acquired thrombosis

Inherited and Acquired Inherited and Acquired Thrombotic DisordersThrombotic Disorders

Augusto B. Federici

Hematology and Transfusion MedicineL. 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 THROMBOSISThe Virchow Triad

A) Vessel Wall Damage:• Endothelial damage • AtherosclerosisB) Blood reology:• Increased stasis and viscosityD) Blood components:• Cell adhesion (platelets)• Hyper-coagulation• Hypo-fibrinolysis

CAUSES OF THROMBOSISRisk Factors

A) Hereditary Thrombophilia: Congenital defects of physiologic inhibitors of blood coagulationB) 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


• Moderate hyperhomocysteinemia• APC Resistance• High factor levels (II, VIII, IX, XI, Fib.)• High TAFI


• Antiphospholipid syndrome• Dysfibrinogenemia

CAUSES OF THROMBOSISInherited 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 THROMBOSISAcquired defects in DVT

Defects associated with Deep Vein Thrombosis (DVT): • Elevated FVIII (16%)• Elevated FXI (11%)• Antiphospholipid antibodies (10%)

AT III DEFICIENCIESBiochemistry 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 DEFICIENCIESClassification 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 DEFICIENCIESAcquired & 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 congenitalAntithrombin deficiency

Inheritance

Values in affected members

Thrombotic symptoms

Possible predisposing factors

Prevalence in patientswith venous thrombosis

Autosomal dominant

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

Deep vein thrombosis

Pregnancy, surgery,oral contraceptives, etc.

2-4%

PROTEIN C DEFICIENCIESBiochemistry 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 DEFICIENCIESBasic 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 DEFICIENCIESAcquired & Associated

• Consumption coagulopathy: DIC • Liver Dysfunction• Renal Disease• Malignancy : Leukemias• Drugs: Estrogens, Hep, L-asparaginase• Other: Vasculitis, hemodialysis, infections, plasmapheresis,

PROTEIN S DEFICIENCIESBiochemistry 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 DEFICIENCIESClassification 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 clottingType III: normal total PS, but low free and functional PS

PROTEIN S DEFICIENCIESAcquired & 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 congenitalProtein C/Protein S deficiency

Inheritance


Thrombotic symptoms



Autosomal dominant

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

Deep vein thrombosis,superficial thrombophlebitis


4-8%

FACTOR V LEIDENDefinitions

• 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 congenitalAPC resistance (FV Leiden)

Inheritance


Thrombotic symptoms



Autosomal dominant

Heterozygous: low APC-ratioHomozygous: very low APC-ratio



20-60%

PROTHROMBIN G20210ADefinitions

• 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 congenitalHyperprothrombinemia

(Prothrombin mutation 20210)Inheritance


Thrombotic symptoms



Autosomal dominant

Heterozygous:110-130%Homozygous > 130%



6-18%

HOMOCYSTEINEMIADefinitions

• 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


Deficiency of CBS, abnormal(absent or thermolabile variant) MTHFR.

Vitamin deficiency (folate, B12),age, gender, chronic renal failure.

Moderate:Medium:Severe:

Moderate: arterial, venous thrombosisSevere: homocystinuria syndrome

10-20%

15-30 µM30-100 µM

> 100 µM

Main characteristics of congenitalDysfibrinogenemia

Inheritance

Main laboratory features

Symptoms


Autosomal recessive

Discrepancy between immunologic and functional fibrinogen, prolonged thrombin clotting time

None, hemorrhage, venousand arterial thrombosis

< 1%

ANTIPHOSPHOLIPID SYNDROMEDefinitions

• 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 SYNDROMEPathogenesis 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 thrombophiliaWhich 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 ofpositive 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

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acquired thrombosis