Pathogenesis Of Thrombotic Thrombocytopenic Purpura Research Articles

Weibel-Palade bodies: function and role in thrombotic thrombocytopenic purpura and in diarrhea phase of STEC-hemolytic uremic syndrome.

Vascular endothelial cells are equipped with numerous specialized granules called Weibel-Palade bodies (WPBs). They contain a cocktail of proteins that can be rapidly secreted (3-5min) into the vascular lumen after an appropriate stimulus such as thrombin. These proteins are ready without synthesis. Von Willebrand factor (VWF) and P-selectin are the main constituents of WPBs. Upon stimulation, release of ultralarge VWF multimers occurs and assembles into VWF strings on the apical side of endothelium. The VWF A1 domain becomes exposed in a shear-dependent manner recruiting and activating platelets. VWF is able to recruit leukocytes via direct leukocyte binding or via the activated platelets promoting NETosis. Ultralarge VWF strings are ultimately cleaved into smaller pieces by the protease ADAMTS-13 preventing excessive platelet adhesion. Under carefully performed flowing conditions and adequate dose of Shiga toxins, the toxin induces the release of ultralarge VWF multimers from cultured endothelial cells. This basic information allows insight into the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and of STEC-HUS in the diarrhea phase. In TTP, ADAMTS-13 activity is deficient and systemic aggregation of platelets will occur after a second trigger. In STEC-HUS, stimulated release of WPB components in the diarrhea phase of the disease can be presumed to be the first hit in the damage of Gb3 positive endothelial cells.

ADAMTS13-Mediated Proteolytic Cleavage of Unusually Large von Willebrand Factor Polymers on Endothelial Cells in the Absence of Fluid Shear Stress

To investigate the molecular mechanism of proteolytic cleavage of unusually large von Willebrand Factor(ULVWF) on endothelial cells by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) in the absence of fluid shear stress, so as to provide a theoretical basis for the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and other thrombotic disorders. The ADAMTS13-mediated proteolysis of ULVWF on the surface of endothelial cells in the absence of fluid shear stress was observed through immunofluorescence microscopy. The variation in VWF antigen levels in the conditioned media were determined by ELISA assay. The levels of VWF and the proteolytic fragments released into the conditioned media were determined by ELISA assay and Western blot in the absence and presence of fluid shear stress or FVIII. The effect of ADAMTS13-mediated ULVWF cleavage on the normal distribution of plasma VWF multimers was evaluated by multimer analysis. Histamine stimulated human umbilical vein endothelial cells (HUVECs) were incubated with ADAMTS13 and various N- and C-terminally truncated mutants. Then the ULVWF that maintained binding to the cells were observed through immunofluorescence microscopy and the soluble ULVWF released from endothelial cells was determined by ELISA, so as to demonstrate the domains of ADAMTS13 required for proteolysis of ULVWF on endothelial cells. The ULVWF strings on the endothelial cell surface were rapidly proteolyzed by recombinant and plasma ADAMTS13 in the absence of fluid shear stress. This proteolytic processing of ULVWF depended on incubation time and ADAMTS13 concentration, but not shear stress and FVIII. The distribution of VWF releaseded by ADAMTS13-mediated proteolysis was quite similar to that secreted by endothelial cells under histamine stimulation, suggesting the ULVWF cleavage occured at the cell surface. The proteolysis of the ULVWF on endothelial cells required the Cys-rich(CysR) and spacer domains, but not the TSP1 2-8 and CUB domains of ADAMTS13. The ULVWF polymers on endothelial cells are sensitive to ADAMTS13-mediated cleavage even in the absence of fluid shear stress. The findings provide novel insight into the molecular mechanism of ADAMTS13-mediated ULVWF cleavage at the cellular level and may contribute to understanding of the pathogenesis of TTP and other thrombotic disorders.

A Rare Case of Thrombotic Thrombocytopenic Purpura Caused by Acute Pancreatitis

Thrombotic Thrombocytopenic Purpura is a syndrome of microangiopathic hemolytic anemia accompanied by thrombocytopenia, neurological disorders, renal failure and fever. Acute pancreatitis is a rare cause of Thrombotic Thrombocytopenic Purpura and this manifestation, at the same time, is a rare complication of acute pancreatitis. Thrombotic Thrombocytopenic Purpura is induced in acute pancreatitis by poorly understood mechanism, which involves multiple pathways apart from only ADAMTS-13 deficiency. Here, we analyze the case of a 47-year-old female who presented with an acute pancreatitis. She was diagnosed with Thrombotic Thrombocytopenic Purpura and an acute pancreatitis at the same time, with thrombocytopenia and peripheral smear findings at presentation. Therefore, Thrombotic Thrombocytopenic Purpura secondary to the pancreatitis was considered in this case. In this work, we have discussed details of our case and the different mechanisms involved in pathogenesis of Thrombotic Thrombocytopenic Purpura in acute pancreatitis and their outcome with prompt management.

Biological Function of CysR Domain of ADAMTS13

To investigate the biological function of Cysteine rich (CysR) domain of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) on cleavage of von Willebrand factor (vWF) and provide experimental evidence for exploring the pathogenesis of thrombotic thrombocytopenic purpura (TTP). The six amino acids (EDGTLS) in ADAMTS13 CysR domain were point mutated one by one, and the mutant ADAMTS13 proteins were expressed and purified. The cleavage products of vWF polymer by wild-type or mutant ADAMTS13 under denaturing condition or shear stress were separated by 1% SeaKem HGT agarose gel and detected by Western blot. The mutant ADAMTS13 plasmids (M1: Glu515Ala; M2: Asp516Ala; M3: Gly517Ala; M4: Thr518Ala; M5: Leu519Ala; M6: Ser520Ala) were successfully constructed and the proteins of wild-type and mutant ADAMTS13 were purified. Wild-type ADAMTS13 almost completely cleaved the vWF polymer under denaturing condition, while the cleavage activity of M1 mutant was significantly reduced in the same condition (P<0.01). The cleavage activity of M1 mutant of ADAMTS13 was also significantly reduced compared with that of the wild-type under shear stress (P<0.01). The activity of M1 mutant to cleave the FRETS-vWF73 was dramatically reduced compared with that of wild-type ADAMTS13. However, the binding ability of M1 mutant to vWF was similar with that of wild-type ADAMTS13. The CysR domain of ADAMTS13 plays an important role in the digestion of vWF under denaturing condition and shear stress. The Glu515 amino acid residue might be an important site for substrate recognition.

Diagnosis of thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disease, disease, characterised by microangiopathic hemolytic anaemia, consumption thrombocytopenia, and organ dysfunction. The pathogenesis of TTP is attributed to the deficiency in the activity of the metalloproteinase ADAMTS13, specific von Willebrand factor cleaving protease. TTP is suspected when detecting microangiopathic hemolytic anemia, thrombocytopenia, damage to various organs. Diagnosis of TTP is confirmed by the detection of ADAMTS13 activity in plasma less than 10%. Plasma samples for the study of ADAMTS13 activity should be taken before the start of plasma transfusions or plasma exchange. In patients with severe ADAMTS-13 deficiency autoantibodies anti-ADAMTS13 and inhibitor ADAMTS13 should be investigated. Anti-ADAMTS13 antibodies belonging to IgG not always have inhibitory effects. The inhibitory effect of anti-ADAMTS13 antibodies is confirmed by mixing test. All patients with the first established diagnosis of TTP should be examined for mutations of the ADAMTS13 gene.

ADAMTS13 gene; a novel splicing site mutation in a case with thrombotic thrombocytopenic purpura

A plasma protease, ADAMTS13, cleaves the von Willebrand factor (VWF) and its deficiency is associated with the pathogenesis of thrombotic thrombocytopenic purpura (TTP). According to the Human Gene Mutation Database (HGMD), about 150 mutations have been identified in the ADAMTS13 gene. A 23-year-old man, with hematuria and gingival bleeding was admitted to our University Hospital. Four years ago he was diagnosed with a TTP history. During these years, he was under intermittent plasma exchange. A blood sample was taken for genetic study. He effectively responded to one session of fresh frozen plasma replacement and plasma exchange. Genetic study indicated that this case carries two heterozygous mutations in ADAMTS13 gene; a novel splicing variant (c.2610+5G&gt;A) and a nonsense p.Arg910X mutation that previously is reported to relate to TTP. The novel variant predicted to result in an aberrant ADAMTS13 transcript processing.

SUN-445 A NOVEL SPLICING SITE MUTATION IN ADAMTS13 GENE IN A PATIENT WITH THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP)

Deficiency of ADAMTS13, a plasma protease that cleaves the von Willebrand factor (VWF) multimers, results in the persistence of high-molecular-weight multimers of VWF and has a major role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). A 23-year-old man with a clinical history of TTP since 4 years ago was admitted to our University Hospital because of gingival bleeding and hematuria. He was under intermittent plasma exchange during the last four years. He received last treatment for one month. A blood sample for the genetic study was taken. He successfully responds to one session of plasma exchange and FFP replacement. A genetic study found that the patient is carrying two heterozygous mutations in ADAMTS13 gene: a nonsense mutation (p.Arg910X) already associated with TTP and a novel splicing variant (c.2610+5G>A) predicted to cause aberrant processing of ADAMTS13 transcript. Mutations on these sequences can disrupt existing splice sites or splicing regulatory sequences (splicing silencers and enhancers), create new splicing sites, or activate the cryptic splicing sites that may lead to an aberrant transcript of the mutated gene. To estimate the possible effect of the identified changes, bioinformatic tools can be applied. However, it should be noted that the exact effect of the specific mutation should be verified with functional studies.

METALLOPROTEASE ADAMTS-13

Introduction. The signifi cance of ADAMTS-13 extends beyond its key role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP); there is evidence of a relationship between a decrease in the ADAMTS-13 activity and thrombotic events in acute myocardial infarction and ischemic stroke.Aim. To generalise available information on the structure and function of the metalloprotease ADAMTS-13.General findings. The biological function of ADAMTS-13 consists in the cleavage of ultra-large von Willebrand factor (vWF) multimers. The fact that its defi ciency causes the development of TTP provides a basis for understanding the function of vWF–cleaving protease. ADAMTS-13 has a domain structure. The functional roles of most ADAMTS-13 domains, as well as the key role of the ADAMTS-13-vWF interaction in the regulation of haemostasis, are defi ned. The conformational activation of ADAMTS-13 by vWF constitutes an important aspect of its function. After getting into the bloodstream, ultra-large vWF multimers quickly adopt a closed conformation, which becomes very resistant to ADAMTS-13 proteolysis in the absence of shear stress. Ultra-large plasma vWF multimers regain their sensitivity to ADAMTS-13 after being exposed to high fl uid shear stress, which unfolds the central vWF-A2 domain. The unfolding of a vWF molecule under shear stress conditions reveals previously hidden exosites in domain A2, which gradually increase the binding affi nity between ADAMTS-13 and vWF. The mechanism underlying the production of autoantibodies against ADAMTS-13 is unknown and requires further study. The masking of cryptic epitopes in the closed conformation of ADAMTS-13 prevents the formation of autoantibodies. Early antigen recognition of ADAMTS-13 occurs through surface-exposed epitopes in the C-terminal domains. More detailed information on the mechanisms underlying the interaction between ADAMTS-13 and the vWF can improve the understanding of mechanisms involved in the regulation of the coagulation system.Conflict of interest: the authors declare no confl ict of interest.Financial disclosure: the study had no sponsorship.

The Association Between Human Leukocyte Antigens and ITP, TTP, and HIT.

Autoimmune thrombocytopenia in immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), and heparin-induced thrombocytopenia (HIT) is associated with immunologic degradation of platelets and reduced platelet counts in patients, leading to bleeding risk in patients. Considering the role of human leukocyte antigens (HLA) in the development of immune response, in this review, we examine the relationship between HLA and pathogenesis of the above-mentioned diseases. Relevant English-language literature was searched and retrieved from Google Scholar search engine and PubMed database (1979 to 2018). The following keywords were used: "Immune Thrombocytopenic purpura," "Thrombotic Thrombocytopenic Purpura," Human Leukocyte Antigen," and "Heparin-induced thrombocytopenia." In autoimmune thrombocytopenia, HLA molecule presents self-antigens or foreign antigens similar to self-antigens, provoking an immune response against platelets that results in the degradation of platelets in peripheral blood and possible bleeding in the patient. For example, HLA-DRB1 *11 presents the self-antigen and induces an immune response against ADAMTS13, which is associated with thrombocytopenia in TTP patients. HLA alleles can be used as prognostic biomarkers for immunologic disorders of platelet such as ITP, TTP, and HIT. Different DRB1 alleles enable the assessment of resistance to common ITP treatments as well as disease prognosis. Due to the genetic association between HLA-DR1 and HLA-DQ1 alleles and the role of HLA-DRB1 *11 in TTP, the HLA-DQB1 *02: 02 allele may also play a role in TTP pathogenesis.

Beyond plasma exchange: novel therapies for thrombotic thrombocytopenic purpura.

The advent of plasma exchange has dramatically changed the prognosis of acute thrombotic thrombocytopenic purpura (TTP). Recent insights into TTP pathogenesis have led to the development of novel therapies targeting pathogenic anti-ADAMTS13 antibody production, von Willebrand factor (VWF)-platelet interactions, and ADAMTS13 replacement. Retrospective and prospective studies have established the efficacy of rituximab as an adjunct to plasma exchange for patients with acute TTP, either upfront or for refractory disease. Relapse prevention is a major concern for survivors of acute TTP, and emerging data support the prophylactic use of rituximab in patients with persistent or recurrent ADAMTS13 deficiency in clinical remission. Capalcizumab, a nanobody directed against domain A1 of VWF that prevents the formation of VWF-platelet aggregates, recently completed phase 2 (TITAN) and 3 (HERCULES) trials with encouraging results. Compared with placebo, caplacizumab shortened the time to platelet recovery and may protect against microthrombotic tissue injury in the acute phase of TTP, though it does not modify the underlying immune response. Other promising therapies including plasma cell inhibitors (bortezomib), recombinant ADAMTS13, N-acetyl cysteine, and inhibitors of the VWF-glycoprotein Ib/IX interaction (anfibatide) are in development, and several of these agents are in prospective clinical studies to evaluate their efficacy and role in TTP. In the coming years, we are optimistic that novel therapies and international collaborative efforts will usher in even more effective, evidence-based approaches to address refractory acute TTP and relapse prevention.

Thrombotic Thrombocytopenic Purpura with Cardiac Involvement after an Allergic Reaction to Turmeric and Cumin: A Case Report

Background: Cuminum cyminum (Cumin) and Curcuma longa (Turmeric) are plant derived spices which are commonly used as a part of everyday recipes in variety of cuisines. They are known causes of platelet dysfunction but Thrombotic thrombocytopenic purpura (TTP) following their consumption has never been reported in the literature. Primary cardiac involvement in TTP is a rare but fatal complication and may present as AMI, heart failure or arrhythmias. Case Report: We report a case of a 46-year-old male who presented with features of spontaneous bleeding following consumption of Cumin and Turmeric. Laboratory investigations revealed hemolytic anemia, marked thrombocytopenia and blood smear showed schistocytes. In addition, cardiac involvement was noted as per changes seen on the EKG and markedly elevated Troponin I level. At this stage TTP was diagnosed which was subsequently confirmed by reduced ADAMTS13 activity level of <10%. He was successfully treated with Plasma Exchange (PEX) and Rituximab. PEX was complicated by 5 episodes of non-sustained Ventricular Tachycardia which was managed by Amiodarone. Conclusion: Although the link between ADAMTS13 deficiency and idiopathic TTP has been well-established, the role of Cumin and Turmeric in the pathogenesis of TTP is not yet well clarified. We speculate that the acquired ADAMTS13 deficiency might have been triggered by those Spices. Myocardial injury is underdiagnosed in patients with TTP and is associated with higher mortality. Hence, patients diagnosed with TTP should be monitored for adverse cardiac events due to the high incidence of new AMI, arrhythmias and congestive heart failure.

Recombinant thrombomodulin for secondary thrombotic thrombocytopenic purpura.

In the pathogenesis of thrombotic thrombocytopenic purpura (TTP), reductions in the enzyme activity of ADAMTS13, which cuts ultralarge von Willebrand multimers, generates shear stress on the microvascular endothelium, leading to platelet aggregation and the formation of a thrombus. ADAMTS13 activity is markedly decreased in typical TTP, but is only mildly reduced in secondary TTP, which concomitantly develops with primary disease. The latter develops with septic disseminated intravascular coagulation (DIC) and often causes organ failure. Recombinant thrombomodulin (rTM) is a drug that is used to treat DIC and may also remit TTP because it improves vascular endothelial dysfunction. Therefore, we herein investigated the efficacy of rTM in patients treated for the pathology of secondary TTP. Patients who were admitted to the Emergency and Critical Care Center of our hospital and met the following conditions were extracted and retrospectively analyzed: hemolytic anemia accompanied by fragmented red blood cells (Hb < 12 g/dL or lower); thrombocytopenia (<100 × 103/μL); and ADAMTS13 activity <50%. Sixteen patients were included and accompanied by Kidney Disease: Improving Global Outcomes (KDIGO) stage 2 or more severe nephropathy and DIC. Eleven and 5 patients treated with and without rTM (the rTM and non-rTM treatment groups, respectively) were compared, and no significant difference was noted in their basic characteristics, such as background disease and severity. No significant difference was observed in survival rates; however, the platelet count, which is an important outcome of treatments for TTP, significantly increased in the rTM treatment group: 3.3 ± 2.6→11.3 ± 14.6 versus 3.5 ± 3.7→5.7 ± 3.9 (×1000/μL) (P = 0.034). Thrombotic thrombocytopenic purpura originally requires invasive treatments and its prognosis is not favorable. Blood thrombomodulin levels also markedly increase due to vascular endothelial dysfunction, whereas rTM alleviates vascular endothelial dysfunction in TTP patients with high blood TM levels, suggesting the importance of administering rTM. Thus, rTM may be effective for secondary TTP and may be adopted as adjuvant therapy.

Change in plasma a disintegrin and metalloprotease with thrombospondin type-1 repeats-13 and von Willebrand factor levels in venous thromboembolic patients

Objectives: Venous thromboembolism (VTE) is an important cause of morbidity and mortality. A disintegrin and metalloprotease with thrombospondin type-1 repeats-13 (ADAMTS-13) is a metalloprotease that cleaves plasma von Willebrand factor (VWF) multimers. The presence of large VWF multimers in the plasma due to ADAMTS-13 deficiency is the main factor in the pathogenesis of thrombotic thrombocytopenic purpura. The present study aimed to investigate the relation of plasma levels of ADAMTS-13 and VWF antigen with VTE.Methods: The present study included 30 patients with VTE and age- and gender-matched 30 healthy subjects. Patients with any condition (diabetes, icterus, hyperlipidemia, physical, or surgical trauma, acute coronary syndrome, pregnancy, renal insufficiency, liver disease, malignancy, collagen tissue disease, chronic or acute inflammation, drug use affecting thrombocyte function) that could affect plasma VWF antigen or ADAMTS-13 levels were excluded. Plasma ADAMTS-13 and VWF antigen levels in the VTE and control groups were quantitatively determined by enzyme-linked immunosorbent assay method.Results: The median ADAMTS-13 level was 280 ng/ml (minimum–maximum, 70–1120 ng/ml) in the VTE group and 665 ng/ml (minimum–maximum, 350–2500 ng/ml) in the control group; the difference between the groups was significant (P < 0.0001). The mean VWF antigen level was 1750 ± 616 mU/ml in the patient group, which was significantly higher than that of the control group (950 ± 496 mU/ml) (P < 0.0001).Conclusion: Significantly lower ADAMTS-13 levels and significantly higher VWF antigen levels were concluded to be the result of a pathological process rather than an etiological factor for VTE.

Carboxiterminal pro-endothelin-1 as an endothelial cell biomarker in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is characterised by the deficiency of the von Willebrand factor (VWF) cleaving protease (ADAMTS-13). Although several observations indicate an important role of endothelial activation in the pathogenesis of TTP, no reliable endothelial activation markers are available in the clinical management of TTP. Our aim was to investigate the presence of endothelial activation in TTP and to determine its connections with disease activity, therapy and complement activation. We enrolled 54 patients (median age 40.5; 44 females) and 57 healthy controls (median age 34; 30 females),VWF antigen, carboxiterminal-pro-endothelin-1 (CT-proET-1), complement Factor H and complement activation products (C3bBbP and SC5b-9) were measured. In both the acute and remission phase of TTP we found increased CT-proET-1 and VWF levels, while Factor H levels decreased compared with healthy controls. In remission, however, the elevated CT-proET-1 levels showed 22 % decrease when compared with the acute phase in paired samples (p=0.0031), whereas no changes for VWF and Factor H levels were observed. We also found positive correlations between CT-proET-1 levels and alternative pathway activation markers (C3bBbP; p=0.0360; r=0.4299). The data we present here demonstrate a role of endothelium activation in patients with acute TTP. The finding that CT-proET-1 levels decreased in remission compared with the acute phase further supports endothelial involvement. In addition, we show that endothelial activation also correlated with the activation of the alternative complement pathway. The data suggest that complement and endothelium activation jointly contribute to the development of TTP episodes in patients with predisposition to TTP.

Plasma Levels of Human Neutrophil Peptides and Complement Activation Markers in Patients with Acquired Autoimmune Thrombotic Thrombocytopenic Purpura

Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal syndrome, resulting from autoantibodies against the metalloprotease ADAMTS13. Autoantibodies bind and inhibit plasma ADAMTS13 activity, leading to exaggerated platelet agglutination and thrombus formation in small arteries and capillaries. However, plasma ADAMTS13 deficiency or the presence of anti-ADAMTS13 autoantibodies is not sufficient to cause the disease. Infection or inflammation often precedes the acute onset of TTP. We hypothesize that neutrophil activation or complement activation vial alternative pathway may be the trigger for TTP. In this study, plasma samples were collected prior to plasma exchange from 58 adult patients with acute TTP between 2006 and 2015 at the University of Alabama at Birmingham Medical Center. All patients were diagnosed having TTP on the basis of severe thrombocytopenia, microangiopathic hemolytic anemia, and severe deficiency of plasma ADAMTS13 activity (less than 10%) and the presence of ADAMTS13 inhibitors. All patients were treated by plasma exchange. Thirty plasma samples from healthy individuals were collected for controls. Plasma levels of human neutrophil peptides (HNP-1, -2, and -3) released from activated neutrophils and alternative pathway complement activation markers (iC3b, C4d, C5b-9, and Bb) were determined by enzyme-linked immunosorbent assays (ELISAs). We showed that plasma levels of HNP-1, -2, and -3 in TTP patients were elevated on average by 10-fold when compared with those in the healthy controls (44.12 ± 39.53 ng/ml vs. 3.54 ± 2.97 ng/ml, means ± SD) (p <0.0001). Whereas plasma levels of Bb and iC3b were only modestly increased in TTP patients (2.94 ± 1.82 µg/ml vs. 1.39 ± 0.34 µg/ml for Bb, p <0.0001, and 16.21 ± 9.71 µg/ml vs. 12.13 ± 3.07 µg/ml for iC3b, p =0.028). No significant difference was detected in the plasma levels of C4d and C5b-9 between TTP patients and healthy controls (p >0.05). To differentiate HNP-1 from HNP-2 or HNP-3, we developed a LC/MS mass spectrometric assay and showed that all subtypes of HNPs in TTP patients were significantly elevated when compared with those in the healthy controls (p<0.001). In general, there was good concordance between ELISA and LC/MS MS in the measurement of all three HNPs. These results demonstrate that innate immunity (i.e. activation of neutrophils) and to a lesser degree the activation of complements via alternative pathway may play a role in pathogenesis of TTP in light of severe ADAMTS13 deficiency. Our findings provide rationales for developing novel targeted therapies for acquired TTP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Disappearing acts of ADAMTS13

The study by Thomas and co-workers in this issue of EBioMedicine provides detailed characteristics on the pathogenic properties of antibodies that develop in patients with acquired thrombotic thrombocytopenic purpura (TTP) (Thomas et al., 2015). It is now well-established that TTP is due to dysregulation of primary hemostasis. A crucial initial step in this process involves von Willebrand factor-mediated adhesion of blood platelets to a damaged vessel wall. The platelet-adhesive properties of VWF are dependent on its ability to assemble into large polymers which are released from Weibel–Palade bodies, unique storage organelles present in endothelial cells (Valentijn et al., 2011). Upon their release, these polymers form extended parallel aligned strings that recruit platelets to sites of vascular perturbation. The VWF cleaving protease ADAMTS13 controls VWF multimer size by selectively cleaving a peptide bond in the A2 domain of VWF that becomes exposed under the influence of fluidic shear stress (Crawley et al., 2011). In the absence of ADAMTS13 processing of VWF polymers is incomplete, resulting in enhanced platelet adhesion at sites of vascular injury (Fig. 1A). Fig. 1 Pathogenicity of anti-ADAMTS13 antibodies. (A) Release of platelet recruiting von Willebrand factor polymers from endothelial cells. VWF polymers that are released from Weibel–Palade bodies assemble into ultra-large strings on the surface of endothelial ... For reasons that are so far poorly understood, autoantibodies directed against ADAMTS13 can develop in previously healthy individuals. Like many other autoimmune disorders the highly polymorphic MHC class II locus has been implicated in the pathogenesis of TTP. The frequency of HLA-DR11 was significantly increased in patients with acquired TTP (Scully et al., 2010, Coppo et al., 2010). Feeding of antigen presenting cells with ADAMTS13 revealed that a CUB2 domain derived peptide is preferentially presented on HLA-DR11 (Sorvillo et al., 2013). Based on this it was hypothesized that microbial peptides with sequence overlap to this peptide may contribute to the onset of acquired TTP (Verbij et al., 2014). While our knowledge on the underlying cause for development of acquired TTP is still in its infancy, an increasing number of studies have described the binding characteristics of pathogenic antibodies in patients with acquired TTP (for review see Verbij et al., 2014). In the current study Thomas and co-workers take this approach a step further by elegantly integrating functional and binding studies in a well-characterized cohort of patients with acquired TTP (Thomas et al., 2015). Their study confirms that in the majority of patients antibodies are present that are directed towards the spacer domain (Fig. 1B). In a subset of patients antibodies directed towards the TSP2-8 and CUB1-2 domain were identified. Antibodies exclusively targeting the carboxy-terminal TSP2-8 and CUB1-2 domains were observed in 3 out of 92 patients analyzed. These results are in excellent agreement with previous studies on the domain specificity of anti-ADAMTS13 antibodies (Pos et al., 2011, Zheng et al., 2010). A major asset of the current study is provided by combining mapping studies and linking these to the inhibitory properties of IgG purified from patient plasma. Their results show that inhibitory antibodies primarily target the spacer domain of ADAMTS13. Unexpectedly, in about half of the patients only weakly inhibitory antibodies were detected suggesting that functional inhibition of ADAMTS13 alone cannot account for the severe deficiency of ADAMTS13 in patients with acquired TTP. Based on this observation the authors suggest that enhanced antibody-mediated clearance provides a major pathogenic mechanism in acquired TTP. This observation is corroborated by the reduced levels of circulating ADAMTS13 antigen at first presentation in patients with non-inhibitory antibodies which appears to be a major determinant of disease severity. The current study strongly positions antibody-mediated clearance of ADAMTS13 as an important pathogenic mechanism for acquired TTP. Data included in this study show that residual levels of ADAMTS13 antigen at presentation are related to clinical outcome. Circulating ADAMTS13-Ig complexes have been detected in plasma samples of patients with acquired TTP (Ferrari et al., 2014). As yet our knowledge on the size, composition and clearance-rate of ADAMTS13-Ig complexes is insufficient. Given their importance for the pathogenesis of acquired TTP more detailed knowledge on the mechanism of clearance of ADAMTS13-Ig complexes is urgently needed. This may help to further improve current therapeutic approaches for treatment of patients with acquired TTP.

Effect of rituximab on B cell phenotype and serum B cell-activating factor levels in patients with thrombotic thrombocytopenic purpura.

Autoantibodies inhibiting the activity of the metalloproteinase, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), underlie the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Rituximab (RTX) combined with plasma-exchange (PEX) is an effective treatment in TTP. Patients can remain in remission for extended periods following PEX/RTX, and this is associated with continuing reduction in antibodies to ADAMTS13. Factors controlling B cell differentiation to autoantibody production, including stimulation through the B cell receptor and interactions with the B cell-activating factor (BAFF), may thus impact length of remission. In this cross-sectional study, we measured naive and memory B cell phenotypes [using CD19/immunoglobulin (Ig)D/CD27] following PEX/RTX treatment in TTP patients at B cell return (n=6) and in 12 patients in remission 10-68 months post-RTX. We also investigated relationships among serum BAFF, soluble CD23 (sCD23(-) a surrogate measure of acquiring B memory (CD27(+) ) phenotype) and BAFF receptor (BAFF-R) expression. At B cell return after PEX/RTX, naive B cells predominated and BAFF-R expression was reduced compared to healthy controls (P<0.001). In the remission group, despite numbers of CD19(+) B cells within normal limits in most patients, the percentage and absolute numbers of pre-switch and memory B cells remained low, with sCD23 levels at the lower end of the normal range. BAFF levels were correlated inversely with BAFF-R expression and time after therapy. In conclusion, the long-term effects of RTX therapy in patients with TTP included slow regeneration of memory B cell subsets and persistently reduced BAFF-R expression across all B cell subpopulations. This may reflect the delay in selection and differentiation of potentially autoreactive (ADAMTS13-specific) B cells, resulting in relatively long periods of low disease activity after therapy.

ADAMTS13 and von Willebrand factor in thrombotic thrombocytopenic purpura.

Pathogenesis of thrombotic thrombocytopenic purpura (TTP) was a mystery for over half a century until the discovery of ADAMTS13. ADAMTS13 is primarily synthesized in the liver, and its main function is to cleave von Willebrand factor (VWF) anchored on the endothelial surface, in circulation, and at the sites of vascular injury. Deficiency of plasma ADAMTS13 activity (<10%) resulting from mutations of the ADAMTS13 gene or autoantibodies against ADAMTS13 causes hereditary or acquired (idiopathic) TTP. ADAMTS13 activity is usually normal or modestly reduced (>20%) in other forms of thrombotic microangiopathy secondary to hematopoietic progenitor cell transplantation, infection, and disseminated malignancy or in hemolytic uremic syndrome. Plasma infusion or exchange remains the initial treatment of choice to date, but novel therapeutics such as recombinant ADAMTS13 and gene therapy are under development. Moreover, ADAMTS13 deficiency has been shown to be a risk factor for the development of myocardial infarction, stroke, cerebral malaria, and preeclampsia.

Inhibition of ADAMTS13 Activity By Human Neutrophil Peptide 1 (HNP-1): Potential Link Between Inflammation, Onset of Thrombotic Thrombocytopenic Purpura, and Other Thrombosis

Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal syndrome associated with severe deficiency of plasma ADAMTS13 activity resulting from either mutations or autoantibodies. However, patients with severe ADAMTS13 deficiency do not always develop TTP. Rather, a trigger, such as infection or inflammation, often precedes the onset of the TTP syndrome. We hypothesized that antimicrobial human neutrophil peptides 1-3 (HNPα1-3) or α-defensins, the most abundant proteins in the granules of neutrophils, which are released at site of inflammation, activate platelets, and inhibit fibrinolysis, might help to initiate TTP. This question arose because we noted that the amino acid sequences of HNPα1-3, which are nearly identical except for one residue at the N-terminus, all contain a motif (RRY) similar to exosite 3 (659RRYGEEY665) in the spacer domain of ADAMTS13 (Fig. 1) that was shown to be critical for recognition of von Willebrand factor (VWF). Here, we found that both purified and synthetic HNPα1 bind to FRETS-VWF73 and plasma-derived VWF and inhibit proteolytic cleavage of these substrates in a concentration-dependent manner. At the final concentrations of 10 micro mol/L and 150 micro mol/L, HNPα1 completely abolished the cleavage of FRETS-VWF73 (IC50=3.5 micro mol/L) (Fig. 2) and VWF (IC50=75 micro mol/L) (not shown), respectively. Such concentrations are readily attained locally after systemic infection. Deletion or alanine substitution within the RRY motif of HNPα1 completely abolished its ability to inhibit ADAMTS13 activity assessed by FRETS-VWF73 and VWF multimer analysis. This suggests that an interaction of the RRY motif in HNPα1 with the central A2 domain of VWF is required to mediate its inhibition. In support of this hypothesis, HNPα1 interacts with a human monoclonal antibody against ADAMTS13 scFv (the single chain fragment of variable region) designated 4-20, but not scFv3-1, both isolated by phage display from patients with acquired autoimmune TTP. Hydrogen-deuterium exchange mass spectrometry has shown that the binding site for scFv4-20, but not scFv3-1, contains the RRY sequence. These results suggest that HNPα1-3 released from neutrophils following infection or inflammation may inhibit residual plasma ADAMTS13 activity in vivo similar to anti-ADAMTS13 autoantibodies by interfering with its interaction with VWF, thereby triggering the onset of hereditary and acquired autoimmune TTP. Our findings suggest a potential novel link between systemic inflammation and the pathogenesis of TTP and possibility other thrombotic sequelae. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Similar Levels of Complement Activation in Both Patients with Thrombotic Thrombocytopenic Purpura and Atypical Hemolytic Uremic Syndrome: The Report from the Korean TTP Registry

Background: Uncontrolled complement activation has a major role in the pathogenesis of atypical HUS (aHUS) and the restraint of this process by eculizumab is life saving. However, the evidence of complement dysregulation in the pathogenesis of Thrombotic Thrombocytopenic Purpura (TTP) is still unclear. In this study we examined the presence of complement activation biomarkers in patients with aHUS and TTP and the levels were compared to normal healthy controls .Patients and Methods: Patients with thrombotic microangiopathic thrombocytopenia diagnosed either as TTP with low ADAMTS13 activity less than 10% or aHUS with impaired renal function, Cr> 2mg/dL and normal ADAMTS13 activity were chosen from the Korean TTP registry from February 2012 to June 2014. Prospective plasma and serum samples prior to intervention were collected from newly diagnosed patients with TTP (n=20), aHUS (n=20), and 20 healthy controls and frozen at -700C. Complement activation products (C3a, Bb as alternative pathway; C4d as classic pathway; C5a, C5b-9; terminal pathway) were measured by ELISA.Results: Significantly increased levels of Bb and C5b-9 were observed in TTP (median [range], ng/mL; Bb, 1220 [540.0 – 16560], p=0.048; C5b - 9, 390.1 [238.5 - 938.7], p<0.0001) when compared with controls (Bb, 870.0 [630.0 - 2070]; C5b - 9, 190.8 [77.96 - 458.9]). Increased levels of C3a, C5a, C5b - 9, and Factor Bb were observed in HUS (C3a, 231.3 [80.70 - 791.8], p<0.0001; C5a, 21.38 [5.590 - 34.96], p= 0.006; C5b - 9, 0.49 [0.21 - 1.41], p<0.0001; Bb, 1490 [540.0 – 11800], p= 0.0003) as compared with controls (C3a, 108.7 [30.98 - 425.1]; C5a, 8.620 [2.660 - 26.93]; C5b - 9, 0.49 [0.21 - 1.41]; Bb, 870.0 [630.0 - 2070]). These suggested alternative and terminal complement pathways were activated in initial episodes of TTP or HUS. However levels of C4d were not different in HUS and TTP as compared with controls which suggested classic complement pathways were not important in this process. There were no significant differences in complement levels between TTP and HUS although levels of C3a, C4d, C5b - 9 in HUS (C3a, 231.3 [80.70 - 791.8]; C4d, 2140 [10.00 - 960.0]; C5b - 9, 488.4 [212.7 – 1414]) tended to be increased as compared with TTP (C3a, 134.5 [61.97 - 378.4]; C4d, 1330 [2.000 - 699.0]; C5b - 9, 390.1 [238.5 - 938.7]).Conclusion: Complement biomarkers are activated to a similar level in both newly diagnosed cases of TTP and aHUS. Complement activation product levels did not differentiate aHUS from TTP. DisclosuresNo relevant conflicts of interest to declare.