COAGULATION

• ACRONYMS AND DEFINITIONS

• ACCP - American College of Chest Physicians
• APA - Antiphospholipid antibodies
• aPTT - activated Partial Thromboplastin Time
• ASH - American Society of Hematology
• dRVVT - dilute Russell Viper Venom Time
• DVT - Deep vein thrombosis
• DOAC - Direct-acting oral anticoagulant (factor Xa inhibitors, dabigatran)
• ELISA - Enzyme-linked immunosorbent assays
• INR - International Normalized Ratio
• PCR - Polymerase chain reaction
• PE - Pulmonary embolism
• PT - Prothrombin Time
• TIA - Transient ischemic attack
• VTE - Venous thromboembolism
• VWD - Von Willebrand Disease
• VWF - Von Willebrand Factor

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• OVERVIEW

• Coagulation
• Coagulation is a complex process that is not completely understood
• Coagulation involves a number of factors that interact with each other in differing ways depending on their environment
• Because coagulation is an integral part of some very important disease processes, a number of medications have been developed which affect the coagulation process
• The complexity of the process can be confusing for patients and healthcare providers alike


• Processes
• Coagulation can be divided into two processes that interact
• Medications used to alter coagulation work on one of these two processes
• The two processes of coagulation include:
• Platelet activation
• Coagulation Cascade

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• PLATELET ACTIVATION

• Unactivated platelets circulate freely in the blood
• When there is a breach in a blood vessel wall (ex. from a cut), platelets in the blood come in contact with collagen on the outside of the blood vessel
• Tissue factor on the surface of collagen activates platelets. Activated platelets also express tissue factor.
• Activated platelets interact with von Willebrand Factor (VWF) which causes the platelets to adhere to the blood vessel wall
• Activated platelets recruit more platelets to the area
• Tissue factor activates the coagulation cascade
• The coagulation cascade results in the formation of a fibrin mesh at the site of injury
• Activated platelets get caught in the fibrin mesh and a blood clot is formed
• Illustration of platelet activation

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• COAGULATION CASCADE

• Coagulation factors
• The coagulation cascade is a multistep process that involves a number of coagulation "factors" that are denoted by Roman numerals I - XIII
• Coagulation factors circulate unactivated. Once they become activated, they are denoted by a small "a" (Ex. VIIa)
• The four factors in the table below are often referred to by other names

Other names for Factors I, II, III, and IV
Factor	Other name
I	Fibrinogen
II	Prothrombin
III	Tissue Factor
IV	Calcium

• Vitamin K-dependent factors (II, VII, IX, and X)
• Factors II, VII, IX, and X are made in the liver and require Vitamin K for their synthesis
• Patients with significant liver disease may have deficiencies of Vitamin K-dependent factors. This can lead to bleeding disorders. The liver also makes protein C and protein S which are factors that inhibit coagulation. Deficiencies of protein C and protein S can lead to clotting disorders. Depending on the balance in deficiency of these factors, a patient with significant liver disease may have a bleeding or clotting disorder. (see coagulopathy of liver disease for more details)
• Warfarin (Coumadin®) is a widely used anticoagulant. Warfarin acts by inhibiting Vitamin K and thus inhibits the production of Factors II, VII, IX, and X.

• Coagulation cascade pathways
• The coagulation cascade can be divided into two different pathways:
• Intrinsic pathway - The intrinsic pathway is not as well understood as the extrinsic pathway. In the past, the intrinsic pathway was believed to propagate thrombosis, but it was not considered a factor in the initiation of thrombosis because patients with Factor XII deficiency do not have bleeding disorders. A recent study published in the NEJM challenged this hypothesis. The study found that inhibiting Factor XI significantly reduced the risk of postoperative thrombosis when compared to enoxaparin. The authors hypothesized that the release of DNA, RNA, and polyphosphates during surgical tissue damage leads to the initiation of the intrinsic pathway and subsequent thrombosis. [PMID 25482425]
• Extrinsic pathway - The extrinsic pathway is the main coagulation pathway initiated by tissue damage
• Factor VII circulates freely in the blood
• When there is a breach in a blood vessel wall (ex. from a cut), Factor VII in the blood come in contact with collagen on the outside of the blood vessel
• Tissue factor on the surface of collagen activates Factor VII (Factor VII → VIIa)
• Tissue factor-Factor VIIa complex then activates Factor X (Factor X → Xa). Factor IXa-VIIIa complex also converts Factor X to Xa, propagating the process.
• Factor Xa converts prothrombin to thrombin in the presence of Factor Va
• Thrombin converts fibrinogen to fibrin
• Fibrin links together to form a mesh which captures activated platelets and a blood clot is formed
• Factor XIIIa stabilizes the clot by cross-linking fibrin, and in the process, a degradation product called a D-dimer is released
• Factor XI, Factor IX, and Factor VIII propagate the pathway and are required for proper clot formation
• Genetically inherited disorders of these factors can lead to bleeding disorders (see bleeding disorders below)
• Illustration of coagulation cascade

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• COAGULATION INHIBITION (ANTICOAGULANT PATHWAYS)

• Overview
• Once the coagulation process has commenced, it is important to keep it at the site of vascular injury and not allow it to spread throughout the bloodstream
• A number of important factors play a role in containing coagulation
• Genetically inherited disorders of these factors can lead to thrombophilic disorders (see thrombophilic disorders below)


• Factors that inhibit coagulation
• Protein C - Vitamin K-dependent factor made by the liver. Activated by thrombin-thrombomodulin complex. Protein C works with protein S to inactivate Factor Va and VIIIa.
• Protein S - Vitamin K-dependent factor made by the liver. Protein S works with protein C to inactivate Factor Va and VIIIa.
• Antithrombin - Inactivates thrombin and Factor Xa
• Thrombomodulin - Found on the surface of normal vascular cells. Binds to thrombin and inactivates it. Thrombomodulin-thrombin complex activates protein C.
• Tissue Factor Pathway Inhibitor - Inhibits Tissue Factor-VIIa complex and Factor Xa


• Illustration of anticoagulant pathways

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• BLEEDING DISORDERS

• Overview
• Bleeding disorders are typically caused by genetically inherited defects or deficiencies in clotting factors. Acquired disorders may occur, but they are rare.
• Significant liver disease can also affect coagulation (see coagulopathy of liver disease for more)

Most common bleeding disorders
Bleeding disorder	Incidence
Von Willebrand Disease	0.5 - 2% of general population
Hemophilia A and B	1 in 5000 males 1 in 10,000 overall Hemophilia A is 4 times more common than B

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• BLEEDING DISORDERS | Von Willebrand disease (VWD)

• Overview
• VWD is the most common bleeding disorder with an incidence of 0.5 - 2% in the general population. VWD is an inherited disorder that can affect either the quantity (types 1 and 3) or function (type 2) of von Willebrand factor (VWF).
• VWF has two main functions: (1) it attaches platelets to each other and to the site of vascular injury, (2) it binds to Factor VIII in plasma and prevents its degradation and clearance. Because VWF is involved in both platelet function and the coagulation cascade, VWD affects both processes of coagulation.

• Symptoms
• VWD typically presents as excessive bleeding, and the severity will depend upon the disease type. Examples of excessive bleeding from VWD include the following: easily induced and difficult to stop nosebleeds, excessive menstrual bleeding, excessive bleeding after childbirth, bleeding after dental procedures and surgery, easy bruising, prolonged bleeding from minor cuts, gum bleeding. [2]

• Laboratories
• VWF antigen (VWF:Ag) - measures concentration of VWF protein in plasma. Does not measure VWF function.
• VWF ristocetin cofactor activity (VWF:RCo) - measures ability of VWF to interact with platelets. Ristocetin is an antibiotic that causes VWF to bind to platelets.
• Factor VIII coagulant assay - measures ability of VWF to bind and maintain Factor VIII in plasma. Patient's plasma is mixed with Factor VIII-deficient plasma and the clotting time is measured.
• VWF multimers - under normal conditions, molecules of VWF bind together to form large multimers. Large multimers are the most stable and active form of VWF. In VWD type 2, multimers do not always form correctly and this can cause an abnormal distribution in VWF multimer sizes. VWF multimer assays measure VWF multimer size distribution and can help distinguish between the different subtypes of type 2 VWD.
• VWF gene testing - genetic test that looks for variants in the VWF gene. This test is useful for diagnosing type 2 VWD. It is not widely available.
• aPTT - aPTT times will be prolonged if Factor VIII is reduced [2]

• Diagnosis
• The ASH issued guidelines on diagnosing VWD in 2021. Those guidelines are available at the link below.
• ASH 2021 guidelines on the diagnosis of VWD

• References [1,2,21]

Types of VWD and their characteristics
Type 1 VWD Defect: Mild-moderate deficiency (5 - 30% reduction) of VWF and Factor VIII Transmission: Autosomal dominant Bleeding severity: Mild to moderate % of affected individuals: 60 - 80% Lab findings: VWF:Ag < 30 IU/dl VWF:RCo < 30 IU/dl VWF:RCo / VWF:Ag ratio > 0.5 - 0.7 Factor VIII - decreased or normal
Type 2 VWD Defect: Qualitative defect of VWF. Type 2 VWD is divided into 4 subtypes (A, B, M, N) depending upon the defect. Transmission: Autosomal dominant Bleeding severity: Typically moderate % of affected individuals: 10 - 30% Lab findings: Types 2A, 2B, 2M VWF:Ag <30 - 200 IU/dl VWF:RCo < 30 IU/dl VWF:RCo / VWF:Ag ratio < 0.5 - 0.7 Factor VIII - decreased or normal Types 2N VWF:Ag 30 - 200 IU/dl VWF:RCo 30 - 200 IU/dl VWF:RCo / VWF:Ag ratio > 0.5 - 0.7 Factor VIII - decreased
Type 3 VWD Defect: Severe or complete deficiency of VWF and moderately severe Factor VIII deficiency Transmission: Autosomal recessive Bleeding severity: Severe % of affected individuals: 1 - 5% Lab findings: VWF:Ag < 3 IU/dl VWF:RCo < 3 IU/dl Factor VIII < 10 IU/dl

• Treatment
• Most patients with VWD have minor disease, and they are only treated if they have uncontrolled bleeding or require surgery, in which case, they are given prophylactic treatment. A brief review of therapies used to treat VWD is provided below.
• DDAVP (Desmopressin) - DDAVP is a synthetic analog of antidiuretic hormone (ADH). DDAVP stimulates the release of VWF from endothelial cells. Factor VIII levels also increase after DDAVP. Nasal administration is effective for minor bleeding. Intravenous administration is needed for major bleeding or surgery. Effect of daily administration diminishes after 3 - 5 days [2]
• VWF concentrate - plasma-derived concentrates of VWF. Can be administered daily depending on need for surgery/bleeding. [2]
• Recombinant VWF (Vonvendi®) - in 2015, the FDA approved the first recombinant VWF. The drug is marketed as Vonvendi®, and it is approved for the on-demand treatment of bleeding episodes, perioperative management of bleeding, and prophylactic therapy for adults with severe type 3 VWD. [Vonvendi PI]

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• BLEEDING DISORDERS | Hemophilia A and B

• Overview
• Hemophilia A and B are genetically inherited bleeding disorders
• Hemophilia A is caused by a deficiency of Factor VIII. Hemophilia B is caused by a deficiency of Factor IX.
• Factor VIII and IX play a role in propagating the coagulation cascade (see coagulation cascade) and are essential to normal clot formation
• Hemophilias are X-linked chromosome recessive disorders. This means mostly males will be affected, although it is possible for females to have hemophilia, but it is rare.
• The prevalence of hemophilia is 1 in 5000 males, and 1 in 10,000 overall. Hemophilia A is four times more common than hemophilia B.
• Hemophilias are divided into mild, moderate, and severe depending on the degree of factor deficiency. The type of genetic mutation that is inherited will determine this.

• Symptoms
• Severe hemophilia often manifests as spontaneous bleeding into joints and muscles early in life (before 4 years of age). Easy bruising is also present.
• Moderate hemophilia manifests the same way as severe disease, but may appear later (by 5 years of age)
• Mild hemophilia may manifest as excessive bleeding after trauma or surgery later in life [3]

• Diagnosis
• A family history of hemophilia should raise suspicions, although a third of hemophiliacs have no known family history
• Levels of Factor VIII or IX are measured in the blood
• Factor VIII deficiency must be distinguished from Von Willebrand Disease ( see Von Willebrand disease above)

• Treatment
• Overview
• Factor replacement with Factor VIII or Factor IX concentrate infusions is the primary treatment. Infusions may be given on an as-needed basis for surgery or bleeding, and in severe cases, they can be administered weekly for life.
• Hemophilia A
• In hemophilia A, 30% of patients develop antibodies (inhibitors) to Factor VIII, making replacement therapy ineffective. These patients may be treated with immune tolerance protocols using low-dose Factor VIII or bypassing agents (recombinant activated factor VII [factor VIIa] or activated prothrombin complex concentrate).
• Emicizumab (Hemlibra®), a recombinant bispecific monoclonal antibody that bridges activated Factor IX and Factor X to mimic the action of Factor VIII, has been approved to treat hemophilia A. Its effectiveness was demonstrated in several studies - PMID 28691557, PMID 30157389
• Gene therapy has shown promising results in small studies - PMID 31893514, PMID 34788507, PMID 35294811
• Hemophilia B
• In 11/2022, the FDA approved a gene therapy called Hemgenix (etranacogene dezaparvovec) for the treatment of hemophilia B. Hemgenix is a one-time IV infusion that contains a viral vector carrying the gene for Factor IX. The virus delivers the gene to hepatocytes, causing them to produce Factor IX. In one study (N=54), Hemgenix-treated patients had increased Factor IX activity, decreased need for Factor IX replacement, and a 54% reduction in annualized bleeding rate. [FDA press release]
• Another similar product was studied here - PMID 35857660

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• BLEEDING DISORDERS | Acquired disorders

• Acquired bleeding disorders can occur when autoantibodies form against clotting factors. These antibodies are called factor inhibitors, and while antibodies can form against any factor, the most common factor affected is Factor VIII.
• Factor inhibitor syndromes are rare with an estimated annual incidence of the most common syndrome (Factor VIII inhibitor syndrome) being only 1 - 2 persons per million. Conditions that increase the risk for factor inhibitors include pregnancy, postpartum state, other autoimmune diseases, malignancy, advanced age (median age at diagnosis is 78 years), and certain medications. No underlying disorder is identified in up to 50% of patients.
• Acute active bleeding is treated with desmopressin and/or Factor VIII concentrates. Patients with high levels of factor inhibitors may require bypassing agents (see hemophilia treatment above). Long-term antibody suppression is achieved with immunosuppressants (e.g. corticosteroids, cyclophosphamide) and rituximab. [13,14,23]

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• THROMBOPHILIC DISORDERS

• Overview
• Thrombophilic disorders (also called hypercoagulable disorders) are a group of diseases that increase a person's risk of forming a pathological blood clot
• Pathological blood clots include deep vein thrombosis, pulmonary embolism, and in rare cases, arterial thrombosis
• Some thrombophilic disorders are genetically inherited (ex. protein C and S deficiency, Factor V Leiden), while others are acquired (ex. antiphospholipid antibodies)

• Reference [4,5,18,21]

Prevalence of thrombophilic disorders
Disorder	General population	Patients with VTE
High concentration of Factor VIII	11%	25%
Antiphospholipid antibodies	0 - 5%	24%
Factor V Leiden	5%	20%
Hyper-homocysteinemia	5%	10%
Prothrombin 20210A	2%	6%
Protein C deficiency	0.2 - 0.4%	3%
Protein S deficiency	<0.5%	2%
Antithrombin deficiency	0.02%	1%

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• THROMBOPHILIC DISORDERS | High concentration of Factor VIII

• Factor VIII propagates the coagulation cascade
• Elevated levels of Factor VIII appear to be a risk factor for DVT
• This syndrome has only been recognized recently and has not been studied extensively
• Pregnancy and oral contraceptives may raise Factor VIII levels
• The use of oral contraceptives in patients with high concentrations of Factor VIII may increase their risk for blood clots [6]

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• THROMBOPHILIC DISORDERS | Antiphospholipid antibodies

• Overview
• Antiphospholipid antibodies (APAs) are autoimmune antibodies that increase the risk of thrombosis and miscarriage. APAs are found in up to 24% of patients with VTE, 20 - 40% of patients with systemic lupus erythematosus, and 10 - 15% of women with recurrent miscarriages. The overall prevalence of APAs in the general population is 0 - 5%.
• The exact mechanism by which APAs stimulate clot formation and induce miscarriage is not entirely understood, but it is believed to occur through the induction of inflammatory mediators. The two most common APAs are anti-Beta 2 glycoprotein I and anticardiolipin. Antibodies to other phospholipids exist but are less common. The presence of multiple APA types confers a greater risk of thrombosis. [7,8,18]
• Illustration of antiphospholipid antibodies

• Clinical features
• Many patients with APAs have no clinical symptoms, and they are discovered incidentally during workups for autoimmune diseases, prolonged aPTT, and false-positive syphilis tests
• Clinical features of the APA syndrome are listed in the table below

• Reference [18,22,24]

Clinical features associated with APA syndrome
Obstetrical
Miscarriages (see APA criteria for more)
Severe preeclampsia
Intrauterine growth restriction
Hematologic / Vascular
Recurrent VTE, especially at a young age
Stroke and TIA, especially at a young age
Acute and chronic renal thrombosis
Ocular vaso-occlusive disease (e.g. retinal artery thrombosis) (up to 29% with primary APA syndrome)
Hemolytic anemia
Laboratory
Prolonged activated partial-thromboplastin time (aPTT)
False-positive syphilis testing (Antigen in the RPR test contains cardiolipin)
Thrombocytopenia (< 100,000 per mm3)
Other
Systemic lupus erythematosus
Cardiac valve abnormalities (vegetations, thickening) (up to one-third with primary APA syndrome)
Livedo reticularis and livedoid vasculopathy

• Laboratory tests
• Lupus anticoagulant
• The lupus anticoagulant test detects the presence of antiphospholipid antibodies in the blood
• The procedure for the test is explained here - lupus anticoagulant test
• The test should not be performed if the patient is anticoagulated
• Antibody tests
• Anticardiolipin antibodies (IgG and IgM) and anti-Beta 2 glycoprotein I antibodies (IgG and IgM) can be measured directly in the blood via ELISA
• These tests are not affected by anticoagulation

• Diagnostic criteria
• Because APAs are found in up to 5% of healthy individuals and may be transiently present in certain conditions (e.g. rheumatological diseases, infections, cancer), their presence alone does not establish a diagnosis of APA syndrome. Clinical and laboratory criteria for APA syndrome are presented in the table below.

• GPL - IgG phospholipid; MPL - IgM phospholipid
• Reference [22]

Diagnostic criteria for APA syndrome
Antiphospholipid antibody syndrome is present if at least one clinical criteria and one laboratory criteria are met
Clinical criteria (one of the following): Vascular thrombosis - defined as ≥ 1 clinical episodes of arterial, venous (excluding superficial veins), or small vessel thrombosis, in any tissue or organ. Thrombosis must be confirmed by objective criteria. For histopathologic confirmation, thrombosis should be present without significant evidence of inflammation in the vessel wall. Pregnancy morbidity (any of the following) One or more unexplained deaths of a morphologically normal fetus at or beyond the 10th week of gestation, with normal fetal morphology documented by ultrasound or by direct examination of the fetus One or more premature births of a morphologically normal neonate before the 34th week of gestation because of: (i) eclampsia or severe preeclampsia defined according to standard definitions, or (ii) recognized features of placental insufficiency Three or more unexplained consecutive spontaneous abortions before the 10th week of gestation, with maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded.
Laboratory criteria (one of the following): Lupus anticoagulant (LA) - LA present in plasma, on two or more occasions at least 12 weeks apart Anticardiolipin (aCL) antibody - aCL antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (i.e. > 40 GPL or MPL, or > the 99th percentile), on two or more occasions at least 12 weeks apart Anti-Beta 2 glycoprotein I (aB2GI) antibody - aB2GI antibody of IgG and/or IgM isotype in serum or plasma (in titer > the 99th percentile), present on two or more occasions, at least 12 weeks apart NOTE: Positive laboratory testing must occur within 5 years of the thrombotic or pregnancy event

• Treatment
• APA syndrome is treated with anticoagulation, and warfarin is the preferred therapy. For patients who have never had a thrombotic event (e.g. APA diagnosed based on pregnancy morbidity), low-dose daily aspirin may be beneficial. [6,24]
• Several studies have evaluated the Factor Xa inhibitor rivaroxaban in APA syndrome. A small trial (N=120) comparing rivaroxaban to warfarin in patients with triple-positive APA syndrome was stopped early when rivaroxaban was found to be clearly inferior for preventing thrombotic events. [PMID 30002145] Another study (N=190) found rivaroxaban was likely inferior to warfarin for preventing recurrent thrombosis in patients with thrombotic APA syndrome. [PMID 31610549] After these studies were published, the manufacturers of Factor Xa inhibitors and dabigatran updated their prescribing information to recommend against their use in APA syndrome, especially the triple-positive subtype.
• 2021 ACCP recommendations for VTE treatment in APA syndrome
• Patients with triple-positive APA syndrome and a VTE should be treated with a vitamin K antagonist
• If a patient is initiated on a DOAC, they should be transitioned to a vitamin K antagonist once APA syndrome is confirmed
• Among patients who experience new or progressive thrombosis while receiving standard intensity vitamin K antagonist, it is not recommended to transition to a DOAC. For these patients, other treatment options may include increasing the target INR range, standard treatment dose low-molecular-weight heparin, transitioning to fondaparinux, or the addition of antiplatelet therapy. [25]

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• THROMBOPHILIC DISORDERS | Factor V Leiden mutation (activated protein C resistance)

• Overview
• Factor Va is a coagulation factor that is part of the coagulation cascade
• Factor Va is deactivated by activated protein C
• The deactivation of Factor Va by protein C inhibits clot formation
• The Factor V Leiden mutation is a mutation in the gene that codes for Factor V which makes it resistant to deactivation by protein C
• Resistant Factor Va promotes clot formation because it cannot be deactivated as easily
• Factor V Leiden mutation is the most common thrombophilic disorder with a prevalence of 5% in the general population
• Factor V Leiden mutation is inherited in an autosomal dominant fashion with incomplete penetrance
• Patients who are heterozygous for the mutation are at a slightly increased risk for a blood clot (5 - 10 fold)
• Patients who are homozygous for the mutation are at a greatly increased risk for a blood clot (50 - 100 fold)
• Symptoms
• Blood clots in the deep veins of the legs and/or lungs (DVTs and PEs)
• In heterozygous disease, clots often occur when patient is exposed to another risk factor (ex. oral contraceptives, pregnancy)
• Diagnosis
• Factor V Leiden mutation - PCR test that looks directly for the mutation; test is not affected by anticoagulation
• Activated protein C resistance assay - test measures Factor V resistance to inactivation by protein C; can be used as a screening test for Factor V Leiden mutation; anticoagulation will invalidate the test [6,9,10,21]
• Treatment
• Anticoagulation

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• THROMBOPHILIC DISORDERS | Hyperhomocysteinemia

• Overview
• Homocysteine is an intermediate in amino acid metabolism
• Deficiencies in the enzymes that metabolize homocysteine and/or the vitamins involved in its metabolism (folic acid, vitamin B6, vitamin B12) can lead to elevated levels
• Mild-to-moderate elevations of homocysteine levels occur in 5 - 10% of the population
• Elevated levels of homocysteine are associated with an increased risk of both venous and arterial thrombosis
• The mechanism by which homocysteine promotes thrombosis is not exactly known, but may involve a number of processes including: decreased protein C activation; increased Factor V activity; induction of tissue factor; inhibition of thrombomodulin; decreased antithrombin [6]
• Symptoms
• May cause arterial (strokes and TIAs) or venous (DVTs and PEs) blood clots
• Diagnosis
• Homocysteine levels can be measured directly in the blood
• High levels may be due to MTHFR (methylenetetrahydrofolate reductase) genetic mutations. This mutation can be measured directly with PCR.
• Normal levels of homocysteine have not been completely standardized [6]
• Treatment
• Folic acid, Vitamin B12, and Vitamin B6 supplements can lower homocysteine levels, but they have not been shown to decrease the risk of blood clots [6, 11]

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• THROMBOPHILIC DISORDERS | Prothrombin 20210A mutation (prothrombin gene mutation)

• Prothrombin 20210A is a genetic mutation of the prothrombin gene that is associated with an increase in prothrombin levels and a mild increase in the risk for VTE
• Prothrombin 20210A mutation is inherited in an autosomal dominant fashion. The mutation is more common in caucasians and rare in Africans and Asians.
• Patients who are heterozygous for the mutation have a 3 fold increased risk for VTE. Patients who are homozygous have a higher risk.
• The mutation can be detected directly with a PCR test. Anticoagulation does not affect the test. [21]

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• THROMBOPHILIC DISORDERS | Protein C deficiency

• Overview
• Protein C is a Vitamin K-dependent anticoagulant that is made in the liver. Unactivated protein C circulates freely in the blood.
• Protein C works to keep procoagulant (clotting) processes at the site of vessel injury by inhibiting their spread to normal tissue
• Protein C is activated by the thrombomodulin-thrombin complex
• Activated protein C inhibits coagulation by blocking Factor Va and VIIIa
• Protein C deficiency affects 0.2 - 0.4% of the population. People with protein C deficiency are at increased risk for VTE.
• Protein C deficiency may be inherited or acquired. Inherited protein C deficiency follows an autosomal dominant pattern, and it may be either quantitative (decreased levels) or qualitative (defective protein). Acquired protein C deficiency is more common and may be secondary to recent VTE, Vitamin K deficiency, warfarin, advanced liver disease, pregnancy, and sepsis.
• Illustration of protein C activity
• Symptoms
• Blood clots in the deep veins of the legs (DVTs) or lungs (PEs)
• Clots typically occur in the late teenage years
• Diagnosis
• Protein C activity is measured first. If activity is low, an antigen test that measures protein C levels is then performed.
• Anticoagulation will invalidate the protein C activity test. Warfarin will invalidate the antigen test.
• Treatment
• Anticoagulation [6,21]

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• THROMBOPHILIC DISORDERS | Protein S deficiency

• Overview
• Protein S is a Vitamin K-dependent glycoprotein that is made in the liver. Protein S is a cofactor for protein C.
• Protein S and protein C work together to keep procoagulant (clotting) processes at the site of vessel injury by inhibiting their spread to normal tissue
• Protein S/protein C inhibit coagulation by blocking Factor Va and VIIIa
• Protein S deficiency affects < 0.5% of the population. People with protein S deficiency are at increased risk for VTE.
• Protein S deficiency may be inherited or acquired. Inherited protein S deficiency follows an autosomal dominant pattern, and it may be quantitative (decreased synthesis), qualitative (defective protein), or secondary to increased clearance of free protein S, the active form. Acquired protein S deficiency may be secondary to recent VTE, Vitamin K deficiency, warfarin, advanced liver disease, nephrotic syndrome, pregnancy, and sepsis.
• Illustration of protein S activity
• Symptoms
• Blood clots in the deep veins of the legs (DVTs) or lungs (PEs)
• Blood clots in the superficial veins
• Clots typically occur in early adulthood
• Diagnosis
• Free levels of protein S antigen should be measured first. Total levels of protein S antigen are not typically helpful. Protein S activity is a difficult test to perform and is not generally recommended.
• Warfarin therapy will invalidate the free protein S antigen test
• Treatment
• Anticoagulation [4,6,10,21]

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• THROMBOPHILIC DISORDERS | Antithrombin deficiency

• Overview
• Antithrombin (also called antithrombin III) is an anticoagulant made in the liver and in endothelial cells. Antithrombin binds to thrombin and inactivates it.
• Antithrombin deficiency is rare, occurring in about 0.02% of the population. People with antithrombin deficiency are at increased risk for VTE.
• Antithrombin deficiency may be inherited or acquired. Inherited antithrombin deficiency follows an autosomal dominant pattern, and it may be either quantitative (decreased synthesis) or qualitative (defective protein). Acquired antithrombin deficiency is more common and may be secondary to sepsis, heparin therapy, advanced liver disease, and nephrotic syndrome.
• Illustration of Antithrombin activity
• Symptoms
• About 50% of patients with antithrombin deficiency will develop a blood clot during their lifetime
• Two-thirds of patients experience a clot by age 35 years
• Diagnosis
• Antithrombin activity is measured first. If activity is low, an antigen test that measures antithrombin levels is then performed.
• Anticoagulation invalidates the activity test. Heparin may invalidate the antigen test.
• Treatment
• Anticoagulation [4,6,21]

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• THROMBOPHILIA TESTING

• Overview
• When a patient is diagnosed with a VTE, the question often arises as to whether thrombophilia testing should be performed
• Currently, there are no consensus guidelines regarding when, or if, thrombophilia testing should be performed. In addition, there are no good studies that have evaluated the issue.
• Some practitioners routinely order thrombophilia testing on all patients with a VTE while others do not. Thrombophilia testing can be expensive.
• The decision to order thrombophilia testing must be made on an individual basis. Patient characteristics, treatment risks, and individual preferences need to be considered before ordering the testing.
• Since the issue has not been well-studied in prospective trials, the workup can raise more questions than it answers. Some important issues to consider before ordering thrombophilia testing are listed below
• Important considerations
• Studies have shown that patients with a VTE who have a negative workup have the same risk of recurrence as patients with a positive workup. Using negative testing to guide treatment decisions can be misleading.
• Many patients have multiple risk factors, and there is no good guidance for applying the data. For example, a woman on combined oral contraceptive pills who has a VTE is found to have a Factor V Leiden mutation (present in up to 5% of the population). The combined oral contraceptives should be stopped, but whether she would benefit from lifelong anticoagulation is unknown.
• Positive findings often lead to testing in relatives. It's unclear how relatives without a history of VTE who test positive should be managed. [18,20]

• Clinical features associated with thrombophilias
• Thrombophilia syndromes may be inherited genetically or they may be acquired over time
• Clinical features of each type are listed below
• Clinical features of inherited thrombophilias
• VTE at a young age (< 50 years)
• VTE brought on by weak provoking factor (e.g. oral contraceptives, minor surgery, immobility)
• Strong family history of VTE (first-degree family member affected at a young age)
• Recurrent VTE, especially at young age
• VTE in unusual site (e.g. cerebral veins, splanchnic veins) [18]
• Clinical features associated with acquired thrombophilias (APA syndrome)
• Miscarriages (see APA criteria for more)
• Severe preeclampsia
• Intrauterine growth restriction
• Recurrent VTE, especially at young age
• Systemic lupus erythematosus
• Elevated activated partial-thromboplastin time (aPTT)
• False-positive syphilis testing (the antigen in the RPR test contains cardiolipin) [18,19]

• Testing recommendations
• There are no professional guidelines that give detailed recommendations for thrombophilia testing
• A review article on thrombophilia published in the NEJM in 2018 provided an algorithm that can help aid in decision making. The table below summarizes some information from the article.

• FVL - Factor V Leiden mutation, PTG - prothrombin gene mutation, APA - antiphospholipid antibodies
• Reference [18]

Testing for thrombophilia after first VTE
Provoked VTE Do not test
Weakly-provoked VTE No clear recommendation for extended anticoagulation in these patients (see VTE treatment recs) Negative thrombophilia testing does not equate to a reduced risk for recurrence Test results may be useful if patient has a female family member of childbearing age, because positive results may mean the female family member is also affected. This information may be useful when considering estrogen-containing hormone products and pregnancy. Consider FVL, PTG, protein C and S, and antithrombin Consider APA testing especially for arterial thrombosis, extensive VTE, or recurrent VTE
Unprovoked VTE Extended anticoagulation is recommended for most patients (see VTE treatment recs) Negative thrombophilia testing does not equate to a reduced risk for recurrence Test results may be useful if patient has a female family member of childbearing age, because positive results may mean the female family member is also affected. This information may be useful when considering estrogen-containing hormone products and pregnancy. Consider FVL, PTG, protein C and S, and antithrombin Consider APA testing especially for arterial thrombosis, extensive VTE, or recurrent VTE
Cerebral vein VTE Test for FVL, PTG, protein C and S, antithrombin, and APA
Splanchnic vein VTE Test for FVL, PTG, protein C and S, antithrombin, and APA Also test for myeloproliferative neoplasms and paroxysmal nocturnal hemoglobinuria

• Testing on anticoagulation
• Some thrombophilia tests are affected by anticoagulation, so in general, testing should not be performed until anticoagulation has been stopped
• All patients with VTE (except distal DVTs in some cases) should be anticoagulated for at least 3 months (see VTE treatment recs)
• Thrombophilia test results do not help guide acute treatment, so there is no reason to perform them at the time of VTE diagnosis
• Vitamin K antagonists like warfarin should be stopped 2 weeks before tests that are affected by anticoagulation. Factor Xa inhibitors and direct thrombin inhibitors should be stopped for 5 half-lives (typically 3 days) before testing.
• The table below lists common thrombophilia tests and whether they are affected by anticoagulation

• aPTT-LA - activated partial thromboplastin time-lupus anticoagulant, dRVVT - dilute Russell Viper Venom Time
• Reference [18,21]

Test	Anticoagulation effects
Activated protein C resistance (screening test for Factor V Leiden mutation)	Do not perform if anticoagulated Presence of lupus anticoagulant can also invalidate test
Factor V Leiden mutation	Not affected by anticoagulation PCR test that looks directly for the mutation
Prothrombin gene mutation	Not affected by anticoagulation PCR test that looks directly for the mutation
Protein C deficiency	Protein C activity assay is recommended first Do not perform if anticoagulated Presence of lupus anticoagulant can also invalidate test
Protein S deficiency	Free protein S antigen levels are recommended first Warfarin will invalidate test
Lupus anticoagulant tests (aPTT-LA and dRVVT)	Do not perform if anticoagulated
Anticardiolipin antibodies (IgG, IgM)	Not affected by anticoagulation ELISA test that detects the presence of antibodies
Anti-Beta 2 glycoprotein I antibodies (IgG, IgM)	Not affected by anticoagulation ELISA test that detects the presence of antibodies

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• PREGNANCY AND THROMBOPHILIA

• Overview
• During pregnancy and the 12 weeks following delivery (postpartum), a woman's risk for VTE is greatly increased
• In studies, the risk of VTE is 10-fold higher in pregnant women when compared to nonpregnant controls of similar age. The risk during the postpartum period is even greater with some studies showing a risk that is up to 35X higher. [15]
• Women who have a history of VTE or known thrombophilia pose a clinical challenge in pregnancy
• A meta-analysis published in 2017 looked at the risk of VTE during pregnancy and the postpartum period for a number of thrombophilias. The results are detailed below.
• A review that covers all the current guidelines for managing women in pregnancy who are at increased risk of VTE was published in 2016. A link to that review is available here - Treatment guidelines for women at increased risk for VTE during pregnancy


• Absolute risk of VTE during pregnancy and the postpartum period in women who are not anticoagulated:
• Controls (no known thrombophilia) - 0.1-0.5%
• Antithrombin deficiency - 16.6%
• Protein C deficiency - 7.8%
• Homozygous Factor V Leiden mutation - 6.2%
• Protein S deficiency - 4.8%
• Compound heterozygous factor V Leiden and prothrombin G20210A mutation - 2.5%
• Heterozygous Factor V Leiden mutation - 1.1%
• Heterozygous prothrombin G20210A mutation - 0.9% [16]

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• COAGULOPATHY OF LIVER DISEASE

• The liver synthesizes clotting Factors II, VII, IX, and X. The liver also synthesizes the anticoagulant factors protein C, protein S, and antithrombin.
• Patients with significant liver disease (cirrhosis) are often deficient in all of these factors
• Traditionally, patients with cirrhosis were considered to have a bleeding disorder because their clotting assays (PT, INR) were often prolonged. However, these assays do not account for deficiencies in anticoagulant factors (protein C and S, antithrombin), and therefore, they do not reflect the overall coagulable state.
• Patients with cirrhosis may develop both bleeding and thrombophilic disorders depending on the imbalance in procoagulant and anticoagulant factors
• More research is needed in order to determine the optimal evaluation and treatment of patients with cirrhosis [12]

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• COAGULATION LABS

• Prothrombin Time (PT) and International Normalized Ratio (INR)
• Factors measured
• I, II, V, VII, X
• Procedure
• In the PT assay, plasma is taken from the patient and stored in a tube containing citrate
• Citrate stabilizes calcium in the plasma and prevents the plasma from clotting
• In the lab, tissue factor and fresh calcium are added to the plasma
• The time it takes for a clot to form is then measured
• INR International Normalized Ratio
• The INR is typically measured with the PT
• Tissue factor used in measuring the PT comes from different manufacturers. Different batches of tissue factor have different activity levels.
• The INR is a calculation used to standardize PT results by taking variations in tissue factor activity into account

• Activated Partial Thromboplastin Time (aPTT)
• Factors measured
• I, II, V, VIII, IX, X, XI, XII
• Procedure
• In the aPTT assay, plasma is taken from the patient and stored in a tube containing citrate
• Citrate stabilizes calcium in the plasma and prevents the plasma from clotting
• In the lab, phospholipid and fresh calcium are added to the plasma
• The time it takes for a clot to form is then measured

• Bleeding time
• The bleeding time test involves pricking a patient's skin and then measuring the amount of time it takes for bleeding to stop
• The test is often ordered to measure platelet function
• The bleeding time has limited utility because of high variability between individuals performing the test and the devices used to perform it

• Lupus anticoagulant
• Lupus anticoagulant is a confusing term for a number of reasons
• The term "lupus anticoagulant" is used in different ways in the medical literature. It is used to refer to a lab test, a blood entity, and a syndrome.
• In its purest sense, "lupus anticoagulant" is a syndrome where antiphospholipid antibodies (APA) are present in the blood
• APAs that lead to a "positive lupus anticoagulant test" include anti-Beta 2 glycoprotein I antibodies, anticardiolipin antibodies, and several other APAs that are less common (ex. antiprothrombin antibodies)
• The term "lupus anticoagulant" comes from the fact that APAs are common in patients with lupus erythematosus
• Although APAs prolong clotting times (act as anticoagulants) in the lab, the presence of APAs is associated with thrombophilic disorders
• Testing for the lupus anticoagulant syndrome is reviewed in the illustration below
• Illustration of the lupus anticoagulant testing

• Dilute Russell Viper Venom Time (dRVVT)
• The dRVVT is a test for antiphospholipid antibodies
• APAs are present in the lupus anticoagulant syndrome, and the dRVVT is often used to diagnose this syndrome
• In the dRVVT test, Russell's viper venom and phospholipids are added to the patient's plasma. Russell's viper venom activates Factor X, and in the presence of phospholipids, a clot will form.
• If APAs are present, clot formation will be inhibited, and the dRVVT will be prolonged
• Because Russell's viper venom activates Factor X, the test is not sensitive to factor deficiencies upstream of Factor X
• Illustration of the dRVVT test

• Mixing studies
• If antiphospholipid antibodies or other factor inhibitors are suspected based on a prolonged aPTT or dRVVT, then a mixing study may be performed
• Mixing studies involve mixing the patient's plasma with normal plasma
• If APAs or factor inhibitors are present, the normal plasma will not correct the clotting time
• If coagulation factors are deficient, the normal plasma will correct the clotting time [14]
• See lupus anticoagulant illustration for more information

• Anticardiolipin antibodies
• Cardiolipin is a phospholipid found on mitochondrial membranes
• Anticardiolipin antibodies are one of the main antiphospholipid antibodies that are found in the APA syndrome
• Anticardiolipin antibodies (IgG and IgM) can be measured directly in the blood via ELISA
• Illustration of anticardiolipin antibodies

• Anti-Beta 2 glycoprotein I
• Anti-Beta 2 glycoprotein I is a phospholipid found on the surface of cell membranes
• Anti-Beta 2 glycoprotein I antibodies are one of the main antiphospholipid antibodies that are found in the APA syndrome
• Anti-Beta 2 glycoprotein I antibodies (IgG and IgM) can be measured directly in the blood via ELISA
• Illustration of anti-Beta 2 glycoprotein I antibodies

• D-dimer
• Factor XIIIa stabilizes a clot by cross-linking fibrin in the fibrin mesh (see coagulation cascade for an illustration)
• A degradation product called a D-dimer is released when cross-linking occurs
• D-dimer levels can be measured in the blood
• An elevated D-dimer level is a sign of ongoing clot formation
• This test is often used to rule out a DVT or PE

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• MEDICATIONS

• Overview
• A number of medications affect coagulation
• Medications typically work on either platelet activation or the coagulation cascade


• Antiplatelet medications
• Aspirin
• Dipyridamole (Persantine ®, Aggrenox ®)
• GP IIb/IIIa inhibitors (eptifibatide, Integrilin®, tirofiban, Aggrastat®, abciximab, ReoPro®)
• COX inhibitors - NSAIDS (ibuprofen, naproxen, etc.)
• P2Y12 inhibitors (Plavix®, clopidogrel, prasugrel, Effient®, ticagrelor, Brilinta®)
• Vorapaxar (Zontivity®)


• Anticoagulants
• Factor Xa inhibitors (Apixaban, Eliquis®, rivaroxaban, Xarelto®)
• Direct thrombin inhibitors (dabigatran, Pradaxa®)
• Warfarin (Coumadin®)

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• BIBLIOGRAPHY

• 1 - 15306670 - VWD review
• 2 - NHLBI GL on VWD
• 3 - 12781551
• 4 - 10209995 Lancet hypercoagulable review
• 5 - 20822807 Lancet APA review
• 6 - 11700155 AIM hypercoagulable review
• 7 - 23484830 NEJM APA review
• 8 - 20822807 Lancet APA review
• 9 - 10821379 Lancet coag review
• 10 - 10926920 Clin chem review
• 11 - 17470822 homocysteine trial
• 12 - 21751907 Liver dx review
• 13 - 27406351 Acquired deficiency review
• 14 - 27893111 Mixing Study for Evaluation of Abnormal Coagulation Testing, JAMA (2016)
• 15 - 26780741 Guidance for the treatment and prevention of obstetric-associated venous thromboembolism, J Thromb Thrombolysis (2016)
• 16 - 29074563 Pregnancy, thrombophilia, and the risk of a first venous thrombosis: systematic review and Bayesian meta-analysis, BMJ (2017)
• 17 - 29490924 Antiphospholipid antibodies and recurrent thrombosis after a first unprovoked venous thromboembolism, Blood (2018)
• 18 - 28930509 Thrombophilia Testing and Venous Thrombosis, NEJM (2017)
• 19 - 29791828 Diagnosis and Management of the Antiphospholipid Syndrome, NEJM (2018)
• 20 - 15900005 Thrombophilia, clinical factors, and recurrent venous thrombotic events, JAMA (2005)
• 21 - CPL labs website
• 22 - 16420554 International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome, Journal of Thrombosis and Haemostasis, (2005)
• 23 - PMID 34107184 Case 17-2021: An 82-Year-Old Woman with Pain, Swelling, and Ecchymosis of the Left Arm, NEJM (2021)
• 24 - PMID 34665219 - Retinal Artery Thrombosis and Aortic Valve Vegetations, JAMA (2021)
• 25 - PMID 34352278 - Executive Summary: Antithrombotic Therapy for VTE Disease: Second Update of the CHEST Guideline and Expert Panel Report, Chest (2021)