Coagulation Factors And Bleeding Disorder

Components of Hemostasis • Primary Hemostasis (Platelets &

vWF)

• Coagulation Cascade (Intrinsic & Extrinsic) • Inhibitors of Coagulation (Natural & Acquired) • Fibrinolytic System (Activators, Inhibitors & Degradation products)

Intrinsic pathway

Extrinsic pathway TF

XII XIIa Contact Factors XI

VIIa

XIa

thrombin

VII

..

Ca++

TF+VIIa Ca++/PL

IX

IXa

Ca++/PL-

VIII thrombin

VIIIa

tenase Xa

X V

thrombin

Va

Ca++/PL-

prothrombinase XII

Prothrombin (II)

Thrombin (IIa) XIIa

Fibrinogen

Fibrin

cross-linked fibrin

Platelet activation Fibrinogen to fibrin Factor XIII to XIIIa

thrombin

Factor XI

to

FXa

Factor V Factor VIII

Va VIIIa

‫חסרים בגורמי קרישת דם‬ ‫חסר פקטור ‪ - VIII‬המופיליה ‪A‬‬ ‫חסר פקטור ‪ - IX‬המופיליה ‪B‬‬ ‫חסר פקטור ‪ - XI‬המופיליה ‪C‬‬ ‫חסר פקטור ‪VII‬‬ ‫חסרים נוספים – חסר פקטור ‪V ,X ,XIII‬‬ ‫ופיברינוגן נדירים ‪.‬‬

Hemophilia A and B • • • • • •

History- Queen Victoria, Talmud reference Incidence- 1:10,000 births (A), 1:30,000 (B) Hemophilia in Israel- about 400 patients Inheritance- X linked (30% sporadic cases) Females- 1:1x106 (extr.Lion., double hetero) Bleeding symptoms: hemarthrosis, internal organs (muscular, CNS), no cutaneous bleeding • Severity: sever < 1% factor activity (spontaneous bleeds) moderate 1-5% activity (mostly traumatic bleeds) mild 5-20% activity (traumatic bleeds only)

Type of mutations in hemophilia A

Treatment of hemophilia • Goal- prevention of early and late complications by factor replacement Rx • Early factor infusion (before other diagnostic procedure) • On-demand vs prophylactic therapy • Raise factor to hemostatic level (30-50%) 1unit/kg = 2% raise of plasma factor FVIII half life is about 12 hours • Adjuvant therapy- anti fibrinolytics (EACA) • Local hemostasis- fibrin sealant • Physical therapy • Joint replacement • Hemophilia centers- multi disciplinary approach

Acquired hemophilia • Associated with: auto immunity, post partum, cancer and drugs • Bleeding manifestations: muscular, internal, usually not hemarthroses • Diagnosis: prolonged aPTT, mixing FVIII levels, r/o LAC • Responding to immuno suppressive (IVIG) • Control of bleeding by: high dose FVIII, Porcine FVIII, APCC, rFVIIa

Factor XI deficiency • Autosomal recessive (Chromosome 12) • In Ashkenazics 10% carriers,1:400 affected • Clinical manifestations when FXI<15% • Post trauma bleeding • FXI has a central roll in the intrinsic pathway

FVII Activity in homozygotes range from 3.5-9% Factor VII activity in heterozygotes - 19-46% Bleedings are rare. Frequency: 1:42 in Moroccan jews, 1:40 in Iranian jews. Founder effect was found and mutation was dated to more than 2000 years

A Christian-Arab girl was born to first cousins parents. From birth she presented severe bleeding episodes including Brain bleeding. Factor VII activity was found to be less than 1% and antigen 30%. The girl needs frequent therapy with recombinant factor VII.

Three base deletion was found resulting in codon 24 deletion Coding for the phenyl alanine aa in the Gla domain

Carboxylation of clotting factors Glutamic acid

II VII IX X

CO NH xx CH2 CH2 COO- PIVKA Precursors II, VII, IX X

Carboxyglutamic CO NH xx Acid

CH2 CH-COOCOO-

Active coagulation factors bind Ca++ and phospholipids

Leucine394Arginine mutation

Blood, 92:4554, 1998

FACTOR XIII DEFICIENCY

Factor XIII transglutaminase, cross link fibrin monomers. Deficiency of factor XIII is rare (1:100000), involves post trauma Bleeding. Patients suffer problems in wound healing, women with FXIII deficiency have recurrent fetal loss Recent studies show factor XIII involvement in angiogenesis and Blood vessel organization .

COAGULATION FACTOR ASSAYS Normal plasma and the patient's plasma are compared in their ability to correct the PT or aPTT of plasma from an individual severely deficient in the factor of interest. By convention, normal plasma is said to have 100% or 1 unit per ml activity. If a a specimen has the correcting power of a 1:10 dilution of normal plasma, the specimen has 10% or 0.1 unit per ml activity.

Factor Inhibitors The mixing study The patient's serum is mixed with normal serum and the aPTT of this mixture is measured. A 50:50 mixture will correct a factor deficiency (only 30% activity is needed for a normal aPTT). In the presence of an inhibitor, a 50:50 mix will not correct the abnormal coagulation test. If inhibition found, additional tests with diluted patient serum and normal serum will be applied and level of inhibitor will be determined (Bethesda units) by the dilution activity Factor VIII inhibitors IgG mostly, Occur primarily in haemophilia A patients who have received blood component transfusions. They develop a severe coagulopathy that is very difficult to manage May occur in a variety of unrelated conditions with a coagulopathy that is variable and usually disappears spontaneously.

INR - International Normalized Ratio ISI

INR = R R = PT (sec) patient / PT control example: PT coumadin treated patient - 32 sec, PT control - 12 sec R = 2.6 ISI = international standartization index

Activated partial thromboplastin time (aPTT) Derives its name from use of a partial activated Component like the phospholipid cephalin and Negatively charged substance like kaolin for activation Expressed in seconds with normal range (27-40’’)

Prolonged: Heparin therapy Presence of anticoagulant (lupus like) Factor deficiency Massive blood transfusion. Liver disease High dose coumadin anticoagulation

PROTHROMBIN TIME (PT) Activated by tissue factor (TF, thromboplastin) Prothrombin time is expressed in seconds, % and INR A Normal INR is between 0.9 and 1.2 The test is most sensitive to factor VII deficiency and other Vitamin K (II, VII, IX, X) dependent factors Reduced PT: Anticoagulation therapy coumarin Vitamin K deficiency Severe liver disease Presence of anticoagulant Disseminated Intravascular coagulation (DIC) High dose heparin therapy

Thrombophilia and Thrombosis Risk factors and their mechanisms Epidemiology Screening for risk factors Treatment Algorithm

Inhibitors of coagulation and fibrinolysis

History • • • • • • •

1965 – Antithrombin (Egeberg) 1965 – dysfibrinogenemia (Beck) 1981 - Protein C (Griffin) 1984 - Protein S (Comp) 1993- APCR (Dahlback) 1994 – Factor V Leiden (Bertina) 1996 - PT G20210A mutation (Poort)

Mechanisms of thrombosis :Acquired Trauma or surgery Immobilization Increasing age Malignant disorders Myeloproliferative disorders Previous thrombosis Pregnancy and puerperium Use of contraceptives orHRT Activated protein C resistance Mild to moderate hyperhomocystinemia A ntiphospholipid syndrome

Mechanisms of thrombosis (.(Cont : Inherited Common- (Factor V G1691A (Leiden Prothrombin G20210A Increased levels of FVIII and Fibrinogen RareAntithrombin deficiency Protein C deficiency Protein S deficiency Very rare- Dysfibrinogenemia  Homozygous homocystinuria

? Future risk factors

Inhibitors of coagulation and fibrinolysis

Coagulation inhibitors Half life (hrs) Chromosome

Function

Protein C

4

2q13-14

Protease (FV&FVIII)

Protein S

42

3p11.1-11.2

APC-cofactor

Thrombo modulin

ND

20p11.2-cen Receptor for

Protein C receptor Antithrombin

ND

20q11.2

Receptor for prot.C/APC

70

1q23-25

Protease inhibitor

TFPI

ND

2q31-32.1

Protease inhibitor

Heparin Cofactor II

60

22q11

Protease inhibitor

Thrombin/Prot.C

Mechanism of AT-III effect

Protein C&S pathway

Activation and inactivation of FV • Activation by thrombin or FXa • APC inactivates FVa • FV-Leiden has Gln instead of Arg at 506, which confer APCR

Inhibition of FVIIa by TFPI

Homocysteine pathway

Anti phospholipid syndrome (aPLS) Overlapping entities

Nomenclature

Proposed mechanisms for aPLS • Endothelial cell mediated: injury to EC, receptor induction, increased TF expression, induction of apoptosis • Protein C pathway related: aPL binds PC&S inhibition of PC activation, acquired APCR • Inhibition of heparin-AT complexes • Cross reaction with OX-LDL • Increase PAI-1 • Platelet activation

Criteria for the diagnosis of aPLS • Clinical criteria 1.Vascular thrombosis (one or more ATE or VTE) 2. Pregnancy morbidity a. one or more IUFD >10th week b. one or more premature birth, preeclampsia, placental insufficiency, abruption, IUGR c. 3 or more early (10th week) abortions,( >2 late) • Laboratory criteria 1. Anti CL Ab (IgG/M), on two (6w) occasions 2. LAC on two (6w) occasions (aPTT, DRVVT) 3. Exclusion of other coagulopathies Definite aPLS is establish by at least one criterion of each category

Yield of thrombophilia screening Yield (%) 80 70 60 50 40 30 20 10 0

Selected Unselected until 1993

from 1993

Frequency of inherited thrombophilias in healthy subjects and in unselected and selected patients with venous thrombosis Thrombophilia

Healthy subjects

Unselected patients

Selected patients

N

affected %

N

affected %

N

affected %

Protein C

15,070

0.2-0.4

2,028

3.7

880

5.2

Protein S

ND

2,028

2.3

752

7.4

Antithrombin

9,669

0.02

2,028

1.9

752

4.6

Factor V Leiden 16,150

4.8

1,142

18.8

162

40

2,192

0.05

11,932

2.7

2,884

7.1

551

16

1,811

0.06

PT G20210A

.Frequency of inherited thrombophilia- cont Thrombophilia

Healthy subjects

Unselected patients

Selected patients

N

affected %

N

affected %

N

% affected

FVIII

534

11.8

534

23.2

60

56.7

APCR

445

8.1

337

23.4

Hcy

1,153

6.1

856

11.7

Diagnostic tests for identification of thrombophilia and prothrombotic parameters Priority for testing High

Intermediate

Low

APCR

Protein C activity

Factor V Leiden

Free protein S antigen Thrombin time

PT G20210A

Antithrombin activity FIX activity

Homocysteine

Anticardiolipin Abs

Factor VIII Lupus anticoagulant

Fibrinogen

FXI activity C677T MTHFR

Considerations in planning treatment of patients with thrombosis • • • •

Efficacy of treatment Rate of bleeding complications Rate of recurrence Complications due to recurrence

Heparins: mechanism of action

Heparin and LMWH

HIT and skin necrosis

Oral anticoagulants mechanism of action

Coumadin effect on different coagulation factors • PT correlates initially with FVII thus not reflecting actual AC • Protein C fast drop creates initial hypercoagulability • Initiation of coumadin should be “covered” by heparin

Risk of recurrence of thrombosis according to type of thrombophilia High

Intermediate Increased

None

Antithrombin

Protein C

FVIII

FVL

APL Abs

Protein S

Hcy

FII

?Whom should we screen Evaluation of the likelihood of thrombophilia In patients with established venous thromboembolism Most likely Unprovoked event and one of the following: Age less than 45 years Recurrent event Familial history of venous thrombosis Cerebral or visceral vein thrombosis Stillbirth Three or more unexplained spontaneous abortions 6 months of oral anticoagulant therapy Performance of high and intermediate priority thrombophilia tests

Prothrombin F1+2 and soluble fibrin in normal pregnancy

Sarig (Fertility Sterility 2002)

Gris ( Thromb Haemost )1999

‫ב‪.‬א‪ .‬בת ‪ 46‬אושפזה עקב נפיחות וכאבים ברגל‪ .‬באמצעות פלבוגרפיה אובחנה‬ ‫פקקת )‪ (DVT‬והוחל טיפול בהפרין וקומדין‪ .‬במהלך האישפוז תלונות על‬ ‫קוצר נשימה‪ ,‬אובחן תסחיף ראתי‪ .‬אבחנה‪APS :‬‬ ‫כ‪ 10-‬חדשים לאחר מכן אושפזה לניתוח אלקטיבי‪ ,‬הופסק קומדין‪,‬‬ ‫יומיים לאחר הניתוח – קוצר נשימה ‪ -‬תסחיף ראתי‪.‬‬ ‫‪ 5‬שנים לאחר‪ ,‬תלונות על העדר תחושה‪ ,‬בבדיקת ‪ MRI‬נמצאו מוקדים במוח‪.‬‬ ‫‪- Laboratory findings‬‬

‫‪B.E‬‬

‫)‪71” (C. 27-38‬‬ ‫)”‪54” (C. 32‬‬ ‫)‪34 (C < 15‬‬ ‫)‪2.9 (C < 1.3‬‬ ‫‪1.8‬‬ ‫)‪2.14 (C < 1.25‬‬ ‫) ‪1.45 (C > 2‬‬ ‫‪normal‬‬

‫‪Coagulation‬‬ ‫‪aPTT‬‬ ‫‪aPTT mixed with plasma‬‬ ‫‪CAI‬‬ ‫‪RVVT‬‬ ‫‪RVVT confirm‬‬ ‫‪TTI‬‬ ‫‪APCR‬‬ ‫‪Factor V Leiden‬‬

‫‪75u/ml‬‬ ‫)‪(C < 15‬‬ ‫)‪417 u/ml (C < 200‬‬ ‫)‪190 u/ml (C < 50‬‬

‫‪Immunology‬‬ ‫‪anticardiolipin‬‬ ‫‪anti-nuclear ab.‬‬ ‫‪anti-DNA‬‬