ALBERT

Description: Introduction Clearance of infused factor VIII (FVIII) varies approximately 2-fold between persons with severe hemophilia A. This results in significant interpatient differences in factor levels following an infusion of FVIII and contributes to potentially significant differences in protection against spontaneous musculoskeletal bleeding in patients on fixed dose prophylaxis regimens. Aim The aim of this study is to compare two PK protocols: 1) a 6-point PK protocol with a 72 hour washout; and 2) a 2-point, one clinic visit PK protocol with no washout using the following pharmacokinetic (PK) parameters: clearance (Cl) and time to FVIII:C of 1% above baseline (tt1%) in persons with severe hemophilia A. Methods Inhibitor negative males with severe hemophilia A (FVIII

Description: A significant benefit of aspirin (ASA) has been demonstrated in the prevention of arterial thrombotic events in high-risk adult patients. Despite ASA therapy, recurrence of thromboembolic events, or treatment failure, has been reported in 10–20% of patients, and this has been termed ASA ’resistance’. This term has also been applied to the failure of ASA to affect ASA-dependent laboratory tests. As there is little information on ASA ’resistance’ in children, we examined the efficacy of ASA treatment on platelets from pediatric stroke patients using the PFA-100. Pediatric arterial ischemic stroke afflicts 2–3 children per 100,000 per year, and is associated with recurrent arterial ischemic stroke or transient ischemic attack in 20–40% of cases. Current treatment includes ASA prophylaxis (3 – 5 mg/kg/day) to inhibit platelet function and prevent recurrence, but it is not known whether this ASA dosing regimen is adequate to inhibit platelet function. Our study population consisted of 95 consecutive children with an index arterial ischemic stroke at mean age 5.9 ± 4.8 yrs (range: 0.1 – 17.0 yrs) on active ASA therapy (2.9 ± 1.2 mg/kg/day). Citrated blood samples were obtained at mean age 9.8 ± 4.8 yrs (range: 0.9 – 19 yrs), and were used to measure primary, platelet-related hemostasis in the high-shear PFA-100 system; closure times (CTs) were determined with the collagen/epinephrine cartridge that is sensitive to ASA’s inhibition of thromboxane A2 formation via platelet cyclo-oxygenase 1. The mean CT for all 95 patients was 244 ± 68.1 sec, which is greater than 163 sec, the upper limit of the normal range that we have previously determined for healthy children. The majority of patients, 75/95 (79%), had prolonged CTs (172 sec to 〉 300 sec), indicating inhibition of platelet function by ASA, and they were thus responsive to ASA therapy. 45 of the 75 ASA responders (63%) had CTs 〉 300 sec, i.e. CTs greater than the maximum test time of 300 sec, demonstrating aperture non-closure. The remaining 20/95 patients (21%) were ASA ’resistant’, as they did not respond to ASA therapy, having CTs (93 – 163 sec) within the normal range. Mean ASA dosage did not differ between ASA responders (2.8 ± 1.2 mg/kg/day) and ASA ’resistant’ patients (3.0 ± 1.4 mg/kg/day). 6 of the ASA ’resistant’ patients had their ASA dosage increased, and on repeat PFA-100 testing, 5/6 showed increased CTs 〉 163 sec. In conclusion, in children with arterial ischemic stroke, the majority, 79%, demonstrate inhibition of platelet function, as determined using the PFA-100, by ASA therapy at a mean dose of 2.9 mg/kg/day. The reason for the lack of inhibition of platelet function in 21% of pediatric patients, whether increased ASA dosage or alternative anti-platelet agents (e.g. clopidogrel) should be used in ASA ’resistant’ patients, and the relationship between ASA ’resistance’ and recurrence of arterial thrombotic events in children require further studies.

Description: Interaction of auto-antibodies with platelet surface glycoproteins (GP) has been implicated in the accelerated clearance of platelets in ITP. It was recently shown, using a murine model, that the ITP induced by injection of anti-GPIIb is associated with apoptotic-like processes in platelets. These apoptotic-like processes in platelets were similar to those observed during apoptosis in nucleated cells: activation of caspase-3 (aCASP3), loss of mitochondrial inner transmembrane potential and externalisation of phosphatidylserine (PS). Administration of IVIg, that ameliorates the ITP in this model, also inhibited aCASP3 and PS externalization (Leytin et al., 2006, Br J Haematol; 133:78; Song et al., 2003, Blood; 101:3708). Whether these observations in mice are applicable to patients with ITP had not been investigated. Thus, the aim of our study was to investigate whether platelets from paediatric patients with acute ITP show an enhancement of apoptotic-like processes. Further, we examined whether the increase in platelet count in response to IVIg in these patients is accompanied by an inhibition of apoptotic-like processes in platelets. Children with clinical and laboratory diagnosis of acute ITP were enrolled in this prospective study. Severity of bleeding symptoms was assessed according to a paediatric bleeding score for ITP at the time of diagnosis and after IVIg therapy. Blood samples were obtained at these time points for measurement of platelet count and for flow cytometric analyses of platelet apoptotic-like events. Specifically, platelet aCASP3 and PS externalisation were measured in citrated platelet-rich plasma. Platelets were identified as CD42 positive events; aCASP3 was measured as % platelets with bound FLICA-FITC fluorescence; and PS exposure as % platelets with bound annexin A5-FITC fluorescence. The fraction of young, reticulated platelets (RP) was analysed using thiazole orange. All patients (median age 7.5, n = 12) presented with typical symptoms of acute ITP with a bleeding score of 2 – 3. 10 patients had platelet counts below 10’000/micro l, and 2 had platelets counts over 10’000/micro l. ITP patients had increased levels of platelets with aCASP3 (n = 11) and PS exposure (n = 9) compared with healthy control children (see Figure) and increased reticulated platelets (15.6 ± 2.7 %, n = 10; controls: 1.2 ± 0.8, n = 11). The 10 patients with platelet counts below 10,000/micro l were treated with a maximum of 3 doses of IVIg (0.4 – 0.8 g/kg/dose). All patients showed a rise in platelet counts to above 20’000/micro l and amelioration of bleeding symptoms 24 – 72 hours after IVIg administration. Concomitantly, platelet aCASP3 and PS exposure decreased (see Figure), as did RP, to 3.8 ± 1.5 % (n = 9). One patient with an initial platelet count of 18,000/micro l was not treated. This patient showed 5% positive aCASP3 platelets, 3.5 % platelets with PS exposure and 3.5% RP, similar to controls; one month later, the platelet count increased spontaneously to 60’000/micro l with 10% aCASP positive platelets, 4% platelets with PS exposure and 3 % RP. One patient was lost to follow-up. In summary, we have demonstrated enhancement of the platelet apoptotic-like processes of aCASP3 and PS externalisation in paediatric acute ITP, similar to the reported murine model. Consistent with acute ITP, platelet counts were low and levels of RP were high; after administration of IVIg, platelet counts increased whereas levels of RP decreased. IVIg administration also led to a decrease of aCASP3 and PS exposure in the patients’ platelets. Thus, amelioration of acute ITP in children by IVIg is accompanied by inhibition of enhanced apoptosis-like events in platelets. Figure A: aCASP3 in acute ITP expressed as % platelets with FLICA-FITC fluorescence. B: PS exposure in acute ITP measured as % platelets with bound annexin A5-FITC fluorescence. After IVIg administration, fractions of aCASP3 positive platelets as well as platelets exposing PS were decreased. Figure A:. aCASP3 in acute ITP expressed as % platelets with FLICA-FITC fluorescence. B: PS exposure in acute ITP measured as % platelets with bound annexin A5-FITC fluorescence. After IVIg administration, fractions of aCASP3 positive platelets as well as platelets exposing PS were decreased.

Description: Several large trials have demonstrated the benefit of aspirin for the prevention of arterial thrombotic events in adults. Aspirin has also been increasingly used as an anti-platelet agent in children. However, very few well-designed clinical trials assessing the clinical efficacy and dosing of aspirin in children have been performed. In children, aspirin is usually administrated at a dose between 1 and 10 mg/kg/day. The term “aspirin resistance” (AR) describes the inability of aspirin to protect individuals from arterial thrombotic events (clinical AR) or the failure of aspirin to produce an expected response on laboratory measures of platelet activation and aggregation (laboratory AR). In adults, a prevalence of 5%–51% of AR had been reported, depending on the laboratory method used. A recent study using the platelet function analyser PFA-100 and levels of urinary 11-dehydrothromboxane B2 (11-dTXB2) excretion to define AR in paediatric cardiology patients, found a prevalence of 26% of laboratory AR (Heistein et al 2007). Here we studied the frequency of AR in children given aspirin at 3–5 mg/kg/d after interventional cardiac catheterization for atrial or ventricular septal defects or patent ductus arteriosus. Blood samples were taken 1–3 months after the initiation of aspirin therapy. Definition of AR was based on results from platelet aggregometry (% arachidonate-induced aggregation (AAG) of ≥ 20%) and results were compared with collagen/epinephrine closure times using the platelet function analyser PFA-100 and the levels of 11-dTXB2 excretion. As cyclooxygenase 1 (COX-1) is the primary target of aspirin and mutations could lead to a loss of functional protein, the presence of COX-1 was analyzed by western blot. Further, all children were followed clinically and side effects were monitored. Of 56 children included to date (median age: 10 y, range: 10 mo −20 y), 44 have completed the study. AR, was detected in 6 (14%) children. Of these 6 children, 3 had a normal response to aspirin (i.e. decreased AAG to mean+2SD ): Of these 2 children, one child had AR by the AAG definition even after aspirin dose increase and the other revealed AR by the PFA-100, with normal closure time, but not by AAG. While PFA-100 closure times were prolonged in 48% of children already prior to administration of aspirin, all children with AR according to AAG had prolonged closure times during aspirin-therapy. All 6 AR patients and 10 patients with a normal response to aspirin by AAG demonstrated equal amounts of COX-1 by western blot. While 6 children (14%) showed easy bruising and epistaxis during aspirin therapy, no severe bleeding or thrombotic complications were recorded In conclusion, AR was detected in 14% of children after interventional cardiac catheterization by AAG and could be overcome in some patients by increasing the dose of aspirin. These results are in keeping with findings from adults. By the use of the PFA-100 a previous study found a prevalence of 26% of AR in children (Heistein et al 2007). However, the authors did not correlate these results to AAG. In our cohort, the assessment of AR by the PFA100 was hampered by a high rate of abnormal PFA results previous to the initiation of therapy. Although reduction of 11-dTXB2 excretion indicates an inhibition of the COX-1 pathway by aspirin therapy, 11-dTXB2 and AAG results were not always congruent.

Description: Obtaining an accurate bleeding history is an essential step in the diagnosis of a bleeding disorder, which is often made during childhood. A bleeding history is usually taken in an informal manner, with the interpretation of the responses dependent on the experience of the observer. The development of standardized bleeding questionnaires has improved objectivity and allowed the determination of quantitative scores according to the severity of mucocutaneous bleeding. In adults, using the International Society on Thrombosis and Haemostasis (ISTH) Bleeding Questionnaire for Diagnosis of Type 1 VWD, a bleeding score of 〉3 in males and 〉5 in females is considered abnormal (Rodeghiero et al., J Thromb Haemost2005:3;2619). Children have often not been exposed to hemostatic challenges and may have low scores despite significant bleeding disorders. Symptoms specific to childhood, such as post-circumcision bleeding, umbilical stump bleeding and cephalohematoma may be of greater significance in this patient group. Thus, we adapted the ISTH Bleeding Questionnaire for use in pediatrics, with a symptom-specific grade of -1 to 4 (Tosetto et al., J Thromb Haemost2006:4;766). Bleeding scores were determined by interview of parents/children for 80 children with a previous diagnosis of VWD or a platelet function disorder at The Hospital for Sick Children, Toronto, or Kingston General Hospital, Kingston. 62 children had a diagnosis of VWD and 18, a disorder of platelet function (Glanzmann thrombasthenia: 3; dense granule defect: 2; MYH9-related thrombocytopenia: 2; Hermansky-Pudlak syndrome: 1; unspecified: 10). 45 children were female and 35, male (median age: 10 yrs (range: 9 mo-17 yrs)), with a median age of females of 12 yrs (range: 0–17) and of males, 9 yrs (range: 1–17). Bleeding scores ranged from 0–28 (median: 7), with a median score in females of 7 and in males, 8. Bleeding scores according to diagnosis and age are shown in Tables 1 and 2, respectively. The most frequent reasons for a positive score of ≥2 were epistaxis (43% of patients), bleeding from minor wounds (38%), bleeding after dental extraction (31%) and excessive bruising (26%). Menorrhagia requiring treatment occurred in 47% of menstruating females. Bleeding from the umbilical stump, post-circumcision bleeding and cephalohematoma were reported in 10%, 6% and 4% of patients, respectively. In summary, we have used a standardized bleeding questionnaire, adapted for use in pediatrics, with an accompanying score to quantify bleeding symptoms in children with confirmed VWD or a platelet function disorder. Bleeding scores were lowest in the youngest age group (0–3 yrs), and were slightly higher in males than in females. Bleeding occurred early in childhood, i.e. post-circumcision bleeding and bleeding from the umbilical stump. This standardized pediatric bleeding questionnaire/score may be useful in clinical practice in the assessment of children presenting with symptomatic bruising and bleeding. Diagnosis Type 1 VWD Type 2 VWD Type 3 VWD Platelet function disorder Number of patients 48 8 6 18 Median bleeding score (range) 6 (0–18) 10 (0–17) 14 (9–28) 9 (1–19) Age (years) 0–3 4–6 7–9 10–12 13–15 16–18 Number of patients 7 13 18 16 14 12 Median bleeding score (range) 2 (0–9) 10 (0–16) 6 (0–19) 9.5 (1–28) 6 (2–18) 12 (6–21)

Description: Initial adhesion of platelets to the injured vessel wall involves binding of the glycoprotein (GP)Ib-IX-V complex and integrin α2β1 to von Willebrand factor (VWF) and collagen, respectively, in the subendothelium. Polymorphisms have been described in the GPIbα and α2 genes that change the expression of these proteins or alter their configuration such that the efficiency of platelet adhesion is changed. The variable number of tandem repeat (VNTR) size polymorphism of GPIbα may extend the VWF binding domain at variable lengths above the platelet membrane, with variants A, B, C and D having 4, 3, 2 and 1 repeats, respectively; a polymorphism in the Kozak translation consensus sequence for GPIbα appears to predict receptor density. 3 haplotypes of α2 involving C807T have been correlated with α2β1 expression levels. We investigated these polymorphisms in the GPIbα and α2 integrin genes to evaluate the hypothesis that they influence the efficiency of platelet adhesion in the context of the inherited bleeding disorder, von Willebrand disease (VWD). The study population comprised 47 patients with type 1 VWD and 128 controls. Polymorphism genotyping was performed using restriction enzyme digest analysis of genomic DNA. Frequencies of the GPIbα VNTR alleles were: B=0.05, C=0.81, D=0.14 in the type 1 VWD group and B=0.11, C=0.80, D=0.08 in the controls. These frequencies were not significantly different between the 2 study groups. Genotype frequencies at the VNTR locus were: B/B=0.0, B/C=0.09, B/D=0.02, C/C=0.66, C/D=0.21, D/D=0.02 in the type 1 VWD group and B/B=0.0, B/C=0.20, B/D=0.02, C/C=0.63, C/D=0.13, D/D=0.01 in the controls (P=0.31). C and T Kozak allele frequencies were 0.12 and 0.88, respectively, in the type 1 VWD group and 0.08 and 0.91, respectively, in the controls. These allele frequencies were not significantly different between the 2 groups. Genotype frequencies were: C/C=0.02, C/T=0.20, T/T=0.78 in the type 1 VWD group and C/C=0.01, C/T=0.16, T/T=0.84 in the controls (P=0.61). Frequencies of the α2 alleles 1, 2 and 3 were: 1=0.38, 2=0.47, 3=0.15 in the type 1 VWD group, and 1=0.39, 2=0.50, 3=0.11 in the controls; genotype frequencies were 1/1=0.07, 1/2=0.48, 1/3=0.15, 2/2=0.17, 2/3=0.11, 3/3=0.02 in the type 1 VWD group and 1/1=0.15, 1/2=0.38, 1/3=0.11, 2/2=0.26, 2/3=0.10, 3/3=0.01 in the controls. Neither the allele frequencies nor the genotype distributions at the α2 integrin locus were significantly different between the VWD and control groups. In addition, allele frequencies and genotype distributions at all 3 polymorphic sites were similar to those for previously published controls. Thus, we did not observe any associations between the diagnosis of type 1 VWD and the GPIbα VNTR or Kozak polymorphisms or the α2 integrin polymorphism that determines α2β1 receptor density. Although our observation with the α2 integrin polymorphism is in contrast with that of a recent report that the 807C haplotype is associated with increased bleeding severity scores in type 1 VWD (Kunicki et al., Blood, prepublished online June 29, 2004), our observation with the GPIbα Kozak polymorphism is in accord with this study. Finally, our finding of the 2nd-largest B VNTR allele in the type 1 VWD group at a frequency of ½ that in controls suggests that the lower levels of this larger form of the GPIbα receptor may contribute to an increased bleeding risk in the VWD population.

Description: Abstract 484 Background: Plasma fibronectin (pFn) is an abundant protein in the blood. It has long been suspected that pFn plays a role in thrombosis/hemostasis, but this has remained controversial. Our previous study using pFn deficient mice demonstrated that pFn supports thrombosis (PNAS. 2003; 100: 2415-9). Unexpectedly, depletion of pFn in fibrinogen (Fg) and von Willebrand factor (VWF) double deficient (Fg/VWF−/−) mice enhanced, rather than abolished, platelet aggregation and thrombus formation, revealing a functional switch of pFn in the presence and absence of Fg and VWF (Blood. 2009;113:1809-17). However, the mechanism that controls this switch is not known. Furthermore, the hemostatic function of pFn in VW disease (VWD) or afibrinogenemia is unclear. Methods: To address these questions, we bred pFn conditional knockout mice with VWF−/− or Fg−/− mice, establishing 2 new strains of mice: Fg/pFn−/− and VWF/pFn−/−. We also extended our studies of pFn in the triple knockout (TKO, Fg/VWF/pFn−/−) mice. PolyI-polyC was injected into Cre+ and Cre- mice, which resulted in the depletion of plasma pFn (〉98%) and platelet pFn (〉80%) in Cre+ mice but not in Cre- littermate controls. Aggregometry, a perfusion chamber system, thromboelastography (TEG), tail vein bleeding assay and intravital microscopy were used to study these mice. Results: We first observed a significantly higher mortality in TKO (25%, P

Description: Abstract 1292 Poster Board I-314 Introduction Accurate assessment of bleeding symptoms is an essential step in the diagnosis of an inherited mucocutaneous bleeding disorder (IMBD). This is difficult in childhood when symptoms such as bruising and epistaxis are frequently noted in normal children. In addition, children may not have been exposed to hemostatic challenges such assurgery, dental extraction and menarche. Knowledge of the spectrum of presenting features of an IMBD may aid diagnosis. The MCMDM-1 VWD Bleeding Questionnaire, which uses a symptom-specific grade of -1 to 4 (Tosetto et al., J Thromb Haemost 2006:4;766), was recently modified for use in a pediatric population by inclusion of questions related to pediatric-specific bleeding symptoms. The Pediatric Bleeding Questionnaire (PBQ) has been validated in the assessment of bleeding symptoms in children and a score of 2 or greater predicts a diagnosis of VWD (Bowman et al., J Thromb Haemost 2009; doi:10.1111/j.1538-7836.2009.03499.x). This present study applied the PBQ to a cohort of children with a prior diagnosis of an IMBD in order to evaluate the prevalence and severity of specific bleeding symptoms. Patients and Methods The PBQ was used to determine bleeding score for 123 children with a prior diagnosis of VWD or a platelet function disorder (PFD) at The Hospital for Sick Children, Toronto, or Kingston General Hospital, Kingston, by interview of parents/children. A clinically-significant bleeding symptom was defined as one severe enough to require consultation with a healthcare professional, medical/surgical intervention or blood transfusion. Results 100 children had a diagnosis of VWD and 23, a PFD (Glanzmann thrombasthenia: 4; dense granule deficiency: 9; MYH9-related disease: 3; Noonan syndrome: 2; Ehlers-Danlos syndrome: 2; undefined PFD: 3). 65 were male and 58 female with a median age of 10.7 years (range: 0.6-18). Mean bleeding score was 8.3 for the entire cohort (range: 0-29) and varied between diagnostic groups (definite type 1 VWD: 9.5 (range: 2-18); possible type 1 VWD: 3.0 (range: 0-15); type 2 VWD: 11.8 (range: 3-17); type 3 VWD: 14.6 (range: 4-29); PFD: 9.5 (range: 1-20). The prevalence of each of the bleeding symptoms is shown in Table 1. The most frequent clinically-significant bleeding symptoms in the cohort were: menorrhagia, 57% (of menstruating females); epistaxis, 36%; prolonged bleeding from minor wounds, 35%; and bleeding after tooth extraction, 30%. The most frequent clinically-significant bleeding symptoms did not vary greatly between each of the diagnostic groups but oral cavity bleeding, hemarthrosis, gastrointestinal bleeding and hematoma were more frequent in children with type 3 VWD. All symptoms were less frequent in children with possible type 1 VWD. Menorrhagia was a prevalent symptom in menstruating females in all diagnostic groups. Clinically-significant pediatric-specific bleeding symptoms were seen in 20% of children. The most frequent were: post-circumcision bleeding, 35% (of circumcised males); cephalohematoma, 7%; macroscopic hematuria, 4%; and umbilical stump bleeding, 3%. These symptoms occurred most frequently in children with type 3 VWD, type 2 VWD or PFD. Conclusions Children with an IMBD frequently report menorrhagia, epistaxis, prolonged bleeding from minor wounds and bleeding after tooth extraction. Pediatric-specific bleeding symptoms also occur. There are differences in prevalence of symptoms between subtypes of VWD and PFDs. This information can assist the evaluation of children with bleeding symptoms. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3700 Apoptotic-like processes in platelets are similar to those observed during apoptosis in the cytoplasm of nucleated cells: activation of caspase-8, caspase-9, and caspase-3, loss of mitochondrial inner membrane potential, and externalization of phosphatidylserine (PS) (Leytin et al, Br J Haematol 2006; Lopez et al, J Thromb Haemost 2009). We recently showed that platelets in pediatric primary immune thrombocytopenia (ITP) have activated caspase-3 (aCASP3) and externalized PS, both of which were reduced after IVIg administration (Speer et al, Blood 2008;112: 3417). To gain a more complete understanding of the apoptosis that occurs in ITP platelets, in the present study, we investigated whether caspase-8 and caspase-9 are also activated in platelets from children with ITP, and examined whether the increase in platelet count in response to IVIg is associated with a decrease in activated caspase-8 (aCASP8) and -9 (aCASP9) in platelets, as was observed for aCASP3. In addition, we measured the mitochondrial membrane potential in platelets before and after IVIg therapy. Children with primary ITP were enrolled in this prospective study. Severity of bleeding symptoms was assessed according to a pediatric bleeding score for ITP at the time of diagnosis. Blood samples were obtained at the time of diagnosis and after IVIg therapy for measurement of platelet count and for flow cytometric analyses of platelet apoptotic-like events. In citrated platelet-rich plasma, platelets were identified as CD42 positive events; aCASP8 and aCASP9 were measured as % platelets with bound FITC-fluorescent-labeled inhibitors of activated caspases; and mitochondrial membrane potential was measured as mean fluorescence intensity of the membrane potential sensitive fluorescent tetramethylrhodamine ethyl ester (TMRE). All patients (median age 5.4 yrs, n = 8) presented with typical symptoms of acute ITP with a bleeding score of 2 – 3 and had platelet counts 〈 20×109/L. Results from ITP patients were compared with 2 control groups, healthy children (platelet counts: 266–348 × 109/L, median age 6.8 yrs, n = 7) and children with thrombocytopenia as a result of chemotherapy for malignancies (cTP) (platelet counts: 3–51 × 109/L, median age 10.2 yrs, n = 7). ITP patients had significantly higher proportions of platelets with aCASP8 (17.5±5.1%) and aCASP9 (16.9±5.8%) compared with both healthy children (aCASP8 1.0±0.3%; aCASP9 1.1±0.3%) and children with cTP, (aCASP8 2.2±0.4%; aCASP9 1.9±0.4%) (p 20 × 109/L and amelioration of bleeding symptoms by 24 – 72 hours after IVIg administration. Concomitantly, the fractions of platelets with aCASP8 and aCASP9, decreased towards control values (ITP patients after IVIg: aCASP8 7.8±5.3%; aCASP9 6.9±2.1; p=0.5 for both compared to controls). Again no change in mitochondrial potential was observed after IVIg. In summary, we have demonstrated enhancement of the platelet apoptotic-like processes of aCASP8 and aCASP9 specifically in pediatric primary ITP, which were not observed in cTP. However, the platelet mitochondrial membrane potential was unchanged in ITP (before and after IVIg) and did not differ compared cTP and healthy children. Consistent with primary ITP, the patients' platelet counts were low and increased with IVIg administration. In parallel, IVIg led to a decrease of aCASP8 and aCASP9 in the patients' platelets. Together with our previously reported results (Speer et al, Blood 2008;112: 3417), we show that apoptotic events in platelets such as activation of caspases-8, -9, and -3 and PS exposure are increased specifically in ITP but not in cTP, and are decreased after IVIg treatment. As we detected no loss of the mitochondrial membrane potential in platelets from ITP patients, it may be that apoptotic processes in these platelets are not activated by mitochondrial signaling, but rather via an extrinsic signaling cascade including caspase-8, leading to the activation of caspase-3 and caspase-9. However, the complete signaling pathway leading to caspase-8 activation in platelets of pediatric ITP remains to be elucidated. Disclosures: No relevant conflicts of interest to declare.