19.04.2023 koage15.ppt 1

Disorders of haemostasis - IDisorders of haemostasis - IThe new conception of The new conception of

coagulationcoagulation

Disorders of haemostasis - IDisorders of haemostasis - IThe new conception of The new conception of

coagulationcoagulation

Lectures from Lectures from patpathhologicologicalal phphyyssioliolooggyy

Study materials from pathological physiology, school year 2014/2015

© Oliver Racz

19.04.2023 koage.ppt 2

System of haemostasis• Maintains blood fluidity• Local clot formation after vessel injury• Maintains vessel wall integrity• Cooperation of vessel wall cells,

thrombocytes, other blood cells, coagulation/anticoagulation factors & the fibrinolytic system

• Nonhaemostatic functions of the system

– Inflammation, immunity, glucose and lipid metabolism, angiogenesis, etc

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HistoryBleeding after circumcision – TalmudSchmidt 1861 - Morawetz 1905

I. ThromboplastinII. ThrombinIII. FibrinogenIV. Calcium

1937 Quick, laboratory methods1950 – 1959 DIC, Hardaway, McKay1964 MacFarlane, Davie, Ratnoff - classic coagulation

cascade1974 The role of vitamin KXXI. century – new conception, molecular basis of

diseases

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DISTURBEDINTEGRITY OF VESSEL WALL

VASOCONSTRICTIONseconds

PRIMARY HAEMOSTASIS

3 – 7 min.COAGULATION

5 – 10 min.

FIBRINOLYSIS2 – 3 days

RESTOREDINTEGRITY OF VESSEL WALL

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Repetion of physiology• Physiology of haemostasis (factors,

thrombocytes, pathway of activation)• Biochemical characterisation of

coagulation factors and the role of vitamin K, anticoagulants

• The transformation of fibrinogen to fibrin, fibrinolysis and its regulation

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I Fibrinogen fibrinII Prothrombin thrombin vit KIII Tissue factor (thromboplastin)IV Ca2+

V Proaccelerin accelerinVII Proconvertin convertin vit KVIII Antihaemophilic globulinIX Christmas factor vit KX Stuart factor vit KXI Plasma thromboplastin antecedent (PTA)XII Hageman factor (???)XIII Fibrin stabilising factor (FSF)

Prekallikrein kallikreinvon Willebrand factorHigh/low molecular weight kininogen (HMK,

LMK)

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Basic principle - proteolytic cascade

• 7 factors belong to group of serine proteases (inactive zymogens)

• Activation cascade - amplification and possibility of regulation

• Example: transformation of prothrombin to thrombin by activated factor X

S S

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Basic principle - proteolytic cascade

• Example: transformation of prothrombin to thrombin by activated factor X

S S

Xa

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Basic principle - proteolytic cascade

• Example: transformation of prothrombin to thrombin by activated factor X

S S

Xa

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The role of vitamin K• Rats with vitamin K deficiency• PIVKA = protein induced in vitamin K absence• Similar to prothrombin - not active• Vitamin K is the coenzyme of glutamate

carboxylase, which converts

• glutamic acid -carboxyglutamic acid

• Two COO- s bind Ca2+

• In prothrobin there are 10 such places

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O

-NH-CH-C-

CH2

CH2

COO-

O

-NH-CH-C-

CH2

CH- COO-

COO-

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Postsynthetic modification of coagulation factors

S-S

S-S

Enzyme with vit K

Glutamic acids

-carboxyglutamic acids

PIVKA

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New conception• Initiation – minimal transformation

of prothrombin to thrombin (no clot)

• Amplification• Termination• Doubts about the in vivo role of

„contact“ system (XII)

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Initiation of coagulation

Monocyte

TFVII

X II

TF = FIII, (thromboplastin) CD142 A TRANSMEMBRANE RECPTOR

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Amplification of coagulation IActivation of thrombocytes

II vWfVIII

X

XI

IX

V

thrombocyte

flip-flop of

phospholipids

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Amplification of coagulation II

Internal amplification loop

XI

II VIIIvWf

VX

IX

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Amplification of coagulation IIIActivation of IX/VIII, formation of tenase

XIIX/VIII

II vWf

VX

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Amplification of coagulation IVformation of prothrombinase

XI

X/V

IX/VIII

II

vWf

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Amplification & termination

• Activation of thrombocytes, flip-flop of phospholipids

• Activation of XI – amplification loop• Activation of IX and VIII → TENASE• Tenase activates X and V – PROTHOROMBINASE• production of huge amounts of thrombin• Transformation of fibrinogen to fibrin → CLOT• Formation of crosslinks in fibrin network (XIII) →

STABLE CLOT

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Revision of „contact“ system

• John Hageman (deficiency of XII) died in 1968 on thrombembolic disease after a trauma

• He probably did not read the textbooks of physiology!

• According to current opinion the system has rather nonhaemostatic functions

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Structure of fibrinogen

6 chains, 2*

Proteolytic removal of peptides A,B

Polymerisation

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Polymerisation of fibrinogen

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Stabilisation of fibrin and fibrinolysis

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The anticoagulant system

• Serpins – inhibitors of proteases– Inhibitor of initiation - TFPI– Antithrombin (III)

• Protein C together with S are the most important inhibitors of amplification – proteolytic degradation of V and VIII

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The fibrinolytic – plasminogen system

• PLASMINOGEN – 92 kDa glykoprotein• Two step activation to active form, PLASMIN by

tissue PLASMINOGEN ACTIVATOR - tPA• Plasmin degrades fibrin clot (and has a lot of

nonhaemostatic functions)• The process is inhibited by PAI – 1, an

antagonist of tPA Important – the activities of tPA and PAI are

oscillating in a broad range – effects of stress, adrenaline, antidiuretic hormone, obesity (!) and gene polymorphism

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FIBRIN CLOT

PLASMINOGEN

PLASMIN

FDP

D-DIMERS

PAI-1tPA- +

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Disorders of haemostasis - IIDisorders of haemostasis - IIPrinciples of diagnosticsPrinciples of diagnostics

Disorders of haemostasis - IIDisorders of haemostasis - IIPrinciples of diagnosticsPrinciples of diagnostics

Lectures from Lectures from patpathhologicologicalal phphyyssioliolooggyy

Study materials from pathological physiology, school year 2005/2006

© Oliver Racz, 12. 2. 2006

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Diagnostics IRumpel-Leede, test of capillary fragility Bleeding time (standardized methods!)

normal: 4 - 6 min.; pathological: > 10 min.Whole blood coagulation time (Lee-White) normal: 4 - 8 min.Platelet count (automatic analyzers)

norm: 140 - 440*109/l100*109/l thrombocytopenia50*109/l risk of longer bleeding after an injury or

surgery40*109/l spontaneous bleeding (or not)10*109/l danger of fatal bleeding (GIT, NS)

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Diagnostics IIprinciple of coagulation tests

• Step 1: Anticoagulated blood (Ca2+ binding by citrate);• Step 2: Removal of RBC, LE, TH (centrifugation)• Step 3: Addition of surplus Ca2+ & activators to plasma• Step 4: Measurement of time until the first fibrin filaments

are formed• Expression of results

A. In seconds (international standardized calibrators, QC)B. In relative units compared to norm (time patient/control)

C. INR = International Normalized Ratio

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Diagnostics IIIcoagulation tests - examples

Prothrombin time - QuickOral anticoagulant therapy control (OAC)

Plasma + Ca2++ tissue thromboplastinNorm: 11 - 15 secNecessary factors: VII, X, V, II

APTT = activated partial thromboplastin timePlasma + Ca2+ + caolin (negat. charge) + phospholipid Norm: 35 - 45 sec.Necessary factors: XII, XI, IX, and VII, X, V, II

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Prothrombin and APTT assay

Prothrombin (Quick)

APTT

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Diagnostics IV - other assays

• Fibrinogen concentration (2 - 4 g/l)• Fibrin & fibrinogen degradation products

– D-dimer, degradation product of fibrin clot by plasmin

diagnostics of DIC

exclusion of deep venous thrombosis and pulmonary embolism (limit 500 g/l)

• Special assays – aggregometry, adhaesion of thrombocytes

• Direct measurement of factors

•Assays of polymorphisms, mutations

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Disorders of haemostasis - IDisorders of haemostasis - IIIIIThe most important diseasesThe most important diseasesDisorders of haemostasis - IDisorders of haemostasis - IIIIIThe most important diseasesThe most important diseases

Lectures from Lectures from patpathhologicologicalal phphyyssioliolooggyy

Study materials from pathological physiology, school year 2005/2006

© Oliver Racz, 12. 2. 2006

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Syllabus1. Classification2. Symptomatology3. Disorders of primary haemostasis -

thrombocytopenia & thrombocytopathy4. Coagulation disorders5. Vascular purpuras6. Disseminated intravascular coagulation -

DIC7. Thrombembolic conditions

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ClassificationA. HAEMORRHAGIC DIATHESES

A1. PLATELET DISORDERS A2. COAGULOPATHIES A3. DISORDERS OF THE VESSEL WALL

(In each group inherited and acquired conditions)

B. THROMBEMBOLIC CONDITIONS Congenital thrombophilia and othersThe association betweeen coagulation & atherosclerosis

C. DISSEMINATED INTRAVASCULAR COAGULATION

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ThrombocytopeniaThree mechanisms:• A. Insufficient production of

megakaryocytes• B. Increased destruction of

thrombocytes• C. Abnormal distribution of

thrombocytes

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ThrombocytopeniaA. Insufficient production of

megakaryocytes• Bone marrow damage: drugs,

toxins, radiation, infection, tumors, leukaemia, fibrosis

• Congenital insufficiency• Cobalamin and folic acid deficiency

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Thrombocytopenia

B. Increased destruction– Idiopathic thrombocytopenic purpura

(ITP) - autoimmune disease– Rh group incompatibility– DIC– Arteficial heart valves

C. DistributionSplenomegalia and hypersplenism

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Thrombocytopathy• A. Congenital

– thrombastenia - m. Glanzman AR– insufficient aggregation of platetelets

• B. Acquired– Acetylsalicylic acid, phenylbutasone– Uremia

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Hereditary disorders of coagulation

• Hemophilia A (1/10 000)– Unexpected fluctuation of clinical

symptomatology– Long repeated bleedings– Internal bleedings– Intracranial bleeding (can be fatal)– Chronic anaemia, icterus– Severe damage of joints

• Hemophilia B (IX- XR) C (XI - AR), less severe

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f. VIII concentration and symptoms

Concentration, g/l Symptoms

100 – 50

50 - 30 Intensive and long lastingbleeding after big trauma

30 – 5 Long bleeding after surgery andminor trauma

5 – 1 Intensive/long bleeding afterminor trauma

< 1 Spontaneous bleeding

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von Willebrand disease

• In the past considered as a moderate form of hemophilia

• Von Willebrand factor = big multimeric protein • Synthesised in endothel and megakaryocytes• Function – contact of damaged endothel and

platelets, mutual interaction of thromobocytes• stabilisation of factor VIII. • Concentration in people with blood group AB > 0

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von Willebrand

• Deficiency is probably common (1-3 %) mostly asymptomatic

• Together with other factors can manifest (acetylsalicylic acid, OAC therapy) as mucosal bleeding, bleeding after tooth extraction, menorrhagia

• Type I, IIa, IIb, III

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Acquired coagulation disorders

• Vitamin K deficiency– Newborns, malabsorbtion, obstructve

icterus, etc.

• Anticoagulant drugs– Dicumarols (antivitamin K);– Heparin (activator of antithrombin,

inactivation of X, IX & XI)– Streptokinase

• Liver disease

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Vascular purpura

• Relatively common condition• skin eruption in infectious diseases in

children• senile purpura• scorbut, scurvy - deficiency of vitamin

C– Disorder of collagen synthesis (proline -

hydroxyproline) and abnormal platelet function. Perifollicular skin, gingival, mucosa muscle, GIT bleeding, hematuria. Skin hyperkeratosis.

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Hereditary disordes of vessel wall

Hereditary haemorrhagic teleangiectasy (m. Osler-Weber-Rendu), AD, Epistaxis, teleangiectasy Anaemia, DIC, liver cirrhosis Lack of elastic fibers in vessel wall

Other hereditary collagen diseasesMarfan - Lincoln, Ehlers-Danlos - Paganini

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Disseminated intravascular coagulation,

DIC

• Consumption coagulopathy• Life threating disorder (Death Is

Coming !)• Generalised activation of

haemostasis• Thrombosis and bleeding at the

same time, tissue hypoxia

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DIC - causes

Complications of gravidity. Amniotic fluid embolisation. Preeclampsia, eclampsia

Sepsis & septic shock G- infections (endotoxin), meningococci, pneumococci, plasmodium malariaeSy Waterhouse-Friederichsen as a

consequence of meningitis

Neoplastic disease: Lung, gastric, breast, prostata Ca, leukaemia

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DIC - causes

Liver failureHaemolysis (incompatible transfusion)Trauma (haemorrhagic shock, burns)Rejection of transplanted organsTropical snake toxins

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Pathogenesis of DICA. Generalised activation of coagulationB. Thrombosis

Microthrombi in circulation;Deposits of microthrombi in tissues and

their damageC. Decreased haemostasis

Decrease of platelet number, factor concentration

Activation of fibrinolysisD. BleedingE. Disorder of nonhaemostatic functions

of the system

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BLEEDING

GENERALISED ACTIVATION OF COAGULATION

MICROTHROMBI

DECREASE OF PLATELETS & COAGULATION FACTORS

ACTIVATION OF FIBRINOLYSIS

EMBOLISATION

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BLEEDING

GENERALISED ACTIVATION OF COAGULATION

MICROTHROMBS

DECREASE OF PLATELETS & COAGULATION FACTORS

ACTIVATION OF FIBRINOLYSIS

EMBOLISATION

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Symptoms and forms tissue ischaemia, gangrena of fingers bleeding (after injections); kidney failure, anuria; haemorrhagic necrosis of adrenal cortex - sy

Waterhouse-Friderichsen; haemolytic anaemia, haemoglobinuria;Beginning acute or subacuteStages: Compensated, decompensated and

manifest

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Case studyParameter before beginning DIC!

Th 200 60 20

Quick 12 12 23

aPTI 35 35 63

Fibrinogen 3,0 1,5 0,5

FDP negat + +++

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Our case study• As an editor of a medical journal I

visited our publisher, a cca 50 y old lady.

• Complaining about respiratory condition, therapy resistant

• And also swelling of the legs• Nothing serious?

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Our case study• Complaining about respiratory

condition, therapy resistant• And also swelling of the legs• Nothing serious?• Immediately sent to hospital:

– D-dimers extremely high– X-ray repeated small embolisation

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Thrombosis, thrombembolia, thrombophilia

• Factors– endothel (aterosclerosis, inflammatio)– thrombocytes– coagulation pro & contra, fibrinolysis

(tPA/PAI!)– circulation (stasis)

• Forms– venous thrombosis– arterial (MI)– intracardial (mitral stenosis)– diffuse (DIC)

• Occlusive – nonocclusive clots•Embolia – lung, brain, paradox,

microembolisation

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Trombophiliacomplex disease

• Immobilisation• Stasis• Varices• Gravidity, hormonal

anticonception• Trauma, burns• Malignant tumors• ATHEROSCLEROSIS

• DEFICIENCY OF ANTICOAGULANT FACTORSANTITHROMBIN, 1965PROTEIN C, 1981

• INCREASED ACTIVITY OF PROCOAGULANT FACTORS (VII)

• DECREASED FIBRINOLYSIS

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APC resistance(Activated Protein C)

• Factor V Leiden – (R506Q), 2 – 10 % europoid population– 4 – 7fold increase risk of thrombosis in

heterozygotes (SK 5 %), – Geographic gradient form south to north– Also other mutations - F V Cambridge,

Hongkong• Polymorphism of protein C gene and other genes

of coagulation/fibrinolysis factors• Functional APC resistance

– Increase of f VIII, decrease of protein S in gravidity, during hormonal anticonception, inflammation, obesity, malignant tumors

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Amplifi cation of coagulation I Vf ormation of prothrombinase

XI

X/V

IX/VIII

II

vWf

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Amplifi cation of coagulation I Vf ormation of prothrombinase

XI

X/V

IX/VIII

II

vWf

X/V Leiden

APC

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Trombophiliacomplex disease

• Immobilisation• Stasis• Varices• Gravidity, hormonal

anticonception• Trauma, burns• Malignant tumors• ATHEROSCLEROSIS

• DEFICIENCY OF ANTICOAGULANT FACTORSANTITHROMBIN, 1965PROTEIN C, 1981

• INCREASED ACTIVITY OF PROCOAGULANT FACTORS

• DECREASED FIBRINOLYSIS

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Bleeding vs clotting !

• Haemophilia: 1 in 10 000 (Sk 400)• Other bleeding disorders (Th) not so

rare or clinically silent (vWd)• Thrombophilia 1 in 10 or even more

in older, obese, sick people!!!• In EU more than 540 000 deaths,

often nondiagnosed!!!

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Bleeding vs clotting !

• Thrombophilia 1 in 10 or even more in older, obese, sick people!!!

• (ATRIAL FIBRILLATION, MALIGNANCIES, BURNS, DIABETES, INFECTIONS, POST SURGERY…..

• Both in DIC• Both in MI secondary prevention –

anticoagulant treatment – WALKING THE TIGHTROPE