Management Of Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenic purpura (TTP) is not a common disorder, but the young age, acute onset, fulminant course and sometimes fatal nature of the disease make it remarkable. Although first described by Dr Eli Moschkowitz in 1924, the pathogenesis of the disorder has only recently begun to be truly understood ( Moschkowitz, 1924). Moschkowitz (1924) reported the presentation of a 16-year-old girl with fever, anaemia, central nervous system impairment, renal dysfunction and cardiac failure. The patient died after 2 weeks, with the autopsy showing hyaline thrombi in the terminal arterioles of the majority of organs – a finding considered to be characteristic of the disorder. Over the years, a classical textbook description of a pentad of symptoms for TTP, consisting of microangiopathy, haemolytic anaemia, thrombocytopenia, fluctuating central nervous system abnormalities, fever and renal impairment, has developed. However, it now appears that the classical pentad is infrequently present in the early stages of disease. Only after there is widespread formation of microthrombi and a resultant impact on various organ systems does the full pentad express. In a series of 135 patients that we have recently reported, all patients had schistocytic haemolytic anaemia and thrombocytopenia ( Rock et al, 1998 ). However, only 30 had fever and 86 had neurological abnormalities. Renal impairment was present in 18% of patients. These findings are supported by the literature; a review of published cases by Ridolfi & Bell (1981) reported that 98% of patients had microangiopathic haemolytic anaemia (MHA), 83% thrombocytopenic purpura, 84% neurological symptoms and 76% had renal disease. We have therefore proposed that TTP should be redefined as a syndrome of Coomb's negative microangiopathic haemolytic anaemia and thrombocytopenia in the absence of other possible causes of these manifestations. In our experience, TTP occurs, for the most part, in previously healthy, relatively young individuals who suffer the sudden onset of a thrombotic disorder in which platelet microaggregates deposit in the arterial microvasculature. In our largest series of patients, 85 were women and 50 were men, with a mean age of 41·7 years (range 18–72) ( Rock et al, 1998 ). Occlusion of small arterioles by platelet plugs containing variable quantities of von Willebrand factor (VWF) characterizes the disease. Electron microscopy has shown the thrombi to be composed of degranulated and altered platelets with little fibrinogen or fibrin ( Asada et al, 1985 ). Although TTP is uncommon, more than 200 cases have been reported each year for the last 5 years to the Canadian Apheresis Group, which captures the data on most TTP patients in the country. This is somewhat higher than the data from a recent US epidemiological study, which indicated an incidence of 3·7 cases per 1 000 000 residents ( Torok et al, 1995 ), but may be reflective of an enhanced awareness of the disorder in Canada because of our several TTP studies and the fact that the incidence of TTP appears to be rising recently. It is possible that the true mortality of the disease is underestimated as the majority of deaths occur within 48 h after presentation ( Brailey et al, 1999 ). Clinical Multiple forms of this syndrome are recognized, ranging from those in which the causative factor has been identified, such as in Shiga toxin-induced TTP/haemolytic uraemic syndrome (HUS), to the more obscure idiopathic forms. Most commonly seen is the acute single-incidence episode which, with appropriate therapy, generally resolves within weeks. Chronic relapsing forms of the disease are also seen. In four such patients, there has been documentation of the presence of unusually large factor VIII multimers in the intervals between relapse ( Moake et al, 1982 ). A juvenile chronic relapsing form has also been seen in which these patients appear to respond to infusion of plasma administered on a periodic schedule established to prevent relapses. Four of these patients have been shown to have a congenital deficiency of a protease which reduces high molecular weight forms of VWF ( Furlan et al, 1998 ). Unfortunately, the initiating agent(s) of most forms of the syndrome is not yet known. Laboratory Generally, the earliest initial presentation is purpura owing to thrombocytopenia. We found that the initial platelet count correlated with mortality: 32% of patients with a platelet count of ≤ 20 × 109/l died compared with 18% with a higher platelet count ( Rock et al, 1998 ). However, others have not found this association ( Brailey et al, 1999 ). Although it is well known that platelet microaggregates are formed, the evidence is variable regarding the activation of platelets in TTP. Several studies have shown an increase in CD62, an activation marker, but other studies have found this not to be the case ( Ahn et al, 1996 ). Inevitably, there is a Coombs'-negative schistocytic anaemia and usually an increased serum lactate dehydrogenase (LDH). It is notable that, in 1998, Cohen et al (1998) characterized the elevated serum LDH and the isoenzyme profile in 10 consecutive patients with classic acute TTP, reporting that the elevated LDH was due to release of LDH5 from a variety of tissues damaged as a result of systemic ischaemia and not the direct result of an increase in isoenzymes LDH1 and 2 attributable to erythrocytes. It is important to recognize that this is not a primary disorder of coagulation. Despite the formation of platelet microthrombi, disseminated intravascular coagulation (DIC) or other overt clinical or laboratory abnormalities of the coagulation system are rarely seen and the prothrombin time (PT), partial thromboplastin time (PTT) and fibrinogen levels are generally not altered. In our 135 patients, the PT and PTT were normal at entry in 128 patients ( Rock et al, 1998 ). Although the patients presented at variable points in their disease, the majority had a quantitative elevation of von Willebrand Factor. This points to the generally accepted concept that TTP is ultimately related to some form of endothelial cell damage. This fact is strongly supported by both histological evidence and altered endothelial function, including decreased prostacyclin production, impaired fibrinolytic activities, release of thrombomodulin and, most recently, the finding of induction of apoptosis in microvasculature endothelial cells by TTP plasma ( Dang et al, 1999 ). Recently, a considerable amount of evidence has accumulated for an immunological basis for at least some forms of TTP. The Italian Co-operative TTP group ( Porta et al, 1999 ) has just reviewed these data, which include evidence showing the presence of anti-C36 antibodies as well as an anti-von Willebrand factor cleaving metalloproteinase. Although the initiating factor(s) of the syndrome is not yet certain, it is apparent that there is a final common pathway of disease manifestation, involving the presence of increased amounts of von Willebrand factor both in the plasma and on the surface of the platelets and the formation and deposition of platelet aggregates in the microvasculature. A number of possible pathophysiologies have been considered to account for this, as follows. The presence of platelet aggregating factors In 1988, Siddiqui & Lian (1988) isolated a 37-kDa protein from plasma of some acute TTP patients. This protein agglutinates platelets through interaction with CD36, a platelet membrane glycoprotein ( Lian et al, 1991 ). Another group has demonstrated a proteolytic enzyme, calpain, in TTP plasma during active phases of the disease ( Murphy et al, 1987 ). Calpain is an intercellular enzyme that is found normally in many tissues, including platelets. It is thought to have a role in proteolysing platelet membrane GP1b and activating GPIIb/IIIa, the membrane binding site for VWF. Calpain also proteolyses VWF, producing characteristic multimer patterns on electrophoresis. This altered VWF is highly reactive with activated platelets and binds to GPIIb/IIIa, and has been shown to cause formation of platelet aggregates ( Moore et al, 1990 ). von Willebrand Factor VWF is normally present in plasma at ≈ 1 U/ml and is involved in platelet adhesion to subendothelium and aggregation. A series of VWF multimers can be demonstrated on electrophoresis. Unusually large molecular weight (ULHMW) multimers are normally found in the Wiebel-Palade bodies of endothelial cells and in platelet alpha granules. These multimers, but not normal plasma VWF forms, can induce aggregation of platelets at high shear stress ( Moore et al, 1990 ). Both quantitative and qualitative abnormalities of plasma VWF have been reported for patients with TTP. These involve the appearance in plasma of very high molecular weight multimers during remission of a chronic relapsing form of TTP ( Moake et al, 1982 ) and loss of the larger forms of normal multimers during acute or relapsing TTP. VWF is clearly important in the pathogenesis of the intravascular platelet aggregation and thrombus formation, as supported by the finding of VWF in the thrombi formed in TTP. Mannucci et al (1989) found that VWF proteolysis was enhanced in acute TTP, but did not lead to loss of the larger multimers. Members of the Italian TTP Registry also found that in familial and sporadic TTP cases there was enhanced fragmentation of VWF during acute disease as demonstrated by a decrease in the native 225-kDa VWF subunits, suggesting that abnormal cleavage of VWF might occur ( Galbusera et al, 1999 ). In our study of 135 patients with acute TTP ( Rock et al, 1991 ), we found VWF to be elevated but with a variable presentation of VWF multimers. Few patients had ULVWF forms, most had a normal distribution of multimers or lacked the larger normal forms. VWF multimers did not correlate with outcome, treatment or any other variable. Certainly, proteolytic enzymes including plasmin, calpain and elastase could all be responsible for the loss of larger multimers which are highly susceptible to proteolytic cleavage. At this time, the mechanisms governing the appearance and interaction of the VWF multimers are unclear, with a variety of reports giving different information. As various patients with chronic relapsing disease are seen to have ULVWF multimers in their plasma in quiescent times, some secondary or quantitative effect may be necessary to initiate the acute disease. Antibodies to platelets and/or endothelial cells An aetiological role for antiplatelet antibodies was first suggested by elevated platelet-associated IgG (PAIgG) which resolved as the patient recovered ( Morrison & McMillan, 1977). It was suggested that this represented immune complexes possibly resulting from the response to bacterial or viral infection which interacted with platelets and caused them to aggregate and release. Subsequently, PAIgG has been reported to be elevated in many TTP patients ( Morrison & McMillan, 1977; Neame, 1980), but, as is the situation with most other markers in TTP, it has been reported as normal in others ( Ansell et al, 1978 ). However, the concept that certain antiplatelet antibodies lead to autothrombotic activity is now well appreciated and exemplified by heparin-induced thrombocytopenia (HIT) and the lupus anticoagulant with thrombosis. Antibody binding to specific platelet epitopes can cause activation, leading to episodic thrombotic events via FcγRII receptors and complement pathway activation. We have found antiplatelet antibodies in the sera of 80 out of 102 patients studied ( Tandon et al, 1994 ). Protein blotting of patients' sera demonstrated a significant number of antibodies directed against CD36. Of these, 23/27 (85%) reacted by immunoprecipitation and 17/28 (60%) by dot blots. Human CD36 is a single-chain integral membrane polypeptide (also known as GPIV and IIIb). It is expressed in platelets as well as many other cells, organs and tissues. Many functions have been described for CD36, including a role as a cell-surface receptor interacting with a large number of ligands and implicated in intercellular signalling transcription ( Huang et al, 1991 ) . Interestingly, CD-36 is characteristically restricted to capillary endothelial cells and is not seen in the endothelium of large vessels ( Sverlick et al, 1992 ), thereby corresponding to the pattern of microthrombi deposition in TTP. We found that these anti-CD36 antibodies activated platelets in most cases and that this reaction was enhanced in the presence of purified VWF. The antibodies appear to be a part of a spectrum of autoantibodies arising from immune system hyperactivity or in response to platelet membrane changes in these TTP patients. Recently, Schultz et al (1998) reported their study in which they also found anti-CD36 antibodies in 8/11 TTP patients using a PAIgG assay and 10/14 by immunoblot. Of note, they report two different forms of CD36 with both the classic 88-kDa form and an 85-kDa (less glycosylated) form. Patient sera reacted more strongly to the latter; monoclonal antibodies to the former. This may help to explain the variable results seen in different studies. We have also found anti-CD36 autoantibodies in patients with the lupus anticoagulant and thrombotic complications, but not those with antiphospholipid antibodies without thrombosis ( Rock et al, 1994 ). Antiendothelial cell antibodies have also been reported in TTP ( Leung et al, 1988 ). Burns & Zucker-Franklin (1982) showed that plasma from three patients with TTP caused time-dependent immune destruction of cultured endothelial cells and spontaneous aggregation of normal platelets in vitro. In our experience, the majority of TTP sera show reactivity by protein blotting against microvascular endothelial cells, again demonstrating interaction with CD36 ( Rock et al, 1998 ). Immune injury to vascular endothelial cells could expose thrombogenic subendothelial surfaces and release VWF from intracellular stores which could then potentiate platelet agglutination through some secondary interaction, such as high shear rate or platelet antibodies. Changes in platelet function Early on it was suggested that patients with TTP have either a deficiency of platelet prostacyclin (PGI2) or of a precursor that promotes the formation of this prostaglandin which normally inhibits platelet adherence, aggregation and release ( Remuzzi et al, 1978 ). In our study ( Rock et al, 1998 ), the level of 6-keto prostaglandin F3α (PGFlα) was measured for the first 77 patients entered into the study. However, the results were highly variable and it was not considered useful to continue this assay. Two other specific platelet abnormalities have been described: Murphy et al (1987) and Rock et al (1988) indicated that the quantity of platelet membrane GPIb was decreased in some patients with TTP. Murphy et al (1987) attributed this to the activation of the enzyme calpain. It should be noted that if platelets are treated in vitro with proteolytic enzymes to remove the glycocalicin (GPIb peptide) these platelets subsequently become hypo- rather than hyperaggregable. However, our finding that absorption of normal VWF multimers from plasma was impaired in three out of three patients with TTP gives support to an alteration in a surface membrane receptor which is either quantitatively or functionally diminished ( Rock et al, 1988 ). This suggestion that VWF is bound to the platelets in TTP has recently been confirmed by others using flow cytometry ( Chow et al, 1998 ). Chow et al (1998) also found that although both single episode and recurrent adult TTP patients had platelet aggregates in their blood and increased VWF on single platelets the platelet α-granule protein P-selectin was not increased in most TTP blood samples. This suggests that the VWF bound to the platelets arises from plasma rather than from the α-granules. As the platelets themselves are not activated, this would appear to indicate that the VWF is coming from the damaged endothelium and binds to the platelets externally without specific activation. This binding of VWF to platelets during disease manifestation may account for some of the variability seen in the plasma pattern for VWF multimers. Antibodies against VWF cleaving metalloproteinase VWF is secreted by endothelial cells as a very large polymer of polypeptides joined by disulphide bonds ( Tsai et al, 1989 ). Subsequently, it is cleaved in the circulation between tyrosine at position 842 and methionine at position 843 by a 200-kDa metalloproteinase into smaller functionally low-adhesive dimers of 176 kDa and 140 kDa respectively ( Dent et al, 1990 ). Furlan et al (1997) reported that two brothers with a relapsing form of TTP had a constitutional deficiency of this VWF cleaving protease without any inhibitor. After plasma exchange, both patients had normalized platelet counts and LDH. The biological half life of the VWF cleaving protease was determined to be 3·3 and 2·1 d in these patients. Recently, Tsai & Lian (1998) in New York and Furlan et al (1998) in Switzerland have independently found that the level of plasma VWF cleaving metalloproteinase activity is greatly reduced or absent during acute TTP episodes, with a return to baseline values after recovery. They have detected an autoantibody directed against the enzyme which appears to account for the lack of metalloproteinase activity during the acute disease. Furlan et al (1999) further reported that although TTP patients had impaired metalloproteinase activity this was not the case for familial or acquired HUS, and suggested that this may provide a useful tool to distinguish between the two diseases. However, another report ( Galbusera et al, 1999 ) of enhanced fragmentation of VWF in many TTP patients suggests factors other than ULVWF forms as being important in TTP. Nevertheless, whatever the form, it is clear that the presence of excess VWF, and its binding to platelets, perhaps as a result of alterations in VWF structure, play a crucial role in the evolution of TTP. In most cases, TTP has an acute onset with no apparent initiating factors. However, whereas the majority of cases appear to be idiopathic, secondary associations with other disorders are well documented. These include Escherichia coli 0157:H7 infection ( Chart et al, 1991 ), pregnancy ( Caggiano et al, 1983 ), hormone contraceptive therapy ( Kwaan, 1987), bone marrow transplantation, chemotherapeutic agents including cisplatin and mitomycin ( Murgo, 1987) and human immunodeficiency virus (HIV) infection ( Ucar et al, 1994 ). Haemolytic uraemic syndrome (HUS) is a disorder overlapping with TTP in that its final manifestation is the formation of platelet microthrombi with a haemolytic schistocytic anaemia, but differing in that the predominant affect is seen in the kidneys with deposition of platelet microthrombi in the glomeruli. Many consider TTP and HUS to be essentially the same disorder, perhaps with different initiating events but both having the same result of formation and deposition of platelet aggregates. In childhood HUS, a specific association with verotoxin producing E. coli has been determined ( Karmali et al, 1983 ). This verotoxin is known to have several direct effects, including the release of high molecular weight VWF from endothelial cells. It acts as an endothelial cell cytotoxin and inhibits protein synthesis. there has been relatively little evidence of an in TTP, although some cases of TTP have been to due to E. coli ( et al, 1990 ) and, in association with infection ( et al, ), TTP has also been seen with infection and The association between syndrome and is well established ( & Kwaan, In to the microangiopathic haemolytic anaemia, thrombocytopenia and renal which are is commonly seen. The is to be characteristic of this variety of it from the other TTP is most commonly in by of the Immune complexes are present in the whereas the is In a recent & (1999) that the presence of immune complexes in ≈ of cases was an of TTP. However, in our early series of 102 patients with TTP, we found immune complexes in all patients, although it was necessary to three different to this suggesting some variability in the of complexes which are This may not be if it is considered that the primary and therefore the may be different in different cases of TTP, but that there is a final common pathway of immune formation and endothelial injury with platelet An important by & (1999) is that and may in patients and may the also appear to have a to TTP ( Murphy et al, 1992 ), although the of chemotherapeutic agents such as mitomycin and the is to The endothelial caused by certain of the chemotherapeutic agents is well as is the effect of certain a has also been with cases of TTP. in the in the platelet function by the binding of to its receptor the effect to d after is to prevent and formation after cardiac ( et al, 1988 ). Recently, et al (1998) reported on patients who TTP during These patients were years who had for than 1 The by which this syndrome is not known. These have out that TTP has been during its first years on the This is an important in that that is related to has now of the antiplatelet in the The presence of the in many cases of or TTP and the of the syndrome in patients these suggests that an may be TTP has been reported to be common in some series ( Torok et al, 1995 ). In our Canadian experience, this is not seen. have been reports of a between and TTP ( et al, 1996 ), and have been to consider this in of the TTP may also during it is more commonly seen in the et al reported that women who are either or in the make of TTP patients, and that the disease during the pregnancy it to in In our series of 102 patients to our patients were or had just with and syndrome may TTP has also been described in association with systemic lupus However, this association is and the may be mechanisms including platelet antibodies may be with ( et al, 1998 ). In series of patients with the lupus anticoagulant and we have demonstrated the presence of antibodies to CD36. These antibodies were not present in other patients that had the lupus anticoagulant but no thrombosis ( Rock et al, 1994 ). many years, TTP an fatal disorder. than a of a after the disease was first it was that plasma infusion was to the course of disease, a fact that was attributed to the presence of a in plasma that the factor responsible for platelet aggregation ( & 1977). Early with plasma infusion to of plasma on the that there might be to the of any factors. The of cell which the to plasma exchange, it possible to large plasma that a plasma could be within a of with of the plasma with appropriate other A review ( & showed that widespread plasma the mortality rate was very In the Canadian Apheresis reported on the results of a in which 102 patients were to either plasma with plasma or plasma infusion on of the first d of the ( Rock et al, 1991 ). The plasma plasma for the first three by plasma The infusion patients 30 h then each We found that plasma was to plasma infusion in the treatment of TTP, with at 1 In the that the of plasma administered in the was than that which was if therapy is the of a large quantity of a plasma may have been more by a larger of this study was to the between two a factor in the of plasma infusion is the which can be by the although not the of the of of of the study was in the of two forms of plasma plasma infusion The fact that some was seen with only with for a or altered factor in the Bell et al have also reported that plasma with has been most primary therapy for TTP. data from several studies using for the first half of the by suggests that this is or than using In our study, we the of plasma which is the plasma after and This therapy was first suggested by & and is on the that high molecular weight VWF multimers are involved in the of the disease. It is that as is relatively in the higher molecular weight multimers of factor VIII this could provide the not to the of ULVWF forms. A of the VWF multimer patterns in and the from that plasma using is shown in The quantity of VWF is decreased in compared with of plasma through the we have found an of of VWF in and in This with blood with from group patients having the level of VWF ( et al, 1998 ). that we have out with Dr Tsai New that the metalloproteinase that has described is present in in the same as in plasma – it is not in the VWF multimer patterns of and Two different were from to plasma platelet was from normal blood and was to the of the Canadian with the for VWF multimers were using a and was from Protein were with and with As an initial study, patients with TTP who had not to plasma with were with

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