In this section the various coagulation pathway factors are discussed individually, with emphasis on abnormalities for which laboratory tests are useful.

Factor I (fibrinogen)

Fibrinogen is a glycoprotein that is synthesized in the liver, the liver being a major source of many coagulation factors. Conversion of fibrinogen to fibrin under the influence of thrombin is the final major step in coagulation. Besides its role in coagulation, fibrinogen is a risk factor for coronary heart disease and stroke. Increased plasma fibrinogen most often is temporary and involves the important role it plays as part of the body’s “acute-reaction” response to trauma or to onset of a variety of severe illnesses. This initial response to illness results in fibrinogen production increase and therefore serum level increase. Fibrinogen levels also can be increased by cigarette smoking and by genetic influences. Decreased plasma fibrinogen levels may occurfrom decreased liver production, from the action of fibrinolysins (enzymes thatdestroy fibrin and may attack fibrinogen), and from conversion of fibrinogen tofibrin that is too extensive to permit adequate replacement of the fibrinogen. Decreased fibrinogen production is usually due to liver cell damage, as occurs in acute hepatitis or cirrhosis. However, production mechanisms are efficient, and a severe degree of liver damage is required before significant hypofibrinogenemia develops. Fibrinolysins may be primary or secondary. Primary fibrinolysins are rare and usually attack only fibrinogen. Secondary fibrinolysins are more common and attack primarily fibrin but can attack fibrinogen. Fibrinolysins are discussed in more detail in the section on anticoagulants. The major etiology of hypofibrinogenemia other than severe liver disease is fibrinogen depletion caused by DIC.

Current therapeutic sources of fibrinogen are fresh frozen plasma or cryoprecipitate (Chapter 10).

Factor II (prothrombin)

Prothrombin is a proenzyme synthesized by liver cells. That portion of the prothrombin molecule that permits the molecule to function in the coagulation pathway is formed with the help of vitamin K. Without vitamin K a variety of prothrombin molecules are produced that are inactive. Vitamin K exists in two forms, one of which is preformed in green vegetables and certain other foods and oneof which is synthesized by GI tract bacteria. Both forms are fat soluble and depend on bile salts for absorption. A deficiency of vitamin K may thus result from malabsorption of fat (as seen with lack of bile salts in obstructive jaundice, or primary malabsorption from sprue; see Chapter 26) or from failure of GI tract bacteria to synthesize this substance (due to prolonged oral antibiotic therapy). It usually takes more than 3 weeks before the body vitamin K stores are exhausted and the deficiency of available vitamin K becomes manifest. Dietary lack may be important but ordinarily does not causea severe enough deficiency to produce clinical abnormality unless other factors(e.g., the anticoagulant vitamin K inhibitors) are present.

Neonatal hemorrhage due to vitamin K deficiency may occur during the first 24 hours of life, from 1-7 days after birth, and uncommonly, 1-3 months later. Neonatal deficiency may be idiopathic or associated with certain maternal medications (anticonvulsants, warfarin, antibiotic therapy). It has been reported that up to 3% of infants at birth are vitamin K deficient. Thereafter, dietis important. Breast milk and certain infant milk formulas are relatively low in vitamin K and predispose to clinical deficiency. One dose of vitamin K at birth is frequently used as a prophylactic measure.

Assuming that normal supplies of vitamin K are available, the other main limiting factor in prothrombin formation is the ability of the liver to synthesizeit. In severe liver disease (most often far-advanced cirrhosis), enough parenchyma is destroyed to decrease prothrombin formation in a measurable way, eventually leading to a clinical coagulation defect. Whereas a deficiency of availablevitamin K responds promptly to administration of parenteral vitamin K, hypoprothrombinemia due to liver parenchymal disease responds poorly, if at all, to parenteral vitamin K therapy.

Vitamin K is also necessary for synthesis of factors VII, IX, and X. The greatest effect seems to be on prothrombin and factor VII.

Factor III (tissue thromboplastin)

Tissue thromboplastin is composed of phospholipids and lipoproteins and can be extracted from most tissues. Tissue thromboplastin forms a complex with factor VII and calcium ions that initiates the extrinsic coagulation pathway. This complex is used as the reagent for the PT test.

Factor IV (calcium ions)

Calcium is necessary as a cofactor in several steps of the coagulation pathway. The common hematology and blood bank anticoagulants oxalate, ethylenediamine tetraacetic acid (EDTA), and citrate exert their anticoagulant effect by blocking calcium function through chelation of the calcium ions.

Factor V (labile factor)

Factor V is synthesized by the liver and is decreased in severe liver disease. Factor V is found in plasma but not in serum. Factor V is unstable and becomes markedly reduced in 2-3 days when refrigerated anticoagulated blood is stored in the blood bank. Refrigeration helps preserve factor V in laboratory specimen plasma, but even at 4°C factor V is considerably reduced within 24 hours, and freezing soon after the specimen is obtained is necessary to maintain activity. In factor V deficiency the PT is abnormal; the APTT test is abnormal in severe deficiency but may be normal in mild deficiency.

Factor VII (stable factor)

Factor VII is synthesized in the liver and is dependent on vitamin K for itsactivity. The biologic half-life of factor VII is only 4-6 hours, making it theshortest lived of the vitamin K–dependent coagulation factors. The coumarin vitamin K antagonists produce a more rapid and profound depressive effect onfactor VII than on any of the other vitamin K–dependent factors (discussed at greater length in the section on Coumadin anticoagulants). Factor VII is present in plasma and in serum and is stable in both. In factor VII deficiency the PT is abnormal; the APTT and bleeding time are normal.

Factor VIII/von Willebrand factor complex

Nomenclature of Factor VIII/von Willebrand factor (factor VIII/vWF) complex has been confusing. Until recently, the entire complex was known as “Factor VIII.” In 1985 the International Committee on Thrombosis and Haemostasis proposed a new nomenclature system that is adopted here. Research onFactor VIII over many years demonstrated that the factor was not a single molecule but a complex with at least two components. One is the substance whose deficiency produces classic hemophilia. This component previously was called antihemophilic globulin (AHG) and is now called factor VIII (or antihemophilic factor). For coagulation test purposes, factor VIII (antihemophilic factor) is separated into (1) the factor protein molecule, and (2) its coagulant activity. Antibodies have been produced against the factor VIII protein, and this antigen is now called factor VIII antigen, or VIII:Ag (formerly this was designated VIII C:Ag). The coagulant activity of factor VIII is now designated VIII:C. The secondcomponent of the complex is associated with von Willebrand’s disease and is called von Willebrand factor, or vWf (formerly, this was called factor VIII-related antigen, or VIIIR). Antibodies have been produced against von Willebrandfactor protein, and this antigen is now called von Willebrand factor antigen, or vWf:Ag (formerly, this was called VIII R:Ag). In addition, the von Willebrandfactor has at least two coagulant actions or activities: an effect on platelet adhesion demonstrated by the glass bead column retention test and an effect on platelet aggregation demonstrated by the ristocetin test. The von Willebrand–ristocetin effect on platelets was formerly called the von Willebrand ristocetin cofactor and designated VIIIR:RC; but in the new nomenclature system no formal name or abbreviation is assigned to any vWf function such as the ristocetin cofactor or the vWf effect on platelet adhesion. The site where factor VIII is synthesized in humans is not known, but the von Willebrand factor is apparently synthesized in endothelial cells and megakaryocytes.

The hemorrhagic disease known as hemophilia is usually associated with factor VIIIC deficiency. A similar disease may result from factor IX deficiency (anda similar, but milder, disease from factor XI (deficiency), so factor VIIIC deficiency is sometimes referred to as hemophilia A and factor IX deficiency as hemophilia B. About 90% of clinical hemophilia cases are due to hemophiliaA and about 9% to hemophilia B.

The gene for factor VIII is located on the long arm of the female sex chromosome (Xq28) and is transmitted as a sex-linked recessive trait. Females with the hemophilia A gene are usually carriers (heterozygous, xX), and males with thegene have clinical disease (xY, the hemophilic x gene not being suppressed by anormal X gene as it is in a carrier female). However, about one third of cases are due to fetal gene mutation, not gene inheritance. The gene for vWf is usually transmitted as an autosomal incomplete dominant trait and uncommonly as an autosomal recessive trait. Rare cases of x-linked recessive inheritance have also been reported.

The VIII/vWf complex is found in plasma but not in serum. Both VIII:C and vWf activity (or vWf:RC) are unstable. Refrigeration helps preserve factor VIII activity (VIII:C), but even at 4°C the coagulant activity decreases considerably within 24 hours in laboratory plasma specimens. Freezing maintains factor VIII:C activity if it is done within a short time after the specimen is obtained.

Differentiation of hemophilia A and von Willebrand’s disease. In hemophilia A, male patients have decreased factor VIII coagulant activity, whereas vWf coagulant activity, as measured by platelet function tests, is normal. Factor VIII:Ag is decreased by antibody techniques, whereas vWf: Ag levels are normal. The majority of female hemophilia A carriers have also been shownto have reduced VIII:Ag levels, although usually not depressed to a marked degree (since one chromosome has a normal X gene and apparently can ensure some production of factor VIII). In contrast to hemophilia, patients with classic von Willebrand’s disease have reduced VIII:C activity and vWf activity, both reduced about equal in degree, and demonstrate corresponding decreases in both antigens using antibody techniques. Further discussion of von Willebrand’s disease will be deferred to the section on platelet disorders.

CLINICAL ASPECTS OF HEMOPHILIA A. Hemophilia A exists in varying degrees of factor VIII:C activity deficiency that roughly correlate with clinical severity. Severe (classic) hemophiliacs have 1% or less of normal factor VIII:C activity levels on assay, moderate hemophiliacs have 2%-5%, and mild hemophiliacs have 5%-25% of normal levels. Hemophilia with levels between 25% and 50% of normal is sometimes called subhemophilia or borderline hemophilia. In one study, 55% (literature range, 40%-70%) of factor VIII:C–deficient male patients had classic (severe) hemophilia, about 25% had moderately severe disease, and about 20% had mild disease. Most carrier females have about 50% of normal factor VIII:C activity. About one third of carrier females have factor VIII:C activitylevels similar to those in the borderline decreased group, although usually thecarriers are asymptomatic. About 5% of female carriers have been reported to have mild clinical disease with factor VIII:C activity also in the mildly decreased range; (both X chromosomes affected) and rare homozygous females withclassic disease have been reported.

The major symptom of hemophilia is excessive bleeding. Most bleeding episodes follow trauma. In classic cases the trauma may be very slight. In mild cases the trauma must usually be more significant, such as a surgical operation or dental extraction. Bleeding into joints (hemarthrosis) is a characteristic finding in classic and moderate factor VIII:C activity deficiency, with the more repeated and severe hemorrhagic episodes being found in the more severely factor-deficient patients. Bleeding from the mouth, urinary tract, and GI tract and intracranial hemorrhage are also relatively common in severe cases. In mild cases there may be only an equivocal history of excessive bleeding or no history at all before severe trauma or a surgical procedure brings the condition to light. Another complication of severe hemophilia is hepatitis virus infection from factor replacement therapy. Before laboratory screening of donor blood was possible, transmission of human immunodeficiency virus-I (HIV-I) infection through blood product transfusion affected many hemophiliacs.

The biologic half-life of factor VIII:C activity is approximately 8-12 hours. Factor VIII:C activity is unstable, and in freshly drawn and anticoagulated blood stored in the refrigerator the activity decreases to 40% by 24 hours. The current therapeutic sources of factor VIII:C include fresh frozen plasma, factor VIII:C concentrate, and cryoprecipitate (Chapter 10). Factor VIII:C in therapeutic material is measured in terms of units that equal the amount of factor VIII:C activity in 1 ml of normal plasma. As a rule of thumb, one factor VIII unit in therapeutic material per kilogram of body weight should increase factor VIII activity levels by 2% of normal with a half-life of 12 hours.

About 15% (range, 5%-25%) of patients with hemophilia Adevelop antibodies (acquired inhibitors) against transfused factor VIII, whether obtained from pooled or synthetic (recombinant) plasma. In addition, some persons without factor deficiency develop inhibitors against a coagulation factor (usually factor VIII) for unknown reasons. The most common associated conditions are rheumatoid-collagen diseases, medications, pregnancy or postpartum, malignancy, and Crohn’s disease. Whereas hemophilics typically hemorrhage into joints, factor VIII inhibitors in nonhemophiliacs tend to produce hemorrhage in nonjoint areas.

In either hemophiliacs or nonhemophiliacs, factor VIII inhibitors elevate the APTT just as factor VIII does; and similar to factor VIII deficiency, a 1:1 mixture of patient plasma and fresh normal plasma should decrease the APTT into or nearly into APTT reference range. Incubation of the mixture at 37°C for 1 hour shows relatively little change (less than 8 seconds) in factor VIII deficiency but a significant increase (8 seconds or more) when an inhibitor is present. A 2-hour incubation is necessary with weak inhibitors. About 10%-15% of patients with antiphospholipid antibodies (“lupus anticoagulant,” discussed later in detail) have the same response as a factor VIII inhibitor. The strength of the inhibitor can be quantitated by testing its effect on a standardized preparation of factor VIII, with the result reported in Bethesda units. This test is available mostly in large reference laboratories, university medical centers, and hemophiliac care centers. Factor VIII inhibitors may interfere with those factor VIII assays and other factor assays that use theAPTT as the assay endpoint. Elevation of the APTT due to action of the inhibitor similates the result due to factor deficiency.

Factor IX (prothrombin complex) concentrate has been used with some success in factor inhibitor patients on an empirical basis.

Mild hemophilia A (factor VIII levels >5%) can also be treated with desmopressin (DDAVP), which was found to act as a stimulant to factor VIII and to vWf production. Intravenous injection of desmopressin produces peak action in about 30 minutes, resulting in average twofold increases in factor VIII that last about as long as the effect of cryoprecipitate. Since desmopressin is synthetic, the possible infectious complications of blood products are avoided. However, patients with severe hemophilia A cannot respond sufficiently.

Laboratory tests in hemophilia A.— The PT test results are normal in all factor VIII:C activity–deficient groups. The bleeding time is usually normal; but it will be elevated in about 20% of patients, more often those with severe deficiency. The major screening test for hemophilia A is the APTT. The APTT becomes abnormal when factor VIII:C activity is <30%-35% of normal (range, <20%-<49%). The APTT detects 99%-100% of patients with severe and moderate factor VIII:C activity deficiency. In mild cases, reported detection rate varies from 48%-100% (depending on the particular reagents used and the population selected). Therefore, some of the milder (“subhemophilia”) cases produce borderline or normal results. Definitive diagnosis of hemophilia A usually requires assay of factor VIII:C activity. This used to require the thromboplastin generation test but now is ordinarily carried out with the APTT test and commercially obtained factor VIII-deficient plasma reagent. There is a substantial variation in results in average laboratories, and the reference range is usually considered to be 50%-150% of a normal pooled plasma. The APTT alone detects patients with factor VIII:C less than 30%-35% of normal. This will miss some mild and borderline hemophiliacs and most carriers. Factor VIII:C assay can detect severe, moderate, and mild male hemophiliacs; a considerable number of borderline male hemophiliacs; and about 35% of female carriers. Studies using a combination of factor VIII:C activity assay and vWf protein assay (obtaining a ratio of the two results) are more sensitive than standard factor VIII activity assays alone in detection of hemophilia A, with results of the combined studies (ratio) positive in approximately 50%-75% of female carriers (literature range, 48%-94%). Deoxyribonucleic acid (DNA) probes using multiple targets have been reported to detect 90%-95% of female carriers, making it the most sensitive screening or diagnostic method.

Increased estrogen levels (pregnancy or estrogen-containing contraceptive medication) increase factor VIII:C activity and could mask a mild deficiency. Factor VIII protein is one of the so-called “acute reaction” proteins (Chapter 22) whose synthesis is increased in response to acute illness or stress. Surgery, acute bleeding, pregnancy, severe exercise, inflammation (infectious or noninfectious), and severe stress of other types may raise factor VIII:C activity levels somewhat (within 1-2 days after stress onset) and may temporarily mask a mild factor deficiency state if testing is delayed that long. Another problem is therapy given before test specimens areobtained. There are some technical details that affect tests for factor VIII:C activity. The specimen must be fresh or preserved by short-term refrigeration or by freezing. Most methods quantitate results by comparing the patient resultsagainst those of a reference plasma. Commercially available lyophilized reference plasma gives more reliable results than fresh “normal” plasma. Photooptical instruments in general give better results than mechanical clot timers.

Factor IX (plasma thromboplastin component)

Factor IX deficiency is inherited as an X-linked recessive trait. Deficiencyoccurs in variable degree, and clinical severity varies accordingly, similar tohemophilia due to factor VIII deficiency. Factor IX deficiency produces a hemophilia syndrome comparable to that of factor VII:C deficiency, but hemorrhage into joints is not as frequent as in factor VII:C deficiency, even in the severe form of factor IX disease. The factor IX deficiency syndrome is also called hemophilia B, or Christmas disease (named after the first patient studied in detail). Factor IX is found in plasma or serum and is stable in either.

The APTT test is the standard screening method; however, some reagents only detect deficiency when <20% of normal. It is also used for diagnosis and quantitative activity assay with factor IX-deficient plasma reagent. As in factor VIII deficiency, the PT is normal. Quick differentiation of factor IX deficiency from factor VIII deficiency can be provided by addition of normal serum or normal fresh plasma to patient serum; the APTT abnormality is corrected byserum in factor IX deficiency, whereas fresh plasma but not serum is required to correct a factor VIII deficiency. Increased estrogens may falsely increase factor IX. Diagnosis can also be made by DNA probe methods, similar to hemophilia A.

Current therapeutic sources of factor IX include ordinary blood bank plasma, fresh frozen plasma, and factor IX (prothrombin complex) concentrate. Cryoprecipitate is not useful because it does not contain any factor IX. As a rule of thumb, one unit of factor IX in therapeutic material per kilogram of body weightshould increase factor IX levels by 1% of normal with a half-life of 24 hours. Occasionally, patients develop antibodies against transfused factor IX.

Factor X (Stuart factor)

This factor is a part of both the extrinsic and the intrinsic coagulation pathways. Deficiency is rare and is inherited as an autosomal recessive trait. The clinical syndrome produced is usually mild compared with classic hemophilia A. Factor X is present in plasma and in serum and is stable in both. Both the APTT and the PT are abnormal; the bleeding time is normal.

Factor XI (plasma thromboplastin antecedent)

This factor deficiency is uncommon and is inherited as an autosomal incompletely recessive trait. Deficiency varies in degree according to whether the patient is homozygous or heterozygous. Clinical symptoms from either degree of deficiency are milder than those of comparable cases of hemophilia A or B. Factor XI is present in plasma and serum and is stable in both. The APTT is abnormal; the PT and bleeding time are normal.

Factor XII (Hageman factor)

Factor XII deficiency is very uncommon and is inherited as an autosomal recessive trait. Clinical manifestations (bleeding) are rare, and most of the interest in this disorder derives from the role of factor XII as a surface contact activator of the intrinsic coagulation pathway. The extrinsic pathway is not affected. Factor XII deficiency produces clotting deficiency in laboratory tests that is particularly marked when activation enhancement materials are included in coagulation test reagents (such as the APTT) or glass tubes are used (glass ordinarily would strongly activate factor XII). The APTT and whole blood clotting time are abnormal; the PT and the bleeding time are normal. Diagnosis can be confirmed by means of the APTT with commercial factor XII–deficient plasma reagent. It has been reported that patients with factor XII deficiency have an increased incidence of myocardial infarction and thrombosis.

High molecular weight kininogen (HMWK; Fitzgerald factor)

This factor is a part of the kallikrein system, which affects factor XII in the coagulation intrinsic pathway and also plays a role in the body inflammatory response. Deficiency of HMWK is rare, and no clinical bleeding is induced. The APTT and whole blood clotting time are abnormal; the PT and bleeding time are normal.

Prekallikrein (Fleher factor)

This proenzyme is the central part of the kallikrein system. Prekallikrein deficiency does not predispose to abnormal bleeding. The whole blood clotting time is abnormal; the PT and bleeding time are normal. The APTT is abnormal if particulate activators such as silica or kaolin are used but is normal if solubleactivators such as ellagic acid are used. The APTT abnormality with particle activators may be corrected by prolonged incubation (10-15 minutes vs. the usual 1-3 minutes).

Factor XIII (fibrin stabilizing factor)

Factor XIII deficiency is transmitted as an autosomal recessive trait. Bleeding frequently is first noted in newborns. In later life bleeding episodes are usually mild except when related to severe trauma or surgery. Secondary deficiency may occur in a variety of conditions, especially malignancy or severe liverdisease, but abnormality is usually subclinical. All of the usual coagulation tests (PT, APTT, and bleeding time) are normal, even in congenital deficiency. Diagnosis can be made because fibrin clots from factor XIII–deficient persons are soluble in certain concentrations of urea, whereas clots from normal persons are not. Several other assay methods have been reported.