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] Jaspinder Kaur, MBBS[3]

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.

Pathophysiology

• Coagulation is an inherent property of the hematologic system and normal blood flow is maintained by the balance between the pro-coagulant and anti-thrombotic factors under healthy conditions. A hypercoagulable state and subsequent thromboembolism is a result of overactivity of pro-coagulant factors or a deficiency in anti-coagulants. Anticoagulants that regulate thrombin include antithrombin, protein C, and protein S. The primary mechanism for thrombus formation in common inherited thrombophilic states involves thrombin dysregulation. However, the interplay of these factors is complicated process consisting of coagulation activators and inhibitors and their production and degradation (quantitative) and functional properties (qualitative) influencing the thrombosis process.

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

Figure 1: Thrombus formation in inherited thrombophilias. Adapted from: N Engl J Med. 2001 Apr 19;344(16):1222-31.

Antithrombin III (ATIII) deficiency

• Antithrombin (previously called antithrombin III) is synthesized by the liver but is not vitamin K-dependent. Its inhibitory effect is not confined to thrombin. It also inhibits the activated clotting factors IXa, Xa, XIa, XIIa and tissue factor-bound factor VIIa. Antithrombin III binds to heparin on endothelial cells and forms a complex between antithrombin and the serine proteases and thus, inhibiting coagulation. Mutations in antithrombin can lead to increased thrombus formation.
• The prevalence may be 1 in 500 in the general population. Affected patients have antithrombin levels 40–60% of normal, and 70% of those affected experience thrombo-embolic events before the age of 50. Thrombotic episodes are rare before puberty in AT-deficient individuals. They start to occur with some frequency after puberty, with the risk increasing substantially with advancing age.
• Type of inheritance: Antithrombin (AT) deficiency is a heterogeneous disorder. It is usually inherited in an autosomal dominant fashion, thereby affecting both sexes equally. Homozygous ATIII deficiency is incompatible with life unless affecting the heparin-binding site.
• Causes: 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). Usually these patients present with venous thrombosis and less likely with arterial thrombosis.
• Classification: Three major types of heritable AT deficiency are recognized as follows:
  • Type I: It is characterized by a quantitative reduction of qualitatively (functionally) normal antithrombin protein and thereby reducing both the antigenic and functional activity of AT in the blood. The values are reduced by approximately 50 percent in the heterozygote. The 1997 antithrombin mutation database included 80 distinct mutations in patients with type I deficiency. The database shows that the molecular basis for this disorder is usually a small deletion or insertion (less than 22 base pairs) or a deletion of a major segment of the AT gene.
  • Type II: It is produced by a discrete molecular qualitative mutational defect within the protein which either affect the heparin-binding site (HBS), the reactive site (RS) or result in pleiotropic effects (PE). While the AT immunologic activity is normal in this deficiency, plasma AT functional activity is markedly reduced leading to risk of thrombosis. It is subclassified according to the site of the molecular defect:
    • Reactive site (RS) and abnormalities residing in the reactive (thrombin binding) site.
    • Heparin binding site (HBS) and abnormalities residing in the heparin binding site.
    • Pleiotropic effect (PE) and abnormalities residing in both reactive and heparin binding sites.
  • Type III: This type is characterized by normal functional and antigenic antithrombin levels but impaired interaction between AT and heparin.

Protein C deficiency

• Protein C is a vitamin K-dependent protease synthesized in the liver and circulates in plasma at low concentrations which serves a critical role in the regulation of thrombin. Protein C becomes activated to form activated protein C (APC) via interactions with thrombin. APC acts as one of the major inhibitors of the coagulation system by cleaving and inactivating clotting factors V and VIII which are necessary for efficient thrombin generation, and thereby exerting potent anticoagulant activity. Moreover, APC also reduces platelet prothrombinase activity by degrading platelet bound factor Va at the receptor for factor Xa. Additionally, the inhibitory effects of APC are facilitated through the cofactor activity of protein S, another vitamin K-dependent protein.
• Protein C deficiency is a rare acquired or congenital disorder characterized by a reduction in the activity of protein C, a plasma serine protease involved in the regulation of blood coagulation which results in the excessive formation of blood clots (thrombophilia).
• Congenital protein C deficiency results from mutations in the PROC gene and inherited in an autosomal dominant manner. The gene for protein C is located on chromosome 2 (2q13–14) and appears to be closely related to the gene for factor IX.
• Mode of inheritance:
  • Heterozygous: Mutations in a single copy in heterozygous individuals cause mild protein C deficiency.
  • Homozygous: Individuals with homozygous mutations present with severe protein C deficiency and thrombotic tendency in infancy characterized as purpura fulminans.
• Inherited: Two major subtypes of heterozygous protein C deficiency have been delineated using immunologic and functional assays. Over 160 different gene abnormalities have been associated with the two subtypes.
  • Type I: This state is more common with a reduced plasma protein C concentration at approximately 50% of normal in both immunologic and functional assays. Most affected patients are heterozygous and carries an increased risk of developing warfarin-induced skin necrosis. More than half of the mutations are missense and nonsense mutations and other includes promoter mutations, splice site mutations, in-frame deletions, frameshift deletions, in-frame insertions, and frameshift insertions. Moreover, there is marked phenotypic variability among patients with heterozygous type I protein C deficiency. Similar mutations have been found among symptomatic and asymptomatic individuals which suggests that the nature of the protein C gene defect alone does not explain the phenotypic variability.
  • Type II: It has normal plasma protein C antigen levels with decreased functional activity; and a variety of different point mutations affecting protein function have been identified in this disorder.
• Acquired: Protein C is known to have a role in the regulation of inflammation and sepsis which demonstrates its cytoprotective functions. Reduced protein C activity is observed in DIC, liver disease, coumarins use, and adverse pregnancy outcomes such as DVT, preeclampsia, intrauterine growth restriction and recurrent pregnancy loss.
• Additionally, 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 can be seen; and thereby need the bridging with parenteral heparin to avoid warfarin-induced skin necrosis.

Protein S deficiency

• 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, remains uncomplexed and is the active moiety; while about 60% is in a high-affinity complex with the complement regulatory factor C4b-binding protein (C4BP) and has no cofactor activity. The bioavailability of protein S is closely linked to the concentration of C4bBP, which acts as an important regulatory protein in the activated protein C:protein S inhibitory pathway.
• The anticoagulant activity of protein S is two-fold as follows:
  • Protein S operates as a cofactor for activated protein C (APC) and inactivates coagulation Factor Va and Factor VIIIa;
  • 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 deficiency is usually congenital caused by mutations in the PROS1 gene and mainly an autosomal dominant pathology.
• Mode of inheritance:
  • Heterozygous: Mutations in a single copy in heterozygous individuals cause mild protein S deficiency
  • Homozygous: Individuals with homozygous mutations present with severe protein S deficiency
• Inherited: More than 200 PROS mutations have been described and may result in three different forms of protein S deficiency:
  • Type I: It is a quantitative defect presenting with low levels of total protein S (TPS) and free protein S (FPS) with reduced levels of functional protein S activity.
  • Type II (or Type IIb): This type of protein S deficiency is characterized by decreased functional protein S activity with normal levels of TPS and FPS antigens. Interestingly, all five mutations originally described were missense mutations located in the N-terminal end of the protein S molecule consisting of the domains that interact with activated protein C.
  • Type III (or Type IIa): It consists of a quantitative defect presenting with normal levels of TPS, but selectively reduced levels of FPS and functional protein S activity to less than approximately 40 percent of normal.
• Acquired: Causes of temporary acquired fluctuations in protein S levels may include vitamin K-antagonist therapy (coumarins), antiphospholipid antibodies, chronic infections, severe hepatic disease, nephritic syndrome, and DIC.
• Gender predilection: Protein S levels are slightly higher in men than in women. Contrarily, protein S levels fall progressively during pregnancy and are reduced to a lesser extent in women using oestrogen containing oral contraceptives or hormone replacement therapy. Moreover, the risk of VTE is also increased in patients using oral contraceptives and pregnancy.

Factor V Leiden mutation

• The most common inherited thrombophilia is Factor V Leiden which is a polymorphism of Factor V that is resistant to APC inactivation. Other FV mutations include factor V Cambridge and factor V Hong Kong.
• Activated protein C (APC): Protein C interacts with thrombomodulin to become APC which has anticoagulant, anti-inflammatory, and cytoprotective properties. The signal cascade leading to APC can become distorted through many acquired or inherited mechanisms leading to APC resistance. Hence, APC resistance occurs when APC fails to inactivate downstream coagulation factors, specifically Factor V and Factor VIII.
• The factor V Leiden mutation further increases arterial thrombosis risk by enhancing thrombin production. Protein C and S are natural anticoagulants which inhibit thrombin formation. Dysregulation in APC can occur as either defects in the protein C or S molecule (Protein C and S deficiency) or as resistance to APC activity.

Prothrombin G20210A mutation

• 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 hemostasis imbalances.
• It is the second most common inherited thrombophilia which involves a gain of function mutation of the prothrombin gene (Prothrombin G20210A) resulting in increased protein activity and thrombus formation.
• It is due to a single point mutation which involves the G to A transition at nucleotide 20210 in the 30-untranslated region of the prothrombin gene and thereby associated with elevated plasma prothrombin levels and demonstrating a higher risk for arterial and venous thrombotic events.

Hyperhomocysteinemia

• Homocystinuria and hyperhomocysteinemia are rare metabolism disorder associated with the marked elevations of plasma and urine homocysteine concentrations resulting from an impaired intracellular metabolism of homocysteine which can be due to both genetic and acquired abnormality.
• Homocysteine is an amino acid derived from methionine which is metabolized by the body in two possible following pathways:
  • Transsulfuration of homocysteine produces cysteine, and this reaction is catalyzed by cystathionine-β-synthase which requires pyridoxal phosphate (Vitamin B) as a cofactor.
  • Remethylation of homocysteine produces methionine which is catalyzed either by methionine synthase or by betaine homocysteine methyltransferase. Vitamin B12 (cobalamin) is the precursor of methylcobalamin, which is the cofactor for methionine synthase.
• Nutritional deficiencies in vitamin cofactors such as vitamin B6, B12, and folate or genetic defects of enzymes such as cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) decrease the efficiency of homocysteine metabolism. Furthermore, chronic medical conditions such as renal failure, hypothyroidism, and drugs such as methotrexate, phenytoin, and carbamazepine increase homocysteine levels.
• Hence, premature atherosclerosis and arterial thrombosis is associated with severe hyperhomocysteinemia.

Elevated factor VIII (FVIII)

• Higher levels: African-Americans appear to have its higher levels. It further increases the risk of thrombosis, and found to be correlated with acute phase reactions, estrogen usage, pregnancy, and aerobic exercise.
• Lower levels: Individuals with blood group "O" tend to have lower levels of FVIII and correlated with bleeding in hemophilia A patients.

Dysfibrinolysis

• Plasminogen deficiency, dysfibrinogenemia, tissue plasminogen activator (tPA) deficiency, plasminogen activator inhibitor (PAI) increase, and factor XII deficiency impairs plasmin generation.
• Dysfibrinogenemia: The patients with structural or functional changes to fibrinogen result in dysfibrinogenemia through an abnormal thrombin-mediated conversion to fibrin and thereby, developing the risk for thrombosis or bleeding. Most patients are clinically asymptomatic inspite of having predisposition for bleeding, thrombosis or both.

Antiphospholipid syndrome (APS)

• It is the most common acquired thrombophilia in which antibodies are directed against natural constituents of cell membranes, the phospholipids.
• These antiphospholipid antibodies (APLA) consisting of lupus anticoagulant, anticardiolipin, and anti-beta-2-glycoprotein occur in 3 to 5% of the population and may cause arterial or venous thrombosis and fetal loss.
• APLA may occur secondary to other diseases such as collagen vascular disease or infections or drugs like phenytoin.
• Hence, any patient presenting with stroke, deep vein thrombosis, and rheumatological disorder should be screened for underlying antiphospholipid antibody syndrome.

Malignancy

• It is the second most common acquired hypercoagulability which 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 further associated with vascular stasis from tumor compression, paraproteinemia, and cytokine release.
• In 85% cases, cancer procoagulant (CP) is elevated which activates factor X, and thus causing hypercoagulability in cancer patients.
• Migratory thrombophlebitis known as Trousseau syndrome and Polycythemia vera poses a thrombotic risk in addition to hyperviscosity.
• The interaction of malignancy and coagulation not only favors thrombosis but also the hemostatic system which influences angiogenesis and support tumor growth and spread. Hence, targeting the hemostatic system might offer treatment options for anticancer therapy.

Smoking

• Smoking tobacco contains various toxins such as nicotine which 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.
• Hence, arterial bypass grafts fail prematurely in smokers.

Exercise

• Exercise influences coagulation, fibrinolysis, and platelet aggregation which is usually kept in balance; however, in some cases the immediate postexercise period is characterized by a hypercoagulable state with an increase of factor eight (intrinsic pathway activation) and platelet activation.
• Although exercise improves the cardiovascular risk profile; but 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.

Pregnancy

• The physiological changes that occur during pregnancy presents a time of hypercoagulability extending from 2 months of gestation into the postpartum period 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.
• Hematological changes: A number of clotting factors including factor VII, factor VIII, Factor X, von Willebrand factor, and fibrinogen are elevated as a result of hormonal changes. At the same time, resistance to activated protein C increases in the second and third trimesters and the activity of protein S is decreased due to changes in the total protein S antigen level. There is also an increase in a number of inhibitors of the fibrinolytic pathway such as activatable fibrinolytic inhibitor (TAFI) and plasminogen activator inhibitor 1 and 2 (PAI-1 and PAI-2).
• Physical changes: An increased pressure on the pelvic veins from the gravid uterus and decreased flow in the lower extremities result in increased stasis and thrombotic state. Relative compression of the left iliac vein by the right iliac artery as it courses across the vessel leads to an increase of clots in the left iliac vein. Although stasis increases throughout the course of pregnancy and leg pain and swelling are more frequent during the third trimester, incidence of DVT is distributed relatively equally across trimesters.
• Concomitant diseases such as systemic lupus erythematous or sickle cell disease along other risk factors including obesity, decreased mobility, increased age, and smoking further elevates the thrombosis risk.
• Predisposing factors: It has been observed that the pregnant women over 35yrs of age have a 1.38 fold increased risk of having a clotting event during the peripartum period. Additionally, women who have had spontaneous clotting events in the past have an increased risk of developing a second event with an estimated rate of recurrence of 10.9% during pregnancy.
• Overall, both the physiologic and anatomic changes of pregnancy take several weeks to resolve after delivery, and the risk of thrombosis remains elevated compared to pregnancy until approximately 6 weeks postpartum.

Heparin-induced thrombocytopenia (HIT)

• Heparin is a commonly used anticoagulant and under certain circumstances, arterial and venous thrombosis concomitantly with thrombocytopenia paradoxically results from its prolonged administration which is called heparin-induced thrombocytopenia (HIT). 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.
• Type-I HIT: Platelets show a weak reduction and have little clinical consequences.
• Type-II HIT: It characterizes the strong reduction of thrombocytes and serious clinical sequelae.

Trauma

• Trauma causes the procoagulant disbalance which 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 correlated with elevated tissue factor.

Inflammatory and hypercoagulable state

• There is an interplay between inflammation and the coagulation system as inflammation triggers a hypercoagulable state which can be observed clinically in patients with purpura, vasculitis, and septic thromboembolism.
• Endotoxin activates the complement system leading to thrombocytopenia and hypercoagulability. Moreover, coagulation helps to limit the expansion of infection and some bacteria use fibrinolytic properties to oppose this response. The cytomegaly virus (CMV) has correlations to atherogenesis through a change of the cellular lipid metabolism and leukocyte adherence.
• 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. Other conditions associated with a hypercoagulable state include myeloproliferative disorders, multiple myeloma, paroxysmal nocturnal hemoglobinuria, heart failure.
• Cardiac events: The endothelium of the left atrial appendage (LAA) 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.