Factor XII

Factor XII is a coagulation factor that initiates the intrinsic (contact) coagulation system. It is for this reason that it is also known as contact factor. It was described in 1955 in a patient John Hageman and hence it is also known as Hageman’s factor. It does not have a role in normal coagulation but may provide a link between inflammation and coagulation. It may have a role in pathological thrombosis.



The Gene for factor XII is situated on chromosome 5 (5q33-qter). The gene is 12 kilobases in length and contains 13 introns and 14 exons. It has an oestrogen responsive element in it’s promoter.


Biochemistry of Factor XII

Factor XII is a 596 amino acid single chain β-globin zymogen with a molecular weight 80kDa. It is primarily produced in the liver. Factor XII is 17% glycosylated. The plasma concentration of factor XII is 40 μg/ml (500 nmol/L) and it has a half-life of 2 to 3 days.


It is the following domains (from the N-terminal to the C terminal)

  1. N-terminal fibronectin domain type II (exons 3,4)
  2. Epidermal-growth-factor like domain (exon 5)
  3. Fibronectin domain type I (exon 6)
  4. Epidermal growth factor like domain (exon 7)
  5. Kringle domain (exon 8 and 9),
  6. Proline rich region
  7. The catalytic domain (exon 10-14) at the COOH end.

Kallikrein splits the bond between Val352 and Arg353 activating factor XII to XIIa (see activation). activation converts factor XII into a two chain structure held together by a disulfide bond between Cys340-Cys367.  The heavy chain is responsible for binding of factor XIIa to anionic surfaces (52 kDa) and a light chain (28 kDa) that has the enzymatic activity.



Factor XII can be activated to XIIa by the following

  1. Contact with negatively charged surfaces: Factor XII is activated by contact with negatively charged surfaces e.g. glass, kaolin, dextran sulphate, ellagic acid and bismuth subgallate. This rection forms the basis of the activated partial thromboplastin test. As none of these activators are encountered under physiological circumstances it is not the mechanism by which factor XII is activated in vivo for normal haemostasis. It has been hypothesized that contact with anionic surfaces provided by polyphosphates results in a conformational change and activation of factor XII. The mechanism of this activation is not clear.
  2. Activation of By Kallikrein: Factor XII, prekallikrein and high molecular weight kininogen can form a complex on anionic phospholipids of membranes. This, as discussed above, leads to a conformational change in factor XII leading to it’s activation. XIIa splits kallikrein from prekallikrein that in turn activates factor XII leading to a mutual activation loop. Activation involves a cleavage of the peptide bond between Arg 353-Val354 that converts a single chain factor XII to a two chain factor XIIa that are connected by a single disulphide bond between Cys340-Cys367.



Actions of Activated Factor XIIa

Factor XIIa activates coagulation by the intrinsic pathway by activating factor XI. The ability of factor XIIa to split prekallikrein to kallikrein links it to inflammation and fibrinolysis. Kallikrein is converts plasminogen to plasmin, pro-u-PA to u-PA and cleaves high-molecular weight kininogen to give bradykinin. Factor XII is pro-inflammatory and promotes thrombolysis by these actions.




The principle inhibitor of factor XIIa is C1-inhibitor. Antithrombin, α1-antitrypsin, α1-antiplasmin and α2-macroglobulin are the other inhibitors of factor XIIa


Role of Factor XII in Haemostasis

Patients with factor XII deficiency do not suffer bleeding following trauma or surgery despite having a prolonged aPTT. The role of factor XII in haemostasis is complex and unclear. There are many divergent pieces of evidence from experimental and epidemiological studies. These include:

  1. Deficiency in factor XII in mice has been shown to protect against thrombosis induced by injury.
  2. Anti-factor XII monoclonal antibody has been shown to reduce fibrin formation in collagen coated tubes perfused by human blood.
  3. The same antibody has been shown to reduce platelet and fibrin deposition in a baboon graft model
  4. Epidemiological studies have shown an inverse relation between factor XII levels from between all cause mortality and factor XII levels for factor XII levels between 10-100% but no increase in patients with factor levels ≤10%.

Factor XII and Disease

Mutations in factor XII have been associated with type III hereditary angioedema (HE) type III. Unlike patients of type I and II HE who have a defect of C1 inhibitor, patients of Type III HE have point mutation in factor XII resulting in substitution of threonine at position 309 with arginine or lysine.




Evaluation of a Bleeding Patinet

From the point of evaluation of a bleeding patients haemostasis can be divided into the following parts.

  1. Platelets,
  2. Vessel wall
  3. Coagulation system: The coagulation system has the three parts intrinsic pathway, extrinsic pathway and common pathway.  The framework of intrinsic pathway, extrinsic pathway and common pathway are useful to understand how to interpret coagulation tests but does not reflect how coagulation proceeds in vivo.
    1. Intrinsic pathway: Coagulation by the intrinsic pathway starts by contact activation of factor XII, this in turn sequentially activates factors XI and IX. Activated factor XI with its cofactor activated factor VIII lyse and activate factor Xa. Factor XII has no role in coagulation in vivo.
    2. Extrinsic pathway: Coagulation by the extrinsic pathway involves activation of factor VII by tissue factor.  Activated factor VII then activates factor X.
    3. Common Pathway: Common pathway includes factor X, its cofactor factor V, prothrombin and thrombin
  4. Thrombolytic system.

The first step in the evaluation of a patients with a bleeding tendency is to perform screening tests to determine which part of the clotting system  is involved. This can be done with the use of the following tests.

  1. Prothrombin time: Prothrombin time asses the efficiency of extrinsic coagulation pathway.
  2. Activated Partial Thromboplastin Time: Activate partial Thromboplastin time assess the efficiency of intrinsic coagulation pathway
  3. Thrombin Time: Thrombin time asses the amount and quality of fibrin.
  4. Platelet Counts

The results of first-line tests are interpreted as follows.

    1. All tests deranged (Prolonged PT, APTT and TT and Low platelets): Derangement of all screening tests for coagulation is seen in patients with disseminated intravascular coagulation, coagulopathy of chronic liver disease, and acute live cell failure. Patients who have received a massive transfusion may also show this pattern of first line tests. In addition to there being a history of massive transfusion, these patients do not show increase in fibrin degradation products (D-dimer).
    2. Global coagulation defect with normal platelet counts (prolonged PT, prolonged APTT, prolonged PT normal platelets): All three first-line tests are abnormal in patients with primary fibrinogenolysis, fibrinogen deficiency, dysfibrinogenaemia, liver disease or treatment with conventional heparin.
    3. Global coagulation defect with normal fibrinogen and platelets (Prolonged PT and APTT normal TT and Platelets):
      1. Fibrinogen synthesis is not vitamin K dependent. Lack of vitamin K coenzyme activity, as is seen in vitamin K deficiency or use of warfarin like anticoagulants, affects the factors of the intrinsic system (factor II and IX ) and extrinsic system (factor VII). The PT and APTT prolong but TT which depends only on fibrinogen remains normal.
      2. The components of the common pathway include factor V, X and factor II. Deficiency of these factors or the presence of inhibitors against any of these factors causes prolongation of PT and APTT but a normal TT.  A rare autosomal recessive disorder, combined factor V and VIII deficiency can cause prolongation of PT and APTT with a normal TT.
      3. Some patients with liver disease may have a prolonged PT and TT.
    4. Isolated prolongation of APTT (Normal PT, Prolonged APTT, Normal TT, Normal Platelets counts): The commonest cause of isolated prolongation of APTT is deficiency or inhibitors of factor VIII or factor IX. Deficiency of other components of the intrinsic pathway including factors XI and XII and prekallikrein can cause isolated prolongation of APTT. Of these only deficiency of factor IX deficiency causes bleeding. Deficiency on factor XII and prekallikrein are not associated with bleeding.
    5. Isolated Prolongation of PT (Prolonged PT, Normal APTT, Prolonged TT and normal platelet count):
      1. The commonest cause of isolated prolongation of PT is a deficiency of factor VII. This may be seen in
        1. Vitamin K deficiency/Use of Warfarin type oral anticoagulants: Factors II, VII, IX and X are vitamin K dependent and have half-lives of 65hrs, 5hrs, 25hrs and 40hrs respectively. When vitamin K co-factor activity is not available either as a result of vitamin K deficiency or the use of warfarin type of oral anticoagulants, the synthesis of vitamin K dependent factors decreases.  The half-life of factor VII is considerably shorter than that of other factors and the levels of factor VII fall the earliest. This results in a prolonged PT but a normal APTT. With time other factor levels fall and the APTT also prolongs.
        2. Early Liver disease
      2. The other causes of isolated prolongation of PT include inherited factor VII deficiency, dysfibrinogenaemia or mild factor VIII, IX or IX deficiency
    6. Isolated prolongation of TT: Isolated prolongation of TT is seen in fibrinogen deficiency or dysfibrinogenaemia
    7. Coagulopathy with normal first-line coagulation tests: May be seen in platelet function defects, vascular coagulation defects, mild von Willebrand’s disease, factor XIII deficiency, disorders of fibrinolysis, dysfibrinogenaemia and α2-Plasmin inhibitor deficiency.
    8. Isolated thrombocytopenia: Isolated thrombocytopenia may be seen in bone marrow failure syndromes, immunethrombocytopenia, following viral infection and in patients with hypersplensim or inherited thrombocytopenia.

Thrombin Time

Thrombin time is the clotting time of plasma on addition of thrombin. It measures the amount and quality of fibrinogen. Thrombin time is prolonged when the fibrinogen levels fall below 70-100mg/dL. The levels of other coagulations factors do not affect thrombin time. Thrombin time is also prolonged by the presence of fibrin/fibrinogen degradation products. It can also be prolonged with a very high fibrinogen levels. Dilution of test plasma normalizes the thrombin time in such patients.


The tests is performed on platelet poor plasma. Thrombin solution is added to PPP and the time for the clot to form is measured. The nature of the clot is also observed. The clot may be transparent or opaque. The test is performed with commercially available bovine thrombin. The concentration of thrombin is adjested to get a thrombin time of 15 on normal plasma. If a more concentrated thrombin is used mild defects in fibrinogen may not be detected. The test is performed with paired test as well as control samples. The thrombin time is the mean of the two readings. Normal thrombin time is two ±2 seconds from the control.

Causes of Prolongation

  1. Therapy with unfractionated heparin. Low molecular weight heparin does not alter the thrombin time appreciably.
  2. Hypofibrinogenaemia and occasionally hyperfibrinogenaemia may prolong thrombin time. The mechanism of prolongation by hyperfibrinogenaemia is unclear but hyperfibrinogenaemia may interfere with fibrin polymer formation.
  3. Dysfibrinogenaemia
  4. Paraproteins may interfere with polymerization of fibrin and prolong thrombin time.

Short Thrombin Time

The thrombin time is shortened when the coagulation is activated.

Nature of the Clot

Transparent fluffy clots indacetes an anomaly in fibrin polymerization. This seen with dysfibrinogenaemias either congenital on seen in liver disease.

Reptilase Clotting Time

Reptilase clotting time is a variation of thrombin time. Clotting is induced by reptilase, an enzyme prepared from snake venom. This test is unaffected by heparin. In samples contaminated with heparin the thrombin time is prolonged where as the reptilase time is normal. On the other hand dysfbrinogenaemia affects the reptilase clotting time more than thrombin time. In patinets with hypofibrinogenaemia the prolongation is equal.

Activated Partial Thromboplastin Test

Activated partial thrombocplastin time (aPTT) measures the efficiency of the contact activation of clotting. It depends on factor XII, XI, IX, VIII, V and X.


Plasma is incubated with a substance like kaolin or elagic acid to bring about contact activation of factor XII to XIIa. XIIa cleaves XI to XIa. The subsequent steps in coagulation are calcium dependent and take place only when calcium is added. Phospholipid is added to provide a platelet sunstitute.


The test involves mixing platelet poor plasma and a factor XII activator e.g. silica, celite, kaolin, micronized silica or ellagic
acid with phospholipid containing reagent. Calcium chloride is then added. The time take for a clot to form after adding calcium chloride is the activated partial thromboplastin time. The test is performed with paired samples one test and one control.


Mixing Studies

Prolongation of a coagulation test may be as a result of a deficiency in coagulation factors or because of the presence of in inhibitor of coagulation. Mixing studies are performed to differentiate between the two.

Principle of Mixing Studies

Inhibitors affecting activated partial prothrombin time (aPTT) are more common in clinical practice and the discussion that follows is in relation to aPTT. A mixing study with prothrombin time (PT) may be performed using the same principle. Prolongation of aPTT indicates decrease in the coagulation factors the test is dependent on. aPTT prolongs when the factor levels fall to less than 40% of normal.

When the test plasma is mixed with normal pooled plasma in a 1:1 ratio the factor levels are and average of that in the two samples. The coagulation factor levels in pooled plasma are close approximately 100%. Patients with severe coagulation factor deficiency typically have <1% of normal coagulation factors levels. When plasma from such patients is mixed with normal pooled plasma the factor levels in the plasma are will be 50%. For patients with a lesser degree of coagulation factor deficiency the coagulation factor levels in mixed plasma will be greater than 50%. As a normal aPTT needs 40-50% coagulation factor activity and mixing a factor deficient plasma with normal plasma  would result in normalization of a prolong aPTT even in the most severe form of coagulation factor deficiency.

Inhibitors of coagulation are antibodies directed against coagulation factors. Presence of the antibody is associated with diminished coagulation factor activity and a prolonged aPTT.  When the plasma from a patient carrying an inhibitor is mixed with equal amount of normal plasma the inhibitor present in the patients plasma inhibits the activity of the coagulation factors present in pooled plasma. The prolonged aPTT of patient’s plasma fails to correct because the activity of the mixture falls to below that needed for a normal aPTT. Some antibodies act with a lag period. The aPTT of these patients may correct immediately on mixing but will prolong after 2 hours of incubation.


Performance of the Mixing studies

Mixing studies involves performance of duplicate aPTT on the following samples

  • Tube 1: Pooled plasma – Plasma from at least 20 normal individuals
  • Tube 2: Patients plasma
  • Tube 3: Patients plasma and pooled plasma incubated separately for 2 hours at 37°C. The two are mixed and the mixture immediately tested.
  • Tube 4: Patients plasma and pooled plasma mixed and incubated for 2 hours at 37°C



Interpretation of Mixing Studies

Normal Plasma (Tube 1)
Test Plasma (Tube 2)
Incubated separately and mixed (tube 3)
Mixed and Incubated (tube 4)
Normal Normal Normal Normal Normal
Immediate Inhibitor Normal Prolonged Prolonged Prolonged
Delayed inhibitor Normal Prolonged Minimally prolonged if at all Prolongrd

Prothrombin Time

Prothrombin time was described by Quick in 1935. It is the time taken by re-calcified plasma to clot in the presence of tissue procoagulant extract known as thromboplastin. It asses the efficiency of the extrinsic coagulation system. The test depends on activation of factor X by factor VII by tissue factor.

The Reagents

1. Thromboplastin: Thromboplastin is a mixture of tissue procoagulants. Thromboplastins have different sources e.g. placenta, human brain, rabbit brain. Recombinant thromboplastins are available.

2. Calcium Chloride: 25mM calcium chloride.


The details of the method are beyond the scope of this text. The outline is as follows. Thromboplastin is added to plasma that has been separated from blood collected in sodium citrate and allowed time to mix. To this mixture calcium chloride is added. The time taken for the plasma to clot is the prothrombin time. The end point (clotting) may be determined manually or using automated (optical or magnetic) methods.

Reporting Results

The results is reported as the number of seconds taken for the clot to form along with a normal value and as INR.

The Concept of International Normalized Ratio

Thromboplastin is a mixture of tissue procoagulants. The source of thromboplastin affects the test results. From the time Quick described the prothrombin time till the 1950s the cadaveric human brain was the source for thromboplastin. Each laboratory had to make it’s own thromboplastin.

In the 1950s commercially available thromboplastins became available. These were derived from animal tissue. Contamination by serum resulted in the presence of some coagulation factors in the commercially available thromboplastin. Oral anticoagulants reduce the levels of vitamin K dependent factors (II, VII, IX, X) prolonging the prothrombin time in proportion to the decrease in the levels of coagulation factors. Use of thromboplastins contaminated with coagulation factors made the test less sensitive to the decrease in coagulation factors in the test plasma. An important clinical consequence of the insensitivity of prothrombin time performed with use of animal thromboplastin was increased risk of bleeding in patients on oral anticoagulant therapy.

A survey of average warfarin dose showed that the average dose of warfarin was higher in North America where commercially manufactured thromboplastins were used compared to UK that used human thromboplastin. Anticoagulation monitored by human thromboplastin at PT ratio (Patient’s PT/Normal or control PT) of 1.5-2 was compared with anticoagulation monitored with animal thromboplastin and a target PT ratio of 2-2.5. The two were found to be equally effective. Also the patients monitored by PT preformed by using human thromboplastin had a 20% lower risk of bleeding.

These observations suggested the need for standardization of prothrombin time to adjust for variations in the reagents used. The concept of international normalized ratio was introduced by WHO in 1983 to fulfil this need.

International Normalized ratio (INR) is defined as

INR = (Patient’s PT/Mean normal PT)ISI

Mean normal PT is the geometric mean of prothrombin time of plasmas collected from at least 20 normal individuals. The group should include members of both sexes. The ISI is the international sensitivity index of the thromboplastin that is calculated as a part of caliberating of thromboplastin by comparing the thromboplastin against a the WHO reference thrombopastin (Internationla Referenmce Preperation, IRP).

Importance of International Sensitivity Index (ISI)

ISI CurveINR depends of the prothrombin ratio and ISI. The difference between prothrombin ratios at INR 2 and 3 for thromboplastin with ISI between 1 and 2 are plotted in the graph above. The INR is maintained between 2 and 3 fro most indications. With a thromboplastin with an ISI of 1 the difference between prothrombin ratio at INR 2 and 3 is 1. This reduces to just over 0.3 for thromboplastin of ISI 2. Thromboplastins with a low ISI give a wider prothrombin time range for a given INR range allowing a more accurate control of anticoagulation. Higher ISI thromboplastins also give a less precise measurement of INR with a high coefficient of variation. It is recommended that the ISI of the thromboplastin used for prothrombin time should be < 1.7 (preferably between 0.9-1.2).

Target INRs for Anticoagulant Therapy

There are many recommendations for target INR for anticoagulation. In general these recommend that therapeutic anticoagulation in most situations needs a target INT of 2.5. Patients at high risk e.g. those having thrombosis on oral anti coagulants or those having mechanical prosthetic valves with moderate to high thrombotic risk (see British Committee for Standardization in Haematology Oral Anticoagulant with Warfarin Guidelines – Fourth Edition and Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines)

Pre-Analytic Variables in Prothrombin Time

Sample collection is critical for prothrombin time. Faulty collection may result in activation of coagulation while collection. Sample for prothrombin time must be collected from a large vein with free flow of blood with a reasonably large calibre needle. The sample should not be a part of a large collection. If evacuated blood collection tubes are used then the prescribed order of draw must be followed (see the BD order of draw chart). If the sample is to be drawn from indwelling catheters 10-15ml blood is drawn and discarded before collection of sample for prothrombin time. Lipaemic plasma may interfere with the function of photo-electric measurements. Samples should not be obtained after meal. Samples should be collected in 3.2% buffered sodium citrate. Nine parts of blood should be mixed with 1 part of anticoagulant. It is not recommended toThe amount of blood in patients with very high or very low haematocrit may be calculates as follows

Amount of blood for every 0.5ml 3.2% sodium citrate =

[60/{100-haematocrit}] X 4.5


Related links in this site

  1. Bleeding Time
  2. Pharmacogenetically Guided Warfarin Therapy


Further Reading

  1. Poller L. International Normalized Ratio (INR): The First 20 Years. J Thromb Haemost 2004; 2: 849–60. – One of the most comprehensive articles discussing INR and it’s evolution I have come across