Week 5: Primary Hemostasis

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Week 5 Primary Hemostasis

• Hemostasis
• Platelet function
• Platelet anatomy
• Megakaryocyte
• Platelet kinetics
• Platelet count
• Aggregation studies
• Von Willebrands
• Bernard-Soulier
• Aspirin (salicylate)

• Vascular problems
• Allergy
• Viral infection
• Collagen disorders
• Vitamin C deficiency
• Ehlers-Danlos
• Render-Osler-Weber telangiectasia
• Petechiae, ecchymoses
• Bleeding time

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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
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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)
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Platelet Function

• 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

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Platelet FunctionGlycoprotein 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

• GMP-140 mediates adhesion of neutrophils and
  monocytes to PLT

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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

• 1o aggregation with agonists ADP, epinephrin,
  serotonin, PF4 (anti-platelet)
• Release or secretion facilitated by TxA2
• Dense body for 2o aggregation and
  vasoconstriction (ADP, Ca, serotonin)
• ?-granule for heparin neutralization and clot
• Platelet derived growth factor (PDGF) for healing
• Retraction

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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
  (Osler-Rendu-Weber) dilated superficial
  capillaries
• Ehlers-Danlos collagen disorder
• Marfan syndrome connective tissue
• Osteogenesis imperfecta

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Acquired Vascular Problems

• Senile purpura (Batemans) 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
• 1980s disposable devices (e.g., Simplate,
  Surgicutt)

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Bleeding Time
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Quantitative Platelet Disorders

• Thrombocytopenia
• lt100,000/ml BT prolonged
• 10,000 Bleeding in trauma or OR
• lt10,000 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

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About Thrombotic Thrombocytopeneic Purpura (TTP)

• 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

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Quantitative Platelet Disorders

• Thrombocytopenia due to decreased production
• Aplastic anemia (e.g., Fanconis)
• 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)

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Platelet Satellitosis in EDTA
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Quantitative Platelet Disorders

• Thrombocytosis
• Primary with dysfunctions (e.g., CML, ET)
• Post splenectomy also see HJ, etc.
• Hemolytic anemia
• Acute hemorrhage and surgery

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Pseudo Thrombocytosis

• Red cell abnormalities
• HJ bodies
• Clumped Pappenheimer bodies
• nRBC
• Malaria
• Microspherocytes and schistocytes
• White cell abnormalities
• Unlysed WBC
• WBC fragments and necrobiotic cells

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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

• Berhard-Soulier GP-Ib deficiency, adhesion
  problem
• Von Willebrands vWF deficiency, adhesion
  problem
• Glanzmanns thrombasthenia GP-IIb/IIIa
  deficiency, aggregation problem -- cannot bind
  vWF and Fib
• Storage pool disease dense body defect,
  secretion problem

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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 a-granule defect
• Hypofibrinogenemia

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Aggregation Studies

• ADP
• reversible 1o wave
• if ADP is released, then 2o 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

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Platelet Aggregometry
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Platelet Aggregation