Dr.

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

Hemostasis
BV Injury

Neural
Contact

Damage/contact.

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

Anticoagulants
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 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, 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 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 Dukes 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 doesnt 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

Hemophilia
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

Anticoagulants
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