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Thrombosis

The risk of development of a venous thromboembolism is directly related to three pathologic factors first identified by Virchow in the 19th century, and now known as Virchows Triad:

• Vessel damage
• Blood hypercoagulability
• Stasis.

Over the last 30 years, the mechanisms by which the procoagulants are regulated and excessive clotting inhibited have become increasingly better understood.  The naturally occurring proteins that take part in the system of clot regulation are called the inhibitors of coagulation.  The Fibrinolytic system is the mechanism through which the clot is removed and normal architecture restored.   In patients presenting with thromboembolic disease, the molecular explanation could either be a hyperactive procoagulant pathway, hypoactive anticoagulant pathways, or a defect in the Fibrinolytic system.

Abnormalities in coagulation may be acquired or inherited. Inherited defects in the inhibitors of coagulation lead to a life-long increased risk of thrombotic disease. Defining an inherited abnormality in this system clearly has important implications for any individual's care and life. 

This discussion will include the following:

1. A brief overview of the history and incidence of thromboembolic disease
2. The clinical signs and symptoms of thromboembolic disease
3. The pathophysiology of the naturally occurring inhibitors (including those factors which are part of the Fibrinolytic pathway) and the procoagulants that when deficient or abnormal contribute to changing the delicate balance of hemostasis towards thrombosis
4. The diagnostic evaluation for these patients
5. An overview of the medical therapeutic armamentarium available for therapeutic intervention and prevention.

History
Inherited thrombophilia is defined as a genetically determined tendency for venous thromboembolism that characteristically occurs at a young age, prior to 40 or 45 years, without apparent cause and with a tendency to recur.  Antithrombin(AT) deficiency was first identified in a kindred in 1965 and for many years this was the only known protein regulating excessive clot formation.  In the 1980's the protein C(PC) and S(PS) pathway was described and individuals with deficiencies of these proteins identified.  Together, however, these three proteins accounted for less than 15% of selected cases of juvenile and/or recurrent thrombosis, and less than 10% of unselected cases.  In 1993, it was observed that plasma samples of some members of a kindred with inherited thrombophilia without a deficiency of AT or protein C or S were resistant to the anticoagulant action of activated protein C(APC).  APC resistance has now been shown to be the most common cause of inherited thrombophilia accounting for somewhere between 20-50% of cases.  In 1994, mild hyperhomocysteinemia was found in 19% of jevenile venous thrombosis and family studies revealed that this abnormality was inherited.  Inherited hyperhomocysteinemia may be caused by defects in several genes encoding for enzymes involved in the metabolism of the amino acid.  In 1996 a defect in the prothrombin gene (P20210) that results in an increased expression of prothrombin was identified and linked to an increased of thrombosis.  Severeal other coagulation abnormalities have also been described and linked to an increased risk of thrombosis, specifically increased levels of factors VIII, IX and XI. 

Incidence
Venous thromboembolism is a major medical problem constituting the third most common cardiovascular disease after ischemic cardiac syndrome and stroke.  As such it is the most frequent cause of post-operative death after major surgery.  In adults, thrombosis is a common problem representing approximately 2,000,000 cases per year of deep venous thrombosis (DVT).  Prospective studies suggest that less than 50% of cases are symptomatic.  Between 2.5% and 5% of the population has been estimated to suffer from at least one thrombotic episode during their lives.  Approximately 600,000 people experience a pulmonary embolism (PE) yearly and about 60,000 of these are fatal.  About 70% of all patients with PE have a DVT.  Estimates, believed by some to be conservative, are that there are 100,000 to 200,000 deaths each year from pulmonary emboli that begin as thrombi in the deep veins of the legs.  Estimates of the incidence of and prevalence of PE are less reliable than those for DVT because the diagnosis is difficult and many patients with PE go unrecognized. In a study of Horowitz and Tatter, who analyzed the results of 11,000 autopsies, 316 showed macroscopic PE, but this diagnosis had been made in only 11% ocases prior to death. 

In childhood, there is a decreased rate of thromboembolic complications from either inherited or acquired risk factors that cause thrombotic events in the adult population.  Newborns and infants under one year of age are at the greatest risk for thromboembolic complications in childhood.  Idiopathic venous thrombotic disease occurs in less than 5% odren compared to approximately 40% ots.  Further, the majority of children have several associated risk factors prior to the thrombotic event.  Central venous catheters are the single most important risk factor for thrombotic events in children but account for only a few percent of the adults with venous thrombi.  There are some similarities for the underlying diagnoses that include cancer, trauma/surgery and systemic lupus erythematosus(SLE).  The most commonly identified congenital disorders are APC resistance or a deficiency of PC, PS, or AT.  For the majority of children with an inherited prethrombotic disorder, an acquired risk for thromboembolic complications unmasks the deficiency. 

A Canadian registry of DVT and PE in children aged 1 month to 18 years has been established since 1990, and since that time 137 patients have been reported.  The incidence of DVT and PE was 5.3/10,000 hospitalized admissions or 0.07/10,000 children in Canada.  Infants under 1 year of age and teenagers predominated with equal numbers of both sexes.  DVT were located in the upper (50), lower (70) venous system, or as PE alone (8).  CVL were present in approximately 33% odren with DVT (45).  Associated conditions (cancer, CHD, trauma, parenteral nutrition, infection, nephrotic syndrome, surgery, oral contraceptives, etc.) were present in 96% odren and 90% odren had two or more associated conditions.  22 of the 31 V/Q scans performed were interpreted as high probability for PE.  2.2% ochildren died as a result of their thromboembolic disease.  DVT recurred in 23 children and post-phlebitic syndrome occurred in 26.

Symptoms
The symptoms of thrombosis are related to whether the clot is venous or arterial, totally or partially occlusive and the location of the affected vessel.  Venous thrombi that form in regions of slow to moderate flow are composed of a mixture of red cells, platelets, and fibrin and are known as mixed platelet fibrin thrombi.  Partially occlusive venous thrombosis of the deep veins in the legs or the abdomen may present with subtle symptoms and sometimes not until significant collateral circulation has developed.  Pain, swelling of the affected extremity, and discoloration (bluish or suffused color), are common symptoms.  Physical examination may reveal a positive Homans sign (pain on dorsiflextion of the foot), swelling, pain to palpation, presence of a cord, and evidence of collateral circulation, usually manifested by increased prominence of superficial veins. 

Arterial thromboses tend to present with symptoms such as claudication or those associated with acute arterial occlusion (pain, pallor, paresthesia, pulselessness, and paralysis).  Arterial thrombi are composed predominantly of platelets and fibrin. 

Embolic disease, meaning pulmonary emboli, may also present subtly with the following complaints listed in order of frequency: dyspnea, pleural pain, apprehension, cough, hemoptysis, sweats and syncope.  On physical examination one may find tachypnea, tachycardia, rales, fever, sweating, thrombophlebitis, accentuation of the pulmonary closure sound, gallop heart sounds  and cyanosis.  Proximal vein thrombosis is much more likely to lead to fatal PE than is calf vein thrombosis. The incidence of fatal PE can be markedly reduced if DVT is treated with anticoagulant therapy. 

The clinical features of patients with inherited deficiencies of AT, PC, PS and APC resistance include venous thromboembolism (>90% os) including deep vein thrombosis of the lower limbs (common), pulmonary embolism (common), superficial thrombophlebitis, and mesenteric vein thrombosis (rare but characteristic).  Also, there is often a family history of thrombosis, first thrombosis at a young age (usually less than 40 years of age), frequent recurrence, and rarely neonatal purpura fulminans which is seen with homozygous protein C and S deficiencies.

Pathophysiology
This section is divided into the following areas:

• Abnormal Inhibitors of coagulation including antithrombin, protein C and S, thrombomodulin, and heparin cofactor II
• Abnormal or increased procoagulants including activated protein C resistance, factor VIII, XI, VII and finrinogen
• Abnormalities of fibrinolysis
• Other causes of thrombosis including hyperhomocysteinemia, pregnancy, inflammatory disorders, myeloproliferative disorders, post-surgery, nephrotic syndrome, estrogens and OCPs, malignancy and PNH
• Acquired syndromes leading to prothrombotic states including lupus anticoagulants and antiphospholipid antibody syndrome

Abnormal Inhibitors Of Coagulation
Antithrombin:

• Single chain glycoprotein 432 amino acids with 4 oligosaccharide side chains
• Molecular weight ~ 60,000
• Plasma concentration 125 ug/ml
• Half-life ~ 65 hours. 
• Synthesized in liver 
• Gene located at chromosome 1q23-25 and spans 16 kilobases. 

AT belongs to the family of serpins, which are serine protease inhibitors.  AT inactivates thrombin, Xa, IXa, XIa, and XIIa.  Heparinoids are proteoglycans, and are the naturally occurring anti-coagulants found on the endothelial cell surface and in numerous tissues including lungs and intestines.  The inhibition of serine proteases by AT is accelerated by heparin.  Heparin promotes a tertiary complex with AT and its serine protease bringing the active site of AT in closer proximity to its serine protease greatly decreasing the time for inhibition of thrombin from approximately 1 minute to milliseconds. 

The frequency of symptomatic AT deficiency is estimated at 1:2,000 to 1:5,000.  Asymptomatic deficiency may occur as frequently as 1:600.  In patients with a history of thrombotic disease, the incidence of AT deficiency ranges from 0.5% to 4.9%. AT deficiency is inherited as an autosomal dominant trait.  Homozygous deficient patients have been described very rarely and only in those individuals with defects in the heparin-binding site.  These individuals have a severe thrombotic tendency that presents early in life, often including arterial thrombotic disease.  AT deficiency is sub-categorized into Type I and II.  Type I deficient patients demonstrate a decrease in both the antigenic and activity level of the protein, which most often is due to an unexpressed protein from the mutant allele.  Type II deficiency is characterized by a decrease in the activity of the protein with a normal antigenic level, indicating a dysproteinemic state where the mutant allele produces an abnormally functioning protein.  Type II is further sub classified into two subtypes; those with abnormalities affecting the unfractionated heparin binding site and those that reduce the neutralizing capacity of antithrombin for thrombin activity in the absence of UFH.  Homozygous Type I deficiencies have been reported once in infants dying within the first few weeks of life leading to the hypothesis that the homozygous deficiency state is a lethal event. 

AT Levels
In affected heterozygous the levels of AT range between 40% a omal.    Levels of AT are gestationally dependent with normal adult levels achieved at 6 months of age.  AT levels are also decreased in liver dysfunction, consumptive coagulopathy, obstetric complications, the newborn, pregnancy, OCP, renal disease, malignancies, malnutrition and GI loss and drugs.  Levels may increase while on coumarins.  The molecular defects leading to AT deficient states have been well described, and include major deletions (less common) and the more common single nucleotide changes, and short insertions or deletions.

PC, PS , Thrombomodulin Protein C

• Vitamin K dependent glycoprotein
• Molecular weight ~ 62,000, 23% of which is carbohydrate
• Plasma concentration of 48 - 80 nmol/l
• Half-life of 6 to 8 hours
• PC gene localized at chromosome 2q13-q14 & spans over 11 kilo bases & comprises 9 exons 
• PC is synthesized in the liver, as a 461 amino acid single chain polypeptide composed of a leader sequence, a propeptide recognition site for the vitamin K dependent gamma carboxylase, a light and a heavy chain.  Functional PC is a double chain resulting from intracellular proteolytic cleavage and removal of the prepropeptide and cleavage of the single chain. 

PC is slowly activated by thrombin to APC.  The activation is increased 20,000 fold when PC complexes with thrombin bound to an endothelial receptor, thrombomodulin.  APC inactivates membrane bound factor Va and VIIIa.

PC Levels
Levels of this protein are gestationally dependent and do not reach adult levels until late adolescence.  Protein C levels are also decreased in liver disease, DIC, thrombosis, OCP, oral anticoagulants, childhood, (?RSD)RDS, preeclampsia, acquired purpura fulminans, SLE and Ulcerative colitis.

Protein S
Vitamin K dependent single chain glycoprotein

• Molecular weight ~ 69,000, ~ 7% cdrate residues. 
• Plasma concentration 20 to 25 ug/ml 
• Half life ~ 42 hours 
• Two homologous genes for PS localized at chromosome 3.  The active gene is located on the region 3p11.1-3q11.2 & spans over 80 kilo bases & comprises 15 exons. 
• Synthesized by liver, also localized to endothelial cells, megakaryocytes, & Leydig cells
• Synthesized as a 676 amino acid chain with a leader sequence which is cleaved before secretion

Protein S acts as the principle cofactor to protein C. PS exists in the circulation in two forms, in equilibrium with each other, a free form and a non-covalently bound form complexed to C4 binding protein, a component of the complement pathway.  About 60-65% ototal PS exists in the circulation in the bound form with approximately 35-40% ifree form.  Free PS is the only form involved in APC anticoagulant activity. 

PS Levels
The levels of PS are gestationally related with adult levels achieved at approximately 6 months to a year.  Physiologic variations include a lower mean free PS level in normal females than males, lower free PS in pregnancy and women on oral contraceptives, and lower free and total PS in newborn infants.  Levels in heterozygotes are approximately 40-70% of the normal level.

Defects of the PC, and PS pathway lead to a thrombotic tendency.  The deficient states are transmitted as an autosomal dominant trait.  The frequency of PC deficiency ranges from 1.4% to 8.6% in patients studies.  In the general population the frequency of the deficiency extrapolated from data obtained from cohorts of patients is approximately 1:16,000 to 1:36,000.  However, in a study of healthy subjects the frequency of the deficiency was found to be 1:200 to 1:300.  A study of almost 10,000 blood donors confirmed this increased frequency of 1:500 to 1:700.  Abnormal assays of PC in this study were corroborated by genetic analysis.  PC deficiency is subtyped into two categories.  Type I deficiency PC activity and antigen are decreased to the same extent, whereas in type II deficiency there is a decreased activity with a normal antigenic analysis. 

The frequency of PS deficiency is roughly the same as PC deficiency with a range of 1.4% to 7.5%. Relatively few mutations of the PS gene have been identified, due to the technical difficulties related to the size of the gene and the presence of a pseudo gene.  The majority of defects described to date are point mutations.  There is no data on the frequency of PS deficiency in the general population.  Extrapolation from cohorts of patients with thrombotic disease gives a frequency of 1:33,000, which based upon the above discussion with PC is most likely an underestimate.  There are three subtypes of PS deficiency.  Type I has a decreased in the total and free PS.  Type II deficiency has normal levels of both free and total protein S, but abnormal functional activity is present.  Type III deficiency has normal levels of total PS, but the bound fraction is greater than the free.

Homozygous deficiency of PC and PS has been described and usually present in the neonatal period with purpura fulminans that will lead to severe morbidity if not death unless identified and treated.  The purpura fulminans in these patients results from microvascular thrombosis with cutaneous and subcutaneous ischemic necrosis.  Homozygous deficiencies with very low levels of PC, ranging from 5% to 20%, also been identified and may not be associated with purpura fulminans, but can be associated with a severe thrombotic tendency at an early age.  The parents of homozygous deficient patients are both heterozygotes, but often and oddly do not have a personal or family history of thrombosis.  The frequency of thrombosis is 5% irozygous in kindred's of homozygous patients, whereas the frequency is approximately 50% irozygotes in kindred's without homozygous individuals.  The frequency of homozygous deficiency has been estimated to be 1:160,000 to 1:360,000. 

Thrombomodulin is the endothelial receptor for thrombin which when bound activates protein C.  Thrombomodulin soluble fragments can be measured in plasma and are present in healthy subjects at a level of 2.2 to 4.8 ng/ml.  Increased levels of these fragments are seen in patients with DVT, pulmonary embolism, arterial thrombosis, cerebral infarction, retinal thrombosis, and DIC.  A patient with a documented history of thromboembolic disease has been identified in which baselines levels of TM soluble fragments were decreased, and a point mutation was identified in the thrombomodulin gene.

Heparin cofactor II
HCII is a serpin, closely related to AT that rapidly inactivates thrombin in the presence of dermatan sulfate.  Only a few cases of HCII deficiency have been described.

TFPI Deficiency
Tissue Factor Pathway Inhibitor (TFPI)
TFPI is a kunitz-type inhibitor that regulates the initiation of coagulation.  TFPI inhibits the complex of tissue factor:Factor VIIa:Factor Xa that starts the coagulation cascade.  The majority of TFPI (60-80%) is bound to the vascular endothelium, with only 20% floating freely in the blood.  Recent evidence suggests that low levels of TFPI are a risk factor for VTE. (Dahm et al. Blood. 2003:4387-92).  Interestingly, polymorphisms have been found in the TFPI gene that result in elevated levels of TFPI in the circulation.  One report suggests that these elevated levels “correct the balance” in patients with Factor V Leiden, and normalize their risk for a thrombotic event. (Ameziane et al, Thromb Haemost. 2002;88: 195-199)

Abnormal Or Increased Procoagulants
APC resistance:
First described by Dr. Dahlback in 1993 as a novel mechanism for thrombophilia.  The APC resistance phenotype was discovered in a middle-aged man with recurrent deep venous thrombosis.  It was observed that he addition of exogenous APC to the patient's plasma did not result in the expected prolongation of the clotting time.  In this family, APC resistance was found to be inherited as an autosomal dominant trait and to cosegregate with thrombosis that suggested a new genetic cause of inherited thrombophilia.  It was also observed that a crude protein fraction of normal plasma corrected the APC resistance of the patient’s plasma, whereas the corresponding fraction of another patient's plasma with APC resistance did not correct the abnormality.  The protein correcting the defect was purified and found to be factor V, suggesting APC resistance to be caused by a molecular defect in factor V.  A Dutch group then determined the point mutation responsible in the factor V gene responsible for the APC resistance. 

The factor V gene is located on chromosome 1q21-25, contains 25 exons and spans over 80 kilo bases.  APC resistant individuals were originally screened for mutations affecting the factor Va cleavage site (Arg 506-Gly 507), or the APC binding region of factor V and were found to have a single point mutation in the factor V gene (Arg 1865-Ile 1874) leading to a substitution of Arg 506 by Gln and causing resistance to APC inactivation.  This defect has been identified in a further 80%-100% of patients investigated with similar APC resistance.  APC resistance in individuals lacking this point mutation may be secondary to an as yet unidentified abnormality in either factor V or VIII.

The frequency of APC resistance ranges in studies from 10%-64% with an average frequency of 17%.  The high frequency of APC resistance in patients with venous thromboembolism mirrors the high rate of carrier status within populations of European descent, estimated to be from 2-10%.  This mutation is so frequent as to suggest that it may have conferred some evolutionary advantage.  The mutation is seen in lower frequency in individuals of African and Asian descent. 

As the APC resistance gene is a frequent finding in some populations, one would predict that individuals would be identified with double deficiencies.  This has been clearly documented in kindred's who also show separate segregation for defects in both the PC and PS gene.  Individuals who are affected with double deficiency states are at increased risk of thrombosis compared to their singularly affected family members and warrant special consideration.  It is therefore reasonable to perform a complete evaluation on patients who present with their first or recurrent episode of thromboembolic disease, especially those who present in childhood.  APC resistance leads to a dramatically increased risk of thrombosis associated with pregnancy or estrogen containing oral contraceptives.  Because of the high frequency of APC resistance in the general population, homozygous deficient patients may be predicted in a frequency of 1:5,000. 

The overall  thrombotic risk for homozygotes is

• estimated to be 4-11-fold higher than for heterozygotes, and
• 80 fold higher than for normal individuals. 

For homozygotes, the probability of a thrombotic episode before the age of 33 years is twice that for heterozygotes (40% versus 20%). Whether homozygotes have an increased risk of arterial thrombosis remains to be further investigated.

Other mutations leading to APC resistance and abnormal APC resistance due to other factors
Several other alterations in the Factor V gene have demonstrated APC resistance as well.  The Factor V Cambridge mutation (Williamson et al. Blood.  1998; 91:1140-44) and the Factor V Hong Kong mutation (Chan et al. Blood.  1998; 91:1135-39) have both demonstrated mild APC resistance.  The HR2 haplotype of Factor V has several polymorphisms that also contribute to APC resistance (Bernardi et al. Blood.  1997; 90: 1552-7).  While these genetic alterations in the Factor V gene due contribute to APC resistance, they lead to much less resistance then the Factor V Leiden mutation.   Acquired APC resistance may contribute to the hypercoagulable states of pregnancy, estrogen therapy, and anti-phospholipid antibody syndrome.

Prothrombin 20210
A nucleotide change (G π A) at position 20210 was identified in the sequence of 3’-UT region by Poort in 1996 and found to be present in the normal control population 1% versus 18% of patients with venous thromboembolism.  In a population based study this allele was found to have a prevalence of 1.2% and increases the risk of thrombosis almost three-fold.  This allele is associated with elevated levels of prothrombin usually > 1.15 U/ml, a 25% increase compared to the normal range.  This was the first strong evidence of a quantitative trait locus mutation in the prothrombin gene that influenced prothrombin activity levels and susceptibility to thrombosis.  Other associated thrombotic episodes linked to this mutation include:

• Coronary artery disease especially in young women
• Stroke
• Venous thrombosis
• Mesenetric vein thrombosis
• Central retinal arterial thrombosis
• Portal vein thrombosis

Elevated factor VIII
Factor VIII is present on the X chromosome and is a known acute phase reactant.  It is often difficult to determine whether an elevated factor VIII level is due to acute event or precedes it.  Elevated levels of factor VIII as defined as > 1.5 IU/ml or 150%, represent a constitutional and independent risk factor for venous thromboembolism.  An odds ratio of 4.8 was determined for the first DVT with levels >150% compared to those with a level <100% in the Leiden thrombophilia study.  (Koster T, Blann AD, Briet E. Lancet 1995; 345:152-5)  Another study (Kraaijenhagen RA. Throm Haemost 2000:83:5-9) showed that levels >175% may be found in 25% of unselected patients with symptomatic venous thromboembolism and is a dose dependent risk factor with each increment of 10 IU/ml increasing the risk of VTE by 10%, and for recurrent disease this figure is 24% (i.e. elevated factor VIII is an even stronger risk factor for recurrent VTE).  The likelihood of recurrence of a thrombotic event at 2 years was found in another study to be 37% as compared with 5% among patients with a lowered factor VIII level.  (Kyrle PA NEJM. 2000 Aug 17;343(7):457-62.)     

Elevated factor IX
Factor IX is also located on the X chromosome, and with its cofactor, factor VIII, activates factor X.  Elevated levels of factor IX may also play a role in venous thromboembolism.  The Leiden thrombophilia study also found that levels of factor IX in the 90th percentile and higher increased by 2-3 fold the risk of VTE.(Van Hylckama Vlleg A, van der Linden IK, Bertina RM, Rosendaal FR.  Blood.  2000;95:3678-82).

Elevated factor XI
The role of factor XI in hemostasis is a combination of procoagulant action in the contribution to the formation of fibrin, and an antifibrinloytic action in its protection of fibrin.  Through feedback mechanisms (secondary thrombin generation), thrombin levels increase which are not only necessary for the formation of fibrin, but also to prevent its dissolution.  Theexcess thrombin  activates thrombin-activatable fibrinolysis inhibitor (TAFI, also called procarboxypeptidase B or procarboxypeptidase U), which once activated inhibits fibrinolysis.  TAFI removes the C-terminal lysine residues from fibrin, which are essential for the binding and activation of plasminogen, and subsequent fibrinolysis. 

Elevated factor XI has been associated with an age and sex adjusted increased odds ratio of 2.2 for development of DVT when the level is greater than the 90th percentile (> 120%). There also appears to be a dose response relationship between the factor XI level and risk of thrombosis. (Meijers J NEJM 342(10) March 9, 2000.696-701)   Elevated levels of factor XI have also been associated with an increased risk of cardiovascular disease in women.  (Berliner JI. Thromb Res 2002 Jul 15;107(1-2):55-60)

Elevated factor VII
Significant associations of FVII with increased coronary risk have been documented but not as an independent risk factor after controlling for cholesterol, LDL-cholesterol, and triglycerides. (Junker R et al. Arterioscler Thromb Vasc Biol. 1997 Aug;17(8):1539-44.)  Elevated fVIIa levels have been documented in retuinal vein occlusion. (Kadayifcilar S et al. Br J Ophthalmol. 2001 Oct;85(10):1174-8.)

Elevated von Willebrand factor
Von Willeband factor is produced in endothelial cells and as such those events leading to endothelial damage or inflammation lead to increased vWF levels.  FVIII circulates in conjunction with vWF and often the levels of these two clotting factors are concordant when stress, inflammatory states or endothelial injury occurs.  Sustained elevations in factor VIII levels lead to an increased risk of VTE; therefore it might be reasonable to assume that elevated vWF levels would also be associated with and contribute to increased risk of VTE.  Additionally, vWF, in particular the high molecular weight mulitmers, plays an integral role in platelet adherence to areas of damaged endothelium and elevated vWF levels may have more than one pathogenic mechanism by which they contribute to VTE.

Fibrinogen
Fibrinogen has a molecular weight of 340,000 and is coded for on chromosome 4 bands q23 to 32 spanning a 50 kilo base region.  Fibrinogen is a dimeric molecule consisting of three pairs of disulfide bonded chains with a total of approximately 1482 amino acids.  Fibrinogen is produced in the liver with the capacity for a 20 fold increase in production when required.  The half life of Fibrinogen is 3 to 5 days.  An additional pool of Fibrinogen exists in platelets and is produced by megakaryocytes. 

Fibrinogen binds thrombin and various proteins involved in fibrinolysis.  Abnormalities at binding sites or at sites at which plasmin degrades fibrin may lead to decreased inactivation of thrombin or to impaired fibrinolysis, and a tendency to thrombosis.  The primary manifestations of thrombophilia associated with dysfibrinogenemia are deep vein thrombosis and PE.  Arterial thrombosis in these patients have been reported.  It is inherited as an autosomal dominant trait, homozygous states have not been reported.  Abnormalities of binding of plasminogen or tPA to fibrin or lysis of fibrin by plasmin are not detected by routine screening tests of coagulation.  It is probable then that dysfibrinogenemias associated with thrombosis are under diagnosed.  Fibrinogen levels are at adult levels at birth and may increase in concentration as an acute phase reactant in response to a variety of stresses such as trauma, pregnancy, tissue inflammation, etc. 
Elevated levels of fibrinogen are a risk factor for atherothrombotic events. (Folsom AR. Thromb Haemost 2001 Jul 86(1):336-73) but perhaps not for venous thromboembolism (Tsai AW. Am J Med 2002 Dec 1;113(8): 636-42)

Platelet gylcoprotein receptor abnormalities
Abnormalities Of Fibrinolysis Plasminogen

• Single chain glycoprotein consists of 791 amino acids with 24 disulfide bonds
• Molecular weight ~ 90,000
• Plasma concentration ~ 200 mg/L
• Half-life ~ 2.2 days
• Synthesized in liver, but also present in other cells & found in extravascular space of most tissues, some of which may be capable of synthesizing it such as eosinophils and kidney. 
• Gene located on 6q26-q27 & spans 52.5 kilo bases.

Plasminogen is converted to a proteolytic enzyme, plasmin by plasminogen activators tissue-plasminogen activator (tPA) and urokinase-plasminogen activator(uPA).  The main action of plasmin is the degradation of fibrin through a series of proteolytic cleavages.  Defective fibrinolysis has been known to be associated with thrombovascular disease.  There are two types of plasminogen deficiency, Type I, in which antigenic and activity levels of plasminogen are reduced (hypoplasminogenemia), and Type II deficiency where in which there exist normal antigenic levels in the face of decreased activity assays (dysplasminogenemia).  Familial plasminogen deficiency appears to be an uncommon but recognized cause of inherited thrombophilia.  The thrombovascular complications of this deficiency are predominantly venous and include thrombophlebitis, PE, and stroke.  It is interesting to note that in affected individuals, there have not been reports of thrombotic disease in association with pregnancy or oral contraceptive use, and even more intriguing is the report of normalization of plasminogen levels in a deficient patient during pregnancy indicating that in heterozygotes the normal allele is able to respond to factors that stimulate plasminogen synthesis.    Higher levels of plasminogen levels are found in women in the last trimester of pregnancy, and approximately one-half the adult levels are found in newborns.  The normal adult level is not attained until late adolescence. 

Homozygous deficiency has been identified and is associated with ligneous conjunctivitis but not thrombosis.  This deficiency is associated with a variety of systemic manifestations including conjunctival lesions, ear, sinus, pulmonary, oral and GU.  Removal of the lesions is not curative and may exacerbate recurrence.  The lesions are responsive to systemic replacement of plasminogen or to local therapy such as Eminase (plasminogen and streptokinase) in the eyes.  The incidence of this disorder is not well established and may be underestimated as Ophthalmologists, Dentists, and OB/GYN and ENT physicians may see these patients and not refer or recognize the local manifestation to be plasminogen deficiency.  The fact that homozygous patients infrequently develop thromboses, especially spontaneous events, makes the diagnosis of heterozygous plasminogen deficiency as a cause for thrombotic events to be suspect.

Decreased TPA
TPA is synthesized by endothelial cells and when released converts plasminogen to plasmin.  Theoretically, decreased release of tPA could lead to a hypercoaguable state due to decreased fibrinolysis. 

Increased PAI-1
PAI-1 functions as the primary inhibitor of plasminogen activator in plasma.  Increased levels of PAI-1 could lead to excessive inhibition of tPA leading to a decrease in activation of fibrinolysis and a thrombotic tendency.  Increased PAI-1 levels have been shown in some kindred's to be an inherited trait.  In mice engineered to produce increased levels of PAI-1 newborns have developed thrombosis in peripheral vessels resulting in ischemic necrosis of the tails and feet.  Well controlled data clearly linking increased PAI-1 levels to a thrombotic tendency remain to be elucidated.

Elevated TAFI
TAFI as stated above helps inhibit fibrinolysis by preventing plasminogen from binding to the fibrin clot.  Elevated levels of TAFI would prevent the onset of normal fibrinolysis and therefore should theoretically predispose to a prothrombotic state.  One report from the Leiden Thrombophilia Study does suggest that high levels of TAFI may be a mild risk factor for a hypercoagulable state.  (Tilburg, Rosendaal, Bertina.  Blood.  2000;95:2855-59).  However, these results need to be confirmed.

Other Causes Of Thrombosis

Hyperhomocysteinemia
Homocysteine is a sulfydryl amino acid derived from metabolic conversion of methionine.  It is intracellularly metabolized through remethylation to methionine or transulfuration to cysteine.  There are two remethylation pathways.  In that catabolized by methionine synthetase, the methyl group is donated by methyltetrahydrofolate (MTHFR) with cobalamin as a cofactor.  In the other pathway, betaine is the methyl donor and the reaction is catalyzed by betaine-homocysteine methyltransferase.  In the transulfuration pathway, Homocysteine is transformed by cystathionine-beta-synthase into cystathionine, with pyridoxine acting as a cofactor.  Homocysteine is oxidized in plasma to the disulfides homocysteine-homocysteine and the mixed disulfide homocysteine-cysteine.  Homocysteine and the two disulfides exist both in the free and protein bound forms and are referred to globally as total homocysteine, which has a concentration range in normal plasma of 5 to 16 umol/L. 
 
Legend
Homocysteine’s intracellular metabolism occurs through remethylation to methionine or transulfation to cysteine.  Numbered circles indicate the principle enzymes involved:

• methionine synthase
• 5, 10-methylenetetrahydrofolate reductase
• betaine-homocysteine methyltransferase
• cystathionine-beta-synthase

De Stefano V et al. Blood  Vol 87(9)1996:3531-3544. Pg 3534.

Several inherited or acquired conditions can cause an increase in homocysteine levels that can be severe (>100 umol/L), moderate (25-100 umol/L) and mild (16 to 24 umol/L).  The homozygous inherited deficiency of cystathionine-beta-synthase (a frequency in the population of about 1:200,000 to 1:335,000) is the most frequent cause of severely elevated levels of homocysteine.  Arterial vascular disease and venous thromboembolism are included the clinical stigmata of this disorder.  A smaller number of cases, 5% t associated with inherited defects of the remethylation pathway.  Homozygous deficiency of the wild form of methylenetetrahydrofolate reductase is the most common cause of these defects that also is characterized by premature vascular disease and thromboembolism. 

Mild or moderate elevations in homocysteine can be found in individuals with gene defects and in some individuals with acquired diseases.  In the general population, heterozygous cystathionine-beta-synthase deficiency has a high frequency of 0.3 to 1.4%.  A common defect of the remethylation pathway (thermolabile mutant of methylenetetrahydrofolate reductase) has about 50% of the normal enzyme activity and a prevalence in the general population of 5% in the homozygous state.  The most common causes of acquired hyperhomocysteinemia are nutritional deficiencies of cobalamin, folate, or pyridoxine, the essential cofactors for homocysteine metabolism.  Elevated levels of homocysteine are common in the elderly population.  Chronic renal insufficiency and compounds that interfere with the metabolism of folate (e.g. methotrexate and anticonvulsants) or that of cobalamin, such as nitrous oxide, may also cause mild to moderate elevations of homocysteine.

Mild to moderate hyperhomocysteinemia is an independent risk factor for stroke, myocardial infarction, peripheral arterial disease, and extracranial carotid artery stenosis.  High plasma levels are associated with enzymatic defects or deficiencies of folate or vitamin B6, particularly in the elderly.  Mild or moderate hyperhomocysteinemia has been associated with venous thrombosis in the young and recurrent venous thrombosis and has been shown to have a high frequency (10%) ients with first episodes of venous thrombosis.  In family studies of these individuals, there have been at least one first-degree relative with hyperhomocysteinemia.  These data suggest the inherited nature of this disorder.  The clinical manifestations of venous thromboembolic disease in patients with hyperhomocysteinemia do not differ from those of other deficiency sates in thrombophilic patients.  These manifestations have included DVT with or without PE in 64% of cases, superficial thrombophlebitis (24%), thrombosis of cerebral or mesenteric veins (12%), and are often associated with other risk factors such as oral contraception, trauma/surgery, pregnancy, the puerperium, and immobilization. 

Pregnancy
The most common cause of maternal death in pregnancy is from thromboembolism, with an incidence of 1-5/1,000 pregnancies including the post-partum period.  Women with a previous history of a DVT the risk with pregnancy is 12-35% and increases to 75% in women with prothrombotic disorders such as antithrombin deficiency.  Etiologies include decreased levels of total and free protein S, antithrombin and venous pressure and stasis.  Also increased levels of factors VGII, VIII, von Willebrand factor and fibrinogen are seen and may contribute to these events.

Inflammatory disorders
Disorders such as inflammatory bowel disease, infections increase inflammatory mediators, monocyte procoagulants and C4 binding protein hence decreasing free protein S and may all contribute to a prothrombotic state.  Large and/or unusual site thrombotic episodes have been reported in inflammatory bowel disease including large vessel thrombosis of the IVC and carotid and mesenteric and portal veins.
 
Myeloproliferative disorders
A leading cause of death in myeloproliferative disorders is thromboembolism.  This may result from abnormal platelets and increased viscosity.  In fact, thrombotic episodes may occur prior to the diagnosis of the myeloproliferative syndrome.

Post-surgery
Surgical interventions and sometimes the conditions requiring them may lead to an increase of inflammatory mediators, venous stasis and a suppression of fibrinolytic activity contributing to a prothrombotic state.

Nephrotic syndrome
Nephrosis leads to an increase in inflammatory mediators and a decrease of free protein S due to an increase of C4 binding protein. Also urinary loss of antithrombin, protein C and S are increased.  Thrombocytosis and increased platelet aggregability may also be linked and contribute to thromboembolism in nephrosis.  

Estrogens and BCPs
Estrogens and BCPs hormonally may mimic the pregnant state and lead to coagulation changes known to be associated with pregnancy and therefore be associated with an increased risk of thrmoembolism especially in individuals with abnormalities of coagulation such as factor V Leiden.

Malignancy
Malignant cells may produce procogulant substances (i.e. tumor tissue factor) that lead to an increased risk of thrombnoembolism.  Malignancies may also lead to venous obstruction and an increase in inflammatory mediators also contributing to a thrombotic potential.

Paroxysmal nocturnal hemoglobinuria
PNH is known to be associated with an increased risk of thrombosis.  PNH is am acquired disorder of hematopoetic stem cells and is caused y a mutation in a somatic cell of the phophatidylinositol glycan class A (PIG A) gene which is located on the X chromosome.  This disorder usually presents in adulthood and less commonly in childhood.  In adults the most common presentation is a hemolytic anemia with nocturnal exacerbations while in children bone marrow failure is the most common presentation.  Thrombosis may occur in 39% of adults and 31% of children with PNH.  Venous thrombosis predominates with a predilection towards the hepatic veins (Budd-Chiari syndrome), but also include the portal veins, CNS, and peripheral venous system.  Increased circulating activated platelets have been implicated in thrombosis but no consistent fibrinolytic or coagulation abnormality has been documented.  The PIG A gene is associated with the synthesis of the glycosyl phosphatidylinositol anchor to which many cell surface proteins attached. 

Lipoprotein a
Lp(a) Is a large lipoprotein similar to LDL.  LP(a) is made up of two proteins, apoB-100 anda glycoportein apo(a) attached to apoB-100 through a disulfide bond.  The structure of this polypeptide is similar to plasminogen.  Increased levels of Lp(a) are inherited and expressed during childhood, and are a risk factor for CAD in early adult life, similar to heterozygous familial hypercholestrolemia. There is a suggestion that elevated levels of Lp(a) may contribute to venous thromboembolism, especially in patients with Factor V Leiden. (von Depka et al.  Blood.  2000; 96: 3364-68), but this correlation needs further evaluation.

Acquired Syndromes Leading To A Pro-Thrombotic State:

Lupus anticoagulants / antiphospholipid syndromes

History
Antiphospholipid-protein syndrome (APS) is a syndrome that is comprised of a variety of clinical and laboratory abnormalities.  Patients with APS have laboratory abnormalities in either coagulation assays or various solid phase ELISAs or radioimmunoassays.  These assay system originally were thought to detect anti-phospholipid antibodies.  The first of these antiphospholipid antibodies was reagin first described by Wasserman in 1906 when they developed a complement fixation test to detect syphilis.  Following the initial description of serologic tests for syphilis (STS), additional assays were developed including the VDRL, which used alcohol extracts from bovine heart as the source of antigen, also named cardiolipin.  Cardiolipin is a major component of the VDRL reagent.  Routine STS performed on Army recruits found STS positive individuals without evidence of underlying syphilis (false positives).  These false positives were seen in a number of settings including recent infections, following vaccinations, in association with underlying SLE and in cases without an underlying cause or explanation. 

The second member of the APA family described by Conley and Hartmen in 1952 when they identified a circulating anticoagulant (inhibitor) in patients with SLE.  Although originally thought to be associated with bleeding, this anticoagulant was later paradoxically found to be associated with thrombosis.  Because of the frequency of the association between this anticoagulant and SLE, it was called a lupus anticoagulant by Feinstein and Rappaport.  LA is a misnomer, as the vast majority of patients identified with this anticoagulant do not have SLE.  LA may be a transient phenomenon particularly in the setting of a recent infection.  LA are immunoglobulins (either IgG, IgM, IgA, or mixtures) which interfere with one or more in vitro phospholipid dependent tests of coagulation (APTT, KCT, dRVVT, etc).  The third member of the APA family is the anticadiolipin antibody (ACA). Harris and coworkers developed a more sensitive assay for ACA compared to the standard STS. Subsequent studies clearly have demonstrated differences between LA and ACA.

The concept of the APS was initiated in papers in 1983 which described an association between thrombosis, both arterial and venous, recurrent spontaneous abortion (RSA), thrombocytopenia, and various neurologic manifestations together with positive laboratory tests for LA and/or ACA. Harris described criteria for diagnosis in 1987. Patients with APS should have at least one clinical and one laboratory finding during their disease process. The APA tests should be positive on at least two occasions greater than 8 weeks apart. In many individuals this diagnosis may occur in the setting of underlying SLE, and in other patients without an underlying systemic disease. This latter group of patients are referred to as having primary antiphospholipid-protein syndrome (PAPS). Some patients with PAPS may progress to SLE. APS may also be induced by drugs or an underlying malignancy.

The association between thrombosis and APA was first reported by Bowie in 1963. In their original patients, who had SLE, the dermatologic finding of livido reticularis was also described. In patients with LA, 70% of thrombotic events are venous, and the remaining arterial. The most common site for arterial thromboembolic events is the cerebral circulation. Ischemic episodes may be transient to permanent and are often recurrent. Positive APA are found in 18-46% of unselected young patients, and in older patients the range is 10 to 18%.

Thrombocytopenia, which can commonly be seen as part of this syndrome, is felt to be immune mediated. The incidence of thrombocytopenia in patients with APS has ranged from 20% to 46%. In patients with secondary APS (those with a demonstrable underlying disease (SLE, systemic sclerosis, etc) there is a more frequent occurrence of autoimmune cytopenias. The incidence of thrombocytopenia, thromboembolic events, RSA are similar in both primary and secondary APS patients.

APA have a variety of pathophysiologic effects through which a prothrombotic state is created. APA inhibit mobilization of arachidonic acid from endothelial cell membranes mediated by inhibition of phospholipase A2, and therefore inhibits prostacyclin production, a potent vasodilator and inhibitor of platelet aggregation. Sera of patients with LA also induce expression of Thrombomodulin. This endothelial cell receptor for thrombin, when complexed with thrombin activates protein C. It may be up or down regulated by a variety of stimuli. Inhibition of protein C activation leads to a prothrombotic state. Heparin sulfate is found on the surface of endothelial cells and acts as an endogenous anticoagulant through its interaction with AT. Sera of patients with LA also interact with a disaccharide sequence found in the critical AT binding region of heparin and Heparin sulfate. APA may also alter the fibrinolytic system by decreasing tPA activity, increased production of PAI-1, but these data are poorly supported to date.

Treatment of patients with transiently positive ACA or LA is unecessary. Treatment should be reserved for patients who have clinical evidence of the APS syndrome. Patients who are persistently positive with either LA or ACA and have a thrombotic history appear to be at increased risk for recurrent (approximately 50% over a five year period) thrombosis. Thus, if an individual initially experiences a venous thrombosis, the subsequent recurrence typically is venous. The intensity oral anticoagulation (INR 2 to 3) required to suppress venous recurrences is still controversial. The existing consensus is that patients with APA should be treated more aggressively with oral anticoagulants (high intensity) with an INR of 2.5 to 3.5. However, a recent report indicates that standard therapy(goal INR 2.0-3.0) may be adequate therapy. (Crowther et al. NEJM. 2003; 349:1133-8).

Diagnostic Evaluation

Venous thrombosis has usually been detected by three types of screening tests.

Tests using radio-labels

• Radioactive fibrinogen leg scanning
  • This test is moderately sensitive and specific for calf and popliteal vein thrombosis, but less sensitive to superficial femoral or ileac vein thrombosis.
• The Acutect Venogram
  • The Acutect is a synthetic peptide radiopharmaceutial that binds preferentially to the glycoprotein (GP IIa/IIIb receptor) found in activated platelets. The AcuTect venogram is indicated for acute venous thrombosis in the lower extremities of patients with signs and symptoms of acute venous thrombosis. AcuTect appears to detect acute and not chronic venous thrombosis.

Tests using Ultrasound

• Doppler-augmented ultrasound and impedance plethysmography (IPG)
  • Insensitive to calf vein thrombosis and non-occlusive proximal vein thrombi, although sensitive to occlusive proximal thrombi
• Doppler ultrasonography
  • Most widely used and available noninvasive test for DVT often used with B mode imaging of the lower extremities.
  • Has become the dominant test since the 1980's and has largely replaced impedance plethysmography for noninvasive testing.
  • The positive predictive value of impedance plethysmography is slightly less than that of Doppler ultrasonography.

Tests using X-Rays or CTs

• Venography
  • Can detect both calf and proximal vein thrombi.
• V/Q scans
  • Most studies investigating the incidence of PE in hospitalized patients have used lung perfusion scanning as the diagnostic test. Perfusion lung scanning is non-specific with a false positive rate of over 50%, so studies based upon this diagnostic method alone produce falsely elevated estimates for PE. The specificity of the perfusion scanning for PE is improved by adding ventilation scanning; however in the largest study of V/Q scanning in patients referred because of suspicion of PE, 81% of whom had angiography as well, the specificity was 97% in those with high probability scans, but the sensitivity was only 41%. PE occurred in 12% of patients with low-probability scans. Overall, in patients with high or low probability scans, the sensitivity was 98%, but the specificity only 10%. However, the value of V/Q scanning should not be minimized.
• Helical spiral computed tomography (spiral CT)
  • Has a positive predictive value if >95% in clinically relevant PE and a large number of alternative diagnoses in symptomatic patients with a non-diagnostic or high probably VP scan. This is an accurate method for detection and exclusion of PE with the exception for isolated subsegmental PE.

Laboratory Testing

• D-Dimer
  • The ability of D- Dimer assays to exclude the diagnosis if thromboembolic disease is controversial. The accuracy of this test has been shown to correlate with the patient setting. In outpatients, the negative predictive value was 98% (95% confidence interval (CI), 93%-100%) and 99% (94-100%) for the microlatex and Elisa Methods respectively at the recommended cutoffs. In contrast, in hospitalized patients, the confidence intervals for the areas under the ROC curves included 0.5 (6.0 [95% CI, .50-.71] and .56 [.44-0.67]). The accuracy of the elevated D-Dimer in the hospitalized patient presumably reflecting thrombosis and comorbidies other than pulmonary embolism that led to an increase in D-dimer concentration. (Schrecengost JE et al. Clin Chem 2003 Sep;49(9):1483-90.)
  • D-dimer levels may also be predictive of recurrence of VTE after discontinuation of oral anticioagulation. Normal levels obtained one month after discontinuation has a high negative predictive value for recurrence. Also normal D-dimers measured 1 month after oral anticoagulation withdrawal have a high negative predictive value for recurrence in subjects with previous unprovoked VTE whether or not they are carriers of a form of congenital thrombophilia. (Palareti G et al. Circulation 2003 Jul22;108(3):313-8.)
    
    

Treatment/Anticoagulation
Management of inherited thrombophilia encompasses the treatment of acute thromboembolic events, primary prophylaxis in asymptomatic individuals, and secondary post-thrombotic prophylaxis of recurrences. Data from controlled randomized studies to address these issues in this vulnerable patient population are not available and guidelines for therapy are based upon cumulative experience in the care of small series of deficient patients.

In general, as thrombotic episodes do not occur in a continuous fashion except in homozygous PC and PS deficient patients, lifelong prophylaxis should not be considered for asymptomatic patients not exposed to risk factors. The cost of lifelong anticoagulation, poor compliance, the lack of adequate target anticoagulation levels, and the long-term bleeding risk of anticoagulation in general make this approach impractical. At the present time there is no definitive way to identify those asymptomatic affected individuals destined to develop thrombosis. However, prophylactic measures should be undertaken in affected asymptomatic patients during those situations placing them at increased risk. Concomitant risk factors for development of a thrombosis are:

• Pregnancy, BCP, puerperium
• Surgery/trauma
• Dehydration
• Sepsis
• Immobility
• Fractures
• CHF
• Increasing age
• Chronic inflammatory states
• Air Travel: As many as 10% of airline passengers traveling without prophylaxis for long distances my develop venous thrombosis (Jacobson BF et al. S Afr Med J. 2003 Jul;93(7):522-8.)