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Week 5: Primary Hemostasis
HemostasisHemostasis Platelet functionPlatelet function Platelet anatomyPlatelet anatomy MegakaryocyteMegakaryocyte Platelet kineticsPlatelet kinetics Platelet countPlatelet count Aggregation studiesAggregation studies Von Willebrand’sVon Willebrand’s Bernard-SoulierBernard-Soulier Aspirin (salicylate)Aspirin (salicylate)
Vascular problemsVascular problems AllergyAllergy Viral infectionViral infection Collagen disordersCollagen disorders Vitamin C deficiencyVitamin C deficiency Ehlers-DanlosEhlers-Danlos Render-Osler-Weber Render-Osler-Weber
telangiectasiatelangiectasia Petechiae, ecchymosesPetechiae, ecchymoses Bleeding timeBleeding time
Vascular Injury
Serotonin and thrombaxane ASerotonin and thrombaxane A2 2 (TxA(TxA22) for ) for
vasoconstrictionvasoconstriction Prostacyclin PGI-2 for arteriole relaxation Prostacyclin PGI-2 for arteriole relaxation
to increase blood flowto increase blood flow Exposure of basement membrane and Exposure of basement membrane and
collagen (negatively charged surface)collagen (negatively charged surface)
Process of Hemostasis
Vascular injuryVascular injury Platelet adhesion and activationPlatelet adhesion and activation Platelet aggregation (1Platelet aggregation (1oo hemostatic plug) hemostatic plug) Fibrin formation via cascade (2Fibrin formation via cascade (2oo
hemostasis)hemostasis) Clot retraction (thrombasthenin)Clot retraction (thrombasthenin) Fibrinolysis and healingFibrinolysis and healing
Role of Platelets
Surveillance for Surveillance for vascular integrityvascular integrity
Formation of 1Formation of 1oo hemostatic plughemostatic plug
Activation of 2Activation of 2oo hemostasishemostasis
HealingHealing
Platelet Formation
Megakaryoblast undergoes endomitosisMegakaryoblast undergoes endomitosis Intermiediate stage promegakaryocyte Intermiediate stage promegakaryocyte
without granuleswithout granules Megakaryocyte (2N to 64N) with over Megakaryocyte (2N to 64N) with over
100µ diameter100µ diameter IL3, GM-CSF, thrombopoietinIL3, GM-CSF, thrombopoietin 20% of platelet stored in spleen20% of platelet stored in spleen
Platelet
2 - 4µ diameter2 - 4µ diameter Round or ovalRound or oval Hyalomere - clear peripheral zoneHyalomere - clear peripheral zone Granulomere - highly stained area with Granulomere - highly stained area with
granulesgranules
Platelet Anatomy
Peripheral zone with glycoprotein Peripheral zone with glycoprotein receptorsreceptors
Structural zone with contractile Structural zone with contractile microtubules (thrombasthenin)microtubules (thrombasthenin)
Organelle zone with granulesOrganelle zone with granules Membrane with open cananicular and Membrane with open cananicular and
tubule systems for increased surface tubule systems for increased surface area and rapid releasearea and rapid release
Electron micrograph of a platelet x 25,000
Longitudinal peripheral microtubule (brown), endoplasmic reticulum (blue), mitochondria (green), glycogen (black)
Platelet Function
Adhesion to basement membrane or collagen with Adhesion to basement membrane or collagen with vWF and GP-IbvWF and GP-Ib
ActivationActivation Shape change from discoid to distortedShape change from discoid to distorted Exposure of GP-IIb/IIIa and other receptorsExposure of GP-IIb/IIIa and other receptors TxATxA22 synthesis (cyclo-oxygenase dependent) synthesis (cyclo-oxygenase dependent)
Membrane phospholipid Membrane phospholipid Arachidonic acid Arachidonic acid
Arachidonic acid Arachidonic acid TxA TxA22 and prostaglandins and prostaglandins
Platelet Function:Glycoprotein Group or Gene Families
IntegrinsIntegrins Leucine rich glycoprotein familyLeucine rich glycoprotein family Selectin familySelectin family Quadraspanin familyQuadraspanin family Immunglobulin supergene familyImmunglobulin supergene family
Integrins
Integral to membraneIntegral to membrane CaCa++++ dependent GPIIb/IIIa most abundant dependent GPIIb/IIIa most abundant Cell-cell or cell-substrata interactionCell-cell or cell-substrata interaction Receptor to Fib, vWF, vitronectin, Receptor to Fib, vWF, vitronectin,
fibronectinfibronectin
Leucine Rich GP Family
Adhesion to subendothelial collagen (COL) Adhesion to subendothelial collagen (COL) mediated by vWF by transmembrane mediated by vWF by transmembrane complex GPIb/IXcomplex GPIb/IX
Stabilizes PLT membrane by interaction Stabilizes PLT membrane by interaction with cytoskeletonwith cytoskeleton
Selectin Family
GMP-140 mediates adhesion of neutrophils GMP-140 mediates adhesion of neutrophils and monocytes to PLTand monocytes to PLT
Quadraspanin Family
Plasma membrane protein p24/CD9 Plasma membrane protein p24/CD9 interacts with GPIIb/IIIa, modulating interacts with GPIIb/IIIa, modulating adhesion moleculesadhesion molecules
Leads to CaLeads to Ca++++ increase and subsequent PLT increase and subsequent PLT activation and aggregationactivation and aggregation
Immunoglobulin Supergene Family
Functional role unclearFunctional role unclear Has role in cellular interactionsHas role in cellular interactions
Platelet Activation
11oo aggregation with agonists: ADP, epinephrin, aggregation with agonists: ADP, epinephrin, serotonin, PF4 (anti-platelet)serotonin, PF4 (anti-platelet)
Release or secretion facilitated by TxA2Release or secretion facilitated by TxA2 Dense body for 2Dense body for 2oo aggregation and aggregation and
vasoconstriction (ADP, Cavasoconstriction (ADP, Ca++++, serotonin), serotonin) -granule for heparin neutralization and clot-granule for heparin neutralization and clot Platelet derived growth factor (PDGF) for Platelet derived growth factor (PDGF) for
healinghealing RetractionRetraction
Signs and Symptoms of 1o Hemostasis Problems EcchymosesEcchymoses PetechiaePetechiae Mucus membrane bleedingMucus membrane bleeding HematomaHematoma Prolonged bleeding after minor surgeryProlonged bleeding after minor surgery
Hereditary Vascular Problems
Hereditary (spider) telangiectasis (Osler-Hereditary (spider) telangiectasis (Osler-Rendu-Weber): dilated superficial Rendu-Weber): dilated superficial capillariescapillaries
Ehlers-Danlos: collagen disorderEhlers-Danlos: collagen disorder Marfan syndrome: connective tissueMarfan syndrome: connective tissue Osteogenesis imperfectaOsteogenesis imperfecta
Acquired Vascular Problems
Senile purpura (Bateman’s): altered Senile purpura (Bateman’s): altered connective tissue supportconnective tissue support
Cushing syndrome: metabolicCushing syndrome: metabolic Scurvy: abnormal collagenScurvy: abnormal collagen Allergy: vascular inflammationAllergy: vascular inflammation Viral infectionViral infection
Bleeding Time
For vascular and platelet functionsFor vascular and platelet functions Duke (1910) on earlobesDuke (1910) on earlobes Ivy (1941) on arm with 1mm x 3mm Ivy (1941) on arm with 1mm x 3mm
incisionincision Mielke (1969) with 1mm x 10mm templateMielke (1969) with 1mm x 10mm template 1980’s: disposable devices (e.g., Simplate, 1980’s: disposable devices (e.g., Simplate,
Surgicutt)Surgicutt)
Quantitative Platelet Disorders
ThrombocytopeniaThrombocytopenia<100,000/<100,000/ll BT prolongedBT prolonged≈≈10,00010,000 Bleeding in trauma or ORBleeding in trauma or OR<10,000<10,000 Spontaneous, CNS bleedingSpontaneous, CNS bleeding
Thrombocytopenia due to destructionThrombocytopenia due to destruction ITP (acute in children, chronic in young women) with ITP (acute in children, chronic in young women) with
anti-glycoproteinanti-glycoprotein Drug reactionDrug reaction Heparin induced thrombocytopeniaHeparin induced thrombocytopenia DIC and TTPDIC and TTP
About Thrombotic Thrombocytopeneic Purpura (TTP) Disorder of systemic platelet aggregation in Disorder of systemic platelet aggregation in
microvasculaturemicrovasculature Stimulus: unusually large vWfStimulus: unusually large vWf In children: likely to be deficiency in vWf In children: likely to be deficiency in vWf
metalloproteinase to break down vWfmetalloproteinase to break down vWf In adults: vWf metalloproteinase inhibited by In adults: vWf metalloproteinase inhibited by
autoantibodiesautoantibodies Low PLT count, intravascular hemolysis, RBC Low PLT count, intravascular hemolysis, RBC
fragmentation, high LDHfragmentation, high LDH
Quantitative Platelet Disorders Thrombocytopenia due to decreased productionThrombocytopenia due to decreased production
Aplastic anemia (e.g., Fanconi’s)Aplastic anemia (e.g., Fanconi’s) FibrosisFibrosis Acute leukemiaAcute leukemia Megaloblastic anemiaMegaloblastic anemia Hereditary (e.g., May-Hegglin, Wiscott-Aldrich, Bernard-Hereditary (e.g., May-Hegglin, Wiscott-Aldrich, Bernard-
Soulier)Soulier) Splenic sequestrationSplenic sequestration HELLP syndrome (hemolysis, elevated liver enzyme, low PLT) in HELLP syndrome (hemolysis, elevated liver enzyme, low PLT) in
pre-eclampsiapre-eclampsia Dilution (massive transfusion)Dilution (massive transfusion)
Quantitative Platelet Disorders
ThrombocytosisThrombocytosis Primary with dysfunctions (e.g., CML, Primary with dysfunctions (e.g., CML,
ET)ET) Post splenectomy: also see HJ, etc.Post splenectomy: also see HJ, etc. Hemolytic anemiaHemolytic anemia Acute hemorrhage and surgeryAcute hemorrhage and surgery
Pseudo Thrombocytosis
Red cell abnormalitiesRed cell abnormalities HJ bodiesHJ bodies Clumped Pappenheimer bodiesClumped Pappenheimer bodies nRBCnRBC MalariaMalaria Microspherocytes and schistocytesMicrospherocytes and schistocytes
White cell abnormalitiesWhite cell abnormalities Unlysed WBCUnlysed WBC WBC fragments and necrobiotic cellsWBC fragments and necrobiotic cells
Platelet Count
Rees-Ecker with brilliant cresyl blueRees-Ecker with brilliant cresyl blue Brecker-Cronkite with ammonium oxalateBrecker-Cronkite with ammonium oxalate Unopette: similar to BCUnopette: similar to BC Electronic countersElectronic counters
Qualitative Platelet Disorders
Berhard-Soulier: GP-Ib deficiency, adhesion Berhard-Soulier: GP-Ib deficiency, adhesion problemproblem
Von Willebrand’s: vWF deficiency, adhesion Von Willebrand’s: vWF deficiency, adhesion problemproblem
Glanzmann’s thrombasthenia: GP-IIb/IIIa Glanzmann’s thrombasthenia: GP-IIb/IIIa deficiency, aggregation problem -- cannot bind deficiency, aggregation problem -- cannot bind vWF and FibvWF and Fib
Storage pool disease: dense body defect, secretion Storage pool disease: dense body defect, secretion problemproblem
Qualitative Platelet Problems
Aspirin: inhibits cyclo-oxygenase (COX), Aspirin: inhibits cyclo-oxygenase (COX), secretion problem, no TxAsecretion problem, no TxA22
Plavix (Clopidogrel) inhibits ADP receptorPlavix (Clopidogrel) inhibits ADP receptor Other medications affect GPIIa/IIIb Other medications affect GPIIa/IIIb
interaction with Fibinteraction with Fib Uremia, secretion problemUremia, secretion problem Gray platelet syndrome: Gray platelet syndrome: -granule defect-granule defect HypofibrinogenemiaHypofibrinogenemia
Aggregation Studies
ADPADP reversible 1reversible 1oo wave wave if ADP is released, then 2if ADP is released, then 2oo wave wave abnormal with aggregation and release problemsabnormal with aggregation and release problems
EpinephrinEpinephrin similar to ADPsimilar to ADP
CollagenCollagen direct release so only one wave of aggregationdirect release so only one wave of aggregation
RistocetinRistocetin antibioticantibiotic aggregation only with vWF and GP-Ibaggregation only with vWF and GP-Ib
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