Lecture 8, fall 2014
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The document discusses thrombophilia, highlighting the causes and effects of arterial and venous thrombosis. It details the pathogenesis of atherosclerosis leading to conditions such as stroke and myocardial infarction, as well as the complexities of venous thrombosis and risk factors associated with it. Additionally, the document outlines inherited and acquired thrombophilic disorders, emphasizing the need for testing in patients with unexplained thrombosis.
Lecture 8, fall 2014
- 2. Thrombophilia • Arterial Thrombosis • Stroke and myocardial infarc:on are major causes of death ▫ Every 45 seconds someone in the US suffers a new or recurrent stroke ▫ 800,000/year – Every 34 seconds someone in the US suffers a new or recurrent MI – 1.5 million/year à ~ 1/3 will die 2
- 3. Atherosclerosis • Atherosclerosis – thickening of the arterial wall – primary cause of coronary artery disease and cerebrovascular disease – Arterial wall thickens to form an atherosclero:c plaque – Reduces the blood supply to the organ (heart and brain – most common) • Atheroma – accumula:on of intracellular and extracellular lipid in the in:ma of large and medium sized arteries
- 4. Atherosclerosis • Mechanism 1. A lesion begins as a faTy streak (preatheroma) that protrudes into the in:ma • LDL enters the in:ma – modified by oxida:on and aggregates within the extracellular in:ma space • Smooth muscle cells, T-‐lymphocytes , and macrophages migrate into the area – macrophages phagocy:ze the oxidized lipids • Proteoglycans, collagen and elas:c fibers migrate into the area 2. Fibro-‐faTy lesion forms – diffuse in:mal thickening occurs • Atheromatous plaque 3. Complicated plaque • Eggshell briTleness, ulcera:on of the luminal surface, micro-‐emboli released into the blood stream, decreased blood flow results in more thrombus forma:on
- 5. Pathogenesis 1. Chronic inflammatory response of the vascular wall to endothelial injury or dysfunc:on 2. Elevated plasma LDL levels causing the deposit of LDL in the subendothelium of blood vessels 3. Oxida:on of transmigrated LDL 4. Ac:va:on of endothelial cells 5. Recruitment of monocytes/macrophages which ingest ox-‐LDL through scavenger receptors 6. Forma:on of foam cells – faTy streaks 7. Prolifera:on of smooth muscle cells 8. Deposi:on of extracellular matrix proteins
- 7. • Coronary Atherosclerosis hTp://upload.wikimedia.org/wikipedia/commons/9/9a/ Endo_dysfunc:on_Athero.PNG
- 8. Mechanism of Arterial Thrombosis hTp://www.drugs.com/health-‐guide/images/205452.jpg8
- 9. CORONARY ARTERY DISEASE 1. Artery narrowed by cholesterol containing atheroma – note how the tube which the blood flows through has been narrowed and restricted 2. Once the surface of the vessel is damaged, platelet clot accumulates restric:ng flow – this may resolve or worsen 3. Platelets may accumulate so that blood flow is limited by the clot and this causes starva:on of oxygen death of muscle and a heart aTack
- 10. Pathogenesis of coronary heart disease (CHD)
- 12. Thrombophilia ▫ Venous Thrombosis ▫ DVT/PE à ~ 900,000 to 2,000,000/year ▫ 60,000-‐100,000 individuals will die of DVT/PE ▫ 10-‐30% will die within one month of diagnosis ▫ ~25,000 of these deaths result from VTE contracted in hospitals ▫ >25X the number of deaths from MRSA ▫ >Combined total deaths from BC + AIDS + MVA • Ironically – fatal PE caused by DVT may be the most common preventable cause of hospital death in the US – only 1/3 of hospitalized paBents with risk factors for VTE receive preventaBve measures 12
- 13. Mechanism of Venous Thrombosis 13 • Most common manifesta:ons – Deep vein thrombosis – Pulmonary embolism – Postphlebi:c syndrome • Mechanism – Endothelial damage • Trauma, surgery • TF • Thrombin genera:on • Primary hemosta:c plug with fewer platelets • Venous stasis – Red clots Nature, 451(21) Feb 2008
- 14. What Causes a Thrombus to Form? Venous Thrombogenesis – Thrombi begin in regions of slow/disturbed blood flow Damaged veins, valve cusp pockets – Inherited/acquired hypercoagulable states important – Stasis is a major risk factor – Variable response to thrombus • Classical signs/symptoms DVT • Minimal signs/symptoms
- 15. DVT/PE 15
- 17. Venous Clot
- 18. Thrombosis (Blood Clots) Intracoronary Clot DVT PE
- 19. Hemorrhage (Bleeding) Intracranial Bleed Hemorrhagic Stroke Gross specimen, coronal sec:on of brain, large subcor:cal hypertensive ICH
- 20. Post phlebitic syndrome Chronic venous ulceration Very difficult to manage Pain (dull and aching), leg cramps, heaviness, itching and altered sensation
- 21. Arterial versus Venous Thrombosis Arterial Thrombosis Venous Thrombosis — Arterial thrombosis — Occur under high shear condi:ons — Rich in platelets — Involved disrupted atherosclero:c plaque — Platelet adhesion, ac:va:on, and aggrega:on prior to ac:va:on of coagula:on cascade — White clots — Myocardial infarcBon and stroke — AnBplatelet agents — AnBfibrinolyBc and anBthromboBc agents • Venous thrombosis – Under low shear stress – Fewer platelets involved – TF generates thrombin prior to platelet ac:va:on – Red clot – DVT, PE – AnBcoagulaBon agents for venous thrombosis 21
- 22. Arterial vs Venous Clot hTp://www.emedicinehealth.com/ slideshow_pictures_deep_vein_thrombosis_dvt/ar:cle_em.htm hTps://www.med.unc.edu/wolberglabl/scien:fic-‐images/arterial %20thrombosis.jpg/view
- 23. Virchow’s Triad 23 Stasis Thrombosis Changes in Blood Composi;on Vascular Injury Arterial Rudolph Virchow Post-‐operaBve state CasBng/splinBng Sedentary state Leukostasis syndrome (AML) Congenital heart disease Central line, Sepsis Trauma, APA Chemotherapy/toxins Hyperhomocysteinemia Inherited thrombophilia Acquired thrombophilia
- 24. Virchow’s Triad Thrombosis involves 3 interrelated factors: 1. Abnormali:es of the blood vessel wall 2. Abnormali:es in blood flow 3. Abnormali:es in the blood cons:tuents • Cells -‐ Erythrocytes, leukocytes, platelets • Plasma proteins
- 25. Risk Factors 25 • Mul:ple risk factors—mul:-‐factorial process – Hereditary – Acquired • Mul:-‐hit hypothesis – Most hereditary and acquired risk factors have a rela:vely small individual effect – Risk is greatly increased when two or more risk factors combine • Classifica:on of Thrombophilia – Inherited – Acquired—Physiologic, Environmental
- 26. Thrombophilia 26 • Acquired or inherited causes • Venous and arterial events • Occurs when the cloqng system is ac:vated 1. Excessive genera:on of prothrombo:c factors 2. Failure in the regulatory mechanisms to down-‐ regulate the coagula:on cascade 3. Inhibi:on of the fibrinoly:c system
- 27. Thrombophilic Risk Factors Congenital Risk Factors Mechanism 27 ¤ Protein C ¤ Protein S ¤ AT ¤ FVL ¤ PG20210 ¤ FVIII ¤ Homocysteine (acquired also) ¨ Non-‐modifiable Inhibitory Prothrombotic
- 28. Acquired Risk Factors 28 ¨ Acquired risk factors ¤ Pregnancy ¤ Malignancy ¤ Surgery ¤ Immobiliza:on ¤ Hormone therapy (HRT, OCT) ¤ An:phospholipid an:bodies ¤ Trauma ¤ Obesity ¨ Physiologic risk factors ¤ Gender (hormonal changes) ¤ Age –Increases ~1%/year of age n Childhood = 1/100,000 n 40 years = 1/1000 n 75 years = 1/100 Modifiable Non-‐modifiable ¨ IdenBfy a populaBon at risk but have low predicBve value for individuals
- 29. Prevalence of Risk Factors Congenital Risk Factors Acquired Risk Factors 25% 20% 15% 10% 5% 0% % Risk Prevalence of Inherited Risk Factors FVL PG AT PC PS FVIII Risk Factor General Population Selected: 1st Thrombotic Event Prevalence of Acquired Risk Factors 90 80 70 60 50 40 30 20 10 0 Fractures Hip Cancer APAS OCT Pregnancy HRT Hcys FVIII Risk Factor Increase 29
- 30. Who should be tested 30 ¨ Pa:ents presen:ng with ¤ Venous thrombo:c event before 40-‐50 years of age ¤ Unprovoked or Recurrent thrombosis at any age ¤ Thrombosis at unusual site ¤ Posi:ve family history of thrombosis ¤ Unexplained abnormal laboratory test (PT, aPTT) ¨ Age of first episode ¤ 0-‐12 years Rare ¤ 13-‐45 years Highly probable ¤ 45-‐60 years Probable ¤ 60+ years Possible Congenital Risk Factors
- 31. When to test 31 ¨ Op:mal Times for Tes:ng • Asymptoma:c • Not on an:coagulant therapy • Any:me for DNA tes:ng ¨ To establish • Pathologic basis for the thrombo:c event • Dura:on and intensity of therapy • Prophylaxis for high risk pa:ents • To alert the pa:ent's immediate family members to the presence of possible inherited risk factors
- 32. Laboratory Assays for Thrombophilia 32 • Plasma-‐based assays – AT – PC – PS – APC-‐R – Lupus An:coagulant/An:phospholipid An:bodies • Dilute Russell Viper Venom Test (dRVVT) • An:cardiolipin An:bodies • An:-‐β2-‐Glycoprotein An:bodies – Factor VIII – Homocysteine • DNA-‐based assays – FVL – PG20210 – MTHFR
- 33. An:thrombin Deficiency 33 • Eggberg, 1965 • Reported the first inherited thrombophilic state • Func:ons as a naturally occurring inhibitor of the coagula:on cascade • Most severe of the inherited condi:ons • Rela:vely uncommon (~1% of first DVT) • Clinical Manifesta:ons – Increased incidence of venous thrombosis – AT levels <40-‐50% – Ini:a:ng events leading to thrombosis • Surgery • Trauma • Pregnancy • OCT
- 34. Protein C Deficiency 34 ¨ Griffin, early 1980’s ¨ 75% of individuals will experience one or more events ¨ Thrombosis may be spontaneous ¨ Func:ons as a naturally occurring inhibitor of the coagula:on cascade ¨ 50% of heterozygotes will experience VTE by 40 years of age ¨ Common Manifesta:ons ¤ DVT ¤ PE ¤ Superficial thrombophlebi:s ¤ Cerebrovascular events ¤ Myocardial events
- 35. Protein S Deficiency ¨ Described in 1984, Comp ¨ TOTAL PS circulates in 2 forms: ¤ Bound PS—60% n C4B-‐BP—nonfunc:onal ¤ Free PS—40%-‐func:onal ¨ Serves as a cofactor for PC ¨ Binds aPC to the phospholipid surface ¨ 50% of heterozygotes will experience VTE by 40 years of age Free PS 35 Total PS C4bBP
- 36. 36 aPC-‐Resistance—Screening assay • aPC-‐resistance – Dahlbäck et al in 1993 – Func:ons as a natural an:coagulant – Poor an:coagulant response of aPC to degrade FVa and VIIIa – Ra:o of 2 aPTT’s—(+/-‐ APC) __(aPTT plus APC)__ (aPTT minus APC) • “Screening assay” for FVL muta:on • http://www.wardelab.com/arc_2.html Approximately 90% of APC Resistance is caused by a defect in the Factor V molecule, known as the Factor V Leiden gene muta:on
- 37. Factor V Leiden—Confirmatory Assay for FVL Muta:on – Muta:on later described in 1994 by Ber:na et al – Caused by single point muta:on in the FV gene • A single nucleo:de subs:tu:on of adenine for guanine at nucleo:de 1691 of the FV gene • Replacement of Arg (R) with Gln (Q) at pos 506 in F.V protein – Higher risk for thrombosis – Venous thrombosis most common manifesta:on 37
- 38. PG20210 Muta:on ¨ Poort et al, 1996 ¨ Single nucleo:de subs:tu:on G20210A in the 3’ UT region of the prothrombin gene ¤ G → A subs:tu:on at nucleo:de 20210 in prothrombin gene ¨ Results in elevated levels of prothrombin (~30% increase) ¨ No screening test available ¨ Occurs primarily in Caucasians -‐-‐~3% in general popula:on ¨ 2-‐5-‐fold increased risk of VTE 38
- 39. Homocysteine 39 ¨ McCully suggested an associa:on between elevated levels of homocysteine in plasma and arterial disease ¨ Most common congenital form due to: 1. (C677T)* in MTHFR gene 2. B-‐cystathionine synthase gene ¨ Acquired form due to deficiencies in Folate, B-‐6, B-‐12 ¨ Gene:c tes:ng* is controversial ¤ Homocysteine levels may provide more informa:on ¨ Normal values increase with age ¤ Higher in males www.naturaleyecare.com/ar:cles/elevated-‐homocysteine-‐and-‐eye-‐...
- 40. 40 An:phospholipid An:bodies • An:phospholipid an:bodies – Acquired thrombophilic disorder – An:bodies directed against proteins that bind to phospholipid membrane surfaces – Autoimmune process • Subgroups of APLAs – Lupus An;coagulant – An;-‐ Cardiolipin an;bodies – An;-‐Beta-‐2-‐glycoprotein I an;bodies – An:-‐Prothrombin an:bodies – An:-‐Phospha:dylserine an:bodies – An:-‐Phospha:dylethanolamine an:bodies – An:-‐ Phospha:dylinositol an:bodies hTp://circ.ahajournals.org/cgi/reprint/112/3/e39
- 41. Clinical Diagnosis APAS • Acquired disorder which occur in 1-‐5% of the general popula:on • Pa:ent must present with one clinical and one laboratory criteria • Clinical Manifesta:on – Vascular thrombosis • One or more clinical episodes of arterial, venous or small vessel thrombosis in any :ssue or organ – Pregnancy Morbidity • One or more spontaneous abor:ons, severe preeclampsia, eclampsia, death of a normal fetus at or near 10 months gesta:on • Laboratory Criteria – Posi:ve test in the APA test panel on 2 separate occasions, > 6-‐12 weeks apart • Lupus An:coagulant • An:cardiolipin An:body • An: –B2-‐Glycoprotein I An:body 41
- 42. Lupus An:coagulant • Heterogeneous group of an:bodies (IgG, IgM, or both) that prolongs phospholipid-‐dependent coagula:on tests – Immunoglobulin that acts as a coagula:on inhibitor – Does not recognize a “specific” coagula:on factor – Retards the rate of thrombin genera:on and clot forma:on in vitro by interfering in phospholipid-‐ dependent reac:ons • Detected in in vitro coagula:on assays only 42
- 43. Lupus An:coagulant • Affect 2-‐4% of the U.S. popula:on • Discovered accidentally—prolonged aPTT found during a pre-‐opera:ve evalua:on • O|en cause a variety of clinical and laboratory effects – O|en there are no clinical consequences other than the need to explain the reason for the long APTT – Minority of pa:ents with LA have a hypercoagulable state manifested by: • Recurrent thromboses • Mul:ple spontaneous miscarriages • Migraine headaches • Stroke • Rarely pa:ents may experience bleeding – Bleeding due to an:bodies to prothrombin • Lupus an:coagulants (LA) are a heterogeneous group of an:bodies that cause a variety of clinical and laboratory effects 43
- 44. Lupus An:coagulant • Results in prolonga:on of phospholipid-‐dependent assays • LA is o|en iden:fied during rou:ne screening with the standard aPTT – In vitro à results in a prolonged aPTT • Prolonga:on due to reagent sensi:vity to lupus an:coagulant • Usually does not result in clinical bleeding – In vivo à usually results in thrombosis rather than clinical bleeding • Abundance of phospholipid – These neutralize the an:body – May explain why bleeding does not occur in vivo • An:body may be persistent or transient 44
- 45. Lupus An:coagulant • All lupus an:coagulants are APAs, but not all APAs are lupus an:coagulants • Targets specific proteins • B2GPI • Prothrombin • Proteins C, S • Annexin V 45 ANTIBODY-‐MEDIATED THROMBOSIS Phospholipid Associated Proteins: •Protein C,S •β2GPI •Prothrombin •and others Phospholipid Membrane Antibody: •lupus anticoagulant •anticardiolipin •antiphosphatidylserine •anti b2GPI •anti Annexin V •anti Prothrombin
- 46. Lupus An:coagulant and Thrombosis The Paradox... How does an anticoagulant in vitro, become a risk factor for hypercoagulability in vivo ? Coagulation Factor/Protein = Phospholipid CA+2 Calcium Anchor 46 =PL 46
- 47. Clinical Significance of the APAs/LA 47 • Prevalence in venous and arterial thrombosis • DVT ~32% • Stroke ~15% • Superficial thrombophlebi:s ~9% • Pulmonary embolism ~9% • Fetal loss ~8% • TIA ~7% • Associated with 2 broad categories of phospholipids an:bodies – An:cardiolipin an:bodies • Most likely to be clinically significant with high :ters for IgG and IgA – β2-‐GPI an:bodies • More specific for thrombosis and other clinical complica:ons of the APAS
- 48. E:ology of LA • Exact e:ology of LA is unclear • An:bodies are commonly found in asymptoma:c elderly individuals • Pa:ents with autoimmune disorders • SLE have the highest incidence (20-‐45%) • Pa:ents with HIV infec:on have a high incidence of LA at some :me in the course of their disease • A number of drugs are known to induce LA, most notably – Procainamide – Hydralazine – Isoniazid – Dilan:n – Phenothiazines – Quinidine – ACE inhibitors are known to induce LA 48
- 49. Lupus An:coagulant and Thrombosis • LA are one of the most common acquired predisposing causes of thrombosis – cerebral thrombosis – deep venous thrombosis – renal vein thrombosis – pulmonary emboli – arterial occlusions – stroke • Reports indicate that LA are found in: – 8-‐14% of pa:ents with deep venous thrombosis – 1/3 pa:ents with stroke <50 years of age – Evidence that recurrent thrombo:c events tend to be persistent over :me in the same pa:ent 49
- 50. LA in Thrombocytopenia and Pregnancy • LA and thrombocytopenia – An immune type thrombocytopenia has been observed in a small percentage of pa:ents with LA – This may be due to reac:ons between an:bodies and platelet membrane-‐associated phospholipids • LA and Pregnancy – increased risk of fetal loss due to pre-‐eclampsia, placental abrup:on, intrauterine growth retarda:on, and s:llbirth – Some evidence suggests that this may be due to an:bodies against the placental an:coagulant protein, annexin V – Placental infarc:on has been suggested as the cause of the failure to carry to term but pathological analysis has not definitely supported this conten:on 50
- 51. Mechanisms of the Reduc:on of Annexin V Levels and the Accelera:on of Coagula:on Associated with An:phospholipid An:bodies Rand J, NEJM 1997;337:154-‐160 • Annexin V – Phospholipid dependent an:coagulant proper:es on cell membranes – Placental an:coagulant (shield on placental villi) – Vascular an:coagulant 51
- 52. Proposed Mechanisms of Thrombosis • Impaired Fibrinolysis • Inhibi:on of Protein C and S system • Inhibi:on of Prostacyclin release from endothelial cells • Inhibi:on of Annexin V – Tissue Pathway Down Regula:on • Inhibi:on of B2GPI – may affect Protein S 52
- 53. An:cardiolipin An:body ELISA 53