Apr 28, 2023 1

HEMOSTATIC PHYSIOLOGY

Mansyur ArifDept. of Clinical Pathology

Fac.of Medicine,Hasanuddin University, Makassar

Apr 28, 2023 2

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

Apr 28, 2023 3

Blood clotting results from :1. Interaction between endothelial cells and platelets

the primary hemostatic plug.2. The coagulation phase thrombin is generated &

fibrin develops3. Formation of peptide bonds stabilization of the

fibrin network.

Apr 28, 2023 4

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

Apr 28, 2023 5

PLATELET STRUCTURE

Major Structure features:• A typical cell membrane.• Circumferential microtubular system• Dense tubular system• Various granules• Externally communicating open

canalicular system.

Apr 28, 2023 6

Platelet Membrane

• Bilipid membrane and membrane pro-tein.

• 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.

Apr 28, 2023 7

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.

Apr 28, 2023 8

Microtubules and Microfilaments

• Microtubules are composed of tubulin and participate in cytoskeletal support and in contraction of the stimulated cell.

• Microfilaments contain actin and participate in platelet pseudopod forma-tion.

Apr 28, 2023 9

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.

Apr 28, 2023 10

Granules

• Dense granules contain high concentrations of ADP and Ca as well as serotonin. These substances are released upon PLT stimulation, enhance PLT aggregation.

• α Granules store a variety of proteins that are secreted by stimulated PLT. These includes PF4, β-thromboglobulin, PDGF, Fibrinogen, F V, vWf and various glycoproteins important to adhesion (thrombospondin & fibronectin)

Apr 28, 2023 11

Canaliculi

• Open canalicular system is a complex network of surface membrane invagi-nations that look like vacuoles.

• Increase the PLT surface area.• The contents of PLT granules are

released through this system

Apr 28, 2023 12

Platelet Physiology

When 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 arachidonic acid

Apr 28, 2023 13

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.

Apr 28, 2023 14

PhospholipidPhospholipase

Arachidonic acid Cyclo-oxygenase (aspirin inhibits)

PGG2

Peroxidase PGH2

Thromboxane synthetase Prostacyclin synthetase (platelets) (endothelium)

TxA2 PGI2

H2O TxB2 6-keto PGIa

Fig. 1. Arachidonic acid metabolism in platelets endothelium.

Apr 28, 2023 15

Endothelium Contribution

• Metabolize AA to Prostacyclin (PGI2).• PGI2 has major contribution as

antithrombotic in intact endothelium.• Low dose aspirin completely block TxA2

production.

Apr 28, 2023 16

Coagulation phase :

1. 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.

Apr 28, 2023 17

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.

Apr 28, 2023 18

Table 1. Plasma coagulation factors

Factor Alternative name Path-way

Half-life (hours)

IIIIIIV

VIIVIIIIXXXIXII

XIII HMW kininogen

Prekallikrein

Fibrinogen ProthrombinTissue factorProaccelerinProconvertinAntihemophilic factorChristmas factorStuart - Prower factorPlasma thromboplastin antecedentHageman factorFibrin - stabilizing factorFitzgerald factorFletcher factor

CCICEII

I,E,CIIIII

90-12048-120

Not available12-24

2-610-1218-3024-6045-8040-70

72-200150

48-52

Apr 28, 2023 19

Coagulation systems : 1. 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).

Apr 28, 2023 20

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).

TFVIIa

XaV

Lipid (TF)

Apr 28, 2023 21

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)

Apr 28, 2023 22

d. - 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).

Apr 28, 2023 23

Contact factors Prothrombin

XIaIX

(Ca 2+)

X

Test : 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 XIIa

HMWK

XI

IXaVIIIPF3

XaV

Lipid (PF3)

Apr 28, 2023 24

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.

Apr 28, 2023 25

f. 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)

g. 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)

Apr 28, 2023 26

Prothrombin Intrinsic system Extrinsic system

Thrombin

Fig.3. Autocatalytic action of thrombin. (TF= tissue fct ; PF3 = platelet fct 3)

IXa VIII VIIIa

PF3

XaV Va

PF3

XaVa V

TF

Apr 28, 2023 27

Prothrombin Intrinsic system Extrinsic system

XIa

(Ca2+) IX

(Ca2+)

X Xa

ThrombinFig. 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.

TFVIIa

IXaVIIIPF3

Apr 28, 2023 28

3. Fibrin generation

When 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)

Apr 28, 2023 29

ThrombinFibrinogen Fibrin Fibrin Polymer

monomer (hydrogen bonded)

Fig. 5. Fibrin generation

a. Screening test : Thrombin time (TT) TT is prolonged due to : - fibrinogen concentration is very low (<80 mg/dl)- interfere with polymerization of fibrin monomer (fibrin (ogen) degradation product/FDP, parapro-teinemias, uremia)- heparin and abnormal fibrinogen (dysfibrinoge-nemia) are present.

Apr 28, 2023 30

b. Additional test

In 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.

Apr 28, 2023 31

Fibrin stabilization

• Final stage of coagulation• F 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.

Apr 28, 2023 32

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.

Apr 28, 2023 33

Fibrinolysis• Deposition of fibrin is associated with activati-

on of fibinolysis• Fibrin 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.

Apr 28, 2023 34

• 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).

.

Apr 28, 2023 35

Physiologic inhibitors

• Procoagulant & fibrinolytic activities are homeostatically regulated by counter-balancing natural inhibitors

• In the coagulation system, antithrom-bin III inhibits not only thrombin but other serine protease as well (F IXa, Xa, XIa, XIIa).

Apr 28, 2023 36

• Protein C along with its cofactor, protein S, degrades F VIIIa and Va.

• Plasmin is neutralized primarily by

2 - antiplasmin.

Apr 28, 2023 37

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.

Kallikrein TPA UrokinaseStreptokinase

Apr 28, 2023 38

INTRINSIC SYSTEMHMWK

XII 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

XIII XIIIa Stable fibrin clot

Ca 2+

Apr 28, 2023 39

II. TESTS OF COAGULATION SYSTEM

A. Screening Test 1. Partial thromboplastin time (PTT) and

activated partial thromboplastin time (aPTT)2. Prothrombin time (PT)3. Quantitative fibrinogen4. Thrombin time (TT)5. Screening test for factor XIII

B. Spesific factor assays

Apr 28, 2023 40

THANK YOU

Apr 28, 2023 41

Apr 28, 2023 42

Apr 28, 2023 43

Apr 28, 2023 44

Apr 28, 2023 45

Apr 28, 2023 46

Apr 28, 2023 47

III. CONGENITAL HEMORRHAGIC DISORDER

A. Hemophilia A (factor VIII deficiency) and Hemophilia B (factor IX deficiency)

1. Pathophysiology :X chromosome, gene defect.

2. Clinical features :mild, moderate and severe

disease3. Diagnosis :

a. screening testsb. specific factor assay

Apr 28, 2023 48

4. Therapy : to raise the deficient factor

a. Pharmacologic therapy :

1. Hemophilia A : Desmopressin (dDAVP)

2. Hemophilia B : no effective drugs

b. Replacement therapy :

1. Beware of adverse effects

2. The choice of blood product is critical

Apr 28, 2023 49

c. Treatment options :

1. Fresh frozen plasma (FFP)

2. Cryoprecipitate

3. Factor concentrates

5. Complication : Arthropathy, Inhibitors, Liver disease, HIV infection.

6. Interdisciplinary care hemophilia center medical care, psychosocial care and

genetic counseling

Apr 28, 2023 50

B. von Willebrand’s disease

1. Physiology of vWF :- HMW glycoprotein, 250 kD- In plasma and in platelets- as a carrier protein for coag. F VIII- important for primary hemostasis- mediates adhesion of platelets to the s.endot- vWF – F VIII complex : F VIII : C, F VIII : Ag, F VIII : Cag, F VIII : RCof.

Apr 28, 2023 51

2. Clinical features : >> mucous membrane bleeding, epistaxis, menorrhagia

3. Diagnosis : - Combination of prolonged BT and a decreased F VIII : C level classically- Combination of abnormalities of the functional measures of the vWF – F VIII complex variants of vWF (see table 2)- Ristocetin aggregation - Diff. of type F VIII multimer analysis :type I, IIA, IIB, IIC, III.

Apr 28, 2023 52

Table 2. Laboratory Findings in Hemophilia A and Severe vWF Disease

Labotatory test Finding Hemophilia A vWF disease

Bleeding timeVIII : CVIII : AgVIII : RCof

NormalDecreased

NormalNormal

ProlongedDecreasedDecreasedDecreased

Apr 28, 2023 53

4. Therapy a. dDVAPb. Cryoprecipitatec. F VIII concentrates

C. Other inherited factor deficiencies 1. F XII, prekallikrein and HMWK2. F I, II, V, VII, X, XI and XIII

Apr 28, 2023 54

IV. ACQUIRED HEMORRHAGIC DISORDER

A. Vitamin K deficiency1. Etiology

a. dietary deficiencyb. malabsorptionc. antibiotic therapyd. hemorrhagic disease of the new born

2. Clinical features : severe bruising or excessive bleeding or asymptomatic

Apr 28, 2023 55

3. Diagnosis - severe : prolonged PT and PTT- early or milder def. : prolonged PT 4. Therapy - Parenteral vit. K- FFP

B. Liver disease1. Etiologya. decreased synthesis of coag. factorsb. Vit. K def

Apr 28, 2023 56

c. Functionally abnormal fibrinogensd. Disseminated Intravasc. Coagulation (DIC) and consumption of coag. factors2. Clinical features - vary with the course of the patient’s liver disease- >> GIT bleeding3. Diagnosis - Lab. findings varies widely

Apr 28, 2023 57

4. Therapy - trial of parenteral vit K therapy- Replacement therapy FFP

C. Clotting factor inhibitors autoantibodies1. Inhibitors in hemophilia2. Inhibitors in patients without

preexisting bleeding disorders a. Etiology : drugs, autoimmune or

lymphoproliferative disorders, spontaneous a.coagulants

Apr 28, 2023 58

b. Diagnosis : mixing study, factor assays

c. Therapy : supportive

3. Lupus anticoagulant cardiolipin

a. Prolonged PTT, false + serologic test

b. associated with recurrent

spont.abortions.

Apr 28, 2023 59

COAGULATION REGULATION AND

HYPERCOAGULABLE STATES

Apr 28, 2023 60

I. CONTROL MECHANISMS IN COAGULATION

A. Naturally occuring a.coagulants

1. Antithrombin III (AT III) acts as a serine protease inhibitor

2. Protein C, Protein S Vit K – dependent

3. Other plasma protease inhibitor : heparin

cofactor II, 2- macroglobulin.

Apr 28, 2023 61

B. Fibrinolitic system

1. Activation :

a. Intrinsic activation : a poorly understood mechanism of activation

b. Extrinsic Activation : Tissue plasminogen activator (t-PA), urokinase

c. Exogeneous (therapeutic) activation : streptokinase, urokinase and t-PA

Apr 28, 2023 62

2. Sites of action a. Fibrinogen b. Fibrin

II. THROMBOTIC DISORDER

A. Congenital thrombotic disorder

1. AT III deficiency

2. Protein C deficiency

3. Protein S deficiency

Apr 28, 2023 63

B. AQUIRED THROMBOTIC DISORDER THROMBOEMBOLIC DISEASE

1. Risk factors :a. abnormalities of blood flowb. abnormalities of the vasculaturec. abnormalities of the coagulation system

2. Clinical manifestations :a. Venous thrombosis

- superficial thrombosis - deep vein thrombosis

Apr 28, 2023 64

b. Pulmonary embolismc. Arterial thrombosisd. Other

4. Therapya. Agents treatment and prevention 1. Anticoagulants :- Heparin- Warfarin 2. Thrombolytic agents :- Streptokinase- Urokinase- t-PA

Apr 28, 2023 65

b. Treatment approaches

1. Deep vein thrombosis : >> heparin, thrombolytic agent

2. Pulmonary embolus : heparin, thrombolytic agent

3. Myocardial infarction : thrombolytic agent

4. Peripheral artery and catheter thrombosis :

thrombolytic agent.

Apr 28, 2023 66

III. THROMBOHEMORRHAGIC DISORDER

A. Disseminated intravascular coagulation(DIC)1. Pathophysiology (see table 3)

a. Initiation : pathologic activation of the coag. cascade

b. Thrombosis c. Consumption d. Fibrinolysis

e. Hemolysis

Apr 28, 2023 67

Infectious condition Gram negative septicemiaOther endotoxin-related condition

Obstetric conditionsAbruptio placentaeAmniotic fluid embolismRetained dead fetus

Vascular conditionsAneurysmGiant cavernous hemangioma

Hematologic conditions Massive hemolysisPromyelocytic leukemiaSnake venom

Trauma

Table 3. Conditions that commonly precipitate DIC

Apr 28, 2023 68

2. Clinical features varies widely a. diffuse bleeding from multiple sites b. thrombotic lessions

3. Diagnosis lab. findings vary with time & circumstances Severe DIC :

a. Thrombocytopenia b. Prolonged PT, PTT, TTc. Decreased fibrinogend. FSPse. Microangiopathic hemolytic anemia

Apr 28, 2023 69

4. Therapy :

a. Low grade DIC treatment may not be necessary

b. Clinically significant bleeding replacement of depleted coagulation factors and cells

c. Thrombosis : heparin

Apr 28, 2023 70

B. Thrombotic thrombocytopenic purpura

(TTP) and hemolytic-uremic syndrome

(HUS)

C. Heparin thrombocytopenia

Apr 28, 2023 71

THANK YOU