Introduction to Special Coagulation Ahmad Sh. Silmi Hematology Lecturer Medical Tech. Dept IUG

Hemostasis Hemostasis: The balance between clotting and bleeding Components of Hemostasis: Vasculature Coagulation proteins Platelets Clot Formation Clot Dissolution

Blood Vessels Intact endothelium forms a thromboresistant surface endothelial cells have additional synthetic functions.

Vascular Endothelium Function Prostacyclin Thromboxane A2 ELAMs, ICAMs von Willebrand factor Vasodilation, inhibition of platelet aggregation From platelets, constrict muscular arteries Cytokines induce synthesis to promote leukocyte adhesion Promote platelet-collagen adhesion to exposed sub-endothelium

Vascular Endothelium Function Tissue factor pathway inhibitor Thrombomodulin Tissue plasminogen activator Heparan sulfate proteoglycans Tissue factor Anticoagulant- Inhibits coagulation extrinsic pathway Anticoagulant- Inhibits coagulation by activating protein C system Anticoagulant- Inhibits coagulation by activating fibrinolysis Anticoagulant- Inhibits coagulation by activating antithrombin Procoagulant- Inflammatory cytokines (IL-1, TNF) induce expression

Blood Platelets Platelets are formed from the cytoplasm of bone marrow megakaryocytes and are the smallest of the blood cells.   •Normal platelet count lies between 150-400 x 109/L •Disc-shaped, anucleated cells with complex internal structure reflecting the specific hemostatic functions of the platelet.

Blood Platelets (cont’d) Two major types of intracellular granules:  --a-granules contain: coagulation factors (fibrinogen, von Willebrand Factor, and coagulation factors V and VIII); and platelet-derived growth factor (PDGF).   --dense granules (because of their appearance on EM) contain: ADP, ATP and serotonin. a- and dense granules contents released by platelet activation.

Diagrammatic Representation of the Platelet

PLATELET ACTIVATION PATHWAYS

Platelet Activation Pathways (1) ADP Adrenaline COLLAGEN THROMBIN ADP GpIIb/IIIa Aggregation GpIIb/IIIa Aggregation GpIIb/IIIa Aggregation GpIIb/IIIa Adhesion Platelet GpIb Adrenaline Adhesion vWF Exposed Collagen Endothelium

What happens when you have an injured blood vessel? Endothelial cells Coagulation factors… Platelet Exposed sub-endothelium Tissue factor (TF)

How does a platelet plug a hole under high shear stress conditions? -GP Ib binds the exposed vWF on sub-endothelial cells Platelet Platelets roll on vWF and become activated… vWF

Platelet activation Thrombin Agonist eg. thrombin Negative charge (Priming and propagation (Intrinsic pathway) of coagulation) Agonist eg. thrombin Negative charge Activates the coagulation factors and propagates the production of higher levels of thrombin required to support haemostasis PS PS Upon platelet activation, phosphatidyl serine (PS) flips to the outer leaflet of the plasma membrane. PS PS Activation PS PS Thrombin Fibrinogen Fibrin

Platelet activation (continued from previous slide) also results in the release of the constituents of the alpha and dense granules Activation of aIIbb3 allows binding to fibrinogen Alpha granules P selectin (tm) -fibrinogen -PDGF -VWF ADP COX AA TXA2 Aspirin, inactivates cyclooxygenase (COX) Activates platelets Thromboxane A2 Arachidonic acid (AA)

Platelet activation (cont.): - aIIbb3 becomes activated and capable of binding fibrinogen Fibrinogen The co-crosslinking of two or more platelets by aIIbb3 and fibrinogen is termed ‘aggregation’ Platelet

Coagulation Cascade

Coagulation Cascade – Then & Now “Waterfall” theory developed in the 1960’s Believed that clotting occurs through a series of reactions in which serine protease zymogens are converted into active enzymes in a step-wise process For many years, intrinsic pathway was believed to be the more important clotting mechanism; didn’t take into account the significance of the extrinsic pathway (TF/FVII pathway)

Coagulation Cascade Vascular damage initiates the coagulation cascade. Results in the generation of thrombin at the site of injury. Thrombin catalyzes the conversion of fibrinogen to an insoluble fibrin (clot) matrix.

Anticoagulation proteins: Protein C, Protein S, Antithrombin III, TFPI Coagulation Cascade Intrinsic Pathway Extrinsic Pathway “Contact Activation” IX “TF Pathway” Tissue Factor + VII X XI TF-VIIa Prekallikrein HMW Kininogen Ca2+ PL Common Pathway XIIa Prothrombin XIa PL, Ca2+ (Tenase) IXa VIIIa PL, Ca2+ Xa XIII Va Anticoagulation proteins: Protein C, Protein S, Antithrombin III, TFPI (Prothrombinase) Thrombin XL- Fibrin Polymer XIIIa Fibrinogen Fibrin Monomer

Coagulation Cascade Abnormal activation of blood coagulation and/or depressed fibrinolytic activity may lead to the formation of a thrombus (clot). In contrast, a defect or deficiency in the coagulation process and/or accelerated fibrinolysis is associated with a bleeding tendency.

Coagulation Cascade The cascade scheme is organized into the INTRINSIC and EXTRINSIC pathways, converging into the COMMON pathway.

Intrinsic Pathway Intrinsic Pathway “Contact Activation”: IX Initiated by the activation of FXII involving contact factors on negatively-charged phospholipid surfaces (glass or kaolin in vitro) Factors XII, XI, IX, VIII, prekallikrein, HMW kininogen Measured with aPTT clotting assay Contact Activation IX X XI Prekallikrein HMW Kininogen XIIa Ca2+ XIa IXa Xa

Intrinsic Pathway - APTT The Activated Partial Thromboplastin Time (APTT): The clotting time in seconds of a mixture of citrated plasma, Ca2+, contact activator, and phospholipid Tests for deficiencies of pro-coagulant factors in the INTRINSIC and COMMON pathways Heparin, Warfarin, Factor Inhibitors, Lupus Anticoagulant can prolong the APTT

Extrinsic Pathway Extrinsic Pathway “Tissue Factor Pathway” Initiated when blood is exposed to TF released from damaged endothelium Tissue Factor (TF), FVII Measured with PT clotting assay IX “TF Pathway” X TF +VIIa Ca2+ PL PL, Ca2+ (Tenase) IXa VIIIa Xa

Extrinsic Pathway - PT Prothrombin Time (PT): clotting time in seconds of a mixture of thromboplastin (Tissue Factor) reagent and citrated plasma in the presence of Ca2+ Tests for deficiencies of pro-coagulant factors of the EXTRINSIC and COMMON pathways

Common Pathway X Common Pathway: Factors V, X, XIII, II (prothrombin), Fibrinogen Common Pathway Prothrombin PL, Ca2+ (Tenase) IXa VIIIa PL, Ca2+ Xa XIII Va (Prothrombinase) Thrombin XL- Fibrin Polymer XIIIa Fibrinogen Fibrin Monomer

Activated Protein C, Protein S Coagulation Cascade Intrinsic Pathway Extrinsic Pathway “Contact Activation” IX “TF Pathway” TFPI Antithrombin X XI TF:VIIa Prekallikrein HMW Kininogen PL Common Pathway XIIa Ca2+ Prothrombin XIa PL, Ca2+ (Tenase) IXa VIIIa PL, Ca2+ Xa XIII (Prothrombinase) Va Activated Protein C, Protein S Thrombin XL- Fibrin Polymer XIIIa Fibrinogen Fibrin Monomer

Anticoagulation Pathways - Antithrombin Antithrombin is the major inhibitor of thrombin, accounting for approximately 80% of thrombin inhibitory activity in plasma Antithrombin primarily inhibits Thrombin and FXa

Anticoagulation Pathways - Antithrombin FX TF FVIIa Prothrombin TFPI PL FXa Heparin (cofactor) Va Antithrombin III Thrombin

Antithrombin Antithrombin inhibits thrombin and FXa, as well as FIXa, FXIa, FXIIa and the complement enzyme C1. Antithrombin forms a 1:1 complex with the inhibited protease. The inhibition is enhanced by heparan sulphate, a heparin like substance on the endothelial cells, lining the blood vessels. Binding of heparan sulphate to antithrombin induces a conformational change in the antithrombin molecule at the reaction site. This facilitates its reaction with the enzyme.

Proposed Mechanism of AT III-Heparin System Th Lysine sites Serine site H Arginine site Heparin Thrombin Antithrombin III H AT III Th

Anticoagulation Pathways – Protein C Protein C Inhibitor (PAI-3) Trypsin Inhibitor a2-Macroglobulin FX Prothrombin APC Protein S FVIIIa FV PL, Ca2+ FXa FVa Thrombin Thrombin-Thrombomodulin Complex Protein C Fibrinogen Fibrin

The Protein C Anticoagulant Pathway Thrombus at site of injury Blood Flow Thrombomodulin Protein C APC Anticoagulation downstream Thrombin Thrombin Thrombus Thrombus at site of injury

The Protein C Anticoagulant Pathway Blood Flow VIIIa Va Thrombus Vai VIIIai Factor V Leiden PS APC APC

Activated Protein C (APC) cofactors APC has two known cofactors: Protein S and Factor V. Protein S: Protein S enhances binding of APC to the phospholipid of platelets and endothelial cells. Only free protein S has a APC cofactor function. 60% of protein S is bound to C4bBP. Factor V Factor V together with Protein S makes APC degrade FVIIIa and FVa more effectively.

Fibrinolytic Pathway Fibrinolysis is initiated when fibrin is formed and eventually dissolves the clot.

degradation products (FDP) Fibrinolytic Pathway PAI-1 Plasminogen Tissue Plasminogen Activator (t-PA) Urokinase (uPA) Exogenous: streptokinase Plasmin Inhibitor XL-Fibrin, fibrinogen Plasmin XL- fibrin degradation products (FDP)

Degradation of Fibrin/Fibrinogen Fibrinogen or Fibrin Fragment X Small Peptides Fragment Y Fragment D Fragment E Plasmin Plasmin Plasmin

Approach to evaluate Fibrinolysis D-Dimer, a measure of fibrin degradation products, is the final product formed during the fibrinolysis process by plasmin

Approach to evaluate Fibrinolysis cont, Elevated levels of D-Dimer are indicative of on-going fibrinolysis Found high in : (DVT) (PE) (DIC) D-Dimer levels also rise during the normal pregnancy and very high levels are associated with complications.

Bleeding Disorders

Clinical Features of Bleeding Disorders Platelet disorders Coagulation factor disorders Site of bleeding Skin Deep in soft tissues Mucous membranes (joints, muscles) (epistaxis, gum, vaginal, GI tract) Petechiae Yes No Ecchymoses (“bruises”) Small, superficial Large, deep Hemarthrosis / muscle bleeding Extremely rare Common Bleeding after cuts & scratches Yes No Bleeding after surgery or trauma Immediate, Delayed (1-2 days), usually mild often severe

Hematologic disorders causing bleeding Coagulation factor disorders Platelet disorders

Coagulation factor disorders Inherited bleeding disorders Hemophilia A and B Von Willebrands disease Other factor deficiencies Acquired bleeding disorders Liver disease Vitamin K deficiency/warfarin overdose DIC

Hemophilia A and B Hemophilia A Hemophilia B Coagulation factor deficiency Factor VIII Factor IX Inheritance X-linked X-linked recessive recessive Incidence 1/10,000 males 1/50,000 males Severity Related to factor level <1% - Severe - spontaneous bleeding 1-5% - Moderate - bleeding with mild injury 5-25% - Mild - bleeding with surgery or trauma Complications Soft tissue bleeding

Hemophilia Clinical manifestations (hemophilia A & B are indistinguishable) Hemarthrosis (most common) Fixed joints Soft tissue hematomas (e.g., muscle) Muscle atrophy Shortened tendons Other sites of bleeding Urinary tract CNS, neck (may be life-threatening) Prolonged bleeding after surgery or dental extractions

Hemarthrosis (acute)

von Willebrand Disease: Clinical Features von Willebrand factor Synthesis in endothelium and megakaryocytes Forms large multimer Carrier of factor VIII Anchors platelets to subendothelium Bridge between platelets Inheritance - autosomal dominant Incidence - 1/10,000 Clinical features - mucocutaneous bleeding

Laboratory evaluation of von Willebrand disease Classification Type 1 Partial quantitative deficiency Type 2 Qualitative deficiency Type 3 Total quantitative deficiency Diagnostic tests: vonWillebrand type Assay 1 2 3 ___________________________________________________ vWF antigen ß Normal ßß vWF activity ß ß ßß Multimer analysis Normal Normal Absent

Vitamin K deficiency Source of vitamin K Green vegetables Synthesized by intestinal flora Required for synthesis Factors II, VII, IX ,X,Protein C and S Causes of deficiency Malnutrition Biliary obstruction Malabsorption Antibiotic therapy Treatment Vitamin K Fresh frozen plasma

Common clinical conditions associated with Disseminated Intravascular Coagulation Activation of both coagulation and fibrinolysis Triggered by Obstetrical complications Amniotic fluid embolism Abruptio placentae Vascular disorders Reaction to toxin (e.g. snake venom, drugs) Immunologic disorders Severe allergic reaction Transplant rejection Sepsis Trauma Head injury Fat embolism Malignancy

Disseminated Intravascular Coagulation (DIC) Mechanism Systemic activation of coagulation Depletion of platelets and coagulation factors Intravascular deposition of fibrin Thrombosis of small and midsize vessels with organ failure Bleeding

Release of thromboplastic material into Pathogenesis of DIC Release of thromboplastic material into circulation Consumption of coagulation factors; presence of FDPs  aPTT  PT  TT  Fibrinogen Presence of plasmin  FDP Intravascular clot  Platelets Schistocytes Coagulation Fibrinolysis Fibrinogen Thrombin Plasmin Fibrin Monomers Fibrin(ogen) Degradation Products Fibrin Clot (intravascular) Plasmin

Classification of platelet disorders Quantitative disorders Abnormal distribution Dilution effect Decreased production Increased destruction Qualitative disorders Inherited disorders (rare) Acquired disorders Medications Chronic renal failure Cardiopulmonary bypass

Laboratory Evaluation of Bleeding Overview CBC and smear Platelet count Thrombocytopenia RBC and platelet morphology TTP, DIC, etc. Coagulation Prothrombin time Extrinsic/common pathways Partial thromboplastin time Intrinsic/common pathways Coagulation factor assays Specific factor deficiencies 50:50 mix Inhibitors (e.g., antibodies) Fibrinogen assay Decreased fibrinogen Thrombin time Qualitative/quantitative fibrinogen defects FDPs or D-dimer Fibrinolysis (DIC) Platelet function von Willebrand factor vWD Bleeding time In vivo test (non-specific) Platelet function analyzer (PFA) Qualitative platelet disorders and vWD Platelet function tests Qualitative platelet disorders

Laboratory Evaluation of the Coagulation Pathways Partial thromboplastin time (PTT) Prothrombin time (PT) Surface activating agent (Ellagic acid, kaolin) Phospholipid Calcium Thromboplastin Tissue factor Phospholipid Calcium Intrinsic pathway Extrinsic pathway Common pathway Thrombin time Thrombin Fibrin clot

Pre-analytic errors Problems with blue-top tube Partial fill tubes Vacuum leak and citrate evaporation Problems with phlebotomy Heparin contamination Wrong label Slow fill Underfill Vigorous shaking Biological effects Hct ≥55 or ≤15 Lipemia, hyperbilirubinemia, hemolysis Laboratory errors Delay in testing Prolonged incubation at 37°C Freeze/thaw deterioration

Initial Evaluation of a Bleeding Patient - 1 Normal PT Normal PTT Abnormal Urea solubility Factor XIII deficiency Normal Consider evaluating for: Mild factor deficiency Monoclonal gammopathy Abnormal fibrinolysis Platelet disorder (a2 anti-plasmin def) Vascular disorder Elevated FDPs

Initial Evaluation of a Bleeding Patient - 2 Normal PT Abnormal PTT 50:50 mix is abnormal Repeat with 50:50 mix Test for inhibitor activity: Specific factors: VIII,IX, XI Non-specific (anti-phospholipid Ab) 50:50 mix is normal Test for factor deficiency: Isolated deficiency in intrinsic pathway (factors VIII, IX, XI) Multiple factor deficiencies (rare)

Initial Evaluation of a Bleeding Patient - 3 Abnormal PT Normal PTT 50:50 mix is abnormal Repeat with 50:50 mix Test for inhibitor activity: Specific: Factor VII (rare) Non-specific: Anti-phospholipid (rare) 50:50 mix is normal Test for factor deficiency: Isolated deficiency of factor VII (rare) Multiple factor deficiencies (common) (Liver disease, vitamin K deficiency, warfarin, DIC)

Initial Evaluation of a Bleeding Patient - 4 Abnormal PT Abnormal PTT 50:50 mix is abnormal Repeat with 50:50 mix Test for inhibitor activity: Specific : Factors V, X, Prothrombin, fibrinogen (rare) Non-specific: anti-phospholipid (common) 50:50 mix is normal Test for factor deficiency: Isolated deficiency in common pathway: Factors V, X, Prothrombin, Fibrinogen Multiple factor deficiencies (common) (Liver disease, vitamin K deficiency, warfarin, DIC)

Coagulation factor deficiencies Summary Sex-linked recessive  Factors VIII and IX deficiencies cause bleeding Prolonged PTT; PT normal Autosomal recessive (rare)  Factors II, V, VII, X, XI, fibrinogen deficiencies cause bleeding Prolonged PT and/or PTT  Factor XIII deficiency is associated with bleeding and impaired wound healing PT/ PTT normal; clot solubility abnormal  Factor XII, prekallikrein, HMWK deficiencies do not cause bleeding

Thrombin Time Bypasses factors II-XII Measures rate of fibrinogen conversion to fibrin Procedure: Add thrombin with patient plasma Measure time to clot Variables: Source and quantity of thrombin

Causes of prolonged Thrombin Time Heparin Hypofibrinogenemia Dysfibrinogenemia Elevated FDPs or paraprotein Thrombin inhibitors (Hirudin) Thrombin antibodies

Approach to the thrombocytopenic patient History Is the patient bleeding? Are there symptoms of a secondary illness? (neoplasm, infection, autoimmune disease) Is there a history of medications, alcohol use, or recent transfusion? Are there risk factors for HIV infection? Is there a family history of thrombocytopenia? Do the sites of bleeding suggest a platelet defect? Assess the number and function of platelets CBC with peripheral smear Bleeding time or platelet aggregation study

Bleeding time and bleeding 5-10% of patients have a prolonged bleeding time Most of the prolonged bleeding times are due to aspirin or drug ingestion Prolonged bleeding time does not predict excess surgical blood loss Not recommended for routine testing in preoperative patients

Special Coagulation is a specialized, complex and dynamic field! Conclusion Special Coagulation is a specialized, complex and dynamic field!