An update on the pathogenesis and management of acquired thrombotic thrombocytopenic purpura

PURPOSE OF REVIEW: Thrombotic thrombocytopenic purpura, a clinical syndrome characterized by thrombocytopenia and microangiopathic haemolytic anaemia, was almost universally fatal until the introduction of plasma exchange therapy in the 1970s. Current outcomes have improved dramatically with the initiation of prompt plasma exchange, a treatment routinely used without any real understanding of why it is effective. RECENT FINDINGS: Recent advances suggest that a deficiency of a specific plasma metalloprotease, responsible for the physiological processing of von Willebrand factor multimers, plays a substantial role in the pathogenesis of congenital and acquired idiopathic thrombotic thrombocytopenic purpura. The von Willebrand factor-cleaving protease has now been identified as a new member of the ADAMTS family of metalloproteases, designated ADAMTS13. The acquired form of thrombotic thrombocytopenic purpura is associated with inhibitory autoantibodies against ADAMTS13, and the congenital chronic relapsing form is caused by mutations in the ADAMTS13 gene, resulting in a constitutional deficiency. Plasma exchange has been proved to be the most important therapy in thrombotic thrombocytopenic purpura, but clinical data for adjunctive therapies, such as corticosteroids, antiplatelet drugs and other immunosuppressive agents often used in combination with plasma exchange, are less well defined. SUMMARY: Recent advances in our understanding of the pathological mechanisms of thrombotic thrombocytopenic purpura not only provide a rationale for the previously empirical plasma exchange therapy (removal of the inhibitory antibodies and replacement of the deficient protease from the plasma infused), but may also help in developing more rational and targeted treatment strategies. This review discusses the clinical presentation, pathophysiology and current management of thrombotic thrombocytopenic purpura.     

A new approach to detect reticulated platelets stained with thiazole orange in thrombocytopenic patients

Recent studies have shown that reticulated platelets stained with Thiazole Orange (T.O.) are useful markers for thrombopoiesis. The percentage of T.O. positive platelets tends to be inconsistent using the original method, especially when the peripheral blood platelet count is very low. We measured T.O. positive platelet levels in patients with severe thrombocytopenic disorders, using concentrated platelet-rich plasma and carrying out a two-color analysis involving T.O. and an anti-glycoprotein IIb/IIIa monoclonal antibody. This method allowed us to obtain consistent T.O. positive platelet rates in patients with thrombocytopenia whose platelet counts were below 20 approximately 30×10⁹/l. By this method, the T.O. positive rates of platelets from idiopathic thrombocytopenic purpura patients were found to be significantly higher than in the control group. The T.O. positive rates of other thrombocytopenic disorders were similar to those of the control group. These results are consistent with those previously reported. We conclude that our technique of measuring of T.O. positive platelets using platelet-rich plasma is useful for analyzing severe thrombocytopenic disorders.     

Management of children with bleeding disorders

Bleeding and easy bruising in children may be due to one of the many inherited or acquired disorders of hemostasis. An understanding of normal hemostasis is essential for evaluation of a child with abnormal bleeding. A complex interaction between vascular endothelium, von Willebrand factor and procoagulant protein Factor I to XIII results in formation of a fibrin clot at the site of injury and bleeding stops. Conditions causing quantitative or qualitative defects of platelets, von Willebrand factor or various procoagulant protein will result in abnormal bleeding. Patients with hemophilia experience more severe bleeding and are diagnosed early in life. von Willebrand disease on the contrary has a variable presentation and therefore the diagnosis is delayed in some cases. Morbidity and mortality due to bleeding may be reduced with appropriate management in the majority of patients with bleeding disorders. A systematic approach to a patient with bleeding is discussed. Management of hemophilia and von Willebrand disease is also reviewed.     

Immunoglobulin for concurrent Guillain-Barré and immune thrombocytopenic purpura

Guillain-Barré syndrome, or acute inflammatory demyelinating polyradiculoneuropathy, and immune thrombocytopenic purpura are both autoimmune disorders thought to result from molecular mimicry in response to an antecedent introduction of foreign antigen. Guillain-Barré syndrome is an ascending motor paralysis that can lead to respiratory compromise. Immune thrombocytopenic purpura is an isolated disorder of platelet destruction leading to mucocutaneous bleeding. Guillain-Barré does not typically occur with other autoimmune disorders, and concurrent Guillain-Barré and immune thrombocytopenic purpura has only rarely been reported. We present a patient with both conditions who experienced prompt resolution of neurologic and hematologic sequelae after intravenous immunoglobulin therapy was initiated within 12 hours of presentation. The case provides further evidence that Guillain-Barré syndrome and immune thrombocytopenic purpura can occur simultaneously, possibly caused by a similar pathogenic mechanism, as well as suggesting that the prompt initiation of intravenous immunoglobulin is an effective monotherapy leading to prompt resolution of both conditions and prevention of further sequelae.     

A review of current methods for assessing hemostasis in vivo and introduction to a potential alternative approach

A validated method for assessing hemostasis in vivo is critical for testing the hemostatic efficacy of therapeutic agents in preclinical animal models and in patients with inherited bleeding disorders, such as von Willebrand disease (VWD) and hemophilia A, or with acquired bleeding disorders such as those resulting from medications or disease processes. In this review, we discuss current methods for assessing hemostasis in vivo and the associated challenges. We also present ARFI-Monitored Hemostatic Challenge; a new, potentially alternate method for in vivo hemostasis monitoring that is in development by our group.     

Pharmacology and clinical use of recombinant activated factor seven in neurosciences

Recombinant activated factor VII (rFVIIa, NovoSeven, NovoNordisc, Danemark) has been approved for the treatment of patients with hemophilia with inhibitors, further indications, at least in some countries, include the treatment of factor VII deficiency and Glanzmann thrombasthenia refractory to conventional therapy. Apart from these indications, the agent is increasingly used for the treatment of severe and potentially life-threatening bleeding manifestations, irrespective of the underlying hemostatic abnormality. The agent has successfully been used for the treatment of both inherited and acquired coagulopathies as well as thrombocytopathia or thrombocytopenia, however, most information on off-label use derives from case reports and retrospective studies and therefore publication bias can-not be excluded. In this review, we focus on the use of rFVIIa for the treatment of spontaneous and perioperative intracranial hemorrhage as well as trauma patients. We review the current knowledge regarding the physiology of hemostasis, the pharmacology of rFVIIa, and its clinical use in neurosciences. Further studies are urgently needed to define the efficiacy and safety of recombinant activated factor VII in patients without hemophilia, factor VII deficiency, or Glanzmann thrombasthenia. At time, its use can be justified in life-threatening bleeding situations refractory to conventional treatment.     

Inhibitors in mild/moderate haemophilia A: an update

The development of inhibitors in patients with mild/moderate hemophilia A is an increasingly recognized occurrence and is manifested by the patients' bleeding pattern becoming more severe. Inherited (hemophilia genetic mutations) and acquired (type and delivery of factor VIII replacement therapy) factors have been associated with an increased likelihood of developing factor VIII inhibitors. Although the use of bypassing agents (i.e. activated prothrombin complex concentrates and recombinant factor VII activated) has been demonstrated to be effective in controlling bleeding episodes in patients who develop factor VIII inhibitors, the limited data available in the literature are insufficient to determine the optimal approach to the eradication of inhibitors (i.e. immune tolerance induction, immunosuppression or both) for this group. Particular attention should be directed to the prevention of this complication in those patients with mild/moderate hemophilia recognized to be at increased risk of developing a factor VIII inhibitor. In conclusion, large prospective trials are warranted in order to elucidate the many still unclear pathogenic and therapeutic aspects of the development of inhibitors in patients with mild/moderate hemophilia A.     

Central nervous system inflammatory demyelination after rituximab therapy for idiopathic thrombocytopenic purpura

A 53-year-old female developed unilateral shoulder girdle pain, entire body paresthesiae and imbalance starting 10 days after completing a course of rituximab for chronic idiopathic thrombocytopenic purpura. Neuroimaging studies showed inflammatory demyelinating lesions of the cervical spinal cord and cerebellum.Immunocytochemistry of spinal fluid lymphocytic pleocytosis showed an absolute B cell depletion and a polyclonal/inflammatory T cell response. Intravenous immunoglobulin infusions were associated with clinical and radiological improvements. Anti-CD20-therapy possibly resulted in an imbalance between B cell and T cell populations in the central nervous system, and may have triggered a predominantly cell-mediated immune attack against unidentified nervous system antigens.     

Challenges in the laboratory analyses of bleeding disorders

Standard coagulation assays such as the activated partial thromboplastin time and prothrombin time are sufficient to detect deficiencies in coagulation factors contributing to the intrinsic and extrinsic pathways, respectively. Deficiencies in factors VIII and IX can also be detected by one-stage and two-stage clotting assays. While these assays are instrumental in assessing the initiation of clot formation, sufficient formation of a clot is a continuous process that may be better studied by the use of a global hemostasis assay. Several global assays are currently being studied, including the thrombin generation assay, thromboelastography, clot waveform analysis, clot formation and lysis (CloFAL) assay, euglobulin clot lysis assay, thromboplastin generation assays and simultaneous thrombin and plasmin generation assays. This review will concentrate on the thrombin generation test, thromboelastography, the activated partial thromboplastin time waveform analysis and the CloFAL which measure the production of thrombin, as well as the kinetics of clot formation. As such, these global assays can provide greater insight into deficiencies in the mechanisms mediating hemostasis and fibrinolysis in patients with hemophilia, other bleeding disorders or even thrombophilia. These assays have been shown to be clinically relevant for assessing the response to treatment with bypassing agents (recombinant activated FVII and plasma-derived prothrombin complex concentrate) used for the hemostatic control of acute bleeding in patients with congenital hemophilia A/B. The limitations of standard coagulation assays and the use of global hemostasis assays to assess bleeding disorders and the clinical efficacy of bypassing agents will be discussed in detail.     

The use of rFVIIa in non-haemophilia bleeding conditions in paediatrics. A systematic review

From its original envisioned use in patients with hemophilia and inhibitors, recombinant factor VIIa has been increasingly used in a variety of non-hemophilia bleeding/hemorrhagic situations with great efficacy. Most of the reported work has been in adult patients. This paper sets out to review its use in the pediatric non-hemophilia patients and the varied conditions it has been tried and used. Most of the published literature has shown that this agent is efficacious, safe and can be used as an adjunctive measure in the achievement of hemostasis. However, most of the published work is mainly anecdotal, case reports or small series. Randomized trials in children are eagerly awaited.     

An open-label trial of rituximab (Rituxan®) in multifocal motor neuropathy

Multifocal motor neuropathy (MMN) is an acquired demyelinating motor neuropathy. Intravenous immunoglobulin (IVIg) has been found to be safe and effective. Rituximab, a genetically engineered monoclonal antibody against CD-20 antigen, has been reported effective in immune disorders including MMN. We performed an open-label trial to determine if rituximab would reduce the IVIg requirement, disability, and impairment, and be safe in patients with MMN. Six MMN patients, who were on periodic IVIg treatments, received two doses of IV rituximab (1,000 mg) given 2 weeks later. Assessments were performed at baseline and at 2, 4, 6, 8, 10, and 12 months. The primary outcome was total amount of IVIg used during the 12-month study compared to the 12 months prior. Secondary outcomes included changes in Medical Research Council (MRC) sum scores, grip strength, disability and handicap scores, and safety. There was no significant change in IVIg use, MRC sum score, grip strength, overall disability sum score, or Rotterdam handicap scale. One patient developed a hypersensitivity reaction during the first infusion that responded to adjustments in the infusion rate and treatment with diphenhydramine and acetaminophen. We conclude that rituximab can be safely given to people with MMN but in this pilot study we were unable to reduce the amount of IVIg required, at least in the regimen used.     

Psychogenic purpura, idiopathic thrombocytopenic purpura, and platelet dysfunction in the same patient

A patient whose peculiar and painful purpura seemed to be strongly related to psychogenic factors is described. The skin bleeding pattern in this patient was consistent with the diagnosis of psychogenic purpura (autoerythrocyte sensitization). In addition, idiopathic thrombocytopenic purpura and platelet storage pool deficiency were present. These somatic conditions are considered to be of minor importance in the pathogenesis of this hemorrhagic syndrome. The association of these various types of bleeding disorders in the same patient has not been previously described.     

Novel recombinant glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 and variants for assessment of anti-ADAMTS13 autoantibodies in patients with thrombotic thrombocytopenic purpura

Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). We report here a novel cell-based assay using glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 or variants expressed on cell membrane for assessment of autoantibodies in patients with TTP. We showed that IgGs from all 26 patients with acquired TTP bound to cells expressing a GPI anchored full-length ADAMTS13 (gFL) and a variant truncated after the spacer domain (gS). Also, IgGs from 25/26 (96.7%) of these TTP patients bound to cells expressing a GPI-anchored C-terminal fragment, TSP1 2-8 plus CUB (gT2C). In contrast, none of the 20 healthy blood donors showed detectable binding of their IgGs to the cells expressing gFL, gS, and gT2C. A moderate, but statistically significant correlation was observed between plasma concentrations of anti-ADAMTS13 IgG and positive cells expressing gFL (r=0.65), gS (r=0.67), and gT2C (r=0.42). These results suggest that the microtiter-plate assay and the cell-based assay may detect differential antigenic epitopes. Moreover, antigens clustered on cell membranes may enhance antibody binding affinity, thereby increasing analytical sensitivity. Finally, our assay was able to determine kinetic changes of plasma levels of anti-ADAMTS13 IgGs in TTP patients during plasma therapy. Together, our findings suggest that the novel cell-based assay may be applicable for rapid identification and mapping of anti-ADAMTS13 autoantibodies in patients with acquired TTP.     

Addition of rituximab to standard therapy improves response rate and progression-free survival in relapsed or refractory thrombotic thrombocytopenic purpura and autoimmune haemolytic anaemia

Treatment of relapsed or refractory autoimmune mediated haemolytic syndromes, such as autoimmune haemolytic anaemia (AIHA) and thrombotic thrombocytopenic purpura (TTP), represents a therapeutic challenge. Here we report on our experience with the monoclonal anti-CD20 antibody rituximab (R) compared to standard treatment in these diseases. Patients with non-familial TTP or AIHA and no underlying malignancy were included in our analysis. Safety and efficacy of R-treatment were compared to results obtained in standard treatment approaches. Altogether, 27 patients were analyzed, comprising 15 patients with TTP and 12 patients with AIHA. The patients' average age at the time of diagnosis was 54 years. Eleven patients received antibody treatment (8 TTP, 3 AIHA). No acute or late WHO grade III/IV toxicity associated with rituximab was noted. With standard therapy, the overall response rate (ORR) was 66.7% for AIHA and 65.8% for TTP, respectively. For the R-containing regimens the ORR was 100%. In patients with TTP, median progression free survival (PFS) with R-treatment was 3.8 years, as compared to 0.1 years in the standard-treatment group. In patients with AIHA median PFS was not reached for R-containing treatment; all patients are in sustained remissions with a median follow up of 12.5 months. In the absence of prospective trials, our data underline the safety and efficacy of rituximab in relapsed and refractory autoimmune anaemias with favourable response rates and promising long-term progression-free survival. Therefore, prospective clinical trials evaluating rituximab as salvage- and first-line-therapy are clearly warranted.     

Intrakranielle Blutung bei erworbener Hemmkörperhämophilie

Acquired hemophilia is a rare complication in autoimmune disorders and malignancies. It can result in bleedings into skin and muscle, whereas intracranial hemorrhage in adults has so far not been described. We report a patient with acute intracerebral hemorrhage due to acquired hemophilia with factor VIII inhibition. The patient was treated with recombinant factor VIIa and open hematoma evacuation followed by administration of cortisone and cyclophosphamide. After good initial recovery, intracerebral rebleeding occurred and the patient died from brainstem compression.     

Pathogenic effects of anti-glycoprotein Ib antibodies on megakaryocytes and platelets

Antibodies directed against the glycoprotein (GP) Ib have been identified as the potential cause of various platelet disorders: Immune thrombocytopenic purpura (ITP) may be caused by such autoantibodies; Anti-thrombotic drugs targeting GPIb also induce thrombocytopenia. In order to elucidate the potential mechanism(s) of the anti-GPIb effects, we have examined by electron microscopy (EM) the effect of several antibodies directed against GPIb and GPIIb-IIIa on human culture megakaryocytes (MK). Virtually all antibodies to GPIb enhanced the interaction of newly formed platelets with MK when compared to other antibodies. These effects were retrieved when antibodies were tested on platelets. We conclude that antibodies to GPIb can potentially inhibit platelet release by MK, and can also induce homotypic platelet adhesion. These results may have implications in the pathophysiology of thrombocytopenia and platelet recovery in ITP, and shed light on the pathological effect of anti-GPIb antibodies used as antithrombotic drugs.     

Haematology and neurology

This review aims to update the reader on advances in the understanding of haematological conditions that may arise in neurological practice. Thrombophilia, antiphospholipid antibody syndrome, thrombotic thrombocytopenic purpura, sickle cell and clonal disorders associated with neuropathy are discussed.     

Improved coagulation in bleeding disorders by Non-Anticoagulant Sulfated Polysaccharides (NASP)

Additional therapeutic options are needed for patients with bleeding disorders such as hemophilia A, hemophilia B, severe von Willebrand disease, and other rare factor deficiencies. A novel approach to improve coagulation in such clotting disorders has been identified that, parodoxically, involves heparinlike sulfated polysaccharides. Select molecules of this broad class are largely devoid of anticoagulant activity and are here denoted Non-Anticoagulant Sulfated Polysaccharides (NASPs). A mechanism involving blockade of the extrinsic pathway downregulator, Tissue Factor Pathway Inhibitor (TFPI) by NASPs, was conceived as an approach for improving procoagulant behavior in hemophilic settings. A subset of NASPs, including pentosan polysulfate (PPS) and fucoidan inhibited both full-length and Kunitz 1 and 2 (K1K2) TFPI and, at concentrations from 4-500 nM, improved (i.e. accelerated) the clotting time of human hemophilia A and hemophilia B plasmas or plasma with reduced factor VII levels when tested in dilute prothrombin time (dPT) assays. Fucoidan did not reduce normal plasma APTT times implying specificity for extrinsic pathway control. Improved hemostasis in vivo was observed in mice with hemophilias A or B following low dose subcutaneous administration of PPS or fucoidan, or a combination of NASP plus factor supplement. Increased survival of factor deficient mice following a bleeding challenge was observed. Accordingly, administration of select NASP(s), via mechanism(s) not fully understood, represents a unique means of improving coagulation in bleeding disorders.     

Distribution of Th1- and Th2-induced anti-factor VIII IgG subclasses in congenital and acquired hemophilia patients

Development of antibodies (Ab) that inhibit the procoagulant function of factor VIII (fVIII) seriously complicates the treatment of hemophilia A patients. It also causes acquired hemophilia, a rare yet serious autoimmune disease. The design of effective fVIII-specific tolerizing procedures will require lucidation of the role of the different CD4(+) T cell subsets that drive inhibitor synthesis. To examine the contribution of Th1 and Th2 cells in the anti-fVIII Ab response, we measured the concentration of Th1- and Th2-driven anti-fVIII IgG subclasses in 17 patients with severe hemophilia A and 18 patients with acquired hemophilia. We found that both congenital and acquired hemophilia patients had similar and comparable proportions of Th1- and Th2-induced anti-fVIII Ab, suggesting a more important role of Th1 cells in the immune response to fVIII than previously appreciated. The distribution of anti-fVIII IgG subclasses was stable for periods of up to six months. More intense anti-fVIII Ab responses and higher inhibitor titers correlated with a predominance of Th2-driven subclasses. In contrast, Th1-driven anti-fVIII Ab were predominant in patients who had low anti-fVIII Ab concentrations, even when this was the result of successful immune tolerance or immunosuppressive therapy, which had caused drastic reduction or disappearance of inhibitors. Thus, synthesis of Th2-driven inhibitors occurs when the anti-fVIII Ab response is intense, while Th1 cells may be involved in the long-term maintenance of anti-fVIII Ab synthesis.     

Inhibition of extrinsic pathway inhibitor shortens the coagulation time of normal plasma and of hemophilia plasma.

An increasing amount of evidence suggests that coagulation factors VIII and IX play a role not only in the intrinsic but also in the extrinsic pathway of coagulation. In this context the influence of the Extrinsic Pathway Inhibitor (EPI) on the coagulation time of hemophilia plasma lacking FVIII or FIX has been investigated. The coagulation time was measured in a dilute thromboplastin assay. Addition of recombinant EPI (rEPI) prolonged the coagulation time of normal plasma while the addition of an inhibitory antibody against EPI shortened the coagulation time. At low concentrations of thromboplastin the coagulation time of hemophilia plasma was prolonged and at all dilutions of thromboplastin, addition of anti-EPI IgG normalized the coagulation time of a hemophilia plasma. Analysis of 10 individual donor plasma samples and 8 individual hemophilia samples showed that addition of anti-EPI IgG shortened the coagulation time more in hemophilia plasma than in normal plasma. This illustrates the importance of a powerful extrinsic FVII dependent pathway to achieve hemostasis in the case of FVIII or FIX deficiency (hemophilia A and B).