ALBERT

Description: Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals. (Blood. 2004;103: 3412-3419)

Description: Hemophilia and von Willebrand disease are the most common congenital bleeding disorders. Treatment of these disorders has focused on replacement of the missing coagulation factor to prevent or treat bleeding. New technologies and insights into hemostasis have driven the development of many promising new therapies for hemophilia and von Willebrand disease. Emerging bypass agents including zymogen-like factor IXa and Xa molecules are in development and a bispecific antibody, emicizumab, demonstrated efficacy in a phase 3 trial in people with hemophilia A and inhibitors. Tissue factor pathway inhibitor, the protein C/S system, and antithrombin are targets of novel compounds in development to alter the hemostatic balance and new approaches using modified factor VIII molecules are being tested for prevention and eradication of inhibitor antibodies in hemophilia A. The first recombinant von Willebrand factor (VWF) product has been approved and has unique VWF multimer content and does not contain factor VIII. These new approaches may offer better routes of administration, improved dosing regimens, and better efficacy for prevention and treatment of bleeding in congenital bleeding disorders.

Description: A family history (FH) of thrombophilia may prompt a referral to a pediatric thrombophilia clinic. The International Society of Thrombosis and Haemostasis recommends a step-wise thrombophilia evaluation for children with venous thrombosis, but there are no screening recommendations for asymptomatic children with a FH of thrombophilia. There is limited data on how many children are evaluated by a pediatric hematologist for a FH of thrombophilia or how these children are managed. We utilized the Centers for Disease Control and Prevention (CDC) Thrombosis and Hemostasis Centers Pilot Sites Registry to characterize children without a personal history of thrombosis who are referred to Thrombosis Centers (TCs). The CDC initiated the registry between eight TCs, including four pediatric sites. We queried the database for pediatric subjects, who enrolled in the registry between August 2003-March 2006. Data collection included referral patterns, laboratory and radiological tests ordered, diagnosis, and treatments. Three hundred thirty-two subjects were identified, ages 0–17 years (average 9 years). Fifty-five (16.6%) have no personal history of thrombosis. Among the 55 asymptomatic children 4 (7.3%) were referred exclusively for a FH of thrombosis without thrombophilia, 12 (21.8%) were referred exclusively for a FH of thrombophilia without a FH of thrombosis, 11 (20.0%) were referred for a FH of both thrombosis and thrombophilia, and 28 (50.9%) did not have a FH of thrombosis or thrombophilia. The latter subjects may have been referred to the TCs for anticoagulation management or for evaluation of a condition associated with thrombophilia such as migraine. No asymptomatic subjects were referred from the in-patient setting, 48 (87.3%) were referred from the out-patient setting, and 7 (12.7%) were self-referred. Of the 55 asymptomatic children, 19 (34.5%) were referred with a prior diagnosis of thrombophilia. Table 1 compares testing at the TCs of symptomatic and asymptomatic subjects. Three asymptomatic subjects with a FH of thrombosis and thrombophilia received anticoagulation prophylaxis, and 3 subjects received a recommendation for prophylaxis for future high risk situations. In summary, 16.6% of children enrolled in the CDC registry have no personal history of thrombosis. A significant proportion of these asymptomatic children have testing for prothrombotic risk factors prior to referral and at the TCs. We are conducting research to determine who and when to screen for thrombophilia, potential complications of such testing, when to provide genetic counseling, and optimal clinical application of the results. Thrombophilia Testing at Pediatric Thrombosis Centers % subjects tested for Genetic tests Anticoagulants/Antiphospholipid antibodies Clotting factors Screening tests Lipids/Homocysteine All (n=332) 42.5 54.8 43.7 56.6 36.5 Symptomatic (n=277) 38.6 52.7 44.0 57.8 32.1 Asymptomatic (n=55) 61.8 65.5 41.8 50.9 58.2 Among asymptomatics with: FH of thrombosis (n=4) 100 100 25 100 100 FH of thrombophilia (n=12) 75 75 41.7 41.7 58.3 FH of thrombosis and thrombophilia (n=11) 72.7 63.6 27.3 36.4 81.8 No FH (n=28) 46.4 57.1 50 53.6 42.9

Description: Key Points Coagulation fVIII binds to a protein complex, including fibrin, on stimulated platelets rather than to membrane PS. Anti-fVIII antibodies inhibit function on platelets differently than on phospholipid vesicles used in clinical assays.

Description: OBJECTIVE: The prevalence of post-thrombosis sequelae (PTS) is increasing in children due to an increase in venous thromboembolic events (VTEs). Pediatric PTS involves extremity and non-extremity manifestations depending on the location of VTEs. Existent PTS assessment scales assess the severity of extremity PTS only. The aim of this study was to develop a clinically significant Expanded PTS Assessment Scale (EPTSAS) which will incorporate both extremity and non-extremity manifestations and provide a framework for interventions. METHOD: All children with objectively confirmed VTEs that were prospectively followed in the Pediatric Coagulation clinic from 2004 to 2006 were included in the study. The clinical consequences that could be directly attributed to intravenous hypertension due to VTEs were carefully recorded and an EPTSAS was developed. This scale included 4 major (varicosities, ulceration, non-extremity manifestations, radiological findings) and 9 minor criteria (pain, swelling, venous collaterals, limb-length and limb-circumference discrepancies, facial swelling, increase in head circumference, activity limitation, tenderness, skin hyperpigmentation). The interpretation was categorized into 3 grades: clinically non-significant, clinically significant and clinically severe (Table). Three independent investigators classified the patients using EPTSAS, one of whom was a pediatric hematologist whose responses were considered gold standard. The clinical diagnosis of PTS was reinforced by an observed response to treatment and decrease in PTS score on follow up visits. RESULTS: Fifty children were eligible for assessment. Median age at diagnosis of PTS: 4.6 years (90 days to 22 years). Median follow up: 1 year (180 days to 10 years). Median interval between diagnosis of VTE and development of PTS:180 days (90 to 3 years). Anatomical locations of VTEs were as follows: lower extremity,23; upper extremity,12; intrathoracic,4; intra-abdominal,3; head and neck,8 (neck:3, sinovenous thrombosis:5); multiple locations,5. Frequently observed symptoms were: collaterals 20/50(40%), swelling 13/50(26%), pain 7/50(14%) and activity limitation 10/50(20%). Six(12%) children developed non-extremity manifestations as a consequence of regional thrombosis: portal hypertension,3; chylothorax,1, communicating hydrocephalus, 1; loss of kidney,1. Twenty-seven (54%) children received a score ≥1 on either a major or minor criteria. Based on EPTSAS aggregate scores, PTS were classified as: clinically non-significant:8/27(30%); clinically significant:6/27(22%), clinically severe:13/27(48%). Three of 6 with non-extremity PTS were missed by existing PTS scales. The positive predictive value, negative predicitve value, sensitivity and specifity of EPTSAS at 1 year after initial assessment was 75%(15/20), 100%(30/30),100%(15/15) and 86%(30/35) respectivley. Inter-observer agreement was good, 78%. Intra-observer agreement was very good, 90%. CONCLUSIONS: This study shows that EPTSAS allows the evaluation of PTS to better cover the full scope of venous thrombotic complications in children. The validity of this EPTSAS will be evaluated prospectively with a larger sample size. Expanded Post-Thrombosis Sequelae Assessment Scale Score Interpretation Management Major 0 + Minor

Description: Background Factor VIII (fVIII) functions as a co-factor for factor IXa on the membranes of stimulated platelets. Binding sites for fVIII(a) are expressed at two levels; thrombin induces 3,000 – 20,000 sites/platelet while the combination of collagen and thrombin or A28137 induce 〉50,000 sites/platelet. Hypothesis We hypothesized that binding sites for fVIII(a) on thrombin-stimulated platelets, are distinct from phosphatidylserine (PS), while those on maximally stimulated platelets are predominantly PS-containing sites. Corollaries were 1) that epitopes on fVIII interact with the non-PS sites and 2) that a macromolecule or a macromolecule complex comprises the binding sites on thrombin-stimulated platelets. Methods Platelets were purified on a density gradient and binding of fluorescein-labeled fVIII (fVIII-fluor) was measured by flow cytometry using a Becton Dickinson LSR-Fortessa flow cytometer. Factor VIII activity was measured in a discontinuous factor Xase assay using extruded phospholipid vesicles of composition PS:PE:PC 4:20:76 or platelets as the membrane source. Oligomeric fibrin was immobilized by incubating thrombin, 1 u/ml, with fibrinogen, 10 µg/ml for 10 min without mixing prior to addition of 59D8-Superose beads. Binding of fVIII-4 Ala to platelets was measured in complex with Alexa-488 labeled mAb GMA-8021, against the A2 domain. Polyphosphate was size-fractionated and recombinant PPX-MBD produced as previously described. Results Lactadherin, a phosphatidyl-L-serine-binding protein, competed for 97% of factor VIII-fluorescein (fVIII-fluor) binding sites on A23187-stimulated platelets but only 30% of binding sites on thrombin-stimulated platelets. Unlabeled fVIII competed with fVIII-fluor for all binding sites. A fVIII C2 domain mutant, with no measurable phospholipid binding - M2199A/F2200A/L2251A/L2252A (fVIII-4Ala) bound to only 3,000 – 5,000 sites on platelets stimulated with A23187 but to a similar number on thrombin-stimulated platelets with a KDof 7 nM. These data indicate that non-PS sites are dominant on thrombin-stimulated platelets but that PS-containing sites comprise at least 95% of sites on A23187-stimulated platelets. We evaluated a panel of mAb’s against the fVIII-C2 domain for platelet-specific inhibition of binding and function. mAb’s ESH4 and I54, with overlapping epitopes, blocked binding of fVIII to thrombin-stimulated platelets but only decreased affinity for PS-containing membranes. In 1-stage and 2-stage commercial aPTT assays ESH4 inhibited 28-33% of fVIII activity. In contrast, ESH4 inhibited 80% of fVIII activity on thrombin-stimulated platelets. mAb’s ESH8 and G99, with partially overlapping epitopes, decreased the affinity of fVIII-fluor for thrombin-stimulated platelets approx. 70% but had no effect on phospholipid binding. ESH8 inhibited 58 ± 8% of fVIII activity on thrombin-stimulated platelets but did not decrease activity supported by phospholipid vesicles. Because oligomeric fibrin is required for expression of most fVIII binding sites on thrombin-stimulated platelets (Phillips et al 2004; JTH 2:1806) we hypothesized that oligomeric, platelet-bound fibrin is a constituent of fVIII binding sites. fVIII-fluor bound to fibrin monomers and oligomers immobilized on mAb 59D8-Superose, detected in solution by flow cytometry. Binding was enhanced by mixing polyphosphate (polyP) with fibrinogen prior to thrombin, with a maximum gain in affinity at 0.1 µM elemental phosphorous. The apparent affinity of fibrin-polyP for fVIII-fluor was 2-12 nM, based on competition studies with unlabeled fVIII. Like binding to platelets, specific binding of fVIII to fibrin-polyP was blocked by mAb’s ESH4, I54 and diminished by ESH8, and G99. Thrombin-stimulated platelets, but not resting platelets, exhibited bound polyP, as detected by PPX-MBP, specific for polyP. Thus, bound polyP is present on thrombin-stimulated platelets under conditions that lead to binding of oligomeric fibrin. Conclusions These data indicate that thrombin-stimulated platelets bind fVIII via a non-PS binding site and that the binding is mediated by epitopes that have greater functional importance on platelets than on phospholipid vesicles. Platelet-bound oligomeric fibrin with polyP is a candidate for the non-PS binding site. These findings have clinical relevance to detection of inhibitory antibodies against fVIII. Disclosures: No relevant conflicts of interest to declare.