Haemolytic Uraemic Syndrome (HUS) and Thrombotic Thrombocytopaenic Purpura (TTP), two different classes of Thrombotic Angiopathies, these are disorders that lead to the occlusion of the microvasculature of an organ, or multiple organs, within the body (Hoffbrand et al, 2001). They are characterized by systemic or intrarenal aggregation of platelets, thrombocytopaenia, and microangiopathic haemolytic anaemia (Desch and Motto, 2007). In TTP, the systemic aggregation of platelets results in formation of thrombi, and consequent ischaemia of various tissues (Moake, 2002.) In contrast, HUS, the occlusion of blood vessels, particularly of the renal circulation, is a result of platelet-fibrin clot production.
It was believed both disorders shared the same pathogenesis due to the similarities of the clinical presentations of both disorders. Many decades lie between the recognition and the discovery of the first pathological clues which ultimately led to the finding of the molecular mechanism. In 1982, unusually large accumulations of von Willebrand factor, released from endothelial cells, were revealed in the plasma of patients suffering from chronic relapsing thrombotic thrombocytopaenic purpura. In 1985, a strong link between HUS and infections with Shiga toxin producing strains of E.coli was recognised (Moake, 2002.)
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It is now known that the pathogenesis of each disorder varies greatly. This paper aims to describe the molecular pathogenesis of both Thrombotic Thrombocytopaenic Purpura and Haemolytic Uraemic Syndrome, and consider the effect of these differences with regards to diagnosis and treatment of two diseases.
The Coagulation pathway in health
HUS and TTP are haematological disorders in which much of their effect is produced by deregulation of the coagulation process. The normal process of coagulation is as follows:
Damage of the endothelial cells initiates vasoconstriction. Vasoconstrictors are released by damaged local nerves and endothelial cells. In addition myogenic constriction also occurs as a result of the contraction of damaged smooth muscle cells within the wall of the vessel. This reduces the blood flow to decrease blood loss (Silverthorn et al, 2010).
The next step involves formation of a primary haemostatic plug:
The injured endothelial cells produce von Willebrand factor (vWF), which is stored in Weibel-Palade bodies of endothelial cells and is released when the vessel wall has been damaged. vWF is secreted as an ultra large form but is cleaved by ADAMTS-13 to smaller vWF monomers seconds after being released (Petri et al, 2010). In areas of high shear stress it is thought that it is advantageous not to cleave the ULvWF as their enhanced affinity for platelets aids clotting in these circumstances (McGrath et al, 2010). The vWF adheres to the exposed collagen fibres in the extracellular matrix. The glycoprotein 1B receptors (GP1b) expressed on the circulating platelets then binds to the vWF. (Reinger, 2008).
Once bound the platelets release a number of chemicals:
Thromboxin A.2 acts as a vasoconstrictor and increases platelet aggregation (when platelets bind together). It does this by binding to receptors on other platelets and induces a conformational change increasing their affinity for other platelets.
Adenosine diphosphate (ADP) is also released and works in a similar way to thromboxin A.2 but binds to different receptors.
Serotonin acts as a vasoconstrictor and increases aggregation again by a similar mechanism to ADP (Minors, 2007)
Once the primary haemostatic plug is formed it needs to be reinforced. This is done with a fibrin 'net'. This is formed via a series of reactions in which the coagulation factors activate one another on the phospholipid membrane of the platelet plug. Eventually the activated factor X converts prothrombin to thrombin. Thrombin then goes on to turn fibrinogen to fibrin, and also activates factor XII which produces cross linking of the fibrin strands in the platelet plug. This leads to the formation of the secondary haemostatic plug (Delyaeye and Conway, 2009; Silverthorn et al, 2010). This can occur through the extrinsic pathway; this is when tissue factor from the damaged endothelial cells sets off the chain of events, or it can occur via the intrinsic pathway which is triggered when factor XII comes into contact with the primary haemostatic plug. The common pathway is the collective name for steps which occur in both the extrinsic and intrinsic pathways (FIG. 1) (Silverthorn et al, 2010)
Figure 1: How the intrinsic and extrinsic pathways activate components of the common pathway.
Always on Time
Marked to Standard
Von Willebrand factor (vWF) is particularly important in HUS and TTP. It is a multimer glycoprotein, composed of monomers of vWF linked by disulphide bonds (Hoffbrand et al, 2001). It is synthesised in endothelial cells and megakaryoctyes, bone marrow cells which are responsible for platelet production in the blood (Furlan and Lammle, 2001 ; Hoffbrand et al, 2001). vWF circulating in normal plasma, exhibits very weak binding affinity (Furlan and Lammle, 2001) and haemostatic activity can only be observed in polymeric forms of vWF.
However in patients with TTP and HUS ultra large multimers of vWF (ULvWF) are found in the plasma, secreted from the Weibel Palade bodies of endothelial cells (Moake, 2002.) These large multimers cause adhesion of platelets to the sub endothelium of damaged blood vessel walls (Furlan and Lammle, 2001). They are more effective at inducing platelet aggregation than the largest possible vWF polymer found circulating in normal plasma, with a binding affinity that is up to ten times greater (Moake, 2002.). ULvWF under normal circumstances is broken down by ADAMTS 13, a protease to prevent this from happening.
HAEMOLYTIC URAEMIC SYNDROME
In 1955 Gasser and colleagues used the term Haemolytic Uraemic Syndrome to describe a similar condition to Thrombotic Thrombocytopaenic Purpura seen in children presenting primarily with acute renal failure (Hosler et al, 2003.) There are two distinct types of HUS. The first is D+HUS (diarrhoeal associated) or E-coli associated HUS and the second is D-HUS (not diarrhoeal associated) or atypical HUS. D+HUS is far more common and is the classical acquired form accounting for 95% of cases of HUS in children (Tan, 2010,) although it can occur at any age. D+HUS is caused by shiga-toxin producing bacterium, most commonly by E-coli 0157 serotype (Ayyash and Ogundele, 2010.) Other infectious agents include Shigella and Streptococcus pneumonia (Scheiring et al, 2010.)D-HUS accounts for the remaining 5% of cases and its aetiology, age of onset and clinical presentations are far more varied.
D+ Haemolytic Uraemic Syndrome
Escherichia Coli 0157
E-coli 0157 are commonly found in the gut flora of cattle and other farm animals and therefore are able to contaminate meat, milk, cheese and other types of insufficiently cooked or pasteurised food (Joel and Moake, 2002). E-coli 0157 enters the host via the faecal-oral route therefore a common history, for a child presenting with D+HUS, would be that they have eaten an under-cooked hamburger or have made a recent visit to farm (Tan, 2010). The incubation period for E-coli 0157 is between 1-6 days and the subsequent clinical features of HUS present 5-14 days following the onset of diarrhoea (Borton, 2010).
Other serotypes of E-coli have also been identified in association with D+HUS, such as 0103, 0111, 026 and rarer bacteria (Scheiring, et al, 2010).
Pathogenesis of D+HUS
Shiga toxins are a group of protein toxins which have the ability to bind to cell surfaces and enter the cells causing inhibition of protein synthesis leading to cell death (Sandvig, 2001). The Shiga toxins that are able to be encoded on bacteriophage DNA and present in the mentioned E-coli serotypes are Shiga toxins 1 and 2. Each Shiga toxin consists of 6 parts, 1 A subunit and 5 B, or binding subunits. Each subunit binds with high affinity to globotriaosylceramide ( Gb3) receptors in the membranes of glomerular, colonic and cerebral epithelial or microvascular endothelial cells, (eg. renal mesangial and tubular cells) monocytes and platelets (Joel and Moake, 2002). Renal glomerular endothelial cells are the primary target for Shiga toxins since they are particularly rich in glycolipid Gb3, which is the predominant membrane receptor for Shiga toxin (Franchinia et al, 2005).
What causes the bloody diarrhoea?
The bloody diarrhoea is produced by the invasion and replication of the bacteria within the colon, which then releases Shiga toxins. These cause damage to the underlying tissues and also the associated vascular supply, resulting in haemorrhagic and ulcerative lesions and thus bloody diarrhoea (Razzaq, 2006).
Pathway of the Shiga Toxins.
Shiga toxins are able to enter the blood supply and travel to target organs including the kidneys. They may also travel attached to platelets, monocytes and polymorphonuclear leukocytes as well as free in the plasma (Franchinia et al, 2005).
Cause of platelet aggregation-destroying endothelial cells
In the kidneys the Shiga toxin binds itself to glycolipid Gb3 on the endothelial cells. This binding internalises the A subunit of the Shiga toxin which, through ribosome inactivation, suppresses protein synthesis (Franchinia et al, 2005). This results in cell death as seen in the colon endothelium. However; in the kidney, it also results in the endothelial cells being separated from the basement membrane. This separation activates platelets as they come into contact with the exposed collagen and ULvWF in the subendothelium (Franchinia et al, 2005). Fibrinogen binds itself to activated platelets causing the aggregation of platelets in conditions of high flow, for example the glomerular microcirculation, and is converted to fibrin (Joel and Moake, 2002). Subsequently the aggregation of platelets results in reduced amount of circulating platelets, hence thrombocytopaenia.
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It also causes renal failure due to the destruction of the endothelial wall. Finally the platelet and fibrin aggregation begin to narrow and block the microcirculation in the kidneys, causing damage to RBCs and thus giving rise to microangiopathic haemolytic anaemia.
Cause of platelet aggregation-stimulation of cytokines
Before the endothelial cells are destroyed it is thought the Shiga toxins stimulate the release of a number of different cytokines and immunological messengers. Shiga toxin 1 stimulates the release of tumour necrosis factor Î±, interleukin-1 and interleukin-6 from monocytes and renal epithelial cells (Joel and Moake, 2002).This increases the Shiga toxins ability to adhere to the renal endothelium and stimulate ULvWF release. These ultra large vWF multimers are thought to up-regulate the expression of adhesion molecules on cell surfaces (Franchinia et al, 2005). Finally the local exposure of tissue factor and activation of factor VII may activate the coagulation cascade which subsequently lead to thrombin generation and microvascular thrombosis in D+HUS (Franchinia et al, 2005).
A rare and unique form of acquired HUS may occur following infection of streptococcus pneumoniae (SPA-HUS). The infection that follows is usually severe and invasive with children presenting with septicaemia, meningitis and pneumonia with empyema (Scheiring et al, 2010).
Atypical Haemolytic Uraemic Syndrome
Atypical Haemolytic Uraemic Syndrome (aHUS) is defined as HUS where E. coli producing Shiga-toxin are absent (Hirt-Minkowski et al, 2010). The aetiology of aHUS is unknown, though the disease often has a gradual onset, and can occur in familial or sporadic forms. Sporadic forms of aHUS (figure 2), can be triggered by conditions such as HIV infection and cancer, but there are still a large proportion of idiopathic cases (Noris M & Remuzzi G, 2009).
Figure 2. Classification of Atypical Haemolytic Uraemic Syndrome.
As figure 2 suggests there are disorders in the complement system, a component of the innate immunity, which can lead to the development of D-HUS. The abnormalities, which specifically affect the alternative complement pathway, are due to mutations in the genes encoding complement regulatory proteins.
Half of D-HUS patients are found to have these mutations, either alone or in combination, which can be classed as "loss of function" or "gain of function" mutations. "Loss of function" mutations are mutations in genes encoding proteins which protect host cells from complement activation.
These proteins are:
Complement Factor H (CFH)
Complement Factor I (CFI)
Membrane Co-Factor Protein (MCP or CD46)
"Gain of function" mutations are those mutations in genes encoding proteins that activate the complement system. These proteins are:
Complement Factor B (CFB)
Complement component 3 (C3)
(Hirt-Minkowski et al, 2010)
In addition a further 10% of D-HUS patients have CFH deficiency due to anti-CFH antibodies (Hirt-Minkowski et al, 2010.)
There are 3 pathways (classical, lectin & alternative) found in the complement system (Sánchez-Corral and Melgosa, 2010). In the alternative pathway, when microbes enter the blood, enzymes are activated, and C3 is cleaved by C3 convertase to give C3a and C3b. C3b deposits on foreign surfaces where it is then bound by CFB. CFB is cleaved by factor D, which produces C3bBb (the C3 convertase of the alternative pathway). This produces more C3b, which can then cleave C5 to give C5a and C5b.
C5b and complement components 6-9 form the terminal complex C5b-9, or Membrane Attack Complex (MAC), which lyses the microbe. However; C3b can also deposit on host cells e.g. endothelial cells, which would lead to these cells being attacked by the MAC, leading to inflammation and platelet activation (figure 3). . Protection of host cells is provided by fluid-phase (plasma) regulator proteins e.g. CFH and membrane-bound regulator proteins e.g. MCP/CD46. These regulators have 2 possible actions: decay acceleration activity (breaking up the C3 convertase) and cofactor action. CFH is the most important complement regulator, having cofactor activity with CFI and decay acceleration activity. CFI depends on cofactor activity (either CFH or MCP/CD46) to cleave C3b to inactive C3b (iC3b). MCP/CD46 is the other cofactor for CFI and also decays C3 convertase (Hirt-Minkowski et al, 2010). When the alternative pathway is impaired, as in D-HUS, there is no inactivation of C3b into iC3b and excessive generation of C3b lead to production of the MAC and endothelial damage (Sánchez-Corral, P & Melgosa, M, 2010).
Figure 3. Model for the Mechanisms Leading from Impaired Regulation of the Alternative Pathway to Thrombotic Microangiopathy.
"Loss of function" mutations
CFH mutation is the most common mutation of alternative complement pathway regulation that is found in D-HUS in 10-30% of cases.
CFH is primarily synthesized in the liver. It is a plasma glycoprotein composed of 20 units of 60 amino acids, which are known as Short Consensus Repeats (SCR). At the N-terminus of CFH, SCR sites 1-4 are important for CFI cofactor activity and decay acceleration.
At the C-terminus of CFH, SCR sites 19-20 are important for stopping alternative pathway activation on host cells e.g. endothelial cells, which CFH can bind with.
Most mutations are found in the C-terminus of the protein, so they have reduced ability to bind to cell surfaces and cannot protect them from alternative pathway activation. However, in these types of mutations they usually retain normal regulatory function in the plasma as a cofactor (Hirt-Minkowski et al, 2010).
In addition to CFH, there are 5 proteins that are genetically and therefore structurally related to CFH. These proteins are known as Complement Factor H-related Proteins 1-5 (CFHR1 -CFHR5). The genes for these proteins are found on the same chromosome as the CFH gene, which can lead to nonallelic recombinations. Therefore, in 3-5% of cases of aHUS, a hybrid gene from a recombination between the genes for CFH and CFHR1 gives a protein with decreased regulatory function (Noris and Remuzzi, 2009).
CFH deficiency can also present due to anti-CFH autoantibodies, in 6 to 10% of patients with D-HUS (Noris and Remuzzi, 2009). The autoantibodies have been shown to recognise SCR 20 at the C-terminus of CFH, and it is thought that the antibodies cause the same dysfunction as mutations of the CFH gene at SCR 20 i.e. cannot bind to cell surfaces to prevent complement activation.
Patients with anti-CFH autoantibodies present with homozygous deletion of the genes for CFHR1 and CFHR3. These findings have been investigated further, and it has been shown that homozygous deficiency of CFHR1 only can predispose to anti-CFH autoantbodies (Sánchez-Corral and Melgosa, 2010).
MCP (CD46) is a transmembrane glycoprotein that is found on the surfaces of most cells and tissues. Mutation of the MCP gene is found in 10-15% of cases of D-HUS. There are 4 SCR at the N-terminus of MCP, which are the site for binding C3b. Most of the mutations are found in this region. The majority of these mutations result in reduced MCP expression on cell surfaces, which leave these surfaces vulnerable to complement attack (Hirt-Minkowski et al, 2010).
CFI is a serine protease which is mostly synthesized by the liver, but it circulates in the plasma converting C3b to iC3b in the presence of a cofactor (CFH or MCP). Mutation of the CFI gene is found in 5-10% of cases of aHUS.
CFI is a 2-chain protease (light and heavy chain) in which mutations are primarily found in the catalytic domain of the light chain (Hirt-Minkowski et al, 2010).
"Gain of function" mutations
CFB is a zymogen, carrying the site of catalysis for the alternative pathway convertase C3bBb. It is activated when it interacts with C3b and is cleaved by factor D to give Ba and Bb. Bb is bound to C3b to form C3bBb, which further cleaves C3 to C3b. Mutations affecting the CFB gene generally results in accelerated formation of the C3bBb convertase or increased resistance to CFH or MCP trying to inactivate C3bBb. This mutation presents in 0-3% of aHUS cases (Hirt-Minkowski et al, 2010).
C3 is the main component of the complement system, found in high concentrations in the plasma and acting as a substrate for C3 convertases. Mutations in the gene for C3 have been shown to result generally in low levels of C3 (Sánchez-Corral and Melgosa, 2010.)
Mutations in THBD, the gene encoding TM, have been found in 5% of D-HUS patient in the absence of mutations for the genes encoding complement proteins. TM usually binds C3b and enhances its inactivation to iC3b in the presence of CFI and CFH. Mutations found in the THBD gene reduce this inactivation function.
However, the mutations have been shown to increase TM binding to C3b, so whether the aHUS pathogenesis in THBD gene mutations involves the complement system or is related to TM function in the fibrinolytic pathway is unknown (Sánchez-Corral and Melgosa, 2010)
Although all these mutations are undoubtedly factors in the pathogenesis of aHUS, they are predispositions rather than the actual cause of the disease. This is proved by the fact that aHUS is only expressed in 50% of carriers of mutations of genes of complement regulator proteins. Therefore, the presence of these mutations on their own cannot be used to definitively predict whether you will develop aHUS or not. When these mutations are found in combination (often CFH with either MCP or CFI), which would obviously mean extensive deregulation of the alternative pathway, there may still need to be a risk polymorphism present for aHUS to develop (Noris M & Remuzzi G, 2009).
The pathology of aHUS is indistinguishable from the pathology of typical HUS, where lesions are usually found in the kidney. The glomerular endothelium is at increased risk because it is fenestrated (has perforations), so the subendothelial matrix is exposed. Therefore, when the alternative pathway is activated in people with mutations in genes for complement regulator proteins or complement activator proteins, the MAC is formed and the endothelium injured, leading to the formation of thrombus as platelets are activated, which leads to haemolysis as erythrocytes pass through the glomerular circulation. This then presents the triad of conditions seen in all HUS patients: haemolytic anaemia, thrombocytopaenia and acute renal failure (Noris M & Remuzzi G, 2009).
Diagnosis of HUS
HUS is the most common cause of acute renal damage in children (Trachtman et al 2003). D+HUS often presents with:
Diarrhoea (bloody stool in 70%) (Tan A, 2010)
Neurological symptoms in 30% of cases making it sometimes difficult to distinguish from TTP.
Fever as it often follows an infection
In the late stages it can even cause: bruising, pallor, petechiae, decreased urine output, decreased consciousness and jaundice (Dugdale, 2010).
The atypical HUS is distinguished by its chronic relapsing course and similarity to TTP, as there is haemolytic anaemia and thrombocytopaenia as well as renal dysfunction and neurological symptoms (Bolton-Maggs, 2010).
Investigations in establishing a diagnosis in patients presenting with D+HUS include the observation of the following:
Full blood count (FBC) is likely to show reduced red cell count.
Peripheral blood smear would show the presence of schistocytes, fragmented or helmet shaped RBC's and giant platelets.
Haemoglobin level of less than 8g/dL suggestion haemolytic anaemia.
Lactate dehydrogenase level is increased due to the intravascualar lysis of red blood cells.
Creatinine and urea will be elevated.
Stool cultures show Shiga toxin producing E coli.
Hypertension and reduced urine output suggesting reduced kidney function.
Blood cultures will be E coli negative as only the toxin circulates in the blood.
Protein and haemoglobin present in urine. (Tan et al 2010)
Investigations in establishing a diagnosis in patients presenting with D-HUS include the observation of the following:
Serum levels of C3, C4, factor H and factor I to determine the severity, prognosis and treatment options.
Genetic analysis for mutated complement genes CFH,Â CD46,Â CFI,Â CFBÂ andÂ C3 although this often takes a long time it is necessary when considering transplantation.
Enzyme-linked immunosorbent assay to detect autoantibodies against factor H.
Fluorescent-activated cell sorting (FACS) analysis: a quick screen for decreased or mutated expression of the transmembranous protein CD46.
Consider other rarer causes of aHUS for example pregnancy, malignancy, HIV, drugs etc. (Taylor et al 2010)
Treatment of HUS
In a review of recent randomised controlled trials no intervention has been shown to be more effective than supportive therapy for D+HUS (Micheal et al, 2009). Although this was a small review and the trials had a limited number of patients, it was supported by a Cochrane review. This also stated that supportive therapy included:
Fluid and electrolyte control,
Blood transfusion if required. (Micheal et al, 2009)
Possible treatments of fresh frozen plasma (Stanworth et al, 2004), steroids (Perez et al, 1998), heparin (with and without dipyridamole)( Van Damme-Lombaerts et al, 1988 and Vitacco et al, 1973) and a Shiga toxin binding agent (Tractman et al, 2003) have been trialled but have been shown to have no clinical benefit.
In the case of D-HUS, guidelines suggest that plasma exchange should be started along with general supportive measures, to replace abnormal factors as soon as the disease is suspected over D+HUS, until the anaemia is reversed (Taylor et al, 2010). Although, this is based more on expert opinion rather than evidence from clinical trials and individuals with different complement deregulations respond differently (Bolton-Maggs, 2010 ; Taylor et al, 2010).
Kidney transplantation has varying success rates depending on the cause of the D-HUS. For example it is not recommended in patients with abnormal CFH or CFI, as it is associated with an 80% recurrence in disease, (Taylor et al 2010) whereas patients with isolated CD46 dysfunction are unlikely to respond to plasma exchange alone.
Patients with autoantibody associated D-HUS respond better to plasma exchange and immunosupression although they may also require a kidney transplant. However immunosuppression should be continued throughout (Bolton-Maggs, 2010).
As factor H and I are produced in the liver, liver and combined liver/ kidney transplantation should be considered in patients with known CFH or CFI abnormalities, as part of clinical trials, as it has been undertaken with some success with prophylactic plasma exchange (Taylor et al, 2010).
New therapies for D-HUS are currently being suggested(Bolton-Maggs, 2010; Taylor et al, 2010). This includes the use of eculizumab, a humanized monoclonal antibody that blocks complement activity (NÏ‹rberger et al, 2009), for which there is currently an open label controlled trial for patients with plasma sensitive aHUS in the USA (Alexion Pharmeceuticals, 2010).
A successful case has been observed in a patient using eculizumab after failed plasma exchange and renal transplantation (NÏ‹rberger et al, 2009).
THROMBOTIC THROMBOCYTOPAENIC PURPURA
TTP was first described in 1924 by Dr Moschocowitz who suspected that a powerful agglutinative and haemolytic poison was responsible for this disease (Lammle et al, 2005.) in the years ahead, identification of ULvWF in patients with TTP and with advent of gene cloning, ADAMTS 13 was identified as the enzyme responsible for the breakdown of ULvWF. TTP exists in two forms, congenital and acquired TTP. Despite the similar clinical presentations with HUS the pathogenesis varies greatly.
Congenital Thrombotic Thrombocytopaenic Purpura
In TTP it is suggested, and plausible, that vWF multimer unfolds and goes under a stress induced conformational change, that is from a globular structure, to a molecule with extended chains. This promotes the attachment of platelets to its surface, resulting in the formation of platelet- rich thrombi in the blood, which is observed in patients with TTP. The result is the occlusion of blood vessels, and consequent ischaemia of the tissues supplied (Furlan and Lammle, 2001). In explaining the pathogenesis of TTP it is also worth noting the structure and the role of ADAMTS-13.
ADAMTS 13 (FIG 4) consists of 1427 amino acids and is a single peptide. Constituents of the protease include:
a short propeptide terminating sequence (PQRR)
a reprolysin-like metalloprotease domain
a disintegrin like domain
athrombospodin-1 repeat (TSP1)
a Cys-rich domain
an ADAMts spacer
seven additional thrombospondind-1 repeats
Figure 4: Structure of ADAMTS-13 gene.
2 CUB domains
Under normal circumstances ULvWF is broken down in the circulation, by ADAMTS-13, as soon as it is released from the endothelial cells (Feys et al, 2009; Lotta et al, 2009). However; in patients with congenital TTP, decreased activity of ADAMTS-13, due to a genetic mutation is consistently found. Congenital TTP accounts for approximately 5% of all TTP cases (Lotta et al, 2009).
It is an autosomal recessive disorder, caused by a mutations occurring on the ADAMTS-13 gene, (Lotta et al, 2009) which lies on chromosome 9q34, expressed in endothelial cells and the liver (Senior, 2001). The connection between ADAMTS-13 and congenital TTP was first established in 2001. (Lotta et al, 2009) Since then another 75 mutations have been identified the majority of which are heterozygous mutations identified in: disintegrin, cysteine-rich/spacer, TSPI-2, TSPI-3, TSPI-5, TSPI-6, TSPI-7, TSPI-8, CUB-1 and CUB-2 domains of the gene (Feys et al, 2009).
The existence of middle aged individuals with severe deficiency of the protease, but with no symptoms of TTP previously, suggests a multifactorial mechanism rather than genetics alone in precipitating an attack of TTP in patients with genetic protease deficiency. It has been suggested that acute episodes of TTP require triggers which may consequently initiate activation or apoptosis of the endothelial cells present within the microcirculation (Furlan and Lammle, 2001).
Most cases of acquired TTP are due to auto-antibodies which inhibit the proteolytic activity of ADAMTS 13. These antibodies usually occur transiently and are associated with sporadic TTP. In some cases, however; the auto-antibodies may persist during remission and cause chronic relapses. Antibodies IgG and IgM have been identified to cause the inhibitory activity of ADAMTS 13. Other rare causes of TTP include drugs such as clopidogrel and oral contraceptive pills.
It has been found that pre-incubation of IgG molecules which contain an inhibited Fab region, of the IgG antibody, but not anti-Fc region reverses the inhibition of ADAMTS-13 activity. This suggests involvement of antigen-antibody interaction (Tsai, 2003).
Although the physiological functions of the domains of ADAMTS-13 are not understood, it has been demonstrated that cysteine rich, the spacer domain, the CUB domains and the first TSP1 repeat are the major epitopes (binding region on the enzyme). However; they are not exclusive for auto-antibodies against ADAMTS-13 in patients with TTP (Klaus et al, 2003; Lammle et al, 2005).
Various antigenic regions for auto-antibodies suggest there may be other inhibitory mechanisms other than neutralisation of ADAMTS-13 that occur which prevent ULvWF breakdown. In accordance to this; a study has demonstrated the presence of bound IgG and IgM to ADAMTS-13 where the enzymes activity is not neutralised (Scheiflinger et al, 2003).
The activity of the protease could be inhibited by preventing interaction between the endothelial cells and the enzyme by the auto-antibodies. In contrast the antibodies could inhibit the activity of modulators that are involved bringing about the enzymatic activity. Another proposed mechanism is that theses auto-antibodies could be decreasing the half life of the circulating enzyme in plasma and increasing its clearance (Scheiflinger et al, 2003).
Diagnosis of Thrombotic Thrombocytopaenic Purpura
Clinical presentation ofÂ TTP has considerable overlap with HUS, however typical characteristics are:
2. Neurological symptoms, which may or may not be present or may fluctuate e.g. seizures, hemiparesis, bizarre behaviour, headache and reduced consciousness.
3. Renal Failure (plasma urea >8mmol/l andÂ creatine >140Âµm/l).
5. Thrombocytopaenia (platelets < 150 x 109/l).
6.Â Microangiopathic haemolytic anaemia (MAHA) low or falling haemoglobin or fragmented red blood cells (schistocytes(Figure 4.)) .
7. Liver function tests showÂ hyperbilirubinaemia and raisedÂ transaminase levels.
8. Raised lactateÂ dehydrogenase (LDH) levels.
(Longmore et al, 2010; Pollock et al, 2008)
Figure 5. Blood film showing a schistocyte.
Haematological investigations specific toÂ TTP include detection of ultra-largeÂ vWF multimers, which are not normally found in circulating plasma due to the activity of the metalloproteinase ADAMTS-13.Â ADAMTS-13Â would normally cleave the peptide bonds between residues Tyr 1605 and Met 1606 preventing the formation ofÂ ULvWFÂ multimers (Alford et al, 2003).Â
ADAMTS-13 auto-antibodies are present in acquiredÂ TTP withÂ IgG antibodies present in 97%Â of patients with acuteÂ TTP and plasmaÂ ADAMTS-13 < 10% (Shelat et al, 2005).Â This elucidates to the potential use ofÂ Rituximab (see below for more detail) as a chemotherapeutic agent in the treatment of acquired TTP.Â ADAMTS-13 deficiency can be evaluated and indeed in patients with idiopathicÂ TTP this has been considered as a useful prognostic indicator of an increased likelihood of relapse (Sadler E,Â 2008).
A retrospective diagnosis can be made when the platelet count has normalised, with a renal biopsy showing arteriolar and capillary thrombosis; the composition of which is mostly platelets with the presence of vWF.Â This contrasts with HUS, which demonstratesÂ subendothelial widening of the capillary wall withÂ glomerular andÂ arteriolar fibrin thrombi (Remuzzi and Ruggenenti, 1995).
Treatment of Thrombotic Thrombocytopaenic Purpura
First line treatment ofÂ TTP is plasma exchange (PE) with fresh frozen plasma (FFP), which removes the patient's own ADAMTS-13 auto-antibodies and replenishes functional ADAMTS-13, which will cleaveÂ vWF in the normal fashion.Â Alternative first line therapies have been associated with failure of remission and increased mortality (Michael et al, 2009). PE should be continued for at least two days after remission (Alford et al, 2003).
A new approach which shows some promise is use of the immune modulatorÂ Rituximab, a monoclonal antibody, which destroys B cells with aÂ CD20 protein on their surface including those responsible for ADAMTS-13 inhibition (Ling et al, 2009).Â By tackling the immune mediated mechanism it is possible to prevent the production of furtherÂ ADAMTS-13 autoantibodies. However, the exact role ofÂ Rituximab is not clear, but its use as an adjuvant therapy seems increasingly beneficial in some forms ofÂ TTP (Sadler E,Â 2008).
For patients with relapsing or refractoryÂ TTP Luc Dubois et al (2010) suggested splenectomy, which has been tried with some success; however, these conclusions are based upon very small numbers of patients in case studies with no control groups and do not conform to the consensus of opinion in the literature.
TTP and HUS are disorders which are clinically difficult to distinguish due to similar clinical presentations. However the mechanism of pathogenesis varies and it is imperative that this difference is recognised for allowing effective treatment to be established.
Congenital or acquired TTP results in the deficiency of ULvWF-cleaving protease, ADAMTS-13 which ultimately leads to the formation of platelet rich thrombi within the microvasculature and the consequent thrombocytopaenia and organ damage. Most causes of the acquired form are idiopathic but may also be triggered by pregnancy, drugs or infections. In any case; however, the deficiency of the ADAMTS-13 enzyme is mediated by the production of auto-antibodies.
In contrast D+HUS occurs as a result of infection by Shiga toxin producing bacteria, most commonly by E.coli 0157, and is predominantly seen in children. Ultimately the toxins cause endothelial damage and cytokine secretion which results in activation of the coagulation cascade and the subsequent fibrin rich thrombi formation. In contrast to TTP, HUS affects the kidneys predominantly. In contrast D-HUS is mediated by the activation of the complement system which results in symptoms shown by the typical form.
Despite the significant progress in understanding of the mechanisms underlying both disorders there still remain small uncertainties that need to be revealed which in return will allow for more effective pharmacological treatment.