Susan Russell

Platelet glycoprotein Ibα supports experimental lung metastasis

The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the α-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy.

Efficient generation of neural stem cell lines from adult bone marrow using serum free defined media.

Here we report the characterization of a mouse model of the Bernard-Soulier syndrome generated by a targeted disruption of the gene encoding the glycoprotein (GP) Ibβ subunit of the GP Ib-IX complex. Similar to a Bernard-Soulier model generated by disruption of the mouse GP Ibα subunit, GP IbβNull mice display macrothrombocytopenia and a severe bleeding phenotype. When examined by transmission electron microscopy, the large platelets produced by a GP IbβNull genotype revealed α-granules with increased size as compared with the α-granules from control mouse platelets. Data are presented linking the overexpression of a septin protein, SEPT5, to the presence of larger α-granules in the GP IbβNull platelet. The SEPT5 gene resides approximately 250 nucleotides 5′ to the GP Ib β gene and has been associated with modulating exocytosis from neurons and platelets as part of a presynaptic protein complex. Fusion mRNA transcripts present in megakaryocytes can contain both the SEPT5 and GP Ibβ coding sequences as a result in an imperfect polyadenylation signal within the 3′ end of both the human and mouse SEPT5 genes. We observed a 2- to 3-fold increase in SEPT5 protein levels in platelets from GP IbβNull mice. These results implicate SEPT5 levels in the maintenance of normal α-granule size and may explain the variant granules associated with human GP Ibβ mutations and the Bernard-Soulier syndrome.

Long-term outcomes in children with high-risk neuroblastoma treated with autologous stem cell transplantation.

We retrospectively analysed the outcomes of children transplanted for high-risk neuroblastoma (NB) at a single institution predominantly transplanted with total body irradiation and chemotherapy. The aims of this study were to determine the prognostic impact of clinical and biological features and to document long-term health outcomes. Forty patients were transplanted with a single unpurged autograft. Fourteen patients died from disease progression and two from late complications of treatment. Twenty-three patients are alive at a median of 4.6 years from diagnosis. Kaplan–Meier estimates of overall survival at 2, 5 and 10 years are 76±7.0, 60.2±8.4 and 54.7±9.3% following transplant. Response to induction therapy was significantly associated with survival (P<0.01). Long-term complications included growth (100%) and pubertal failure (83%), hearing impairment (73%), orthopaedic complications (63%), renal impairment (47%) and thyroid abnormalities (36%). Intrinsic and acquired resistance to chemotherapy remains the major obstacle to improving outcomes in high-risk NB. Although patients with chemo-sensitive disease are less likely to experience a relapse, substantial therapy-related toxicities result in poor long-term health outcomes for survivors.

Transplant-Related Mortality Following Allogeneic Hematopoeitic Stem Cell Transplantation for Pediatric Acute Lymphoblastic Leukemia: 25-Year Retrospective Review

Over the last 25 years, donor source, conditioning, graft‐versus‐host disease prevention and supportive care for children undergoing hematopoeitic stem cell transplantation (HSCT) have changed dramatically. HSCT indications for acute lymphoblastic leukemia (ALL) now include high‐risk patients in first and subsequent remission. There is a large burden of infectious and pre‐HSCT morbidities, due to myelosuppressive therapy required for remission induction. We hypothesized that, despite these trends, overall survival (OS) had increased.

Visualizing the von Willebrand factor/glycoprotein Ib-IX axis with a platelet-type von Willebrand disease mutation

Platelet-type von Willebrand disease (PT-VWD) is a bleeding disorder of the platelet glycoprotein Ib-IX/von Willebrand factor (VWF) axis caused by mutations in the glycoprotein Ib-IX receptor that lead to an increased affinity with VWF. In this report, platelets from a mouse expressing a mutation associated with PT-VWD have been visualized using state-of-the art image collection and processing. Confocal analysis revealed that VWF bound to the surface of single platelets and bridging micro-aggregates of platelets. Surface-bound VWF appears as a large, linear structure on the surface of 50% of the PT-VWD platelets. In vivo thrombus formation after chemical injury to the carotid artery revealed a severe impairment to occlusion as a consequence of the PT-VWD mutation. In vitro stimulation of PT-VWD platelets with adenosine diphosphate or thrombin demonstrates a significant block in their ability to bind fibrinogen. The impairment of in vivo thrombus formation and in vitro fibrinogen binding are more significant than might be expected from the observed platelet binding to VWF polymers over a small portion of the plasma membrane. Visualization of the receptor/ligand interaction and characterization of a severe antithrombotic phenotype provide a new understanding on the molecular basis of bleeding associated with the PT-VWD phenotype.

Allogeneic bone marrow transplant improves outcome for juvenile myelomonocytic leukaemia

Objective: To correlate clinical presentation and therapeutic outcomes in children with a diagnosis of juvenile myelomonocytic leukaemia.

Chronic localized intravascular coagulation complicating multifocal venous malformations

We present two female children aged 7 years with extensive multifocal venous malformations complicated by chronic localized intravascular coagulation. In both cases ultrasonography and magnetic resonance imaging revealed extensive venous malformations involving the skin, mucous membranes and pharynx, which were not apparent on clinical examination. Haematological investigations demonstrated persistent elevation of the D‐dimer, consistent with chronic localized intravascular coagulation. The course of one patient was complicated by the development of multiple painful thromboses at distant sites following percutaneous sclerotherapy. Persistent elevation of the D‐dimer occurring in association with large venous and veno‐lymphatic malformations has been termed chronic localized intravascular coagulation, and is thought to occur due to thrombosis at sites of stagnant blood flow within venous malformations. It is of clinical concern due to the potential for transformation into serious thrombohaemorrhagic coagulation disorders, including disseminated intravascular coagulation. While previously described in association with large segmental venous malformations, these cases demonstrate the occurrence of chronic localized intravascular coagulation as a complication of disseminated multifocal venous malformations.

Localised intravascular coagulation complicating venous malformations in children: Associations and therapeutic options: Localised intravascular coagulation

Venous malformations are slow‐flow congenital vascular malformations that enlarge as the child ages and may be associated with localised intravascular coagulation, a consumptive coagulopathy characterised by elevated D‐dimer and decreased fibrinogen levels. The authors review the known correlations between localised intravascular coagulation and venous malformation number, size and planes involved, and call attention to the concept of the progression of localised intravascular coagulopathy as the child ages and their venous malformations enlarge. The authors also discuss the identified therapeutic options for its investigation, management and treatment, including compression garments, anti‐coagulation therapy, sclerotherapy, endovascular laser, surgical excision and sirolimus (rapamycin). Evidence for protocol improvements that may be instigated for the optimal physical and medical therapy of venous malformations complicated by localised intravascular coagulopathy is reviewed.

The clinical efficacy and safety of the FVIII/VWF concentrate, BIOSTATE®, in children with von Willebrand disorder: a multi-centre retrospective review.

Summary.  Factor replacement with BIOSTATE®, a factor VIII (FVIII)/von Willebrand factor concentrate, forms the mainstay of treatment for children with von Willebrand disorder (VWD) in Australia and New Zealand. However, published data on the clinical efficacy and safety of BIOSTATE in the VWD paediatric population are limited. We retrospectively assessed the efficacy and safety of BIOSTATE in 43 children with VWD who received treatment for surgery, non‐surgical bleeds or continuous prophylaxis at eight paediatric haemophilia centres in Australia and New Zealand. Data were collected on patient demographics, disease history, treatment history, dosage, administration, adverse reactions, concomitant medications and excessive bleeding events. BIOSTATE provided excellent/good haemostatic efficacy in 90% of surgical procedures (n = 42) with a mean daily FVIII dose of 47 IU FVIII:C kg−1 and a median treatment duration of 3 days. Excellent/good haemostatic efficacy was achieved in 94% of non‐surgical bleeding events (n = 72) with a mean FVIII dose of 45 IU FVIII:C kg−1 day−1 and a median treatment duration of 1 day. There were no bleeding events attributable to lack of efficacy. One case of nausea, possibly related to BIOSTATE administration, was reported. These results suggest that BIOSTATE is safe and effective for the treatment and prophylaxis of bleeding in children with VWD.

Goats' milk quackery

An 11-month-old male infant was referred to the emergency department by his local doctor with a 1-day history of fever. The child had increasing pallor for 3 weeks, and had become increasingly lethargic in the week before presentation. As a result of these symptoms, his local doctor had empirically commenced iron supplementation 3 days before presentation. Apart from his fever, there were no other localizing symptoms of infection such as cough, rhinnorhoea, vomiting, diarrhoea, or rash. The patient was the first child of 22-year-old parents who lived in the inner city area of a large Australian city. He was born by normal vaginal delivery at 39 weeks gestation following an uneventful pregnancy. There were no neonatal problems and no significant prior illnesses. He was unimmunized due to parental objection. He had been breast-fed for the first 2 weeks of life and was changed to a commercial formula after his mother developed mastitis. The formula feeds were discontinued after 1 week based on his mother’s belief that he did not like the taste. Following this, he was fed on a homemade formula consisting of barley water, corn syrup and goats’ milk. At time of presentation, he had not been started on solids, apart from the occasional spoonful of rice cereal. His mother’s diet was reported to be normal. Developmental history was appropriate. At 11 months, he was able to pull to stand and was cruising well. He was able to walk a couple of steps on his own, waved good-bye, and had three words in his vocabulary. Physical examination showed a very unwell, pale and lethargic child. His weight was between the 3rd and 10th percentiles, length on the 3rd percentile and head circumference between the 50th and 98th percentile. He was febrile and tachycardic, but well perfused and normotensive. There were no dysmorphic features. He had a systolic flow murmur, but no evidence of cardiac failure. Respiratory, abdominal and ENT examinations were within normal limits. Full blood count showed a macrocytic anaemia with a haemoglobin of 40 g/L and a mean corpuscular volume (MCV) of 99 fL (normal range for age 70–83). He was thrombocytopenic with a platelet count of 42 × 109/L, but had a normal white cell and neutrophil count. Examination of his blood film showed oval macrocytes, hypersegmented neutrophils and circulating megaloblastoid erythroblasts (Fig. 1). Further investigations showed a low folate level of 2.1 nmol/L (normal range: 5.5–33.3) and a borderline low vitamin B12 level of 100 pmol/L (normal range: 97–394). Urine metabolic screen was normal. Iron studies were within normal limits; however; in the context of his acute illness and 3 days of oral iron supplementation, could not exclude an underlying iron deficiency.