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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?