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)

Inside of a vessel SEM x 2,500

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)

Bleeding Time

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)

Platelet Satellitosis in EDTA

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

Platelet Aggregometry

Platelet

Aggregation