Platelet dysfunction in homozygous beta-thalassemia

This report details the results of studies performed in nine patients with homozygous beta-thalassemia evaluating platelet function and prostaglandin formation. Platelet malonyldialdehyde (MDA) formation in the presence of N-ethyl maleimide (NEM 1 mM) or thrombin (0.5 u/ml) was used as an indicator of platelet prostaglandin synthesis. The data on the nine patients revealed two distinct subgroups of patients. Six of nine thalassemics, demonstrated platelet abnormalities. Their mean bleeding time was 7.5 +/- 2.5 min (1 SD), significantly prolonged (P less than 0.005) when compared to a value of 3.5 +/- 1.0 min in normal controls. MDA formation in the presence of NEM was significantly decreased (P less than 0.005) to 2.41 +/- 0.49 (1 SD) when compared to a control value of 3.24 +/- 0.33 nmoles MDA/10(9) platelets. Similarly, the mean value for thrombin induced MDA was 0.98 +/- 0.18 nmoles which was decreased (P less than 0.02) when compared to a value of 1.26 +/- 0.2 in the controls. Platelet aggregations with adenosine diphosphate (ADP), epinephrine, and collagen were abnormal in all six patients. However, when platelets from these patients were mixed with platelets from donors who had ingested aspirin 2-8 hr before donation mutual correction and secondary irreversible aggregation of the mixture resulted. No mutual correction was observed when the thalassemic platelets were preincubated with aspirin in vitro before mixing with platelets from donors who had recently ingested aspirin. Although the total amount of platelet malonyldialdehyde formed by the thalassemic platelets in response to NEM and thrombin was decreased when compared to normal controls, this reduction was not the cause of the platelet aggregation abnormalities. This appears to be so because the amount of MDA, and, thus, prostaglandin endoperoxides synthesized by these platelets in response to external stimuli was sufficient to cause irreversible aggregation of platelets from donors who had recently ingested aspirin, and were, therefore, unable to synthesize their own endogenous platelet endoperoxides. In the remaining three patients, bleeding times, platelet aggregation, and MDA formation was normal. No correlation was observed between the platelet abnormalities noted and the magnitude of iron overload, presence of fibrin degradation products, liver function abnormalities, or the use of iron chelators in the individual patient. Family studies were normal. Although the platelet dysfunction does not appear to be of major significance in the usual patient with thalassemia major under normal circumstances, antiplatelet aggregating agents should be used with caution. Aspirin inhibits platelet endoperoxide and prostaglandin formation and this effect may potentiate the platelet dysfunction present in some patients with thalassemia major.     

Giant platelet disorders in African-American children misdiagnosed as idiopathic thrombocytopenic purpura.

A retrospective chart review of six African-American children with a diagnosis of macrothrombocytopenias (MTCP) was performed to evaluate the accuracy of their diagnosis. The following was diagnosed in the six children with MTCP: Fechtner syndrome (two children), Sebastian syndrome (one child), and unnamed MTCP (three children). In five families, chronic idiopathic thrombocytopenic purpura (ITP) was diagnosed in the propositus, which resulted in therapy using steroids, intravenous immunoglobulin (IVIG), and in one case splenectomy. Bleeding symptoms were generally mild. All six patients had thrombocytopenia ranging from 10 to 125 x 10(9)/L with mean platelet volume of 8 to 20 fL. Bleeding times were abnormal in two of three patients, and platelet aggregation was abnormal in three of four patients tested. Bone marrow aspirates were reported as increased megakaryocytes in the three patients on whom the procedure was performed. Ultrastructural morphology of platelets and leukocytes was performed in all six patients demonstrating giant platelets in all six patients and leukocyte inclusions in three patients. Differentiating MTCP from the more common ITP can be difficult but important in avoiding unnecessary diagnostic studies and potentially harmful therapy associated with ITP.     

Bartter's syndrome in Arabic children: review of 13 cases

BACKGROUND: Bartter's syndrome (BS) is an inherited disease of renal potassium wasting characterized by hypokalemic alkalosis, normal blood pressure, vascular insensitivity to pressor agents and elevated plasma concentrations of renin and aldosterone. It is caused by generalized hyperplasia of the juxtaglomerular apparatus at the site of renin production caused by mutations in the Na-K-2Cl cotransporter gene, NKCC2. The objective of our study is to establish the prevalence and incidence of BS in Kuwait and to assess treatment modalities for it. METHODS AND RESULTS: Bartter's syndrome was diagnosed in 13 Kuwaiti children over a 14 year period (1981-1995) with the estimated incidence of 1.7/100,000 live births. The mean age at diagnosis was 9.3 months (range 2-32 months). There were five males and eight females (ratio 1:1.6). The mean duration of follow up was 5.6 years (1-14 years). Both consanguinity and familial history among our patients were high (69 and 54%, respectively). All patients had hypokalemia, hypochloremia with metabolic alkalosis, hyperreninemia and were normotensive. Clinical presentation was essentially similar to that in other series. Eleven patients (85%) had growth failure, two had nephrocalcinosis (15%) and one had renal failure. All patients were treated with supplemental potassium, an aldosterone antagonist (spironolactone) and a prostaglandin synthetase inhibitor (indomethacin or aspirin) sequentially. Significant catch-up of growth (four patients) and increases in serum potassium (eight patients) were recorded after administration of indomethacin therapy. One patient died of severe pneumonia with respiratory failure from hypokalemic myopathy. Clinical presentation, inheritance, complications and therapy of BS are briefly discussed. CONCLUSION: Bartter's syndrome is a rare disease, but should be considered in the differential diagnosis of other disorders with growth failure and/or hypokalemia. Early diagnosis, close follow up and compliance with treatment may lead to appropriate growth and development.     

Hemostasis in Glanzmann's thrombasthenia (GT): GT platelets interfere with the aggregation of normal platelets

The transfusion requirements for a 6-year-old Glanzmann's thrombasthenia (GT) patient undergoing tonsillectomy and adenoidectomy were studied. Transfusion of pheresed platelets from a single normal donor increased the platelet count by 63 x 10(9)/L but did not correct the bleeding time. Since the ratio of normal:GT platelets in vivo was approximately 1:5, it was possible that GT platelets interfered with the function of normal platelets. To test this hypothesis, mixtures of platelet-rich plasma (PRP) from a normal donor and the patient were studied to determine a ratio of normal:GT platelets that would yield acceptable in vitro aggregation. Normal:GT ratios of 1:4 and 3:2 resulted in 25% and 59% aggregation, respectively. Mixtures of normal and nonfunctional ethylene glycol tetra-acetic acid-treated platelets gave similar results. Aggregates from mixtures of normal and patient platelets were also examined morphologically by light microscopy and were proportional in size to the normal:GT platelet ratio. Transfusion of platelets from the pheresis of four donors increased the patient's platelet count by greater than 300 x 10(9)/L (normal:GT ratio 1:1), produced 53% aggregation, and resulted in satisfactory postoperative hemostasis. The platelet transfusion requirement for this GT patient was much greater than would have been expected in the absence of aggregation-defective platelets.     

Long-term use of propranolol, ibuprofen, and spironolactone in the management of Bartter's syndrome.

This report concerns two patients with Bartter's syndrome who were treated with propranolol, spironolactone, and potassium supplements. When ibuprofen was added to this regimen, potassium supplements were no longer required. In both patients, plasma renin activity decreased, plasma volumes increased, and a "catch-up" in linear growth ensued. This report confirms others that indicate prostaglandin synthetase inhibitors are a useful adjunct in the therapy of Bartter's syndrome.     

Bartter's syndrome. Differentiation into two clinical groups.

We were able to separate Bartter's syndrome patients into two groups: Group 1 patients are seen at a young age, with hyponatremia and episodes of extracellular volume contraction. Group 2 patients are older and most have tetany; plasma sodium values and renal sodium balances are normal. Plasma potassium values are lower and plasma renin activity and plasma magnesium levels are higher in group 1 than in group 2. It may be that all patients with Bartter's syndrome manifest the features of both groups, group 1 patients with time developing into group 2 patients. These observations have important implications in the treatment of patients with Bartter's syndrome.     

Studies on the Effect of Long-Term Use of Low Dose Aspirin in Kawasaki Disease

The inhibitory action of long-term low dose aspirin (1–2 mg/kg/day for over 10 months) on the cyclooxygenase pathway in platelets and vascular endothe-lium was evaluated in 10 patients with Kawasaki disease. The results were compared with those obtained after taking aspirin at 5–10 mg/kg/day during the acute phase of the illness. Platelet aggregations induced by adenosinedi-phosphate (ADP), epinephrine and collagen were inhibited by aspirin doses of 1–2 and 5–10 mg/kg/day, when compared with those of controls (p < 0.05). Platelet synthesis of thromboxane B₂ (TXB₂) under doses of 1–2 and 5–10 mg/kg/day was 0.57 ± 0.07 and 0.72 ± 0.09 ng/ml platelet-rich plasma (PRP) /10⁵ platelets, respectively (p > 0.1). These values were significantly lower than those of the control group (22.88± 3.42 ng/ml PRP/10⁵ platelets) (p < 0.05). No differences were found in platelet aggregation and TXB₂ productivity between the two aspirin doses. Levels of 6 keto-prostaglandin F₁α (6k-PGF₁α) in platelet-poor plasma (PPP) did not differ significantly in these 3 sets of data. The results indicate that long-term administration of low dose aspirin (1–2 mg/kg/day) inhibits platelet aggregation by inhibiting synthesis of thromboxane A₂ (TXA₂), without interfering with prostacyclin production probably in the endothelium of blood vessels.     

Gray platelet syndrome]

BACKGROUND. Gray platelet syndrome is a rare (about 40 cases published), inherited disorder characterized by a marked decrease or absence of platelet alpha-granules and platelet specific alpha-granule proteins. CASE REPORT. A boy, aged 4 years, presented with frequent ecchymoses. Acute idiopathic thrombocytopenia purpura was diagnosed because of his reduced platelet count (36,000/mm3) and recent viral infection. Intravenous gammaglobulin infusion was followed by a small rise in the platelet count (125,000/mm3). The patient was reinvestigated a few months later because of persistent thrombocytopenia and the failure of the immunologic treatment. The bleeding time was long and the platelets on blood smears appeared gray. Electron microscopy revealed numerous vacuoles and very few or no alpha-granules. Platelet aggregation and adhesion were normal, but stimulated platelets failed to liberate factor 4 and beta-thromboglobulin, while the plasma levels of beta-thromboglobulin were elevated. CONCLUSION. The frequency of gray platelet syndrome is probably underestimated in those diseases resulting in thrombocytopenia and this will continue until blood smears are thoroughly examined. Synthesis of platelet specific alpha-granule proteins seems normal; but these proteins cannot be stored as there are very few, or no alpha-granules. This abnormality could lead to increased levels of such proteins in the plasma.     

Platelet dysfunction in the neonate with essential fatty acid deficiency.

Platelet aggregation in response to ADP was studied in five EFA-deficient sick premature newborn infants who were receiving fat-free parenteral nutrition. EFA deficiency was diagnosed by analysis of plasma lipid fatty acid values. The deficient infants had impaired platelet aggregation when compared to other low-birth-weight infants fed orally who served as control subjects (maximum 18.1% versus 43.4% at 2.5 muM ADP; 34.5% versus 52.2% at 5.0 muM ADP). In addition, the platelets from EFA-deficient infants demonstrated clearly evident disaggregation. On recovery from their deficient state, the low-birth-weight infants had platelet functions similar to those of apparently healthy premature infants. Clinical hemorrhage occurred in four of the EFA-deficient infants. Thus a deficiency of arachidonic acid, the precursor of thromboxane A2, is correlated with an impairment of the aggregation of platelets, a phenomenon mediated by thromboxane A2. This correlation provides the basis for a hypothesis that the observed EFA deficiency is causally related to the platelet dysfunction.     

Treatment of Bartter's syndrome in early childhood with prostaglandin synthetase inhibitors.

The diagnosis of Bartter's syndrome was made in a 9-month-old boy investigated for poor weight and height gain. Initial treatment with oral potassium supplements and later spironolactone had little or no effect on his growth, although plasma potassium rose to normal after spironolactone. At 33 months indomethacin therapy was started with dramatic results. His symptoms went and his height and weight accelerated into the normal range. In view of the toxicity of indomethacin it was replaced after 12 months by another prostaglandin synthetase inhibitor, ketoprofen, with a satisfactory result. During the change-over period from indomethacin to ketoprofen the expected deterioration in clinical well-being was observed, accompanied by a rise in urinary prostaglandins and plasma renin activity. Prostaglandin synthetase inhibitors provide the best available treatment for Bartter's syndrome.     

Platelet aggregation in iron deficiency anemia

In-vitro platelet aggregation with ADP, adrenaline and collagen was studied in pediatric patients with iron deficiency before and after iron therapy, and in normal controls. Hyporeactivity of platelets to all agonists was seen in the untreated patients. This returned to normal following correction of anemia by iron therapy. There was a significant correlation between hemoglobin or serum iron and transferrin saturation on the one hand, and parameters of platelet aggregation on the other (P<0.001). Results of zero order correlation coefficient however, showed that platelet reactivity was actually dependent on the hemoglobin levels rather than the iron parameters per se. This may explain the prolonged bleeding time in patients with iron deficiency, particularly those associated with thrombocytopenia. Anemic individuals may be protected against thrombotic disorders due to this reduced platelet reactivity.     

Effect of indomethacin in a patient with Bartter's syndrome: evidence against a primary role of prostaglandin in this disorder

It has been claimed that in patients with Bartter's syndrome, enhanced prostaglandin synthesis causes the blunted vasopressor response to angiotensin II. The present study was undertaken to test this hypothesis by administering indomethacin to a patient with Bartter's syndrome. In domethacin corrected the subnormal pressor response to angiotensin II and lowered plasma renin activity from 115 to 15 ng/ml/hr. This effect was associated with renal sodium retention and a 7% increase in body weight. In contrast, when indomethacin was given but the sodium retention prevented by concomitant administration of furosemide, the blunted vasopressor response to angiotensin II and the hyperreninemia were not corrected. It is concluded that the effectiveness of indomethacin to correct the hyperreninemia and the blunted vasopressor to angiotensin II in the preset patient was due in large part to the ability of the drug to correct sodium balance rather than by inhibition of prostaglandin synthesis.     

Type IIB von Willebrand's Disease: Report on the First Case in Japan

Three members of a family are reported with type IIB von Willebrand's disease (vWD), characterized by prolonged bleeding time, increased ristocetin-induced platelet aggregation, fast anodal migration of factor VIII-related antigen on crossed immunoelectrophoresis and an absence in plasma and presence in platelets of the larger multimers of factor VIII/von Willebrand factor (FVIII/vWF). Binding ability of the patient's FVIII/vWF to washed normal platelets is significantly increased in the presence of low concentrations of ristocetin, whereas binding ability of the patient's platelets to normal FVIII/vWF is not increased. These later findings are contrary to those in patients with pseudo-vWD or platelet type vWD. The administration of DDAVP gives a shortening of bleeding time and an increase in plasma levels of factor VIII-related activities. In addition, there is a transitory appearance of the larger multimers and a moderate diminution of the platelet count.     

Erythrocyte Sodium Transport in Bartter's Syndrome

ABSTRACT. Erythrocyte sodium transport was evaluated by measurement of intracellular Na concentration (ICNa), ²²Na efflux rate constant (NaERC) and 3H-ouabain binding (BMax) (reflecting the number of Na/K ATPase pump sites) in 9 children with Bartter's syndrome compared to controls (children and adults) and children with various forms of salt wasting disease. There were no differences between control children and adults. In untreated Bartter's syndrome ICNa was significantly increased with NaERC and BMax significantly decreased compared to findings in controls and patients with other salt wasting disease. On prostaglandin synthetase inhibitor (Indomethacin) therapy, ICNa decreased but remained higher than in controls, NaERC increased to normal values but BMax remained low. These data support the view that there is a widespread defect in membrane electrolyte transport in Bartter's syndrome but suggest that the benefit of indomethacin therapy is not manifest via an effect on Na/K ATPase.     

Erythrocyte sodium transport in Bartter's syndrome

Erythrocyte sodium transport was evaluated by measurement of intracellular Na concentration (ICNa), 22Na efflux rate constant (NaERC) and 3H-ouabain binding (BMax) (reflecting the number of Na/K ATPase pump sites) in 9 children with Bartter's syndrome compared to controls (children and adults) and children with various forms of salt wasting disease. There were no differences between control children and adults. In untreated Bartter's syndrome ICNa was significantly increased with NaERC and BMax significantly decreased compared to findings in controls and patients with other salt wasting disease. On prostaglandin synthetase inhibitor (Indomethacin) therapy, ICNa decreased but remained higher than in controls, NaERC increased to normal values but BMax remained low. These data support the view that there is a widespread defect in membrane electrolyte transport in Bartter's syndrome but suggest that the benefit of indomethacin therapy is not manifest via an effect on Na/K ATPase.     

阿司匹林对川崎病患儿抗血小板聚集功能的研究

目的　评估阿司匹林在川崎病患儿中的抗血小板聚集功能。方法　回顾性分析2016年9月至2018年9月北京大学首都儿科研究所教学医院收治的川崎病患儿的临床资料。所有患儿常规给予阿司匹林治疗,急性期为大剂量（30～50） mg/（kg·d）,恢复期为小剂量（3～5） mg/（kg·d）, 应用光学比浊法（light transmission aggregometry, LTA）测定应用不同剂量阿司匹林的血小板聚集率以评价其抗血小板聚集功能, 并用统计学方法分析发生阿司匹林抵抗（aspirin resistance,AR）的危险因素。结果　（1）川崎病患儿口服大剂量和小剂量阿司匹林治疗后的血小板聚集率（AA%）分别为 3.3 %（1.2%, 7.1%）及2.9%（1.5%, 6.4%）, 不同剂量阿司匹林对血小板的抑制作用（AA%）差异无统计学意义（P＝0.174）。（2）大剂量阿司匹林组AR发生率为9.75%（23/236）, 小剂量阿司匹林组AR发生率为8.05 %（19/236）, 二者差异无统计学意义（P＝0.617）。（3）小剂量阿司匹林组中19例存在AR与217例阿司匹林敏感（aspirin sensitivity, AS）患儿的年龄、 性别及凝血、 生化等相关指标差异无统计学意义。结论　阿司匹林对川崎病患儿抗血小板聚集功能与剂量无关。在川崎病治疗中存在AR,AR发生率与剂量无关。     

Effect of intravenous immune globulin on the coagulopathy of Kawasaki syndrome

We studied the effects of immune globulin and aspirin versus aspirin alone on platelet count, platelet activation, and factor-mediated coagulation in patients with Kawasaki syndrome. Coagulation tests were performed on the day of admission to the study and 4 to 6 days later. Twenty-three patients were enrolled; 12 received immune globulin intravenously plus aspirin, and 11 received aspirin alone. At initiation of the study the groups were comparable with regard to age, sex, race, and time from onset of illness to study entry. Coagulation values were similar at entry with the exception that the aspirin group had a geometric mean platelet count that was higher than the platelet count in the aspirin-immune globulin group (p = 0.02). Four days after entry there were no significant differences between the two groups in any coagulation studies. Although the immune globulin preparation used has been effective in reducing the prevalence of coronary artery aneurysms, it appears to have no early effect on reduction of platelet activation or other measures of coagulopathy. The mechanism of action of immune globulin in patients with Kawasaki syndrome remains to be elucidated.     

Comparative effects of mezlocillin and carbenicillin on platelet function and thromboxane generation in patients with cancer

Carbenicillin and mezlocillin are widely used for treatment of Pseudomonas infections in patients with cancer. Carbenicillin has been reported to cause platelet dysfunction and bleeding diathesis in some individuals. We evaluated whether carbenicillin causes deterioration of platelet function in patients with cancer and whether mezlocillin causes similar effects on platelets from normal subjects or from patients with cancer. In these in vitro studies, carbenicillin and mezlocillin decreased ADP and epinephrine-induced platelet aggregation and thromboxane A2 generation similarly, but only in concentrations of 3.2 mg/ml or higher. In contrast, carbenicillin was more potent than mezlocillin in decreasing ristocetin-induced platelet aggregation. We also evaluated effects of these antibiotics on platelet function in 19 patients with cancer who developed fever and neutropenia. These patients received either mezlocillin (10 patients) or carbenicillin (nine patients) in combination with nafcillin and gentamycin. Neither carbenicillin nor mezlocillin had any significant effect on platelet aggregation or thromboxane A2 generation. Lack of effects in vivo was due to defective platelet function in these patients prior to any antibiotics. These defects were most probably related to underlying disease and/or prior chemotherapy. Thus, carbenicillin and mezlocillin can both safely be used in patients with cancer who develop fever and neutropenia, and neither seems to have advantage over the other in terms of platelet function.     

Increased neonatal platelet deposition on subendothelium under flow conditions: the role of plasma von Willebrand factor

In vitro platelet function of umbilical cord blood and neonatal peripheral vein blood from full-term newborns was compared with that of adults. Citrated whole blood was subjected to shear stress (1300 s(-1)) on subendothelial extracellular matrix (ECM)-coated wells in a cone and plate(let) analyzer. Adhered platelets on the ECM were quantitated by image analyzer. Both umbilical cord and neonatal peripheral blood platelets demonstrated more extensive adhesion than adult platelets, and similar aggregate formation on ECM. The ability of neonatal platelets to form aggregates on ECM was confirmed by scanning electron microscopy. Similar activation of neonatal and adult platelets after subjection to shear stress, in the suspension phase, was established by flow cytometry, which showed an increase in fibrinogen binding and a decrease in glycoprotein Ib expression on platelet membrane. The difference in adhesion rates between neonatal and adult platelets was preserved even when the hematocrit level of the neonatal blood was adjusted to that of adults. Reconstitution of neonatal or adult platelet-rich plasma with autologous or heterologous red packed cells yielded no change in adhesion and aggregation. When von Willebrand factor-covered plates were used to prevent deposition of plasma von Willebrand factor on the surface, no difference in platelet adhesion was seen between neonatal and adult blood. In conventional aggregometry assay, the response to ristocetin of washed platelets of either neonatal or adult source was higher on addition of plasma from neonates than from adults. Our data suggest that the extensive neonatal platelet deposition on ECM is mediated by plasma von Willebrand factor, which is known to be more multimerized and, therefore, more active in neonates than in adults. This mechanism may provide balanced primary hemostasis in neonates despite the platelet hyporeactivity to agonists without application of shear stress.     

Sustained engraftment post bone marrow transplant despite anti-platelet antibodies in Glanzmann thrombasthenia

BACKGROUND: Patients with Glanzmann thrombasthenia (GT) have normal platelet counts but abnormal platelet aggregation and carry the risk of life-threatening bleeding. We report three patients who received bone marrow transplantation (BMT) for type I GT and discuss the risk and management of anti-platelet antibodies. PATIENTS AND RESULTS: Diagnosis of GT was made through abnormal platelet aggregation studies or the absence of GPIIb/IIIa by flow cytometry. All patients had severe bleeding requiring multiple red blood cell transfusions. One patient received an unrelated donor transplant and two received matched sibling donor transplants following conditioning therapy with busulfan, cyclophosphamide, and fludarabine. Two patients developed an anti-platelet antibody, treated in one with intravenous immune globulin (IVIG). Engraftment of white blood cells and platelets was achieved on day +13 to +14 and +17 to +25, respectively. Complete donor chimerism and GPIIb/IIIa+ platelets are sustained at +22 to +30 months post transplant. CONCLUSIONS: In summary, patients with GT and history of severe hemorrhage can be cured with BMT, but the presence of anti-platelet antibodies should be sought and platelet transfusions minimized prior to transplant. IVIG may be helpful in cases of refractory immune thrombocytopenia related to anti-platelet antibodies. Improvement in transplant-related complications with current transplant regimens allows consideration of BMT for life-threatening non-malignant disorders such as GT.