1. PATHOPHYSIOLOGY OF THROMBOSIS “Virchow’s Triad” 1.Injury to blood vessels Trauma, atherosclerosis, surgery 2.Stasis of blood Immobility, 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

2. INHERITED 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)

3. DEEP VENOUS THROMBOSIS

4. PULMONARY EMBOLISM Arrow points to large clot in pulmonary artery Clot dissolved after administration of fibrinolytic drug

5. THERE ARE MANY POTENTIAL GENETIC CAUSES OF THROMBOPHILIA “If something can go wrong, it will” (Murphy)

6. THERE ARE FIVE KNOWN CAUSES OF INHERITED THROMBOPHILIA Defects in physiologic anticoagulant pathways Increased production of procoagulant 1.Antithrombin deficiency 2.Protein C deficiency 3.Protein S deficiency 4.Factor V Leiden 5.Prothrombin G20210A gene mutation

7. QUANTITATIVE VS QUALITATIVE DEFICIENCY OF CLOTTING 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

8. ANTITHROMBIN 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

9. THE ANTITHROMBIN SYSTEM

10. Serine protease mechanism

11. ANTITHROMBIN-HEPARIN INHIBITORS 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

12. INHERITED ANTITHROMBIN DEFICIENCY Prevalence: 0.2-0.4% of population; 2-5% of inherited thrombophilia Dominant inheritance with variable penetrance –No homozygotes known (lethal?)

13. ANTITHROMBIN 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

14. THE 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

15. THE PROTEIN C SYSTEM IIa E C IIa E C P C APC P S + Va Vi VIIIa VIIIi TM

16. PROTEIN 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

17. DOMINANT INHERITANCE OF PROTEIN C DEFICIENCY 2217181383026213230222419 Protein C deficient Protein C normal History of thrombosis

18. RECESSIVE INHERITANCE OF PROTEIN C DEFICIENCY

19. HOMOZYGOUS PROTEIN C DEFICIENCY CAUSES NEONATAL PURPURA FULMINANS

20. PROTEIN C LEVELS DROP FASTER THAN LEVELS OF OTHER VITAMIN K-DEPENDENT PROTEINS DURING WARFARIN TREATMENT Protein C Prothrombin

21. WARFARIN-INDUCED SKIN NECROSIS IN A PROTEIN C-DEFICIENT PATIENT

22. PROTEIN 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

23. PROTEIN S Crossed immunoelectrophoresis showing bound and free forms Bound (inactive) Free (active)

24. PROTEIN S DEFICIENCY Dominant inheritance, prevalence unknown Found in about 5% of inherited thrombophilia Three patterns of deficiency 1.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

25. PROTEIN 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

26. PROTEIN C AND S Acquired 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)

27. MEASURING PROTEIN C AND S IN WARFARIN-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

28. 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 Leiden

29. IIa E C IIa E C P C APC P S + Va Vi VIIIa VIIIi TM

30. MODIFIED FUNCTIONAL ASSAY FOR FVL 4. APC ratio = aPTT with APC aPTT without APC 1.Mix patient plasma with factor V deficient plasma (1:4) 2.Plasma mixtureaPTT 3.aPTTMixture + APC

32. THE FACTOR V LEIDEN MUTATION

34. DNA TESTING FOR FACTOR V LEIDEN NORMALHOMOZYGOUSHETEROZYGOUS FVL DNA AMPLIFIED BY PCR, DIGESTED WITH RESTRICTION ENZYME

35. 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 MUTATION

36. COMPARISON OF INHERITED THROMBOPHILIAS PhenotypeNumber of genotypes Approx prevalence in thrombophilia Approx relative risk of thrombosis Antithrombin deficiency Many5% or lessUp to 10 (varies with mutation) Protein C deficiency Many5%Up to 10 (varies with mutation) Protein S deficiency Many5% or lessUp to 10 (varies with mutation) Factor V LeidenOne40-50%3-7 Prothrombin G201210A One5-10%2-3

37. INHERITED THROMBOPHILIA: GENE DOSE Relative risk of thrombosis in heterozygous and homozygous factor V Leiden Rosendaal et al, Blood 1995;85:1504 GenotypeRelative Risk Normal1 Heterozygous7 Homozygous80

38. INHERITED THROMBOPHILIA: GENE INTERACTIONS Co-inheritance of protein C deficiency and factor 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 C5 (31)11 (69) Factor V2 (13)11 (87) None011 (100)

39. RISK OF VENOUS THROMBOSIS Factor V Leiden plus oral contraceptive Vandenbroucke et al, Lancet 1994;344:1453 RISK FACTOR RELATIVE RISK OF THROMBOSIS Oral contraceptive4 Factor V Leiden8 Both35

40. INHERITED THROMBOPHILIA IS A RISK FACTOR, NOT A DISEASE Thrombophilia is a weak (not statistically significant) predictor of recurrence in patients with venous thrombosis

41. RISK OF VENOUS THROMBOSIS IN AFFECTED VS UNAFFECTED RELATIVES 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

42. TESTING 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?

43. 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 THROMBOPHILIA What's next?