PFA-100 System: A New Method for Assessment of Platelet Dysfunction.

This is a celebratory reprint of a historical paper published in STH in 1998. The original Abstract follows.The PFA-100 system is a platelet function analyzer designed to measure platelet-related primary hemostasis. The instrument uses two disposable cartridges: a collagen/epinephrine (CEPI) and a collagen/ADP (CADP) cartridge. Previous experience has shown that CEPI cartridges detect qualitative platelet defects, including acetylsalicylic acid (ASA)-induced abnormalities, while CADP cartridges detect only thrombocytopathies and not ASA use. In this seven-center trial, 206 healthy subjects and 176 persons with various platelet-related defects, including 127 ASA users, were studied. The platelet function status was determined by a platelet function test panel. Comparisons were made as to how well the defects were identified by the PFA-100 system and by platelet aggregometry. The reference intervals for both cartridges, testing the 206 healthy subjects, were similar to values described in smaller studies in the literature (mean closure time [CT] of 132 seconds for CEPI and 93 seconds for CADP). The use of different lot numbers of cartridges or duplicate versus singleton testing revealed no differences. Compared with the platelet function status, the PFA-100 system had a clinical sensitivity of 94.9% and a specificity of 88.8%. For aggregometry, a sensitivity of 94.3% and a specificity of 88.3% were obtained. These values are based on all 382 specimens. A separate analysis of sensitivity by type of platelet defect, ASA use versus congenital thrombocytopathies, revealed for the PFA-100 system a 94.5% sensitivity in identifying ASA users and a 95.9% sensitivity in identifying the other defects. For aggregometry, the values were 100% for ASA users and 79.6% for congenital defects. Analysis of concordance between the PFA-100 system and aggregometry revealed no difference in clinical sensitivity and specificity between the systems (p > 0.9999). The overall agreement was 87.5%, with a Kappa index of 0.751. The two tests are thus equivalent in their ability to identify normal and abnormal platelet defects. Testing 126 subjects who took 325 mg ASA revealed that the PFA-100 system (CEPI) was able to detect 71.7% of ASA-induced defects with a positive predictive value of 97.8%. The overall clinical accuracy of the system, calculated from the area under the receiver operating characteristic curve, was 0.977. The data suggest that the PFA-100 system is highly accurate in discriminating normal from abnormal platelet function. The ease of operation of the instrument makes it a useful tool to use in screening patients for platelet-related hemostasis defects.

The American Society of Pediatric Hematology Oncology workforce, productivity, and fellowship assessment: Current state of the workforce.

The American Society of Pediatric Hematology Oncology conducted follow-up workforce surveys in 2017 and 2021 as well as a Pediatric Hematology Oncology Fellowship Program Directors Survey in 2020 to provide an updated review of the current workforce. We provide a comprehensive review and analysis of these results with the goal to provide better understanding of the current landscape in pediatric hematology oncology.

When Screening for Severe Combined Immunodeficiency (SCID) with T Cell Receptor Excision Circles Is Not SCID: a Case-Based Review.

Newborn screening efforts focusing on the quantification of T cell receptor excision circles (TRECs), as a biomarker for abnormal thymic production of T cells, have allowed for the identification and definitive treatment of severe combined immunodeficiency (SCID) in asymptomatic neonates. With the adoption of TREC quantification in Guthrie cards across the USA and abroad, typical, and atypical SCID constitutes only ~ 10% of cases identified with abnormal TRECs associated with T cell lymphopenia. Several other non-SCID-related conditions may be identified by newborn screening in a term infant. Thus, it is important for physicians to recognize that other factors, such as prematurity, are often associated with low TRECs initially, but often improve with age. This paper focuses on a challenge that immunologists face: the diagnostic evaluation and management of cases in which abnormal TRECs are associated with variants of T cell lymphopenia in the absence of a genetically defined form of typical or atypical SCID. Various syndromes associated with T cell impairment, secondary forms of T cell lymphopenia, and idiopathic T cell lymphopenia are identified using this screening approach. Yet there is no consensus or guidelines to assist in the evaluation and management of these newborns, despite representing 90% of the patients identified, resulting in significant work for the clinical teams until a diagnosis is made. Using a case-based approach, we review pearls relevant to the evaluation of these newborns, as well as the management dilemmas for the families and team related to the resolution of genetic ambiguities.

Rubella Virus-Associated Cutaneous Granulomatous Disease: a Unique Complication in Immune-Deficient Patients, Not Limited to DNA Repair Disorders.

The association of immunodeficiency-related vaccine-derived rubella virus (iVDRV) with cutaneous and visceral granulomatous disease has been reported in patients with primary immunodeficiency disorders (PIDs). The majority of these PID patients with rubella-positive granulomas had DNA repair disorders. To support this line of inquiry, we provide additional descriptive data on seven previously reported patients with Nijmegen breakage syndrome (NBS) (n = 3) and ataxia telangiectasia (AT) (n = 4) as well as eight previously unreported patients with iVDRV-induced cutaneous granulomas and DNA repair disorders including NBS (n = 1), AT (n = 5), DNA ligase 4 deficiency (n = 1), and Artemis deficiency (n = 1). We also provide descriptive data on several previously unreported PID patients with iVDRV-induced cutaneous granulomas including cartilage hair hypoplasia (n = 1), warts, hypogammaglobulinemia, immunodeficiency, myelokathexis (WHIM) syndrome (n = 1), MHC class II deficiency (n = 1), Coronin-1A deficiency (n = 1), X-linked severe combined immunodeficiency (X-SCID) (n = 1), and combined immunodeficiency without a molecular diagnosis (n = 1). At the time of this report, the median age of the patients with skin granulomas and DNA repair disorders was 9 years (range 3-18). Cutaneous granulomas have been documented in all, while visceral granulomas were observed in six cases (40%). All patients had received rubella virus vaccine. The median duration of time elapsed from vaccination to the development of cutaneous granulomas was 48 months (range 2-152). Hematopoietic cell transplantation was reported to result in scarring resolution of cutaneous granulomas in two patients with NBS, one patient with AT, one patient with Artemis deficiency, one patient with DNA Ligase 4 deficiency, one patient with MHC class II deficiency, and one patient with combined immunodeficiency without a known molecular etiology. Of the previously reported and unreported cases, the majority share the diagnosis of a DNA repair disorder. Analysis of additional patients with this complication may clarify determinants of rubella pathogenesis, identify specific immune defects resulting in chronic infection, and may lead to defect-specific therapies.

Origins and organization of the NHLBI State of the Science Workshop: Generating a national blueprint for future research on factor VIII inhibitors.

INTRODUCTION: The major complication of protein replacement therapy for haemophilia A is the development of anti-FVIII antibodies or inhibitors that occur in 25%-30% of persons with severe haemophilia A. Alternative therapeutics such as bypassing agents or immune tolerance induction protocols have additional challenges and are not always effective. AIM: Assemble a National Heart, Lung and Blood Institute (NHLBI) State of the Science (SOS) Workshop to generate a national blueprint for research on inhibitors to solve the problem of FVIII immunogenicity. METHODS: An Executive Steering Committee was formed in October 2017 to establish the scientific focus and Scientific Working Groups for the SOS Workshop in May 2018. Four working groups were assembled to address scientific priorities in basic, translational and clinical research on inhibitors. RESULTS: Working Group 1 was charged with determining the scientific priorities for clinical trials to include the integration of non-intravenous, non-factor therapeutics including gene therapy into the standard of care for people with haemophilia A with inhibitors. Working Group 2 established the scientific priorities for 21st-century data science and biospecimen collection for observational inhibitor cohort studies. The scientific priorities for acquiring an actionable understanding of FVIII immunogenicity and the immunology of the host response and FVIII tolerance were developed by Working Group 3. Working Group 4 designed prospective pregnancy/birth cohorts to study FVIII immunogenicity, inhibitor development and eradication. CONCLUSION: The NHLBI SOS Workshop generated a focused summary of scientific priorities and implementation strategies to overcome the challenges of eradicating and preventing inhibitors in haemophilia A.

Parent proxy assessment of sibling quality of life following pediatric hematopoietic cell transplantation.

BACKGROUND: When a child undergoes hematopoietic cell transplantation (HCT), the impact extends to the entire family, including siblings. Assessment of the quality of life (QoL) of siblings is challenged by their general lack of availability for regular assessment by clinical providers. Thus, the use of parent proxy reporting may be useful. Our aim was to describe the QoL of siblings of HCT survivors, as reported by their parents, as well as to identify parent and family factors associated with lower sibling QoL. METHODS: A cross-sectional study was utilized to assess parent-reported QoL of the HCT recipient's sibling (Short Form (SF)-10 Health Survey for Children and the Pediatric Symptom Checklist (PSC)-17). Parent QoL was assessed using the SF-12. Multivariable linear regression was used to explore hypothesized predictors of sibling QoL, including parent QoL, family impact/function (Impact on Family Scale, Family Adaptability and Cohesion Evaluation Scales, IV, and a question asking about financial problems) while adjusting for demographic and HCT characteristics. RESULTS: Ninety-seven siblings (55% males) with a mean age of 12 years (standard deviation [SD] 4 years) were assessed, representing HCT survivors, who were an average of 5 years (SD 4 years) post-HCT. Neither sibling psychosocial (mean 49.84, SD 10.70, p = 0.87) nor physical health scores (mean 51.54, SD 8.42, p = 0.08) differed from norms. Parent proxies reported behavioral/emotional problems (PSC-17 total score > 15) in 24% of siblings. While parental ratings of their own physical health (SF-12 were higher than norms (mean 53.04, SD 8.17, p = 0.0005), mental health scores were lower (mean 45.48, SD 10.45, p < 0.0001). In multivariable analysis, lower parent emotional functioning and adverse family function were associated with lower sibling QoL, as reported by parents. CONCLUSIONS: While proxy-reported QoL of siblings did not differ significantly from normative data, both parent QoL and family function were associated with sibling QoL. Future research is needed to understand how siblings themselves perceive their QoL following HCT.

Use of Factor XIII (FXIII) concentrate in patients with congenital FXIII deficiency undergoing surgical procedures.

BACKGROUND: Patients with congenital Factor XIII (FXIII) deficiency have impaired fibrin stabilization and are at high risk for surgical bleeding. Data regarding the use of FXIII concentrates before and during surgery are lacking. The objective of this study was to report the use of plasma-derived FXIII concentrate (Corifact in the United States; Fibrogammin P in other countries) in patients with congenital FXIII deficiency undergoing surgical procedures. STUDY DESIGN AND METHODS: FXIII concentrate at preoperative doses ranging from 25 to 40 U/kg was administered to six patients with congenital FXIII deficiency undergoing major or minor surgeries. RESULTS: FXIII concentrate was administered immediately before surgery for five surgical cases; three of these patients achieved excellent hemostasis during and after surgery, while two had intraoperative bleeding. In one surgical case, a regular prophylactic dose of FXIII concentrate was administered to the patient 1 week before minor surgery. FXIII concentrate provided rapid replacement of FXIII activity. In all but one of the patients given a dose of FXIII designed to increase FXIII levels more than 50%, there was satisfactory intraoperative and postoperative hemostasis. One patient undergoing aortic valve replacement on cardiopulmonary bypass (CPB) was the exception. Intraoperative bleeding in this patient was associated with lower-than-expected blood levels of FXIII. CONCLUSION: Preoperative plasma-derived FXIII concentrate allowed for sufficient hemostasis in most patients with FXIII deficiencies. Additional doses were necessary to achieve hemostasis in one patient who underwent a CPB procedure.

Effects of hematopoietic stem cell transplantation on acyl-CoA oxidase deficiency: a sibling comparison study.

OBJECTIVE: Acyl-CoA oxidase (ACOX1) deficiency is a rare disorder of peroxisomal very-long chain fatty acid oxidation. No reports detailing attempted treatment, longitudinal imaging, or neuropathology exist. We describe the natural history of clinical symptoms and brain imaging in two siblings with ACOX1 deficiency, including the younger sibling's response to allogeneic unrelated donor hematopoietic stem cell transplantation (HSCT). METHODS: We conducted retrospective chart review to obtain clinical history, neuro-imaging, and neuropathology data. ACOX1 genotyping were performed to confirm the disease. In vitro fibroblast and neural stem cell fatty acid oxidation assays were also performed. RESULTS: Both patients experienced a fatal neurodegenerative course, with late-stage cerebellar and cerebral gray matter atrophy. Serial brain magnetic resonance imaging in the younger sibling indicated demyelination began in the medulla and progressed rostrally to include the white matter of the cerebellum, pons, midbrain, and eventually subcortical white matter. The successfully engrafted younger sibling had less brain inflammation, cortical atrophy, and neuronal loss on neuro-imaging and neuropathology compared to the untreated older sister. Fibroblasts and stem cells demonstrated deficient very long chain fatty acid oxidation. INTERPRETATION: Although HSCT did not halt the course of ACOX1 deficiency, it reduced the extent of white matter inflammation in the brain. Demyelination continued because of ongoing neuronal loss, which may be due to inability of transplant to prevent progression of gray matter disease, adverse effects of chronic corticosteroid use to control graft-versus-host disease, or intervention occurring beyond a critical point for therapeutic efficacy.

Tissue factor inflammatory response regulated by promoter genotype and p38 MAPK in neonatal vs. adult microvascular endothelial cells.

OBJECTIVE AND DESIGN: Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18-basepair deletion (D) or insertion (I) at -1,208. We sought to determine the relationship between these haplotypes and interleukin-1α (IL-1α)-induced TF expression in neonatal versus adult HMVEC. RESULTS: IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed threefold higher levels of TF mRNA, twofold higher TF protein, and fourfold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates, but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC. CONCLUSIONS: Upregulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants.

Successful treatment of post-transplant thrombocytopenia with romiplostim in a pediatric patient with X-linked chronic granulomatous disease.

Thrombocytopenia is a frequent complication following HSCT in pediatric patients. Romiplostim is a TPO receptor agonist that has been utilized successfully in the treatment of pediatric patients with immune thrombocytopenia. We describe a three-yr-old male with X-linked CGD treated with an unrelated donor bone marrow transplant. His course was complicated by the development of symptomatic thrombocytopenia. He was started on romiplostim with prompt improvement in his thrombocytopenia. We found the use of romiplostim to be an effective and safe alternative to the potential complications as well as morbidity and mortality associated with the use of immunosuppressive agents such as corticosteroids.

A randomized, double-blind study of romiplostim to determine its safety and efficacy in children with immune thrombocytopenia.

Romiplostim, a thrombopoietin-mimetic peptibody, increases and maintains platelet counts in adults with immune thrombocytopenia (ITP). In this first study of a thrombopoietic agent in children, patients with ITP of ≥ 6 months' duration were stratified by age 1:2:2 (12 months-< 3 years; 3-< 12 years; 12-< 18 years). Children received subcutaneous injections of romiplostim (n = 17) or placebo (n = 5) weekly for 12 weeks, with dose adjustments to maintain platelet counts between 50 × 10(9)/L and 250 × 10(9)/L. A platelet count ≥ 50 × 10(9)/L for 2 consecutive weeks was achieved by 15/17 (88%) patients in the romiplostim group and no patients in the placebo group (P = .0008). Platelet counts ≥ 50 × 10(9)/L were maintained for a median of 7 (range, 0-11) weeks in romiplostim patients and 0 (0-0) weeks in placebo patients (P = .0019). The median weekly dose of romiplostim at 12 weeks was 5 µg/kg. Fourteen responders received romiplostim for 4 additional weeks for assessment of pharmacokinetics. No patients discontinued the study. There were no treatment-related, serious adverse events. The most commonly reported adverse events in children, as in adults, were headache and epistaxis. In this short-term study, romiplostim increased platelet counts in 88% of children with ITP and was well-tolerated and apparently safe. The trial was registered with http://www.clinicaltrials.gov as NCT00515203.

Mean platelet volume and integrin alleles correlate with levels of integrins α(IIb)ß(3) and α(2)ß(1) in acute coronary syndrome patients and normal subjects.

OBJECTIVE: The interindividual variation in platelet α(2)ß(1) exceeds a 2-fold variance in platelet α(IIb)ß(3) level. Our objective was to parse the contribution of mean platelet volume (MPV) and integrin gene alleles to this variation in large cohorts of patients with acute coronary syndrome (ACS) and normal subjects. METHODS AND RESULTS: Platelet α(IIb)ß(3) and α(2)ß(1) levels were measured by flow cytometry in whole blood from 320 ACS patients and 128 normal subjects and compared with MPV, platelet count, ITGA2 rs1126643, and ITGB3 rs5918 alleles. In all subjects, a strong direct correlation was found between MPV and α(IIb)ß(3) level (P<0.001). Neither MPV nor α(IIb)ß(3) level correlated with ITGB3 rs5918 alleles. In the case of α(2)ß(1) level, MPV contributed modestly, whereas ITGA2 rs1126643 exerted a greater effect. An inverse correlation was found between MPV and the rs1126643 minor allele. CONCLUSIONS: MPV is the major effector of platelet α(IIb)ß(3) level, whereas the ITGA2 rs1126643 alleles influence α(2)ß(1) level more than MPV does. The rs1126643 minor allele, associated with lower MPV, likely exerts this effect via the influence of α(2)ß(1) on megakaryocyte maturation. Because of the hyperactivity of larger platelets, MPV is an accurate metric of risk for adverse outcome in ACS.

Genetic variants that affect platelet function.

PURPOSE OF REVIEW: This review summarizes our current knowledge of common gene variants (polymorphisms) that have small individual effects on platelet function in humans, but can cumulatively lead to hyperreactive platelets and increase risk for negative outcomes in thrombotic disorders. RECENT FINDINGS: Candidate gene association and genome-wide association studies (GWAS) have identified loci that include single nucleotide polymorphisms, which exert a cumulative effect on platelet function by modifying basic platelet parameters, such as mean platelet volume (MPV) or platelet count, by altering the expression or activity of key platelet receptors, or by influencing downstream effector pathways utilized by these receptors. SUMMARY: Variation in MPV between normal individuals is responsible for roughly a two-fold range in platelet protein content, including key surface receptors and reactive granule constituents, the association of ADRA2, GP1BA, GP6, ITGA2 and P2Y12 variants with platelet reactivity, initially identified by candidate gene analyses, has now been validated by genome-wide approaches in much larger individual cohorts, and GWAS have identified novel gene variants, most notably PEAR1, that participate in variation in platelet reactivity among normal individuals, all of which contribute to a genetic basis for differences in platelet reactivty among normal individuals.

Late effects in hematopoietic cell transplant recipients with acquired severe aplastic anemia: a report from the late effects working committee of the center for international blood and marrow transplant research.

With improvements in hematopoietic cell transplant (HCT) outcomes for severe aplastic anemia (SAA), there is a growing population of SAA survivors after HCT. However, there is a paucity of information regarding late effects that occur after HCT in SAA survivors. This study describes the malignant and nonmalignant late effects in survivors with SAA after HCT. A descriptive analysis was conducted of 1718 patients post-HCT for acquired SAA between 1995 and 2006 reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). The prevalence and cumulative incidence estimates of late effects are reported for 1-year HCT survivors with SAA. Of the HCT recipients, 1176 (68.5%) and 542 (31.5%) patients underwent a matched sibling donor (MSD) or unrelated donor (URD) HCT, respectively. The median age at the time of HCT was 20 years. The median interval from diagnosis to transplantation was 3 months for MSD HCT and 14 months for URD HCT. The median follow-up was 70 months and 67 months for MSD and URD HCT survivors, respectively. Overall survival at 1 year, 2 years, and 5 years for the entire cohort was 76% (95% confidence interval [CI]: 74-78), 73% (95% CI: 71-75), and 70% (95% CI: 68-72). Among 1-year survivors of MSD HCT, 6% had 1 late effect and 1% had multiple late effects. For 1-year survivors of URD HCT, 13% had 1 late effect and 2% had multiple late effects. Among survivors of MSD HCT, the cumulative incidence estimates of developing late effects were all <3% and did not increase over time. In contrast, for recipients of URD HCT, the cumulative incidence of developing several late effects exceeded 3% by 5 years: gonadal dysfunction 10.5% (95% CI: 7.3-14.3), growth disturbance 7.2% (95% CI: 4.4-10.7), avascular necrosis 6.3% (95% CI: 3.6-9.7), hypothyroidism 5.5% (95% CI: 2.8-9.0), and cataracts 5.1% (95% CI: 2.9-8.0). Our results indicated that all patients undergoing HCT for SAA remain at risk for late effects, must be counseled about, and should be monitored for late effects for the remainder of their lives.

The genetics of normal platelet reactivity.

Genetic and environmental factors contribute to a substantial variation in platelet function seen among normal persons. Candidate gene association studies represent a valiant effort to define the genetic component in an era where genetic tools were limited, but the single nucleotide polymorphisms identified in those studies need to be validated by more objective, comprehensive approaches, such as genome-wide association studies (GWASs) of quantitative functional traits in much larger cohorts of more carefully selected normal subjects. During the past year, platelet count and mean platelet volume, which indirectly affect platelet function, were the subjects of GWAS. The majority of the GWAS signals were located to noncoding regions, a consistent outcome of all GWAS to date, suggesting a major role for mechanisms that alter phenotype at the level of transcription or posttranscriptional modifications. Of 15 quantitative trait loci associated with mean platelet volume and platelet count, one located at 12q24 is also a risk locus for coronary artery disease. In most cases, the effect sizes of individual quantitative trait loci are admittedly small, but the results of these studies have led to new insight into regulators of hematopoiesis and megakaryopoiesis that would otherwise be unapparent and difficult to define.

Platelet adhesion to decorin but not collagen I correlates with the integrin α2 dimorphism E534K, the basis of the human platelet alloantigen (HPA)-5 system.

A single nucleotide polymorphism in the integrin α2 gene ITGA2 (rs1801106; G1600A) creates the non-conservative amino acid substitution E534K, the basis of the human platelet alloantigen system HPA-5. Yet HPA-5 alleles do not influence binding of α2ß1 to its primary ligand collagen I, and the effect of HPA-5 on platelet function has not been determined. We used a direct platelet adhesion assay to evaluate whether differential inheritance of HPA-5 alleles influences platelet adhesion to collagen I or an alternative ligand, decorin. Platelets from donors bearing one or more minor allele HPA-5b showed attenuated adhesion to purified decorin but not collagen I. Adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by the collagen I sequence GFOGER or α2-specific inhibitory monoclonal antibodies. The minor allele 534K attenuates platelet adhesion to decorin but not collagen I, providing the first evidence of a functional effect of HPA-5 alleles.

Response to steroids predicts response to rituximab in pediatric chronic immune thrombocytopenia.

BACKGROUND: Treatment choice in pediatric immune thrombocytopenia (ITP) is arbitrary, because few studies are powered to identify predictors of therapy response. Increasingly, rituximab is becoming a treatment of choice in those refractory to other therapies. METHODS: The objective of this study was to evaluate univariate and multivariable predictors of platelet count response to rituximab. After local IRB approval, 565 patients with chronic ITP enrolled and met criteria for this study in the longitudinal, North American Chronic ITP Registry (NACIR) between January 2004 and October 2010. Treatment response was defined as a post-treatment platelet count ≥ 50,000/µl within 16 weeks of rituximab and 14 days of steroids. Treatment response data were captured both retrospectively at enrollment and then prospectively. RESULTS: Eighty (14.2%) patients were treated with rituximab with an overall response rate of 63.8% (51/80). Univariate correlates of response to rituximab included the presence of secondary ITP and a positive response to steroids. In multivariable analysis, response to steroids remained a strong correlate of response to rituximab, OR 6.8 (95% CI 2.0-23.0, P = 0.002). Secondary ITP also remained a strong predictor of response to rituximab, OR 5.6 (95% CI 1.1-28.6, P = 0.04). Although 87.5% of patients who responded to steroids responded to rituximab, 48% with a negative response to steroids did respond to rituximab. CONCLUSION: In the NACIR, response to steroids and presence of secondary ITP were strong correlates of response to rituximab, a finding not previously reported in children or adults.

Unaffected Li-Fraumeni Syndrome Carrier Parent Demonstrates Allele-Specific mRNA Stabilization of Wild-Type TP53 Compared to Affected Offspring.

Li-Fraumeni Syndrome (LFS) is an autosomal dominant disorder where an oncogenic TP53 germline mutation is inherited by offspring of a carrier parent. p53 is a key tumor suppressor regulating cell cycle arrest in response to DNA damage. Unexpectedly, some mutant TP53 carriers remain unaffected, while their children develop cancer early in life. To begin unravelling this paradox, the response of dermal fibroblasts (dFb) isolated from a child with LFS was compared to those from her unaffected father after UV exposure. Phospho-Chk1[S345], a key activator of cell cycle arrest, was increased by UV induction in the LFS patient compared to their unaffected parent dFb. This result, along with previous findings of reduced CDKN1A/p21 UV induction in affected dFb, suggest that cell cycle dysregulation may contribute to cancer onset in the affected LFS subject but not the unaffected parent. Mutant p53 protein and its promoter binding affinity were also higher in dFb from the LFS patient compared to their unaffected parent. These results were as predicted based on decreased mutant TP53 allele-specific mRNA expression previously found in unaffected dFb. Investigation of the potential mechanism regulating this TP53 allele-specific expression found that, while epigenetic promoter methylation was not detectable, TP53 wild-type mRNA was specifically stabilized in the unaffected dFb. Hence, the allele-specific stabilization of wild-type TP53 mRNA may allow an unaffected parent to counteract genotoxic stress by means more characteristic of homozygous wild-type TP53 individuals than their affected offspring, providing protection from the oncogenesis associated with LFS.

The low-frequency isoform of platelet glycoprotein VIb attenuates ligand-mediated signal transduction but not receptor expression or ligand binding.

The 2 most common haplotypes of human GP6, GP6a and GP6b, generate the allelic isoforms glycoprotein VI (GPVI)a and GPVIb that differ by 5 amino acids: S219P, K237E, and T249A in the ectodomains, and Q317L and H322N in the cytoplasmic domain. By quantitative Western blot, we found no association between GP6 genotype and total platelet GPVI content among 132 normal subjects. When expressed as soluble products or as membrane-associated receptors, GPVIa and GPVIb have identical affinities for type I collagen, collagen-related peptide, or convulxin. However, the cytoplasmic domain substitutions in GPVIb have a significant effect on GPVI-dependent subcellular associations and ligand-induced signal transduction. L317 increases binding to calmodulin, whereas N322 attenuates binding to Fyn/Lyn. Consistent with the latter finding, convulxin-induced Syk phosphorylation is significantly attenuated in Dami cells stably transfected with GPVIb, relative to GPVIa. This represents direct evidence that haplotype-related GPVI functional differences are inherent in the cytoplasmic domain substitutions, whereby GPVIb binds less strongly to Fyn/Lyn and attenuates the rate and extent of Syk phosphorylation. These allelic differences in GP6a and GP6b explain functional differences in the respective isoforms, but the molecular basis for the several-fold range in GPVI levels of human platelets remains to be determined.