Week 5: Primary Hemostasis
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Hemostasis
Platelet function
Platelet anatomy
Megakaryocyte
Platelet kinetics
Platelet count
Aggregation studies
Von Willebrand’s
Bernard-Soulier
Aspirin (salicylate)
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Vascular problems
Allergy
Viral infection
Collagen disorders
Vitamin C deficiency
Ehlers-Danlos
Render-Osler-Weber
telangiectasia
Petechiae, ecchymoses
Bleeding time
Vascular Injury
Serotonin and thrombaxane A2 (TxA2) for
vasoconstriction
 Prostacyclin PGI-2 for arteriole relaxation
to increase blood flow
 Exposure of basement membrane and
collagen (negatively charged surface)
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Inside of a vessel SEM x 2,500
Process of Hemostasis
Vascular injury
 Platelet adhesion and activation
 Platelet aggregation (1o hemostatic plug)
 Fibrin formation via cascade (2o
hemostasis)
 Clot retraction (thrombasthenin)
 Fibrinolysis and healing
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Role of Platelets
Surveillance for
vascular integrity
 Formation of 1o
hemostatic plug
 Activation of 2o
hemostasis
 Healing
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Platelet Formation
Megakaryoblast undergoes endomitosis
 Intermiediate stage promegakaryocyte
without granules
 Megakaryocyte (2N to 64N) with over
100µ diameter
 IL3, GM-CSF, thrombopoietin
 20% of platelet stored in spleen
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Platelet
2 - 4µ diameter
 Round or oval
 Hyalomere - clear peripheral zone
 Granulomere - highly stained area with
granules
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Platelet Anatomy
Peripheral zone with glycoprotein
receptors
 Structural zone with contractile
microtubules (thrombasthenin)
 Organelle zone with granules
 Membrane with open cananicular and
tubule systems for increased surface area
and rapid release
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Electron micrograph of a platelet x 25,000
Longitudinal peripheral microtubule (brown), endoplasmic reticulum
(blue), mitochondria (green), glycogen (black)
Platelet Function
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Adhesion to basement membrane or collagen with
vWF and GP-Ib
Activation
 Shape change from discoid to distorted
 Exposure of GP-IIb/IIIa and other receptors
 TxA2 synthesis (cyclo-oxygenase dependent)
Membrane phospholipid  Arachidonic acid
Arachidonic acid  TxA2 and prostaglandins
Platelet Function:
Glycoprotein Group or Gene Families
Integrins
 Leucine rich glycoprotein family
 Selectin family
 Quadraspanin family
 Immunglobulin supergene family
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Integrins
Integral to membrane
 Ca++ dependent GPIIb/IIIa most abundant
 Cell-cell or cell-substrata interaction
 Receptor to Fib, vWF, vitronectin,
fibronectin
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Leucine Rich GP Family
Adhesion to subendothelial collagen (COL)
mediated by vWF by transmembrane
complex GPIb/IX
 Stabilizes PLT membrane by interaction
with cytoskeleton
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Selectin Family
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GMP-140 mediates adhesion of neutrophils
and monocytes to PLT
Quadraspanin Family
Plasma membrane protein p24/CD9
interacts with GPIIb/IIIa, modulating
adhesion molecules
 Leads to Ca++ increase and subsequent PLT
activation and aggregation
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Immunoglobulin Supergene Family
Functional role unclear
 Has role in cellular interactions
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Platelet Activation
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1o aggregation with agonists: ADP, epinephrin,
serotonin, PF4 (anti-platelet)
Release or secretion facilitated by TxA2
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 Dense body for 2 aggregation and
vasoconstriction (ADP, Ca++, serotonin)
 -granule for heparin neutralization and clot
 Platelet derived growth factor (PDGF) for
healing
Retraction
Signs and Symptoms of 1o
Hemostasis Problems
Ecchymoses
 Petechiae
 Mucus membrane bleeding
 Hematoma
 Prolonged bleeding after minor surgery
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Hereditary Vascular Problems
Hereditary (spider) telangiectasis (OslerRendu-Weber): dilated superficial
capillaries
 Ehlers-Danlos: collagen disorder
 Marfan syndrome: connective tissue
 Osteogenesis imperfecta
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Acquired Vascular Problems
Senile purpura (Bateman’s): altered
connective tissue support
 Cushing syndrome: metabolic
 Scurvy: abnormal collagen
 Allergy: vascular inflammation
 Viral infection
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Bleeding Time
For vascular and platelet functions
 Duke (1910) on earlobes
 Ivy (1941) on arm with 1mm x 3mm
incision
 Mielke (1969) with 1mm x 10mm template
 1980’s: disposable devices (e.g., Simplate,
Surgicutt)
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Bleeding Time
Quantitative Platelet Disorders
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Thrombocytopenia
<100,000/ml
BT prolonged
≈10,000
Bleeding in trauma or OR
<10,000
Spontaneous, CNS bleeding
Thrombocytopenia due to destruction
 ITP (acute in children, chronic in young women)
with anti-glycoprotein
 Drug reaction
 Heparin induced thrombocytopenia
 DIC and TTP
About Thrombotic Thrombocytopeneic
Purpura (TTP)
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Disorder of systemic platelet aggregation in
microvasculature
Stimulus: unusually large vWf
In children: likely to be deficiency in vWf
metalloproteinase to break down vWf
In adults: vWf metalloproteinase inhibited by
autoantibodies
Low PLT count, intravascular hemolysis, RBC
fragmentation, high LDH
Quantitative Platelet Disorders
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Thrombocytopenia due to decreased production
 Aplastic anemia (e.g., Fanconi’s)
 Fibrosis
 Acute leukemia
 Megaloblastic anemia
 Hereditary (e.g., May-Hegglin, Wiscott-Aldrich,
Bernard-Soulier)
Splenic sequestration
HELLP syndrome (hemolysis, elevated liver enzyme,
low PLT) in pre-eclampsia
Dilution (massive transfusion)
Platelet Satellitosis in EDTA
Quantitative Platelet Disorders
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Thrombocytosis
 Primary with dysfunctions (e.g., CML,
ET)
 Post splenectomy: also see HJ, etc.
 Hemolytic anemia
 Acute hemorrhage and surgery
Pseudo Thrombocytosis
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Red cell abnormalities
 HJ bodies
 Clumped Pappenheimer bodies
 nRBC
 Malaria
 Microspherocytes and schistocytes
White cell abnormalities
 Unlysed WBC
 WBC fragments and necrobiotic cells
Platelet Count
Rees-Ecker with brilliant cresyl blue
 Brecker-Cronkite with ammonium oxalate
 Unopette: similar to BC
 Electronic counters
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Qualitative Platelet Disorders
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Berhard-Soulier: GP-Ib deficiency, adhesion
problem
Von Willebrand’s: vWF deficiency, adhesion
problem
Glanzmann’s thrombasthenia: GP-IIb/IIIa
deficiency, aggregation problem -- cannot bind
vWF and Fib
Storage pool disease: dense body defect, secretion
problem
Qualitative Platelet Problems
Aspirin: inhibits cyclo-oxygenase (COX),
secretion problem, no TxA2
 Plavix (Clopidogrel) inhibits ADP receptor
 Other medications affect GPIIa/IIIb
interaction with Fib
 Uremia, secretion problem
 Gray platelet syndrome: -granule defect
 Hypofibrinogenemia
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Aggregation Studies
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ADP
 reversible 1o wave
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 if ADP is released, then 2 wave
 abnormal with aggregation and release problems
Epinephrin
 similar to ADP
Collagen
 direct release so only one wave of aggregation
Ristocetin
 antibiotic
 aggregation only with vWF and GP-Ib
Platelet Aggregometry
Platelet
Aggregation