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Background Severe ADAMTS13 deficiency is a critical component of the pathogenesis of idiopathic thrombotic thrombocytopenic purpura but is found only in about 60% of patients clinically diagnosed with this disease. Design and Methods Over a period of 8 years and six episodes of thrombotic thrombocytopenic purpura we studied the evolution of the anti-ADAMTS13 antibody response in a patient using different ADAMTS13 assays and epitope mapping. Results Anti-ADAMTS13 autoantibodies were found in all episodes but were inhibitory only in the last two episodes. In a flow-based assay, normal ADAMTS13 activity was found only during the first disease episode, while ADAMTS13 activity was normal using a static assay in episodes 1 and 3, and severely deficient in the last two episodes. Fluorescence evolution in a modified fluorescence resonance energy transfer assay using a von Willebrand factor A2 domain peptide substrate was linear in episodes 1, 5 and 6, but increased exponentially in episodes 3 and 4. Despite the variable functional characteristics of the anti-ADAMTS13 autoantibodies, their principal epitope was the ADAMTS13 spacer domain in all episodes. Conclusions The patient is unique as he displayed features of maturation or shaping of the anti-ADAMTS13 autoantibody response during the course of multiple episodes of thrombotic thrombocytopenic purpura. Anti-ADAMTS13 autoantibodies may be important in vivo despite normal ADAMTS13 activity in routine assays. Consequently, treatment decisions should not be based solely on activity assay results.

Background The current gold-standard for diagnosing heparin-induced thrombocytopenia is the detection of platelet-activating antibodies by means of functional assays which, since they are time consuming and not widely available, are not suited to guiding acute treatment decisions. The objective of our study was to assess the ability of more rapid immunoassays to predict the presence of functionally relevant anti-platelet factor 4/heparin-antibodies.Design and Methods We analyzed 1,291 of 1,383 (93.4%) patients consecutively evaluated for suspected heparin-induced thrombocytopenia at our institution. Clinical pre-test probability was defined by the 4T-score. Anti-platelet factor 4/heparin-antibodies were measured with three immunoassays (ID-H/PF4-PaGIA, Asserachrom-HPIA, and GTI-PF4) and their functional relevance was assessed by a two-point heparin-induced platelet aggregation test. Performance of the immunoassays was evaluated by receiver operating characteristic analysis.Results Among 1,291 patients, 96 (7.4%) had a positive heparin-induced platelet aggregation-test: 7 of 859 (0.8%) with a low, 50 of 358 (14.0%) with an intermediate, and 39 of 74 (52.7%) with a high 4T-score. Receiver operating characteristics analysis indicated that best immunoassay thresholds for predicting a positive platelet aggregation test were: Titer of 4 or more (ID-H/PF4-PaGIA), optical density more than 0.943 (Asserachrom-HPIA) and more than 1.367 (GTI-PF4). A 100% negative predictive value was observed at the following thresholds: Titer of 1 or under (ID-H/PF4-PaGIA), optical density less than 0.300 (Asserachrom-HPIA) and less than 0.870 (GTI-PF4). A 100% positive predictive value was reached only by ID-H/PF4-PaGIA, at titers of 32 or over. Positive and negative likelihood ratios were calculated for results between the thresholds with 100% negative or positive predictive value.Conclusions We show that: i) negative and weak positive results of immunoassays detecting anti-platelet factor 4/heparin-antibodies exclude heparin-induced thrombocytopenia; ii) anti-platelet factor 4/heparin-antibody titers of 32 or over (ID-H/PF4-PaGIA) have a 100% positive predictive value for functionally relevant antibodies; iii) combining the clinical pre-test probability with the likelihood ratio of intermediate immunoassay results allows assessment of post-test probability for heparin-induced thrombocytopenia in individual patients.

Introduction: The aim of the present work was to verify whether calculating a ratio between clotting times obtained with the sensitive PTT-LA and a less sensitive activated partial thromboplastin time (aPTT)-reagent may represent a valuable aPTT-based screening strategy for lupus anticoagulants (LA). Methods: For the pilot study, plasma samples from normal subjects (n = 15) and from patients with LA (n = 10), therapeutic anticoagulation with vitamin K-antagonists (VKA) (n = 15) or unfractionated heparin (n = 15), coagulation factors deficiency (n = 16), and inhibitory antibodies against factor VIII or IX (n = 11) were studied. For the evaluation study, 1553 consecutive plasma samples from nonanticoagulated patients investigated for LA between January 2005 and December 2007 at our institution were studied. Following screening strategies were employed: Pathromtin-SL (aPTT-SL), PTT-LA (aPTT-LA), ratio aPTT-LA/aPTT-SL (aPTT-ratio), and Russell's viper venom (RVV) based LA-Check. LA positive samples were identified by mixing studies and diluted RVV confirmation test (LA-Check/LA-Sure). Results: Pilot study: All screening strategies had a 100% sensitivity, and the aPTT-ratio reached the highest specificity (82%; 95%CI: 74-90%). Within the evaluation study, following sensitivities for LA screening were observed: aPTT-SL 59.0% (95%CI: 57-61%), aPTT-LA 82.1% (95%CI: 80-84%), aPTT-ratio 92.3% (95%CI: 91-94), and LA-Check 83.3% (95%CI: 82-85%). Conclusion: Calculating a ratio between the LA-sensitive PTT-LA and the less sensitive Pathromtin-SL improves the performance of the PTT-LA itself and represents a simple and sensitive aPTT-based integrated strategy for LA screening.

Summary. Background: Recent studies have shown that a low clinical pretest probability may be adequate for excluding heparin-induced thrombocytopenia. However, for patients with intermediate or high pretest probability, laboratory testing is essential for confirming or refuting the diagnosis. Rapid assessment of anti-PF4/heparin-antibodies may assist clinical decision-making. Objectives: To evaluate the performance of rapid ID-H/PF4-PaGIA. In particular, we verified reproducibility of results between plasma and serum specimens, between fresh and frozen samples, and between different ID-H/PF4-polymer lots (polystyrene beads coated with heparin/PF4-complexes). Patients/Methods: The samples studied were 1376 plasma and 914 corresponding serum samples from patients investigated for suspected heparin-induced thrombocytopenia between January 2000 and October 2008. Anti-PF4/heparin-antibodies were assessed by ID-H/PF4-PaGIA, commercially available ELISAs and heparin-induced platelet aggregation test. Results: Among 914 paired plasma/serum samples we noted discordant results (negative vs. low-titre positive) in nine instances (1%; 95%CI, 0.4–1.6%). Overall, agreement between titres assessed in plasma vs. serum was highly significant (Spearman correlation coefficient, 0.975; P

Forty patients with severe sepsis or septic shock recently received C1 inhibitor. In the present study we studied the effect of C1 inhibitor therapy on circulating elastase-α1-antitrypsin complex (EA) and lactoferrin (LF) levels in these patients to gain further insight about agonists involved in the activation of neutrophils in human sepsis. Elevated levels of EA and LF were found in 65 and 85% of the septic patients, respectively. Patients with elevated EA levels had higher organ dysfunction scores, higher levels of cytokines, and higher levels of complement activation products than patients with normal EA levels. C1 inhibitor therapy reduced EA as well as complement activation and IL-8 release in the patients with elevated EA on admission. We conclude that neutrophil activation in human sepsis correlates with the severity of organ dysfunction and involves complement and interleukin-8 as agonists. The effect of C1 inhibitor therapy on neutrophils may provide an explanation for the beneficial, although mild, effects of this treatment on organ dysfunction in sepsis.

Hereditary plasma prekallikrein (PK) deficiency was diagnosed in a 71-year-old man with an 8-year history of osteomyelofibrosis. PK deficiency was suspected in view of a severely prolonged activated partial thromboplastin time (aPTT) that nearly normalized following prolonged preincubation (10 min) of patient plasma with kaolin-inosithin reagent. Hereditary PK deficiency was demonstrated by very low PK values in the propositus (PK clotting activity 5%, PK amidolytic activity 5%, PK antigen 2% of normal plasma, respectively) and half normal PK values in his children. Normalization of a severely increased aPTT (>120 s) after prolonged preincubation with aPTT reagent occurred in plasma deficient in PK but not in plasma deficient in factor XII (FXII), high-molecular-weight kininogen (HK), factor XI (FXI), factor IX, factor VIII, Passovoy trait plasma or plasma containing lupus anticoagulant. Autoactivation of FXII in PK-deficient plasma in the presence of kaolin paralleled the normalization of aPTT. Addition of OT-2, a monoclonal antibody inhibiting activated FXII, prevented the normalization of aPTT. We conclude that the normalization of a severely prolonged aPTT upon increased preincubation time (PIT), characteristic of PK deficiency, is due to FXII autoactivation.

Bei einer Patientin ohne Blutungsneigung und ohne anamnestische Thromboembolien wurde auf Grund einer isoliert stark verlängerten aPTT bei normaler Thromboplastinzeit (Quick) und normaler Thrombinzeit ein schwerer Faktor XII (FXII)-Mangel diagnostiziert. Ein Mangel eines der Faktoren des Kontaktaktivierungssystems (FXII, Präkallikrein, hochmolekulares Kininogen) wird meist zufällig, z.B. anläßlich eines präoperativ durchgeführten Gerinnungsstatus, anhand einer isolierten Verlängerung der aPTT entdeckt. Ein partieller oder schwerer Mangel von Faktor XII, Präkallikrein oder hochmolekularem Kininogen ist nicht mit einer verstärkten Blutungstendenz assoziiert. Personen mit einem Faktor XI-Mangel können unter einer meist milden hämorrhagischen Diathese leiden, FVIII-Mangel (Hämophilie A, Autoimmunhämophilie, von Willebrand Krankheit) und FIX-Mangel (Hämophilie B) sind mit einer Blutungsneigung assoziiert, deren Schweregrad vom FVIII- bzw. FIX-Spiegel abhängt. Eine isolierte aPTT-Verlängerung durch ein Lupus Antikoagulans ist demgegenüber oft mit einer Neigung zu arteriellen und/oder venösen Thrombosen assoziiert. Bei einer isolierten Verlängerung der aPTT muß deshalb immer eine weiterführende Abklärung erfolgen.

Abstract —Increased levels of hemostatic factors and genetic mutations of proteins involved in coagulation may play a role in the pathogenesis of coronary artery disease. We investigated clotting activity of factors II (FII:C), V (FV:C), VII (FVII:C), and X (FX:C), the prothrombin gene 20210G→A transition, and the factor V Leiden mutation in 200 survivors of myocardial infarction and in 100 healthy controls. FV:C ( P P

SummaryIn this study we prospectively assessed the reliability of a new fibrin monomer assay in 106 outpatients with clinically suspected deep venous thrombosis of the lower limb. According to the results of the objective tests and using different cut-off points we calculated the sensitivity, specificity and negative predictive value of the fibrin monomer assay. The prevalence of deep vein thrombosis was 44.3% (31.1% proximal, 13.2% distal). Using a cut-off level of plasma fibrin monomer of 3.5 μg/ml, a sensitivity, specificity and negative predictive value of 100% (95% CI: 94-100%), 35.6% (95% CI: 23-48%) and 100% (95% CI: 86-100%), respectively, were obtained. The exclusion rate was 19.8% (95% CI: 12-27%) of all referred patients. These accuracy indices compared favourably with the respective results of a routine D-dimer ELISA used for comparison. Conclusion: This new fibrin monomer assay appears to be a reliable method for the exclusion of deep vein thrombosis in symptomatic outpatients.

SummaryThe role of plasminogen (plg) deficiency in the pathogenesis of venous thromboembolism is debated in the literature. In the present study we evaluated the prevalence of plg deficiency in our thrombophilia patients and aimed to elucidate the thrombosis risk of plg deficiency as a single defect or in combination with other defects, with special focus on APC resistance.The study cohort included 1192 consecutive patients with a history of clinically or objectively diagnosed venous and/or arterial thromboembolism and/or positive family history who were referred to our department for thrombophilia investigation from 02/1988 to 03/1997. All available family members of patients with plg deficiency were tested for plg, APC resistance and other thrombophilic defects that were established in the propositus.23/1192 propositi were plg-deficient corresponding to an overall prevalence of 1.9%, i.e. 2.2% in patients with venous thrombosis and 1.4% in those with arterial events. Out of the 23 plg-deficient propositi, 8 showed one or multiple additional thrombophilic defects, and in 4 patients relevant circumstantial risk factors were present. Of the 53 available family members, 28 were plg-deficient including 5 with additional APC resistance, and 4 subjects had isolated APC resistance. Ten of the 53 family members had already suffered thromboembolic events, i.e. 5 (18%) in the plg-deficient group and 5 (20%) in the non-deficient group, both groups showing an almost identical median age at the time of investigation (28.9 years and 27.1 years, respectively).Based on our data, plg deficiency is a rare defect in thrombophilic patients and as a single defect it does not seem to be a strong thrombotic risk factor, as 11 of 23 propositi had additional thrombophilic defects or circumstantial risk factors, and in the family members thrombotic events were equally frequent in the plg-deficient and non-deficient subjects.

Resistance to activated protein C (APC resistance) due to the factor V mutation 506 Arg-->Gln (factor V Leiden) is the most prevalent inherited risk factor for venous thromboembolism. Its association with arterial thromboembolic disease, however, is still controversial. In the present study we found no difference between the prevalence of APC resistance (assessed by the ratio of the aPTT with and without added APC) in 134 non-anticoagulated survivors of myocardial infarction and that in 100 controls of similar age and sex distribution (2.2% and 2.0%, respectively). Patients showed a significantly higher median value for the aPTT ratio than controls (2.85 and 2.66, respectively), a fact we could not explain by our data.

This pilot study seeks to determine whether contact system activation (CSA) occurs in human sepsis patients and to characterise blood levels of the 47kD light chain of high-molecular weight kininogen (47kD HK).Six consecutive patients with clinical suspicion of sepsis were evaluated on days 1, 2, 3 and 6-8 for 47kD HK blood levels expressed in U/ml of whole blood and as percent of total HK. 47kD HK was measured in whole blood by quantitative immunoblot analysis.On study day 1 or 2, analysis of 47kD HK in U/ml of whole blood identified CSA in 3/6 patients. When 47kD HK levels were expressed as percent of total HK, 4/6 patients were identified with CSA before day 3. The degree of CSA as assayed by the presence of 47kD HK correlated with the severity of the systemic inflammatory syndrome (SIRS), i.e. mean CSA increased progressively from basal levels in healthy controls (0.08 U/ml or 10.4%) to patients without SIRS (0.10 U/ml or 15.1%), to patients with sepsis (0.12 U/ml or 15.0%), and finally to patients in a combined category of severe sepsis and septic shock (0.13 U/ml or 17.4%).CSA, defined by increased 47kD HK, occurred early on in the course of sepsis in a subset of sepsis patients. 47kD HK levels, an indicator of bradykinin release, correlated with sepsis severity. Future larger studies will need to evaluate the role of 47kD HK as a biomarker for both prognosis and treatment response in human sepsis..

SummaryMany preanalytical variables affect the results of coagulation assays. A possible way to control some of them would be to accept blood specimens shipped in the original collection tube. The aim of our study was to investigate the stability of coagulation assays in citrated whole blood transported at ambient temperature for up to two days after specimen collection. Blood samples from 59 patients who attended our haematology outpatient ward for thrombophilia screening were transported at ambient temperature (outdoor during the day, indoor overnight) for following periods of time

Background. Autoantibodies (Abs) neutralizing and/or accelerating clearance of the von Willebrand factor-cleaving protease-ADAMTS13 are the major culprits in acquired Thrombotic Thrombocytopenic Purpura (aTTP). Despite the first-line treatment with daily plasma exchange (PEX), acute aTTP is still today associated with a mortality rate of ~20% leaving survivors at high risk of relapse. To improve patient care and/or treatment of aTTP we explored the capacity of anti-idiotypic small molecules to specifically bind and potentially neutralize pathogenic anti-ADAMTS13 autoantibodies thereby restoring plasma-derived ADAMTS13 activity in acquired TTP patients. Method: As selecting tools we used previously generated spleen-derived inhibitory human monoclonal anti-ADAMTS13 antibodies consisting of the 4 antigen-binding CDR3 motifs shared by the two aTTP patients (Schaller et al, Blood 2014;124(23):3469-79). Either 3 mAbs representative for CDR3 Motif-1 or 3 mAbs for CDR3 Motif-3 or 2 mAbs representing CDR3 Motif-4 were pooled at equimolar concentrations by ribosomal display to screen for binders in two combinatorial small protein libraries (DARPins from Molecular Partners, Schlieren, Switzerland) containing either two (N2C) or three (N3C) randomized ankyrin repeat modules, respectively From each of the 6 libraries single anti-idiotypic DARPins were cloned into a pMPAG6 expression vector and purified by His-tag affinity chromatography. In total 22 unique anti-idiotypic DARPins, 4 N2C-DARPins and 2 N3C-DARPins selected by Motif-1, 9 N2C-DARPins by Motif-3 and 7 N2C-DARPins by Motif-4 mAbs were identified. Inhibitor targeting Strategy: The neutralization potential towards plasma-derived anti-ADAMTS13 Abs, was tested by pre-incubating the DARPins prior measuring the residual activity by functional FRETS assay, in a cohort of 50 acute aTTP patients with pathological inhibitor titers >0.4 BU/ml (diagnosed at our Center 2006-2014). First, equimolar pools (1000 nM) of anti-idiotypic DARPins (Motif-1, Motif-3 or Motif-4) were tested in 11/50 patients, secondly the contribution of the single DARPins from the responsive pools was assessed in 18/50 patients to finally test the entire cohort with a combination of all neutralizing anti-idiotypic DARPins. Results: The pool of all Motif-1 anti-idiotypic DARPins (n=6) neutralized inhibitor titers below the pathological threshold in 9/11 (82 %) aTTP patients tested, whereas only a slight reduction of the inhibitor titers (in average 15%) was observed for Motif-3 and Motif-4 DARPin pools, revealing the Motif-1 DARPins as the most universal neutralizers. To test the contribution of each Motif-1 anti-idiotypic DARPin to the observed inhibitor neutralization, single DARPins were tested in 18/50 aTTP revealing a reduction of the inhibitor of 20-50% (N2C-19) or 20% (N3C-74) in 5/18 patients tested. In contrast 90% (45/50) aTTP patients incubated with the pool of all 6 Motif-1 DARPins caused inhibitor titers to drop either completely or below the pathological threshold. The inhibitor titer was reduced below 1BU/ml in 3/50, but was irresponsive in 2/50 patients. Conclusions: Our data clearly show that anti-idiotypic DARPins can potently and universally neutralize inhibitory anti-ADAMTS13 Abs of aTTP patients. However an effective neutralization, restoring ADAMTS13 activity above 10-15% in most patients was only achieved when pooling all Motif-1 DARPins. If a combination of Motif-3 and/or Motif-4 DARPins with Motif-1 DARPins can additionally increase neutralization capacity also in relapsing patients is currently under investigation. Disclosures No relevant conflicts of interest to declare.

About 60% of patients diagnosed with acute thrombotic thrombocytopenic purpura (TTP) display a severe ADAMTS13 deficiency. Recently, Raife et al. concluded from a small case series, that factor V Leiden (FVL) might constitute a risk factor for acute thrombotic microangiopathy (TMA) without severe ADAMTS13 deficiency. Therefore, we determined ADAMTS13 activity and FVL carrier-ship in 256 consecutive patients presenting with various forms of acute TMA, including patients diagnosed with TTP or hemolytic-uremic syndrome (HUS). The overall prevalence of FVL was 8.2% (6.25% among patients diagnosed with TTP, and 9% among those with HUS) concordant with the FVL prevalence reported in Europe. FVL was present in 9.9% of patients with ADAMTS 13 activity10% and in 9.7% of those with normal ADAMTS13 activity (50%). We conclude that FVL is not more prevalent in TMA patients without as compared to those with severe ADAMTS13 deficiency. The prevalence of FVL carriers in certain HUS subgroups (HUS with ADAMTS 13 activity50%) reaching 12.3% suggests that a contributory role of FVL in the pathogenesis of defined forms of HUS needs further study.

Hereditary plasma prekallikrein (PK) deficiency was diagnosed in a 71-year-old man with an 8-year history of osteomyelofibrosis. PK deficiency was suspected in view of a severely prolonged activated partial thromboplastin time (aPTT) that nearly normalized following prolonged preincubation (10 min) of patient plasma with kaolin-inosithin reagent. Hereditary PK deficiency was demonstrated by very low PK values in the propositus (PK clotting activity 5%, PK amidolytic activity 5%, PK antigen 2% of normal plasma, respectively) and half normal PK values in his children. Normalization of a severely increased aPTT (120 s) after prolonged preincubation with aPTT reagent occurred in plasma deficient in PK but not in plasma deficient in factor XII (FXII), high-molecular-weight kininogen (HK), factor XI (FXI), factor IX, factor VIII, Passovoy trait plasma or plasma containing lupus anticoagulant. Autoactivation of FXII in PK-deficient plasma in the presence of kaolin paralleled the normalization of aPTT. Addition of OT-2, a monoclonal antibody inhibiting activated FXII, prevented the normalization of aPTT. We conclude that the normalization of a severely prolonged aPTT upon increased preincubation time (PIT), characteristic of PK deficiency, is due to FXII autoactivation.

Increased levels of hemostatic factors may play a role in the pathogenesis of myocardial infarction by triggering thrombin formation. We measured factor XII (FXII), factor XI (FXI), plasma prekallikrein (PK) and high-molecular-weight kininogen (HK) in 200 patients having survived myocardial infarction for at least 2 months, and in 100 healthy controls. We found significantly elevated levels of FXI clotting activity (FXI:C), HK:C and of the amidolytic activity of PK (PK:Am) among the patients as compared to the controls. Plasma levels of FXI:C, HK:C and PK:Am in the highest quartile were associated with an odds ratio of 1.9 (95% CI: 1.0–3.8), 2.0 (95% CI: 1.0–4.0) and 5.4 (95% CI: 2.6–11.2), respectively, compared to the respective plasma levels in the lowest quartile. After correction for established clinical and laboratory risk factors, the association between PK:Am plasma levels and myocardial infarction remained significant ( P =0.0007). Combination of high PK:Am plasma levels and smoking or arterial hypertension, respectively, resulted in a more than additive relative risk for myocardial infarction.

A therapy aiming at neutralization of inhibitory autoantibodies (Abs) causing autoimmune thrombotic thrombocytopenic purpura (aTTP) would be highly desirable. We screened a large combinatorial small protein library of Designed-Ankryin-Repeat-Protein (DARPins, Molecular Partners AG, Switzerland; library size 1015-23) for anti-idiotypic binders to an equimolar pool of three spleen-derived inhibitory anti-ADAMTS13 monoclonal Abs of two aTTP patients containing 1 of 4 shared CDR3 motifs. Four unique anti-idiotypic DARPins were purified and their neutralization potential tested against 8 spleen-derived anti-ADAMTS13 Abs (200 nM), belonging to the same, shared CDR3 motif used for selection. Anti-idiotypic DARPins restored ADAMTS13 activity up to 100% (2000 nM). By ELISA we found Abs in plasma of 27 of 37 randomly selected aTTP patients that bound specifically to the anti-idiotypic DARPins. Pre-incubation with anti-idiotypic DARPins (960 nM) prevented binding of plasma anti-ADAMTS13 Ab to recombinant ADAMTS13 in a competition ELISA (n=5). Our findings of anti-idiotypic DARPins able to neutralize several, although similar (belonging to the same CDR3 motif) pathogenic anti-ADAMTS13 Abs and the fact that the DARPins are also specifically recognized by plasma-derived anti-ADAMTS13 Abs of different aTTP patients are promising and suggest that possibly only a limited number of the selected DARPins are required to develop a general treatment for aTTP.

Abstract 3299 Background. Thrombotic thrombocytopenic purpura (TTP) is a rare disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia with schistocytes on the peripheral blood smear and a variable degree of ischemic organ dysfunction due to microvascular thrombosis. In the case of acquired TTP, circulating autoantibodies inhibit ADAMTS13 activity and/or increase ADAMTS13 clearance. The first-line treatment of an acute TTP bout is plasma exchange (PEX) therapy with the replacement of fresh frozen plasma. Up to 30% of TTP patients show an exacerbation under PEX. After initial improvement, platelet counts drop again which is typically associated with a strong increase of ADAMTS13 inhibitor titers, and therefore the phenomenon is also referred to as inhibitor boosting. Interestingly it is apparently not seen in other autoimmune diseases treated with PEX, like acquired hemophilia A or myasthenia gravis. The aim of this study was therefore to investigate if inhibitor boosting in acquired TTP is a general or ADAMTS13 specific phenomenon. Methods. Plasma samples, obtained day-to-day during PEX therapy supplemented with steroids until remission, of 9 patients suffering from acute TTP due to severe acquired ADAMTS13 deficiency, were analyzed for the following laboratory parameters: (a) C-reactive protein (CRP) and total IgG, (b) ADAMTS13 activity, (c) anit-ADAMTS13 antibody titers by ELISA, (d) functional inhibitor titers by FRETS assay, and (e) anti-TG and anti-TPO antibody titers. Results. At initial presentation inhibitor titers ranged from 0.7 - >10 BU/ml (a positive functional inhibitor titer is defined as >0.4 BU/ml by FRETS). All patients initially improved when PEX was started, however, in 7/9 cases a drop in platelet count was noted on day 7 (n=5), 8 or 9 (each n=1), which was associated with a substantial increase in anti-ADAMTS13 antibody and functional inhibitor titer in all seven patients. CRP was increased (>30mg/L) in one patient only, during the three days before the raise in inhibitor titer and a drop in platelet count was noted, while total IgG remained stable in all 9 patients throughout PEX. At presentation none of the patients had a positive result for the thyroid antibodies, however, all 9 patients developed positive anti-TPO antibody titers during treatment and three patients tested positive for anti-TG antibodies at the same time. Anti-TPO antibody peaks occurred after the anti-ADAMTS13 antibody peak in all patients (range days 9–17), except the two patients where no raise in anti-ADAMTS13 antibody titers was observed (anti-TPO peak on day 3 in both). The two patients without inhibitor boosting required only five and six PEX sessions, respectively to get into remission, of the other seven patients two didn't overcome the inhibitor boosting and became refractory and were subsequently successfully treated by splenectomy or Rituximab, and one patient died of sepsis on day 11. Conclusions. Inhibitor boosting in acquired TTP due to antibody-mediated severe ADAMTS1313 deficiency treated with PEX is common (78%) and cannot be overcome by standard first line treatment in half of the patients. The fact that significant titers of other autoantibodies than anti-ADAMTS13 antibodies occur during the acute disease episode is striking and points to a more general dysregulation of the immune response in acquired TTP. Investigation of further autoantibodies, cytokines, etc. to explore the role of B-cells and T-cells in this phenomenon in more detail are underway. Disclosures: No relevant conflicts of interest to declare.

Abstract 1431 Background. Thrombotic Thrombocytopenic Purpura (TTP) is a severe, life-threatening disease and results from an impaired regulation of the von Willebrand factor multimer size due to a deficiency of its cleaving protease ADAMTS13. In case of acquired TTP, inhibitory autoantibodies neutralize and/or accelerate ADAMTS13 clearance. To better understand the role of these autoantibodies in the pathogenesis of TTP and to develop more specific therapies, molecular analysis of the pathological relevant isotype IgG4 is necessary. Rituximab, a B-cell depleting anti-CD20 mAb is used to treat various autoimmune diseases with variable success. Increasing evidence points at the spleen, rather than the bone marrow as the major reservoir of memory B cells, which are thus accessible to Rituximab. The analysis of the humoral immune response of splenic B-cells towards ADAMTS13 in TTP patients treated with/without Rituximab offers a unique opportunity to address resistance to Rituximab. Methods. Two patients suffering from acute TTP with high titers of anti-ADAMTS13 antibodies were splenectomized because of frequent relapses, which occurred in one case even after several courses of Rituximab (patient B). Splenic mononuclear cells were isolated and used to clone the patient's entire IgG4 repertoire using phage display technology. Two IgGk-IgGλFab libraries of ∼2 × 1010 members each were constructed and selected against recombinant ADAMTS13 coated on ELISA plates. Additionally an existing naïve IgM Fab library constructed from cord blood RNA was screened identically. To analyze the memory repertoire of Rituximab-resistant and non-resistant anti-ADAMTS13 IgG, the subset of switched memory B cells (CD19, CD27 IgG) was isolated by magnetic beads (Miltenyi Biotec) from the splenic mononuclear cells of both patients prior to immortalization by Epstein-Barr virus (EBV) transformation to generate monoclonal antibodies. Results. Phage display. After 5 rounds of selection a 105-fold enrichment in eluted phages was observed. An ELISA screening after the fourth panning round showed 16/34 clones displayed a strong binding to ADAMTS13. Sequencing of DNA encoding the heavy chain variable region (IgVH) of strong binding clones revealed that the Fabs of patient A were most homologous to germline genes IgVH1-3*01 (7/11) and IgVH4-28*01 (4/11). The usage of these germline genes was also observed in patient B (1/5 and 2/5 clones IgVH1 and IgVH4, respectively), in addition 2/5 clones used IgVH1-69*01 germline gene, which had been previously observed in other acquired TTP patients. Six out of 14 analyzed anti-ADAMTS13 Fabs of the naïve IgM Fab library showed the same restricted gene usage (VH1-2, VH1-69 and VH4-28), whereas 4/14 clones were found to comprise heavy chains encoded by IgVH6-1*02, 2/14 were encoded by IgVH3-48*02 and 2/14 by IgVH3-24*04. The homology to the closest germline gene for all VH genes was in the range of 81.6–99%, with the lowest mutation rate found in cord blood anti-ADAMTS13 IgM Fabs (range 1–7.7%) followed by the anti-ADAMTS13 Fabs of patient B (after Rituximab; range 2.9–13.6%, average 10%) and finally patient A (range 8.3–18.6%, average 15%), the mutation rate being in the range of an antigen-driven immune response. EBV transformation. Screening of the supernatant of single clones of EBV transformed memory B-cells for the presence of anti-ADAMTS13 IgG antibodies was positive in 5/125 (4%) of clones of patient A and in 8/109 (7.3%) of patient B. Sequencing of IgVH encoding DNA of 3 single clones to date revealed gene usage of IgVH1-3*01 in 2 clones (one patient A and B) with a mutation rate of 15.4% and one (patient A) being homolog to IgVH3-30*022 with a lower mutation rate of 8.2%, in the upper limit of the naïve repertoire. Conclusions. VH gene usage of specific anti-ADAMTS13 B-cells, including Rituximab-resistant specific anti-ADAMTS13 B-cells is genetically restricted to VH1, VH3 and VH4. Rituximab may deplete circulating specific anti-ADAMTS13 B cells. However, newly differentiated specific anti-ADAMTS13 B cells characterized by a lower degree of somatic mutation may evolve from the memory cell pool giving rise to pathological anti-ADAMTS13 antibody. Functional characterization (inhibitory capacity, affinity and epitope mapping) of these anti-ADAMTS13 Fabs are underway. Disclosures: No relevant conflicts of interest to declare.

Abstract 4199 INTRODUCTION In recent years, diagnostic tests for ADAMTS-13 and respective autoantibodies became available and are routinely used to support thrombotic thrombocytopenic purpura (TTP) diagnosis and therapy. ADAMTS-13 abnormalities have also been described in other pathological situations such as liver cirrhosis (Uemura, 2008), sepsis- induced DIC (Ono, 2006) or inflammatory bowel disease (Feys, 2009), where the relationship between ADAMTS13 parameters and state of disease is less clear than in TTP. A clear-cut distinction between normal and pathologic levels of ADAMTS-13 parameters (activity, antigen and autoantibodies) is only available in most TTP, with ADAMTS-13 activity levels < 10% and antibody titers well above normal levels. Pathological ranges for other diseases are not yet well defined. As normal ranges for ADAMTS-13 levels seem to be very broad, differentiation between normal and pathologic levels in cases other than TTP will depend on the size of the population used to establish the normal range and presumably also on age, gender, and likely also on other parameters present in this population. AIM The aim of this study was therefore to establish normal values for ADAMTS-13 parameters by analyzing normal populations in five different European laboratories using the same commercial assays. Material and Methods ADAMTS-13 autoantibodies were measured using the TECHNOZYM®ADAMTS-13 INH ELISA (anti-IgG). ADAMTS-13 activity and antigen concentrations were measured using TECHNOZYM®ADAMTS-13 ELISA, a combined assay for activity and antigen. Samples were frozen citrated plasma samples used in the respective laboratories as normal controls (approximately 40 in each laboratory; all together 193 normal controls). RESULTS The normal range for anti-ADAMTS-13 IgG was compared between the 5 different labs. For ADAMTS-13 INH, the median in these different populations varied between 6 and 9 U/ml which is well below the previously defined borderline value of 12 – 15 U/ml in this assay. The median for all samples was 6.7 U/ml; the number of “false-positives” (>15U/ml) varied from 5 to 10%. To analyze a possible age dependency, three groups were considered : 50 y.o. No age dependent differences between these groups could be found (p>0.6). No significant differences were either found between male and female controls (p=0.55). For ADAMTS-13 activity and antigen only data from 140 normal subjects and 60 TTP patients are currently available. For normal subjects, the median for ADAMTS-13 activity was 103%, for antigen 101%. Both parameters showed a wide distribution of values between 53% and 205% for activity and 34% and 217% for antigen. In comparison, the median values in TTP patients were significantly lower (activity 12%, antigen 49%). Conclusion We can show that a substantial variation in normal values for ADAMTS-13 activity and antigen exists in normal subjects, while the range for ADAMTS-13 autoantibodies is rather narrow. All values in normal subjects are well separated from those obtained in TTP patients. The broad range of ADAMTS-13 activity and antigen in normal subjects has to be considered when these parameters are measured in diseases other than TTP. Disclosures: Geiter: Technoclone GmbH: Employment. Graf:Technoclone GmbH: Employment. Tschokert:Technoclone GmbH: Employment. Vetr:Technoclone GmbH: Employment.

Anlaslich der Abklarungen von unerklarten venosen oder arteriellen Thrombosen und Embolien wurde in den Jahren 1988 bis 1990 bei samtlichen zugewiesenen Patienten neben den ublichen gerinnungsphysiologischen Parametern der Thrombin-Antithrombin-III-Komplex (TAT-Komplex) bestimmt. Das Ziel war, festzustellen, ob bei mittelgradigen bis schweren Mangelzustanden an Protein C oder Protein S Hinweise auf eine dauernde in-vivo-Thrombin-Generation als Ausdruck einer latenten Thromboseneigung bestehen. Die Patienten wurden stets mehrere Wochen bis Monate nach einer akuten Thromboembolie untersucht bzw. nach Absetzen einer Antikoagulation.

Background: A severe ADAMTS13 deficiency ( Methods: 74 consecutive patients with acute acquired idiopathic TTP-HUS from the Oklahoma TTP-HUS registry (partly published by Vesely et al. [Blood2003;102:60–8]) were included into this study. For laboratory investigation, serum samples had been withdrawn before initiation of any form of treatment. ADAMTS13 activity was determined by quantitative immunoblotting, ADAMTS13 autoantibodies were evaluated by a routine inhibitor screening test and the TECHNOZYM ADAMTS13 INH ELISA, and patients’ VWF multimers were analyzed by SDS-agarose electrophoresis to assess endogenous VWF proteolysis arisen in vivo. Results: An ADAMTS13 activity Conclusions: Most patients suffering from acute idiopathic TTP-HUS and high titers of ADAMTS13 autoantibodies had impaired VWF proteolysis in line with severely decreased ADAMTS13 activity. Patients without ADAMTS13 antibodies as well as the majority of patients with intermediate antibody titers demonstrated normal or even increased VWF proteolysis. (Severely) impaired proteolysis of endogenous VWF was observed in 3 patients with high titer anti-ADAMTS13 antibodies and normal ADAMTS13 activity (50–100%). Thus, our data suggest a pathophysiological role of ADAMTS13 in at least some patients despite normal ADAMTS13 activity by standard assay.

Background: Severe deficiency of ADAMTS13, the VWF-cleaving protease ( Methods: Between January 2001 and July 2003 ADAMTS13 activity was determined in 396 consecutive patients referred to our laboratory for diagnostic purposes. Based on the clinical and patient data provided by the referring clinicians 131 patients were excluded from this study (no clinical information available [n=45]; no acute TMA [50]; no plasma left [26]; non-Caucasians [3] and various reasons [7]). The remaining 265 patients were assigned to three principal clinical categories: idiopathic TMA (n=204, among them 80 patients with the clinical diagnosis of TTP and 124 cases with hemolytic-uremic syndrome [HUS]), secondary TMA (30) and TMA not further specified (31). ADAMTS13 activity was assessed by a quantitative immunoblotting assay. FVL carrier-ship was determined by a commercial aPTT-based APC resistance assay and in cases with prolonged base-line aPTT (>42s), decreased factor V clotting activity ( Results: A severe ADAMTS13 deficiency was found in 22% (59/265) of patients investigated, among them none of the HUS cases but 49 patients diagnosed as having TTP. Twenty-one (7.9%) of all patients were FVL carriers consistent with the FVL prevalence reported for Europe (0.9–14%), especially however, for Germany (4–8.9%) and Switzerland (3–7%), who referred most patients (176 [66.4%] and 52 [19.6%], respectively). Similar allele frequencies were observed among patients diagnosed as TTP (6.3%, 5/80) or HUS (8.9%, 11/124). The difference between the latter two was statistically not significant (p=0.497, chi-square test). FVL was found in 6.8% (4/59) of patients with severe ADAMTS13 deficiency and in 8.3% (17/206) without severe ADAMTS13 deficiency (p=0.712, chi-square test). Data did not change when the cut-off level for severe ADAMTS13 deficiency was raised to an ADAMTS13 activity of 10%. Conclusions: We conclude, that FVL is not more prevalent in patients with acute TMA without severe ADAMTS13 deficiency than in TMA patinets with severe ADAMTS13 deficiency. Furthermore, there is no difference in the FVL prevalence in patients diagnosed as having HUS or TTP. Based on our results, FVL is not a risk factor for acute TMA and pathogenetically not involved in acute TMA without ADAMTS13 deficiency.

H eparin-induced skin necrosis, first described by O'Toole [1] in 1973 has since been recognized as a rare side effect of heparin therapy. This phenomenon may accompany the more frequently occurring heparin-associated thrombocytopenia and thrombosis syndrome (HATT) [2] and seems to be independent from dose [3,4]. In most reported cases, heparin-induced skin necrosis occurred after subcutaneous heparin administration, but in a few instances it has also been observed with intravenous use of the drug [5]. Bovine heparin was found to bear a greater potential of causing H A T T than do porcine mucosal dr low-molecularweight heparins [3,6,7]. Heparin-associated thrombocytopenia presents in two distinct forms [8]: the first is a mild, transient thrombocytopenia beginning within two to five days after starting heparinization; the pat ient remains asymptomatic. The second, severe form occurs after an interval of at least six days from initiating heparin administration and is accompanied in more than half of the cases by thromboembolic complications. Unlike the former, this late-onset heparinassociated thrombocytopenia does not resolve unless the drug is discontinued. Pathogenetically, an IgG antibody causing heparin-dependent platelet aggregation has been found in plasma from patients with lateonset thrombocytopenia [8]. Intravascular platelet aggregation in the presence of therapeut ic heparin concentrations may be responsible for the frequently fatal thromboembolic complications seen with continued heparinization [4]. We describe a patient with heparin-associated skin necrosis, thrombocytopenia, and deep vein thrombosis. The patient's plasma consistently induced fast and pronounced heparin-dependent aggregation of normal platelets from several donors. In order to assess the specificity of this in vitro test, 159 control plasma samples were examined.

A patient with von Willebrand's disease underwent cholecystectomy after replacement therapy with a factor VIII concentrate that had been sterilized by treatment with tri(n-butyl)phosphate and Tween 80. The patient received 53 units of factor VIII per kg of body weight prior to operation. In addition, a total of 280 units of factor VIII per kg was infused within 10 days after operation. This replacement regimen prevented excessive bleeding during surgery and supported normal hemostasis during the postoperative period. Analysis of the multimeric pattern and the functional assay of von Willebrand factor in factor VIII concentrates indicated that the procedures utilized for virus inactivation had no significant deleterious effect upon the quality of von Willebrand factor molecules.

Abstract 194 Background. Acquired Thrombotic thromobcytopenic purpura (TTP) is the result of autoantibodies neutralizing and-or accelerating clearance of ADAMTS13. We have previously isolated mononuclear cells of two spleens donated to our laboratory by patients (A and B) splenectomized for frequently relapsing TTP to generate human monoclonal anti-ADAMTS13 antibodies. Preliminary characterization of the patient's entire IgG4 repertoire using phage display technology and Epstein Barr virus (EBV) transformation of switched memory B cells (CD19+, CD27+, IgG+) showed that the memory repertoire of Rituximab-resistant (B) and non-resistant (A) anti-ADAMTS13 IgG used the same restricted VH germline genes, namely VH1-3 (55%), VH1-69 (17%), VH3-30 (7%) and 4–28 (21%). In contrast, peripheral blood-derived IgG1 anti-ADAMTS13 antibody repertoire from 2 acquired TTP patients, inhibiting ADAMTS13 activity by maximal 15–40% used VH1-69 germline gene segment only (Luken et al, JTH 2006 and Pos et al, JTH 2009). To evaluate if the generated spleen-derived monoclonal anti-ADAMTS13 antibodies are of pathological relevance we started with their functional characterization. Methods. Both patients (A and B) were suffering from acute TTP with high titers of anti-ADAMTS13-Ab and therefore needed to be splenectomized because of frequent relapses, which occurred even after several courses of Rituximab (B). Both patients have remained in remission since splenectomy. Previous screening of the IgGk-IgGλ IgG4-Fab phage-display libraries and EBV-transformed clones on recombinant ADAMTS13 by ELISA revealed high ADAMTS13 specificity for 16/34 and 13/234 antibodies, respectively. Functional characterization of the phage-display clones included (a) determination of their inhbitiory capacity by functional FRETS inhibitor assay, (b) assessment of their affinity performed by either fluid-phase competition ELISA and/or plasma surface resonance (Biacore), and (c) epitope mapping by dot blot analysis testing their recognition of recombinant full-length wildtype ADAMTS13 and a truncated ADAMTS13 variant. Results. Detailed sequence analysis of all human monoclonal anti-ADAMTS13 analyzed showed seven unique CDR3 signatures in the variable heavy region of all clones generated using both techniques (phage display and EBV-transformation), three of which were seen in both patients. These CDR3 signatures are different from signatures found in peripheral blood-derived antibodies (Luken et al, JTH 2006 and Pos et al, Blood 2010). Strikingly, the anti-ADAMTS13 antibodies of EBV-transformed memory B cells used λ light chain exclusively, whereas the phage-display-derived clones showed usage of either κ- (A: 6/11 and B: 3/5) or λ-light chain (A: 5/11 and B: 2/5). Anti-ADAMTS13 IgG in supernatants of EBV-immortalized switched memory B-cell clones were mainly of subclass IgG4 (A: 60%; B: 25%) and IgG1 (A: 40%; B: 75%) thus IgG1 being the prevailing subclass in patient B, who relapsed despite several courses of Rituximab. Functional characterization of the anti-ADAMTS13 IgG4 antibodies revealed that 15/16 phage display clones are strongly inhibitory, showing 0–100% (A) and 60–100% (B) inhibition at 200 nM and even 0–92% (A) and 0–16% (B) inhibition at 20 nM. This represents a 5–6 times increase of inhibitory capacity when compared to previouse findingy by others, in the range of 2×10−7-7×10−10 M (A) and 1×10−9-6×10−10M (B) as measured by Biacore or/and competition ELISA. Epitope mapping revealed that 3/16 clones recognized a novel epitope at the C-terminus of ADAMTS13, in contrast to the previously reported N-terminally located spacer domain. Conclusions. The identification of several spleen-derived anti-ADAMTS13 clones displaying highly similar CDR3 variable heavy chains, in 2 unrelated acquired TTP patients is striking and novel. Our data points at the discovery of unique CDR3 signatures that characterize the pathological relevant, inhibitory antibodies paving thus the road to define their triggering ADAMTS13 epitope(s), while providing insight into the mode of success of splenectomy in relapsing TTP patients. Further functional characterization of the anti-ADAMTS13 EBV-derived clones and confirmation of our data by analysis of the immunological memory from two additional spleens are underway. Disclosures: No relevant conflicts of interest to declare.