A new protocol for assessing action observation and imitation abilities in children with Developmental Coordination Disorder: A feasibility and reliability study.

AIMS: To develop a new protocol for the assessment of action observation (AO) abilities and imitation of meaningful and non-meaningful gestures, to examine its psychometric properties in children with DCD and typically developing (TD) children. BACKGROUND: For learning manual skills, AO and imitation are considered fundamental abilities. Knowledge about these modalities in children with DCD is scarce and an assessment protocol is lacking. METHOD: The protocol consists of 2 tests. The AO test consists of two assembly tasks. The imitation test includes 12 meaningful and 20 non-meaningful gestures. Items of both tests are rated on a 4-point scale. Twelve children with DCD (mean age 8y3m, SD, 1.30) and 11 TD children (mean age 8y2m, SD 1.52) were enrolled. For inter-rater reliability, intraclass correlation coefficients (ICC) were calculated for the total score, weighted kappa and percentage agreement for single items. Known group validity was assessed by comparison of DCD and TD group (Wilcoxon rank sum test). For construct validity, the mABC-2 test was used. The protocol was adapted and confirmed by an intra and inter-rater reliability study (new sample of 11 DCD children, mean age 7y5m, SD 1.37). RESULTS: Excellent ICCs were reported for intra and inter-rater reliability for the final protocol. A significant difference between DCD and TD group was found for AO abilities (p < .01), for nonmeaningful gestures (p < .001). A significant correlation was reported between the AO test and the mABC-2 test (r = 56;p ≤0.0001). No significant correlations were revealed for the imitation tests. DISCUSSION AND CONCLUSION: The results support the psychometric properties of this protocol. When fully validated, it may contribute to map the deficits in AO abilities and imitation, to evaluate treatment effects of imitation and AO interventions.

Comparison of three commercially available buffy coat pooling sets for the preparation of platelet concentrates.

BACKGROUND: A disposable set for platelet concentrate (PC) preparation by the buffy coat method allows pooling of buffy coats, centrifugation and cell separation with in-line leucocyte filtration. This study compares three commercially available pooling sets in combination with INTERCEPT pathogen inactivation (PI). MATERIALS AND METHODS: Sets for pooling of buffy coats were from Fresenius Kabi (FRE), Macopharma (MAC) and Terumo BCT (TER). Platelet yield, recovery and concentration were compared before and after PI (n = 20). Platelet quality was assessed by annexin V binding, P-selectin expression and PAC1 binding. RESULTS: The TER pooling set had the highest platelet yield (5·39 ± 0·44 × 1011 ) compared with MAC (4·53 ± 0·77) and FRE (4·56 ± 0·51) prior to PI. This was the result of a significantly higher platelet concentration in the TER storage bag (1·41 ± 0·12 × 106 /µL) compared with MAC (1·18 ± 0·19) and FRE (1·28 ± 0·15). However, the TER platelet content decreased by 15·6% after PI, yielding 4·55 ± 0·47 × 1011 platelets compared with smaller reductions at 9·5% for MAC (4·10 ± 0·69) and 4·4% for FRE (4·36 ± 0·52). None of the individual PC contained >106 leucocytes. The pH in TER PC was lower compared with MAC and FRE caused by a higher lactic acid production rate. Consequently, PAC1 binding after TRAP activation was lowest for TER PC on day 6. P-selectin and annexin V were not different between suppliers. CONCLUSION: This study demonstrates the added value of evaluating the entire component production process when introducing a new consumable. This study helped to inform a decision on what pooling set is ideally suited for routine implementation taking into account PI.

High platelet content can increase storage lesion rates following Intercept pathogen inactivation primarily in platelet concentrates prepared by apheresis.

BACKGROUND: Pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide. In vitro studies have demonstrated its effects on storage lesion, but little routine quality control data on blood banking outcomes have been reported. MATERIALS AND METHODS: Swirling of distributed products was monitored before and after implementation of Intercept pathogen inactivation. Metabolic parameters pH, glucose and lactic acid were determined in a random cohort of expired pathogen-inactivated products. Storage lesion indicators in apheresis concentrates with premature low swirling were compared to concentrates with normal swirling. RESULTS: During validation for implementing Intercept pathogen inactivation, pH and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pathogen-inactivated pooled buffy coat-derived products. In routine products, glucose exhaustion was more often found in apheresis compared to buffy coat-derived platelet concentrates despite 3-7% more plasma carryover in the former. Annual incidence of premature low swirling increased significantly by 50% following implementation of pathogen inactivation implementation for apheresis but not for pooled buffy coat platelet concentrates. In addition, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5·0 × 1011 ) than unaffected products (3·5 × 1011 ). CONCLUSION: The risk of increased storage lesion rates following Intercept pathogen inactivation is higher for apheresis than for buffy coat-derived platelet concentrates, especially when platelet contents are higher than 5·0 × 1011 .

Persistent aggregates in apheresis platelet concentrates are commonly collected from donors with a history of aggregate donation.

Platelet apheresis sometimes causes persistent aggregates (PA). This study (n = 211) shows that changing the apheresis settings to reach fixed product volumes instead of yields does not influence PA incidence, even though PA products on average contain more platelets than controls. Furthermore, logistic regression was used to model if PA can be predicted on the basis of certain predonation parameters. PA donation history was the only parameter retained, proving a strong determinant of predictability [AUC = 0.735 (SE = 0.022)]. Consequently, donations from a donor with previous PA history are 7.8 times more likely to contain PA than from a donor without preceding history.

The contribution of von Willebrand factor-GPIbα interactions to persistent aggregate formation in apheresis platelet concentrates.

BACKGROUND AND OBJECTIVES: Apheresis platelet concentrates sometimes contain persistent aggregates (PA). Because apheresis involves extracorporeal circulation, we hypothesized that interactions between GPIbα and von Willebrand factor (VWF) underlie their origin. MATERIALS AND METHODS: Platelets in donations with PA were compared to aggregate-free (AF) controls. Flow cytometry was used to determine platelet bound VWF. Degranulation was measured using P-selectin expression in flow cytometry and cytokine release using immunosorbent assays. Platelet adhesion to VWF was assessed in hydrodynamic flow and real-time video microscopy. RESULTS: Platelets in PA concentrates had significantly more (P = 0·009, n ≥ 8) bound VWF compared to AF platelets, but differences in VWF concentration, VWF collagen binding, activated VWF or GPIbα expression were not found. Degranulation was higher (P = 0·030, n = 7) in PA than AF concentrates on day 1 of storage, but adhesion to immobilized VWF under hydrodynamic flow conditions was normal at that moment. On day 6, however, significantly less VWF adhesion (P = 0·009, n ≥ 6) was found for PA platelets compared to AF, indicating accelerated storage lesion in PA products. In a model that mimicks PA formation by chemically induced binding of VWF to platelets, we found that degranulation, phosphatidylserine expression and metabolism did not differ with paired controls at any time during subsequent storage. CONCLUSION: Accelerated storage lesion is found in concentrates with PA, but this cannot be explained solely by increased platelet bound VWF following apheresis. Therefore, additional stressors are probably responsible for the increases observed in platelet degranulation and storage lesion in products with PA.

Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13.

BACKGROUND: Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF). OBJECTIVES: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13. METHODS: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy. RESULTS: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. CONCLUSION: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.

An evaluator-blinded randomized controlled trial evaluating therapy effects and prognostic factors for a general and an individually defined physical therapy program in ambulant children with bilateral spastic cerebral palsy.

BACKGROUND: Cerebral palsy (CP) is characterized by a heterogeneous nature with a variety of problems. Therefore, individualized physical therapy might be more appropriate to address the needs for these children. AIM: The first aim was to compare the effectiveness of an individually-defined therapy program (IT) and a general therapy program (GT) on gait and gross motor function in children with CP. The second aim was to evaluate interaction-effects, time-effects, treatment with botulinum toxin A, age, gross Motor Function Classification Scale (GMFCS), treatment frequency and quality as factors influencing outcome. DESIGN: An evaluator-blinded, randomized controlled trial. SETTING: Outpatient rehabilitation unit. POPULATION: Forty ambulant children with spastic bilateral CP (mean age 6 years 1 month). METHODS: All children were randomly assigned to receive either IT or GT over a 10 week period. Nineteen of these children were enrolled into a second and/or third program, resulting in 60 interventions. Primary outcome was assessed with the Goal Attainment Scale (GAS) for gross motor function goals and z-scores for goals based on specific 3D gait parameters. Secondary outcome included the Gross Motor Function Measure-88 (GMFM-88) scores, time and distance gait parameters, Gait Profile Score, Movement Analysis Profiles and time needed to complete Timed-Up-and-Go and Five-Times-Sit-To-Stand tests. RESULTS: There were higher, but non-significant GAS and z-score changes following the IT program compared to the GT program (GAS: 46.2 for the IT versus 42.2 for the GT group, P=0.332, ES 0.15; z-score: 0.135 for the IT compared to 0.072 for the GT group, P=0.669, ES 0.05). Significant time-effects could be found on the GAS (P<0.001) and the GMFM-88 total score (P<0.001). Age was identified as a predictor for GAS and GMFM-88 improvement (P=0.023 and P=0.044). CONCLUSION: No significant differences could be registered between the effects of the IT and the GT. The favorable outcome after the IT program was only a trend and needs to be confirmed on larger groups and with programs of longer duration. CLINICAL REHABILITATION IMPACT: Both programs had a positive impact on the children's motor functioning. It is useful to involve older children more actively in the process of goal setting.

Persistent aggregates in apheresis platelet concentrates.

BACKGROUND: Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing product wastage. This study assessed product quality of apheresis concentrates containing persistent aggregates (PA) and aimed to identify the factors that contribute to their formation. METHODS: Donation (n = 180) and platelet indices (n ≥ 10) from apheresis concentrates with PA were compared with aggregate-free products. RESULTS: The proportion of donors with at least one previous PA donation was twofold higher in the PA group (P < 0·0001) indicating a donor dependence. Significantly higher donor whole blood platelet counts (286 ± 50 vs. 266 ± 49 × 10(3) /µl, P < 0·0001) and higher apheresis yields (6·0 ± 1·6 vs. 5·4 ± 1·5 × 10(11) , P < 0·0001) were noted in the PA group. Haematocrit was also slightly higher, but age, gender and body mass were similar. The pH of PA products on day six postdonation was significantly lower (P < 0·001), in line with higher lactic acid concentrations. Flow cytometry showed no differences in GPIbα levels or phosphatidylserine exposure. However, there was slightly more integrin activation as well as increased degranulation measured by P-selectin expression. Cytokine concentrations were also significantly higher in PA concentrates. Aggregation was normal in response to SFLLRN peptide and collagen stimulation, but agglutination at low-dose ristocetin was significantly higher (P = 0.01) in PA products. Finally, PA were disintegrated by plasmin-mediated thrombolysis but not by integrin αIIb ß3 inhibition. CONCLUSION: Products with PA have acceptable quality parameters, but additional functional studies are warranted. Furthermore, PA are more likely to recur in certain donors who have higher platelet counts.

Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro.

BACKGROUND: Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultraviolet light illumination reduces the proliferation potential of pathogens by damaging biomolecules. MATERIALS AND METHODS: The impact of riboflavin (RF-PRT)- and amotosalen (AS-PCT)-based pathogen inactivation on platelets was studied using microfluidic flow chambers on immobilized collagen using standard platelet concentrates prepared from buffy coats in additive solution. Flow cytometry, metabolic parameters and light transmission aggregometry with thrombin-related peptide, collagen and ristocetin were determined concurrently. RESULTS: Both PCTs significantly decreased the platelet surface coverage kinetics in flow chambers over the course of the 7-day study. Platelet aggregation was affected following RF-PRT in response to all agonists, while AS-PCT mainly impacted low-dose ristocetin agglutination. RF-PRT induces premature platelet activation because integrin αII b ß3 was spontaneously activated, and α-degranulation, phosphatidylserine/-ethanolamine exposure and anaerobic metabolism significantly increased following treatment, which was not the case for AS-PCT. On the other hand, AS-PCT significantly diminished thrombus growth onto von Willebrand factor under shear flow. This defect was caused by fewer integrin αII b ß3 interactions, not by defective GPIbα-VWF binding as shown by adhesion experiments in the presence of tirofiban. Moreover, integrin αII b ß3 activation was also affected following the activation of platelets via GPVI-FcγRIIa or PAR1. Finally, amotosalen illumination as such is sufficient to induce platelet damage, with no additional measurable effect of the chemical adsorption step. Gamma irradiation caused no significant difference compared to controls on any time-point or for any parameter. CONCLUSION: Both PCTs significantly reduce thrombus formation rate but by different biochemical mechanisms.

Respiratory morbidity in children with profound intellectual and multiple disability.

BACKGROUND: 'Profound intellectual and multiple disability' (PIMD) is defined as a profound cognitive disability with severe sensory and motor impairments. The aim of this study was to evaluate the respiratory morbidity in children with PIMD and investigate possible risk factors. METHODS: In 10 specialized facilities for daily care of patients with PIMD, children underwent a standardized clinical assessment evaluating respiratory and motor function. Additional medical information was obtained. RESULTS: One hundred and twenty seven children aged 2-21 years were tested (median age 12 years; IQR 8-16). 72% had epilepsy, 42% were gastrostomy fed. The median number of lower airway infection per years was four (IQR 1-4). While 68% of patient had no hospital admissions for respiratory disease, 12% of patients were admitted three times or more. Chronic antibiotic therapy was prescribed to nine patients (7%), and 19 patients (15%) were chronically treated with mucolytics, inhaled corticosteroids and/or bronchodilators. Chest physiotherapy was given daily to 26 patients (22%). Gastroesophageal reflux, swallowing problem and aspiration increased the risk for hospital admissions. Additionally risk factors were the severity of disability, axial hypotonia, presence of epilepsy, scoliosis, limited shoulder movement, paradoxical breathing and absence of a spontaneous cough reflex. CONCLUSION: The overall respiratory morbidity in our sample of children with PIMD was lower than anticipated. While a subgroup of children are prone to recurrent severe airway problems, the majority of children did not experience severe airway infections.

Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion.

BACKGROUND AND OBJECTIVE: Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT). METHODS: Activity and antigen of plasma components were determined following RF-PRT in the presence or absence of dissolved molecular oxygen. RESULTS: Employing ADAMTS13 as a sentinel molecule in plasma, our data show that its activity and antigen are reduced by 23 ± 8% and 29 ± 9% (n = 24), respectively, which corroborates with a mean decrease of 25% observed for other coagulation factors. Western blotting of ADAMTS13 shows decreased molecular integrity, with no obvious indication of additional proteolysis nor is riboflavin able to directly inhibit the enzyme. However, physical removal of dissolved oxygen prior to RF-PRT protects ADAMTS13 as well as FVIII and fibrinogen from damage, indicating a direct role for reactive oxygen species. Redox dye measurements indicate that superoxide anions are specifically generated during RF-PRT. Protein carbonyl content as a marker of disseminated irreversible biomolecular damage was significantly increased (3·1 ± 0·8 vs. 1·6 ± 0·5 nmol/mg protein) following RF-PRT, but not in the absence of dissolved molecular oxygen (1·8 ± 0·4 nmol/mg). CONCLUSIONS: RF-PRT of single plasma units generates reactive oxygen species that adversely affect biomolecular integrity of relevant plasma constituents, a side-effect, which can be bypassed by applying hypoxic conditions during the pathogen inactivation process.

Three-dimensional head and trunk movement characteristics during gait in children with spastic diplegia.

This study uses a recently developed trunk model to determine which head and trunk kinematic parameters differentiate children with spastic diplegia from typically developing (TD) children while walking. Differences in head and trunk parameters in relation to the severity of the motor involvement (GMFCS levels) were additionally examined. The trunk model consisted of five segments (pelvis, thorax, head, shoulder line, spine). Discrete kinematic parameters (ROM, mean position) and angular waveforms were compared between 20 children with spastic diplegia (age 9.8 years±2.9 years; GMFCS I: n=10, GMFCS II: n=10) and 20 individually age-matched TD children (9.7 years±3 years). A new measure for overall trunk pathology, the trunk profile score (TPS), was proposed and included in the comparative analysis. Compared to TD children, children with GMFCS II showed a significantly higher TPS and increased ROM for pelvis tilt, for thorax and head in nearly all planes, and the angle of kyphosis. In children with GMFCS I, only ROM of thorax lateral bending was significantly increased. Sagittal ROM differentiated best between GMFCS levels, with higher ROM found in children with GMFCS II. Current results provide new insights into head and trunk kinematics during gait in children with spastic diplegia.

Reliability of head and trunk kinematics during gait in children with spastic diplegia.

This study describes the reliability of a clinically oriented model for three-dimensional movement analysis of head and trunk movements in children with spastic diplegia. The model consists of five rigid segments (head, thorax, pelvis, shoulder line, spine) and includes a detailed analysis of spinal segmental movements. Within and between session reliability during gait was tested in 10 children with spastic diplegia (6-14yrs). Reliability of discrete parameters was assessed with the intraclass correlation coefficient (ICC) and similarity of thorax and pelvis waveforms with the coefficient of multiple correlation (CMC). Measurement errors were calculated for all parameters (SEM, σ). Results indicated acceptable within and between session reliability of discrete parameters for thorax, pelvis, shoulder line, angle of kyphosis and the majority of the spinal segmental angles, reflected by low SEMs (<4°) and most ICCs>0.60. Within and between session waveform errors were below 4°. CMCs ranged from poor to very good, with highest values for movements in the frontal and transversal planes. The angle of lordosis showed lower between session reliability for several discrete parameters, although waveform errors were still below 5°. Head parameters showed lower overall reliability. The results of this study support the reliability of the proposed model. Head kinematic parameters should be interpreted with caution, due to difficulties in standardization. Accurate palpation of the spinal markers, especially the lumbar spine, is critical and demands thorough training of the assessor.

Bimanual force coordination in children with spastic unilateral cerebral palsy.

In this study bimanual grip-force coordination was quantified using a novel "Gripper" system that records grip forces produced while holding a lower and upper unit, in combination with the lift force necessary to separate these units. Children with unilateral cerebral palsy (CP) (aged 5-14 years, n=12) were compared to age matched typically developing (TD) children (n=23). Compared to TD, the CP-group is much slower and takes 50% more time to generate grip and lift forces with more fixating force before lifting the upper unit. In addition the coordination between forces in both hands is reduced. The CP-group increases the lift force in the upper hand 2.5 times more than the holding force when pulling the two units apart, while this is only 1.5 times in TD. Moreover, the correlation between forces generated in both hands in the CP-group is lower. The lack of fine tuning of the forces, measured by the linearity error is increased, especially when the magnet load keeping the unit together is low. The results indicate an impaired pull-hold synergy between upper and lower hand and the lift force. Bimanual tasks evaluating bimanual grip and lift forces in children with CP and can give us new insights in the underlying force control mechanisms of the spastic hand.

Is the good-imitator-poor-talker profile syndrome-specific in Down syndrome?: evidence from standardised imitation and language measures.

The emergence of the Down syndrome (DS) behavioural phenotype during early development may be of great importance for early intervention. The main goal of this study was to investigate the good-imitator-poor-talker developmental profile in DS at preschool age. Twenty children with Down syndrome (DS; mean nonverbal mental age NMA 1 y10 m) and 15 children with non-specific mental retardation (NS-MR; mean NMA 1 y11 m) participated in this study. The Preschool Imitation and Praxis Scale (PIPS) and the Dutch version of the MacArthur-Bates Communicative Development Inventories (N-CDI) were used to determine absolute and relative (contrasted to a nonverbal mental age reference) imitation and language abilities. Results revealed that there was clear evidence for a good-imitator-poor-talker profile in preschoolers with DS. However, only the advanced bodily imitation ability seems to be syndrome-specific. Clinical implications of these findings are considered.

A systematic review of arm activity measures for children with hemiplegic cerebral palsy.

OBJECTIVE: To identify psychometrically sound and clinically feasible assessments of arm activities in children with hemiplegic cerebral palsy for implementation in research and clinical practice. DATA SOURCES: PubMed, CINAHL, Cochrane Library, Web of Science and reference lists of relevant articles were searched. REVIEW METHODS: A systematic search was performed based on the following inclusion criteria: (1) evaluative tools at the activity level according to the International Classification of Functioning, Disability and Health; (2) previously used in studies including children with hemiplegic cerebral palsy aged 2-18 years; (3) at least one aspect of reliability and validity in children with cerebral palsy should be established. Descriptive information, psychometric properties and clinical utility were reviewed. RESULTS: Eighteen assessments were identified of which 11 met the inclusion criteria: eight functional tests and three questionnaires. Five functional tests were condition-specific, three were generic. All functional tests measure different aspects of activity, including unimanual capacity and performance during bimanual tasks. The questionnaires obtain information about the child's abilities at home or school. The reliability and validity have been established, though further use in clinical trials is necessary to determine the responsiveness. CONCLUSIONS: To obtain a complete view of what the child can do and what the child actually does, we advise a capacity-based test (Melbourne Assessment of Unilateral Upper Limb Function), a performance-based test (Assisting Hand Assessment) and a questionnaire (Abilhand-Kids). This will allow outcome differentiation and treatment guidance for the arm in children with cerebral palsy.

The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet-decorated VWF strings in vivo.

BACKGROUND: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. METHODS: Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13(-/-) mouse. RESULTS: Murine MDTCS (devoid of T2-8 and CUB domains) retained full enzyme activity in vitro towards FRETS-VWF73 and the C-terminal T6-8 (del(T6-CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet-decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6-CUB)) or complete (delCUB) addition of the T2-8 domains gradually attenuates its activity. CONCLUSIONS: Our data demonstrate that the ADAMTS13 CUB and T2-8 domains influence proteolysis of platelet-decorated VWF strings in vivo.

Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura.

BACKGROUND: ADAMTS13 deficiency causes accumulation of unusually large von Willebrand factor molecules, which cross-link platelets in the circulation or on the endothelial surface. This process of intravascular agglutination leads to the microangiopathy thrombotic thrombocytopenic purpura (TTP). Most TTP patients have acquired anti-ADAMTS13 autoantibodies that inhibit enzyme function and/or clear it from the circulation. However, the reason for ADAMTS13 deficiency is not always easily identified in a subset of patients. OBJECTIVES: To determine the origin of ADAMTS13 deficiency in a case of acquired TTP. METHODS: Western blotting of ADAMTS13 in plasmas from acute and remission phases was used. RESULTS: The ADAMTS13 deficiency was not caused by mutations or (detectable) autoantibodies; however, an abnormal ADAMTS13 truncated fragment (100 kDa) was found in acute-phase but not remission-phase plasma. This fragment resulted from enzymatic proteolysis, as recombinant ADAMTS13 was also cleaved when in the presence of acute-phase but not remission-phase plasma. Inhibitor screening showed that ADAMTS13 was cleaved by a serine protease that could be dose-dependently inhibited by addition of exogenous α2 -antiplasmin. Examination of the endogenous α2-antiplasmin antigen and activity confirmed deficiency of α2 -antiplasmin function in acute-phase but not remission-phase plasma. To investigate the possibility of ADAMTS13 cleavage by plasmin in plasma, urokinase-type plasminogen activator was added to an (unrelated) congenital α2 -antiplasmin-deficient plasma sample to activate plasminogen. This experiment confirmed cleavage of endogenous ADAMTS13 similar to that observed in our TTP patient. CONCLUSION: We report the first acquired TTP patient with cleaved ADAMTS13 and show that plasmin is involved.