Niranjan Murthy H L Asst Prof of Physiology ESIC-MC & PGIMSR, Bangalore

BV Injury



Blood Vessel Constriction

Platelet Aggregation Primary hemostatic plug Platelet Activation

Coagulation Cascade

Reduced Blood flow

Fibrin formation

Stable Hemostatic Plug

1. Constriction of vessel:  Nervous reflexes  Response of smooth muscles in vessel wall  Serotonin, TXA2 and other vasoconstrictors 2. Temporary platelet plug formation 3. True clot formation 4. Stabilization of clot and clot retraction 5. Activation of anticoagulant system

Clotting factors
I Fibrinogen II Prothrombin III Thromboplastin IV Calcium V Proaccelerin; Labile factor VI VII Proconvertin; Stable factor

VIII Antihemophilic factor IX Christmas factor; Antihemophilic factor B X Stuart-Prower factor XI Plasma thromboplastin antecedent; AHF C XII Hageman factor; Glass factor XIII Fibrin-Stabilizing factor; Laki-Lorand factor HMW-K High Molecular Weight Kininogen Pre-Ka Prekallikrein; Fletcher factor Ka Kallikrein PL Platelet phospholipid

• • • • • Thrombomodulin Antithrombin III Heparan sulphate Protein C Protein S

Fibrinolytic system
• Plasmin • Tissue plasminogen activator • Urokinase

I Fibrinogen• Synthesized in liver • Present in platelets • Formation of fibrin threads II Prothrombin• Produced in liver and Vitamin K dependent • Converted active thrombin • Activates factors I, XIII, V, VII, VIII, XI • Activates platelets • Combines with Thrombomodulin to activate protein C • Α thrombin is potent mitogen- tissue repair & remodeling

III Thromboplastin: • Phospholipid and glycoprotein combination • Not expressed in resting platelets and endothelial cells • Non-enzymatic cofactor for factor VIIa • Activate factors IX & X IV Ionic Calcium: • Cofactor • Requirement in all steps except initial 2 steps of intrinsic pathway

Factor V: • Present in plasma and platelets • Acts as receptor for Xa Factor VI: • Activated factor V Factor VII: • Initiator of extrinsic pathway • Activated by thrombin • Synthesized in liver • Vitamin K dependent

Factor VIII: • Circulate in combination with vWF • vWF increases half-life of VIII to 12hrs • Forms intrinsic thenase with IXa, Ca2+ & PL and activates factor X Factor IX: • Vitamin K dependent • Activates factor X Factor X: • Activated by extrinsic & intrinsic tenases • Xa-Va- Ca2+ form prothrombinase complex • Vitamin K dependent

Factor XI: • Combination with HMWK • Activated by XIIa, α thrombin, & autoactivation • Activates factor IX Factor XII: • Initiates coagulation on contact with negative surfaces like glass, kaolin, etc • Increased levels in pregnancy and post menopausal women • Activates factor XI Factor XIII: • Stabilizes fibrin

Plasma Kallikrein: • Link between coagulation & inflammation • Activates factor XII • Activates plasminogen • Converts HMWK to bradykinin • Activates neutrophils HMWK: • Cofactor for activation of XII and Prekallikrein • Source of bradykinin

Phases of coagulation
1. 2. 3. 4. 5. Initiation of coagulation Propogation of α Thrombin function Termination of procoagulant response Elimination of fibrin clot Tissue repair & regeneration

Coagulation pathways
Intrinsic / Contact / Accessory pathway: • Activated by contact with subendothelial collagen in the presence of HMWK and Kallikrein • Thought to have key role in DIC and promotion of thrombus stability • Factor XII, HMWK, Kallikrein, Factor XI

Extrinsic / Primary / Tissue factor pathway: • Tissue factor is expressed in extravascular cell surfaces • It is also expressed in endothelial cells and peripheral blood cells under the influence of inflammatory cytokines • Factor VII

Anticoagulant proteins
Protein C: • Proteolysis of Va and VIIIa • Activated by thrombin in combination with thrombomodulin • Deficiency causes thrombosis • Vitamin K dependent Protein S: • Enhances inactivation of Va and VIIIa of APC

Thrombomodulin: • Activates protein C & TAFI • Present on surface of endothelial cells • Inflammation reduces thrombomodulin expression- hypercoagulable states Antithrombin III: • Inhibits IIa, Xa, IXa, VIIa-tissue factor, XIa, XIIa, Kallikrein, HMWK • Anti-inflammatory & antiproliferative • Heparin increases its activity

Factors keeping blood in fluid state
Endothelial factors: 1. Antiplatelet activities• Smooth and negatively charged glycocalyx • Secrete 3 antiplatelet factors- PGI2, NO, & adenine nucleotides 2. Anticoagulant activities• Heparin in combination with antithrombin III inactivates thrombin • Protein S as a cofactor of APC inactivates Va & VIIIa • TFPI is synthesized by endothelial cell Flow of blood: • Prevents accumulation of clotting factors Fibrinolytic system:

Fibrinolytic system
Plasminogen: • Synthesized in liver • Vitamin K dependent • Activated to Plasmin / Fibrinolysin by the action of t-PA, u-PA Tissue plasminogen activator: • Secreted from endothelial cells under the stimulation of thrombin, histamine, acetylcholine, bradykinin, shear stress

Inhibitors of Fibrinolysis
• Thrombin Activable Fibrinolysis inhibitor (TAFI)

• Plasminogen activator inhibitor 1 (PAI 1)

Tests for coagulation
I Tests for vascular and platelet phases 1. Bleeding time • Duke’s method; Ivy method • Does not distinguish between vascular defects, thrombocytopenia, and platelet dysfunction • It is also determined by hematocrit, some coagulation components, skin quality and technique • Normal BT doesn’t exclude bleeding disorder

2. Platelet enumeration: • Counting chamber and automated methods • Difficult to count because of small size and tendency to adhere & aggregate • 1.5 to 4 lakhs / cubic millimeter of blood 3. Platelet volume measurement: • Increased volume in accelerated platelet turnover 4. Platelet function assays: • Platelet aggregation by using aggregometers 5. Platelet function analyzer (PFA-100)

II Tests of coagulation phase: 1. Activated partial thromboplastin time (PTT): • Test for intrinsic and common pathway • Done on citrated blood • Mixture of platelet phospholipid substitute and ionic calcium are added • Soya bean phosphatides, chloroform extracts of brain • Used to monitor heparin anticoagulation • Can detect hypercoagulable states

2. Prothrombin time: • Tests extrinsic and common pathways • Citrated blood • Plasma is recalcified in presence of excess tissue factor • Factors V, VII, X, II & I • Coumarin derivatives inhibit vitamin K dependent factors • Used to assess anticoagulant therapy

• International Normalized Ratio for PT • INR = [ PT test/PT normal]ISI • ISI = International Sensitive Index for tissue factor • Normal Range = 0.9 to 1.3 • Warfarin therapy = 2.0 to 3.0

3. Stypven time: • One step Prothrombin time • Venom from russell viper directly activates factor X 4. Assay of plasma fibrinogen 5. Thrombin time: • Diagnosis of DIC • Prolonged by heparin & abnormal fibrinogen

III Tests for fibrin degradation products (FDP) • Diagnose DIC IV Tests for factor XIII activity: • Clot stability after 24hrs V Tests for fibrinolysis VI Bio-assays of coagulation factors VII Tests fro inhibitors of coagulation

Clotting time
• Capillary tube method • Lee white method • 2-8 mins

Disorders of bleeding
• Vitamin K deficiency • Hemophilia • thrombocytopenia

Vitamin K deficiency
• Necessary for synthesis of factors II, VII, IX, X, & Protein C • Synthesized by colonic bacteria • Deficiency due to liver disorder leading to reduced bile flow, malabsorption, antibiotic therapy, colectomy • Preventive administration in newborns • Coumarins

• • • • • Deficiency of factor VIII X-linked recessive Females are carriers Hemophilia B due to factor IX deficiency Treatment by cryoprecipitate

Von willebrands factor
vWF: • Acute phase reactant (pregnancy, stress) • Glycoprotein • Stabilizes factor VIII • Forms bridge between platelets • Present in endothelial cells and platelets • Von Willebrands disease

Disseminated Intravascular Coagulation
• Defibrination syndrome / consumption coagulopathy • It is an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes. It can originate from and cause damage to the microvasculature, which if sufficiently severe, can produce organ dysfunction.

• Infections, obstetric complications, massive tissue injury, carcinomas • Test for FDP and D-dimer • Treatment: Heparin therapy; Fibrinogen and platelet infusion

Thromboembolic disorders
• Thrombus: Abnormal clot developed within the blood vessel • Embolus: detached thrombus • Causes: atherosclerosis, infections, trauma, sluggish flow of blood • Venous thrombosis, coronary thrombosis • Treatment: t-PA infusion

Therapeutic: • Low molecular weight heparin: facilitates antithrombin action • Coumarins (Dicumarol & Warfarin): competitive inhibitors of vitamin K In vitro: • Ca2+ chelating agents: citrates, oxalates • Heparin • Siliconized containers

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