Plasma beta-thromboglobulin: differentiation between intravascular and extravascular platelet destruction.

To elucidate the usefulness of beta-thromboglobulin (beta TG) in the differentiation of the mechanism of thrombocytopenia, plasma beta TG concentration was measured in one patient with amegakaryocytic thrombocytopenia, four patients with autoimmune thrombocytopenia (ATP), two patients with thrombotic thrombocytopenia (TTP), and one patient with thrombocytopenia secondary to disseminated intravascular coagulation (DIC). Plasma beta TG was not measurable in amegakaryocytic thrombocytopenia, was normal in ATP, and was increased in TTP and DIC. These data indicate that in thrombocytopenic patients, increased plasma beta TG concentration may result from intravascular platelet consumption with release of platelet constituents in contrast to extravascular platelet destruction by the macrophage-monocyte system.     

Decreased von Willebrand factor protease activity associated with thrombocytopenic disorders

Recent studies investigating thrombotic thrombocytopenic purpura (TTP) have implicated abnormal plasma von Willebrand factor (vWF)-cleaving metalloprotease activity in this disorder. It has been proposed that a metalloprotease cleaves unusually large (UL) multimers of vWF, which enter the circulation from the endothelium. Abnormal metalloprotease activity could result in ULvWF, which could participate in TTP. However, the diagnostic specificity of abnormalities in the plasma metalloprotease activity has not been established. A prospective study of vWF protease activity was performed using samples from 20 healthy controls, 20 patients with acute TTP, 20 patients with immune idiopathic thrombocytopenic purpura (ITP), 10 patients with disseminated intravascular thrombocytopenia (DIC), 10 patients with systemic lupus erythematosus (SLE,) and 5 thrombocytopenic patients with leukemia. Studies were performed blinded to the diagnosis. Samples from hospitalized patients with normal platelet counts were also tested. The vWF digests and multimer analysis were done using previously described methods. Six laboratory personnel independently scored each of the multimer gels. Reduced protease activity was observed in 9 of 20 patients with TTP. Reduced activity was also observed in 6 of 20 patients with ITP, 6 of 10 patients with DIC, 5 of 10 patients with SLE, 1 of 5 patients with leukemia, 2 of 20 healthy controls, and 3 of 25 hospitalized patients. This study indicates that abnormalities of vWF protease activity are not restricted to patients with the diagnosis of TTP.     

Extracorporeal Plasma Treatment in Thrombotic Thrombocytopenic Purpura and Hemolytic Uremic Syndrome: A Review

Abstract: This review summarizes the state of the art of apheresis in hemolytic uremic syndrome (HUS) and in thrombotic thrombocytopenic purpura (TTP). Both entities are characterized by thrombotic microangiopathy, hemolytic anemia, and thrombocytopenia. While HUS often presents with renal insufficiency, cerebral involvement is more common in TTP. Recently, in TTP, a primary or secondary lack of activity of a von Willebrand factor (vWF) degrading enzyme was made responsible for the presence of unusually large vWF multimers causing platelet aggregation and thrombus formation in the microvasculature. In contrast, in familial HUS, a factor H deficiency with uninhibited complement activation seems to play a role. Therapeutic plasma exchange (TPE) using fresh frozen plasma or cryosupernatant as the substitution fluid is indicated in acute TTP and atypical HUS without antecedent diarrhea. As a rule, it will show good effectiveness, especially in the former entity. HUS in pregnancy should be treated by instant delivery whereas postpartum HUS may resolve using protracted courses of TPE. In contrast, in thrombotic microangiopathy after bone marrow transplantation as well as in HUS due to cancer, mitomycin C, or after renal transplantation, TPE is of questionable value and indicated only as a last resort treatment.     

Pathophysiology and treatment of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS)

The central pathogenic feature of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) is the formation of platelet aggregates, perhaps by damaged endothelial cells. The evidence for endothelial cell damage has been supported by multiple findings, including the harmful effects of TTP/HUS plasma, which induces endothelial cell apoptosis. Ultra-large multimers of von Willebrand factor (vWf), which activates platelets, are found in TTP/HUS patients, presumably after being released into circulation from damaged endothelial cells. It has been reported that loss or dysfunction of the vWf-cleaving protease is related to the development of acute or chronic TTP/HUS. Detection of platelet activation in TTP/HUS patients, which previously had been difficult, is now possible with a particle-counting technique using light scattering. TTP/HUS occurring after bone marrow transplantation (BMT), similar to that in classic TTP/HUS, appears to represent a separate facet of the disease. We observed that BMT-TTP/HUS might be predicted at an early stage by determining any increase in plasma interleukin-12 at the time of leukocyte recovery. It is known that plasma treatment is effective for TTP/HUS patients; we found that a high-molecular-weight fraction (HMW-F) of plasma is effective in treating chronic TTP/HUS patients. HMW-F of plasma may contain the main factor necessary for improvement of TTP/HUS syndrome.     

Anti-CD36 autoantibodies in thrombotic thrombocytopenic purpura and other thrombotic disorders: identification of an 85 kD form of CD36 as a target antigen

The presence of anti-CD36 antibodies in plasma of patients with thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), and heparin-induced thrombocytopenia without/with thrombosis (HIT/HITT) has been examined by immunoblots, and a monoclonal antibody capture assay, the platelet-associated IgG characterization assay (PAICA). Results with PAICA showed that 73% (8/11) of patients with TTP were positive, and 71% (10/14) by immunoblots. With ITP, 20% (6/30) were positive by PAICA and 19% (3/16) by immunoblots; HIT, 30% (3/10) were positive by PAICA and 60% (6/10) by immunoblot; HITT, 50% (2/4) by PAICA and 100% (4/4) by immunoblot. Purification of CD36 by fast protein liquid chromatography (FPLC) from Triton X-100 extracts of normal platelet membranes resulted in the isolation of two different forms: the classic 88 kD form, and a second, lighter 85 kD form. Our data indicated that the patients' plasma autoantibodies reacted strongly with the 85 kD form. Conventional monoclonal and polyclonal antisera produced to the 88 kD form reacted strongly with the 88 kD form but weakly with the 85 kD form. These results confirm the possible importance of anti-CD36 antibodies in the pathophysiology of TTP and other thrombocytopenias and demonstrate the presence of a previously unrecognized target antigen for these antibodies.     

Clinical analysis on thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, with plasma level of cytokine

We studied 10 patients with thrombotic thrombocytopenic purpura (TTP) and 5 patients with hemolytic uremic syndrome (HUS). Common cold symptoms were observed in 2 with TTP and 3 with HUS, and SLE was noted or suspected in 3 with TTP, and the onset was after operation in on with TTP and one with HUS. All TTP patients had coma and high fever. Renal failure was noted in 3 with TTP and their prognosis was poor. Seven patients with TTP and 4 patients with HUS survived. Autoantibody was highly positive in TTP but slightly positive in HUS. High molecular weight multimer of von Willebrand factor was decreased in 3 of 6 with TTP, platelet aggregating factor was positive in 4 of 6 with TTP, and microthrombus was observed in 7 of 8 with TTP. Tumor necrosis factor was increased in 5 of 9 with TTP and HUS, Interleukin-1 beta was increased in all TTP and HUS patients, and soluble interleukin 2 receptor and interferon alpha were also increased. Although plasma exchange was generally effective, some patients required combination therapy with steroids. We speculated that an autoimmune mechanism was involved in the on onset of TTP.     

Thrombocytopenic conditions-autoimmunity and hypercoagulability: commonalities and differences in ITP, TTP, HIT, and APS

Immune thrombocytopenia purpura (ITP), thrombotic thrombocytopenia purpura (TTP), heparin-induced thrombocytopenia (HIT), and antiphospholipid syndrome (APS) are clinical conditions associated with significant morbidity and mortality. These well-defined clinical syndromes have in common several properties: (1) their pathogenesis is immune mediated, specifically by autoantibodies; (2) thrombocytopenia is a hallmark in these four conditions; (3) except for the case of ITP, platelet and endothelial cell activation occurs in TTP, HIT, and APS, resulting in a prothrombotic state and an increased risk of thrombosis. Although these four immune-mediated syndromes are well-defined diseases, several case reports and studies have documented the association of two diseases in the same patient, illustrating the concept of the kaleidoscope of autoimmunity.     

Plasma sFas and sFas ligand levels in patients with thrombotic thrombocytopenic purpura and in those with disseminated intravascular coagulation.

Fas, a member of the tumor necrosis receptor superfamily, is 36 kD surface protein containing a single transmembrane region and induces apoptosis by Fas-Fas ligand binding. Soluble Fas (sFas) is produced as the form lacking 21 amino acid residues containing the transmembrane domain by alternative splicing. We found that the plasma sFas levels of 33 patients with thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), 19 patients with disseminated intravascular coagulation (DIC), and 10 non-DIC patients with multiple organ failure (MOF) were significantly higher than those of 21 non-DIC patients without organ failure and those of 25 healthy volunteers. The plasma sFas ligand levels of the TTP/HUS patients, the DIC patients, and the non-DIC patients with MOF were significantly higher than those of the non-DIC patients without organ failure and those of the healthy volunteers. The plasma sFas levels were significantly correlated with the plasma sFas ligand levels in all subjects. The plasma thrombomodulin (TM) levels were increased significantly in the TTP/HUS patients, the DIC patients, and the non-DIC patients with MOF compared with the levels of the non-DIC patients without organ failure and the healthy volunteers. The plasma sFas antigen levels were correlated significantly with the plasma TM levels in all subjects. These findings suggest that the increases of sFas and sFas ligand that cause apoptosis might be related to the vascular endothelial cell injuries in TTP and DIC with organ failure.     

Thrombotic thrombocytopenic purpura plasma enhances platelet–leucocyte interaction in vitro

In thrombotic thrombocytopenic purpura (TTP), intravascular platelet aggregation and formation of platelet-rich thrombi impair the microcirculation. TTP plasma has been shown to induce aggregation of normal platelets in vitro . The present study investigates the formation of activated platelet aggregates (aPAg) induced by TTP plasma, with particular attention to their binding to leucocytes (LPAg). Results were compared with the effects of plasmas from normal controls (CTL) and from patients with immune thrombocytopenic purpura (ITP) or thrombosis (THR). Following addition of test plasma to normal whole blood (WB), aPAg and LPAg were assayed by flow cytometry using mAbs against CD41 (platelet marker), CD62p (platelet activation marker) and CD45 (pan-leucocyte marker). Compared to control plasma, TTP plasma was more potent than ITP or THR plasma in increasing aPAg; only TTP plasma significantly promoted leucocyte binding to give increased LPAg. Prior removal of neutrophils (PMN) from WB by beads coated with anti-CD15 mAb largely prevented formation of aPAg and LPAg. However, TTP plasma added to normal platelet-rich plasma significantly increased aPAg, which suggested possible hindrance of aPAg formation by erythrocytes and other leucocytes in PMN-depleted blood. We concluded that TTP plasma was most potent in the induction of aPAg and unique in promoting LPAg formation in WB. Neutrophils, and not other leucocytes, appear to be essential for LPAg formation. Enhanced PMN–platelet interaction in the microcirculation may facilitate platelet adhesion to vessel walls and promote the formation of platelet-rich microthrombi in TTP.     

Plasma thrombomodulin as a marker of vascular disorders in thrombotic thrombocytopenic purpura and disseminated intravascular coagulation.

Plasma thrombomodulin (TM) levels were significantly elevated at disease onset in patients with thrombotic thrombocytopenic purpura (TTP) and disseminated intravascular coagulation (DIC), but was not in those with essential thrombocythemia and idiopathic thrombocytopenic purpura. However, in patients with TTP and DIC, TM levels decreased significantly after they achieved complete remission. In both TTP and DIC patients, plasma TM levels at onset in those with poor prognosis were higher than that in those with good prognosis. Among DIC patients, the plasma TM level was higher in those with organ failure than in those without, but there were no differences among patients with various underlying diseases associated with DIC. It is speculated that the plasma TM level reflects damage to vascular endothelial cells or organ failure and that it is useful in assessing prognosis for patients with DIC and TTP.     

Coagulation changes associated with the hemolytic uremic syndrome

The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, acute renal failure, and thrombocytopenia. The pathological correlate is thrombotic microangiopathy of glomerular capillaries and arterioles in the kidneys and almost every other organ. The presence of platelet thrombi without extensive soluble coagulation system activation is a constant feature of HUS and thrombotic thrombocytopenic purpura (TTP). Damage to the endothelial cell seems to be a central event in the pathogenesis of HUS and TTP, resulting in loss of fibrinolytic properties and subsequent thrombotic occlusion of the microvasculature. According to earlier and recent studies, a variety of hemostatic alterations have been described. Among the many findings, low platelet counts, increased von Willebrand's factor (vWF), and normal fibrinogen are almost invariably observed. The dubious long-term outcome, even of postdiarrheal HUS, which is believed to have a more favorable prognosis than HUS of other etiopathogenic origin, necessitates further investigation of the pathophysiology of thrombotic microangiopathy and meticulous reevaluation of treatment strategies aimed at interfering with the process of thrombosis early in the disease course. The intention of this article is to highlight findings possibly relevant for disease management and to give an overview of the putative pathomechanisms involved.     

Thrombotic thrombocytopenic purpura/haemolytic uraemic syndrome associated with clopidogrel: report of two new cases

Clopidogrel has been reported to be safe and effective in reducing vascular events. Nevertheless, there is growing evidence that clopidogrel may cause thrombotic thrombocytopenic purpura/haemolytic uraemic syndrome (TTP/HUS). This association has been debated, since in several cases alternative causes could not be excluded. Two new cases of TTP/HUS associated with clopidogrel are reported here. After discontinuation of clopidogrel and treatment with plasma exchange, both patients had a complete and sustained recovery from TTP/HUS. These cases corroborate previous observations that clopidogrel may indeed be a rare cause of TTP/HUS.     

Thrombotic microangiopathies and HIV infection: Report of two typical cases, features of HUS and TTP, and review of the literature

Summary Haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are thrombotic microangiopathies increasingly reported in patients with HIV infection. However, characteristic features of thrombotic microangiopathies associated with HIV disease have not been defined yet. The typical courses of HUS and TTP in two patients are presented. The data as well as the analysis of cases published in the literature demonstrate the association of thrombotic microangiopathies with late-stage HIV disease. Moreover, differences between HUS and TTP can be detected. Patients with HUS present with more severe immunologic deterioration. Although clinical symptoms are fewer, HUS implicates a very poor prognosis. Life expectancy rarely exceeded 1 year after diagnosis. HUS and TTP should therefore be added to the international AIDS classification.     

Elevated endothelial microparticles in thrombotic thrombocytopenic purpura: findings from brain and renal microvascular cell culture and patients with active disease

Endothelial injury is believed to be a key initiating event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), leading to platelet activation and formation of platelet-rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and clinical assays to rapidly and reliably monitor endothelial damage are not readily available. Using flow cytometry, we measured endothelial microparticles (EMPs) generated from cultured renal and brain microvascular endothelial cells (MVECs) during activation and apoptosis, and evaluated the effect of TTP plasma on them. EMPs were measured using positivity for monoclonal antibodies (mAbs) CD31 and CD51, and their procoagulant activity was assessed using a Russell viper venom assay. Both cell lines generated procoagulant EMPs when cultured with inducers of activation (tumour necrosis factor alpha; TNF-alpha) or apoptosis (mitomycin C). TTP plasma induced a five- to sixfold increase of EMP generation and a two- to threefold increase of procoagulant activity in cultured brain and renal MVECs. TTP plasma induced a threefold and 13-fold increase of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, respectively, on renal MVECs. Procoagulant activity tended to parallel EMP numbers. The effect of TTP plasma on cell viability was similar to that of TNF-alpha, implying that it induced activation rather than apoptosis. Control plasma and idiopathic thrombocytopenic purpura (ITP) plasma had little effect. In the clinical study, EMP assay of blood from acute TTP patients showed levels markedly elevated compared with normal controls, but values returned to normal in remission. In conclusion, TTP plasma activated and induced injury to MVECs in culture, judged by production of EMP and expression of activation markers. Released procoagulant EMP may play a role in the pathogenesis of TTP. Assay of EMP may be a useful marker of disease activity and endothelial injury in TTP and possibly other thrombotic disorders.     

Haemolytic-uraemic syndrome during severe lupus nephritis: efficacy of plasma exchange

Systemic lupus erythematosus (SLE) has been described as a cause of thrombotic microangiopathy, especially thrombotic thrombocytopenic purpura (TTP). Haemolytic-uraemic syndrome (HUS) is less frequent in SLE. We report a case of such an association during an episode of severe lupus nephritis in a young woman, who was successfully treated with steroids, cyclophosphamide and especially plasma exchange with plasma replacement. This report highlights the importance of recognising atypical HUS in SLE patients by looking for schistocytes in case of haemolytic anemia with a negative antiglobulin test, in order to begin plasma exchange.     

Thrombotic thrombocytopenic purpura/haemolytic uraemic syndrome: a new index predicting response to plasma exchange

Despite the favourable response of thrombotic thrombocytopenic purpura/haemolytic uraemic syndrome (TTP/HUS) to plasma exchange, an early level of mortality persists. Non-response has been associated with a low frequency of exchange. The Rose index of TTP/HUS severity, occasionally used to predict the response of TTP/HUS to plasma exchange, remains unsatisfactory. The purpose of this study was to develop a new index predicting response of TTP/HUS to plasma exchange and to compare it with the Rose index. Retrospective analysis of 171 cases of TTP/HUS from 39 apheresis units across Canada between 1980 and 2001 was conducted. Logistic regression analysis was used to derive a model predicting 6-month mortality from presenting characteristics. The reduced model contained age >40 years, haemoglobin <9.0 g/dl and the presence of a fever at presentation. Gender, platelet count, creatinine and neurological signs were not part of the final model. This model predicted 13.4% of outcome variance. Predictive scores of 0, 2, 4 and 6 correlated with 6-month mortality rates of 12.5%, 14.0%, 31.3% and 61.5% respectively in our source population. This simple model may help identify those patients who would benefit from higher treatment intensity.     

Drug-induced thrombotic thrombocytopenic purpura/hemolytic uremic syndrome: a concise review

An extensive variety of drugs have been associated with thrombotic thrombocytopenic purpura and hemolytic uremic syndrome (TTP/HUS). Although a direct causal effect has usually not been proven, the cumulative evidence linking several drugs with TTP/HUS is strong. This paper reviews several categories of drugs including antineoplastics, immunotherapeutics and anti-platelet agents that have been reported to induce TTP/HUS. The pathogenesis of drug-induced TTP/HUS and the effectiveness of treatment regimens are also reviewed. A consensus on diagnostic criteria to accurately and consistently diagnose drug-induced TTP is needed.     

Thrombotic thrombocytopenic purpura/hemolytic uremic syndrome secondary to pancreatitis

Pancreatitis is a rare (～2.0%) complication of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS). The opposite finding has rarely been reported. We present a case of an 18 years old obese male with alcohol associated pancreatitis (amylase 840 IU/L) who three days after onset, as the pancreatitis subsided (amylase 341 U/L), developed TTP/HUS. The TTP/HUS was marked by oliguria and severe renal failure (creatinine 1,326 μmol/L), was treated with daily plasma exchanges, obtained a complete response, and recovered renal function (creatinine 115 μmol/L). Similarly, in six of seven other cases from the medical literature the TTP/HUS occurred within 2–3 days of the onset of pancreatitis.     

Rituximab for the treatment of refractory idiopathic thrombocytopenic purpura (ITP) and thrombotic thrombocytopenic purpura (TTP): report of three cases

Three patients (one with idiopathic thrombocytopenic purpura [ITP] and two with thrombotic thrombocytopenic purpura [TTP]) were treated with rituximab (anti-CD20 chimeric antibody) at a dose of 325 mg/m2 administered weekly after they failed standard therapies. The patient with ITP who did not respond to steroids and anti-D antibody administration achieved augmentation of her platelet counts up to 180 x 10(3)/microL after four doses of rituximab. Six months later, when her counts started to decrease, she received maintenance therapy with an additional course of 4 standard doses of antibody that resulted in consolidation of her platelet counts around 100 x 10(3)/microL. One patient with TTP and concurrent idiopathic nephropathy who was previously treated with plasmapheresis, steroids, and vincristine improved only after 4 weekly administrations of the antibody. Moreover, his nephrotic-range proteinuria resolved after he received rituximab. The other patient with chronic TTP who still relapsed after splenectomy received 5 doses of rituximab with concomitant plasmapheresis. His thrombocytopenia improved slowly, and his platelet count stabilized at 300 x 10(3)/microL. All three patients showed evidence of response to anti-CD20 antibody with improvement in clinical outcome as well as augmentation of platelet counts to normal levels. We conclude that rituximab is a useful immunomodulating adjunct in the treatment of refractory ITP and TTP.     

Plasmapheresis for thrombotic thrombocytopenic purpura following bone marrow transplantation.

Recognition of thrombotic thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS) following bone marrow transplantation (BMT) has increased in recent years. The use of plasma exchange has greatly improved the outlook of de novo TTP. Fewer data are available on its use in post-BMT TTP but small uncontrolled series showed poor results with low response rates. We present here a case of a young patient who developed manifestations of TTP 10 months after BMT with complete recovery following treatment with plasma exchange for 1 month. This case suggests that plasma exchange could be life saving and should be tried in every patient with post-BMT TTP.