Mechanisms of inhibition of human monoclonal antibodies in immune thrombotic thrombocytopenic purpura.

Antibody binding to a plasma metalloprotease, a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13), is necessary for the development of immune thrombotic thrombocytopenic purpura (iTTP). Inhibition of ADAMTS13-mediated von Willebrand factor (VWF) cleavage by such antibodies clearly plays a role in the pathophysiology of the disease, although the mechanisms by which they inhibit ADAMTS13 enzymatic function are not fully understood. At least some immunoglobulin G-type antibodies appear to affect the conformational accessibility of ADAMTS13 domains involved in both substrate recognition and inhibitory antibody binding. We used single-chain fragments of the variable region previously identified via phage display from patients with iTTP to explore the mechanisms of action of inhibitory human monoclonal antibodies. Using recombinant full-length ADAMTS13, truncated ADAMTS13 variants, and native ADAMTS13 in normal human plasma, we found that, regardless of the conditions tested, all 3 inhibitory monoclonal antibodies tested affected enzyme turnover rate much more than substrate recognition of VWF. Hydrogen-to-deuterium exchange plus mass spectrometry experiments with each of these inhibitory antibodies demonstrated that residues in the active site of the catalytic domain of ADAMTS13 are differentially exposed to solvent in the presence and absence of monoclonal antibody binding. These results support the hypothesis that inhibition of ADAMTS13 in iTTP may not necessarily occur because the antibodies directly prevent VWF binding, but instead because of allosteric effects that impair VWF cleavage, likely by affecting the conformation of the catalytic center in the protease domain of ADAMTS13. Our findings provide novel insight into the mechanism of autoantibody-mediated inhibition of ADAMTS13 and pathogenesis of iTTP.

Race, rituximab, and relapse in TTP.

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.

Protein arginine methyltransferase 1 in the generation of immune megakaryocytes: A perspective review.

Megakaryocytes (Mks) in bone marrow are heterogeneous in terms of polyploidy. They not only produce platelets but also support the self-renewal of hematopoietic stem cells and regulate immune responses. Yet, how the diverse functions are generated from the heterogeneous Mks is not clear at the molecular level. Advances in single-cell RNA seq analysis from several studies have revealed that bone marrow Mks are heterogeneous and can be clustered into 3 to 4 subpopulations: a subgroup that is adjacent to the hematopoietic stem cells, a subgroup expressing genes for platelet biogenesis, and a subgroup expressing immune-responsive genes, the so-called immune Mks that exist in both humans and mice. Immune Mks are predominantly in the low-polyploid (≤8 N nuclei) fraction and also exist in the lung. Protein arginine methyltransferase 1 (PRMT1) expression is positively correlated with the expression of genes involved in immune response pathways and is highly expressed in immune Mks. In addition, we reported that PRMT1 promotes the generation of low-polyploid Mks. From this perspective, we highlighted the data suggesting that PRMT1 is essential for the generation of immune Mks via its substrates RUNX1, RBM15, and DUSP4 that we reported previously. Thus, we suggest that protein arginine methylation may play a critical role in the generation of proinflammatory platelet progeny from immune Mks, which may affect many immune, thrombotic, and inflammatory disorders.

Synergistic effects of ADAMTS13 deficiency and complement activation in pathogenesis of thrombotic microangiopathy.

Severe deficiency of plasma ADAMTS13 activity is the primary cause of thrombotic thrombocytopenic purpura (TTP) whereas overwhelming activation of complement via an alternative pathway results in atypical hemolytic uremic syndrome (aHUS), the prototypes of thrombotic microangiopathy (TMA). 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, which is 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 (ie, cfh W/R ) alone remain asymptomatic despite the presence of occasional microvascular thrombi in various organ tissues. However, mice carrying both Adamts13 -/- and cfh W/R exhibit thrombocytopenia, low haptoglobin, increased fragmentation of erythrocytes in peripheral blood smear, increased plasma levels of lactate dehydrogenase activity, blood urea nitrogen, and creatinine, as well as an increased mortality rate, consistent with the development of TMA. Moreover, mice with a homozygous mutation of cfh (ie, cfh R/R ) with or without Adamts13 -/- developed severe TMA. The mortality rate in mice with Adamts13 -/- cfh R/R was significantly higher than that in mice with cfh R/R alone. Histological and immunohistochemical analyses demonstrated the presence of disseminated platelet-rich thrombi in terminal arterioles and capillaries of major organ tissues in these mice that were either euthanized or died. Together, our results support a synergistic effect of severe ADAMTS13 deficiency and complement activation in pathogenesis of TMA in mice.

Histone-induced thrombotic thrombocytopenic purpura in adamts13 -/- zebrafish depends on von Willebrand factor.

Thrombotic thrombocytopenic purpura (TTP) is caused by severe deficiency of ADAMTS13 (A13), a plasma metalloprotease that cleaves endothelium-derived von Willebrand factor (VWF). However, severe A13 deficiency alone is often not sufficient to cause an acute TTP; additional factors may be required to trigger the disease. Using CRISPR/Cas9, we created and characterized several novel zebrafish lines carrying a null mutation in a13-/- , vwf, and both. We further used these zebrafish lines to test the hypothesis that inflammation that results in neutrophil activation and release of histone/DNA complexes may trigger TTP. As shown, a13-/- zebrafish exhibit increased levels of plasma VWF antigen, multimer size, and ability of thrombocytes to adhere to a fibrillar collagen-coated surface under flow. The a13-/- zebrafish also show an increased rate of occlusive thrombus formation in the caudal venules after FeCl3 injury. More interestingly, a13-/- zebrafish exhibit ~30% reduction in the number of total, immature, and mature thrombocytes with increased fragmentation of erythrocytes. Administration of a lysine-rich histone results in more severe and persistent thrombocytopenia and a significantly increased mortality rate in a13-/- zebrafish than in wildtype (wt) ones. However, both spontaneous and histone-induced TTP in a13-/- zebrafish are rescued by the deletion of vwf These results demonstrate a potentially mechanistic link between inflammation and the onset of TTP in light of severe A13 deficiency; the novel zebrafish models of TTP may help accelerate our understanding of pathogenic mechanisms and the discoveries of novel therapeutics for TTP and perhaps other arterial thrombotic disorders.

Murine systemic thrombophilia and hemolytic uremic syndrome from a factor H point mutation.

Complement plays a key role in host defense, but its dysregulation can cause autologous tissue injury. Complement activation is normally controlled by regulatory proteins, including factor H (FH) in plasma and membrane cofactor protein (MCP) on the cell surface. Mutations in FH and MCP are linked to atypical hemolytic uremic syndrome, a type of thrombotic microangiopathy (TMA) that causes renal failure. We describe here that disruption of FH function on the cell surface can also lead to disseminated complement-dependent macrovascular thrombosis. By gene targeting, we introduced a point mutation (W1206R) into murine FH that impaired its interaction with host cells but did not affect its plasma complement-regulating activity. Homozygous mutant mice carrying this mutation developed renal TMA as well as systemic thrombophilia involving large blood vessels in multiple organs, including liver, lung, spleen, and kidney. Approximately 30% of mutant mice displayed symptoms of stroke and ischemic retinopathy, and 48% died prematurely. Genetic deficiency of complement C3 and factor D prevented both the systemic thrombophilia and renal TMA phenotypes. These results demonstrate a causal relationship between complement dysregulation and systemic angiopathy and suggest that complement activation may contribute to various human thrombotic disorders involving both the micro- and macrovasculature.

Clinical factors and biomarkers predict outcome in patients with immune-mediated thrombotic thrombocytopenic purpura.

Immune-mediated thrombotic thrombocytopenic purpura is characterized by severe thrombocytopenia and microangiopathic hemolytic anemia. It is primarily caused by immunoglobin G type autoantibodies against ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor. However, reliable markers predictive of patient outcomes are yet to be identified. Seventy-three unique patients with a confirmed diagnosis of immune-mediated thrombotic thrombocytopenic purpura between April 2006 and December 2017 were enrolled from the Univeristy of Alabama at Birmingham Medical Center. Clinical information, laboratory values, and a panel of special biomarkers were collected and/or determined. The results demonstrated that the biomarkers associated with endothelial injury (e.g., von Willebrand factor antigen and collagen-binding activity), acute inflammation (e.g., human neutrophil peptides 1-3 and histone/deoxyribonucleic acid complexes), and activation of the complement alternative pathway (e.g., factors Bb and iC3b) were all significantly increased in patients with acute immune-mediated thrombotic thrombocytopenic purpura compared to those in the healthy controls. Moreover, failure to normalize platelet counts within 7 days or failure to markedly reduce serum lactate dehydrogenase by day 5, low total serum protein or albumin, and high serum troponin levels were also predictive of mortality, as were the prolonged activated partial thromboplastin time, high fibrinogen, and elevated serum lactate dehydrogenase, Bb, and sC5b-9 on admission. These results may help to stratify patients for more intensive management. The findings may also provide a framework for future multicenter studies to identify valuable prognostic markers for immune-mediated thrombotic thrombocytopenic purpura.

Transfusion of Platelets Loaded With Recombinant ADAMTS13 (A Disintegrin and Metalloprotease With Thrombospondin Type 1 Repeats-13) Is Efficacious for Inhibiting Arterial Thrombosis Associated With Thrombotic Thrombocytopenic Purpura.

Objective- ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cleaves VWF (von Willebrand factor). This process is essential for hemostasis. Severe deficiency of plasma ADAMTS13 activity, most commonly resulting from autoantibodies against ADAMTS13, causes thrombotic thrombocytopenic purpura. Therapeutic plasma exchange is the standard of care to date, which removes autoantibodies and replenishes ADAMTS13. However, such a therapy is often ineffective to raise plasma ADAMTS13 activity, and in-hospital mortality rate remains as high as 20%. Approach and Results- To overcome the inhibition by autoantibodies, we developed a novel approach by delivering rADAMTS13 (recombinant ADAMTS13 ) using platelets as vehicles. We show that both human and murine platelets can uptake rADAMTS13 ex vivo. The endocytosed rADAMTS13 within platelets remains intact, active, and is stored in α-granules. Under arterial shear (100 dyne/cm2), the rADAMTS13 in platelets is released and effectively inhibits platelet adhesion and aggregation on a collagen-coated surface in a concentration-dependent manner. Transfusion of rADAMTS13-loaded platelets into Adamts13-/- mice dramatically reduces the rate of thrombus formation in the mesenteric arterioles after FeCl3 injury. An ex vivo transfusion of rADAMTS13-loaded platelets to a reconstituted whole blood containing plasma from a patient with immune-mediated thrombotic thrombocytopenic purpura and the cellular components (eg, erythrocytes and leukocytes) from a healthy individual, as well as a fresh whole blood obtained from a patient with congenital or immune-mediated thrombotic thrombocytopenic purpura also dramatically reduces the rate of thrombus formation under arterial flow. Conclusions- Our results demonstrate that transfusion of rADAMTS13-loaded platelets may be a novel and potentially effective therapeutic approach for arterial thrombosis, associated with congenital and immune-mediated thrombotic thrombocytopenic purpura.

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)γRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.

Human neutrophil peptides inhibit cleavage of von Willebrand factor by ADAMTS13: a potential link of inflammation to TTP.

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 <.001). There is a good correlation between measurement of HNPs1-3 by ELISA and by LC-MS/MS (Spearman ρ = 0.7932, P < .0001). Together, these results demonstrate that HNPs1-3 may be potent inhibitors of ADAMTS13 activity, likely by binding to the central A2 domain of VWF and physically blocking ADAMTS13 binding. Our findings may provide a novel link between inflammation/infection and the onset of microvascular thrombosis in acquired TTP and potentially other immune thrombotic disorders.

Management of chronic myeloid leukemia in the setting of pregnancy: when is leukocytapheresis appropriate? A case report and review of the literature.

BACKGROUND: Chronic myeloid leukemia (CML) is a common hematologic malignancy; however, its occurrence during pregnancy is unusual due to its low prevalence in females of childbearing age. There are conflicting reports of how to best manage CML in pregnancy, particularly in the setting of leukocytosis. HEMAPHERESIS: A 30-year-old female was diagnosed with CML at 18 weeks' estimated gestational age. On initial presentation she reported fatigue, night sweats, and early satiety, and was found to have a white blood cell (WBC) count of 69.3 × 109 /L and platelet count of 366 × 109 /L. Her disease was managed during pregnancy using interferon-α alone despite persistent leukocytosis. CONCLUSION: CML may be effectively managed during pregnancy, even in the setting of leukocytosis, without the application of leukocytapheresis. Management relies not only upon the coordination of drug therapy and fetal monitoring, but requires close communication between multiple medical disciplines. Leukocytapheresis has been safely performed during pregnancy and may be a suitable adjunct management strategy in pregnant patients diagnosed with CML with specific clinical presentations, such as hyperleukocytosis (WBC count > 150 × 109 /L) and/or symptomatic leukostasis.

Expression of ADAMTS13 in Normal and Abnormal Placentae and Its Potential Role in Angiogenesis and Placenta Development.

OBJECTIVE: ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 repeats, member 13) is primarily synthesized in liver. The biosynthesis of ADAMTS13 and its physiological role in placenta are not known. APPROACH AND RESULTS: We used real-time polymerase chain reaction, immunohistochemistry, and Western blotting analyses, as well as proteolytic cleavage of FRETS (fluorescent resonance energy transfers)-VWF73, to determine ADAMTS13 expression in placenta and trophoblasts obtained from individuals with normal pregnancy and patients with severe preeclampsia. We also determined the role of ADAMTS13 in extravillous trophoblasts using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound scratch assay, transwell migration assay, tube formation assay, and tissue outgrowth assays. We showed that full-length and proteolytically active ADAMTS13 was expressed in normal human placenta, primarily in the trophoblasts and villous core fetal vessel endothelium during pregnancy. Placental expression of ADAMTS13 mRNA, protein, and proteolytic activity was at the highest levels during the first trimester and significantly reduced at the term of gestation. Additionally, significantly reduced levels of placental ADAMTS13 expression was detected under hypoxic conditions and in patients with preeclampsia. In addition, recombinant ADAMTS13 protease stimulated proliferation, migration, invasion, and network formation of trophoblastic cells in culture. Finally, knockdown of ADAMTS13 expression attenuated the ability of tube formation in trophoblast (HTR-8/SVNEO) cells and the extravillous trophoblast outgrowth in placental explants. CONCLUSIONS: Our results demonstrate for the first time the expression of ADAMTS13 mRNA and protein in normal and abnormal placental tissues and its role in promoting angiogenesis and trophoblastic cell development. The findings support the potential role of the ADAMTS13-von Willebrand factor pathway in normal pregnancy and pathogenesis of preeclampsia.

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome.

Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.

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.

ADAMTS13, lucky to have a hydrophobic pocket.

In this issue of Blood, de Groot et al identify a hydrophobic pocket in the Cys-rich domain of ADAMTS13 that appears to interact with the hydrophobic pocket in the central A2 domain of von Willebrand factor (VWF) for its proteolysis.

Platelet-delivered ADAMTS13 inhibits arterial thrombosis and prevents thrombotic thrombocytopenic purpura in murine models.

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.

ADAMTS13 test and/or PLASMIC clinical score in management of acquired thrombotic thrombocytopenic purpura: a cost-effective analysis.

BACKGROUND: The ADAMTS13 test distinguishes thrombotic thrombocytopenic purpura (TTP) from other thrombotic microangiopathies (TMAs). The PLASMIC score helps determine the pretest probability of ADAMTS13 deficiency. Due to inherent limitations of both tests, and potential adverse effects and cost of unnecessary treatments, we performed a cost-effectiveness analysis (CEA) investigating the benefits of incorporating an in-hospital ADAMTS13 test and/or PLASMIC score into our clinical practice. STUDY DESIGN AND METHODS: A CEA model was created to compare four scenarios for patients with TMAs, utilizing either an in-house or a send-out ADAMTS13 assay with or without prior risk stratification using PLASMIC scoring. Model variables, including probabilities and costs, were gathered from the medical literature, except for the ADAMTS13 send-out and in-house tests, which were obtained from our institutional data. RESULTS: If only the cost is considered, in-house ADAMTS13 test for patients with intermediate- to high-risk PLASMIC score is the least expensive option ($4,732/patient). If effectiveness is assessed as measured by the number of averted deaths, send-out ADAMTS13 test is the most effective. Considering the cost/effectiveness ratio, the in-house ADAMTS13 test in patients with intermediate- to high-risk PLASMIC score is the best option, followed by the in-house ADAMTS13 test without the PLASMIC score. CONCLUSIONS: In patients with clinical presentations of TMAs, having an in-hospital ADAMTS13 test to promptly establish the diagnosis of TTP appears to be cost-effective. Utilizing the PLASMIC score further increases the cost-effectiveness of the in-house ADAMTS13 test. Our findings indicate the benefit of having a rapid and reliable in-house ADAMTS13 test, especially in the tertiary medical center.

Plasma levels of complement activation fragments C3b and sC5b-9 significantly increased in patients with thrombotic microangiopathy after allogeneic stem cell transplantation.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is an uncommon but severe complication in patients undergoing allogeneic stem cell transplantation (allo-SCT). However, the mechanism is unclear. From 2011 to 2014, 20 patients with TA-TMA, 20 patients without, and 54 patients with various other complications, including veno occlusive disease (VOD), graft-versus-host disease (GVHD), and infection, were recruited in the study. Plasma vWF antigen (vWFAg), vWF activity (vWFAc), and ADAMTS13 activity were determined in these patients by ELISAs and FRETS-vWF73 assay, respectively. Plasma C3b, sC5b-9, and CH50 were also determined by ELISAs. Plasma levels of C3b were significantly increased in patients with either TA-TMA (p < 0.0001) or GVHD (p < 0.01). Plasma sC5b-9 and CH50 levels in patients with TA-TMA were also significantly increased (p < 0.001). Plasma ADAMTS13 activity was lower in patients with VOD, but normal with other complications. Both plasma vWFAg and vWFAc levels were not elevated in patients with TA-TMA or VOD compared with those of other groups. Complement activation likely via an alternative pathway (increased C3b, sC5b-9, and CH50) may play a role in the pathogenesis of TA-TMA. ADAMTS13 activity is reduced in VOD, but the ADAMTS13/vWF axis appears to be unaffected in patients with TA-TMA.

Knockout of Adamts7, a novel coronary artery disease locus in humans, reduces atherosclerosis in mice.

BACKGROUND: Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. METHODS AND RESULTS: We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7(-/-)/Ldlr(-/-) and Adamts7(-/-)/Apoe(-/-) mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-α stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. CONCLUSIONS: These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Exploiting the kinetic interplay between GPIbα-VWF binding interfaces to regulate hemostasis and thrombosis.

Platelet-von Willebrand factor (VWF) interactions must be tightly regulated in order to promote effective hemostasis and prevent occlusive thrombus formation. However, it is unclear what role the inherent properties of the bond formed between the platelet receptor glycoprotein Ibα and the A1 domain of VWF play in these processes. Using VWF-A1 knock-in mice with mutations that enhance (I1309V) or disrupt (R1326H) platelet receptor glycoprotein Ibα binding, we now demonstrate that the kinetic interplay between two distinct contact surfaces influences the site and extent to which platelets bind VWF. Incorporation of R1326H mutation into the major site shortened bond lifetime, yielding defects in hemostasis and thrombosis comparable to VWF-deficient animals. Similarly, disrupting this region of contact with an allosteric inhibitor impaired human platelet accrual in damaged arterioles. In contrast, the I1309V mutation near the minor site prolonged bond lifetime, which was essential for the development of a type 2B-like VWD phenotype. However, combining the R1326H and I1309V mutations normalized both bond kinetics and the hemostatic and thrombotic properties of VWF. These findings broaden our understanding of mechanisms governing platelet-VWF interactions in health and disease, and underscore the importance of combined biophysical and genetic approaches in identifying potential therapeutic avenues for treating bleeding and thrombotic disorders.