Background and Purpose Pediatric stroke, birth-18 years, is a significant cause of long-term disability in the United States, however there is currently little experimental data on the pathophysiology of childhood stroke due to lack of animal models. We developed a novel mouse model of experimental childhood-onset arterial ischemic stroke (AIS) in order to characterize the sex-specific response of the adolescent brain to cerebral ischemia and assess the neuroprotective effect of estrogen at this developmental stage. Methods Postnatal day 20–25 (P20-25) mice were subjected to 90 minutes experimental stroke via the intraluminal filament middle cerebral artery occlusion (MCAO) model and ischemic damage assessed 22 hr after reperfusion. Real-time quantitative RT-PCR (qPCR) was performed 22 hr after MCAO to determine the effects of ischemia and estrogen treatment on the pro-apoptotic gene Bax. Results Ischemic injury did not differ between male and female juvenile (P20-25) mice following MCAO. However, estrogen reduced ischemic injury in female mice, while having no effect in juvenile males. No differences in estrogen receptor expression was observed between P20 males and females. In contrast, estrogen minimized the ischemia-induced increase in the pro-apoptotic gene Bax in female mice, while having no effect on Bax induction in the male brain. Conclusions Focal ischemia has fundamentally different effects in the juvenile brain compared to the adult, as evidenced by the lack of gender difference in ischemic injury in the murine P20-25 MCAO model and the sexually dimorphic response to estrogen neuroprotection.

3477 Poster Board III-414 Background Congenital afibrinogenemia (CA) is a rare bleeding disorder. While thrombosis is a recognized complication of dysfibrinogemia, the risk for thrombosis in CA has not been well studied. Life-threatening thrombosis with onset shortly after puberty has developed in 2 of 3 patients with CA followed through the coagulation program at The Children's Hospital, Colorado. Patient 1 suffered recurrent peripheral arterial thrombosis following cryoprecipitate administration during adolescence, massive myocardial infarction (MI) at age 19 years and death from spontaneous intracranial hemorrhage at age 20 years. Patient 2 has had superficial thrombophlebitis following intravenous (IV) infusion of fibrinogen concentrate, as well as bilateral pulmonary embolism with infarction 1 month following last infusion, without further recurrence over the course of 6 months of judicious use of anticoagulation and fibrinogen replacement. Patient 3 is 14 years old and receives fibrinogen replacement episodically for bleeding events, without thrombotic complications to date. Objective The object of this study was to investigate overall thrombin and plasmin generation potential and overall coagulative and fibrinolytic potential in CA, in order to generate hypotheses regarding pathophysiology of bleeding and thrombotic complications. Methods Plasma was collected from 2 CA patients at asymptomatic baseline states and following fibrinogen concentrate replacement. Plasma was isolated from whole blood within 1 hour, via double centrifugation at 2500 x g at 4°C x 15 minutes, and stored at -70°C until time of assay. Standard assays included fibrinogen activity by Clauss clotting assay and thrombin-antithrombin complexes (TAT) by ELISA (Siemens, Marburg, GE). Measurement of overall coagulative and fibrinolytic capacity in plasma was performed by Clot Formation and Lysis (CloFAL) global assay, as previously described (Goldenberg et al., Haemophilia, 2008). Simultaneous Thrombin and Plasmin generation assay (STP) was performed by fluorometric method with the same reagents as used in the CloFAL assay (dilute tissue factor, phospholipid, tissue plasminogen activator), as previously reported (Grunzke et al., J Thromb Haemost 2009 (abstract)). Results TAT was elevated to twice the upper limit of normal (9.4 mcg/L) in patient 1 six months following MI. In patient 2, TAT increased progressively from age 15 (2.9 mcg/L) to 20 years (13.7 mcg/L) while STP showed progressive shortening of time to peak thrombin generation by 60% over these 5 years (Figure 1). Baseline plasmin generation was nearly undetectable in both patients studied. As shown in Table I, TAT increased significantly at peak post infusion of fibrinogen concentrate in both patients evaluated during half-life and recovery studies. As shown in Figure 2, panels A and B, coagulative capacity by CloFAL assay and velocity of plasmin generation by STP showed dose dependence to fibrinogen following IV replacement with concentrate in patient 3. Discussion These data suggest that baseline and post infusion thrombin generation may be increased in post pubertal patients with CA. Furthermore, given undetectable plasmin generation in the absence of fibrinogen, the hemostatic balance in CA is simultaneously hemorrhagic and prothrombotic. These preliminary findings call for further prospective evaluation in other identified patients with CA. Disclosures: Grunzke: National Hemophilia Foundation/Baxter Clinical Fellowship Award: Research Funding. Manco-Johnson:CSL Behring: Honoraria, Research Funding.

2988 Poster Board II-964 BACKGROUND: In pediatric venous thromboembolism (VTE), duration of hypercoagulability—which has implications for recurrence risk and duration of therapy–is not readily assessed despite a battery of laboratory assays for comprehensive thrombophilia testing. Global clotting assays offer the possibility to better understand duration of hypercoagulability on an individualized basis. OBJECTIVE: We sought to evaluate overall coagulability and fibrinolytic potential over time in children with acute VTE and to compare these findings with D-dimer and established thrombophilia traits. METHODS: The Clot Formation and Lysis (CloFAL) global assay was performed in platelet-poor plasma in 58 children enrolled in a single-institutional prospective inceptional cohort study of VTE at The Children's Hospital, Colorado, between March 2006 and June 2009. This spectrophotometric fibrin registration assay employing clotting activation with dilute tissue factor and phospholipid and fibrinolytic enhancement with tissue plasminogen activator has been previously shown analytically sensitive to physiologic and pathologic alterations in multiple components of the coagulation and fibrinolytic systems (Goldenberg et al., Thromb Res 2005). Hypercoagulability was defined by an area under the curve (AUC) of the CloFAL waveform that exceeded the upper limit of age-appropriate reference values established in healthy children (n=26) using the non-parametric method of Tukey, and hypofibrinolysis was similarly defined as a fibrinolytic index (FI, which relates to the slope of decline in absorbance over the period of 30 minutes following maximal amplitude of clot formation) that was below the lower limit of normal. Coagulation index (CI, measured as the AUC over the first 30 minutes of the assay, indexed to the pooled normal plasma standard) was also evaluated, and compared to established reference ranges. Analyses were grouped by period post-diagnosis, as follows: acute (0-1 month; n=10), subacute (1-3 months; n=12), early chronic (3-6 months; n=10), late chronic (≥1 year; n=32). Comprehensive testing for genetic and acquired thrombophilia states was performed in all subjects, along with serial assessment of D-dimer and automated euglobulin lysis time (ELT). RESULTS: Of the 58 children with VTE evaluated for global coagulative and fibrinolytic capacity, 25% underwent repeat testing in follow-up. A positive relationship with factor VIII activity (FVIII) was demonstrated for both CI and AUC (r=0.37, P=0.006 and r=0.52, P<0.0001). In addition, AUC correlated inversely with FI and directly with ELT (r=-0.58, P<0.0001 and r=0.40, P=0.003), underscoring the interrelatedness between the coagulative and fibrinolytic systems (as has been previously shown, for example, via thrombin-activatable fibrinolytic inhibitor). Figure 1 demonstrates a decreasing trend in values over time post-diagnosis for CloFAL AUC, D-dimer, and FVIII, and increasing trend in CloFAL FI. The proportion of patients with abnormally elevated values for AUC, D-dimer, and FVIII, and abnormally low values for FI, also progressively decreased with time (Figure 2). Notably, however, persistent hypercoagulability was demonstrated in 28% , and hypofibrinolysis in 12.5%, of patients studied ≥1 year post-event (n=32). CONCLUSIONS: These findings demonstrate net hypercoagulability and hypofibrinolysis acutely in the majority of children with VTE, with a trend toward normalization in hemostasis over time across the study population. Further prospective studies are underway to determine whether persistence of hypercoagulability and/or hypofibrinolysis (as observed by global clotting assay in a subset of patients studied here), serves as a prognostic factor for recurrent VTE, and hence may contribute to future risk-stratified approaches to anticoagulant therapy duration. Disclosures: Off Label Use: The presentation refers to the use of anticoagulants as a drug class in general in the treatment of venous thromboembolism (VTE) in children. Despite their use in the standard care for pediatric VTE, all anticoagulants remain off-label for this indication in children.