A. Overview of Thrombophilic Disorders [13,26]

1. Also called thrombophilia or hypercoagulability
   1. Clots occur under inappropriate conditions
   2. Clots are intravascular and cause significant morbidity or mortality
   3. Venous thrombophilia includes deep vein thrombosis (DVT) and pulmonary embolism [9]
   4. Arterial thrombosis includes myocardial infarction, stroke, peripheral arterial clots
2. Classification of Thrombophilic Disorders
   1. Hereditary (Genetic) - predisposition to clotting
   2. Hereditary divided into reduced anticoagulants or increased procoagulants [13]
   3. Acquired - malignancy and pregnancy are most common
   4. Most acquired thrombophilias probably have a genetic predisposition component
   5. Risk factors are usually associated with EITHER venous or arterial thromboses
   6. Coagulation pathway, endothelium, inflammatory pathway and platelets involved
   7. Many risk factors are associated with clots in specific vascular beds
   8. Platelets likely play major role in arterial but not venous biased thrombophilic events
3. Malignancy
   1. Venous clotting much more common than arterial
   2. Trousseau Syndrome - migratory thrombophlebitis
   3. DVT
   4. Hypercoagulability is related to acquired resistance to anti-clotting factors
   5. May also be related to endothelial cell dysfunction with loss of anti-clotting functions
   6. Anti-phospholipid antibody related thrombosis may also occur
4. Pregnancy
   1. Environment (pregnancy) interactions with genetic predisposition [22]
   2. Congenital abnormalities of Protein S, Protein C, or antithrombin (AT III)
   3. Antiphospholipid (anticardiolipin) antibodies
   4. Over 20% of pregnant women with pregnancy related venous thromboembolism have Factor V Leiden (see below)
   5. Mutations in other genes correlated with increased thrombophilia in pregnancy:
   6. Methylenetetrahydrofolate reductase (MTHFR) mutations - homocysteine metabolism
   7. Prothrombin (Factor II) gene mutations
   8. Venous thromboembolism more common than arterial
5. Oral Contraceptives
   1. Overall thromboembolic relative risk ~3 fold
   2. Strong interactions with genetic factors [18]
   3. Appears to increase risk of venous thromboembolism in air travelers [6]
   4. Venous thromboembolism more common than arterial
6. Estrogen Replacement Therapy
7. Hyperhomocysteinemia
   1. Hyperhomocysteinemia usually due to inadequate folate, Vitamin B6 or B12 intake
   2. Some patients have specific genetic mutations (see below)
   3. Levels >18.5µmol/L have 2.5X risk thrombosis
   4. Levels >20µmol/L have 3.5X risk thrombosis
   5. Both venous and arterial thromboembolisms associated with hyperhomocysteinemia
   6. Smoking increases homocysteine levels (along with CRP and fibrinogen) [1]
8. Atherosclerosis
   1. Deposition of cholesterol-laden plaque in arteries (not found in normal veins)
   2. Plaque rupture leads to exposure of highly thrombogenic surface
   3. Large thrombus forms above ruptured plaque leading to vessel occlusion
   4. Genetic components clearly contribute to plaque friability and arterial thrombosis
9. Diabetes Mellitus [29]
   1. Insulin resistance and hyperinsulinemia themselves increase clotting factors
   2. Levels of plasminogen activator inhibitor 1 (PAI-1), fibrinogen, Factor VII, and tissue plasminogen activator (TPA) increase with increasing fasting insulin levels
   3. Hyperglycemia leads to endothelial dysfunction and hyperlipidemias
   4. Result is overall pro-coagulant state with highly excess vascular disease risk
   5. Patients with only insulin resistance, not frank diabetes, are at increased risk for vascular events
   6. Elevated levels of PAI-1 stimulate atherosclerosis and plaque rupture
10. Other Acquired Hypercoagulable States
    1. PNH
    2. Myeloproliferative Diseases
    3. Warfarin Induced Skin Necrosis (see below)
    4. Thrombotic Thrombocytopenic Purpura (TTP)
    5. Abnormal von Willebrand factor (vWF) processing protease implicated in TTP [21]
    6. Elevated vWF levels are a risk factor for DVT [11]
    7. Obesity - possible association with increased thromboembolic disease
    8. Chronic renal failure - elevated fibrinogen, prothrombin fragments, and D-dimer [33]
    9. Elevated fibrinogen associated with increased clot risks [1]
    10. Systemic lupus erythematosus (SLE) - particularly associated with certain antibodies
    11. Homozygous variant alleles of mannose binding lectin (MBL2) associated with 7X increased risk for arterial (but not venous) thrombosis in SLE [5]
11. Thrombotic Storm [16]
    1. Thrombosis perpetuates thrombosis
    2. Most common in above disorders
    3. Microangiopathic Hemolytic Syndromes also associated with thrombotic storm
    4. Antiphospholipid Storm has also been documented
12. Cryofibrinogenemia (see below) [12]

B. Anti-Phospholipid Syndrome (APLS)

1. Components of APLS
   1. Recurrent arterial and/or venous thromboembolic events
   2. Thrombocytopenia
   3. Recurrent Fetal Loss
2. Appears to be an acquired condition, though underlying genetic predisposition likely
   1. May be primary syndrome existing alone
   2. Secondary APLS: associated with Systemic Lupus or other autoimmune disorders
3. Classical Description of Anti-Phospholipid Antibodies (APL Ab)
   1. Lupus Anticoagulant - can cause an elevated APTT blood test
   2. False positive test for syphilis (positive RPR, rapid plasma reagin test)
   3. Anti-cardiolipin Ab - often specific for ß2-glycoprotein
4. Mechanism of APL Ab Induced Thrombophilia
   1. APL Ab reduce levels of annexin V and accelerate plasma coagulation
   2. Annexin V also called placental anticoagulant protein 1, vascular anticoagulant alpha
   3. Appears to be important endothelial and placental anticoagulant protein
   4. May explain bias of syndrome towards fetal loss and endothelial dysfunction
5. Anticardiolipin Ab positivity after first DVT predicts recurrence [15]

C. Overview of Hereditary Coagulation Protein Disorders [3,4,9,13,30]

1. Antithrombin (AT; formerly AT III) Deficiency
2. Protein C Disorders
   1. Protein C Deficiency - venous and arterial thromboses
   2. Activated Protein C Resistance (Factor V Leiden Mutation - mainly venous thrombosis
3. Protein S Deficiency - venous thrombosis
4. Plasminogen Deficiency
5. Elevated Levels of Procoagulants
   1. Fibrinogen (Factor I; also called dysfibrinogenemia)
   2. Factor VIII
   3. Factor IX
   4. Factor XI (polymorphism also associated with hypercoagulability [4])
6. Hyperhomocysteinemia
7. Polymorphisms
   1. Prothrombin (Factor II) Mutation - leads to elevated prothrombin levels
   2. Factor VII H7H7 Genotype (H6H6 high risk)
8. These genetic factors interact with each to increase clotting risk [3]

D. Antithrombin (AT) Deficiency [13]

1. Previously called antithrombin III (AT III) deficiency
2. Autosomal dominant inheritance, 1/2000 persons
3. Risk increased for venous thromboembolic disease
4. Increased risk for fetal loss (~5X normal risk)
5. Increased risk for thromboembolism in third trimester and peripartum
6. ~50% of patients have thrombotic events by age 50
7. Purified antithrombin now available

E. Protein C Deficiency

1. Symptoms may occur in persons with <50% of normal levels
   1. Autosomal recessive low factor levels
   2. Resistance to activated Protein C (Factor V-Q506 ("Leiden") Mutation)
   3. Acquired resistance to protein C (APC) due to oral contraceptive agents (OCPs) [27]
   4. Acquired resistance to APC is found in cancer patients with venous thromboembolism [34]
2. Effects of Protein C Deficiency [3,10]
   1. Risk for recurrent venous thromboembolic disease is about ~20/1000 patients/year
   2. Increased risk for arterial thrombosis and childhood stroke in homozygous deficiency
   3. Increased risk for fetal loss (~2X normal)
   4. Risk for recurrent venous thromboembolism highest in first year after initial event
   5. Risk for recurrent thromboembolism declines after first year
   6. ~30% of patients have thrombotic events by age 50
3. May accompany disseminated intravascular coagulopathy (DIC) and/or liver dysfunction
4. Treatment is usually warfarin therapy
   1. Usual therapy is lifelong warfarin, but this may not be optimal
   2. Warfarin does reduce risk of recurrent thromboembolism by about 50%
5. Warfarin Induced Skin Necrosis
   1. Warfarin anticoagulant therapy appears to acutely decrease Protein C levels
   2. This precedes the decrease in the other Vitamin K dependent zymogens
   3. Skin necrosis may occur, especially with warfarin therapy
   4. In general, heparin should be given prior to initiation of warfarin therapy
6. Activated protein C (Xigris®) is beneficial in severe sepsis []

F. Factor V "Leiden" (FVL) Mutation [7,8,10]

1. Formerly called Activated Protein C Resistance
2. FLV is a mutation at position 506 in in Factor V, first described in Leiden
   1. Mutation causes an arginine to glutamine (R-->Q) change
   2. Active Protein C is a protease which cleaves Factor V at three positions
   3. FVL reduces ability of Protein C to cleave at one of the three Factor V sites
   4. Overall, 3-7% of the population has Leiden mutation
3. Frequently associated with hyperhomocyteinuria and thrombosis
   1. Increased risk of single and recurrent DVT [8]
   2. Homozygous and heterozygous mutation predicts recurrent DVT in cancer patients
   3. Associated with increased risk (2X normal) of fetal loss
4. Risks of Thromboembolic Events with FVL [7,10]
   1. Alone, carries ~2-5% absolute increased risk of thrombotic event over lifetime
   2. Additive or synergistic with other thrombophilia risks
   3. Increased risk of thrombosis with heterozygous FVL alone is on the order of 3X
   4. Absolute annual incidence of DVT in FVL carriers is 0.58% (5-6X normal population) [36]
   5. Homozygosity for FVL carries 18X increased risk of DVT, 1-5% annual risk [7]
   6. FVL carries ~20% increased risk for coronary artery disease [2]
   7. Oral contraceptives (OCP 2nd generation) alone increase thrombotic risk is ~4 fold [27]
   8. Leiden + OCP have risk of >30 fold
   9. Leiden + estrogen replacement therapy had ~3X increased risk above ERT alone [31]
   10. Over 40% of women with venous thromboembolism during pregnancy have FVL
   11. Increased risk of venous thromboembolism in air travelers with FVL
   12. Does not appear to be a risk factor for cerebrovascular disease or myocardial infarction
   13. Carrying Factor V Leiden or prothrombin mutations do not increase recurrence risk [17]
5. Spontaneous Abortion
   1. Risk of recurrent spontaneous abortion increased ~2X with FVL mutation [19]
   2. Increased risk ~3X of late fetal loss in heterozygotes [32]
6. Activated Protein C assays or DNA sequences must be measured to detect FVL

G. Protein S Deficiency

1. Hereditary disease is best characterized; acquired deficiency may occur in cancers
2. Associated with increased risk of venous thromboembolic disease
3. Much increased risk for venous thromboembolism in third trimester and peripartum
4. May become manifest first with warfarin therapy or with increased fetal loss
5. Functional protein S deficiency may occur in patients with carcinomas
6. This may be related to the etiology Trousseau's Syndrome
7. ~30% of patients have thrombotic events by age 50 [13]

H. Prothrombin (Factor II) [18,20]

1. Elevated levels associated with G20210A mutation
2. Prevalence of G20210A mutation is ~2%
3. This mutation is found in the 3' untranslated region of the mRNA
4. Apparently, G to A mutation raises prothrombin mRNA and therefore protein levels
5. Confers a 3-5 fold increased risk of thrombotic events, mainly venous
6. Women with prothrombin G20210A mutation on ERT and hypertension have >4X increase risk for first myocardial infarction [35]
7. Associated with ~3X increased risk of late fetal loss in heterozygotes [32]
8. Carries ~1.3X risk of coronary artery disease [2]
9. Risk increase is independent of Factor V mutatations
10. No effect on risk of recurrent DVT [17]

I. Factor VIII Levels [25]

1. Levels of FVIII are controlled by at least 3 sets of genes
2. Genes that code for ABO blood group (Group O persons have lower F VIII levels)
3. Genes that code for von Willebrand Factor (carrier protein for F VIII)
4. Unknown third set of genes
5. F VIII levels >150% of normal (>1500U/L) found in ~11% of general population
6. F VIII levels >150% of normal are found in 25% of patients with DVT
7. F VIII elevation is a >3X risk factor for DVT [11]

J. Abnormal Platelets

1. Essential thrombocythemia
2. Heparin induced thrombocytopenia - thromboembolic events
3. Homocysteinuria - possible effect on platelets and vascular endothelium
4. Coagulation Defects - von Willebrand's Disease, Chronic Renal Fialure (dialysis)
5. Polymorphisms in platelet glycoprotein Ia/IIa correlated with MI risk [21]
6. Thrombotic Thrombocytopenic Purpura (TTP)
7. Hereditary Disorder

K. Coagulation Pathway Levels and/or Mutations Implicated in MI [24]

1. Plasmin-alpha-antiplasmin complex levels
2. Plasminogen activator inhibitor 1 (PAI-1)
3. Tissue plasminogen activator antigen
4. D-Dimer Levels
5. Factor VIII levels
6. Thromboplastin
7. von Willebrand Factor
8. Thromboglobulin
9. Prothrombin (Factor II) Levels
10. Polymorphisms in platelet glycoprotein Ia/IIa correlated with MI risk [21]

L. Extracellular Signals Influencing Endothelial Cell Clotting Functions [26]

1. Inflammatory Signals
   1. Tumor necrosis factor alpha (TNFa)
   2. Interleukin 1
   3. Interleukin 6
2. Factors Involved in Angiogenesis and Tissue Repair
   1. Tranforming growth factor ß (TGFß)
   2. Vascular endothelial growth factor (VEGF)
   3. Platelet-Derived growth factor (PGDF)
3. Shear Stress
   1. Increased thrombomodulin (anticoagulant)
   2. Increased tissue-type plasminogen activator (anticoagulant)
   3. Increased nitric oxide synthetase (anticoagulant)
   4. Increased tissue factor (procoagulant)
4. Hypoxia
   1. Increased plasminogen-activator inhibitor type 1 (PAI-1; procoagulant)
   2. Decreased tissue-type plasminogen activator (TPA; anticoagulant)
5. Airplane travel increase coagulability in some persons, particularly after 8 hours [6]

M. Laboratory Evaluation of Procoagulants

1. Prothrombin Time (PT)
   1. Rabbit brain tissue factor (III) added to plasma with calcium
   2. Clotting time depends on levels of factor II, V, VII, X and fibrinogen
   3. Tests extrinsic and common coagulation pathways
   4. Now calculated as International Normalization Ratio (INR) for better interlab correlation
2. Partial Thromboplastin Time (PTT)
   1. Phospholipid and Hageman Factor XII added to blood for 5 minutes; then Calcium added
   2. Senisitive to nearly all clotting deficiencies and inhibitors
   3. Exceptions include factor VII inhibitors and some anticardiolipin antibodies
3. Inhibitor Screens
   1. Mixing normal plasma with test plasma is simplest inhibitor screen
   2. Russel Viper Venom Time (RVVT)
   3. Anti-cardiolipin Antibody Titers
   4. Lupus Anticoagulant
4. Thrombin Time
   1. Time necessary for dilute solution of thrombin to clot plasma
   2. Dependent on normal fibrinogen and prolonged by heparin and other anticoagulants
   3. May be only abnormality in patients with dysfibrinogenemia (abnormal fibrinogen)
5. Reptilase Time
   1. Reptilase is a thrombin-like enzyme unaffected by heparin or fibrin degradation products
   2. Converts fibrinogen to fibrin
   3. A long thrombin time with normal Reptilase time suggests heparin effect

N. Genetic Screening for Thrombophilia

1. Factor V Leiden Mutation Screening
   1. Factor V destruction assay - with activated protein C
   2. DNA sequencing (mutation detection) - G1691A mutation
2. Factor II (Prothrombin) Gene Mutation
   1. G20210A Mutation in 3' untranslated region leads to elevated levels of protein in serum
   2. Increased risk for DVT and cerebral-vein thrombosis [18]
   3. Increased risk for complications of pregnancy [22]
3. Factor VII Genotyping
4. Homocysteine Pathway
   1. Methylenetetrahydrofolate reductase (MTHFR) mutations
   2. Cysteine ß-synthetase (CBS) mutations
   3. Mutations in folate-pathway enzymes
5. Screening for AT, Protein C, Protein S mutations

O. Cryofibrinogenemia [12]

1. Rarely symptomatic disorder with primary (essential) and secondary forms
2. Cryofibrinogen refers to an abnormal, cold-precipitable protein complex which forms clots
   1. Insoluble complexes consisting of fibrin, fibrinogen, fibrin split products with albumin, plasma proteins, immunoglobulins
   2. High plasma levels of protease inhibitors alpha1-antitrypsin (A1AT) and alpha2- macroglobulin have been found
   3. These protease inhibitors fibrinolytic activity of plasmin
   4. Thus, large insoluble protein complexes can form clots
3. Symptomatic disease is uncommon
   1. Prevalance of laboratory defined disorder may be as high as 13%
   2. Symptomatic disease much less frequent, more common in colder climates
4. Symptoms
   1. Usually due to cutaneous ischemia
   2. Purpura, livido reticularis, ecchymosis, ulcerations
   3. Ischemic necrosis and gangrene can occur
5. Diagnosis
   1. Consider in persons no explanation for new onset tissue ischemia and gangrene
   2. Demonstration of cryofibrinogens and absence of cryogobulins required
   3. Blood collected in oxalate, citrate, or EDTA tubes (but not heparin)
   4. Blood stored at 37°C until centrifuged
   5. After centrifugation, plasma stored at 4°C for 72 hours
   6. Cryofibrinogen (cloudy precipitates) develop in 24-72 hours
   7. Cryofibrinogen precipitate can be reduced by streptokinase
   8. Presence of elevated levels of antitrypsins above
6. Treatment
   1. Avoidance of cold exposure and warming in environment at 37°C
   2. Antiseptic wound care
   3. Standard management of gangrene or other ischemic tissue
   4. Streptokinase IV 25,000-200,000 units q24 hours to break up clots
   5. Stanozolol, testosterone derivative, 2-4mg po bid, active after 2-4 day onset
   6. Plasmapheresis of limited benefit

P. Increased Bleeding Disorders

1. Factor XII Deficiency
   1. Increases clotting time of blood in glass
   2. No hemorrhagic problems in patients lacking this (Hageman) factor
   3. Similar findings for patients lacking HMWK and Prekallikrien
2. Factor XI Deficiency: bleeding fairly common, particularly after surgery
3. Hemophilias
   1. Factor VIII Deficiency (Hemophilia A)
   2. Factor IX Deficiency (Hemophilia B)
   3. Acquired Hemophilias
4. Von Willebrand's Disease
   1. vWF is the carrier protein for Factor VIII
   2. It is also involved in platelet activation
5. Disorders of Mixed Bleeding and Thromboembolic Events
   1. Essential Thrombocythemia
   2. Disseminated Intravascular Coagulopathy (DIC)
   3. HUS/TTP
6. Platelet Disorders

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