Factor II, Prothrombin Assay

Prothrombin is the precursor of thrombin in the coagulation pathway; it is synthesized in the liver, much as other vitamin K – dependent proteins are, and has a molecular weight of 72 kd. The plasma half-life of prothrombin is approximately 60 hours. ^[1]

Normal findings: The reference range is between 80% and 120% of normal values. ^[2]

Prothrombin 20210 GA gene mutation leads to increases in prothrombin. Several conditions may lead to decreases in prothrombin, as follows:

• Congenital factor II deficiency

• Vitamin K – antagonist therapy

• Liver diseases

• Massive blood transfusion

• Disseminated intravascular coagulation (DIC)

Specimen collection proceeds as follows:

• Specimen: Blood

• Container: Blue-top vacuum tube

• To ensure compliance with Occupational Safety and Health Administration (OSHA) safety standards, the sample must be sent in a leak-proof sealed container labeled with a biohazard sticker

Description

Prothrombin (factor II) is the precursor of thrombin in the coagulation pathway; it is synthesized in the liver, much as other vitamin K–dependent proteins are, and has a molecular weight of 72 kd. The plasma half-life of prothrombin is approximately 60 hours. ^[1]

The prothrombin gene is found at 11p11-q12) on chromosome 11. ^[3] Mutation at position 20210 of the gene results in thrombophilia. ^[4] The prevalence of factor II G20210A in Caucasian populations is estimated to be in the range of 1-6%. ^[5] The mutation is congenital. ^[6] Worldwide, about 30 people have been diagnosed as having congenital factor II deficiency. ^[7]

Inheritance is heterozygous or, rarely, homozygous. Heterozygous mutations raise the risk of venous thromboembolism about 3-fold to 11-fold. ^{[8, 5]} When homozygous mutations occur, they raise the risk of thrombosis even more than heterozygous mutations do. Factor II gene mutation is not typically associated with other factor mutations; when it is, the associated mutation most commonly involves factor V Leiden.

Complete prothrombin deficiency has not been reported in humans. The observation that prothrombin-null mice die in utero or shortly after birth suggests that complete deficiency of factor II is incompatible with life. ^[9]

Two sites on prothrombin are enxymatically cleaved by activated factor X (factor Xa) to yield thrombin. Factor Xa activity is increased by binding to activated factor V (Va); this binding forms the prothrombinase complex.

Ten glutamic acids on prothrombin are converted by vitamin K to gamma-carboxyglutamic acid (Gla) residues, the effect of which is to promote prothrombin binding to phospholipid bilayers when calcium is present. The production of Gla residues is inhibited by warfarin or vitamin K deficiency, and this inhibited production slows the activation of the coagulation pathway. ^[1] Prothrombin differs from other coagulation factors in that it is only minimally affected by pregnancy. ^[10]

The activities of tissue factor, as well as factors II, X, and XII, appear to be substantially greater in early atherosclerotic lesions than in stable advanced atherosclerotic lesions. ^[11] One or more of these procoagulants may be involved in atherogenesis (a possibility that is currently the subject of intense research interest).

It was once believed that prothrombin gene mutation might have a relationship with inflammatory bowel disease (Crohn disease or ulcerative colitis), but research efforts have not found evidence of such a relationship. ^[12]

Indication/Applications

Prothrombin testing is indicated in the following situations:

1. Prothrombin deficiency is suspected

2. The clinician needs to differentiate the effects of oral anticoagulants from those of liver disease (on ccasion)

The following conditions or events may lead to decreases in prothrombin:

• Malabsorption

• Warfarin therapy

• Lack of intestinal colonization by bacteria

• Liver disease

• Consumptive coagulopathy

• Massive blood transfusion

• Circulating anticoagulants or deficiencies in vitamin K

• Pathologic fibrinolysis

• Congenital deficiency (transmitted in a recessive fashion)

• Heparin therapy - Although this does not normally decrease prothrombin, a transient decrease may be seen after a heparin bolus

• Technical errors (eg, incomplete filling of the tube during the blood draw)

The G20210A mutation increases prothrombin and raises the risk of thromboembolic disease.

Considerations

The prothrombin time (PT) is used to assess the quality of the extrinsic and common pathways of coagulation. It is defined as the time required for a sample of citrated, platelet-poor plasma to form a fibrin clot after tissue factor (animal-derived or recombinant) is added. Marked prolongation of the PT is indicative of advanced liver disease.

In patients receiving warfarin or related drugs, a substantial increase in the international normalized ratio (INR) denotes excessive anticoagulation and calls for a prompt decision; an INR below 2 indicates insufficient anticoagulation. Concurrent abnormalities in both the PT and the partial thromboplastin time (PTT) could be attributable to the following ^[8] :

• Oral anticoagulants

• Liver disease

• Massive transfusions

• Vitamin K deficiency

• DIC

• Factor II, V, or X deficiency

Warfarin and related drugs have the effect of inhibiting vitamin K-dependent carboxylation of prothrombin, as well as of several other coagulation factors. Prothrombin deficiency states such as intractable bleeding from warfarin use may be correctable by administering prothrombin-rich preparations such as fresh frozen plasma (FFP) or prothrombin complex concentrate (PCC).

Recombinant thrombin, in the form of a reconstitutable powder, is suitable for topical intraoperative application as an aid to hemostasis. Although it helps control minor bleeding from capillaries and small venules, it is of little use for arterial bleeding. ^{[13, 14]} In urgent settings, PCC is effective for correcting warfarin anticoagulation. It achieves more timely (and potentially more complete) correction than FFP does, without volume overload. Such advantages suggest that wider use of PCC in urgent situations should be considered. ^[15]

The following factors may decrease the accuracy of the PT ^[8] :

• Partial clotting of specimens, resulting from improper mixture of the anticoagulant (3:2 sodium citrate, as per the manufacturer’s blue-top tube)

• Overfilling or underfilling of test tubes, either of which alters the blood-to-anticoagulant ratio (9:1)

• Analytical errors (eg, lipemic, icteric, or hemolyzed plasma), which may interfere with photoelectric measuring instruments