Thrombotic Thrombocytopenic Purpura Ttp Biology Essay

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This literature review will attempt to outline thrombotic thrombocytopenic purpura and the role of the von Willebrand factor-cleaving protease in the disease. This work will start by outlining the principal causes of TTP and discuss some of the epidemiology of the disease. In addition, it will go on to explore the methods of TTP diagnosis, outlining criteria used by physicians and general laboratory tests to confirm the diagnosis. A molecular overview of the ADAMTS13 protease will be discussed within the context of TTP, followed by a description of the mechanism whereby enzymatic degradation of its substrate occurs, based on recent published evidence. Then the review will finish by discussing existing treatments for thrombotic thrombocytopenic purpura, dealing with routine treatments and discussing therapeutic options in more severe cases.

Von Willebrand factor

During normal function, the multimeric glycoprotein von Willebrand factor (vWF) binds to other proteins in the bloodstream, most notably inactive factor VIII. During clotting, factor VIII disassociates from vWF and is free to act as a cofactor with other blood factors during the clotting process. VWF also contributes to normal blood clotting by binding to and linking platelets and existing blood clots and binding them to the blood vessel wall (Sadler, 1998).

At the structural level the vWF multimer consists of a series of 2050 amino acid monomers, each of which contains several domains (D', D3, A1, A2, A3, D4, B1, B2, B3, C1, C2 and the 'cysteine knot') with specific binding or cleavage sites (Furlan et al., 1993). After translation, the vWF protein monomers are N-glycosylated and arranged into dimers in the endoplasmic reticulum. These dimers are ultimately linked together via disulfide bonds crosslinking of cysteine residues into functional multimers in the Golgi apparatus.

The hereditary loss of vWF is known as von Willebrand Disease - named after Erik von Willebrand, who first described the disease in 1926 - and is characterised by extensive bleeding, usually from the nose and gums (Willebrand, 1999). Due to the size of the vWF protein, it requires enzymatic degradation in the blood to prevent the formation of spontaneous blood clots. If the large multimers are not able to be degraded, the disease thrombotic thrombocytopenic purpura may occur.

Thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) was first described in 1924 in a sixteen-year-old girl who presented symptoms of anaemia as a result of disrupted red blood cells (haemolytic anaemia), along with thrombocytopenia, neurological dysfunction and renal damage (Moschcowitz, 1924). After her death due to cerebral infarction and cardiac failure, an autopsy was performed revealing widespread thromboses throughout the body, specifically located within the terminal arterioles.

Today, TTP is recognised as a rare blood-coagulation disorder that results in the formation of microscopic thromboses in small blood vessels throughout the body. Molecular and clinical studies have revealed that these clots result from large multimeric vWF molecules binding platelets as illustrated in figure 1, which in turn bind fibrin, causing thrombotic microangiopathy (TMA) (Sadler, 2008). These thrombi can disrupt the blood flow, especially in narrow capillaries where their presence subjects passing red blood cells to shear stress, causing damage to the cell membranes. Eventually, this shear stress will fragment the red blood cells forming schistocytes and causing capillary necrosis and anaemia (Imoto, 2005). The TMA and subsequent decrease in the concentration of platelets leads to bleeding or purple coloured bruising (purpura) under the skin. In cases of TTP, the organ most frequently affected is the brain, giving rise to headaches, confusion, difficulty speaking, paralysis, numbness or fits, in addition to the more common symptoms of malaise, fever, and diarrhoea that accompany the disease (Amorosi, 1966).

Generally five broad categories are used to diagnose TTP: low platelet count, leading to purpura; anaemia with schistocytes present in the blood; neurologic symptoms; kidney failure; and fever. It should be noted that in the majority of cases these categories do not arise together; by way of example, some forms of TTP have been reported that exhibit anaemia in the absence of schistocytes (Daram, 2005). Due to the severity of the symptoms and the possibility of renal failure, treatment for TTP is regarded as a medical emergency.

TTP is a rare disease and only about four to six cases arise per million people per year (Sadler, 2008). The disease is much more prevalent in children (1 in 25,000) than adults. It appears that TTP is more common in pregnant women, affecting approximately one in 25,000 pregnancies (Sadler, 2008). This occurrence during pregnancy may be one reason for the increased incidence (three-fold) of chronic TTP in women aged between twenty and forty years compared with males of the same age (Tsai, 2006). During pregnancy, the general likelihood of thrombosis increases as maternal blood pressure becomes elevated (Flessa et al., 1974) and as the platelet count naturally decreases in the third trimester, any risks associated with TTP increase considerably. It is also possible for women in the postpartum stage to contract TTP, with cases reported up to three months after giving birth (Fujimura et al., 2008).

Table 1.show the ADAMTS13 and the characteristics of TTP

ADAMTS13 metalloprotease

Characteristics of TTP

Mutation in children

Upshaw Schulman syndrome

Mutation during adult onset

Congenital :chronic relapsing

Deficiency / autoantibodies

Acquired: transient

Deficiency /underlying disease

secondary

Deficiency unknown

Idiopathic

There are two major forms of TTP as stated in Glatzel (2004) and table 1 idiopathic and secondary TTP. Idiopathic (or immune) thrombocytopenic purpura (ITP) is classified as an autoimmune disease, as antibodies are raised against clotting bodies such as platelet membrane glycoproteins or the von Willebrand factor-cleaving protease (ADAMTS13), which increases the blood clotting activity. ADAMTS13 is required to break down large vWF molecules, but enzyme targeting by antibodies results in a severely decreased ADAMTS13 activity. By definition the causes of ITP are unknown, and it is therefore not possible at present to prevent the disease

Around 40% of TTP sufferers are diagnosed with secondary TTP, as a result of primary conditions such as cancer or HIV, or after treatment with platelet aggregation inhibitors, quinine or following bone marrow transplantation (Moake, 2002). Secondary TTP shares the symptoms of idiopathic TTP but usually ADAMTS13 activity is not as decreased as in cases of ITP. In addition to the types of TTP mentioned, figure 1 on page 11 illustrates an inherited deficiency of ADAMTS13, known as Upshaw-Schülman syndrome that results from a frameshift or point mutation in the ADAMTS13 gene (Tseng, 2011), located on the ninth chromosome (9q34) (Levy et al., 2005). It is interesting to note that sufferers of Upshaw-Schülman syndrome generally exhibit few symptoms, but are likely to develop TTP if the vWF concentration increases, as seen during an infection. It has been hypothesised that Upshaw-Schülman syndrome is inherited in an autosomal recessive fashion, with parents of sufferers exhibiting few symptoms and only having a moderately decreased ADAMTS13 activity (Kinoshita et al., 2001).

ADAMTS13

The von Willebrand factor-cleaving protease (ADAMTS13) is a 1,427 amino acid, 145kDa zinc-containing metalloprotease (Zheng et al., 2001). ADAMTS13 is one of the nineteen-member ADAMTS (A Disintegrin And Metalloproteinase with a Thrombospondin type 1 motif) family of peptidases that have a biological function in cleaving proteins in humans (Zheng et al., 2001). As the name suggests, ADAMTS13 contains a protease domain (amino acid motif: 'arginine, glutamine, arginine, arginine') for protein hydrolysis; an adjacent disintegrin domain that inhibits platelet aggregation; and eight thrombospondin domains, which may have a role in cleaving the disulfide bond located between vWF dimers (Wang et al., 2010). Experiments have shown that activation of the metalloprotein is not solely dependent on zinc ions but can also function in the presence of calcium and barium ions, although the latter cation only activates ADAMTS13 in the presence of citrate (Anderson et al., 2006). ADAMTS13 is secreted in blood and binds to a ≥73 amino acid fragment (encompassing aspartic acid1596 to arginine1668) within the vWF A2 domain (Kokame et al., 2004), cleaving multimers between the amino acids tyrosine1605 and methionine1606. Through co-purification experiments, it has been hypothesised that there is at least one additional ADAMTS13 binding site on the vWF protein (Fujikawa et al., 2001).

ADAMTS13 belongs to the metzincin metalloprotein superfamily, but unlike other protein members does not require cleavage prior to biological activation (Frederici and Lee, 2011). Despite being secreted into the blood plasma in a fully active form, ADAMTS13 does not immediately cleave vWF multimers. Studies have concluded that in the fully folded vWF structure, the amino acid bond target within the A2 domain is 'cryptic' - that is, inaccessible to cleavage by ADAMTS13. It is hypothesised that during circulation in the blood plasma, external shear forces act to slightly denature the folded vWF protein, providing access to the tyrosine1605 - methionine1606 residues for cleavage (Tsai, 1994). Interestingly, when blood factor VIII is bound to vWF under shear forces, ADAMTS13 cleavage activity has been shown to increase in vitro by an unknown mechanism (Cao, 2008). During the fully folded conformation of vWF, concealing the A2 domain, ADAMTS13 has recently been shown to bind to the exposed D4 domain on the surface of the vWF protein (Zanardelli et al., 2009).

Immunoglobin G (IgG), IgA and IgM antibodies have been shown to target ADAMTS13 in patients, leading to the development of TTP in a majority of cases not attributed to Upshaw-Schülman syndrome (Levy et al, 2005). In addition, there are around eighty mutations in the ADAMTS13 gene that can cause Upshaw-Schülman syndrome (Fujimura et al., 2008), and these are not concentrated to any particular enzymatic 'hot-spot', suggesting that each of the domains are important for proper protein function. Many of the mutations result in impaired secretion of the enzyme into the blood plasma although some prevent the function of proteolytic domains or the formation of disulphide bonds in the molecule (Frederici and Lee, 2011). Some more drastic frameshift mutations have resulted in protein truncation that prevents protein secretion from MDCK cells, as demonstrated in vitro (Shang et al., 2006). Autoantibodies against ADAMTS13 contribute to the majority of cases of TTP. Predominantly, these antibodies inhibit ADAMTS13 enzymatic function but can also act to degrade the enzyme itself (Scheiflinger et al., 2003). Antibody-mediated degradation preferentially targets the cysteine-rich spacer region of the protein (Soejima et al., 2003), specifically at threonine572, asparagine579, valine657 and glycine666 sites (Luken et al., 2006); although many of the TTP patients have polyclonal antibodies that target various regions of the ADAMTS13 protein (Klaus et al., 2004). Although much focus has been directed towards mutations in the ADAMTS13 gene, it should be noted that some in vitro experiments have identified polymorphisms in vWF that result in an increased rate of cleavage by wild-type ADAMTS13 (Bowenm and Collins, 2004), suggesting the substrate structure may also contribute to TTP susceptibility or resistance.

Diagnosis of TTP

In cases of idiopathic TTP, adult patients usually present with a chronic disease whereas children between the ages of one and seven years present with an acute disorder, usually following an infection. As mentioned, the loss of platelets in TTP leads to bruising and bleeding that can range from mild purpura to massive haemorrhage, which can be identified during a physical examination. Idiopathic TTP is identified by a decrease in concentration of blood platelets without a change in other cell types. Due to the severity of TTP, a presumptive diagnosis may be made if symptoms relate to any type of haemolytic anaemia; a practice that has resulted in an eight-fold increase in diagnoses of TTP in recent years (Clark, 2003). Traditionally the 'classic pentad' (low platelet count, anaemia, neurological symptoms, kidney failure, and fever) developed by Amorosi and Ultmann in the mid-1960s is used to diagnose TTP (Amorosi and Ultmann 1966), although some reports claim that the infrequency of incidence exhibiting all symptoms renders the classic pentad irrelevant to current practice (George, 2010).

Although concentrations and activity of ADAMTS13 can be measured, generally this test is not required prior to beginning treatment. Neurological symptoms relating to the formation of microscopic thromboses in blood vessels serving the brain (including coma, stroke, seizures, or focal abnormalities) may indicate TTP (Vesely et al., 2003). Blood tests of suspected TTP sufferers often reveal high serum concentrations of lactate dehydrogenase, released from fragmented red blood cells (Cohen et al., 1998) and a high creatinine level if the kidneys are affected (Vesely et al., 2003). Some patients present with heart problems, although a myocardial infarction is usually absent. In TTP patients with cardiac involvement, an increase in serum troponin I may be detected (Hawkins et al., 2008). It should be noted that several diseases cause TTP-like symptoms including: preeclampsia, some autoimmune disorders (e.g. systemic lupus erythematosus or antiphospholipid syndrome), systemic infection and malignant hypertension (George, 2010). If these TTP-mimicking diseases are diagnosed after plasmapheresis has been initiated, the treatment is immediately stopped.

TTP- and ITP-like diseases

There have been cases of patients presenting with TTP-like symptoms, often causing confusion during diagnosis. Often the similarity between types of thrombocytopenia results in the illness recorded as TTP and the treatment by plasmapheresis being initiated. The distinguishing factor between types of TTP is the concentration of the ADAMTS13 protease, which in TTP-like disorders is usually at a normal concentration. In two recorded cases, the classic pentad of symptoms for TTP was observed, including symptoms related to life-threatening kidney disease (uremia). In this case, the treatment recommended was several rounds of haemodialysis, which resulted in an attenuation of symptoms and restoration of the platelet count. Subsequent analysis revealed a normal concentration of ADAMTS13, giving the first indication that this was a TTP-like disease (Nakamura et al., 2007). As discussed briefly, secondary TTP can arise from a number of scenarios, usually related to medication (e.g. drugs affecting platelet formation) or resulting from primary disorders. In some case,s organ transplantation has resulted in a TTP-like disease being reported - a scenario that can be severe repercussions and pose a medical dilemma in terms of treatment. Such disorders can be referred to as transplant associated (or post-transplant) microangiopathies and are most likely to develop after solid-organ or stem-cell transplantation. Although these microangiopathies are characterised by TTP-like symptoms, the mechanism is likely to be quite different. Most crucially, although the same treatments are used for transplant associated microangiopathies and TTP, the former does not respond well and consequently, the patients have a poor prognosis (Crowther, 2008). As an example, a TTP-like disorder can arise after a kidney transplant manifesting in systemic symptoms. The severity of disorder may lead to a recommendation to withdraw the standard immunosuppressive therapy, which in turn may lead to host rejection of the donor organ, requiring an emergency removal of the kidney (a nephrectomy).

In an attempt to characterise some of these TTP-like diseases in the light of new and improved molecular and diagnostic techniques, a spectrum of TTP-related syndromes has been developed that reveals a wide range of contributing factors, causes and responses to treatments. At one end of this spectrum is the 'pure' form of TTP discussed earlier resulting from the functional loss of the ADAMTS13 protease with transplant associated microangiopathies forming a distinct, but closely-related part of the spectrum. Next is likely to be so-called catastrophic antiphospholipid antibody syndrome (CAPS), which results in blockages of blood vessels to major organs and has a high (~50%) mortality rate (Asherson, 2005). Further away from TTP on the spectrum is pregnancy-associated microangiopathic Hemolytic anaemia and finally epidemic haemolytic-uremic syndrome (HUS) is at the farthest end of the spectrum. HUS has similar symptoms to TTP but is caused by bacterial infection and requires different treatment (Crowther, 2008).

Treatment of TTP

TTP has a high mortality rate (95%) if left untreated. Treatment is routinely performed via donor blood plasma exchange (plasmapheresis) that works according to the Furlan-Tsai hypothesis (Furlan et al.,1998). This hypothesis suggests that removing the antibodies to ADAMTS13 leads to an increased vWF cleaving activity and ameliorates the TTP condition. Plasmapheresis also serves to replace the ADAMTS13 enzyme, giving a very good prognosis (80-90% survival) if the treatment is repeated daily for one-to-eight weeks (Tsai, 2006). If plasmapheresis cannot be administered immediately, plasma infusion may be performed in the short-term, although comparisons of both methods suggest that plasmapheresis is the superior treatment (Rock, 1991). In patients exhibiting a severe hemorrhage, a platelet transfusion may be required to prevent further bleeding.

Patients who do not respond to plasmapheresis may require immunosuppressant drugs such as corticosteroids to decrease production of all antibodies, including anti-ADAMTS13. Corticosteroid treatment usually involves high doses, such as methylprednisolone (1000 mg/day for 3 days) or Rituximab (375 mg/m2) weekly for 4 weeks (George, 2010). Once the patient's condition improves and the platelet count begins to increase, plasma exchange can be resumed. In rare cases, all treatments may be insufficient, in which case additional immunosuppressants such as vincristine or cyclosporine may be used (George, 2010).

In rare cases of TTP, removal of the spleen (a splenectomy) is necessary. Previous experiments have demonstrated that 51Cr-chromate-labeled platelets are accumulated predominantly in the spleen (Aster, 1969). The spleen has also been identified as an important site of production of platelet antibodies in patients with ITP (Kuwana et al., 2002) and its removal allows the platelet concentration to increase and normal clotting to occur.

Continued patient testing involves monitoring concentrations of lactate dehydrogenase, platelets, ADAMTS13, or antibodies against ADAMTS13. Platelets usually reach normal levels with one week of treatment and signs of purpura subside over time. In patients with neurological symptoms, the clearing of headaches, confusion or paralysis are often the first sign of successful treatment. Conversely, renal complications are often the last to be resolved and the outcomes of kidney damage are somewhat uncertain (George, 2010). As plasmapheresis involves multiple donors, to ensure that no blood-borne disease has been introduced into the patient, vaccinations are often required, especially for hepatitis B. Once successful, the patient platelet count increases to normal levels (1.5 x 1011/L) (Kumar and Clark, 2005), aspirin-based drugs may be administered to prevent the risk of future TTP relapse.

After treatment, relapses of TTP are relatively frequent and it is difficult to predict their timing, although many incidences occur within the first month after treatment. One study has quantified the risk of relapse as 36% over 10 years post-treatment (Shumak et al., 1995). Ongoing research into treatments for TTP are focusing around ADAMTS13, however there is likely to be alternative defects causing TTP - as demonstrated by the relatively few symptoms seen in patients with Upshaw-Schülman syndrome, that often do not have a functioning ADAMTS13 enzyme, but do not have the anti-ADAMTS13 antibodies present (Murrin and Murray, 2006). Other treatments involve the use of the chimeric monoclonal antibody, rituximab, commonly used in autoimmune diseases and the alkylating agent cyclophosphamide which acts as an immunosuppressant. A study has shown a small number of patients with resistant TTP have shown improvements after treatment with these drugs used together (Stein et al., 2004). This research has been promising thus far and further work is on-going to develop additional novel treatments.

In conclusion, this review has discussed the role and the structure of ADAMTS13 gene including focusing on the mutations in ADAMTS13 gene that caused TTP. TTP is a severe disorder and treatment is regarded as a medical emergency. There are different treatment methods mentioned in the review that showed good progress with time. In order to get the appropriate treatment TTP has to be diagnosed properly because there are view diseases which have TTP-like symptoms often causing confusion at choosing the treatment for example Uremia, life threatening kidney disease.

Table2: outline the clinical symptoms 18 patients with severe ADAMTS13 deficiency.

Patient

Year of examination

Sex

Age

Race

BMI (kg/m2)

ADAMTS13 inhibitor

Symptoms

Neurologic symptoms

Platelet (x109/L)

Hematocrit %

LDH U/L

Creatininemg/dL

PEX no

Diagnosis to death d

Remission to replace death

1

1995

F

41

AA

33.7

Moderate

Abdominal pain, diarrhoea

None

4

15

1363

1.2

24

-

58

2

1996

M

34

AA

22.8

Strong

Chest pain, hematura

None

5

18

3423

1.0

19

-

133

3

1996

F

58

AA

29.8

Moderate

Nausea, vomiting and diarrhoea

None

6

19

1018

1.5

42

488

318

4

1996

F

39

W

48.1

Strong

Disoriented, diarrhoea, numbness in face and right hand

Numbness on right hand

18

22

1976

1.1

8

-

-

5

1996

M

43

W

35.4

Absent

Abdominal pain, chest pain, confusion and ataxia

Confusion

7

24

1668

3.9

71

-

950

6

1999

F

71

W

20.9

Trace

Nausea, vomiting and ataxia

Stroke

6

21

1156

1.0

48

-

121

7

1999

M

30

AA

28.1

Strong

Confusion, disorientation

Seizure, coma

11

21

2231

1.9

3

3

Died

8

1999

F

25

W

27.2

Mild

Confusion, hematuria

Confusion

11

20

2379

1.5

22

-

38,39,99,55

9

1999

M

55

AA

26.7

Mild

Right hand weakness, aphasia

Right hand weakness, aphasia

24

30

436

1.2

20

-

319

10

2000

F

22

W

42.5

Strong

Weakness, nausea, vomiting

None

11

22

1367

1.0

74

-

-

11

2000

F

33

AA

43.0

Moderate

Abdominal pain, nausea, vomiting

stroke

20

22

1613

5.5

15

-

-

12

2001

F

39

W

28.0

Strong

Abdominal pain

None

11

23

1434

0.9

10

-

-

13

2001

F

50

W

27.3

Strong

Seizure dysarthria

Seizure, dysarthria

17

19

2712

2.9

7

17

Died

14

2001

F

33

W

41.6

Strong

weakness

None

7

24

3909

2.2

10

-

-

15

2001

F

19

W/NA

22.2

Trace

Headache, abdominal and chest pain,

None

7

18

1113

0.9

5

-

-

16

2001

F

38

AA

42.2

Moderate

Right hand numbness, dysarthria

Right hand numbness, dysarthria

11

18

1862

1.1

33

-

-

17

2001

F

20

AA

51.4

Strong

Dyspnea, abdominal pain

None

5

18

2901

1.7

24

-

-

18

2001

F

34

AA

32.8

Moderate

Right sided weakness and aphasia

Right sided weakness and aphasia

27

15

1428

1.0

14

-

-

Figure 1: Thrombotic thrombocytopenic purpura (TTP) is disease characterized by ADAMTS 13 deficiency. As on figure 1a: Platelets attach to ultra large vWF multimers. Because vWF-CP (ADAMTS 13) is inhibited this leads to massive vWF:platelet thrombosis as in figure2 b.

Endothelium

Platelets

TTP

vWF-CP

vWF vWF-CP Ab

Endothelium

A clot formation due to large multimeric vWF molecules binding platelets and fibrin.

Fibrin

Platelets

vWF

Figure 2: shows the structure of ADAMTS13 and the 20 exons. It also shows the 12 familial mutations on the gene in TTP.

S

Metallo protease

P

1 2 3 456 7 8 91011 121314 15 16 1718 19 20 21 22 23 24 25 26 27 28 29 22229

CUB

CUB

5

8

7

6

4

3

2

Spacer

Cys

1

Dis

H96D

R102C

T1961

C951G

C1024G

C1213Y

Frameshift

R398H

R528G

Splice site

R692C

Frameshift

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