Platelets/Introduction to Hemostasis Monday, April 21 – 10:00 am HEMOSTASIS Primary vs. Secondary vs. Tertiary • Primary Hemostasis – Platelet Plug Formation Hemostasis/Thrombosis I – Dependent on normal platelet number & function – Initial Manifestation of Clot Formation •Secondary Hemostasis Normal Hemostasis/Thrombosis; – Activation of Clotting Cascade Î Deposition & Stabilization of Assessment of Clotting System Fibrin • Tertiary Hemostasis – Dissolution of Fibrin Clot – Dependent on Plasminogen Activation HEMOSTATIC DISORDERS HEMOSTASIS & THROMBOSIS Suspicions • Spontaneous bleeding •Platel ets •Prolonged or excessive bleeding after • Coagulation Cascade procedures or trauma • Regulation of Coagulation • Simultaneous bleeding from multiple sites HEMOSTATIC DEFECTS CIRCULATORY SYSTEM Bleeding Platelet Coagulation Problem Disorder Abnormality • Low volume, high pressure system Petechiae + - • Efficient for nutrient delivery to tissues • Prone to leakage 2º to endothelial surface Bleeding M ucous Deep Tissue damage Sites MbM em b rane • Small volume loss large decrease in Î Time of Immediate Delayed nutrient delivery onset of • Minimal extravasation in critical areas bleeding Î irreparable damage/death of organism Ecchym oses ++ /Hem atom as 1 Platelets/Introduction to Hemostasis Monday, April 21 – 10:00 am HEMOSTATIC ABNORMALITIES "Screening Tests" (History) • Dental Extractions • Prior Surgery/Biopsy • EbiiEasy bruising • Heavy menses • Deliveries • Blood transfusion PLATELET ACTIVATION PLATELETS • Anucleate cellular fragments; multiple granules, multiple organelles • Synthesis controlled by IL-6, IL-3, IL-11, & thrombopoietin • Circulate as inactive, non-binding concave discs • On stimulation, undergo major shape change • Develop receptors for clotting factors • Develop ability to bind to each other & subendothelium Resting vWF Platelets Platelet Adhesion Initial Thrombin Formation IIa TR II IIa X Xa VIIa -TF 2 Platelets/Introduction to Hemostasis Monday, April 21 – 10:00 am PLATELET ACTIVATION II IIa X Xa VIIa Platelet -TF Activation Platelet Release TxA2, ADP, Serotonin, Fibrinogen, Thrombospondin II IIa X Xa VIIa -TF PLATELET PLUG FORMATION COAGULATION CASCADE General Features • Zymogens converted to enzymes by limited proteolysis • Complex formation requiring calcium, phospholipid surface , cofactors • Thrombin converts fibrinogen to fibrin monomer • Fibrin monomer crosslinked to fibrin • Forms "glue" for platelet plug 3 Platelets/Introduction to Hemostasis Monday, April 21 – 10:00 am COAGULATION CASCADE Platelet Release TxA2, ADP, Serotonin, Fibrinogen, Thrombospondin VIII VIIIa V Va II IIa IX IXa X Xa VIIa VIIa -TF -TF THROMBIN Tenase/Prothrombinase complex assembly • Serine protease X Xa • Cleaves fibrinogen to fibrin II IIa VIIIa/IXa • Activates V to Va, VIII to VIIIa Va/Xa VIIIa R • Activates platelets-cleaves thrombin receptor Va R • Activates XIII to XIIIa • In presence of thrombomodulin activates Protein C to APC FIBRIN FORMATION A A COAGULATION CASCADE B B T F XIIIa 4 Platelets/Introduction to Hemostasis Monday, April 21 – 10:00 am VITAMIN K DEPENDENT CARBOXYLASE BLEEDING TIME vs. PLATELET COUNT • Post-translational modification 400 • Factors II, VII, IX, X; proteins C & S 350 • Converts 1st 7-12 glutamic acids to 300 250 γ-carboxyglutamic acid nt (x 1000) u 200 • Confers calcium binding and lipid binding on these 150 proteins 100 50 Platelet co • Without vitamin K, secrete des- 0 γ-carboxyglutamic acid containing proteins 3.5 4 4.5 5 5.5 7 9 12 15 25 30 (inactive in coagulation) Minutes γ -CARBOXYGLUTAMIC ACID COAGULATION CASCADE COO- COO- COO- CH2 CH O2,CO2 CH2 CH2 NH + CH NH + CH 3 Vit K 3 Carboxylase COO- COO- Glu γ-carboxy Glu PLATELET FUNCTION STUDIES Clotting Tests • Recalcification times • Blood collected in sodium citrate, a weak • Bleeding Time calcium chelator • Plate let C ount • AtdidtdliAmount designed to drop calcium • Platelet Aggregation Studies concentration to < 1 mM • At that level, blood won’t clot spontaneously • RBC’s & platelets centrifuged off, leaving plasma (unclotted liquid portion of blood) 5 Platelets/Introduction to Hemostasis Monday, April 21 – 10:00 am COAGULATION CASCADE INTRINSIC PATHWAY Prothrombin Time FXII FXIIa Surface Active aPTT Components •Mixture of: FXI HMWK – 50% patient’s platelet poor plasma FXIa FIX Ca+2 FIXa +2 – 25% Mixture of Tissue Factor & phospholipid T Ca species VIII VIIIa VIIIa/IXa/PL Middle Components FX +2 – 25% Calcium chloride (to bring final calcium Ca FXa Ca+2 concentration to c. 3-5 mM) T – Time to clot formation measured V Va Va/Xa/PL PT Ca+2 Common Pathway T FG F COAGULATION CASCADE COAGULATION CASCADE EXTRINSIC PATHWAY Contact Phase Prothrombin Time (PT) TF FVII Prekallikrein FVIIa Ca+2 Kallikrein VIIa/TF F XII Middle Components FX Ca+2 HMWK FXa Ca+2 T F XIIa V Va Va/Xa/PL PT Ca+2 Common Pathway T FG F XI F F XIa COAGULATION CASCADE Intermediate Phase aPTT (activated partial thromboplastin time) F VII/TF Ca+2 • 50% patient’s platelet-poor plasma F VIIa/TF • 25% mixture of phospholipid & surface active agent (Celite, Kaolin) XIa VIIa/TF/PL • 25% Calcium Chloride to bring calcium F IX Ca+2 Ca+2 concentration to 3-5 mM T • Time to clot formation measured VIII VIIIa + F IXa IXa/VIIIa/PL Tenase Complex +2 F X Ca F Xa 6 Platelets/Introduction to Hemostasis Monday, April 21 – 10:00 am COAGULATION CASCADE Common Pathway IXa/VIIIa/PL VIIa/TF/PL F X +2 F X Ca+2 Ca V Va+ FXF Xa X/V/PLXa/Va/PL PT Ca+2 Prothrombinase Complex T Fibrinogen Fibrin monomer CLOTTING ASSAYS Specific factors/Inhibitors • Deficiency states of every clotting protein described • 50% of any clotting factor will yield normal PT and/or aPTT • Mix 1/2 patient plasma with 1/2 factor deficient plasma; then perform PT and/or aPTT • Factor deficient plasma with missing factor will Î prolonged clotting time; all others WNL • If more than one clotting assay prolonged, implies inhibitor to clotting protein 7.