activates ciagulation with factor VII
Fibrinolytic properties
- tissue plasminogen activator (TPA): released by endothelial cells
> during clot formation, TPA and plasminogen bind to the clot
> TPA activates plasminogen to plasmin
> plsmin breaks down fibrin and restores blood flow
platelet roles in hemostasis
- maintain vascular integrity
- platelet plug formation for initial stopping of bleeding
- promoting fibrin formation to stabilize the clot
must be normal in number and function to do these
these nourish the endothelial cells lining the vascular system
- PLTs!
- help with maontoanong vascular integrity
- accomplished by releasing platelet-derived growth factor (PDGF) from alpha granules
what does platelet plug formation inolve?
- adhesion
- shape change
- release rxn/secretion
- aggregation
adhesion of PLTs
- sitck to foreign surface; blood vessel injury expose collagen
- requires GP Ib/IX/V on PLT surface & vWF (from endothelial cells + megs)
PLT shape change
- normally discoid
- agonists = collagen, ADP, and thromboxane A2 alter levels of Ca 2+ promoting shape change
- activated PLTs = spherical
PLT dense granules
PLT activating molecules; small molecules
- ADP
- ATP
- Ca 2+
- serotonin
- epinephrine
PLT dense granules
PLT activating molecules; small molecules
- ADP
- ATP
- Ca 2+
- serotonin
- epinephrine
PLT alpha granules
PLT-specific proteins
- PDGF
- platelet factors
procoagulants
- fibrinogen
- vWF
- factor V
T or F. alpha granules are released first
F! dense then alpha
where do PLTs aadhered to exposed subendothelium release their granular contents into?
open canalicular system
serotonin
vasoconstrictor
ADP cause PLTs to __________
aggregate
Ca 2+
aplifies aggregation by activating PLT membrane phopholipases which lead to formation of thromboxane A2
thromboxane A2
enhances release rxn and aggregation, powerful vasoconstrictor
Role of TXA2
- TXA2 binds membrane receptors, deceases conctn of cyclic AMP by suppressing adenylate cyclase
- allows release of ionic calcium from DTS
- increased calcium = contraction of actin microfilaments, activating PLT = degranulation
PLT aggregation
PLTs sitcking to each other
- ADP released from dense granules causes initial aggregation of PLTs
- requires GPIIb/IIIa, fibrinogen, and Ca 2+
a stable platelet plug is required to stop bleeding
procoagulation comes in
> fibrinogen bound to PLTs must be converted to fibrin
> role of PLTs in fibrin formation = provide phospholipids required for intrinsic coagulation system
what is PF3?
- platelet membrane phospholipid exposd when PLts are activated
- provides a charged surface for conctn and activation of coagulation factors
- results in localization of the rxn to the injury site and increases the rate of rxn
- interacts with vit K-dependent coagulation factors (II, VII, IX, X)
