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
XII XIIa TF VII
Contact Factors
XI XIa VIIa
thrombin
.. TF+VIIa
Ca++ Ca++/PL

IX IXa
Ca++/PL-
VIII thrombin VIIIa
tenase

X Xa
Ca++/PL-
V Va
thrombin
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 Va
Factor VIII 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 II VII IX X

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

Carboxyglutamic CO NH xx
Acid CH2
CH-COO-
COO-

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
Rare- Antithrombin 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 ND 20p11.2-cen Receptor for

modulin Thrombin/Prot.C

Protein C ND 20q11.2 Receptor for

receptor prot.C/APC

Antithrombin 70 1q23-25 Protease

inhibitor
TFPI ND 2q31-32.1 Protease
inhibitor
Heparin 60 22q11 Protease
Cofactor II inhibitor
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 Selected
0
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

PT G20210A 11,932 2.7 2,884 7.1 551 16

1,811 0.06
 .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 Fibrinogen

Factor V Leiden Free protein S antigen Thrombin time

PT G20210A Antithrombin activity FIX activity
Homocysteine Anticardiolipin Abs FXI activity
Factor VIII C677T MTHFR
Lupus anticoagulant
 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.‬‬

‫‪B.E‬‬ ‫‪- Laboratory findings‬‬

‫‪Coagulation‬‬
‫‪aPTT‬‬ ‫)‪71” (C. 27-38‬‬
‫‪aPTT mixed with plasma‬‬ ‫)”‪54” (C. 32‬‬
‫‪CAI‬‬ ‫)‪34 (C < 15‬‬
‫‪RVVT‬‬ ‫)‪2.9 (C < 1.3‬‬
‫‪RVVT confirm‬‬ ‫‪1.8‬‬
‫‪TTI‬‬ ‫)‪2.14 (C < 1.25‬‬
‫‪APCR‬‬ ‫) ‪1.45 (C > 2‬‬
‫‪Factor V Leiden‬‬ ‫‪normal‬‬

‫‪Immunology‬‬
‫‪anticardiolipin‬‬ ‫‪75u/ml‬‬ ‫)‪(C < 15‬‬
‫‪anti-nuclear ab.‬‬ ‫)‪417 u/ml (C < 200‬‬
‫‪anti-DNA‬‬ ‫)‪190 u/ml (C < 50‬‬