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

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)

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

Role of Platelets
Surveillance for vascular integrity Formation of 1o hemostatic plug Activation of 2o hemostasis Healing

Platelet Formation
Megakaryoblast undergoes endomitosis Intermiediate stage promegakaryocyte without granules Megakaryocyte (2N to 64N) with over 100 diameter IL3, GM-CSF, thrombopoietin 20% of platelet stored in spleen

Platelet
2 - 4 diameter Round or oval Hyalomere - clear peripheral zone Granulomere - highly stained area with granules

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

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 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

Integrins
Integral to membrane Ca++ dependent GPIIb/IIIa most abundant Cell-cell or cell-substrata interaction Receptor to Fib, vWF, vitronectin, fibronectin

Leucine Rich GP Family

Adhesion to subendothelial collagen (COL) mediated by vWF by transmembrane complex GPIb/IX Stabilizes PLT membrane by interaction with cytoskeleton

Selectin Family

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

Immunoglobulin Supergene Family

Functional role unclear Has role in cellular interactions

Platelet Activation

1o aggregation with agonists: ADP, epinephrin, serotonin, PF4 (anti-platelet) Release or secretion facilitated by TxA2 o 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

Hereditary Vascular Problems

Hereditary (spider) telangiectasis (OslerRendu-Weber): dilated superficial capillaries Ehlers-Danlos: collagen disorder Marfan syndrome: connective tissue Osteogenesis imperfecta

Acquired Vascular Problems

Senile purpura (Batemans): altered connective tissue support Cushing syndrome: metabolic Scurvy: abnormal collagen Allergy: vascular inflammation Viral infection

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)

Bleeding Time

Quantitative Platelet Disorders

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)

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

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)

Platelet Satellitosis in EDTA

Quantitative Platelet Disorders

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

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

Platelet Count
Rees-Ecker with brilliant cresyl blue Brecker-Cronkite with ammonium oxalate Unopette: similar to BC Electronic counters

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

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

Aggregation Studies

ADP reversible 1o wave o 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