James G. White

In Vitro Bactericidal Capacity of Human Polymorphonuclear Leukocytes: Diminished Activity in Chronic Granulomatous Disease of Childhood *

Diminished bactericidal capacity was found to be characteristic of polymorphonuclear leukocytes (PMN) from five children with the clinical syndrome of granulomatous disease of childhood. The PMN from these children demonstrated nearly normal phagocytic capacity, and the majority of viable bacteria, after 2 hours of incubation in the phagocytosis system, were found associated with leukocytes. The morphology of the unstimulated polymorphonuclear leukocytes from patients with chronic granulomatous disease was similar to those from normal persons of similar ages by light and electron microscopy. In addition, the total lysozyme and phagocytin activity of leukocyte extracts from these patients was similar to those from equal numbers of leukocytes from controls.A striking difference in the cytoplasmic response after phagocytosis characterized the PMN of the patients with granulomatous disease. Whereas degranulation, vacuole formation, and rapid bacterial digestion were the rule in the PMN from controls, little degranulation and persistence of intact bacteria in the cytoplasm characterized disease. The deficiency of bactericidal capacity and the minimal degranulation after active phagocytosis by the PMN of these children with an inherited syndrome suggest that separate metabolic processes are involved in phagocytosis and in intracellular digestion. Continuing study of the metabolic function of leukocytes from these children should provide an opportunity for increased understanding of the metabolic basis for degranulation and intracellular digestion in phagocytic cells.

Complement (C5-a)-induced granulocyte aggregation in vitro. A possible mechanism of complement-mediated leukostasis and leukopenia.

Activated plasma complement will induce biphasic aggregation of human granulocytes dectable by standard nephelometric techniques. The responsible active component was suggested to be C5a by molecular weight and heat-stability assays; moreover, aggragating activity was ablated by anti-C5 but not anti-C3 antibodies. C5a prepared by trypsinization of purified C5 reproduced the aggregating activity of whole activated plasma, whereas plasma from a C5-deficient donor did not support aggregation. Embolization of granulocyte aggregates might be a previously unsuspected cause of leukostasis and pulmonary damage in various clinical situations where intravascular complement activation occurs.

Familial dyserythropoietic anaemia and thrombocytopenia due to an inherited mutation in GATA1.

Haematopoietic development is regulated by nuclear protein complexes that coordinate lineage-specific patterns of gene expression. Targeted mutagenesis in embryonic stem cells and mice has revealed roles for the X-linked gene Gata1 in erythrocyte and megakaryocyte differentiation. GATA-1 is the founding member of a family of DNA-binding proteins that recognize the motif WGATAR through a conserved multifunctional domain consisting of two C4-type zinc fingers. Here we describe a family with X-linked dyserythropoietic anaemia and thrombocytopenia due to a substitution of methionine for valine at amino acid 205 of GATA-1. This highly conserved valine is necessary for interaction of the amino-terminal zinc finger of GATA-1 with its essential cofactor, FOG-1 (for friend of GATA-1; refs). We show that the V205M mutation abrogates the interaction between Gata-1 and Fog-1, inhibiting the ability of Gata-1 to rescue erythroid differentiation in an erythroid cell line deficient for Gata-1 (G1E). Our findings underscore the importance of FOG-1:Gata-1 associations in both megakaryocyte and erythroid development, and suggest that other X-linked anaemias or thrombocytopenias may be caused by defects in GATA1.

Abnormal Adherence of Sickle Erythrocytes to Cultured Vascular Endothelium: POSSIBLE MECHANISM FOR MICROVASCULAR OCCLUSION IN SICKLE CELL DISEASE

The abnormal shape and poor deformability of the sickled erythrocyte (RBC) have generally been held responsible for the microvascular occlusions of sickle cell disease. However, there is no correlation between the clinical severity of this disease and the presence of sickled RBC. In searching for additional factors that might contribute to the pathophysiology of sickle cell disease, we have investigated the possibility that sickle RBC might be less than normally repulsive of the vascular endothelium. After RBC suspensions are allowed to settle onto plates of cultured human endothelial cells, normal RBC are completely removed by as few as six washes. In contrast, sickle RBC remain adherent despite multiple washes. On subconfluent culture plates, normal RBC are distributed randomly, whereas sickle RBC cluster around endothelial cells. Sickle RBC adherence is not enhanced by deoxygenation but does increase with increasing RBC density. The enzymatic removal of membrane sialic acid greatly diminishes the adherence of sickle RBC to endothelial cells, suggesting that sialic acid participates in this abnormal cell-cell interaction. Although net negative charge appears normal, sickle RBC mainfest an abnormal clumping of negative surface charge as demonstrated by localization of cationized ferritin. These abnormalities are reproduced in normal RBC loaded with nonechinocytogenic amounts of calcium. We conclude that sickle RBC adhere to vascular endothelial cells in vitro, perhaps caused by a calcium-induced aberration of membrane topography. This adherence may be a pathogenetic factor in the microvascular occlusions characteristic of sickle cell disease.

Nitric oxide donors increase blood flow and reduce brain damage in focal ischemia : evidence that nitric oxide is beneficial in the early stages of cerebral ischemia

We studied whether administration of nitric oxide (NO) donors reduces the ischemic damage resulting from middle cerebral artery (MCA) occlusion in spontaneously hypertensive rats (SHRs). In halothane-anesthetized and ventilated SHRs, the MCA was occluded. CBF was monitored using a laser-Doppler flowmeter. Three to five minutes after MCA occlusion, the NO donors sodium nitroprusside (SNP; 3 mg/kg/h) or 3-morpholino-sydnonimine (SIN 1; 1.5–6 mg/kg/h) were administered into the carotid artery for 60 min. As a control, the effect of papaverine (3.6 mg/kg/h), a vasodilator that acts independently of NO, was also studied. The hypotension evoked by these agents was counteracted by intravenous infusion of phenylephrine. At the end of the infusion, rats were allowed to recover. Stroke size was determined 24 h later in thionin-stained sections. In sham occluded rats, SNP (n = 5), SIN 1 (n = 5), and papaverine (n = 5) produced comparable increases in CBF (p > 0.05 from vehicle). After MCA occlusion, SNP (n = 5) and SIN 1 (n = 5), but not papaverine (n = 5), enhanced the recovery of CBF (p < 0.05 from vehicle) and reduced the size of the infarct by 28 ± 12 and 32 ± 7%, respectively (mean ± SD; p < 0.05 from vehicle). To determine whether NO donors could act by inhibiting platelet aggregation, we studied the effect of SNP on collagen-induced platelet aggregation. Intracarotid administration of SNP (3 mg/kg/h for 60 min) did not affect platelet aggregation to collagen, suggesting that the protective effect of NO donors was not due to inhibition of platelet function. We conclude that NO donors increase CBF to the ischemic territory and reduce the tissue damage resulting from focal ischemia. The protective effect may result from an increase in CBF to the ischemic territory, probably the ischemic penumbra. These findings suggest that NO donors may represent a new therapeutic strategy for the management of acute stroke.

Corticosteroids inhibit complement-induced granulocyte aggregation. A possible mechanism for their efficacy in shock states.

Granulocyte (PMN) aggregation and embolization may underlie complement (C)-mediated organ dysfunction in such syndromes as hemodialysis neutropenia and Purtschers ischem;c retinopathy. Because of clinical and pathologic parallels, we have further suggested a role for this phenomenon in the genesis of the adult respiratory distress syndrome (ARDS). Because corticosteroids are commonly used in immune diseases, and have particularly been claimed efficacious in shock and ARDS, we tested the capability of methylprednisolone (MP), hydrocortisone (HC), and dexamethasone (DEX) to inhibit PMN aggregation. Aggregation engendered in vitro by zymosan-activated plasma (ZAP) was inhibited by MP and HC at concentrations approximating plasma levels achieved with the large bolus (30 mg/kg i.v) therapy advocated in shock states; DEX was almost without effect. Using intravital fluorescence microscopy, we observed PMN aggregation and embolization in the mesenteric vessels of rats given intra-arterial infusions of ZAP; this was also prevented by pretreatment with 30 mg/kg MP. Steroid inhibition of aggregation seemed not to involve disruption of receptor function, because aggregation induced by alternative agents, n-formyl-Met-Leu-Phe and the ionophore A23187, was also inhibited by MP. Moreover, corticosteroid inhibition of PMN prostaglandin synthesis is also an unlikely explanation for our results, since aspirin and ibuprofen failed to block aggregation and arachidonic acid neither effected aggregation itself nor ameliorated the steroid effect. Our studies provide a plausible rationale for the empiric observation that high-dose corticosteroids may benefit patients with syndromes associated with microvascular leukostasis.

Proteomic analysis of platelet α-granules using mass spectrometry

Summary.  Background: Platelets have three major types of secretory organelles: lysosomes, dense granules, and α‐granules. α‐Granules contain several adhesive proteins involved in hemostasis, as well as glycoproteins involved in inflammation, wound healing, and cell–matrix interactions. This article represents the first effort to define the platelet α‐granule proteome using mass spectrometry (MS). Methods: We prepared a subcellular fraction enriched in intact α‐granules from human platelets using sucrose gradient ultracentrifugation. α‐Granule proteins were separated and identified using sodium dodecylsulfate polyacrylamide gel electrophoresis and liquid chromatography–tandem MS. Results: In the sucrose fraction enriched in α‐granules, we identified 284 non‐redundant proteins, 44 of which appear to be new α‐granule proteins, on the basis of a literature review. Immunoelectron microscopy confirmed the presence of Scamp2, APLP2, ESAM and LAMA5 in platelet α‐granules for the first time. We identified 65% of the same proteins that were detected in the platelet releasate (J. A. Coppinger et al. [Blood 2004;103: 2096–104]) as well as additional soluble and membrane proteins. Our method provides a suitable tool for analyzing the granule proteome of patients with storage pool deficiencies.

Further studies on the platelet-inhibitory effect of guanidinosuccinic acid and its role in uremic bleeding

The average guanidinosuccinic acid (GSA) level in the serum of seventeen uremic subjects was found to be 2.53 mg per cent (0.15 mM) with a range of 0.86 to 5.4 mg per cent; serum GSA was less than 0.15 mg per cent in each of seventeen normal subjects. Measured GSA levels are sufficient to account for the inhibition of adenosine diphosphate (ADP)-induced platelet factor 3 (PF-3) activation, which we have previously demonstrated in citrated uremic plasma. GSA levels and the degree of inhibitio of ADP-induced PF-3 activation decreased in parallel in the serum and plasma of uremic patients undergoing peritoneal dialysis. In vitro inhibition of ADP-induced PF-3 activation did not occur with urea, creatinine, guanidinoacetic acid or arginine. GSA inhibited the second wave of platelet aggregation induced by ADP or by epinephrine and reduced the degree of aggregation induced by collagen suspensions. At increased concentrations of ADP and l-epinephrine the second wave of aggregation (attributed to release of endogenous platelet ADP) was again demonstrated. The aggregation of uremic citrated platelet-rich plasma (C-PRP) required nearly twice as much ADP as did normal C-PRP for either the second wave of aggregation or a maximal aggregation response. Acetyl salicylic acid (ASA) at equimolar concentrations was as effective as GSA in inhibiting both ADP-induced PF-3 activation and platelet aggregation, but the two inhibitors differed in several respects. Exposure to GSA makes the dense tubular system of normal platelets more prominent and inhibits the internal transformation characteristic of their response to ADP; these ultrastructural changes correlate with the inhibition of the second wave of aggregation. Thus, GSA at levels found in the serum of uremic patients can account for a number of the in vitro abnormalities of uremic platelet function and may be partly responsible for the bleeding syndrome of uremia.

NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet α-granules

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder that is characterized by large platelets that lack α-granules. Here we show that mutations in NBEAL2 (neurobeachin-like 2), which encodes a BEACH/ARM/WD40 domain protein, cause GPS and that megakaryocytes and platelets from individuals with GPS express a unique combination of NBEAL2 transcripts. Proteomic analysis of sucrose-gradient subcellular fractions of platelets indicated that NBEAL2 localizes to the dense tubular system (endoplasmic reticulum) in platelets.

Mutation of a new gene causes a unique form of Hermansky-Pudlak syndrome in a genetic isolate of central Puerto Rico

Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by oculocutaneous albinism and a storage pool deficiency due to an absence of platelet dense bodies. Lysosomal ceroid lipofuscinosis, pulmonary fibrosis and granulomatous colitis are occasional manifestations of the disease. HPS occurs with a frequency of one in 1,800 in north-west Puerto Rico due to a founder effect. Several non-Puerto Rican patients also have mutations in HPS1, which produces a protein of unknown function. Another gene, ADTB3A, causes HPS in the pearl mouse and in two brothers with HPS-2 (refs. 11,12). ADTB3A encodes a coat protein involved in vesicle formation, implicating HPS as a disorder of membrane trafficking. We sought to identify other HPS-causing genes. Using homozygosity mapping on pooled DNA of 6 families from central Puerto Rico, we localized a new HPS susceptibility gene to a 1.6-cM interval on chromosome 3q24. The gene, HPS3, has 17 exons, and a putative 113.7-kD product expected to reveal how new vesicles form in specialized cells. The homozygous, disease-causing mutation is a large deletion and represents the second example of a founder mutation causing HPS on the small island of Puerto Rico. We also present an allele-specific assay for diagnosing individuals heterozygous or homozygous for this mutation.