HEMOSTATIC & COAGULATION PHYSIOLOGY

Regulation of hemostatic mechanism involves complex interaction between vessels walls, blood cell elements and a variety of plasma proteins.

Blood clotting results from :Interaction between endothelial cells and platelets the primary hemo-static plug.The coagulation phase thrombin is generated & fibrin developsFormation of peptide bonds stabilization of the fibrin network.

Fibrinolysis is the process of enzymatic degradation of fibrin clots whereby : coagulation activity is limited to the area surrounding vessels wall injury and patency of vessels is maintained or restored

PLATELET STRUCTUREMajor Structure features:- A typical cell membrane.- Circumferential mcrotubular system- Dense tubular system- Various granules- Externally communicating open canalicular system.

Platelet MembraneBilipid membrane and membrane protein. Bilipid membrane around the platelet contains several important glycoproteins that function as surface receptors. Bilipid membrane is also the site of complex coagulation activities of the platelet.

Bilipid membrane (cont.)1. Glycoprotein Ib (GP Ib). MW of about 140 kD. It serves as the binding site for vWf.2. Glycoprotein IIb-IIIa (GP IIb-IIIa). A prominent Ca-dependent membrane protein complex that function as a fibrinogen receptor.

Microtubules and Microfilaments1. Microtubules are composed of tubulin and participate in cytoskeletal support and in contraction of the stimulated cell.2. Microfilaments contain actin and participate in platelet pseudopod formation.

Dense Tubular System- Electron dense material.- Selectively binds divalent cations and serves as the platelet Ca reservoir.- Site of PLT cyclooxygenase and of prostaglandin synthesis.

Granules1. Dense granules contain high concentrations of ADP and Ca as well as serotonin. These substances are released upon PLT stimulation,enhance PLT aggregation.2. Granules store a variety of proteins that are secreted by stimulated PLT. These includes PF4, -tromboglobulin, PDGF, Fibrinoen, F V, vWf and various glycoproteins important to adhesion (thrombospondin & fibronectin)

Canaliculi Open canalicular system is a complex network of surface membrane invaginations that look like vacuoles.Increase the PLT surface area.The contents of PLT granules are released through this system

Platelet PhysiologyWhen a blood vessels is injured:- Subendothelial tissue is exposed.- PLT adhere to Subendothelial tissue.- Adherence mediated by vWf form a bridge between subendothelial tissue and GP Ib.- Thrombin stimulates membrane phospholipids to release archidonic acid .

Platelet Physiology AA is converted to cyclic endoperoxides and TxA2. Stimulate granules and dense bodies. High concentration locally thrombin, TxA2 and ADP will change GP IIb-IIIa becomes receptor for fibrinogen to forms a bond between adjacent PLT creating a hemostatic plug.

PhospholipidPhospholipase Arachidonic acid Cyclo-oxygenase (aspirin inhibits) PGG2Peroxidase PGH2Thromboxane synthetase Prostacyclin synthetase (platelets) (endothelium) TxA2 PGI2 H2O TxB2 6-keto PGIa

Fig. 8. Arachidonic acid metabolism in pletelets endothelium.

Endothelium ContributionMetabolize AA to Prostacyclin (PGI2).PGI2 has major contribution as antithrombotic in intact endothelium.Low dose aspirin completely block TxA2 production.

Coagulation phase :Conversion of fibrinogen (soluble plasma protein) into insoluble fibrin affected by highly specific enzymatic action of thrombin.Thrombin must be generated from zymogen, prothrombin, by a series of reactions between serine proteases, co factors & lipid moieties.

2. Coagulation factors may be group as follows : contact factors, thrombin - sensitive factors, & vit K- dependent factors.Classically, the generation of thrombin is described as occuring through the extrinsic or intrinsic systems. Table 1 summarizes the features of the coagulation factors.

Table 1. Plasma coagulation factors

Factor Alternative namePath-way Half-life (hours)IIIIIIVVIIVIIIIXXXIXIIXIII HMW kininogenPrekallikreinFibrinogen ProthrombinTissue factorProaccelerinProconvertinAntihemophilic factorChristmas factorStuart - Prower factorPlasma thromboplastin antecedentHageman factorFibrin - stabilizing factorFitzgerald factorFletcher factorCCICEIII,E,CIIIII90-12048-120Not available12-242-610-1218-3024-6045-8040-7072-20015048-52

Coagulation systems : The extrinsic systems : triggered by TF/ tissue factor (complete thromboplastin).- TF + VIIa + Ca activates F X (F Xa) - F Xa + V + Lipid (TF) extrinsic pro- thrombinase (converts prothrombin thrombin). (Fig 1).

Prothrombin

(Ca 2+) X (Ca 2+)Test : PT (Quick) Thrombin

Fig.1.Generation of thrombin via the extrinsic system. (TF = tissue fct ; PT = prothrombin ; = prothrombin complex ; = F X activating complex).TFVIIaXaVLipid (TF)

2. The intrinsic system :a. Contains all the elements necessary for clotting.b.Instead of tissue thromboplastin, the lipid moiety in this system is PF3.c.The contact fcts (F XII, XI, prekallikrein/PK, High Molecular-Weight Kininogen/HMWK) are activated by exposure to negatively charged glass surfaces & other substances (ellagic acid,uric acid crystals,skin,collagen & antibody complexes)

-F XIIa in the presence of PK and HMWK activates F XI. -F XIa activates IX, which in a complex with VIII, lipid (PF3) and Calcium activates F X.-F Xa, V and lipid (PF3) comprise intrinsic prothrombinase. (fig. 2).

Contact factorsProthrombin

XIaIX

(Ca 2+) XTest : aPTT (Ca 2+)Thrombin

Fig.2. Generation of thrombin via the intrinsic system. (PK = prekallikrein; HMWK/ high molecular weight kininogen ; PF3 = platelet factor 3 ; aPTT = activated partial thrombplastin time ; = prothrombinase complex ; = F X activa-ting complex.

PKXII XIIaHMWK

XIIXaVIIIPF3XaVLipid (PF3)

e. -Screening test : aPTT screens for all the coagulation factors except F VII. -intrinsic & extrinsic pathways converge at the F X and V level. -A coagulation factor deficiency (or inhibitor) at this level results in abnormal screening test for both system.

F VIII & V are cofactors for F IXa & Xa. When initial traces of thrombin are genera-ted, F VIII & V are activated (VIIIa & Va). Larger amounts of thrombin results in destruction of these factors. (Fig.3) Interlinkage between the intrinsic & extrinsic systems occurs at several levels. The most important of these is the ability of TF and factor VIIa to activate F IX. (Fig.4)

Prothrombin Intrinsic system Extrinsic system

Thrombin

Fig.3. Autocatalytic action of thrombin. (TF= tissue fct ; PF3 = platelet fct 3)IXa VIII VIIIaPF3XaV VaPF3XaVa VTF

Prothrombin Intrinsic system Extrinsic system

XIa (Ca2+) IX

(Ca2+)

X Xa

Thrombin

Fig. 4. Linkage between extrinsic and intrinsic systems. Several interaction occur at various levels of the two systems. Primary among these is the ability of TF and F VIIa and F IX.

TFVIIaIXaVIIIPF3

3. Fibrin generationWhen thrombin acts on the fibrinogen molecu-le, two pairs of tiny fibrinopeptides (A & B) are cleaved off, yielding activated fibrin mono-mer units. The monomers polymerize to form a loose, unstable fibrin clot, which can be converted to a stable fibrin clot. (Fig.5)

ThrombinFibrinogen Fibrin Fibrin Polymer monomer (hydrogen bonded)

Fig. 5. Fibrin generation

Screening test : Thrombin time (TT) TT is prolonged due to : - fibrinogen concentration is very low (

b. Additional testIn the reptile time test, the snake venom employed selectively cleaves fibrinopeptide A from the fibrinogen molecule. Clotting will proceed even though fibrinopeptide B remains intact.This test can be valuable because it is pro-longed in the same circumstances as the TT except that is not prolonged by the presence of heparin.

Fibrin stabilizationFinal stage of coagulationF XIII, a transaminase, is activated by throm-bin and converts the hydrogen-bonded fibrin strands into more stable, covalent peptide bonds. (Fig.6)Screening test : Deficiency of F XIIIa results in clots that dissolve in 5M urea or 1% monochloro-acetic acid.

XIII Thrombin

XIIIa Fibrin polymer Fibrin polymer (hydrogen bonded) (peptide bonded)Fig. 6. Fibrin stabilization. The initially formed clot of polymers of fibrin monomer is stabilized by thrombin activated F XIII. F XIIIa converts the fibrin strands into covalently bonded, stable fibrin.

FibrinolysisDeposition of fibrin is associated with activati-on of fibinolysisFibrin is a substrate for the proteolytic action of plasmin.Plasmin is normally present in its inactive, zymogen form (plasminogen) in blood, urine and other body fluids.Plaminogen may be activated intrinsically by the contact system of coagulation or extrin-sically by TPA/tissue plasminogen activator.

Exogenous activation may be induced therapeutically by administration of urokinase or streptokinase. (Fig.7)Screening Test :TT may be prolonged due to presence of FDP (may show elevation)..

Physiologic inhibitorsProcoagulant & fibrinolytic activities are homeostatically regulated by counter-balancing natural inhibitorsIn the coagulation system, antithrom-bin III inhibits not only thrombin but other serine protease as well (F IXa, Xa, XIa, XIIa).

Protein C along with its cofactor, protein S, degrades F VIIIa and Va.Plasmin is neutralized primarily by 2 - antiplasmin.

Plasminogen Intrinsic system Extrinsic system

Contact XII XIIa HMWK Exogenous

Prekallikrein

Plasmin

Fig.7.The fibrinolytic system. Plasmin, the active fibrin(ogen)olytic enzyme, is generated by activation of plasminogen as shown. = plasminogen activator.

KallikreinTPAUrokinaseStreptokinase

INTRINSIC SYSTEMHMWKXII XII aKallikrein

XI XIa EXTRINSIC SYSTEM

VII IX IXa + VIII TF Ca 2+ Ca 2+ Ca 2+ PL X Xa + V Ca 2+ PL Prothrombin Thrombin

Fibrinogen Fibrin

XIIIXIIIa Stable fibrin clot Ca 2+

II. TESTS OF COAGULATION SYSTEMA. Screening Test Partial thromboplastin time (PTT) and activated partial thromboplastin time (aPTT)Prothrombin time (PT)Quantitative fibrinogenThrombin time (TT)Screening test for factor XIII

B. Spesific factor assays

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