ADAMTS13 Antigen Research Articles

ADAMTS13 content and VWF multimer and triplet structure in commercially available VWF/FVIII concentrates

ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) into smaller multimers in vivo. This cleavage creates both the typical multimeric size distribution and the characteristic triplet band distribution of VWF. Here we analysed ADAMTS13 content, VWF multimeric size distribution and VWF triplet structure in five commercial VWF/factor VIII (FVIII) concentrates.The relative distribution of ADAMTS13 activity values corresponded well to the ADAMTS13 antigen values for all examined concentrates except Haemate HS®, which had markedly higher ADAMTS13 antigen/activity ratio, with Fanhdi® and Haemate HS® displaying the most intense ADAMTS13 signal. Interestingly, ADAMTS13 levels did not correlate with the high molecular weight multimer content of the concentrates, but did correlate with VWF triplet distribution. Densitometric quantification showed that Wilate®, Immunate® and Willfact® displayed human plasma-like VWF triplet distribution, whereas Fanhdi® and Haemate HS® showed enhanced content of the faster migrating triplet band, which corresponded well to their higher ADAMTS13 content.In summary, Immunate®, Willfact® and Wilate® had lower levels of ADAMTS13 antigen and activity and exhibited a plasma-like VWF triplet structure. Fanhdi® and Haemate HS® had higher ADAMTS13 content and an altered triplet structure. The possible impact of these observations on function and clinical efficacy of VWF/FVIII concentrates is discussed.

Domain Specificity and Mechanism of Action of Anti-ADAMTS13 Antibodies at Presentation and Relapse in Acquired Thrombotic Thrombocytopenic Purpura (TTP)

Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease due to autoantibodies (Auto-Ab) against ADAMTS13, resulting in a severe deficiency in enzyme activity (<10%). Little is known about the pathogenicity of anti-ADAMTS13 IgG directed against different domains of the enzyme, or how domain specificity alters between presentation, remission or TTP relapse. We developed novel assays to explore domain specificity and inhibitory potential of anti-ADAMTS13 IgG using samples from the UK TTP Registry.Full-length ADAMTS13 and ADAMTS13 fragments (MD, MDTC and MDTCS - N-terminal domains; TSR2-8 and CUB1/2 - C-terminal domains) were expressed in HEK293T cells and purified. Microtitre plates were coated with ADAMTS13 or ADAMTS13 fragments, incubated with TTP plasmas and bound IgG detected. Domain specificity was further analysed by preincubation of patient samples with ADAMTS13 fragments, assessing the ability of fragments to compete for full-length ADAMTS13 binding on the plate. From a cohort of 92 acquired TTP patients at presentation, 100% (92/92) contained auto-Ab that recognised ADAMTS13 N-terminal domains (MDTCS). Of these, 39% (36/92) had auto-Ab recognising the N-terminal domains alone, 22% (20/92) also had anti-TSR2-8, and 39% (36/92) had anti-CUB1/2 auto-Abs. There was no correlation between auto-Ab domain specificity and mortality/disease severity (anti-ADAMTS13 IgG titre, plasma exchanges to remission, platelet count and cardiac involvement).There was no evidence for auto-Ab recognising MD or MDTC domains in 25 patients tested, suggesting immunoreactivity for MDTCS was primarily mediated through the spacer domain. Consistent with this, TTP IgG inhibited proteolysis of VWF115 (a recombinant VWF A2 domain fragment), but failed to inhibit VWF106 proteolysis (VWF A2 domain fragment lacking the spacer domain binding exosite).To examine the inhibitory potency of anti-ADAMTS13 auto-Ab, IgG isolated from TTP patient samples were titrated in reactions measuring MDTCS-mediated proteolysis of FRETS-VWF73. Twenty eight out of 43 presentation samples tested had inhibitory IgG (IC50 ranging from <0.2-5μM total IgG). Addition of excess of MDTC to 19/19 of these samples did not alter the IC50, confirming inhibition in these samples is mediated solely by anti-spacer antibodies. Intriguingly, 15/43 patients had no evidence of inhibitory auto-Ab, suggesting the pathogenic mechanism in these cases may be enhanced clearance of ADAMTS13. We therefore determined ADAMTS13 antigen concentrations by ELISA. All patients at presentation exhibited markedly reduced ADAMTS13 antigen levels (n=91, median 58ng/ml (range 0-450) vs. normal n=67, median 951ng/ml (515-1829); p<0.0001). Interestingly, patients with predominantly C terminal auto-Ab and no evidence of inhibitory auto-Ab had significantly lower ADAMTS13 antigen levels at first presentation than the rest of the cohort (median 2ng/ml (0-141) vs. 57ng/ml (0-356); p=0.006).Cases where rituximab failed to normalise ADAMTS13 activity despite clinical remission were associated with the persistence of inhibitory anti-N-terminal auto-Ab. Two patients with persistently high anti-ADAMTS13 IgG but normal ADAMTS13 activity and antigen levels during remission had non-pathogenic anti-TSR2-8 antibodies that seemingly had no effect upon ADAMTS13 function or clearance. Nine out of 16 patients exhibited altered auto-Ab domain specificity upon relapse, with either loss or development of anti-C-terminal auto-Ab.In conclusion, from our functional studies ADAMTS13 inhibition appears to be mediated exclusively by anti-spacer antibodies, with no evidence for the presence of auto-Ab that recognise/inhibit other N terminal domains. Importantly, we demonstrate ADAMTS13 antigen depletion is a crucial pathogenic mechanism in acquired TTP, accounting for a large proportion of the reduced ADAMTS13 activity detected in patient samples. Our results suggest that auto-Ab directed against the C-terminal domains of ADAMTS13 may be particularly important in mediating this clearance. There is continual development of the autoimmune response during treatment of TTP patients that can result in altered domain specificity at relapse. DisclosuresNo relevant conflicts of interest to declare.

The Role of ADAMTS13-Specific Circulating Immune Complexes in Prediction of Recurrence of Acquired Thrombotic Thrombocytopenic Purpura

Introduction. Acquired thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy due to the development of autoantibodies against the VWF-cleaving protease ADAMTS13. We recently developed and validated a new ELISA method for the detection of ADAMTS13-specific circulating immune complexes (CICs) in acquired TTP patients. This study aims at investigating the clinical relevance of ADAMTS13-specific CICs at disease presentation in a large cohort of acquired TTP patients.Methods. We measured ADAMTS13-specific CICs by ELISA in 51 patients from the Milan TTP Registry, at the first episode of acquired TTP. All patients presented anti-ADAMTS13 autoantibodies by western blotting and severe ADAMTS13 deficiency (i.e., <10% of normal) by FRETS-VWF73 or CBA assays. We studied the associations between ADAMTS13-specific CICs levels and (i) ADAMTS13-related measurements (i.e., ADAMTS13 antigen, anti-ADAMTS13 IgG), (ii) clinical and laboratory markers of disease severity (i.e., muco-cutaneous bleeding, neurological, renal and cardiovascular symptoms, number of platelets, LDH, hemoglobin, creatinine), (iii) short- and long-term clinical outcomes (i.e., number of plasma exchange procedures required to attain remission, recurrence). Statistical analyses were performed using linear, logistic and Cox regression models.Results. The prevalence of ADAMTS13-specific CICs in patients experiencing a first episode of acquired TTP was 39% (95% confidence intervals [CI]: 26-52%). ADAMTS13-specific CICs were only associated with ADAMTS13 antigen levels at regression analyses (beta per 10% increase in ADAMTS13 antigen, 95% CI: 0.05, 0.02-0.08). The presence of ADAMTS13-specific CICs was associated with more than a four-fold increase in the risk of recurrence at 2 years after the first TTP episode (hazard ratio, 95%CI: 4.2, 1.1-16.4).Conclusions. ADAMTS13-specific CICs are neither a biomarker of disease severity, nor a predictor of clinical outcome during acute phase. Conversely, ADAMTS13-specific CICs seem to have relevance in predicting the recurrence of acute TTP episodes. DisclosuresPeyvandi:NovoNordisk: Research Funding; Kedrion : Research Funding; NovoNordisk: Speakers Bureau; CSL Behring : Speakers Bureau; Baxter: Speakers Bureau; Bayer: Speakers Bureau.

Circulating Adamts-13 Is Reduced in Patients with Waldenstrom’s Macroglobulinemia and Is Associated with Increased IgM Levels and Features of the Disease but Not with the Increased Levels of Von Willebrand Factor

ADAMTS-13 is a protease, member of the ADAMTS family (A Disintegrin and Metalloproteinase with ThromboSpondin type 1 repeats-13), which cleaves the cell bound large ultrapolymeric von Willebrand factor (vWF) strings. Circulating ADAMTS-13 is primarily synthesized and released from hepatic stellate and endothelial cells. Acquired functional deficiency of ADAMTS-13, usually due to inhibitory IgG autoantibodies, results in excessive platelet aggregation and disseminated vWF/platelet-rich thrombus formation. A possible association of low levels of ADAMTS-13 Ag with arterial thrombosis and endothelial dysfunction has also been reported. Hivert and colleagues (Blood 2012;120:3214-21) and our group have shown that increased levels of vWF, the only known substrate of ADAMTS-13, are associated with poorer prognosis in patients with symptomatic Waldenstrom’s Macroglobulinemia (WM). Thus, our aim was to investigate the possible association of ADAMTS-13 antigen (Ag) levels with features of WM and possible biologic implications of the ADAMTS-13 / vWF interaction in WM patients’ prognosis.Our study included 42 patients with symptomatic WM who were treated and followed in the Department of Clinical Therapeutics of the University of Athens (Greece), from 1999 to 2012, 22 patients with asymptomatic WM and 19 healthy controls of matched gender and age. For the purpose of this study we used stored serum, which had been collected before initiation of any therapy. ADAMTS-13 antigen levels were measured using a commercially available kit (R&D Systems, Minneapolis, MN, USA), which has a detection limit for ADAMTS-13 13 (ng/mL) with intra- and inter-Assay Precisions of <3.7% and <5.7%, respectively.The median age of patients with symptomatic WM was 65 years (range: 37-83 years) and 54% were males. Anemia (Hb <11.5 g/dL) was present in 78% of patients, low platelet counts (<100x109/L) in 17%, beta2-microglobulin >3 mg/dl in 56%, while 7.5% had serum LDH ≥250 U/L and 58% had serum albumin <3.5 g/dL. Median serum IgM was 3340 mg/L (range 246-9563 mg/dL). According to IPSSWM, 22% had low, 43% intermediate and 35% high risk WM, respectively. Main reasons to initiate therapy included cytopenias in 42% of patients, B-symptoms in 15%, hyperviscosity in 12%, neuropathy in 10% and other reasons in 21%. Primary therapy based on rituximab was given in 93% of the patients and 54% achieved at least 50% reduction of IgM.The median levels of ADAMTS-13 in patients with symptomatic WM were 848 ng/ml (range 471-1622 ng/ml), while those of patients with asymptomatic WM were 875 ng/ml (range 280.5-1466 ng/nl) and of healthy controls were 1170 ng/ml (range 770-1598 ng/ml). Thus, there was a statistically significant difference in the levels of ADAMTS-13 between patients with either symptomatic or asymptomatic WM and normal individuals (p<0.001 and p=0.002 respectively), while there was no difference between those with symptomatic and asymptomatic WM (p=0.934). We then evaluated the association of ADAMTS-13 antigen levels with features of symptomatic WM. There was a negative correlation between low levels of ADAMTS-13 with high levels of IgM (r=-0.544, p<0.001), beta2-microglobulin (r=-0.376, p=0.01) and with the infiltration of the BM by lymphoplasmatic cells (r=-0.338, p=0.03), while there was a positive correlation with serum albumin levels (r=0.373, p=0.016). Accordingly, in patients who required therapy because of hyperviscosity syndrome, serum ADAMTS-13 was lower (p=0.04). There was no association of the levels of ADAMTS-13 and vWF Ag either in patients with symptomatic or asymptomatic WM (p>0.5 for both), and thus, a direct link between increased vWF Ag levels, which are found in patients with WM, and compensatory ADAMTS-13 Ag levels cannot be supported by our data. The levels of ADAMTS-13 Ag had no prognostic association with overall or progression free survival in patients with symptomatic WM.In conclusion, this is the first evaluation of ADAMTS-13 Ag levels in the serum of patients with WM, in which we found that the levels of ADAMTS-13 Ag are lower compared to healthy controls and these reduced levels inversely correlate with IgM levels, beta2-microglobulin and the degree of bone marrow infiltration. Levels of ADAMTS-13 do not increase in compensation to increased levels of vWF Ag, a marker of endothelial activation and this fact may have biological implications yet to be investigated. DisclosuresNo relevant conflicts of interest to declare.

Changes of ADAMTS13 activity and vWF antigen level in patients with acute myelogenous leukemia and their significance

This study was purposed to investigate the changes of von Willebrand factor cleaving protease (ADAMTS13) activity and vWF antigen level in patients with acute myelogenous leukemia (AML) before and after treatment and evaluate their clinical significance. Seventy-three AML patients were enrolled in this study, the sodium citrate anticoagulated plasma was collected before and after their induction chemotherapy. Fluorescence resonance energy transfer substrate vWF73 (FRETS-vWF73) assay was established to detect the plasma ADAMTS13 activity while vWF antigen level was measured by ELISA. The results showed that the ADAMTS13 activity in newly diagnosed patients with AML before induction therapy was obviously lower than that in normal controls (63.3 ± 25.5)% vs (105.1 ± 37.7)(P < 0.01), while the vWF antigen level was higher than that in normal controls (226.6 ± 127.0)% vs (111.4 ± 39.7)% (P < 0.01). After standard induction chemotherapy, the ADAMTS13 activity of AML patients in complete remission period was higher than that in AML patients before therapy (P < 0.01), and was not significant difference with that in normal controls; the vWF antigen was significantly lower than that in AML patients before therapy (P < 0.01), but it still was higher than that in controls (P < 0.05). The ADAMTS13 activity in newly diagnosed AML patients complicated with infection before therapy was obviously lower than that in AML patients without infection (52.2 ± 20.6)% vs (73.9 ± 24.7)% (P < 0.01), while the vWF antigen level was significantly higher than that in AML patients without infection (262.2 ± 135.7)% vs (193.8 ± 110.2)% (P < 0.05). The ADAMTS13 activity in AML patients with disseminated intravascular coagulation (DIC) was significantly lower than that in AML patients without DIC (42.0 ± 14.5)% vs (73.4 ± 22.7)% (P < 0.01), while the vWF antigen level was obviously higher that in AML patients without DIC (274.2 ± 140.0)% vs (204.7 ± 115.5)% (P < 0.01). It is concluded that the ADAMTS13 activity in newly diagnosed AML patients befor induction therapy has been confiremed to be lower and the vWF antigen level to be higher, especially in AML patients with infection or DIC. The ADAMTS13 and vWF antigen may play a role in the pathogenesis of AML and the formation of infection and DIC.

Novel markers predict death and organ failure following hemorrhagic shock

BackgroundADAMTS 13, sP-Selectin and HSP27 have been investigated as potential prognostic markers in patients with hemorrhagic shock. MethodsThis study was part of a double-blind, randomized, parallel-group, controlled trial and included seventeen trauma patients presented to ED with severe hemorrhagic. The sera for testing were collected from these patients at the time of admission. Investigators and laboratory personnel performing testing were blinded to the patients' identity and clinical course. ResultsThe prognostic value of ADAMTS13, sP-Selectin, and HSP27 was compared to prognostic value of systolic blood pressure (SBP), base deficit estimation (BD), heart rate (HR), shock index (SI) and tissue oxygen saturation (StO2) by constructing the receiver operation characteristics (ROC). The area under the curve (AUC) of the ROC for HSP27 (0.92) was greater than for SBP (0.45), BD (0.89), HR (0.61), SI (0.45) and StO2 (0.46). AUC for sP-Selectin (0.86) and for ADAMTS13 antigen (0.74) were comparable with BD one, but greater than for the rest of currently used tests. ConclusionSerum concentrations of ADAMTS13, HSP27 and sP-Selectin measured during the admission, appear to be comparable to or better than SBP, BD, SI, HR and StO2 in predicting MODS and death after hemorrhage from trauma. These potential new markers deserve further investigation.

ADAMTS13 deficiency, despite well-compensated liver functions in patients with noncirrhotic portal hypertension.

We have reported A disintegrin and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS13) deficiency in noncirrhotic intrahepatic portal hypertension (NCIPH) patients of European origin with preserved liver function. We aimed to study ADAMTS13-von Willebrand factor (vWF) imbalance in Indian patients with NCIPH. Twenty-nine cases with NCIPH [22 males; 29years (13-58); Child's A, 23; B, 6], 22 disease controls with cryptogenic chronic liver disease [15 males; 46years (18-74); Child's A, 9; B, 9; C, 4] and 17 healthy controls [14 males; 32years (27-45)] were enrolled in the study. We measured ADAMTS13 antigen and activity (by collagen binding assay (CBA) and by fluorescence resonance energy transfer [FRET] assay), and vWF antigen levels in plasma of study patients. ADAMTS13 activity by CBA in NCIPH patients (32 %, 5 % to 100 %; median, range; p-value <0.001) and disease controls (36 %, 5 % to 144 %; p = 0.001) was significantly lower than in healthy controls (87 %; 60 % to 148 %). ADAMTS13 antigen and activity by FRET assay were also lower in cases and disease controls. ADAMTS13 activity (by CBA) to antigen ratio was lower in NCIPH and disease controls than in healthy controls. Of 29 NCIPH patients, 3 (all in Child's A) had severe ADAMTS13 deficiency (<10% ADAMTS13 activity), and 8 (Child's A, 7; B, 1) had moderate ADAMTS13 deficiency (10 % to 25% activity). Conversely, vWF antigen and vWF:ADAMTS13 ratio were higher in patients with NCIPH and in disease controls than in healthy controls. This study validates the finding of ADAMTS13 deficiency in NCIPH despite preserved liver functions in an Indian population suggesting its involvement in pathogenesis of NCIPH.

Measurement and prevalence of circulating ADAMTS13‐specific immune complexes in autoimmune thrombotic thrombocytopenic purpura

The formation of ADAMTS13-specific circulating immune complexes (CICs) may be a pathophysiologic mechanism in autoimmune thrombotic thrombocytopenic purpura (TTP), but has not been systematically investigated. (a) To develop an assay for ADAMTS13-specific CICs; (b) to evaluate their prevalence in autoimmune TTP; and (c) to assess their association with ADAMTS13-related measurements and clinical features in autoimmune TTP patients. We developed and validated an ELISA method for ADAMTS13-specific CICs. ADAMTS13-specific CICs were searched for in 55 patients with autoimmune TTP from the Milan TTP Registry (URL:http://www.ttpdatabase.org/) and 28 controls. The associations between ADAMTS13-specific CIC levels and ADAMTS13 activity, antigen, anti-ADAMTS13 IgGs and acute TTP clinical features were assessed by multivariate linear regression. Intra- and inter-assay coefficients of variation of the new test were 5.3 and 9.6%. In 36 patients with severe ADAMTS13 deficiency and anti-ADAMTS13 autoantibodies, the prevalence of ADAMTS13-specific CICs was 47% (n=17; 95% confidence interval [CI], 32-63%). ADAMTS13-specific CICs were detected also in seven of 19 (37%; 95% CI, 19-59%) patients with reduced ADAMTS13 activity, but apparently negative anti-ADAMTS13 autoantibodies. ADAMTS13-specific CICs were not associated with ADAMTS13 activity, antigen or anti-ADAMTS13 IgGs. In patients with acute TTP, increasing levels of ADAMTS13-specific CICs were associated with a higher number of plasma-exchange procedures required to attain remission (per 0.1 increase in normalized OD values, beta, 2.9; 95% CI, -0.7 to 6.5). Approximately one to two-thirds of patients with autoimmune TTP display ADAMTS13-specific CICs. A thorough investigation of the prognostic relevance of ADAMTS13-specific CIC levels in autoimmune TTP is warranted.

Defective ADAMTS13 synthesis as a possible consequence of NASH in an obese patient with recurrent thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a rare and devastating hematologic disorder frequently associated with multiple organ failure and sometimes death. This syndrome is mainly associated with severe deficiency of ADAMTS13, a disintegrin and metalloprotease with thrombospondin (TSP)-1 repeats, cleaving high molecular weight von Willebrand Factor (ULVWF) multimers. Decreased plasma ADAMTS13 activity results in the accumulation of ULVWF multimers with consequent platelet activation. Recently, obesity has been considered as a potential independent risk factor for TTP, but the reason of this association is still unknown. We describe an unusual case of fatal recurrent TTP in a morbid obese female with non-alcoholic steatohepatitis (NASH) and severe ADAMTS13 activity deficiency due neither to an inhibitory autoantibody nor to a gene mutation. Visceral obesity is associated with non-alcoholic fatty liver disease (NAFLD) and NASH: we hypothesized that these conditions can influence ADAMTS13 antigen and activity. In fact, hepatic stellate cells (HSC) are the main producers of ADAMTS13, and a decrease in ADAMTS13 activity has been reported in liver disease.

Single-nucleotide variations defining previously unreportedADAMTS13haplotypes are associated with differential expression and activity of the VWF-cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura family

Background Although autosomal recessive hematologic disorders are individually rare and difficult to ascertain, studies involving one or more homozygous affected children and their unaffected heterozygous parents have led to expanded understanding of known and discovery of previously unknown processes. The son and daughter of two Salvadoran parents were diagnosed with congenital thrombotic thrombocytopenic purpura (cTTP) at 6 and 2 years of age, respectively, after presenting with fever, respiratory symptoms, hemolytic anemia, and thrombocytopenia and being found to have ADAMTS13 activities Methods Prior to a plasma infusion, blood samples were collected from the children and parents. Genomic DNA was isolated for polymerase chain reaction (PCR), and direct Sanger sequencing of all ADAMTS13 exons and flanking intronic segments was performed; all variants identified were confirmed by bidirectional sequencing of a second, independently generated amplicon. Total RNA was isolated and the steady-state level of ADAMTS13 mRNA was measured using a quantitative real-time PCR (q-RT-PCR)-based assay. ADAMTS13 was characterized enzymatically using the fluorogenic FRETS-VWF73 substrate and antigenically by ELISA. Results Both children were found to be homozygous and parents to be heterozygous for the previously described, cTTP-causing ADAMTS13 single-base-substitution mutation 20506C>T, a missense mutation that encodes cDNA-nucleotide 2518 (c.2518C>T) and ADAMTS13 residue 692 (692Arg>Cys [692R>C]) (Fig. A). As expected, the children9s ADAMTS13 antigen and activity levels were undetectable, although notably, steady-state levels of the ADAMTS13 mRNA were >2.5-fold higher in the daughter than in the son. The re-sequenced regions of the ADAMTS13 loci segregating within this family contained 26 additional SNVs, seven of which were nonsynonymous (ns) including two previously unreported ns-SNVs: 27852C>T (c.3362C>T; 972Arg>Trp) and 33325G>A (c.3733G>A; 1096Arg>His) (Fig. A, left panel). The parents9 genotypes differed at nine positions, including three ns-SNVs, creating two distinct, non-mutant haplotypes (designated I and III) at the gene, mRNA and protein levels. The q-RT-PCR assay revealed >4-fold higher steady-state mRNA levels in the father compared to the mother (p Discussion We capitalized on the fortuitous finding of children with complete homozygosity across ancestrally-related ADAMTS13 alleles harboring a null-type, loss-of-function mutation, as this enabled the substantially different levels of gene expression and function observed in the parents to be attributed to their two previously unreported, SNV-based, ADAMTS13 haplotypes. Additional investigation at the molecular, biochemical, cellular, and organismal levels will be necessary to determine which of the myriad potential individual SNV- and/or haplotype-based mechanisms are responsible for the observed parental differences in circulating ADAMTS13 antigen and activity. Disclosures: Kim: Haplomics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Baxter: Honoraria. Marder: Baxter: Research Funding. Howard: Haplomics, Inc.: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Baxter: Research Funding.

Reduced ADAMTS13 activity is associated with an ADAMTS13 SNP, fever and microparticles in a malaria-like model

BackgroundSevere falciparum malaria (SM) remains a major cause of death in tropical countries. The reduced activity of ADAMTS13, increasing levels of ultra-large von Willebrand factor (ULVWF) in SM patients, are assumed as factors that intensify disease severity. However, the reason why ADAMTS13 activity is reduced in SM remains unclear.ObjectivesTo investigate whether rs4962153, febrile temperature, and microparticles, contribute to reduced ADAMTS13 activity.MethodsGenotypic association of rs4962153 with ADAMTS13 antigen and activity was examined in 362 healthy Thai participants. The collagen binding assay was used to study the effects of febrile temperature and microparticles on ADAMTS13 activity.ResultsADAMTS13 antigen and activity were decreased in participants with AA genotype, compared to AG and GG (antigen: p-value = 0.014, and < 0.001; activity: p-value = 0.036, and < 0.002, respectively). There was significantly reduced ADAMTS13 antigen in AG compared to GG (p-value = 0.013), but not in ADAMTS13 activity (p-value = 0.082). The number of rs4962153 A alleles correlated with the reduced level of antigen and activity (p-value <0.001 and p-value = 0.001, respectively). MPs showed an inhibitory effect on ADAMTS13 activity (p-value = 0.025). Finally, ADAMTS13 activity was decreased in a temperature and time-dependent manner. The interaction between these two factors was also observed (p-value <0.001).ConclusionsThese findings suggest that the A allele of rs4962153, MPs, and febrile temperature, contribute to reduce ADAMTS13 activity in plasma. These data are useful in malaria or other diseases with reduced ADAMTS13 activity.

Decreased von Willebrand factor ristocetin cofactor activity and increased ADAMTS13 antigen increase postoperative drainage after coronary artery bypass grafting

Routine coagulation tests and bleed-scores fail to identify patients at risk of excessive postoperative drainage following coronary artery bypass grafting (CABG). We sought to investigate whether lower von Willebrand factor (VWF) and higher ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) are associated with a high postoperative drainage after CABG. In the prospective cohort study, VWF antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCO), VWF collagen-binding (VWF:CB), ADAMTS13 antigen (ADAMTS13:Ag) and ADAMTS13 activity were measured on the day of elective on-pump CABG in 232 consecutive patients without a prior history of haemorrhagic diathesis, including von Willebrand disease (95% discontinued aspirin preoperatively). Postoperative drainage and blood product use were recorded. A comparison of extreme drainage quartiles (n=56) showed that individuals with the highest drainage volumes have mean VWF:RCO lower by 19% (P<0.0001), median VWF:Ag lower by 19% (P<0.0001), ADAMTS13:Ag higher by 8% (P=0.0002), ADAMTS13 activity higher by 9% (P=0.01) and fibrinogen lower by 14% (P=0.03) than those with the lowest drainage. Linear regression analysis showed that preoperative VWF:RCO (b=-4.83, P=0.002) and fibrinogen (b=-61.52, P=0.04) are the only independent predictors of postoperative drainage. Multivariate logistic regression demonstrated that preoperative VWF:RCO in the lowest quartile and ADAMTS13:Ag levels in the highest quartile increased the risk of high (≥1000 ml) drainage (odds ratio [95% confidence interval]=4.88 [1.83-13.02], P=0.001 and 3.77 [1.49-9.52], P=0.005; respectively). Patients undergoing elective CABG with lower preoperative VWF:RCO are at risk of having larger postoperative drainage, which suggests a novel contributor to increased perioperative bleeding in cardiac surgery.

Single-Nucleotide Variations Defining Previously Unreported ADAMTS13 Haplotypes Are Associated With Differential Expression and Activity Of The VWF-Cleaving Protease In a Salvadoran Congenital Thrombotic Thrombocytopenic Purpura Family

BackgroundAlthough autosomal recessive hematologic disorders are individually rare and difficult to ascertain, studies involving one or more homozygous affected children and their unaffected heterozygous parents have led to expanded understanding of known and discovery of previously unknown processes. The son and daughter of two Salvadoran parents were diagnosed with congenital thrombotic thrombocytopenic purpura (cTTP) at 6 and 2 years of age, respectively, after presenting with fever, respiratory symptoms, hemolytic anemia, and thrombocytopenia and being found to have ADAMTS13 activities <1% without neutralizing IgG antibodies. They remain without long-term neurologic or renal sequelae following prophylactic infusions of fresh plasma (10 mL/kg every 2.5-weeks). The purpose of this study was to characterize and correlate single-nucleotide variations (SNVs) in each parent's, non-mutant ADAMTS13 allele with its mRNA and protein expression, activity, and enzyme kinetics. MethodsPrior to a plasma infusion, blood samples were collected from the children and parents. Genomic DNA was isolated for polymerase chain reaction (PCR), and direct Sanger sequencing of all ADAMTS13 exons and flanking intronic segments was performed; all variants identified were confirmed by bidirectional sequencing of a second, independently generated amplicon. Total RNA was isolated and the steady-state level of ADAMTS13 mRNA was measured using a quantitative real-time PCR (q-RT-PCR)-based assay. ADAMTS13 was characterized enzymatically using the fluorogenic FRETS-VWF73 substrate and antigenically by ELISA. ResultsBoth children were found to be homozygous and parents to be heterozygous for the previously described, cTTP-causing ADAMTS13 single-base-substitution mutation 20506C>T, a missense mutation that encodes cDNA-nucleotide 2518 (c.2518C>T) and ADAMTS13 residue 692 (692Arg>Cys [692R>C]) (Fig. A). As expected, the children's ADAMTS13 antigen and activity levels were undetectable, although notably, steady-state levels of the ADAMTS13 mRNA were >2.5-fold higher in the daughter than in the son. The re-sequenced regions of the ADAMTS13 loci segregating within this family contained 26 additional SNVs, seven of which were nonsynonymous (ns) including two previously unreported ns-SNVs: 27852C>T (c.3362C>T; 972Arg>Trp) and 33325G>A (c.3733G>A; 1096Arg>His) (Fig. A, left panel). The parents' genotypes differed at nine positions, including three ns-SNVs, creating two distinct, non-mutant haplotypes (designated I and III) at the gene, mRNA and protein levels. The q-RT-PCR assay revealed >4-fold higher steady-state mRNA levels in the father compared to the mother (p<0.001; Fig. B). Plasma ADAMTS13 activity and antigen levels were ∼2-fold greater in the father than in the mother (p=0.00164 and p=0.0633, respectively), but the specific activities of these structurally distinct ADAMTS13 proteins were notably almost identical (253.5 vs. 256.2 U/μg). Moreover, initial velocity kinetic analysis using the Michelis-Menten equation demonstrated that the Vmax of the father's ADAMTS13 was twice that of the mother's (1.4 vs. 0.7; p < 0.0001) while its affinity for substrate was one-third that of her ADAMTS13 (Km = 0.3 vs. 0.1; p = 0.0585). DiscussionWe capitalized on the fortuitous finding of children with complete homozygosity across ancestrally-related ADAMTS13 alleles harboring a null-type, loss-of-function mutation, as this enabled the substantially different levels of gene expression and function observed in the parents to be attributed to their two previously unreported, SNV-based, ADAMTS13 haplotypes. Additional investigation at the molecular, biochemical, cellular, and organismal levels will be necessary to determine which of the myriad potential individual SNV- and/or haplotype-based mechanisms are responsible for the observed parental differences in circulating ADAMTS13 antigen and activity. [Display omitted] Disclosures:Kim:Haplomics, Inc.: Membership on an entity's Board of Directors or advisory committees; Baxter: Honoraria. Marder:Baxter: Research Funding. Howard:Haplomics, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Baxter: Research Funding.

Predictive Value Of Markers Of Fibrinolysis and Endothelial Dysfunction In The Post Thrombotic Syndrome: A Systematic Review

IntroductionThe post thrombotic syndrome (PTS) is a chronic condition that develops in 20–40% of deep vein thrombosis (DVT) patients. While risk factors that predispose to the development of venous thromboembolism are widely known, factors that influence the development of PTS after DVT have not been well elucidated. Identification of factors to facilitate individualized risk assessment for PTS and thereby the need for prophylactic intervention is desirable. ObjectivesWe conducted a systematic review to determine whether biomarkers of fibrinolysis and endothelial dysfunction can predict the risk of developing PTS among patients diagnosed with DVT. MethodsStudies were identified by searching the electronic databases PubMed, EMBASE, Scopus and Web of science. Studies published between January 1990 and January 2013 which measured biomarker levels in blood of adult patients with DVT, and reported rates of PTS development in these patients were included. Extracted data included source and baseline characteristics of study subjects, potential confounders that could influence the result of measured marker (e.g. malignancy, use of anticoagulants), type of biomarker, time point(s) and method of measurement, criteria for PTS diagnosis, and measure of association including adjustment variables. Risk of bias was assessed using Newcastle-Ottawa Scale, with some additional quality items specific to this literature added. ResultsOut of 2376 records included in our primary screen of titles and abstracts, 84 articles were assessed for eligibility. Fourteen studies were included in this systematic review: 11 investigated the association between D-Dimer and PTS, three examined fibrinogen level, two measured Von Willebrand factor (VWF) antigen and activity, one study measured plasminogen activator inhibitor (PAI)-1, one assessed A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats (ADAMTS)-13 antigen and activity and one study measured Factor XIII (FXIII) activity.Studies varied with regards to inclusion and exclusion criteria (e.g. first DVT, malignancy, current anticoagulant use), use of validated scales to diagnose PTS (e.g. Villalta scale or clinical, etiological, anatomical and pathological [CEAP] classification), timing of PTS assessment, time point of biomarker measurement after DVT, measurement method, and cut off value used for analysis of association between marker and outcome.Figure 1 summarizes results from 10 studies that reported on the association between D-Dimer and PTS. We used crude Odds Ratios (OR) as the summary measure, as adjusted ORs were available for only a few studies, and adjustment variables differed between studies. Furthermore, as some studies restricted their population on certain clinical characteristics, crude ORs may already reflect a certain degree of control for confounding. In general, adjusted ORs (where available) tended to attenuate the association. Studies were grouped according to the time point of D-Dimer measurement after DVT. Even after such subgrouping, we were still unable to combine the results within subgroups due to marked clinical heterogeneity. [Display omitted] Three studies measured fibrinogen levels, two on the day of DVT diagnosis and one a median of 28 months after DVT. None found a significant association between fibrinogen level and the development of PTS. Similarly, no association with PTS was found in the studies examining ADAMTS-13 antigen level and activity, VWF antigen and activity, or PAI-1.The one available study that measured FXIII activity found it to be significantly lower in patients with PTS. ConclusionsWe systematically reviewed the literature on the association between markers of fibrinolysis and endothelial dysfunction and the development of PTS in patients after acute DVT. An elevated D-dimer level may be an intrinsic marker of residual thrombus and/or persistent activation of clotting or inflammatory pathways that could increase the propensity to develop PTS. Similarly, impaired fibrinolysis and endothelial dysfunction are pathophysiological components of PTS. Whether these markers might be useful to predict the development of PTS after DVT is still unclear. Larger prospective studies using validated scores to diagnose PTS, strict inclusion and exclusion criteria, and accounting for confounders in the design and analysis are needed. Disclosures:No relevant conflicts of interest to declare.

Coagulation Activation in Children with Sickle Cell Disease Is Associated with Cerebral Small Vessel Vasculopathy

BackgroundThrombotic complications in Sickle Cell Disease (SCD) arise since infancy, but the role of the coagulation system in children has been poorly explored. To determine its role in the development of clinical complications in childhood we measured coagulation and endothelial parameters in children with SCD at steady state.MethodsMarkers of thrombin generation, fibrin dissolution and endothelial activation were evaluated in 38 children with SS-Sβ°, 6 with SC disease and 50 age and blood group matched controls. Coagulation variables were correlated with markers of hemolysis and inflammation, with the presence of cerebral and lung vasculopathy and with the frequency of clinical complications.ResultsSS-Sβ° patients presented higher levels of factor VIII, von Willebrand factor antigen (VWF:Ag) and collagen binding activity, tissue plasminogen activator antigen (t-PA:Ag), D-dimer, p-selectin, prothrombin fragment1+2 (F1+2) and lower ADAMTS-13:activity/VWF:Ag (p<0.05) compared to controls and SC patients. In SS-Sβ° patients coagulation variables correlated positively with markers of inflammation, hemolysis, and negatively with HbF (p<0.05). Patients with cerebral silent infarcts showed significant decrease in t-PA:Ag and ADAMTS-13 Antigen and a tendency toward higher D-dimer, F1+2, TAT compared to patients without them. D-dimer was associated with a six fold increased risk of cerebral silent infarcts. No correlation was found between coagulation activation and large vessel vasculopathy or other clinical events except for decreased t-PA:Ag in patients with tricuspid Rigurgitant Velocity >2.5m/sec.ConclusionsSS-Sβ° disease is associated with extensive activation of the coagulation system at steady state since young age. ADAMTS-13 and t-PA:Ag are involved in the development of cerebral silent infarcts.

AAV-mediated expression of an ADAMTS13 variant prevents shigatoxin-induced thrombotic thrombocytopenic purpura

Severe deficiency of plasma ADAMTS13 activity causes thrombotic thrombocytopenic purpura (TTP), a life-threatening syndrome for which plasma is the only effective therapy currently available. As much as 5% of TTP cases are hereditary, resulting from mutations of the ADAMTS13 gene. Here, we report the efficacy and safety of recombinant adeno-associated virus serotype 8 (AAV8)-mediated expression of a murine ADAMTS13 variant (MDTCS), truncated after the spacer domain, in a murine model of TTP. Administration of AAV8-hAAT-mdtcs at doses greater than 2.6 × 10(11) vg/kg body weight resulted in sustained expression of plasma ADAMTS13 activity at therapeutic levels. Expression of the truncated ADAMTS13 variant eliminated circulating ultralarge von Willebrand factor multimers, prevented severe thrombocytopenia, and reduced mortality in Adamts13(-/-) disease-prone mice triggered by shigatoxin-2. These data support AAV vector-mediated expression of a comparable truncated ADAMTS13 variant as a novel therapeutic approach for hereditary TTP in humans.

Deficiency of ADAMTS-13 in pediatric patients with severe sepsis and impact on in-hospital mortality

BackgroundThe enzyme involved in regulating the size of vWF (von Willebrand factor) in plasma is ADAMTS-13 (A disintegrin and metalloprotease with thrombospondin type-1 motives). Deficient proteolysis of ULvWF (ultra large von Willebrand factor) due to reduced ADAMTS-13 activity results in disseminated platelet-rich thrombi in the microcirculation characteristic of thrombotic thrombocytopenic purpura. Reduced ADAMTS-13 has also been observed in severe sepsis and is associated with poor survival. We conducted this study to detect ADAMTS-13 deficiency and its impact on in-hospital mortality in pediatric patients with severe sepsis.MethodsPediatric patients diagnosed with severe sepsis were recruited for the study. Baseline clinical characteristics were noted. ADAMTS-13 antigen levels were assayed by ELISA. According to ADAMTS-13 levels, patients were grouped as deficient and non-deficient. Comparison was done with regard to some clinical and biological characteristics and in-hospital mortality between the two groups.ResultsA total of 80 patients were enrolled in the study. The median age of the patients was 3.1 years (Range: 0.1-15 years). ADAMTS-13 deficiency with levels less than 350 ng/dl was found in 65% patients. In patients with ADAMTS-13 deficiency, 75.6% had low platelets of less than 150 × 109/L. In-hospital mortality was 42.3% and 35.7% in ADAMTS-13 deficient and non-deficient group, respectively.ConclusionMajority of the pediatric patients admitted to hospital with severe sepsis exhibit ADAMTS-13 deficiency. ADAMTS-13 deficiency might play a role in sepsis-induced thrombocytopenia. More studies are needed to evaluate the role of ADAMTS-13 deficiency on in-hospital mortality.

ADAMTS13 Content of Plasma-Derived Factor VIII-Von Willebrand Factor Concentrates

AbstractAbstract 3383 Introduction:Anecdotal and published reports on the use of specific plasma-derived Factor VIII (FVIII)-von Willebrand Factor (vWF) concentrates in the treatment of congenital thrombotic thrombocytopenic purpura (TTP) led to the determination of the functional and antigenic content of ADAMTS13 in a number such concentrates commercially available in the US and/or the EU.Deficiency or inhibition of ADAMTS13 is the putative cause of TTP. TTP is a rare disorder causing microvascular thrombosis resulting in low platelet counts. It affects 1 –5 patients per 1,000,000 population. The current treatment of this disorder consists of regular infusions of fresh frozen plasma (FFP) for inherited TTP and exchange plasmapheresis for the acquired version of the disorder. The possibility of utilizing FVIII-vWF concentrates that incorporate robust virucidal treatments in their manufacturing and the possible utilization of lower infusion volumes prompted the evaluation of these concentrates for ADAMTS13 content. Methods:For this analysis, we obtained 5 lots of Koate®-DVI, 2 lots of Human Bioplasma, and 1 lot of each of the following concentrates: Humate® P, Wilate®, Alphanate®, Emoclot® and Fractogel.® Following reconstitution of these concentrates in 10 mL of water for injection, we determined the concentration of ADAMTS13 antigen by an ELISA method previously described by Feys et al. (J Thromb Haemost. 2006; 4: 955–62) with minor modifications (Peyvandi et al. Haematologica 2008; 93: 232–239). The lower limit of detection was 1% while the lower value of the normal range was 45%. The ADAMTS13 activity was measured using collagen binding (CBA) and FRET assays with minor modifications (Peyvandi et al. Haematologica 2008; 93: 232–239); in both assays, serially diluted normal human plasma (NHP) and plasma samples were diluted 1:10 in assay buffer and incubated 1:1 with vWF substrate in a final volume of 100 μl. In addition to the determination of ADAMTS13, we evaluated the amount of vWF antigen (vWF:Ag) using a commercial kit (Instrumentation Laboratory, IL US, Bedford, Massachusetts, USA) in these concentrates and the multimeric composition of this protein. Utilizing densitometry, we determined the percentage of ultra-large vWF multimers in each of these concentrate. Results:The analysis showed that among the tested concentrates, Koate®-DVI had the highest ratio of ADAMTS13/FVIII. This concentrated presented ratios of 9.1 ± 0.7% (Mean ± SD) and 8.4 ± 1.1% (Mean ± SD) per IU FVIII for ADAMTS13 activity and antigen respectively. The remainder of the concentrates contained only nominal amounts of ADAMTS13; only Alphanate® reached a ratio of 0.23% ADAMTS13 activity/IU FVIII. These data are in agreement with previous reports (Qorraj et al. Blood 2001, 116: 3677). A summary analysis data are shown in Table 1.Factor VIII ConcentrateLot NumberFactor VIII Content (IU/mL)vWF:Ag (IU/mL)UL vWF Ratio (Normal Range 0.85 – 1.21)ADAMTS13 Activity (%)ADAMTS13 Antigen (%)Koate-DVI(5 lots)100281±22*0.39±0.04*908±70*842±12*WilateA014A1814901300.491213Humate P30666911A661830.662261AlphanateB2AC9001111001650.09230387Emoclot460804100540.162258Human BioPlasma86152580.24<3No SampleHuman BioPlasma8617100240.61<3No SampleFractogel230/111001460.211610*Mean ± SD.Koate®-DVI displayed a ratio of vWF:Ag/FVIII of approximately 2.81, very similar to the ratio of 2.77 included in the single lot of Humate®-P. The ratio of ultra-large multimers to total multimers in Humate® P was higher, at 0.66 vs. 0.39 for Koate-DVI. However, the lower content of ultra-large multimers in Koate®-DVI may be advantageous in its possible utilization in the treatment of TTP. Conclusion:On the basis of these results, Koate-DVI may have a role to play in the management of congenital and, possibly, idiopathic TTP, especially on the basis of observations that FVIII accelerates the breakdown of vWF (Cao et al. PNAS 2008, 105: 7416–7421). Additional characterization, testing in pertinent animal models and pilot studies in humans would be required to determine the extent to which this concentrate can assist in the treatment of TTP. Disclosures:Guazzini:Kedrion S.p.A: Consultancy. Retzios:Kedrion Biopharma, Inc.: Consultancy.

Extensive Coagulation Profile in Children with Sickle Cell Disease and Its Role in Cerebral Micro-Vasculopathy

AbstractAbstract 3247Activation of the coagulation system is present in adults with Sickle Cell Disease (SCD) who display higher levels of thrombin-antithrombin complex (TAT), prothrombin fragment F1+2, D-dimer, tissue factor and platelet activity. Whether the activation of the coagulation system is a bystander phenomenon or a main determinant of clinical complications is under investigation. Although thrombotic complications in SCD arise since infancy, the coagulation system in children has been poorly explored. We performed a comprehensive and systematic evaluation of the coagulation system in children with SCD and investigated its possible role in the development of the main clinical manifestations of SCD in childhood.The following markers of hemostatic and endothelial function were evaluated during steady state: factor VIII activity (FVIII), von Willebrand factor antigen (vWF:Ag) and collagen binding activity (vWF:CBA), PAI-1 antigen (PAI-1:Ag), t-PA antigen (t-PA:Ag), D-dimer, P-selectin, Nitric Oxide (NO), F1+2, TAT, ADAMTS-13 antigen (ADAMTS-13:Ag) and activity (ADAMTS-13:act). Markers of hemolysis (hemoglobin, reticulocytes, aptoglobin, bilirubin, LDH), inflammation (white blood cells, neutrophils, C reactive protein) were measured. Children were not in chronic transfusion.Magnetic Resonance (MRI), Angio Magnetic Resonance (MRA), Transcranial Doppler (TCD), Tricuspid Regurgitant Velocity (TRV) data and frequency of clinical complications were correlated with coagulation parameters. Continuous variables were compared using two-sided Student's t-test and Mann-Whitney non-parametric test, categorical variables using Chi-square and Fisher's exact test. Correlations between variables were evaluated by the Pearson or Spearman coefficient.Thirty-five HbS/HbS- 3 HbS/betathalassemia° (20 M and 18 F, mean age 6.49 years), and 6 HbS/HbC patients (3 M and 3 F, mean age 11.2 years) were evaluated. 60% of HbS/HbS and HbS/betathalassemia° children presented higher level of FVIII, D-dimer, F1+2 and lower level of NO, revealing coagulation activation since early age in HbS/HbS and HbS/betathalassemia° but not in HbS/HbC (Table 1). Coagulation variables correlated positively with markers of inflammation, hemolysis, endothelial activation demonstrating a broad involvement of the coagulation system in these processes, while correlated negatively with HbF (Table 2). No correlation was seen between coagulation variables and cerebral large vessel vasculopathy investigated by TCD and MRA nor TRV (p >0.05). Patients with Silent Infarcts on MRI (n.9) showed significant decrease in t-PA (4.53 vs 7.67, p 0.03) and ADAMTS-13 Ag (1.15 vs 1.50, p 0.05) -suggesting the presence of endothelial dysfunction- and a tendency toward higher D-dimer (2879.01 vs 1569.51), F1+2 (319 vs 231), TAT (17.17 vs 10.46) -suggesting a trend towards enhanced clotting activation compared to patients without Silent infarcts (n.21). Increase in D-dimer was associated with a Relative risk of 6 (p <0.05) to develop Silent Infarcts. No correlation was found between coagulation activation and Acute Chest Syndrome or Vaso-occlusive crisis.Table 1Coagulation profile in HbS/HbS-HbS/Betathalassemia° and HbS/HbC patientsHbS/HbS+HbS/Betathalassemia°HbS/HbCpVariableMeanFVIII:C %184.96119.220.003VWF:Ag %150.05100.630.013VWF:CBA %135.38107.360.124PAI:Ag ng/ml8.736.850.833t-PA ng/ml6.914.520.062D-dimer ng/ml1906.39366.480.000NO μmol/L7.188.70.336P-selectin ng/ml47.7629.700.010F1+2 pmol/l254.26118.500.037TAT ng/ml12.345.140.359ADAMTS-13:Ag μg/ml1.371.460.653ADAMTS-13:act %79.4278.940.801ADAMTS-13:act/VWF:Ag0.520.780.003Table 2Correlations between hematologic and coagulation parameters in HbS/HbS- HbS/Betathalassemia° (first value is Pearson or Spearman coefficient, second is p-value)White Blood CellsNeutrophilsPlateletsFetal HemoglobinReticulocytesLDHFVIII:C−0.3740.021VWF:Ag−0.3220.049VWF:CBA0.3680.023PAI:Ag0.4490.3690.3330.3520.0050.0250.0440.033t-PA0.3440.034D-dimer0.5590.438−0.3930.3890.4210.0010.0110.0210.0230.013P-selectin0.4610.4000.520−0.4130.3810.0040.0160.0010.0110.020F1+20.4000.5120.3370.0160.0010.044TAT0.3970.3810.3760.0170.0220.024ADAMTS-13 Ag0.4710.004 Disclosures:No relevant conflicts of interest to declare.

Importance of Measuring Circulating ADAMTS13 Immune Complexes in Addition to Free Antibodies in Patients with Acquired TTP

Abstract 102A severe functional deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13 results in accumulation of uncleaved, highly adhesive VWF multimers in plasma. This VWF multimer accumulation leads to increased platelet aggregation and thrombus formation in the microcirculation. Anti-ADAMTS13 autoantibodies that either neutralize ADAMT13's activity or enhance protein clearance are the main cause of severe ADAMTS13 deficiency in acquired thrombotic thrombocytopenic purpura (TTP). Circulating anti-ADAMTS13 immune complexes (CIC) that may be a factor in disease pathogenesis have been described in plasma of TTP patients. We have developed an ELISA assay to specifically determine the levels of CIC in which the antigen portion is immobilized by a polyclonal anti-ADAMTS13 IgG and the immunoglobulin component detected by a class-specific antibody. Samples (individual or longitudinal) from a total of 100 patients with acute idiopathic TTP were used to compare IC levels with those of the free anti-ADAMTS13 antibodies, ADAMTS13 antigen (ADAMTS13:Ag), ADAMTS13 activity (ADAMTS13:Ac) and inhibitor. This analysis was to provide for the first time a complete picture of the autoantibody response in this population.IgG-containing CIC were present in most of the patient samples, with IgG4-IC being the predominant type of CIC. A correlation between the subclasses of the free and complexed IgG anti-ADAMTS13 antibodies was observed, but the levels of CIC appeared to follow inverse kinetics to the free antibody and the inhibitory titer. We also found samples devoid of free ADAMTS13:Ag, but containing CIC, indicating that ADAMTS13, either newly synthesized or exogenously administered by plasma infusion during plasma exchange, had been quantitatively sequestered through formation of IC. ADAMTS13:Ag was also undetectable in some samples with high levels of free anti-ADAMTS13 antibodies but lacking CIC, suggesting that IC that had formed were already cleared at these time points.Our study suggests that in addition to characterization of autoantibodies against ADAMTS13, ADAMTS13-specific CIC also need to be determined at time of diagnosis and during treatment to obtain a full picture of disease status. The formation of ICs capturing all freely available ADAMTS13 may have a high impact on treatment response, especially in patients who are refractory to treatment. The gathered data may thus help to timely implement alternative treatment strategies in patients unresponsive to standard treatments, thereby reducing unexpected disease relapses with fatal outcome. Disclosures:Ferrari:Baxter Innovations GmbH: Employment. Plaimauer:Baxter Innovations GmbH: Employment. Gruber:Baxter Innovations GmbH: Employment. Palavra:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.