Thrombophilia pathophysiology

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]; Associate Editor(s)-in-Chief: Asiri Ediriwickrema, M.D., M.H.S. [2]

Overview

The pathogenesis of thrombophilia is multi-factorial. It is characterized by hypercoagulability, which by itself or in synergy with endothelial injury or stasis (Virchow's Triad) can predispose to clot formation. Multiple genetic mutations and predisposing conditions have been associated with the increased risk of thrombosis due to abnormalities in the coagulation cascade. The most common genes involved in the pathogenesis of acquired thrombophilias are Factor V Leiden and prothrombin gene mutations.

• Coagulation is an inherent property of the hematologic system and under healthy conditions, normal blood flow is maintained by the balance between the pro-coagulant and anti-thrombotic factors. A hypercoagulable state and subsequent thromboembolism is a result of overactivity of pro-coagulant factors or a deficiency in anti-coagulants. The interplay of factors is complicated - coagulation activators and inhibitors and their production and degradation (quantitative) and functional properties (qualitative) all influence thrombosis. The triad of hypercoagulability, vascular stasis and vascular trauma as described by Virchow in 1856 still holds and remains the harbinger of vascular thrombosis. Arterial thrombosis results from atherosclerotic plaque rupture around which a platelet-rich white thrombus forms. Stasis behind venous valves contributes to venous thrombosis and red thrombus. Mutations influence coagulation depending on whether they are present in heterozygous or homozygous genotype.
• As noted, Virchow's triad (i.e., endothelial damage, hypercoagulability, & venous or arterial blood stasis) plays a significant role in the pathophysiology of thrombosis. Damage to the vessel wall leads to the production of pro-inflammatory (and prothrombotic) cytokines, an increase in available tissue factor, the proliferation of adhesion molecules, and enhanced platelet activation. Cytokines initiate inflammation-promoting interaction between leukocytes and endothelial cells. Inflammation is a normal body reaction to unwanted stimuli such as foreign pathogens or infection and endothelial damage, whether acute (e.g., trauma or surgery) or chronic (underlying inflammatory disorders or peripheral vascular disease). The activation of the leucocytes and endothelial cells causes the formation of adhesion molecules, which will eventually initiate clot formation. The body's endogenous anticoagulants, such as protein C & S and Antithrombin-III, prevent the formation of thrombosis through a complex regulatory mechanism that maintains homeostasis. When an imbalance exists in the formation and lysis of clot, it can generate thrombosis; this explains why patients with thrombophilias such as protein C, protein S, and antithrombin III deficiencies are prone to acquiring blood clots. As noted in the Etiology section, a myriad of additional acquired provoking risk factors and comorbidities (e.g., recent surgery, inflammation, infection, pregnancy, estrogen therapy, morbid obesity, or smoking, among others) also increase a chance of developing thrombosis.
• Thrombosis occurs throughout our arterial system, especially in those with predisposing cardiovascular risk factors. In the heart, microthrombi can develop as a result of blood stasis in the ventricles or atria due to underlying valvular heart disease, cardiomyopathies, or arrhythmias such as atrial fibrillation predisposing to ischemic emboli and CVA. Arterial thrombosis (and microthrombi formation) typically initiates by the accumulation of lipid plaques in the arterial wall, provoking chronic inflammatory cells and platelet activation, as can be seen with coronary artery disease. Platelets play a significant role in the development of arterial thrombosis compared to venous thrombosis, and this explains why antiplatelet agents form a cornerstone of the prevention and treatment of arterial thrombosis. The initial lipid plaques evolve into fibrous plaques. Fibrous plaques could rupture, and the erosion of the surfaces of some of these plaques could lead to the release of additional pro-coagulating factors. This process is called atherosclerosis. Atherosclerosis allows the activation of platelets, causing adhesion and aggregation, which leads to the formation of a clot. The occlusion of vessels due to atherosclerosis and thrombin formation in the coronary arteries of the heart may lead to ischemic heart disease and myocardial infarction.

Pathophysiology

• The primary mechanism for thrombus formation in common inherited thrombophilic states involves thrombin dysregulation.
• Anticoagulants that regulate thrombin include antithrombin, protein C, and protein S.
• Mutations in antithrombin can lead to increased thrombus formation.
• Protein C and S are natural anticoagulants which inhbit thrombin formation. Dysregulation in activated protein C (APC) can occur as either defects in the protein C or S molecule (Protein C and S deficiency) or as resistance to APC activity. APC resistance occurs when APC fails to inactivate downstream coagulation factors, specifically Factor V and Factor VIII.
• The most common inherited thrombophilia is Factor V Leiden, which is a polymorphism of Factor V that is resistant to APC inactivation.
• The second most common inherited thrombophilia involves a gain of function mutation of the prothrombin gene (Prothrombin G20210A) resulting in increased protein activity and thrombus formation.
• Dysfibrinogenemia is a disorder of fibrinogen formation or activity resulting in predisposition for bleeding, thrombosis or both.

Thrombus formation in inherited thrombophilia. In thrombophilia, procoagulant and anticoagulant factors are dysregulated, leading to thrombus formation

Figure: Thrombus formation in inherited thrombophilias. Adapted from: N Engl J Med. 2001 Apr 19;344(16):1222-31.

• Antithrombin III (ATIII) deficiency: Antithrombin III binds to heparin on endothelial cells and forms a complex with thrombin (thrombin-antithrombin (TAT) complex) thus inhibiting coagulation. The prevalence may be 1 in 500 in the general population. Its deficiency may present as early age thrombosis (less than 50 years old) and carries the highest risk for thrombotic events among the inherited thrombophilias. Antithrombin is synthesized in the liver but is not vitamin K-dependent. ATIII deficiency can occur as a consequence of reduced synthesis (liver damage) or increased loss (nephrotic syndrome, enteropathy, DIC, sepsis, burn, trauma, microangiopathy, and cardiopulmonary bypass surgery). Qualitative defects of ATIII (type II deficiency) describe mutations which either affect the heparin-binding site (HBS), the reactive site (RS) or result in pleiotropic effects (PE). Homozygous ATIII deficiency is incompatible with life unless affecting the heparin-binding site. Usually these patients present with venous thrombosis and less likely with arterial thrombosis.

• Protein C deficiency: It present as thrombosis in teenagers. Protein C and S deficiency may be inherited but is also inducable by liver dysfunction, vitamin k antagonists, renal failure, DIC, and active thrombosis. Protein S enhances the effect of activated protein C. Protein S deficiency can be classified as type I (reduced quantity of protein S), type II (low APC activity), and type III (low free protein S due to increased binding to the complement factor C4b). The interaction of protein S with C4, which is an active phase reactant exemplifies the relation of coagulation, inflammation, and autoimmunity. The half-life of protein C is shorter than the half-life of other vitamin K-dependent coagulation factors, hence the risk of increased coagulation with the initiation of vitamin K antagonists and need for bridging with parenteral heparin (warfarin-induced skin necrosis).

Protein C deficiency is a rare disorder, characterized by a reduction in the activity of protein C, a plasma serine protease involved in the regulation of blood coagulation. The active form of protein C, activated protein C (APC), exerts potent anticoagulant activity. A deficiency in protein C is characterized by the inability to control coagulation, resulting in the excessive formation of blood clots (thrombophilia). Protein C deficiency may be acquired or congenital. Congenital protein C deficiency results from mutations in the PROC gene. More than 160 PROC mutations have been described and may result in reduced levels of protein C (Type I) or the production of an altered protein C molecule with decreasing levels of activity (Type II). Protein C deficiency is an autosomal dominant condition. Mutations in a single copy in heterozygous individuals cause mild protein C deficiency, whereas individuals with homozygous mutations present with severe protein C deficiency. Protein C is a vitamin K-dependent protease circulating in plasma at low concentrations and serves a critical role in the regulation of thrombin. Levels of protein C mature later than many other coagulation proteins, with levels increasing from birth until 6 months and into puberty. Protein C becomes activated to form activated protein C (APC) via interactions with thrombin. APC acts to downregulate coagulation by cleaving and inactivating clotting factors V and VIII. A deficiency of protein C, and thus APC activity, leads to an inability to inactivate clotting factors and control thrombin production. Protein C is also known to have a role in the regulation of inflammation and sepsis, with demonstrated cytoprotective functions.

• Protein S deficiency: Protein S deficiency is usually congenital, caused by mutations in the PROS1 gene. More than 200 PROS mutations have been described and may result in three different forms of protein S deficiency:
  • Type I: quantitative defect presenting with low levels of total protein S (TPS) and free protein S (FPS), with reduced levels of protein S activity
  • Type II (also known as Type IIb): Decreased protein S activity, with normal levels of TPS and FPS antigens
  • Type III (also known as Type IIa): quantitative defect presenting with normal levels of TPS, but reduced levels of FPS and protein S activity

Protein S deficiency is an autosomal dominant pathology. Mutations in a single copy in heterozygous individuals cause mild protein S deficiency, whereas individuals with homozygous mutations present with severe protein S deficiency. Causes of temporary acquired fluctuations in protein S levels may include vitamin K-antagonist therapy, chronic infections, severe hepatic disease, nephritic syndrome, and DIC. The risk of VTE is also increased in patients using oral contraceptives and pregnancy. Protein S is a vitamin K-dependent protease that circulates in plasma at low concentrations and serves a crucial role in the regulation of coagulation. In circulation, approximately 40% of protein S is free, and about 60% is in a high-affinity complex with the complement regulatory factor C4b-binding protein (C4BP). The anticoagulant activity of protein S is two-fold: Protein S operates as a cofactor for activated protein C (APC), and inactivating coagulation Factor Va and Factor VIIIa; and Protein S is also a cofactor for the tissue factor pathway inhibitor (TFPI) protein, resulting in the inactivation of Factor Xa and tissue factor (TF)/Factor VIIa. Protein S is a complex protein with multiple structural moieties. The 3-dimensional structure is yet to be resolved but is expected to contribute to the understanding of the complex functional nature of PROS1 mutations.

• Factor V Leiden mutation: Protein C interacts with thrombomodulin to become activated protein C (APC). APC has anticoagulant, anti-inflammatory, and cytoprotective properties and has been proposed for the treatment of sepsis. The signal cascade leading to APC can become distorted through many acquired or inherited mechanisms leading to APC resistance. Activated protein C inactivates coagulation factors V and VIII. The factor V Leiden mutation is a common cause for APC resistance and the most frequent genetic thrombophilia. The FV Leiden mutation is also suspected of increasing the risk of arterial thrombosis. Other FV mutations include factor V Cambridge and factor V Hong Kong. The most common genetic risk factor for thrombophilia is Factor V Leiden mutation. It increases the risk of thrombosis by enhanced thrombin production.

• Prothrombin G20210A mutation:Prothrombin is the precursor of thrombin, which is factor II. The prothrombin G20210A mutation is the second most common inherited risk factor for thrombosis and leads to increased levels of prothrombin which demonstrates a higher risk for arterial and venous thrombotic events. It is due to a single point mutation. It is seen commonly in Caucasians. Prothrombin (factor II) is the precursor to thrombin, the end-product of the coagulation cascade. Prothrombin has procoagulant, anticoagulant and antifibrinolytic activities and thus a disorder involving prothrombin results in multiple imbalances in hemostasis.

• Hyperhomocysteinemia: It is associated with premature atherosclerosis and thrombosis and caused by defects of the methionine metabolic pathway. Deficiencies of cofactors of this pathway such as vitamin B6, B12, and folate or defects of enzymes such as cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) decrease the efficiency of homocysteine metabolism. Furthermore renal failure, hypothyroidism, and drugs such as methotrexate, phenytoin, and carbamazepine increase homocysteine levels. On the other hand, lowering homocysteine levels has not been shown to reduce thrombotic risk.

• Elevated factor VIII (FVIII): It increases the risk of thrombosis. African-Americans appear to have higher levels whereas individuals with blood group O tend to have lower levels of FVIII. High levels of this factor also correlate with acute phase reactions, estrogen usage, pregnancy, and after aerobic exercise. A high FVIII level may cause APC resistance not due to FV mutation. In contrast, low levels of FVIII correlate with bleeding in hemophilia A patients.

• Dysfibrinolysis: plasminogen deficiency, dysfibrinogenemia, tissue plasminogen activator (tPA) deficiency, plasminogen activator inhibitor (PAI) increase, and factor XII deficiency, which is involved in plasmin generation. Deficient plasminogen clinically appears similar to protein c deficiency with thrombosis during the teenage years. PAI increase and deficient tPA has an association with diabetes mellitus, inflammatory bowel syndrome and coronary atherosclerosis. In patients with structural or functional changes to fibrinogen (dysfibrinogenemia) thrombosis or bleeding can occur.

• Antiphospholipid syndrome (APS): The most common acquired thrombophilia is the antiphospholipid syndrome (APS) in which antibodies are directed against natural constituents of cell membranes, the phospholipids. These antiphospholipid antibodies (APLA) occur in 3 to 5% of the population and may cause arterial or venous thrombosis and fetal loss. APLAs being tested for include lupus anticoagulant, anticardiolipin, anti-beta-2-glycoprotein. Lupus anticoagulant leads to prolongation of coagulation (aPTT) in vitro but thrombosis in vivo. Antiphospholipid antibodies may also occur secondary to other diseases (collagen vascular disease or infections) or drugs (phenytoin and cocaine among others). The most common thrombotic event is deep vein thrombosis. Any patient with stroke and rheumatological disorder should be screened for antiphospholipid antibody syndrome.

• Malignancy:he second most common acquired hypercoagulability and leads to a prothrombotic state through the production of procoagulant factors (tissue factor and cancer procoagulant) and the interaction of tumor cells with blood and vascular endothelium. Stasis from tumor compression, paraproteinemia, and cytokine release pose an additional risk. In 85% of cancer patients, cancer procoagulant (CP) is elevated. This enzyme actives factor X thus causing hypercoagulability in cancer patients. Polycythemia vera poses a thrombotic risk in addition to hyperviscosity. Migratory thrombophlebitis as a consequence of visceral malignancy is known as Trousseau syndrome. The interaction of malignancy and coagulation is of interest as not only malignancy favors thrombosis but the hemostatic system influences angiogenesis which support tumor growth and spread. Targeting the hemostatic system might offer treatment options for anticancer therapy.

• Smoking: Arterial bypass grafts fail prematurely in smokers. Smoking tobacco contains various toxins. Nicotine results in endothelial cell damage. The release of tissue plasminogen activator (tPA) and tissue factor pathway inhibitor (TFPI) get reduced. Carbon monoxide increases the permeability of endothelium to lipids thus leading to atheroma formation.

• Exercise: Exercise improves the cardiovascular risk profile, but observations of sudden cardiac death in some individuals led investigators to search for causes. Exercise influences coagulation, fibrinolysis, and platelet aggregation. Usually, this is kept in balance but in some individuals, the immediate postexercise period is characterized by a hypercoagulable state with an increase of factor eight (intrinsic pathway activation) and platelet activation. Older individuals carry more cardiovascular risk factors and are less well trained. Thus they are prone to suffer adverse effects from the temporary hypercoagulable state following exercise. In the Tromso study regular, moderate-intensity exercise did not significantly impact the risk for thrombosis.

• Pregnancy:Through the increase of procoagulants (diverse coagulation factors and the number of platelets) and the decrease of anticoagulants (PAI) in addition to stasis caused by compression of the gravid uterus, pregnancy presents a time of hypercoagulability extending 2 months into the postpartum period. This was under evaluation in the MEGA study and LMWH is being tested to prevent miscarriage in pregnant women with inherited thrombophilias in the ALIFE study.

• Heparin-induced thrombocytopenia (HIT):Heparin is a commonly used anticoagulant. Under certain circumstances, arterial and venous thrombosis concomitantly with thrombocytopenia paradoxically results from prolonged heparin administration, which is called heparin-induced thrombocytopenia (HIT). In type-I HIT platelets show a weak reduction of platelets and have little clinical consequences. This is in contrast to the strong reduction of thrombocytes and serious sequelae of HIT type-II. The conformational change of heparin following heparin binding to platelet factor 4 triggers antibody production to heparin. Subsequently, monocytes become activated and attack the vascular endothelium leading to thrombotic events.

• Trauma:Trauma is another acquired hypercoagulable state. Procoagulant disbalance is more pronounced during the first 24 hours following injury and in women. The onset of respiratory distress syndrome and multiorgan failure following trauma has been associated with elevated tissue factor.

• Inflammatory and hypercoagulable state: There is an interplay between inflammation and the coagulation system. Inflammation triggers a hypercoagulable state. Endotoxin activates the complement system leading to thrombocytopenia and hypercoagulability. The relation between inflammation and coagulation can be observed clinically in patients with purpura, vasculitis, and septic thromboembolism. Coagulation helps to limit the expansion of infection, and some bacteria use fibrinolytic properties to oppose this response. Autoimmune diseases like systemic lupus erythematosus, immune thrombocytopenic purpura, polyarteritis nodosa, polymyositis, dermatomyositis, inflammatory bowel disease, and Behcet's syndrome increase the risk of thrombotic events. The cytomegaly virus (CMV) has correlations to atherogenesis through a change of the cellular lipid metabolism and leukocyte adherence. Other conditions associated with a hypercoagulable state include myeloproliferative disorders, multiple myeloma, paroxysmal nocturnal hemoglobinuria, heart failure. The endothelium of the left atrial appendage showed higher expression of tissue factor and plasminogen activator inhibitor compared to the right atrial appendage. This inherent prothrombotic property of the LAA in addition to flow disturbances of atrial fibrillation leads to thromboembolic events.