Clinical Pathology Coagulation PDF

CLINICAL PATHOLOGY COAGULATION Dr. Mary Anne Bernas | September 19, 2024 OUTLINE which would be rolled onto a break in the vascular...

CLINICAL PATHOLOGY COAGULATION Dr. Mary Anne Bernas | September 19, 2024 OUTLINE which would be rolled onto a break in the vascular lining. I. OVERVIEW OF HEMOSTASIS When it has already covered the vascular breakage, it A. NORMAL HEMOSTASIS will provide receptors for the gp 1b/IX/V of platelets to B. PRIMARY HEMOSTASIS bind to and initiate primary hemostasis. Another C. ENDOTHELIUM important fact to remember for von Willebrand protein is - Prothrombotic Properties that it is responsible for carrying factor VIII in the blood. - Antithrombotic Properties This means that deficiency of this protein will also result - Endogenous Anticoagulants in a decreased concentration of Factor VIII thus causing D. COAGULATION CASCADE symptoms similar to that of HEMOPHILIA A. aPTT - Surface-Bound Proenzymes results will also become prolonged due to the deficiency - vitamin K-Dependent Zymogens of Factor VIII. - Cofactors/Substrates Thrombotic Thrombocytopenic Purpura (TTP)- This E. SECONDARY HEMOSTASIS disease is caused by a deficiency in a regulator protein F. SUMMARY called ADAMTS-13. This regulator protein is responsible G. TYPICAL CLINICAL PRESENTATION OF for cleaving VWF into small pieces. This is important BLEEDING DISORDERS because there will be instances that VWF will be H. SCREENING TESTS FOR COAGULATION activated even without collagen exposure due to a break DISORDERS in the vascular wall which can cause unnecessary I. FIBRINOLYTIC SYSTEM hemostasis which can lead to the decrease of platelets - Fibrinolysis due to excessive consumption hence its name. - Inhibitors Bernard Soulier syndrome - This is due to a deficiency in the platelet receptor IB/IX/V. As mentioned before, this II. PLATELET FUNCTION TESTS glycoprotein is responsible for binding to activated VWF. A. BLEEDING TIME If there is no receptor for VWF, platelet adhesion will not - Duke Method occur even if activated VWF is present - Ivy Method B. BT: INTERPRETATION C. BT: SOURCES OF ERROR C. ENDOTHELIUM Endothelial cells are central regulators of hemostasis III. COAGULATION TESTS The balance between the antithrombotic and A. Lee-White Method prothrombotic activities of endothelium determines B. aPTT whether thrombus formation, propagation, or dissolution C. PT occur. D. TT Endothelial cells are activated by: E. INR → Trauma F. Test for Fibrinolytic Pathway → Infectious agents → Hemodynamic forces → Plasma mediators I. OVERVIEW OF HEMOSTASIS → Cytokines PROTHROMBOTIC PROPERTIES OF A. NORMAL HEMOSTASIS ENDOTHELIUM Components: Platelet Effects: → Vascular Wall (Endothelium) → Interaction with ECM (vWF) after injury → Platelets 2. Procoagulant Effects: → Coagulation Cascade → Tissue Factor → Fibrinolytic System 3. Anti-fibrinolytic effect: → Secretion of Plasminogen Activator Inhibitor (PAI) B. PRIMARY HEMOSTASIS ANTI-THROMBOTIC PROPERTIES OF Primary hemostasis is the formation of the platelet plug. ENDOTHELIUM → Steps in Primary Hemostasis: Antiplatelet Effects: ▪ Disruption of endothelium: → Intact endothelium − exposes von Willebrand factor (vWF) and ▪ Serves as a barrier that shield platelets from collagen: subendothelial vWF and collage − Promote platelet adherence and activation. → Prostacyclin ▪ Activation results to: ▪ Produced by COX-1 − Dramatic shape change of platelets (small → Nitric Oxide rounded discs to flat plates with spiky protrusions ▪ Product of eNOS that markedly increase surface area) → Adenosine diphosphatase − Release of secretory granules ▪ Degrades ADP ▪ Within a few minutes, platelet aggregation happens Anticoagulant Effects: to form a PRIMARY HEMOSTATIC PLUG → Normal endothelium shields coagulation factors from tissue factor in vessel walls Additional information: → Thrombomodulin and Endothelial Protein C Receptor vWD - This is due to missing or deficient von Willebrand ▪ binds to thrombin and protein C, respectively→ protein in the blood. This protein looks like a carpet thrombin loses its ability to activate coagulation factor and platelets → instead it cleaves and Transcribed by: Madera (TC); Barrios, Caspe, Delmar, Manibpel, Mungcal, Pomperada, Usman (TW) NMD 2027  Coagulation activates protein C (requires protein S as cofactor) → Activates protein C/S complex → This complex is a potent inhibitor of coagulation cofactors (Va and VIIIa) → Heparin-like molecules ▪ Bind and activate antithrombin III → it will inhibit thrombin, factors IXa, Xa, XIa, and XIIa → Tissue Factor Pathway Inhibitor ▪ It binds and inhibits tissue factor/factor VIIa complexes ▪ It requires protein S as cofactor Fibrinolytic Effects → tPA (tissue plasminogen activator) ▪ Synthesized by endothelium as a counterregulatory mechanism. ▪ Promotes the dissolution of blood clots by converting plasminogen into plasmin→ breaks down the fibrin mesh within the clot→ prevents excessive clot formation →maintains healthy blood flow EXOGENOUS ANTICOAGULANTS Antithrombin III → inhibit the activity of thrombin and other serine proteases (IXa, Xa, XIa, and XIIa) → activated by binding to heparin-like molecules on endothelial cells Protein C and Protein S → vitamin K–dependent proteins that act in a complex to inactivate factors Va and VIIIa Tissue Factor Pathway Inhibitor → produced by endothelium that inactivates tissue factor–factor VIIa complexes Factor Classification → Can be: ▪ Intrinsic D. COAGULATION CASCADE ▪ Extrinsic Third arm in homeostatic process ▪ Common A series of amplifying enzymatic reactions that lead to the → Or can be: deposition of an insoluble fibrin clot. ▪ Surface-bound zymogens Each step in the series proteolytically cleaves an inactive ▪ Vitamin K-Dependent zymogens proenzyme into an active enzyme, culminating in thrombin ▪ Cofactor/Substrates formation. SURFACE-BOUND PROENZYMES FACTOR INFO FXII, Prekallikrein, FXI I Fibrinogen: a globulin protein; substrate These are also known as the “contact system” for thrombin & precursor of fibrin → because FXII auto activates when associated with a II Prothrombin: converted to thrombin (lla) in negatively charged surface (glass tube and several presence of Ca++ physiologic substances) III Thromboplastin: the tissue factor This autoactivation phenomenon allows for a common IV Ionized Calcium: active form for activation laboratory test, the activated partial thromboplastin time of thromboplastin & conversion of (APTT) prothrombin to thrombin, F XIII to F XIIIa, Assays to assess coagulation proteins (e.g. APTT) are F X to F Xa based on the event of FXII autoactivation. V Proaccelerin: accelerates conversion of prothrombin to thrombin VITAMIN K-DEPENDENT ZYMOGENS VII Proconvertin: activates F X FVII, FIX, FX, FII, Protein C VIII Antihemophilic: F VIIIc/vWF Phospholipid bound IX Plasma thromboplastin component: affects Can be activated upon contact with phospholipids amount instead of rate Make up the physiologically important hemostatic system X Stuart-Power: final common pathway; activity Vitamin K is required for an essential γ-carboxylation related to F VII reaction that takes place on the glutamic acid residue of XI Plasma thromboplastin antecedent: each of these proteins located in their amino terminal intrinsic thromboplastin generation regions XII Hageman: surface contact factor This carboxylation allows binding of these proteins to XIII with Ca++, stabilizes fibrin polymers phospholipids and cell membranes, where they are activated. Without carboxylation, malfunction of the hemostatic system occurs. Protein C: a vitamin K–dependent phospholipid-bound zymogen, but when activated, it functions as an inhibitor Protein S: a vitamin K–dependent cofactor for activated protein C 2 of 8  Coagulation COFACTORS/SUBSTRATES → The disease mentioned above will have normal results HMW Kininogen, FVIII, FV, Fibrinogen, Protein S with RISTOCETIN test but will have abnormal results Considered to act as receptors for coagulation proteins with ACE (ADP, COLLAGEN, EPINEPHRINE) Accelerate the reactions in which they participate Functions as a substrate of one or more enzymes that participate in their formation and inactivation G. TYPICAL CLINICAL Proteins of the Plasma Coagulation System PRESENTATIONS OF BLEEDING SURFACE-BOUND VIT. COFACTOR/ DISORDERS ZYMOGENS K-DEPENDENT SUBSTRATES ZYMOGENS Factor XII Factor VII High molecular weight kininogen Prekallikrein Factor IX Factor VIII Factor XI Factor X Factor V Factor II Fibrinogen Protein C Protein S Additional information: Factors with no ‘a’ after the roman numeral are considered as a zymogen or the inactive form of the coagulation factor. Only after an ‘a’ has been placed will it become the active form of the coagulation factor. Example: → Factor II-prothrombin → Factor IIa- thrombin E. SECONDARY HEMOSTASIS Secondary hemostasis: deposition of fibrin H. SCREENING TESTS FOR Steps in Secondary Hemostasis: COAGULATION DISORDERS → 1. Vascular injury exposes TISSUE FACTOR (a Screening tests are used to diagnose the basis of bleeding membrane-bound procoagulant glycoprotein) problems in patients. → 2. TF binds and activates Factor VII: THROMBIN Primary hemostasis refers to platelet reactivity at the site generation of vessel injury. → 3. Thrombin cleaves circulating fibrinogen into insoluble - Platelet count is an example of a screening assay FIBRIN, creating a fibrin meshwork for primary hemostasis. → 4. Fibrin is a potent activator of platelets leading to Secondary hemostasis involves the coagulation of additional PLATELET AGGREGATION plasma. → 5. Result: CONSOLIDATION of initial platelet plug Coagulation assays, when performed on plasma samples, (AKA: Secondary Hemostasis) help identify almost all diagnostic categories for coagulopathy. The cascade hypothesis for hemostasis categorizes F. SUMMARY coagulation proteins into: 1. Intrinsic system A. Primary Hemostasis (Platelet Plug Formation): - 12,11,9,8,PK,HK → 1. Adhesion: platelet to non platelet interaction 2. Extrinsic system → 2. Platelet shape change and secretion - Tissue Factor (3), 7 → 3. Aggregation: platelet to platelet interaction 3. Common pathway B. Secondary Hemostasis (Fibrin Clot Formation): - 10,5,2,1 → 1. Formation of Thrombin This approach aids in the practical diagnosis of affected → 2. Formation of Stable Fibrin Clot proteins based on screening test results Additional information: Secondary hemostasis is more concerned with PLATELET AGGREGATION which is all about the formation of the stable fibrin clot. This phase also involves the interplay of coagulation factors. → This means that anything involving coagulation factors belongs to secondary hemostasis. ▪ This can be one of your guidelines in memorizing the difference between primary and secondary hemostasis. An example of a disease involving this phase would be Glanzmann’s thrombasthenia. REMEMBER: 3 of 8  Coagulation - Plasma concentration of α2-antiplasmin is only about half the plasma concentration of plasminogen Thrombin activatable fibrinolysis inhibitor (TAFI) - Cleaves the C-terminal lysine residues of fibrin, preventing the co-activation of plasminogen by fibrin II. PLATELET FUNCTION TESTS BLEEDING TIME I. FIBRINOLYTIC SYSTEM Measures the time taken for blood vessels constriction and platelet plug formation Fibrinolysis Detects defective platelet function Fibrinolysis is a process by which cross-linked fibrin is Screening tests for acquired and congenital platelet broken down in order to avoid excessive thrombosis. defects Proteins involved in fibrinolysis: Do not perform if platelet count is < 75, 000 1. Plasmin: - Directly degrades soluble fibrinogen to DUKE METHOD produce fibrinogen degradation products Earlobe - Directly degrades insoluble, cross-linked IVY METHOD fibrin Preferred method 2. Plasminogen: Forearm is used - An inactive protein (zymogen) made in Pressure & incision can be fairly well standardized endothelial cells and found in tissues, Allows multiple testing urine, plasma, lysosomal granules & Easier control of bleeding if it becomes excessive vascular endothelium 3. Tissue plasminogen activator (from endothelial PROCEDURE: cells): Inflate sphygmomanometer to 40 mmHg - Converts plasminogen to its active form, Make 3 cuts on volar aspect of forearm plasmin → 2mm x 2mm Summarized steps in Fibrinolysis (refer to figure 8): → 1 cm apart 1. Zymogen plasminogen is converted to plasmin by → Avoid superficial veins tissue plasminogen activator (tPA), single-chain → Start stopwatch at time of incision urokinase plasminogen activator (ScuPA), and Blot bleeding point every 30 seconds two-chain urokinase plasminogen activator (TcuPA). → Touch only tip of each drop 2. Formed plasmin degrades fibrinogen or fibrin to → Note time when bleeding stops and report to nearest form fibrinogen or fibrin degradation products, 30 seconds respectively BLEEDING TIME: INTERPRETATION Reference range: → Ivy: 1-7 minutes → Duke:1-6 minutes If prolonged, nearly always indicated a clinical abnormality Increased in states with abnormal platelet function: → Thrombasthenia → Von Willebrand’s disease → Storage pool disease → Bernard Soulier syndrome → Prolonged fibrinolytic states Inhibitors of Fibrinolysis Plasminogen activator inhibitor 1 (PAI-1) BLEEDING TIME: SOURCES OF - Major inhibitor of tPA and TcuPA ERROR - Rapidly inhibits tPA α2-Antiplasmin (α2-AP) - Serine protease inhibitor FALSE POSITIVE - Major inhibitor of formed plasmin BP cuff maintained too high - Inactivates plasmin by forming inhibitory complex Incision too deep with circulating plasmin Disturbing clot with filter paper → blotting it frequently 4 of 8  Coagulation → touching the incision wound (touch only the tip of the Called “activated” since the negatively-charged surface blood during blotting to avoid interfering with the activator (glass or silica) speeds up the rate of reaction clotting process) Normal reference range: 20 to 35 seconds Fibrinogen