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Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by recurring episodes of thrombotic microangiopathy, causing ischemic organ impairment. Black patients are overrepresented in iTTP cohorts in the United States, but racial disparities in iTTP outcome and response to therapy have not been studied. Using the United States Thrombotic Microangiopathies Consortium iTTP Registry, we evaluated the impact of race on mortality and relapse-free survival (RFS) in confirmed iTTP in the United States from 1995 to 2020. We separately examined the impact of rituximab therapy and presentation with newly diagnosed (de novo) or relapsed iTTP on RFS by race. A total of 645 participants with 1308 iTTP episodes were available for analysis. Acute iTTP mortality did not differ by race. When all episodes of iTTP were included, Black race was associated with shorter RFS (hazard ratio [HR], 1.60; 95% CI, 1.16-2.21); the addition of rituximab to corticosteroids improved RFS in White (HR, 0.37; 95% CI, 0.18-0.73) but not Black patients (HR, 0.96; 95% CI, 0.71-1.31). In de novo iTTP, rituximab delayed relapse, but Black patients had shorter RFS than White patients, regardless of treatment. In relapsed iTTP, rituximab significantly improved RFS in White but not Black patients. Race affects overall relapse risk and response to rituximab in iTTP. Black patients may require closer monitoring, earlier retreatment, and alternative immunosuppression after rituximab treatment. How race, racism, and social determinants of health contribute to the disparity in relapse risk in iTTP deserves further study.

Immune-mediated thrombocytopenic purpura (iTTP) is a thrombotic microangiopathy characterized by an acquired ADAMTS13 deficiency as a result of the presence of an antibody inhibitor of ADAMTS13 leading to the formation of ultralarge von Willebrand multimers. Treatment of iTTP includes plasma exchange, high-dose glucocorticoids, rituximab, and, more recently, caplacizumab, to prevent the development of exacerbations. There is the risk of both relapse and long-term complications that include neurocognitive deficits and cardiovascular events that occur in patients in remission after recovery from an acute iTTP episode. Data on the risk factors for the development of these complications, the appropriate screening, and treatment are limited due to the paucity of research. This article is a review of the current understanding on the risk factors for exacerbation, relapse, and long-term complications of iTTP and discusses an approach to observing patients with iTTP after hospital discharge and during the long-term follow-up in the outpatient setting.

Trauma-induced organ failure is characterized by endothelial dysfunction. The aim of this study was to investigate the role of von Willebrand factor (VWF) and its cleaving enzyme, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13) in the occurrence of endothelial permeability and organ failure in trauma. In an observational study in a level-1 trauma center, 169 adult trauma patients with clinical signs of shock and/or severe injuries were included. Trauma was associated with low ADAMTS13 and high VWF antigen levels, thus generating an imbalance of ADAMTS13 to VWF. Patients who developed organ failure (23%) had greater ADAMTS13-to-VWF imbalances, persistently lower platelet counts, and elevated levels of high-molecular-weight VWF multimers compared with those without organ failure, suggesting microthrombi formation. To investigate the effect of replenishing low ADAMTS13 levels on endothelial permeability and organ failure using either recombinant human ADAMTS13 (rhADAMTS13) or plasma transfusion, a rat model of trauma-induced shock and transfusion was used. Rats in traumatic hemorrhagic shock were randomized to receive crystalloids, crystalloids supplemented with rhADAMTS13, or plasma transfusion. A 70-kDa fluorescein isothiocyanate-labeled dextran was injected to determine endothelial leakage. Additionally, organs were histologically assessed. Both plasma transfusion and rhADAMTS13 were associated with a reduction in pulmonary endothelial permeability and organ injury when compared with resuscitation with crystalloids, but only rhADAMTS13 resulted in significant improvement of a trauma-induced decline in ADAMTS13 levels. We conclude that rhADAMTS13 and plasma transfusion can reduce organ failure following trauma. These findings implicate the ADAMTS13-VWF axis in the pathogenesis of organ failure. ispartof: BLOOD ADVANCES vol:5 issue:17 pages:3478-3491 ispartof: location:United States status: published

The effects of synonymous single nucleotide variants (sSNVs) are often neglected because they do not alter protein primary structure. Nevertheless, there is growing evidence that synonymous variations may affect messenger RNA (mRNA) expression and protein conformation and activity, which may lead to protein deficiency and disease manifestations. Because there are21 million possible sSNVs affecting the human genome, it is not feasible to experimentally validate the effect of each sSNV. Here, we report a comprehensive series of in silico analyses assessing sSNV impact on a specific gene. ADAMTS13 was chosen as a model for its large size, many previously reported sSNVs, and associated coagulopathy thrombotic thrombocytopenic purpura. Using various prediction tools of biomolecular characteristics, we evaluated all ADAMTS13 sSNVs registered in the National Center for Biotechnology Information database of single nucleotide polymorphisms, including 357 neutral sSNVs and 19 sSNVs identified in patients with thrombotic thrombocytopenic purpura. We showed that some sSNVs change mRNA-folding energy/stability, disrupt mRNA splicing, disturb microRNA-binding sites, and alter synonymous codon or codon pair usage. Our findings highlight the importance of considering sSNVs when assessing the complex effects of ADAMTS13 alleles, and our approach provides a generalizable framework to characterize sSNV impact in other genes and diseases.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal thrombotic microangiopathy caused by autoantibody-mediated severe deficiency of ADAMTS13. Standardized definitions of response, exacerbation, remission, and relapse were initially proposed in 2003 and modified by the International Working Group for TTP in 2017. These definitions, which have been widely used in clinical practice and research, are based primarily on the platelet count and are benchmarked against the timing of discontinuation of therapeutic plasma exchange (TPE). They do not incorporate ADAMTS13 activity or the temporizing effects on the platelet count of caplacizumab, a novel anti–von Willebrand factor (VWF) nanobody. In light of these limitations, the IWG aimed to develop revised consensus outcome definitions that incorporate ADAMTS13 activity and the effects of anti-VWF therapy, by using an estimate-talk-estimate approach. The updated definitions distinguish clinical remission and clinical relapse (defined primarily by platelet count) from ADAMTS13 remission and ADAMTS13 relapse (defined by ADAMTS13 activity). The revised definitions of exacerbation and remission are benchmarked against not only the timing of discontinuation of TPE but also that of anti-VWF therapy. Retrospective validation of the revised definitions is described, although they have yet to be prospectively validated. Clinical implications of the updated outcome definitions are also discussed and an example of their application to clinical practice is provided to highlight their clinical relevance.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. In white individuals, HLA allele DRB1*11 is a predisposing factor for iTTP, whereas DRB1*04 is a protective factor. However, the role of HLA in Asians is unclear. In this study, we analyzed 10 HLA loci using next-generation sequencing in 52 Japanese patients with iTTP, and the allele frequency in the iTTP group was compared with that in a Japanese control group. We identified the following HLA alleles as predisposing factors for iTTP in the Japanese population: DRB1*08:03 (odds ratio [OR], 3.06; corrected P [Pc] = .005), DRB3/4/5*blank (OR, 2.3; Pc = .007), DQA1*01:03 (OR, 2.25; Pc = .006), and DQB1*06:01 (OR,: 2.41; Pc = .003). The estimated haplotype consisting of these 4 alleles was significantly more frequent in the iTTP group than in the control group (30.8% vs 6.0%; Pc < .001). DRB1*15:01 and DRB5*01:01 were weak protective factors for iTTP (OR, 0.23; Pc = .076; and OR, 0.23, Pc = .034, respectively). On the other hand, DRB1*11 and DRB1*04 were not associated with iTTP in the Japanese. These findings indicated that predisposing and protective factors for iTTP differ between Japanese and white individuals. HLA-DR molecules encoded by DRB1*08:03 and DRB1*11:01 have different peptide-binding motifs, but interestingly, bound to the shared ADAMTS13 peptide in an in silico prediction model.

Acute ischemic stroke (IS) and transient ischemic attack (TIA) are associated with raised von Willebrand factor (VWF) and decreased ADAMTS13 activity (ADAMTS13Ac). Their impact on mortality and morbidity is unclear. We conducted a prospective investigation of the VWF-ADAMTS13 axis in 292 adults (acute IS, n = 103; TIA, n = 80; controls, n = 109) serially from presentation until >6 weeks. The National Institutes of Health Stroke Score (NIHSS) and modified Rankin scale (mRS) were used to assess stroke severity. Presenting median VWF antigen (VWF:Ag)/ADAMTS13Ac ratios were: IS, 2.42 (range, 0.78-9.53); TIA, 1.89 (range, 0.41-8.14); and controls, 1.69 (range, 0.25-15.63). Longitudinally, the median VWF:Ag/ADAMTS13Ac ratio decreased (IS, 2.42 to 1.66; P = .0008; TIA, 1.89 to 0.65; P < .0001). The VWF:Ag/ADAMTS13Ac ratio was higher at presentation in IS patients who died (3.683 vs 2.014; P < .0001). A presenting VWF:Ag/ADAMTS13Ac ratio >2.6 predicted mortality (odds ratio, 6.33; range, 2.22-18.1). Those with a VWF:Ag/ADAMTS13Ac ratio in the highest quartile (>3.091) had 31% increased risk mortality. VWF:Ag/ADAMTS13Ac ratio at presentation of ischemic brain injury was associated with higher mRS (P = .021) and NIHSS scores (P = .029) at follow-up. Thrombolysis resulted in prompt reduction of the VWF:Ag/ADAMTS13Ac ratio and significant improvement in mRS on follow-up. A raised VWF:Ag/ADAMTS13Ac ratio at presentation of acute IS or TIA is associated with increased mortality and poorer functional outcome. A ratio of 2.6 seems to differentiate outcome. Prompt reduction in the ratio in thrombolysed patients was associated with decreased mortality and morbidity. The VWF:Ag/ADAMTS13Ac ratio is a biomarker for the acute impact of an ischemic event and longer-term outcome.

Upshaw-Schulman syndrome (USS) or congenital thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal thrombotic microangiopathy caused by a severe deficiency of ADAMTS13 activity resulting from biallelic ADAMTS13 gene mutations.1-5 Approximately 25% to 40% of patients with USS have a neonatal episode of severe hemolytic jaundice with thrombocytopenia that requires exchange blood transfusion, which is the classic hallmark of USS.6-11 This hallmark is distinct from the neonatal alloimmune Coombs-positive hemolytic jaundice caused by feto-maternal ABO or more often Rhesus (Rh) incompatibility that begins during pregnancy. Some patients with USS have onset of disease only during later childhood, adolescence, or even adulthood, suggesting that severe deficiency of ADAMTS13 activity is compatible with life as long as no additional triggers such as infections occur. Thus far, it is unclear what triggers neonatal hemolysis and thrombocytopenia in a substantial proportion of USS patients. The following observation of an exceptional case strongly suggests that severe neonatal hemolytic jaundice and thrombocytopenia in USS is triggered by the blood flow conditions in the ductus arteriosus that is normally patent for the first 48 hours after birth.

Severe deficiency of plasma ADAMTS13 activity is the primary cause of thrombotic thrombocytopenic purpura (TTP) while an overwhelming activation of complement via analternative pathway results in an atypical hemolytic uremic syndrome (aHUS), two prototypes of thrombotic microangiopathy. However, clinical and pathogenic distinctions between TTP and aHUS are often quite challenging. Clinical reports have suggested that complement activation may play a role in the development of TTP caused by severe deficiency of plasma ADAMTS13 activity. However, the experimental evidence to support this hypothesis is still lacking. Here, we show that mice with either Adamts13-/- or a heterozygous mutation of complement factor H (cfh) at amino acid residue of 1206 (i.e. cfhW/R) alone remain asymptomatic despite the presence of occasional microvascular thrombi in various organ tissues. However, mice carrying both Adamts13-/- and cfhW/R exhibit thrombocytopenia, low plasma haptoglobin, increased fragmentation of erythrocytes in peripheral blood smear, increased plasma levels of lactate dehydrogenase activity, BUN, and creatinine, as well as an increased mortality rate, consistent with the development of thrombotic microangiopathy. Moreover, mice with a homozygous mutation of cfh (i.e. cfhR/R) with or without Adamts13-/-developed severe thrombotic microangiopathy. The mortality rate in mice with Adamts13-/-cfhR/R was significantly higher than that in mice with cfhR/R alone. Histological and immunohistochemical analyses demonstrated the presence of disseminated platelet-rich thrombi in terminal arterioles and capillaries of major organ tissues in thesemice that were sacrificed or died. Together, our results support a synergistic effect of severe ADAMTS13 deficiency and complement activation in pathogenesis of thrombotic microangiopathy in mice.

Patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) often experience life-threatening relapses of the disease, and rituximab (RTX) can be used to mitigate relapse risk. However, the predictors of relapse in iTTP and the magnitude and duration of effect of RTX remain key unanswered questions. Using a multi-institutional cohort of consecutive adult patients with iTTP, we used survival analysis to compare relapse rates between patients who received RTX during the index presentation with acute iTTP and those who did not. Of 124 patients, 60 (48%) received RTX and 34 (27%) experienced relapse. Median time to relapse was 3.71 (interquartile range, 1.75-4.9) and 1.33 (interquartile range, 0.43-2.35) years for RTX-treated and untreated patients, respectively. RTX conferred protection from relapse at 1 year of follow-up (P = .01) but not at 5 years of follow-up. Extended Cox regression was then used to identify predictors of relapse and to estimate the protective effect of RTX. The following parameters were independently associated with increased risk for subsequent relapse: presenting in iTTP relapse (hazard ratio [HR], 2.97; 95% confidence interval [CI], 1.4-6.4), age younger than 25 years (HR, 2.94; 95% CI, 1.2-7.2), and non-O blood group (HR, 2.15; 95% CI, 1.06-4.39). RTX initially provided protection from relapse (HR, 0.16; 95% CI, 0.04-0.70), but this effect gradually diminished, returning to the baseline risk for untreated patients at approximately 2.6 years. Patients who are young, have non-O blood group, or present with relapsed iTTP are at increased risk for subsequent relapse. RTX appears to confer short-term protection from relapse.

Congenital thrombotic thrombocytopenic purpura (cTTP) is an ultra-rare thrombomicroangiopathy caused by an inherited deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13). There are limited data on genotype-phenotype correlation; there is no consensus on treatment. We reviewed the largest cohort of cTTP cases, diagnosed in the United Kingdom, over the past 15 years. Seventy-three cases of cTTP were diagnosed, confirmed by genetic analysis. Ninety-three percent were alive at the time of review. Thirty-six percent had homozygous mutations; 64% had compound heterozygous mutations. Two presentation peaks were seen: childhood (median diagnosis age, 3.5 years) and adulthood, typically related to pregnancy (median diagnosis age, 31 years). Genetic mutations differed by age of onset with prespacer mutations more likely to be associated with childhood onset (P = .0011). Sixty-nine percent of adult presentations were associated with pregnancy. Fresh-frozen plasma (FFP) and intermediate purity factor VIII concentrate were used as treatment. Eighty-eight percent of patients with normal blood counts, but with headaches, lethargy, or abdominal pain, reported symptom resolution with prophylactic therapy. The most common currently used regimen of 3-weekly FFP proved insufficient for 70% of patients and weekly or fortnightly infusions were required. Stroke incidence was significantly reduced in patients receiving prophylactic therapy (2% vs 17%; P = .04). Long-term, there is a risk of end-organ damage, seen in 75% of patients with late diagnosis of cTTP. In conclusion, prespacer mutations are associated with earlier development of cTTP symptoms. Prophylactic ADAMTS13 replacement decreases the risk of end-organ damage such as ischemic stroke and resolved previously unrecognized symptoms in patients with nonovert disease.

von Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. In this study, we demonstrate that the shear-dependent unfolding of the VWF A2 domain in the multimeric protein is a major regulator of protein self-association. This mechanism controls self-association on the platelet glycoprotein Ibα receptor, on collagen substrates, and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain unfolding due to disulfide bridging of N- and C-terminal residues (“Lock-VWF”) reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and 2 mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD type 2A mutation), enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain (“ΔA1 proteins”) suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified high-density lipoprotein and apolipoprotein-A1 partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13-mediated proteolysis, with low calcium and A2 disease mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved 2 balancing molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation: ADAMTS13-mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by the development of autoantibodies targeting different domains of ADAMTS13. Profiling studies have shown that residues R568, F592, R660, Y661, and Y665 within exosite-3 of the spacer domain provide an immunodominant region of ADAMTS13 for pathogenic autoantibodies that develop in patients with iTTP. Modification of these 5 core residues with the goal of reducing autoantibody binding revealed a significant tradeoff between autoantibody resistance and proteolytic activity. Here, we employed structural bioinformatics to identify a larger epitope landscape on the ADAMTS13 spacer domain. Models of spacer-antibody complexes predicted that residues R568, L591, F592, K608, M609, R636, L637, R639, R660, Y661, Y665, and L668 contribute to an expanded epitope within the spacer domain. Based on bioinformatics-guided predictions, we designed a panel of N-glycan insertions in this expanded epitope to reduce the binding of spacer domain autoantibodies. One N-glycan variant (NGLY3-ADAMTS13, containing a K608N substitution) showed strongly reduced reactivity with TTP patient sera (28%) as compared with WT-ADAMTS13 (100%). Insertion of an N-glycan at amino acid position 608 did not interfere with processing of von Willebrand factor, positioning the resulting NGLY3-ADAMTS13 variant as a potential novel therapeutic option for treatment of iTTP.

The anti–von Willebrand factor nanobody caplacizumab was licensed for adults with immune-mediated thrombotic thrombocytopenic purpura (iTTP) based on prospective controlled trials. However, few data are available on postmarketing surveillance. We treated 90 iTTP patients with a compassionate frontline triplet regimen associating therapeutic plasma exchange (TPE), immunosuppression with corticosteroids and rituximab, and caplacizumab. Outcomes were compared with 180 historical patients treated with the standard frontline treatment (TPE and corticosteroids, with rituximab as salvage therapy). The primary outcome was a composite of refractoriness and death within 30 days since diagnosis. Key secondary outcomes were exacerbations, time to platelet count recovery, the number of TPE, and the volume of plasma required to achieve durable remission. The percentage of patients in the triplet regimen with the composite primary outcome was 2.2% vs 12.2% in historical patients (P = .01). One elderly patient in the triplet regimen died of pulmonary embolism. Patients from this cohort experienced less exacerbations (3.4% vs 44%, P < .01); they recovered durable platelet count 1.8 times faster than historical patients (95% confidence interval, 1.41-2.36; P < .01), with fewer TPE sessions and lower plasma volumes (P < .01 both). The number of days in hospital was 41% lower in the triplet regimen than in the historical cohort (13 vs 22 days; P < .01). Caplacizumab-related adverse events occurred in 46 patients (51%), including 13 major or clinically relevant nonmajor hemorrhagic events. Associating caplacizumab to TPE and immunosuppression, by addressing the 3 processes of iTTP pathophysiology, prevents unfavorable outcomes and alleviates the burden of care.

Numerous studies have reported significant associations between ABO blood group and risk of cardiovascular disease. These studies have consistently demonstrated that thrombotic risk is significantly reduced in individuals in blood group O. Nevertheless, the biological mechanisms through which ABO influences hemostasis have remained poorly understood. Exciting recent data have provided novel insights into how these ABO effects are modulated and have highlighted that ABO group significantly influences platelet plug formation at sites of vascular injury (primary hemostasis). In particular, ABO affects multiple aspects of von Willebrand factor (VWF) biology. In keeping with their reduced thrombotic risk, plasma VWF levels are ∼25% lower in healthy group O compared with healthy group non-O individuals. In addition, blood group O VWF demonstrates enhanced susceptibility to ADAMTS13 proteolysis. Finally, preliminary findings suggest that the interaction of group O VWF with platelets may also be reduced. Although the molecular mechanisms underlying these ABO effects on VWF have not been fully elucidated, it seems likely that they are mediated in large part by the ABO(H) carbohydrate structures that are carried on both the N- and O-linked glycans of VWF. Interestingly, ABO(H) determinants are also expressed on several different platelet surface glycoprotein receptors. Recent studies support the hypothesis that ABO group not only exerts major quantitative and qualitative effects on VWF, but also affect specific aspects of platelet function. Given the severe morbidity and the mortality associated with thrombotic disorders, defining the mechanisms underlying these ABO effects is not only of scientific interest, but also of direct clinical importance.

Introduction of the nanobody caplacizumab was shown to be effective in the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in the acute setting. The official recommendations include plasma exchange (PEX), immunosuppression, and the use of caplacizumab for a minimum of 30 days after stopping daily PEX. This study was a retrospective, observational analysis of the use of caplacizumab in 60 patients from 29 medical centers in Germany. Immunosuppressive treatment led to a rapid normalization of ADAMTS13 activities (calculated median, 21 days). In 35 of 60 patients, ADAMTS13 activities started to normalize before day 30 after PEX; in 11 of 60 patients, the treatment was extended beyond day 30; and in 5 patients, it was extended even beyond day 58 due to persistent autoimmune activity. In 34 of 60 instances, caplacizumab was stopped before day 30 with a favorable outcome whenever ADAMTS13 activities were >10%. In contrast, 11 of 34 patients with ADAMTS13 activities

Thrombotic thrombocytopenia purpura (TTP) is a rare, life-threatening disease with an incidence of approximately 2 persons per million per year. It is characterized by severe deficiency of the von Willebrand cleaving protease, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), leading to formation of platelet-rich thrombi in the microvasculature. Prompt initiation of appropriate therapy, particularly plasma exchange, may be life-saving. Diagnosis of TTP is challenging because of its diverse clinical manifestations, overlap in clinical presentation with other thrombotic microangiopathies, and limited availability of ADAMTS13 testing. Clinical prediction scores have been developed to estimate the pretest probability of severe ADAMTS13 deficiency and may be used as an adjunct to clinical judgment to guide initial management decisions. An ADAMTS13 activity level of less than 10% supports the diagnosis of TTP in appropriate clinical contexts, but many centers do not offer testing in-house and must send out the test to a reference laboratory with a turnaround time of several days. In such instances, initial management decisions must be made without the benefit of laboratory testing. In patients with TTP, inhibitor tests may be useful for distinguishing immune-mediated from congenital TTP. In this article, we review the epidemiology, natural history, and clinical presentation of TTP and laboratory assays for TTP including ADAMTS13 activity and inhibitor assays. We also describe an evidence-based approach to the evaluation of a patient with suspected TTP that integrates clinical and laboratory assessment.

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.

Immune thrombotic thrombocytopenic purpura (iTTP) is primarily caused by immunoglobulin G (IgG)–type autoantibodies that bind and inhibit plasma ADAMTS13 activity and/or accelerate its clearance from circulation. Approximately 50% of patients with iTTP who achieve initial clinical response to therapy experience recurrence (ie, exacerbation and/or relapse); however, a reliable biomarker that predicts such an event is currently lacking. The present study determines the role of longitudinal assessments of plasma ADAMTS13 biomarkers in predicting iTTP exacerbation/recurrence. Eighty-three unique iTTP patients with 97 episodes from the University of Alabama at Birmingham Medical Center between April 2006 and June 2019 were enrolled. Plasma levels of ADAMTS13 activity, antigen, and anti-ADAMTS13 IgG on admission showed no significant value in predicting iTTP exacerbation or recurrence. However, persistently low plasma ADAMTS13 activity (75th percentile; HR, 2.6; 95% CI, 1.0-6.5; P = .047) at clinical response or remission was also predictive of exacerbation or recurrence. Our results suggest the potential need for a more aggressive approach to achieve biochemical remission (ie, normalization of plasma ADAMTS13 activity, ADAMTS13 antigen, and anti-ADAMTS13 IgG) in patients with iTTP to prevent the disease recurrence.

ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, 13) is a metalloprotease responsible for cleavage of ultra-large von Willebrand factor (VWF) multimers. Severely deficient activity of the protease can trigger an acute episode of thrombotic thrombocytopenic purpura (TTP). Our understanding of the pathophysiology of TTP has allowed us to grasp the important role of ADAMTS13 in other thrombotic microangiopathies (TMAs) and thrombotic disorders, such as ischemic stroke and coronary artery disease. Through its action on VWF, ADAMTS13 can have prothrombotic and proinflammatory properties, not only when its activity is severely deficient, but also when it is only moderately low. Here, we will discuss the biology of ADAMTS13 and the different assays developed to evaluate its function in the context of TTP, in the acute setting and during follow-up. We will also discuss the latest evidence regarding the role of ADAMTS13 in other TMAs, stroke, and cardiovascular disease. This information will be useful for clinicians not only when evaluating patients who present with microangiopathic hemolytic anemia and thrombocytopenia, but also when making clinical decisions regarding the follow-up of patients with TTP.

Increasing evidence indicates that inflammation can cause thrombosis by a von Willebrand factor (VWF)-mediated mechanism that includes endothelial activation, secretion of VWF, assembly of hyperadhesive VWF strings and fibers, cleavage by ADAMTS13, and adhesion and deposition of VWF-platelet thrombi in the vasculature. This mechanism appears to contribute to thrombosis not only in small vessels, but also in large vessels. Inflammation and VWF contribute to atherogenesis and may contribute to arterial and venous thrombosis as well as stroke. Elucidation of the mechanism will hopefully identify new targets and suggest new approaches for prevention and intervention.

In this issue of Blood, Alwan et al report that 21 (29%) of 73 patients with congenital thrombotic thrombocytopenic purpura (TTP) had persistent symptoms of headache, lethargy, and abdominal pain, without thrombocytopenia, that responded to prophylactic plasma.1

Thrombocytopenia develops in 5% to 10% of women during pregnancy or in the immediate postpartum period. A low platelet count is often an incidental feature, but it might also provide a biomarker of a coexisting systemic or gestational disorder and a potential reason for a maternal intervention or treatment that might pose harm to the fetus. This chapter reflects our approach to these issues with an emphasis on advances made over the past 5 to 10 years in understanding and managing the more common causes of thrombocytopenia in pregnancy. Recent trends in the management of immune thrombocytopenia translate into more women contemplating pregnancy while on treatment with thrombopoietin receptor agonists, rituximab, or mycophenylate, which pose known or unknown risks to the fetus. New criteria to diagnose preeclampsia, judicious reliance on measurement of ADAMTS13 to make management decisions in suspected thrombotic thrombocytopenic purpura, new evidence supporting the efficacy and safety of anticomplement therapy for atypical hemolytic uremic syndrome during pregnancy, and implications of thrombotic microangiopathies for subsequent pregnancies are evolving rapidly. The goals of the chapter are to help the hematology consultant work through the differential diagnosis of thrombocytopenia in pregnancy based on trimester of presentation, severity of thrombocytopenia, and coincident clinical and laboratory manifestations, and to provide guidance for dealing with some of the more common and difficult diagnostic and management decisions.

Immune-mediated thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder caused by antibodies against ADAMTS13. From the United Kingdom TTP registry, we undertook a prospective study investigating the impact of the presenting anti-ADAMTS13 IgG antibody and ADAMTS13 antigen on mortality. A total of 312 episodes involving 292 patients over 87 months were included; 68% were female, median age 46 (range, 11-88 years), and median presenting ADAMTS13 of 77%) had a mortality of 16.9% compared with 5.0% for the lowest quartile (antibody 11%) (P = .005). The synergistic effect of anti-ADAMTS13 IgG antibody in the upper quartile and ADAMTS13 antigen in the lowest quartile had the highest mortality of 27.3%. We conclude that both anti-ADAMTS13 IgG antibody and ADAMTS13 antigen levels correlate with outcome in TTP with increased cardiac and neurological involvement and increased mortality.

Key Points Immunological dysregulation may underlie unusual autoimmune diseases, which also deserve to be investigated from a genetic point of view.

With timely and effective treatment, most patients with thrombotic thrombocytopenic purpura (TTP) survive the acute TTP episode. In addition to the risk of relapse, TTP survivors have higher all-cause mortality than the general population and increased rates of chronic morbidities, including hypertension, depression, and mild cognitive impairment. We conducted this retrospective-prospective cohort study to determine the incidence and prevalence of stroke after recovery from acute TTP and to test the hypothesis that lower ADAMTS13 activity after recovery from TTP is associated with an increased risk of stroke during remission. Of 170 consecutive patients treated for TTP at The Johns Hopkins Hospital from 1995 through 2018, 14 (8.2%) died during the index episode and 19 were observed for less than 1 month after recovery. Of the remaining 137 patients, 18 (13.1%) developed stroke unrelated to an acute TTP episode over a median observation period of 3.08 years, which is fivefold higher than the expected prevalence of 2.6% from an age- and sex-matched reference population (P = .002). ADAMTS13 activity during remission was measured in 52 patients and was >70% in 44.2%, 40% to 70% in 23.1%, 10% to 39% in 25%, and 70%) and in 27.6% (8 of 29) of patients with low ADAMTS13 activity (≤70%; P = .007). In conclusion, stroke is common after recovery from TTP and is associated with reduced ADAMTS13 activity during remission.

Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood. Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 μM and 45 μM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (KD = 0.72 μM), soluble VWF (KD = 0.58 μM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼170 ng/mL; range, 58-3570; n = 19) compared with healthy controls (median, ∼23 ng/mL; range, 6-44; n = 18) (P < .0001). Liquid chromatography plus tandem mass spectrometry (LC-MS/MS) reveals statistically significant increases of HNP1, HNP2, and HNP3 in patient samples (all P values

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin motif repeats 13) has antithrombotic properties because it cleaves von Willebrand factor (VWF) in smaller, less active multimers. The aim of our study was to investigate prospectively the association between ADAMTS13 activity and ischemic stroke. We included 5941 individuals ≥55 years without a history of stroke or transient ischemic attack (TIA) of the Rotterdam Study, a population-based cohort study. ADAMTS13 activity was measured at inclusion with the FRETS-VWF73 assay and VWF antigen (VWF:Ag) levels by enzyme-linked immunosorbent assay. We assessed the association among ADAMTS13 activity, VWF:Ag levels, and ischemic stroke by Cox proportional hazard analysis. The added value of ADAMTS13 activity above the traditional risk factors for ischemic stroke risk prediction was examined by the C-statistic and the net reclassification improvement index (NRI). All individuals were followed for incident stroke or TIA. Over a median follow-up time of 10.7 years (56,403 total person-years), 461 participants had a stroke, 306 of which were ischemic. After adjustment for cardiovascular risk factors, individuals with ADAMTS13 activity in the lowest quartile had a higher risk of ischemic stroke (absolute risk, 7.3%) than did those in the reference highest quartile (absolute risk, 3.8%; hazard ratio, 1.65; 95% confidence interval [CI], 1.16-2.32). Adding ADAMTS13 to the model in prediction of ischemic stroke, increased the C-statistic by 0.013 (P = .003) and provided 0.058 (95% CI, -0.002 to 0.119) NRI. Low ADAMTS13 activity is associated with the risk of ischemic stroke and improves the accuracy of risk predictions for ischemic stroke beyond traditional risk factors.

Daily therapeutic plasma exchange (TPE) transformed the historically fatal prognosis of acquired, anti-ADAMTS13 antibody-mediated thrombotic thrombocytopenic purpura (TTP), leading to the current overall survival rates of 80%-85%. However, relapses occur in ∼40% of patients and refractory disease with fatal outcomes still occurs. In this context, the introduction of rituximab has probably been the second major breakthrough in TTP management. Rituximab is now routinely recommended during the acute phase, typically in patients with a suboptimal response to treatment, or even as frontline therapy, with high response rates. In more severe patients, salvage strategies may include twice-daily TPE, pulses of cyclophosphamide, vincristine, as well as splenectomy in more desperate cases. In this life-threatening disease, relapse prevention represents a major goal. Persistent severe acquired ADAMTS13 deficiency in patients who are otherwise in remission is associated with a high risk of relapse and preemptive treatment with rituximab may be considered in this context. In the coming years, the TTP therapeutic landscape should be enriched by original strategies stemming from clinical experience and new agents that are currently being evaluated in large, ideally international, clinical trials. Promising agents under evaluation include N-acetylcysteine, bortezomib, recombinant ADAMTS13, and inhibitors of the glycoprotein-Ib/IX-von Willebrand factor axis.

Liver disease results in complex alterations of all 3 phases of hemostasis. It is now recognized that hemostasis is rebalanced in chronic liver disease. The fall in clotting factor levels is accompanied by a parallel fall in anticoagulant proteins. High von Willebrand factor levels counteract defects in primary hemostasis. Conventional coagulation tests do not fully reflect the derangement in hemostasis and do not accurately predict the risk of bleeding. Global coagulation assays (thrombin generation, thromboelastography) reflect the interaction between procoagulant factors, anticoagulant factors, platelets, and the fibrinolytic system and show promise for assessing bleeding risk and guiding therapy. These assays are not yet commercially approved or validated. Prevention of bleeding should not be aimed at correcting conventional coagulation tests. Thrombopoietin receptor agonists were shown to increase the platelet count in cirrhotic patients undergoing invasive procedures but may increase the risk of thrombosis. Rebalanced hemostasis in liver disease is precarious and may be tipped toward hemorrhage or thrombosis depending on coexisting circumstantial risk factors. Bacterial infection may impair hemostasis in cirrhosis by triggering the release of endogenous heparinoids. There are no evidence-based guidelines for hemostatic therapy of acute hemorrhage in liver disease. There is currently inadequate evidence to support the use of recombinant FVIIa, prothrombin complex concentrates, or tranexamic acid in acute variceal or other hemorrhage.

von Willebrand factor (VWF) is an adhesive ligand, and its activity is proteolytically regulated by the metalloprotease ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat 13). An elevated level of plasma VWF has been widely considered a marker for endothelial cell activation in trauma and inflammation, but its causal role in these pathological conditions remains poorly defined. Using a fluid percussion injury mouse model, we demonstrated that VWF released during acute traumatic brain injury (TBI) was activated and became microvesicle-bound. The VWF-bound microvesicles promoted vascular leakage and systemic coagulation. Recombinant ADAMTS-13 given either before or after TBI reduced the VWF reactivity with minimal influence on VWF secretion. rADAMTS-13 protected the integrity of endothelial cell barriers and prevented TBI-induced coagulopathy by enhancing VWF cleavage without impairing basal hemostasis. Promoting microvesicle clearance by lactadherin had efficacy similar to that of rADAMTS-13. This study uncovers a novel synergistic action between VWF and cellular microvesicles in TBI-induced vascular leakage and coagulopathy and demonstrates protective effects of rADAMTS-13.

Understanding the autoimmune etiology of acquired thrombotic thrombocytopenic purpura (TTP) has provided precision for the diagnosis and a rationale for immunosuppressive treatment. These advances have also allowed recognition of the remarkable clinical diversities of patients’ initial presentations and their long-term outcomes. These diversities are illustrated by the stories of patients from the Oklahoma TTP Registry. The initial presentation of TTP may be the discovery of unexpected severe thrombocytopenia in a patient with minimal or no symptoms. The patient may remain asymptomatic throughout treatment or may die suddenly before treatment can be started. ADAMTS13 activity may be reported as normal in a patient with characteristic clinical features of TTP, or the unexpected report of ADAMTS13 deficiency in a patient with another established disorder may lead to the discovery of TTP. ADAMTS13 activity during clinical remission is unpredictable. ADAMTS13 activity may recover and remain normal, it may remain severely deficient for many years, or it may become normal only many years after recovery. Our treatment of initial episodes and management of patients after recovery and during remission continue to change. The addition of rituximab to the treatment of acute episodes and preemptive rituximab for patients with severe ADAMTS13 deficiency during remission are reported to prevent relapse. Because TTP is uncommon, there are few data to guide these changes. Therefore our patients’ stories are profoundly influential. Their stories are the foundation of our experience, and our experience is the guide for our decisions.

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats member 13) prevents microvascular thrombosis by cleaving von Willebrand factor (VWF) within platelet-rich thrombi, and cleavage depends on allosteric activation of ADAMTS13 by the substrate VWF. Human ADAMTS13 has a short propeptide, metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains (distal domains). Distal domains inhibit the catalytic proximal domains; binding of distal T8-CUB domains to the VWF D4 domain relieves autoinhibition and promotes cleavage of the nearby VWF A2 domain. However, the role of specific ADAMTS13 distal domains in this allosteric mechanism is not established. Assays of plasma ADAMTS13 from 20 placental mammals, birds, and amphibians show that allosteric regulation is broadly conserved, and phylogenetic analysis of 264 vertebrates shows the long propeptide, T3, T4, T6, and T6a domains have been deleted several times in placental mammals, birds, and fish. Notably, pigeon ADAMTS13 has only 3 distal T domains but was activated normally by human VWF D4 and cleaved VWF multimers, preferentially under fluid shear stress. Human ADAMTS13 constructed to resemble pigeon ADAMTS13 retained normal allosteric regulation and shear-dependent cleavage of VWF. Thus, the T3-T6 domains of human ADAMTS13 are dispensable. Conversely, deletion of T7 or T8 abolished allosteric activation. For most species, some sequence changes in the VWF substrate can markedly increase the rate of cleavage, suggesting that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs. These properties may reflect evolutionary pressure to balance the risk for VWF-dependent bleeding and thrombosis.

ADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggregation and arterial thrombosis. An inability to cleave ultralarge VWF resulting from hereditary or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma exchange is the most effective initial therapy for TTP to date. Here, we report characterization of transgenic mice expressing recombinant human ADAMTS13 (rADAMTS13) in platelets and its efficacy in inhibiting arterial thrombosis and preventing hereditary and acquired antibody-mediated TTP in murine models. Western blotting and fluorescent resonance energy transfer assay detect full-length rADAMTS13 protein and its proteolytic activity, respectively, in transgenic (Adamts13(-/-)Plt(A13)), but not in wild-type and Adamts13(-/-), platelets. The expressed rADAMTS13 is released on stimulation with thrombin and collagen, but less with 2MesADP. Platelet-delivered rADAMTS13 is able to inhibit arterial thrombosis after vascular injury and prevent the onset and progression of Shigatoxin-2 or recombinant murine VWF-induced TTP syndrome in mice despite a lack of plasma ADAMTS13 activity resulting from the ADAMTS13 gene deletion or the antibody-mediated inhibition of plasma ADAMTS13 activity. These findings provide a proof of concept that platelet-delivered ADAMTS13 may be explored as a novel treatment of arterial thrombotic disorders, including hereditary and acquired TTP, in the presence of anti-ADAMTS13 autoantibodies.

A 40-year-old obese black woman developed abdominal pain with progressive generalized weakness over several days. Physical examination was normal except for several small bruises on her extremities. Laboratory data revealed the following: hemoglobin, 5.0 g/dL; platelet count, 4000/µL; creatinine, 0

Acquired thrombotic thrombocytopenic purpura (TTP) is the consequence of a severe ADAMTS13 deficiency resulting from autoantibodies inhibiting ADAMTS13 or accelerating its clearance. Despite the success of plasma exchange the risk of relapse is high. From 2 patients (A and B), splenectomized for recurrent episodes of acquired TTP, the splenic B-cell response against ADAMTS13 was characterized through generation of human monoclonal anti-ADAMTS13 autoantibodies (mAbs) by cloning an immunoglobulin G (IgG)4κ- and IgG4λ-Fab library using phage display technology and by Epstein-Barr virus transformation of switched memory B cells (CD19+/CD27+/IgG+). Sequence analysis of the anti-ADAMTS13 IgGs of both patients revealed that the VH gene use was limited in our patients to VH1-3 (55%), VH1-69 (17%), VH3-30 (7%), and VH4-28 (21%) and contained 8 unique and thus far not reported heavy-chain complementarity determining region 3 motifs, of which 4 were shared by the 2 patients. The discovery of several highly similar anti-ADAMTS13 autoantibodies in 2 unrelated TTP patients suggests that the autoimmune response is antigen driven, because the probability that such similar immunoglobulin rearrangements happen by chance is very low (< 10(-9)).

Pregnancy can precipitate thrombotic thrombocytopenic purpura (TTP). We present a prospective study of TTP cases from the United Kingdom Thrombotic Thrombocytopenic Purpura (UK TTP) Registry with clinical and laboratory data from the largest cohort of pregnancy-associated TTP and describe management through pregnancy, averting fetal loss and maternal complications. Thirty-five women presented with a first TTP episode during pregnancy: 23/47 with their first congenital TTP (cTTP) episode and 12/47 with acute acquired TTP in pregnancy. TTP presented primarily in the third trimester/postpartum, but fetal loss was highest in the second trimester. Fetal loss occurred in 16/38 pregnancies before cTTP was diagnosed, but in none of the 15 subsequent managed pregnancies. Seventeen of 23 congenital cases had a missense mutation, C3178T, within exon 24 (R1060W). There were 8 novel mutations. In acquired TTP presentations, fetal loss occurred in 5/18 pregnancies and 2 terminations because of disease. We also present data on 12 women with a history of nonpregnancy-associated TTP: 18 subsequent pregnancies have been successfully managed, guided by ADAMTS13 levels. cTTP presents more frequently than acquired TTP during pregnancy and must be differentiated by ADAMTS13 analysis. Careful diagnosis, monitoring, and treatment in congenital and acquired TTP have assisted in excellent pregnancy outcomes.

Pregnancy may precipitate acute episodes of thrombotic thrombocytopenic purpura (TTP), but pregnancy outcomes in women who have recovered from acquired TTP are not well documented. We analyzed pregnancy outcomes following recovery from TTP associated with acquired, severe ADAMTS13 deficiency (ADAMTS13 activity