Thrombogenesis - Thrombophilia
(Portuguese PDF version)

Fernando L. V. Duque1, N. A. Mello1

1. Department of Angiology, Pontifícia Universidade Católica do Rio de Janeiro. Division of Angiology and Vascular Surgery, Hospital Central da Santa Casa da Misericórdia, Rio de Janeiro.

Correspondence:
Prof. Fernando Luiz Vieira Duque
Rua Sorocaba, 464/201
CEP 22271-110 - Rio de Janeiro - RJ
Tel.: +55 21 2246.5431

ABSTRACT

The incidence and development of thromboembolism has been the object of extensive research since the late 18th century. However, a detailed study of all intrinsic and extrinsic factors that may lead to thrombosis is not available yet. Therefore, after a brief historical review of the findings and hypotheses formulated about thrombophilia, the present article describes the risk factors that have been currently investigated, with the aim of offering efficient prophylactic treatments.

Key words: thrombophilia, thromboembolism, risk factors
Palavras-chave: trombofilia, tromboembolismo, fatores de risco.

J Vasc Br 2003;2(2):105-18

Venous inflammation and thromboses have been cited in the medical literature for centuries. Through the years, the clinical importance of thromboembolic events has progressively increased and, today, thrombosis is a severe condition in all medical areas due to its endemic nature.

Hunter,1 in 1784, in the article "Notes on the inflammation of the internal venous layer," drew attention to thrombosis detected after venipunctures, complex fractures and surgeries. Thereafter, this author observed venous inflammation and regarded it as the cause for concomitant venous thrombosis, a point of view also shared by Cruveilhier.2,3 Later on, the same author found thrombosis without suppuration of vessel walls, and called it spontaneous venous wall inflammation.

In 1877, Trousseau4 associated venous thrombosis with neoplasms. Around the same period (1875), Paget5 and, a little bit later (1884), Schröetter described thrombophlebitis caused by strain/effort, in which there seemed to be no venous inflammation. In the late 18th and early 19th centuries, studies on the topic and on venous thrombosis, which were few and far between, only gained momentum in the second half of the 19th century. These studies were mostly carried out by German pathologists, who described almost all structural vascular disorders known to date. The macroscopic and microscopic morphology of thrombi was described by Virchow,6,7 Zahn,8 Welch,2,3 Aschoff,9 among others. However, the best description of thrombotic injury was only made in the mid-20th century with the use of electronic microscopy.

Besides pathoanatomical studies, almost all these investigators postulated hypotheses about the etiopathogenicity of venous thrombosis. Baille2,3 suggested the importance of hemostasis to thrombus formation, which became the workhorse of Virchow.6,7 Rokitansky10 believed there were two forms of venous thrombosis: one provoked by inflammation of the vessel wall and another one caused by intervessel stasis. In 1898, Welch2,3 reviewed the topic thoroughly and concluded that, in most cases, more than one element is involved in the etiology of thrombosis and that only inflammation and stasis would not explain all cases of thrombosis.

Hayem,8 in 1878, observed platelet agglutination at the puncture site of a vessel; Bizzozero8 confirmed this finding and called it hemostatic clot. In 1885, Eberth & Schimmelbuch11 induced thrombus formation in mesenteric vessels of animals and regarded platelet agglutination as the initial phase of thrombosis. In 1927, Wright & Minot12 found out that platelet viscous metamorphosis may occur before fibrin formation. In 1924, Aschoff9 classified the etiopathogenic components of thrombosis into three groups: parietal factors, hemostasis and blood dyscrasia disorders. In 1890, the plasma origin of fibrin was admitted by Virchow,6,7 who termed its precursor form as fibrinogen. In 1895, Schimidt13 observed there existed a "fibrin ferment" (thrombin), which circulated in the blood as a precursor and inactive form (prothrombin). Prothrombin activation occurred due to the presence of "zymoplastic substances" in the tissues, which Morawitz,14 in 1905, called thrombokinase, and Howell15 later denominated "tissue thromboplastins" (factor III). These enzymes were only activated in the presence of calcium ions (Arthus & Pages;16 Hammarsten16). The presence of intrinsic thromboplastin was only discovered some years later, under the name of tryptase (Ferguson17). The fermentative digestion of fibrin (fibrinolysis) was only discovered in 1903.

For several decades, the clinical and epidemiological observations made by a large number of authors from different countries allowed for the identification of various situations and diseases that preceded or accompanied the signs and symptoms of venous thrombosis (Table 1). This association characterized a cause-and-effect relationship, and these events were considered to enhance or trigger off venous thrombosis and, consequently, arterial thrombosis.

click hereTable 1 - Conditions that predispose to thrombosis (risk factors, acquired disorders)

Adenocarcinoma Intravascular hemolysis Heart failure
Anemia Collagen disease Hyperlipidemia
Prolonged general anesthesia Chronic enteritis Homocystinuria
Lupus anticoagulant Corticosteroids Hyperviscosity
Antiphospholipid antibody Exogenous estrogens Hyperhomocysteinemia
Bacteremia Pregnancy* Estrogen therapy
Cancer Use of contraceptives Age
Surgery (pelvis, hip, extensive) Liver diseases Recent myocardial infarction
Leukosis Paroxysmal nocturnal hemoglobinuria Infections
Lupus Kidney diseases Obesity
Multiple myeloma Hemodilution Total immobilization and/or immobilization of lower limbs
Paraproteinemia Polycythemia vera Polycythemia
Puerperium Burns Sedentary lifestyle
Smoking Trauma Previous thrombosis
Varicose veins Behcet's vasculitis
*With intrauterine growth retardation; abruptio placenta; history of thromboembolism;
preeclampsia; postpartum thrombosis; recurrent fetal loss.

More or less at the same time, it was observed that patients with a larger number of risk factors had more propensity for intervascular thrombosis, which led many authors to develop prognostic evaluation methods that included tables in which each factor receives an absolute or percentage value18-20 (Table 2). If the sum of these partial values in a given patient is above a certain value, he/she is considered to be at risk for thromboembolic disease and, therefore, deserves special care, including occasional prophylactic anticoagulant therapy in the perioperative and postoperative periods (Table 3). These tables are of great importance and are used by several services.

click hereTable 2 - Quantification of risk factors for venous thrombosis (thromboembolic disease)*†

High risk (4 points)
Hip or knee surgery or fracture, prosthesis
Long bone fracture or multiple fracture
Polytrauma
Abdominal surgery due to cancer
Surgery and/or severe disease, with previous TED
Moderate risk (2 points)
Age equal to or above 60 years
Previous history of venous thrombosis (plus disease or small surgeries)
Chronic venous insufficiency, with or without ulceration
Congestive heart failure
Complicated myocardial infarction
Malignant neoplasm
Long bed rest
Ischemic stroke
Extensive burns and traumas
General surgery (over one hour, with other risk factors)
Presence of antiphospholipid antibody
Low risk (1 point)
Age between 40 and 60 years
Obesity
Smoking
Estrogen/contraceptive
Pregnancy or puerperium
Diabetes mellitus
Uncomplicated myocardial infarction
Infections
Large surgery in the last 6 months
Nephrotic syndrome
Small surgery (< 30 min)
Large surgery in young healthy individual
*Adapted from Weinmann20
†If the final score for risk factors of a certain patient is = 5,
prophylactic antithrombotic therapy should be used.

click hereTable 3 - Prophylaxis to be adopted based on the scores of patients who will be submitted to additional risk factors (surgery, prolonged bed rest, certain drugs, etc.)

Low risk (1 point)
Active movement of limbs
Early walking
Wearing of elastic stockings
Breathing exercises
Intensive hydration
Avoid sitting
Avoid Fauwler bed
Moderate risk (2 to 4 points)
Same measures as for low risk
Subcutaneous heparin 5,000 IU every 12 hours
Subcutaneous enoxaparin [Clexane®], 20 mg, once a day
Subcutaneous nadroparin [Fraxiparine®], 0.3 mg, once a day
Subcutaneous dalteparin [Fragmin®], 2,500 UI, once a day
In surgical patients, initiate heparin administration some hours before surgery, and maintain it if necessary
High risk (5 or more points)
Same measures as for low risk.
Heparin, sc, 40 mg, once a day
Enoxaparin, sc, 40 mg, once a day
Nadroparin, sc, 0,6 mg, once a day
Dalteparin, sc, 5,000 IU, once a day

Nonetheless, with time, it was noted that many individuals with increased risk factors did not obligatorily have thromboembolic disease, although they were subject to provably thrombogenic conditions; in other words, not all individuals with risk factors had thrombosis after having additional thrombogenic stress (trauma, delivery, etc.).21 Inversely, one third of the patients with clinical signs and symptoms of venous thrombosis did not show any risk factors.

Given these observations, it was clear that there should be an idiopathic "spontaneous thrombophilia," which would cause vascular thrombosis without any extrinsic pathogenic stimulus or even without any triggering process. The old constitutional medicine aphorism was back: "only those who can develop a disease will do so" or, in other words, "the risk factors are only expressed in an individual who has an adequate genetic pattern." Thrombophilia originates from peculiar characteristics of the blood coagulation system, perhaps from latent individual, and from possibly congenital or familial nature.

Based on these findings, the coagulation phenomenon became important in the study of thrombus formation. The hypothesis of parietal lesion and hemostasis as preponderating factors in thrombus formation was to some extent replaced with the importance of the blood component, the third one in the Virchow6,7 and Aschoff9 triad. The paucity of experimental studies observed at the beginning of the century on blood coagulation were supplanted by a large volume of investigations, resulting in the description of several coagulation factors that composed the framework of the "coagulation cascade" and of the fibrinolytic system.

Almost all crucial findings about blood coagulation data back to this period: accelerin,22 convertin,17 Stuart factor,24 tryptase,17 antihemophilic globulin,25 Hageman factor,26 B factor,27,28 C factor,29 antithrombin III,14 etc. In 1933, Tillet & Garner30 found out streptococcal fibrinolysin. In 1941, Milstone31noted that the action of this substance depended upon a human globulin, which he called lytic factor. In 1944, Kaplan32 found out that this plasma factor was a protease precursor activated by the streptococcal factor. In 1945, Christensen33 termed the streptococcal factor streptokinase, and designated the plasma precursor as plasminogen. Active protease was called plasmin.

These findings allowed us to understand about hemorrhagic states, but did not shed further light on thrombotic events.

The term thrombophilia has been used for a long time meaning "capacity or tendency of blood towards thrombus formation" (due to parietovasal injury, rheologic disorders and blood dyscrasia). With time, the term became restricted, meaning "coagulation, precoagulation and hypercoagulable state disorders" (the latter of which is preferred by U.S. authors). It is known today that this state depends on risk factors (extrinsic conditions) that act on their own or are facilitated by previous existence of inherited coagulation disorders and/or fibrinolysis (intrinsic conditions).

The interest aroused by the possible identification of individuals with hypercoagulable states, that is, individuals who are congenitally prone to thrombosis, through laboratory tests, focused on the investigation of these blood-related disorders and enhanced the use of the term thrombophilia to describe only the presence of inherited bleeding disorders.

Knowledge about hypercoagulable states improves the prognosis of venous and arterial thromboembolic disease and allows us to understand about idiopathic thrombosis, the etiology of "spontaneous" venous thrombosis, recurrent phlebitis, migratory phlebitis, individual susceptibility to hormone therapy, blood transfusion, contraceptives, or generally speaking, about the thrombogenic predisposition to "extrinsic thrombotic risk factors."

Unarguably, the identification of inherited thrombophilic factors is a great step towards the study of thromboembolic events. However, these new findings should be used equitably, without disregarding the importance of extrinsic risk factors, which can cause thromboembolic disease, regardless of previously abnormal inherited conditions, or which are hithertofore unknown.

The detection of this individual inherited thrombophilic substrate, to some extent, hinders the studies carried out on the incidence of arterial and venous thromboembolic diseases in physiological and/or pathological situations such as pregnancy, menopause, obesity, sedentary lifestyle, among others. Since thromboembolic events have variable incidence in congenitally different organisms given the same physiological and/or pathological scenario, the epidemiological investigation about frequent clinical correlations such as menopause/myocardial infarction, arterial hypertension/stroke, dyslipidemia/thrombosclerosis, hormone replacement/angiocardiopathies, among others, has to be preceded by the thrombophilic assessment of study participants.

Along with such advancements, we should consider other pathogenic causes of thrombotic events that are still neglected and that are not yet prominent in clinical and laboratory practice:

  • viscosity of blood has been known and investigated for decades. Nevertheless, it seldom opposes to Aschoff-Virchow6,7,9 thrombogenic triad. The relative difficulty in confirming the detrimental action of hyperviscosity and its complex laboratory investigation contribute to its being poorly advertised, although it basically interferes with all vascular pathophysiological phenomena, especially with regard to microcirculation.
  • the activity of endothelium: among the several newly-found endothelial factors there are several factors that act on the coagulation and anticoagulation systems. The assessment of von Willebrand factor, thrombomodulin and of other endothelial products will soon be included as new latent hypercoagulability factors, constituting a state of "endothelial thrombophilia" in addition to the currently identified hypercoagulation factors. Nearly all endothelial disorders known to date are secondary to extrinsic risk factors, but recent cases of primary endothelial defect have been observed.34,35

Concomitantly, the improved knowledge about endothelial dysfunctions might narrow the divide between the activities and functions of the etiopathogenic elements observed in Virchow6,7 triad. Perhaps, we will acknowledge and extend the statement made by Welch2,3 that in most cases of venous (or arterial) thrombosis more than one factor is known to be involved. Very likely, of all parietal factors (especially endothelial ones), rheologic and hematological factors (including viscosity) simultaneously contribute to a lesser or greater extent to thrombus formation.

INNATE PREDISPOSITION TO THROMBOSIS

Thromboembolic disease often has a hereditary/familial characteristic, which is known for centuries. Only in the last few decades, attempts of laboratory detection of blood disorders occasionally present and/or responsible for thrombosis predisposition have yielded positive results. The joint complex of these blood disorders have received different names: thrombophilia, primary thrombophilia, essential, idiopathic, congenital, hereditary, genetic, familial or innate thrombophilia. Other terms with the same meaning include: prothrombotic state, precoagulation state, prethrombotic state, hypercoagulable state, congenital thrombophilic state, thrombotic risk state and predisposing anomalies. The term "risk factor", as currently used, stemmed from the predisposing factors for coronary heart diseases observed in the first reports of the Framingham Study36 and soon extended to other medical areas, including that of coagulopathies.

Different blood disorders were studied and associated with thrombosis. These disorders seemingly occur in thrombotic stress conditions, such as blood hyperviscosity due to an increase in platelets37 or fibrinogen,38 functional platelet disorders,39 hypercholesterolemia,40 hyperprothrombinemia,23 increase of factor VII,23 of factor Stuart,41 of factor Christmas,42 of blood glucose,43 etc. In patients in whom the thromboembolic process was active, possible blood dyscrasia, which characterizes the "hypercoagulable state," was also investigated: increase of fibrinogen,44 of factor VII,45 of proaccelerin,46 of platelet adhesion47 and of plasmin inhibitors.48 The results of these studies were so contradictory that they led Ratnoff & Botti42 to write that "the evidence provided so far shows that it is not possible to obtain a blood sample from a patient and affirm this blood is hypercoagulable."

More reliable investigation was conducted more or less in the same time period. In 1965, Egeberg49 related antithrombin to thrombophlebitis. Browse et al.50 observed a reduction in fibrinolytic activity in thrombogenic states. Under the same conditions, hypercoagulability of total or diluted blood,51 was observed, which was measured by thromboelastography52 or by coagulation time through impedance.53

In 1981, Griffin et al.54 reported protein C deficiency in cases of recurrent familial thrombotic disease. In 1983, Carrel et al.55 affirmed that congenital dysfibrinogenemia occasionally causes thrombosis. In 1984, Towne et al.56 observed thrombophilic activity in dysgenesis and plasminogen hypofunction. In the following year, Nilsson et al.57 found out that the reduced tPA synthesis, or reduced synthesis of endothelial activators could be accompanied by venous thrombosis.

In 1993, Dahlback et al.58 identified a cofactor for blood coagulation, which was resistant to activated protein C and was thrombogenic. This finding was improved by Bertina et al.59 in the following year when they noted that this factor was actually a factor V mutation, which determined activated protein C resistance (this finding occurred at the University of Leiden, hence the name of this factor). In 1998, Williamson et al.60 described another mutation in the factor V gene still considering the "activated protein C resistance" (factor V Cambridge). In 1996, Poort et al.,61 from the Bertina group, identified genetic alterations in chromosome 20.210, in prothrombin, etc.

The way in which these factors and/or mutational disorders act to form thrombi is still unclear and requires further investigation. In the last decades, several possible thrombophilic factors were described, along with inborn errors of hemostasis, as the cause of hypercoagulability or inherited thrombophilia. Every month, new findings are made, and due to such accelerated dynamics, any lists of thrombophilic substances could become outdated as they are made. At the moment, in clinical terms, we currently work with prothrombotic factors, which will be briefly described next; their frequency is shown in Table 4.

click hereTable 4 - Inherited thrombophilic factors

Mean prevalence according to some values taken from the literature %
Association of inherited disorders 30 - 60
Activated protein C resistance (Leiden) 20 - 50
Fibrinolytic disorders 10 - 15
Prothrombin gene mutation G20210A 5 - 15
Antithrombin III deficiency 4 - 10
Protein C deficiency 3 - 6
Protein S deficiency 5 - 15
Plasminogen deficiency 1 - 2
Heparin cofactor II deficiency 1
Fibrinogen disorders 1
t- PA < 1
Excessive PAI < 1
Alpha-2-macroglobulin < 1
Hyperhomocysteinemia < 1

 

In general, arterial thrombosis predominantly derives from platelet activation, lipid deposition and cell proliferation on the atheromatous plaque. Venous thrombosis is essentially dependent on the hemostatic factors under analysis here.

Fibrinogen (factor I)

Fibrinogen is a glycoprotein synthesized by the liver and which circulates in inactive form in the blood serum at the concentration of 160 to 415 mg/dl. It is activated (for factor Ia = fibrin) by thrombin when it is cleaved (fibrinopeptides). The resulting fragments are monomers constituted by domains (d) which, when cross-linked, form fibrin polymers that trap erythrocytes, leukocytes, platelets and coagulation factors (XIII, IIa), originating the hemostatic clot/thrombus. This polymer is initially reversible, but under the action of factor XIII (found in the clot, which is activated by factor IIa = thrombin), it stabilizes within two or three days.

Some studies indicate that hyperfibrinogenemia is associated with thromboembolic disease.62 The increase of serum fibrinogen accelerates clot/thrombus formation, increases platelet aggregation and causes hypercholesterolemia. Conversely, free fatty acids enhance fibrinogen synthesis in culture media.63 During pregnancy, serum fibrinogen and factors VII, VIII, IX, X and XII are elevated, protein S levels are reduced, acquired activated protein C resistance is enhanced, platelets are hyperactivated and PAI 1 and 2 levels increase. Elevated serum fibrinogen and von Willebrand factor are risk factors for ischemic heart disease (independent and genetic), especially in children born of parents with hyperfibrinogenemia.64 Some forms of dysfibrinogenemia are inherited conditions in which fibrinogen molecules are synthesized incorrectly and manifest themselves clinically through arterial and/or venous thrombosis in 5 % of the cases.

The inclusion of fibrinogen dosage in the research algorithm of intrinsic thrombophilia is controversial.

Protein C

During the hemostatic phenomenon, the catalyzing action of thrombin on fibrinogen (producing fibrin) is limited by several regulatory mechanisms, and also by thrombin-thrombomodulin interaction. In addition to direct inactivation, the thrombomodulin/thrombin complex activates serum protein C, which usually inhibits activated factors V and VIII, thus reducing the action of the coagulation cascade and, consequently, restricting the formation of hemostatic clot to the site of endothelial injury, which prevents the excessive formation of thrombotic clot.

Protein C is a serum protease that is vitamin K-dependent and whose activity is enhanced by protein S. Besides restricting the intrinsic coagulation pathway,65,66 protein C helps the fibrinolytic system by lysing the substance [PAI-1], which blocks the tissue plasminogen activator.67

Inherited or acquired protein C deficiency is associated with vascular thrombotic events.68 The inherited deficiency of the homozygous form is incompatible with life. When the reduction of protein C levels is about 50% (i.e. in heterozygous patients), it often favors the development of thrombosis after the second or third decades of life due to traumas, surgeries, etc.67

Recently, it has been observed that thromboembolic events are more common when activated protein C resistance is present than when the total protein C levels are reduced.69,70

Factor V

Factor V is an essential regulator at the initial stage of the coagulation cascade. Its genetic deficiency produces a rare hemorrhagic disease, known as parahemophilia. A slight alteration to its gene (factor V Leiden) increases its action since it is not naturally blocked by protein C, which favors thrombus formation. This mutation is present in 2% to 7% of the general population and in over 50% of individuals diagnosed with thromboembolism. At present, several gene mutations that occasionally produce alterations similar to the factor V Leiden are being investigated.71

Activated protein C resistance (APCR)

Factor V Leiden
It was observed that in some patients with familial and/or recurrent thromboembolism, the partial thromboplastin time (PTT) test was abnormal and that this was not corrected when normal activated protein C was added; that is, there was resistance of factor V to the action of activated C protein, which explains the tendency towards the increase in thrombin production and the increase in the incidence of thrombosis in these patients.58,69 One of the causes of this resistance, the factor V Leiden, consists of a mutation in factor V itself, in which arginine 506 is turned into glutamine.59 With the replacement of this amino acid, the natural site for factor V cleavage by protein C is blocked, which minimizes its action. Thus, there is full action of factor V, with consequent development of hypercoagulable state.

Leiden factor is the most frequently found genetic alteration in patients with APCR (around 95% of the cases). According to Williamson,60 in the remaining 5%, another mutation in factor V exists, which is called factor V Cambridge (named after the university where it was discovered).

The incidence of factor V Leiden is of 2% to 5% in the general population and of 25% in the cases of recurrent venous thrombosis and/or pulmonary embolism. This mutation is characteristic of whites, being absent among blacks and Asians.

Factor V Leiden heterozygous mutation increases the risk of thromboembolism by seven times, and this risk grows over the years, with pregnancy or with the use of oral contraceptives. The homozygous form of the allele increases thrombogenic risk by twenty times.72 The incidence of thromboembolism is higher among individuals who, besides factor V Leiden deficiency, also suffer from protein C or S deficiency.

Factor V Cambridge; Factor V Hong-Kong
In addition to factor V Leiden, two other factor V gene mutations, which affect the protein C cleavage site, were described: factor V/R306T (Cambridge) and factor V/R306G (Hong-Kong). Some authors believe there are not enough elements to relate these factors with thrombotic events.73,74

Protein S

Protein S is a glycoprotein synthesized by the liver, megakaryocytes, osteoblasts75 and by the endothelium.66 It is partly (40%) produced freely and partly (60%) produced in the form of an inactive precursor that is activated during platelet activation.75 Since it is vitamin K-dependent, its synthesis can be inhibited by coumarin drugs, which explains the development of microvascular thrombosis and occasional necrosis of the skin in patients with protein C deficiency who take these drugs.

Protein S deficiency may cause thrombotic states.77-80 Protein C and S deficiencies are more frequent than AT III deficiency in families with recurrent venous thromboembolism.76 Some patients may show normal serum levels of total protein S and decrease of the free form only, whose action as cofactor for activated protein C is greater.

Protein S deficiency may be inherited or acquired, as occurs with protein C and antithrombin III. The acquired form is found in infectious states, neoplasms, nephropathies, pregnancy, presence of tumor necrosis factor and other conditions. In the inherited deficiency, thrombosis occurs spontaneously in 50% of the cases and after traumas or infections in the other 50%.70

Antithrombin III (AT III)

Thrombin is the main factor in the coagulation cascade mechanism, and antithrombin is its major physiological inactivator.83,84 Antithrombin III is a serine protease produced by the liver which, in addition to blocking thrombin [IIa], inhibits the action of coagulation factors IXa, Xa, and XIa.

Under normal conditions, the restriction of the coagulation mechanism to the site of endothelial injury is made through different processes (protein C and S), but basically through thrombin modulation and block that occur at the site of vascular injury. Excess thrombin is inhibited by fibrin fixation, by thrombomodulin block and by antithrombin III.

Quantitatively, the first mechanism is the most important, since after clot formation newly-formed fibrin absorbs 80% of the thrombin generated by prothrombin activation.

Thrombomodulin, coupled to the endothelial surface, has double action: first, it binds to thrombin, making it inactive; second, the thrombomodulin/thrombin complex activates protein C which, as described, inhibits certain factors that precede thrombin formation (Va and VIIIa), avoiding the formation of additional thrombin.

The third mechanism consists of coupling AT to thrombin, which is not adsorbed by fibrin at the time of clot retraction, forming a thrombin/antithrombin complex that does not act upon the coagulation mechanism and that is readily removed from circulation by the liver. This coupling process is more intense in the presence of heparin, which explains why AT III is also known as heparin cofactor 1 (AT 1). The AT/heparin complex has a less intense but equally clear action in removing other coagulation factors (IX, X, XI and XII).

AT deficiency determines thrombotic events, both venous and arterial,84-86 especially in young patients.87,88 This thrombophilia seems to occur due to different types of deficiency:89 lower AT concentration and activity; lower activity with normal or elevated AT concentration; acquired decrease in AT concentration and activity.

The first situation is perfectly understandable. In inherited thrombophilia, spontaneous thrombotic events are rare, thrombosis often occurs after the second decade of life and is subsequent to other risk conditions (infection, trauma, etc.).56,89 Likewise, occlusions of arterial graft and AV fistulas for hemodialysis are common, occurrence of "spontaneous" thrombosis,90 rapid progression of atherosclerosis obliterans with early thrombosis,91 etc. A significant clinical data concerns the early development of clots in surgical processes despite the use of heparin, given the fact that heparin may act fully in the presence of AT III. It should be underscored that even low AT levels can act as heparin cofactor.

In the second situation, we take for granted that the liver produces qualitatively abnormal antithrombin III. The decrease in acquired AT III concentration and activity occurs in the course of severe liver diseases, nephrotic syndrome, hypoalbuminemia, cachexia, disseminated vascular coagulation, use of contraceptives, and in large surgeries, etc.56,89 Even titers slightly below normal levels result in increased thrombosis risk.

Prothrombin (Factor II) - G20210A mutation

G20210A mutation in the prothrombin gene may favor the incidence of venous or arterial (coronary, cerebral) thrombosis. In individuals with a normal gene and a mutant gene (heterozygotes), the incidence of the disorder ranges between 1% and 4%. This mutation has not been observed in black or Asian patients.

Quite often, prothrombin mutation is associated with other inherited risk factors (factor V Leiden, protein C and S deficiencies and AT III deficiency) or acquired factors (lupus anticoagulant, pregnancy, puerperium, traumas, immobilization, neoplasms).

The mechanism that determines a higher incidence of thrombosis seems to be the elevation of serum prothrombin levels, thanks to the improved stability of the RNA of the mutant gene.

The laboratory diagnosis of this disorder is made by molecular biology; assessment through serum concentration is not possible.92

Hyperhomocysteinemia

Homocysteine is a sulfhydryl amino acid that plays a central role in the regulation of methionine metabolism and also involves the metabolisms of vitamins B6, B12 and folic acid. Elevated serum levels indicate accumulation of methionine, homocysteine and its dimer (homocystine) in several tissues of the body. Morbid consequences include osteoporosis, mental retardation, lens luxation and other organic disorders, among which are vascular injuries and thrombosis. The reasons for these vascular disorders in hyperhomocysteinemia are still unclear, but we presume that platelet, vascular and coagulation factors are involved and, according to Handin,72 thrombosis caused by the increase in serum homocysteine is much more common in arteries than in veins.

Inherited hyperhomocysteinemia is an inborn error of homocysteine metabolism due to the deficiency of one of two enzymes associated with it, as a result of chromosomal recessive inheritance (cystathionine beta-synthase, CBS, and methylenetetrahydrofolate reductase, MTHFR). The most commonly observed anomaly is mutation C677T of MTHFR genes, present in approximately 5% to 15% of Caucasian and Asian populations, in which there is a high prevalence rate of homozygous individuals. Gene mutations make enzymes thermolabile and, consequently, their activity decreases by 50%.93

Acquired hyperhomocysteinemia is found in physiological conditions (age, sex), different habits (alcoholism, smoking, etc.), vitamin deficiencies (B6, B12, folic acid) and in some renal, thyroid, and atherosclerotic diseases, among others.

The methionine overload test helps in the diagnosis of suspicious cases in which fasting blood levels are normal.

Anticardiolipin antibody/Lupus anticoagulant

Lupus anticoagulant and anticardiolipin antibodies are antiphospholipid antibodies and are associated with recurrent spontaneous abortion, thrombocytopenia and thromboembolic diseases.94 These antibodies may be found in healthy individuals; autoimmune diseases (RSI, rheumatoid arthritis); neural diseases (epilepsy, migraine, multiple sclerosis, Guillan Barré syndrome); use of medication (interferon, phenytoin, chlorpromazine, antibiotics, etc.); viral infections; parasite infections.

The association between antibodies and thrombosis is still obscure. Apparently, abnormal coagulation is caused by the binding of the antibody to a plasma protein (apolipoprotein H or beta-2-glycoprotein).95,96 Although antibodies do not originate from proven genetic disorders, Johansson-Hughes syndrome should be included among intrinsic thrombophilias due to severity of thrombotic events and due to the necessity for intensive and prolonged antithrombotic therapy.

Plasminogen/t-PA and PAI-1

The main function of the fibrinolytic system is to dissolve thrombi by fibrin degradation. This system consists of several components, of which the major ones are plasminogen and its activator [t-PA and u-PA] and inhibitor [PAI-1] enzymes.

Plasminogen
Plasminogen is a beta-globulin synthesized by the liver, aka profibrinolysin. It is found in serum at the concentration of 1-20 mg/100 ml. Plasminogen becomes active when it turns into plasmin at the site of the clot, where plasminogen and t-PA are deposited. No circulating plasmin is present. Endothelial injury determines the activation of factor XII. However, it also acts upon the plasminogen by turning it into plasmin.

Activated plasmin acts on coagulation factors [I, V and VIII], proteins (hemoglobin, casein), fibrinogen and, especially on fibrin. Fibrin degradation produces protein segments called "fibrin degradation products" (FDP). These fragments have different metabolic activities (X, Y, D and E).

Plasminogen deficiencies are observed in approximately 2% or 3 % of the cases of venous thrombosis in young individuals, without any other apparent cause.97 Nevertheless, the relationship between plasminogen deficiency and thrombosis is not yet clarified.98

Activators
Tissue plasminogen activator (t-PA), an enzyme released by endothelial cells, especially of prevenules and venules of extremely vascularized organs (liver, lung, uterus, pancreas, thyroid and prostate), is the major physiological plasminogen activator. The mechanism of action of t-PA involves proteolytic cleavage, which transforms plasminogen into plasmin.

Urokinase (u-PA) is another physiological activator of plasminogen, which is found in the urinary tract.
Several stimuli may increase t-PA secretion, enhancing fibrinolytic activity (thrombus, physical exercise, fever, norepinephrine, nicotinic acid).

Inhibitors
Antiplasmins are substances in the blood that inhibit plasmin and, therefore, reduce its proteolytic action upon the thrombin-fibrin complex.

The major antiplasmin is known as plasminogen activator inhibitor 1 (PAI-1). It acts on the plasmin precursor, modulating the t-PA activity and it is produced by liver cells, platelets and, especially, by endothelial cells. When PAI-1 level is high, fibrinolytic activity decreases, which increases the risk of venous and arterial thrombosis. PAI-1 is an independent risk factor for coronary and cerebral artery disease, as well as for venous thrombosis and osteonecrosis.99

Another inhibitor is the plasminogen activator inhibitor-2 [PAI-2], originally found in the placenta and whose action has not been properly clarified yet.98 Polymorphism [4G/5G] in the PAI-1 gene increases PAI-1 activity and is an independent risk factor for pregnancy complications.100

Alpha-2-macroglobulin is one of the main physiological plasmin inhibitors, forming the plasmin/antiplasmin (PAP) complex. This reaction is extremely fast in a free solution. However, it is slower in circulation due to the viability of specific sites of plasmin binding.

Heparin cofactor II

Heparin cofactor II has high specificity and antithrombin activity in the presence of relatively high heparin levels. In thrombin inactivation, the formation of the thrombin/cofactor II is catalyzed by dermatan sulfate.101 Heparin cofactor II deficiency is found in some patients with recurrent thromboembolism, especially those with family history.66

Platelets

Essential thrombocythemia is a clonal disorder of pluripotent hematopoietic stem cells and, just as secondary overproduction of platelet cells, it is sporadically associated with microthrombosis (erythromelalgia, hemicrania, transient ischemic attack). Nevertheless, thrombocythemia is often asymptomatic or associated with hemorrhagic events. Platelet count and platelet function tests do not precisely predict bleeding or thrombosis tendency. At present, the differential diagnosis of polycythemia vera, overproduction of platelet cells and new thrombophilic factors in intrinsic hypercoagulable states have been under investigation.102

Factor VIII

The serum level of factor VIII is reliant on genetic and environmental factors. It is influenced by the genes that encode the ABO group and von Willebrand factor, and is increased by unknown familial factors, probably genetic ones.62 Although these studies are still preliminary, it has been reported that factor VIII levels are as high as 7% in the general population and as much as 10% and 15% in patients with thrombotic disease. The elevation of serum factor VIII levels implies a fourfold increase in thrombosis risk if compared to patients with normal values.103

Factors IX and XI

Recently, some references to elevated levels of factors IX and XI in patients with thromboembolism have been made.104 These studies are under way.

Factor XIII (Factor XIII Val-34-Leu)

Valine/leucine mutation in amino acid 34 (Val-34-Leu), in factor XIII gene, alters the physiological behavior of this factor in the coagulation cascade in a yet unclear manner, apparently acting as an antithrombotic agent.105,106

Von Willebrand factor (FvW)

Von Willebrand factor acts as a bridge between platelets and the endothelium, actively participating in hemostasis. Alterations to the amount and/or quality of the factor cause von Willebrand disease, the most common bleeding disorder in man. On the other hand, the elevated FvW levels increase the risk of hypercoagulation.71 Presumably, thrombotic thrombocytopenic purpura (TTP) is caused by FvW multimers, which bind to platelet glycoproteins forming thrombi.107,108 Usually, ADAMTS-13 enzymes break FvW multimers into nonaggregating monomers. In patients with familial TTP, ADAMTS-13 activity is quite low,109 which facilitates the adhesion of large FvW molecules to the surface of platelets.

ASSOCIATED FACTORS

The presence of risk factors does not necessarily translate into occurrence of thromboembolic events. Inherited thrombophilic disorders are not always accompanied by thrombosis, even with concomitant presence of important extrinsic thrombophilic factors in the same individual.

However, in epidemiological terms, the association of two or more inherited risk factors increases the incidence of thrombosis.62 The occurrence of an acquired risk factor in congenitally deficient individuals, acts cumulatively and increases the risk for thrombotic disease.62

With the consolidation of recent findings about intrinsic risk factors, in the near future, we will be able to construct total risk tables, adding the values obtained for inherited thrombophilia and acquired thrombophilia in order to assess the thrombosis risk in a certain patient and then initiate active and prophylactic anticoagulant therapy.

LABORATORU INVESTIGATION OF THROMBOPHILIC FACTORS

For investigation of inherited thrombophilia, the Consensus Work Group of SBACV (Brazilian Society of Angiology and Vascular Surgery) suggests the following tests:
- antithrombin III [AT];
- proteins C and S;
- fibrinogen;
- factor VQ 506 [Leiden];
- prothrombin G20210A.

Some of these tests are expensive and cannot be performed in certain clinical laboratories. It is recommendable to start with APCR and antithrombin III, since they are more comprehensive, and if necessary, apply the other tests. Routine investigation could be restricted to individuals in whom the first episode of thrombosis occurs before the age of 45 years, in cases of spontaneous thromboembolism (without extrinsic high-risk factors), in individuals with previous thrombosis, in recurrent thrombosis, in thrombosis at unusual sites (sagittal sinus, mesenteric vessels, portal or splenic vein), in cases of thromboembolism in other family members, in older women who want to get pregnant or undergo hormone replacement therapy, in victims of associated extrinsic risk factors (trauma, orthopedic surgery and prolonged immobilization), in cases of thromboembolism before and after delivery, in postoperative vascular occlusions, in women with recurrent fetal loss, and in cases of abruptio placenta.

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