Experience with central venous access devices (CVADs) in the Canadian hemophilia primary prophylaxis study (CHPS).

INTRODUCTION: Haemophilia A treatment with factor VIII concentrates requires frequent venipunctures; a central venous access device (CVAD) may be required to facilitate reliable venous access, especially in young children. While CVADs provide reliable venous access, complications such as infection and thrombosis may occur. AIM: The aim of this study was to assess CVAD use in the Canadian Hemophilia Primary Prophylaxis Study (CHPS), a single-arm, multi-centre prospective study whereby factor use is tailored to individual prophylactic need. METHODS: Participants received a tailored, escalating dose, prophylaxis regimen of increasing frequency of FVIII infusions: step-1: 50 IU kg(-1) once weekly; step-2: 30 IU kg(-1) twice weekly; and step-3: 25 IU kg(-1) on alternate days, according to their level of bleeding. CVAD insertion was at the discretion of the local health care team. Details regarding CVAD use during this protocol were analysed. RESULTS: Fifty six boys were enrolled, 21 required 25 CVADs due to difficult venous access. CVADs were inserted at a median age of 1.3 years (range: 0.6-2.1) and were removed at a median age of 8.7 years (range 6.3-11.8). Six participants experienced non-life threatening CVAD-complications, the most frequent being device malfunction requiring CVAD replacement (n = 4). Two boys were shown to have CVAD-associated thrombosis detected on routine imaging; one required removal due to infusion difficulties and the other was asymptomatic and did not require device removal. No CVAD-related infections were documented. CONCLUSION: Our study shows that the CHPS tailored prophylaxis regimen is associated with a decreased requirement for CVADs and with few device-related complications.

von Willebrand disease and platelet disorders.

The diagnosis and management of bleeding disorders is made difficult by the complexity and variety of disorders, clinical symptoms and bleeding type and severity. von Willebrand disease (VWD) and platelet disorders are disorders of primary haemostasis and together represent the most common inherited bleeding disorders. In this article, we describe the diagnosis of VWD and platelet disorders and the treatment options for VWD.

Refinement of the hereditary xerocytosis locus on chromosome 16q in a large Canadian kindred.

The hereditary stomatocytoses are a group of heterogeneous conditions associated with chronic red cell hemolysis for which the causative genetic mutations are not known. We investigated 137 members of a large Canadian kindred with phenotypic findings consistent with hereditary xerocytosis, one of the most common stomatocytosis syndromes. The objectives of this study were to characterize the clinical hallmarks of the hemolytic process, and to define the chromosomal region carrying the disease locus. The mode of inheritance was autosomal dominant. Affected family members had a well-compensated hemolysis, associated with an elevated MCHC, decreased osmotic fragility, decreased haptoglobin, and indirect hyperbilirubinemia. Cholelithiasis and progressive iron loading were common, despite normal hemoglobin levels. Quantitative erythrocyte morphologic evaluation revealed increased schistocytes, target cells, reticulocytes, and eccentrocytes in affected individuals; stomatocytes were not increased. Genetic linkage analysis confirmed the localization of the disease phenotype to chromosome 16q, and refined the candidate region to 16q24.2-16qter, a 2.4 million base pair interval containing 51 known or predicted genes.

Inherited disorders of platelet function and challenges to diagnosis of mucocutaneous bleeding.

SUMMARY: Platelets play a pivotal role in the arrest of bleeding at sites of vascular injury. Following endothelial damage, they respond rapidly by adhesion to subendothelial matrix proteins resulting in platelet activation, spreading, aggregation, secretion and recruitment of additional platelets to form the primary haemostatic plug. This mass provides a surface for thrombin generation and fibrin mesh formation that stabilizes the clot. Careful study of patients with inherited platelet disorders and, subsequently, of informative animal models, has identified structural platelet abnormalities that have enhanced our understanding of platelet function. The investigations of rare, but severe, inherited platelet disorders have led us to the discovery of causative molecular defects. One of the most informative is the rare autosomal recessive disorder Glanzmann thrombasthenia, caused by defect or deficiency in the platelet integrin alphaIIbbeta3, resulting in absent platelet aggregation and a significant clinical bleeding diathesis. Our new challenge is to understand the mechanisms underlying more common, but less well-defined, mucocutaneous bleeding (MCB) disorders. Present diagnostic testing for platelet function disorders and von Willebrand's Disease often fails to identify the cause of bleeding in individuals with inherited MCB.

Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells.

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H(2)O(2)) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H(2)O(2) or 2-ME-induced cell death. H(2)O(2) and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H(2)O(2) or 2-ME through overexpression of manganese-superoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H(2)O(2)- or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H(2)O(2) or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in non-transformed cells.

Differential effects of spermine on aggregation, inositol phosphate formation and protein phosphorylation in human platelets in response to thrombin, arachidonic acid and lysophosphatidic acid.

Platelet aggregation stimulated by thrombin, arachidonic acid or lysophosphatidic acid is associated with rapid phosphorylation of two platelet proteins, myosin light chain and a 47 kDa protein. The polyamine, spermine, inhibited platelet aggregation stimulated by all three agents. Spermine inhibited thrombin-stimulated phosphorylation of myosin light chain and the 47 kDa proteins as well as thrombin-induced production of the inositol phosphates and phosphatidic acid. In contrast, spermine did not inhibit phosphorylation of either protein or the formation of inositol phosphates and phosphatidic acid in response to arachidonic acid or lysophosphatidic acid. Although spermine has been demonstrated to inhibit both phosphatidylinositol-specific phospholipase C and calcium-dependent protein kinases in cell free systems, these results suggest that, in the intact platelet, spermine does not directly inhibit these enzymes. Inhibition of aggregation stimulated by arachidonic acid and lysophosphatidic acid is secondary to interference with platelet-platelet interaction but not with platelet activation. In contrast, spermine inhibits thrombin-induced platelet activation. This thrombin-specific inhibition may be related to interference with the binding of thrombin to its receptor or to its catalytic substrate on the cell surface.

Laboratory testing for platelet function disorders.

Platelet function testing is both complex and labor intensive. A stepwise approach to the evaluation of patients with suspected platelet disorders will optimize the use of laboratory resources, beginning with an appropriate clinical evaluation to determine whether the bleeding is consistent with a defect of primary hemostasis. Bleeding assessment tools, evaluation of platelet counts, and review of peripheral blood cell morphology can aid the initial assessment. For patients requiring further laboratory testing, platelet aggregometry, secretion assays, and von Willebrand factor assays are the most useful next steps and will direct further specialized testing including flow cytometry, electron microscopy, and molecular diagnostics. Guidelines and recommendations for standardizing platelet function testing, with a particular focus on light transmission aggregometry, are available and can provide a template for clinical laboratories in establishing procedures that will optimize diagnosis and assure quality results. This review outlines an approach to platelet function testing and reviews testing methods available to clinical laboratories.

The lysosomal granule membrane protein, LAMP-2, is also present in platelet dense granule membranes.

Lysosomal Associated Membrane Protein-2 (LAMP-2) is an inherent component of lysosomal granule membranes in diverse cell types, including platelets. We examined platelets for evidence of LAMP-2 in dense granule membranes as CD63 has previously been shown to be present in both lysosomal and dense granule membranes. Immunological techniques were used to examine the localization of LAMP-2 in control platelets and those from an individual with Hermansky-Pudlak syndrome (HPS), a condition characterised by platelet dense granule deficiency. Immunoblotting studies demonstrated that LAMP-2 was enriched in a dense granule preparation. Flow cytometry of thrombin-stimulated control platelets was consistent with biphasic surface expression of LAMP-2. The early expression was accompanied by dense granule, but minimal lysosomal granule, release. The late expression was accompanied by additional lysosomal granule release only. Thrombin stimulation of HPS platelets showed only late, lysosome-associated LAMP-2 expression. Immunoelectron microscopy indicated the presence of LAMP-2 in the membranes of serotonin-containing granules as identified by an anti-serotonin polyclonal antibody. These data indicate that LAMP-2 is present in the membranes of platelet dense granules in addition to lysosomal granules, and has a similar distribution to CD63.

CD63 associates with the alphaIIb beta3 integrin-CD9 complex on the surface of activated platelets.

The tetraspanins are integral membrane proteins expressed on cell surface and granular membranes of hematopoietic cells and have been identified in multi-molecular complexes with specific integrins. In resting platelets, CD63, a member of the tetraspanin superfamily, is present in dense granule and lysosomal membranes and, following platelet activation, translocates to the plasma membrane. In the present study, platelet activation by thrombin leads to incorporation of CD63 into the Triton-insoluble actin cytoskeletal fraction. This incorporation was inhibited by preincubation of platelets with RGDS or EGTA and did not occur in platelets from a patient with Glanzmann's thrombasthenia, suggesting that it was dependent upon alphaIIbbeta3. In activated platelets, the anti-CD63 MoAb, D545, co-immunoprecipitated CD63 with other surface-labeled proteins, including alphaIIbbeta3 and another tetraspanin, CD9. The association of CD63 with CD9 and alphaIIbbeta3, was not inhibited by preincubation of platelets with RGDS or EGTA. D545 did not inhibit the adhesion of activated platelets to purified extracellular matrix proteins, but significantly decreased adhesion of thrombin-activated platelets to neutrophils in a rosetting assay. D545 also caused disaggregation of platelets stimulated by ADP, but had no effect on aggregation induced by other agonists. These results are consistent with the proposal that CD63 becomes part of an alphaIIbbeta3-CD9-CD63 integrin-tetraspanin complex in activated platelets--an association that may modulate the function of alphaIIbbeta3-dependent interaction with other cells such as neutrophils.

Effects of the mitogen-activated protein (MAP) kinase kinase inhibitor 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059) on human platelet activation.

The role of mitogen-activated protein (MAP) kinase cascades in platelet function remains to be determined. Several studies have suggested a role in the activation of phospholipase A2; however, other functions seem likely. The object of the present study was to determine the role of the MAP kinase cascade in platelet function. An inhibitor of the mitogen-activated protein kinase kinase MEK1, 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059), was used, at concentrations consistent with those reported to inhibit MEK1, to examine the role that this enzyme plays in platelet function. PD98059 inhibited aggregation in response to low-dose collagen and arachidonic acid, but not that in response to high-dose collagen, thrombin, thrombin receptor-activating peptide (TRAP), 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F2alpha (U46619), or phorbol ester. Thrombin, thrombin receptor-activating peptide, U46619, collagen, and arachidonic acid each caused the release of [3H]serotonin from dense granules, but only that elicited by low-dose collagen and arachidonic acid was inhibited by PD98059. The release of [3H]arachidonic acid in response to thrombin or collagen was unaffected by PD98059 pretreatment. In contrast, collagen- and arachidonic acid-induced thromboxane formation was inhibited by PD98059. These data suggest that MEK1 is not involved in the platelet response to thrombin or U46619. Furthermore, the inhibitory effects of PD98059 on collagen- and arachidonic acid-induced responses suggest that PD98059 may inhibit the conversion of arachidonic acid to thromboxane, in addition to its reported effects on MEK1.

Role of transforming growth factor-beta in chronic lymphocytic leukemia.

TGF-beta is an important immunoregulator as it suppresses proliferation and function of B- and T-lymphocytes. In the present study we have examined the cellular localization and secretion of TGF-beta in B-cells from normal donors and patients with CLL and have assessed the influence of TGF-beta 1 on DNA synthesis in these cells. Using anti-LC(1-30)--a polyclonal anti-TGF-beta 1 antibody--TGF-beta was localized to discrete sites within the cytoplasm of both normal and malignant lymphocytes. These areas co-localized with areas detected by an antigranule antibody (D545), suggesting that TGF-beta may be stored within cytoplasmic secretory vesicles. Both normal B- and CLL cells contained low or undetectable levels of TGF-beta mRNA and secreted low and equivalent amounts of TGF-beta. Compared to untreated cells, DNA synthesis was reduced by TGF-beta 1 to a mean +/- S. E. of 0.84 +/- 0.07 in CLL cells and this was significantly less (p < 0.001) than that observed in normal B-cells (mean +/- S. E. of control, 0.12 +/- 0.02). In 3 of the 18 patients, TGF-beta 1 stimulated DNA synthesis. The reduced inhibition of leukemic cell DNA synthesis by TGF-beta 1 in CLL may provide these cells with a growth or survival advantage over normal lymphocytes and contribute to their selective accumulation.

Deposition of transforming growth factor-beta in the marrow in myelofibrosis, and the intracellular localization and secretion of TGF-beta by leukemic cells.

The marrows of 10 patients with hematologic malignancies were examined by immunohistochemistry using anti TGF-beta antibody, CC(1-30), which detects secreted TGF-beta, and compared with four normal marrows. TGF-beta was not demonstrated in marrows with a normal level of reticulin fibrosis; however, TGF-beta was observed within collagen in marrows having collagen fibrosis or increased reticulin fibrosis. The extent of TGF-beta deposition paralleled the severity of fibrosis (P < .0001), and occurred even with normal or reduced numbers of megakaryocytes. Using another TGF-beta antibody, LC(1-30), which detects intracellular TGF-beta, TGF-beta was detected by immunofluorescence in discrete sites in the cytoplasm of immature and mature myeloid and large granular lymphocytic leukemia cells. These sites colocalized with areas detected by an anti-granule antibody (D545) suggesting that TGF-beta was stored in granules. However, neither the TGF-beta mRNA content nor the degree of TGF-beta secretion by these leukemic cells correlated with the extent of TGF-beta deposition in the marrow. Thus, TGF-beta deposition in marrow may contribute to myelofibrosis, but the source of this cytokine in the absence of megakaryocytes requires further study.

Platelet dense granules: structure, function and implications for haemostasis.

The advances that have been made over the last decade in microscopic, biochemical, molecular, and genetic techniques have led to substantial improvement in our understanding of platelet dense granule structure and function, and the implications of dense granule deficiencies for haemostasis. However, much has still to be learned. For example, what is the specific mechanism of docking and fusion that occurs during dense granule exocytosis? What are the roles of dense granule membrane proteins during exocytosis or after expression on the surface of activated platelets? Finally, how do the genetic defects identified in HPS and CHS result in the clinical phenotype of these diseases, and what does this tell us about the origin and function of the affected subcellular organelles?

Incorporation of map kinases into the platelet cytoskeleton.

Erk1 (p44) and erk2 (p42) mitogen-activated protein (MAP) kinases are activated in agonist-stimulated platelets, although their role(s) in the activation process is unknown. In the present study, erk1, erk2 and the phosphorylated forms of both enzymes became associated with the contractile cytoskeleton in thrombin-stimulated platelets. Enzyme incorporation was accompanied by an increase in MAP kinase activity in the cytoskeleton, which was inhibited by PD98059. Pretreatment of the platelets with the arginine-glycine-aspartic acid-serine (RGDS) polypeptide enhanced both the cytoskeletal association and the enzyme activity, but cytochalasin D had no significant effect. Platelets from a patient with Glanzmann's thrombasthenia lack the alpha(IIb)beta(3) integrin and form only a rudimentary cytoskeleton, however, this cytoskeleton is enriched with both erk1 and erk2. These data suggest either that MAP kinases play a role in cytoskeletal rearrangement or that the cytoskeleton act as a frame to align MAP kinases with substrates in a highly integrated signal transduction pathway.

Activation of permeabilized platelets by inositol-1,4,5-trisphosphate.

The effect of inositol 1,4,5-trisphosphate (IP3) was studied using human platelets permeabilized with saponin and suspended in a high potassium, Ca2+-free buffer containing 40 microM EGTA and 1.2 mM magnesium. Under these conditions IP3 stimulated aggregation at a concentration of 0.5 microM with maximum aggregation at 5.0 microM. Aggregation was associated with phosphorylation of myosin light chain and a 47,000 dalton protein, and with a change in platelet shape including granule centralization and pseudopod formation similar to changes seen when cytoplasmic calcium is raised by other means. IP3 stimulated [14C]-serotonin release from platelet dense granules, [14C]-arachidonic acid release from platelet phospholipids and production of thromboxane B2. Preincubation of platelets with aspirin which blocked thromboxane formation also inhibited protein phosphorylation, serotonin secretion and partially inhibited aggregation. These results support the concept that IP3 is a major intracellular messenger in platelets and suggests that its effects are mediated both through Ca2+ flux and thromboxane formation.

Studies in patients with bleeding disorders show that platelet-vessel interaction is important for thromboxane formation in bleeding time wounds.

The production of thomboxane B2, the primary metabolite of thromboxane A2, and 6-keto prostaglandin F1 alpha, the primary metabolite of prostacyclin, were measured in response to a standardized vascular injury, the bleeding time, in patients with von Willebrand's disease and in patients with platelet function defects. Compared to controls, thromboxane B2 levels in bleeding time blood were significantly lower in subjects with von Willebrand's disease. In patients with platelet function defects associated with a deficient response to thromboxane A2, thromboxane B2 production in bleeding time blood was similar to controls. In subjects with other platelet function defects, thromboxane production was significantly lower than normal. 6-keto PGF1 alpha production in bleeding time blood was not significantly different in patients compared to controls. The results suggest that bleeding time thromboxane production is influenced by the extent of platelet-vessel interaction.

Primary hypothyroidism with intracranial hypertension and pituitary hyperplasia.

A 12-year-old girl presented with primary hypothyroidism, secondary pituitary hyperplasia, and intracranial hypertension. Cranial computed tomography revealed a sellar mass with suprasellar extension. She responded to medical treatment. Intracranial hypertension may be associated with primary hypothyroidism prior to thyroxine treatment. Because significant pituitary hyperplasia can be associated with primary hypothyroidism, it is vital to have endocrine investigation prior to consideration of surgical removal of an apparent pituitary tumor.

Platelet protein phosphorylation.

As can be seen from this review, protein phosphorylation appears involved in both positive and negative regulation of platelets. To date, good evidence has been presented for the involvement of protein phosphorylation in the regulation of granule centralization (i.e. myosin light chain phosphorylation). It is probable that protein phosphorylation may also be involved in granule labilization, pseudopod formation and ATP synthesis. Protein phosphorylation in association with platelet activation appears mediated through calcium flux, in the case of myosin light chain phosphorylation, and through diglyceride or other substances in the case of 47P phosphorylation. A summary scheme is shown in Figure 1.

Musculoskeletal health of subjects with hemophilia A treated with tailored prophylaxis: Canadian Hemophilia Primary Prophylaxis (CHPS) Study.

BACKGROUND: Full-dose prophylaxis is very effective at minimizing joint damage but is costly. Tailored prophylaxis has been proposed as a way of reducing costs while still protecting joints. OBJECTIVE: To report detailed findings in index joints of 56 subjects with severe hemophilia A entered into the Canadian Hemophilia Prophylaxis Study, and treated with tailored prophylaxis, after 13 years. METHODS: Boys with severe hemophilia A (< 2% factor) and normal joints were enrolled between the ages of 1 and 2.5 years. Initial treatment consisted of once-weekly factor infusions, with the frequency escalating in a stepwise fashion when breakthrough bleeding occurred. During the first 5 years, subjects were examined every 3 months using the modified Colorado Physical Evaluation (PE) scale; subsequently, every 6 months. The Childhood Health Assessment Questionnaire (CHAQ) was administered at each visit. RESULTS: Median age at study entry was 19 months (range 12-30 months); median follow-up was 92 months (range 2-156). The median PE score was 2, 3 and 3 at ages 3, 6 and 10 years. Persistent findings were related to swelling, muscle atrophy and loss of range of motion. The median score for each of these items (for the six index joints) was 0 at ages 3, 6 and 10 years. The median overall CHAQ score was 0 at ages 3, 6 and 10 years, indicating excellent function. CONCLUSIONS: Canadian boys treated with tailored primary prophylaxis exhibit minimal joint change on physical examination and minimal functional disability.