PATHOPHYSIOLOGY OF THROMBOSISPATHOPHYSIOLOGY OF THROMBOSIS“Virchow’s Triad”“Virchow’s Triad”

1. Injury to blood vesselsTrauma, atherosclerosis, surgery

2. Stasis of bloodImmobility, venous incompetence, heart failure

3. Increased coagulability of blood - “thrombophilia”Various inherited and acquired conditions

In general, vessel injury is the most important contributing factor to arterial thrombosis (heart attack, stroke) while stasis and increased coagulability are more important in venous thrombosis

INHERITED THROMBOPHILIAINHERITED THROMBOPHILIA

• Venous >> arterial thrombosis• Most venous thrombi in legs

– Occasionally mesenteric, portal, cerebral,retinal veins

• Prevalence of thrombosis varies between families• Thrombotic problems may begin in 20s and 30s –

rarely in childhood• About half of thrombotic episodes occur in

association with other identifiable risk factors (pregnancy, oral contraceptives, surgery, etc)

DEEP VENOUS THROMBOSISDEEP VENOUS THROMBOSIS

PULMONARY EMBOLISMPULMONARY EMBOLISM

Arrow points to large clot in pulmonary

artery

Clot dissolved after administration of fibrinolytic drug

THERE ARE MANY POTENTIAL GENETIC CAUSES OF THROMBOPHILIA

“If something can go wrong, it will” (Murphy)

THERE ARE FIVE KNOWN CAUSES OF THERE ARE FIVE KNOWN CAUSES OF INHERITED THROMBOPHILIAINHERITED THROMBOPHILIA

Defects in physiologic anticoagulant pathways

Increased production of procoagulant

1. Antithrombin deficiency2. Protein C deficiency3. Protein S deficiency4. Factor V Leiden

5. Prothrombin G20210A gene mutation

QUANTITATIVE VS QUALITATIVE DEFICIENCY OF QUANTITATIVE VS QUALITATIVE DEFICIENCY OF CLOTTING PROTEINSCLOTTING PROTEINS

• Quantitative deficiency: decreased protein production (gene deletion, nonsense mutation, etc)– Both antigen and activity low– “Type I” deficiency

• Qualitative deficiency: normal protein production, decreased activity (missense mutation)– Antigen normal, activity low– “Type II” deficiency

ANTITHROMBINANTITHROMBINAKA “ANTITHROMBIN III”AKA “ANTITHROMBIN III”

• Serine protease inhibitor

• Made in liver

• 20 mg/dl plasma concentration

• Inhibits thrombin, Xa, other clotting enzymes

• Activity enhanced by heparin and heparin-like molecules on endothelium

THE ANTITHROMBIN SYSTEMTHE ANTITHROMBIN SYSTEM

Serine protease mechanismSerine protease mechanism

ANTITHROMBIN-HEPARINANTITHROMBIN-HEPARININHIBITORS OF MULTIPLE STEPS IN THE CLOTTING CASCADEINHIBITORS OF MULTIPLE STEPS IN THE CLOTTING CASCADE

Xa Va

TF VII(a)

IIa

XIIa

XIa

VIIIa IXa

ANTITHROMBIN

HEPARIN

Inhibits all serine protease clotting factors except VIIa

INHERITED ANTITHROMBIN DEFICIENCYINHERITED ANTITHROMBIN DEFICIENCY

• Prevalence: 0.2-0.4% of population; 2-5% of inherited thrombophilia

• Dominant inheritance with variable penetrance– No homozygotes known (lethal?)

ANTITHROMBIN ASSAYSANTITHROMBIN ASSAYS

• Patient plasma + heparin + thrombin– Thrombin activity measured with chromogenic

substrate– Measure decay of thrombin activity with time

• Detects both quantitative and qualitative deficiency

• Other serine protease inhibitors in plasma may contribute to measured activity causing decreased sensitivity

• Alternative assay uses factor Xa rather than thrombin, greater specificity and sensitivity

THE PROTEIN C SYSTEMTHE PROTEIN C SYSTEM• PROTEIN C

– Proenzyme precursor of serine protease– Made in liver, vitamin K-dependent– 0.4 mg/dl in blood– When activated by thrombin, degrades Va and VIIIa

• PROTEIN S– No intrinsic enzymatic activity– Made in liver, endothelium, vitamin K-dependent– Bound/inactive and free/active forms in plasma– Cofactor for protein C

• THROMBOMODULIN– Endothelial cell surface component– Binds thrombin– TM-bound thrombin activates protein C

THE PROTEIN C SYSTEMTHE PROTEIN C SYSTEM

IIa

E C

IIa IIa

E C

P C

P C

APC

P S +

VaVi

VIIIa

VIIIi

TM TM

PROTEIN C DEFICENCYPROTEIN C DEFICENCY

• Dominant form: 30-60% of normal protein C activity in blood– Found in about 5% of inherited thrombophilia– Both quantitative and qualitative deficiency can occur

• Recessive form: < 10% of normal protein C activity– Parents (heterozygous) have about 50% of normal level,

asymptomatic– Rare affected individuals (homozygous) have severe

thrombotic tendency that may begin in infancy

• Biologic basis for dominant vs recessive forms unknown

DOMINANT INHERITANCE OF PROTEIN C DEFICIENCYDOMINANT INHERITANCE OF PROTEIN C DEFICIENCY

22 1718 13 830 26 2132 30 2224 19

Protein C deficient

Protein Cnormal

History ofthrombosis

RECESSIVE INHERITANCE OF PROTEIN C DEFICIENCYRECESSIVE INHERITANCE OF PROTEIN C DEFICIENCY

HOMOZYGOUS PROTEIN C DEFICIENCY HOMOZYGOUS PROTEIN C DEFICIENCY CAUSES NEONATAL PURPURA FULMINANSCAUSES NEONATAL PURPURA FULMINANS

PROTEIN C LEVELS DROP FASTER THAN LEVELS PROTEIN C LEVELS DROP FASTER THAN LEVELS OF OTHER VITAMIN K-DEPENDENT PROTEINS OF OTHER VITAMIN K-DEPENDENT PROTEINS

DURING WARFARIN TREATMENTDURING WARFARIN TREATMENT

Protein C

Prothrombin

WARFARIN-INDUCED SKIN NECROSIS IN A WARFARIN-INDUCED SKIN NECROSIS IN A PROTEIN C-DEFICIENT PATIENTPROTEIN C-DEFICIENT PATIENT

PROTEIN C ASSAYSPROTEIN C ASSAYS

• Immunologic– Detects only quantitative deficiency

• Functional, chromogenic substrate– Snake venom enzyme activates protein C in test plasma– Activated protein C cleaves chromogenic substrate– Detects quantitative, most qualitative deficiency

• Functional, clotting time-based– Detects any deficiency– Not useful in patients taking warfarin

PROTEIN SPROTEIN S Crossed immunoelectrophoresis showing bound and free formsCrossed immunoelectrophoresis showing bound and free forms

Bound(inactive)

Free(active)

PROTEIN S DEFICIENCYPROTEIN S DEFICIENCY

• Dominant inheritance, prevalence unknownFound in about 5% of inherited thrombophilia

• Three patterns of deficiency1. Reduced (30-60%) total protein S antigen with

proportionate reduction in free protein S

2. Reduced free protein S with normal total protein S antigen

3. Reduced protein S activity with normal total and free protein S antigen

PROTEIN S ASSAYSPROTEIN S ASSAYS

• Total protein S (immunologic)– Detects only type 1 deficiency

• Free protein S (immunologic)– Detects type 1 and type 2 deficiency

• Protein S activity– Theoretically should detect any deficiency– Some assays give false positive result in

patients with activated protein C resistance due to factor V Leiden

PROTEIN C AND SPROTEIN C AND SAcquired deficiency statesAcquired deficiency states

• Warfarin treatment

• Vitamin K deficiency

• Liver disease

• Newborn

• DIC (protein C)

• Inflammation (free protein S)

• Pregnancy (protein S)

• Oral contraceptive use (protein S)

MEASURING PROTEIN C AND S IN MEASURING PROTEIN C AND S IN WARFARIN-TREATED PATIENTSWARFARIN-TREATED PATIENTS

• Problem: warfarin causes decreased protein C and protein S level

• Solution: compare levels of these proteins to another vitamin K-dependent protein (factor X)

• Low ratio of protein C or S to factor X suggests underlying deficiency state

• Requires steady state warfarin treatment (same dose for at least a week)

• Only applicable to antigen measurements

• Missense mutation changes amino acid 506 of factor V from arginine to glycine

• Mutation is at preferred protein C cleavage site, slows inactivation of factor Va by protein C

• Factor Va procoagulant activity not affected• Single mutation responsible for almost all

cases• Very common (up to 5% of population

heterozygous)• Accounts for up to 50% of inherited

thrombophilia

Factor V LeidenFactor V Leiden

IIa

E C

IIa IIa

E C

P C

P C

APC

P S +

VaVaViVi

VIIIa

VIIIi

TM TM

MODIFIED FUNCTIONAL ASSAY FOR FVLMODIFIED FUNCTIONAL ASSAY FOR FVL

4. APC ratio =aPTT with APCaPTT without APC

1. Mix patient plasma with factor V deficient plasma (1:4)

2. Plasma mixture aPTT

3. aPTTMixture + APC

THE FACTOR V LEIDEN MUTATIONTHE FACTOR V LEIDEN MUTATION

DNA TESTING FOR FACTOR V LEIDENDNA TESTING FOR FACTOR V LEIDEN

NORMAL HOMOZYGOUSHETEROZYGOUS

FVL DNA AMPLIFIED BY PCR, DIGESTED WITH RESTRICTION ENZYME

• Mutation in 3' untranslated (non-coding) part of prothrombin gene

• No effect on prothrombin structure or function• Heterozygotes have 5-10% higher plasma levels of

prothrombin• Heterozygotes have 2-3 fold risk of venous

thromboembolism Risk in homozygotes uncertain

• About 1-2% of population heterozygous; 5-7% of young patients with DVT/PE

• Diagnosis: DNA testing

PROTHROMBIN G20210A GENE MUTATIONPROTHROMBIN G20210A GENE MUTATION

COMPARISON OF INHERITED THROMBOPHILIASCOMPARISON OF INHERITED THROMBOPHILIAS

Phenotype Number of genotypes

Approx prevalence in thrombophilia

Approx relative risk of thrombosis

Antithrombin deficiency

Many 5% or less Up to 10 (varies with mutation)

Protein C deficiency

Many 5% Up to 10 (varies with mutation)

Protein S deficiency

Many 5% or less Up to 10 (varies with mutation)

Factor V Leiden One 40-50% 3-7

Prothrombin G201210A

One 5-10% 2-3

INHERITED THROMBOPHILIA: GENE DOSEINHERITED THROMBOPHILIA: GENE DOSERelative risk of thrombosis in heterozygous

and homozygous factor V Leiden

Rosendaal et al, Blood 1995;85:1504

Genotype Relative Risk

Normal 1

Heterozygous 7

Homozygous 80

INHERITED THROMBOPHILIA: GENE INHERITED THROMBOPHILIA: GENE INTERACTIONSINTERACTIONS

Co-inheritance of protein C deficiency and Co-inheritance of protein C deficiency and factor V Leiden within a familyfactor V Leiden within a family

Koeleman et al, Blood 1994;84:1031

Gene Mutation

Protein C and Factor V

Thrombosis present (%)

16 (73)

Thrombosis absent (%)

6 (27)

Protein C 5 (31) 11 (69)

Factor V 2 (13) 11 (87)

None 0 11 (100)

RISK OF VENOUS THROMBOSISRISK OF VENOUS THROMBOSISFactor V Leiden plus oral contraceptiveFactor V Leiden plus oral contraceptive

Vandenbroucke et al, Lancet 1994;344:1453

RISK FACTORRELATIVE RISK OF

THROMBOSIS

Oral contraceptive 4

Factor V Leiden 8

Both 35

INHERITED THROMBOPHILIA IS A RISK FACTOR, INHERITED THROMBOPHILIA IS A RISK FACTOR, NOT A DISEASENOT A DISEASE

Thrombophilia is a weak (not statistically significant) predictor of recurrence in patients with venous thrombosis

RISK OF VENOUS THROMBOSIS IN AFFECTED VS UNAFFECTED RISK OF VENOUS THROMBOSIS IN AFFECTED VS UNAFFECTED RELATIVES OF THROMBOPHILIC PATIENTSRELATIVES OF THROMBOPHILIC PATIENTS

Blood 2009;113:5314

Unaffected (no defect)

AT

PC

PS

PT

FVL

FVL, PT mutation: Most carriers remain asymptomatic

PC, PS, AT deficiency: Higher chance of thrombosis, but many carriers asymptomatic

TESTING FOR INHERITED THROMBOPHILIATESTING FOR INHERITED THROMBOPHILIA

• Young patient• Family history• Thrombosis in absence of known risk factors• Warfarin-induced skin necrosis (protein C)• Neonatal purpura fulminans (protein C, S)

When is it indicated?

• Rapid, cheap (?) screening for large numbers of mutations and polymorphisms using DNA chip technology

• More accurate diagnosis of inherited antithrombin, protein C, protein S deficiency

• Discovery of many new genetic conditions that affect thrombotic risk

• More information than we know what to do with

DIAGNOSIS OF INHERITED THROMBOPHILIADIAGNOSIS OF INHERITED THROMBOPHILIA

What's next?What's next?