Inherited and Acquired

Thrombotic Disorders
Augusto B. Federici
Hematology and Transfusion Medicine
L. SACCO University Hospital of Milan
augusto.federici @ unimi.it
Regulation of Hemostasis
Blood hemostasis is composed by a
series of integrated and regulated
processes

Coagulation factors are always
balanced by coagulation inhibitors
and fibrinolysis


Definitions of Thrombosis
Thrombosis is the results of a series
of abnormal reactions of hemostasis,
conducting to increased formation
of clots and vessel occlusion

Arterial and venous thrombi can be
formed and can be spread out into
the circulation (embolism). When
venous system is interested = VTE




CAUSES OF THROMBOSIS
The Virchow Triad
A) Vessel Wall Damage:
Endothelial damage
Atherosclerosis
B) Blood reology:
Increased stasis and viscosity
D) Blood components:
Cell adhesion (platelets)
Hyper-coagulation
Hypo-fibrinolysis



CAUSES OF THROMBOSIS
Risk Factors
A) Hereditary Thrombophilia:
Congenital defects of physiologic
inhibitors of blood coagulation
B) Acquired Thrombophilia:
Immobility
Inflammation
Malignancy
Surgery
Drugs (estrogens)
THROMBOPHILIA
It may be defined as a condition
characterized by an increased risk of
thromboembolism at relatively young age.
It may secondary to congenital, or acquired
causes and some of them may be detected
by laboratory investigation.
Congenital or Acquired
Conditions Associated with VTE
Antithrombin deficiency
Protein C deficiency
Protein S deficiency
Fibrinolysis defects
Heparin cofactor II deficiency
TFPI deficiency
Congenital or Acquired
Conditions Associated with VTE
Moderate hyperhomocysteinemia
APC Resistance
High factor levels (II, VIII, IX, XI, Fib.)
High TAFI

Congenital or Acquired
Conditions Associated with VTE
Antiphospholipid syndrome
Dysfibrinogenemia



CAUSES OF THROMBOSIS
Inherited defects of Inhibitors
Defects associated with thrombosis
Antithrombin III (1%)
Protein C (5%)
Protein S (3%)
Dysfibrinogenemia (0.8%)
Factor V Leiden (20%)
Prothrombin gene mutation (6%)
Homocysteinemia (10%)



CAUSES OF THROMBOSIS
Acquired defects in DVT
Defects associated with Deep Vein
Thrombosis (DVT):
Elevated FVIII (16%)
Elevated FXI (11%)
Antiphospholipid antibodies (10%)



AT III DEFICIENCIES
Biochemistry and Genetics
AT III : a 58 kDa glycoprotein with
plasma concentrations of 150 ug/mL
is a serin protease inhibitor (serpins)
that functions by forming a 1:1
complex with thrombin, Xa, IXa, XIa

The 19 kb gene (7 ex, 6 int) is located
on chromosome 1 (1q23-q25)




AT III DEFICIENCIES
Classification of Inherited Defects
Type I: low functional and low antigen ATIII
1a: normal but reduced synthesis rate
1b: decreased abnormal ATIII (i.e. Pro407Leu)

Type II: low functional, but normal antigen ATIII
2a: Decreased activity (heparin binding)
2b: Decreased activity (Ser394Leu, Ala384Pro)
2c: Isolated low heparin binding activity
(Arg47Cys, Pro41Leu)



AT III DEFICIENCIES
Acquired & Associated
Consumption coagulopathy: DIC
Liver Dysfunction
Renal Disease
Malignancy : Leukemias
Malnutrition & gastrointestinal loss
Drugs: Estrogens, Hep, L-asparaginase
Other: Vasculitis, hemodialysis,
infections, plasmapheresis,
Main characteristics of congenital
Antithrombin deficiency
Inheritance

Values in affected members



Thrombotic symptoms

Possible predisposing factors


Prevalence in patients
with venous thrombosis
Autosomal dominant

Heterozygous: ~ 50%
Homozygous: < 10%
(functional assay)

Deep vein thrombosis

Pregnancy, surgery,
oral contraceptives, etc.

2-4%



PROTEIN C DEFICIENCIES
Biochemistry and Genetics
Protein C: a vitamin K-dependent
glycoprotein with Mr of 62 kD
synthesized in the liver

The 11 kb gene (9 exons) is located
on chromosome 2 (2q14-21)

PC defects are autosomally inherited:
homozygous or compound heterozygous
may be severely affected



PROTEIN C DEFICIENCIES
Basic and Clinical Effects
Reduced proteolytic cleavage of FV
and FVIII: increased thrombin
generation

Venous thrombo-embolism occurs
in patients with PC defects especially
when associated with other defects:
ATIII, PS, FV and II mutations & acquired
defects



PROTEIN C DEFICIENCIES
Acquired & Associated
Consumption coagulopathy: DIC
Liver Dysfunction
Renal Disease
Malignancy : Leukemias
Drugs: Estrogens, Hep, L-asparaginase
Other: Vasculitis, hemodialysis,
infections, plasmapheresis,



PROTEIN S DEFICIENCIES
Biochemistry and Genetics
Protein S (PS): a vitamin K-dependent
glycoprotein with Mr of 69 kD that serves
as a cofactor of APC

PS exists in plasma in two distinct forms:
Free PS (40%) and PS-C4BP

The 80 kb gene (15 exons) is located
on chromosome 3




PROTEIN S DEFICIENCIES
Classification of Inherited Defects
Autosomal dominant defect
Type I: low functional and low antigen PS:
Low PS-free, low clotting
(Most common heterozygous defects of PS)
Type II: low functional, but normal antigen PS:
Normal levels of total PS and free PS, but low
functionally PS clotting
Type III: normal total PS, but low free and
functional PS



PROTEIN S DEFICIENCIES
Acquired & Associated
Conditions with increased C4BP:
Pregnancy, OCP, Diabetes, Inflammation,
SLE, AIDS, Nephrosis
Conditions with increased synthesis:
Pre-term infants, liver disease, vit K defects,
OAT, CT for breast cancer
Conditions with increased cell binding:
PV, ET, Sickle Cell disease

Main characteristics of congenital
Protein C/Protein S deficiency
Inheritance

Values in affected members



Thrombotic symptoms


Possible predisposing factors


Prevalence in patients
with venous thrombosis
Autosomal dominant

Heterozygous: ~ 50%
Homozygous: < 10%
(functional assay)

Deep vein thrombosis,
superficial thrombophlebitis

Pregnancy, surgery,
oral contraceptives, etc.

4-8%



FACTOR V LEIDEN
Definitions
In 1993, Dahlback first described families with
thrombosis whose plasma was resistant to the
effects of activated protein C (APC)

--Arg506--
APC --------I
---Arg562--

FV Leiden: a specific mutation: Arg-Gln506

FVIIIa
F Va
FV Inact
FVIII Inact



FACTOR V LEIDEN
Epidemiology and Clinic
3-8% of Caucasians are heterozygous for the
FV Leiden defect, 1/1000 are homozygous

>90% of subjects who are APC resistant show
the FV Leiden defect

Many clinical studies have shown that the
odds ratio (OR) for VTE in subjects with FV506
is 2-8 fold and is frequently (20%) in VTE
Main characteristics of congenital
APC resistance (FV Leiden)
Inheritance

Values in affected members



Thrombotic symptoms

Possible predisposing factors


Prevalence in patients
with venous thrombosis
Autosomal dominant

Heterozygous: low APC-ratio
Homozygous: very low
APC-ratio

Deep vein thrombosis

Pregnancy, surgery,
oral contraceptives, etc.

20-60%



PROTHROMBIN G20210A
Definitions
In 1996, a genetic defect (nt G20210A) was
discovered in the 3 untranslated region of the
prothrombin gene that was linked to an
increased risk of VTE. The mutation is
associated with elevated levels of Factor II

The increased concentrations of Factor II could
contribute to enhanced risk of VTE by
promoting enhanced thrombin generation



PROTHROMBIN G20210A
Epidemiology and Clinic
2-3% of Caucasians are heterozygous for the
II G20210A. Rare homozygous have been
reported

Many clinical studies have shown that the
odds ratio (OR) for thrombosis in subjects with
Factor II G20210A is 2-6 fold, and has been
identified in 4-8% of subjects with VTE
Main characteristics of congenital
Hyperprothrombinemia
(Prothrombin mutation 20210)
Inheritance

Values in affected members


Thrombotic symptoms

Possible predisposing factors


Prevalence in patients
with venous thrombosis
Autosomal dominant

Heterozygous:110-130%
Homozygous > 130%

Deep vein thrombosis

Pregnancy, surgery,
oral contraceptives, etc.

6-18%



HOMOCYSTEINEMIA
Definitions
Elevated levels of homocysteine in the blood
are strongly associated with premature vascular
disease as well as arterial and VTE

2 enzymes and 3 vitamins play key roles in the
regulation of homocysteine:
Cystationine-beta-synthase (CBS)
MethylenTetra-HydroFolate-Reductase (MTHFR)




HOMOCYSTEINEMIA
Epidemiology and Clinic
Data from a large number of studies strongly
suggest that elevated levels of Homocys are
associated with an elevated risk of arterial
(OR 2-4) and venous (OD 2-7) occlusions

End-stage renal disease, renal transplantation,
and cyclosporin therapy are associated with
high levels of Homocysteinemia
Main characteristics of Hyperhomocysteinemia
Congenital



Acquired


Values in affected
subjects



Symptoms


Prevalence in patients
with venous thrombosis
Deficiency of CBS, abnormal
(absent or thermolabile variant)
MTHFR.

Vitamin deficiency (folate, B
12
),
age, gender, chronic renal failure.

Moderate:
Medium:
Severe:

Moderate: arterial, venous thrombosis
Severe: homocystinuria syndrome


10-20%
15-30 M
30-100 M
> 100 M
Main characteristics of congenital
Dysfibrinogenemia
Inheritance

Main laboratory features



Symptoms



Prevalence in patients
with venous thrombosis
Autosomal recessive

Discrepancy between
immunologic and functional
fibrinogen, prolonged thrombin
clotting time

None, hemorrhage, venous
and arterial thrombosis


< 1%



ANTIPHOSPHOLIPID SYNDROME
Definitions
Antiphospholipid antibodies (APA) are IgG, IgM
or IgA allo-antibodies that occur as a results of
autoimmune disease or as a reaction to
infections or drugs.

APA can be divided into two broad categories:
- anticardiolipin antibodies (ACA)
- lupus like anticoagulant (LLAC)



ANTIPHOSPHOLIPID SYNDROME
Pathogenesis of Thrombosis
Autoimmune APA are associated with
thrombosis and vascular disease, whereas
APA secondary to infections or drugs are
usually asymptomatic

APA may promote thrombosis by inhibiting the
action of APC or by stimulating increased
binding of prothrombin to PL surfaces: the
result is excessive thrombin generation



ANTIPHOSPHOLIPID SYNDROME
Diagnosis
Two different types of assays can be used to
determine the APA:
1) Assays in fluid phase: Lupus Anticoagulant
APTT, DRVVT
Dilute PT
2) Assays in solid phase: ACA
anti-B2-GPI
anti-II
anti-ox-LDL
Patients with history of thrombosis
Family members
No general screening of the population for
APC resistance.
Is prophylactic APC resistance testing
beneficial in association with risk
situation?
Who should be tested
Lab diagnosis of thrombophilia
Laboratory diagnosis of
thrombophilia
After (and far from) a thrombotic episode
After discontinuation of oral
anticoagulation
Is prophylactic APC resistance testing
beneficial in association with risk
situation?
When is it appropriate to test
Laboratory diagnosis of thrombophilia
Which kind of test
Protein C Chromogenic assay with snake venom
Antithrombin Heparin cofactor activity against FXa
Protein S Free antigen
APC-Resistance APTT-based method without and with
FV-deficient plasma. Confirmation of
positive results with FV genotype.
Prothrombin Genotyping
Dysfibrinogenemia Thrombin and reptilase times
APLA PL-dependent tests for LA (KCT and
dRVVT) plus ACA
Homocysteine HPLC, FPIA