Whole blood platelet deposition on extracellular matrix under flow conditions in preterm neonatal sepsis

Platelet function in preterm infants with sepsis was evaluated by measuring their adhesion and aggregation properties using the Cone and Plate(let) Analyser. This may lead to earlier detection of bleeding tendency in septic infants. Platelet function was investigated in 54 preterm infants, of whom 32 had proven neonatal sepsis and 22 were healthy matched controls. Citrated whole blood was subjected to shear stress (1300 s(-1)) for 2 min on tissue culture plates precoated with subendothelial extracellular matrix (ECM). The percentage of ECM surface covered with platelets and the average size of the ECM-bound platelet particles were determined with an image analyser. Assays for von Willebrand factor (vWF) antigen, ristocetin co-factor, and vWF collagen binding activity (CBA) were performed on samples from an additional 47 preterm infants: 38 healthy and 9 septic. Platelets of the preterm infants with sepsis displayed lower adhesion than those of the healthy controls. Mean surface coverage was 16.9+/-8.2% for the septic infants, 15.4+/-7.9% for the septic infants after exclusion of those with coagulase-negative staphylococci sepsis, and 20.8+/-9.6% for the healthy group ( P<0.05). Platelet aggregation, vWF antigen, ristocetin co-factor, and CBA levels were similar between the septic and healthy groups. The most significant factor influencing surface coverage was the presence of sepsis. CONCLUSION: platelet adhesion to extracellular matrix is significantly lower in septic preterm infants than in healthy preterm infants. Intrinsic platelet properties, rather than the concentration or activity of plasma von Willebrand factor, may be responsible for this difference. Surface coverage obtained by the collagen binding activity test under flow conditions, which represents platelet adhesion, may be an earlier, more sensitive indicator of bleeding tendency in neonatal sepsis than decreased platelet count.     

Management of type 2b von Willebrand disease in the neonatal period

Von Willebrand disease (VWD) is the most common inherited bleeding disorder, affecting one in 1,000 people. Type 2b VWD is a less common subtype caused by a gain‐of‐function mutation in von Willebrand factor (VWF) that leads to the formation of large, ineffective VWF‐platelet multimers in circulation. This unique pathophysiology creates diagnostic and treatment dilemmas. There is limited information on the management of type 2b VWD in the neonatal period. This report describes the management of a neonate with type 2b VWD with an emphasis on the added benefit of concomitant platelet transfusion and factor replacement therapy over factor replacement therapy alone.     

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目的观察1型糖尿病患儿血管内皮损害标志物—内皮素(ET)和血管性假血友病因子(von Wille-brand因子,vWF)的变化,分析其与尿白蛋白排泄率(UAER)的关系,从而筛查出更敏感的早期诊断糖尿病肾病(DN)的指标。方法收集1998-06—2005-06在山东省立医院就诊的4~18岁1型糖尿病患儿40例,根据UAER分为正常白蛋白尿组(A组)25例和微量白蛋白尿组(B组)15例,同时以年龄、性别、身高1∶1匹配的健康儿童作为对照组,分别检测其血糖(FBG)、糖化血红蛋白(HbA1c)、血浆ET和vWF的变化,并分析其相关性。结果与正常对照组比较,1型糖尿病患者血浆ET-1和vWF、HbA1c明显增高,尤其是微量白蛋白尿组升高更明显(均P<0·01),血浆ET-1和vWF与UAER、HbA1c均呈正相关。结论1型糖尿病患儿在出现白蛋白尿前已存在血管内皮功能异常,其白蛋白排泄与血管内皮功能障碍程度有一定相关性。血浆ET-1、vWF检测可作为早期筛查糖尿病肾病的可靠指标。     

Plasma levels of the von Willebrand factor-cleaving protease in physiological and pathological conditions in children

The hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are rare disorders characterized by thrombocytopenia, hemolytic anemia, and ischemic organ failure due to thrombotic occlusions in arterioles. The recent observation that a von Willebrand factor-cleaving protease (VWF-CP) is low in the plasma of patients with TTP but normal in those with HUS has potentially offered a new specific tool for differential diagnosis. In this study, the authors evaluated the plasma levels of the VWF-CP during the neonatal state and healthy childhood and in some pathological pediatric conditions. The protease was measured in 16 healthy newborns, 20 healthy children aged 5-18 years, patients with diabetes mellitus type1 ( n = 7), acute viral hepatitis ( n = 10), chronic viral hepatitis ( n = 10), transfusion-dependent β-thalassemia major ( n = 10), acute varicella infection ( n = 11), the nephrotic syndrome ( n = 11), and familial Mediterranean fever ( n = 10). Mean protease levels were significantly lower in newborns than in healthy children (50.5 ±16.1% vs. 83.3 ±16.3%)( p = .0001). In patients with acute viral hepatitis, protease levels were also significantly reduced (40.2 ±27% v s. 83.3 ±16.3% in healthy children)( p = .0001). Other patient groups had normal protease levels. In conclusion, low protease levels are far from being a specific beacon for TTP. The current paradigm that a single laboratory test may enable physicians to distinguish TTP from HUS seems to be challenged by these and other findings.     

新生儿持续肺动脉高压患儿血浆心房利钠肽、内皮素-1及血管性假血友病因子的变化

目的 探讨血浆心房利钠肽(ANP)、内皮素-1(ET-1)、血管性假血友病因子(vWF)水平在新生儿持续肺动脉高压(PPHN)中的变化及意义。方法 PPHN组患儿66例(轻度26例,中度21例,重度19例),对照组为非PPHN同期住院新生儿40例。对照组患儿入院时即行心脏超声检查,PPHN组患儿均于临床出现不易纠正的低氧血症(治疗前)及治疗后7 d行心脏超声检查测定肺动脉平均压(PASP),ELISA方法测定血浆ANP、ET-1、vWF的水平。结果 治疗前PPHN组患儿血浆ANP、ET-1、vWF水平较对照组明显升高(P<0.05),并随PASP增加而递增。治疗7 d后轻度及中度PPHN组患儿肺动脉压力恢复正常,血浆ANP、ET-1、vWF水平与对照组比较差异无统计学意义;重度组各指标较治疗前明显下降但仍高于对照组。治疗前后三指标水平均与PASP呈明显正相关(P<0.01)。结论 ANP、ET-1、vWF在PPHN治疗前后呈动态变化,且可反映肺动脉压力程度,动态监测有助于判断病情指导治疗。     

Hypopituitarism, deficiency of factors V and VIII and von Willebrand factor: an uncommon association

A 9 year-old boy with hypopituitarism and blood coagulation abnormalities is presented and discussed. The association between acquired von Willebrand disease and hypothyroidism has been reported but the combination of hypopituitarism and coagulopathy is unusual. Combined multiple clotting deficiencies are rare and, when present, factors V and VIII is the commonest association. Although it is known that hypothyroid patients may have a decrease in von Willebrand's factor (vWf) and factor VIII, there are no reports of hypopituitarism associated with combined deficiency of factors V, VIII, and vWf.     

新生儿硬肿症血浆血管性假血友病因子的意义

目的 探讨新生儿硬肿症血浆血管性假血友病因子(vWF)的意义.方法 采用免疫浊度法检测39例新生儿硬肿症和11例正常新生儿血浆vWF水平.结果 硬肿症组血浆vWF明显高于对照组(P＜0.005),轻度硬肿组与对照组比较无显著性差异(P＞0.05),中、重度硬肿组血浆vWF均明显高于对照组(P均＜0.05),且血浆vWF升高与硬肿症程度相一致;血浆vWF与血pH呈负相关(r=-0.808 P＜0.001).结论 中、重度硬肿症体内存在以高凝为主的早期DIC,血管内皮细胞损伤参与其病理生理过程.     

Effect of dexamethasone on rat plasma platelet activating factor acetylhydrolase during the perinatal period

It has been previously reported that the administration of dexamethasone (DEX) to adult rats increases the activity of plasma platelet-activating factor acetylhydrolase (PAF-AH) and prevents the development of intestinal necrosis caused by platelet activating factor (PAF) injection. In this report, we examined the effect of DEX administration on plasma PAF-AH activity during the perinatal period. Timed-pregnant rats received DEX (0.2–1.0 mg/kg/d) or normal saline (controls) on days 16–18 (early group) or days 18–20 (late group) of gestation. Maternal plasma PAF-AH activity was lower in late gestation than in postpartum period (P < 0.001). Fetal and neonatal plasma PAF-AH activity was higher than maternal values (P < 0.05). No changes of PAF-AH activity were seen in maternal, fetal or neonatal plasma after prenatal DEX administration at the aforementioned doses. A higher dose of DEX (1.3 mg/kg/d × 4d) or cortisone (200 mg/kg/d) produced an elevation of maternal plasma PAF-AH activity (DEX 79.2 ± 3.0, cortisone 70.5 ± 1.9 vs. controls 49.4 ± 2.3 nmol/min/ml, P < 0.01), but resulted in a high fetal mortality. Treatment of newborn rats with DEX (0.5 mg/kg/d) on days 1–3 after birth, increased plasma PAF-AH activity on day 4 (DEX 292 ± 5 versus controls 140 ± 9 nmol/min/ml, P < 0.001) and day 6 (DEX 302 ± 12 versus controls 136 ± 6 nmol/min/ml, P < 0.001). Postnatal administration of DEX increases the plasma PAF-AH activity in the rat. Only high doses of prenatal corticosteroids that cause fetal death can elevate maternal plasma PAF-AH activity.     

Type 2B von Willebrand disease associated with the release of platelet agglutinates from megakaryocytes in the bone marrow

We report a child with thrombocytopenia since birth, circulating platelet agglutinates, and a tendency to bleed. A bone marrow aspirate revealed large platelet clumps within the bone marrow and megakaryocyte nuclei surrounded by halos of clumped platelets. Laboratory evaluation revealed type 2B von Willebrand disease. Gene sequencing revealed a G to C mutation at base 3923 of the VWF gene. This mutation was previously described in a family with circulating platelet clumps and abnormal megakaryopoiesis with release of clumped platelets in culture. This same mutation was previously described in a family with circulating platelet aggregates and abnormalities of platelet release from megakaryocytes in vitro. Presence of megakaryocytes with halos of clumped platelets in our patient suggests that platelet agglutinate occurs in the bone marrow in some type 2B von Willebrand disease patients.     

Postnatal management of fetal and neonatal alloimmune thrombocytopenia: the role of matched platelet transfusion and IVIG

We evaluated the effects of platelet transfusions and intravenous immunoglobulin (IVIG) in neonates with fetal and neonatal alloimmune thrombocytopenia (FNAIT) with and without antenatal treatment with IVIG. Records of neonates with FNAIT admitted between January 2000 and November 2005 were reviewed. The patients were divided into group I, treated antenatally with IVIG for known FNAIT, and group II, postnatally diagnosed with FNAIT. The primary outcome was the time interval to reach a platelet level above 100 × 10⁹/L in relation to the type of treatment. Nineteen neonates with FNAIT were identified, 13 in group I and 6 in group II. In group I, four children were born with a platelet count above 100 × 10⁹/L and never needed treatment, and four received a single matched platelet transfusion at birth with a maintained response. Five neonates received IVIG and one matched transfusion, with all but one rapidly responding. In antenatally treated cases, postnatal IVIG had no apparent effect on the platelet count. In group II, two neonates died on day 1 with severe intracranial hemorrhage. Two of the four other patients responded to a number of unmatched platelet transfusions, with one neonate rapidly responding after one matched transfusion, while another needed nine matched transfusions before a persistent adequate platelet count was reached after 9 weeks. Postnatal IVIG had no apparent effect on the platelet count in any of our cases. In neonates with FNAIT treated antenatally with IVIG, neonatal management using a single matched platelet transfusion was adequate in all cases. In neonatally diagnosed cases not treated before birth, multiple matched platelet transfusions may be required. We found no evidence to support the use of IVIG in neonates with FNAIT.     

紫癜性肾炎患儿血浆内皮素—1、vWF、D—二聚体的变化及临床意义

对14例紫癜性肾炎(SHN)、17例不伴尿常规改变过敏性紫癜(SHP)和12例正常儿童的血浆内皮素-1(ET-1)、假性血友病因子(vWF)及D-二聚体(D-D)进行了检测。结果显示:SHN患儿的血浆ET-1(88.48±22.96)ng/L、vWF(1.59±0.38)U/ml及D-D(1.45±0.39)mg/L均显著高于对照组,分别为(43.73±17.89)ng/L、(0.99±0.30)U/ml,(0.28±0.23)mg/L.P均<0.01。在治疗后,SHN患儿的血浆ET-1、vWF和D-D水平均显著下降(P均<0.01);SHN患儿的血浆ET-1和D-D水平与血清肌酐呈正相关(分别为r-O.794,P<0.01;r=0.826,P<0.01)。表明肾血管内皮损伤所致ET-1过度生成、血管内凝血及继发性纤溶可能参与本病肾损伤过程。     

Endotoxin and hypoxia-induced intestinal necrosis in rats: the role of platelet activating factor.

We have previously shown that intravascular platelet activating factor (PAF) causes ischemic bowel necrosis in rats morphologically similar to neonatal necrotizing enterocolitis (NEC). Because endotoxin (LPS) and hypoxia are risk factors for NEC, we studied their effect on PAF metabolism and the development of intestinal injury. Young male Sprague-Dawley rats were anesthetized with pentobarbital and divided into six experimental groups: 1) control, 2) LPS alone (2 mg/kg), 3) hypoxia alone (5% O2), 4) LPS+hypoxia, 5) WEB 2086 (PAF antagonist)+LPS+hypoxia, and 6) SRI 63-441 (PAF antagonist)+LPS+hypoxia. Evaluations included blood pressure recording, superior mesenteric artery blood flow, arterial blood gas, white blood cell count, hematocrit, plasma PAF, plasma acetylhydrolase, plasma tumor necrosis factor, intestinal perfusion, and intestinal injury at 3 h. We found that LPS+hypoxia synergistically contributed to hypotension (mean blood pressure 27 +/- 5.6% baseline versus 101 +/- 3.9% control), metabolic acidosis (pH 7.05, base deficit 24 mEq/L), hemoconcentration, decreased superior mesenteric artery blood flow (2.2 +/- 0.3 mL/min versus 5.8 +/- 0.2 mL/min control), and intestinal injury. The morbidities resulting from LPS+hypoxia were partially or completely prevented by PAF antagonists. In addition, animals treated with LPS+hypoxia had neutropenia, elevated plasma acetylhydrolase, and elevated plasma TNF. These results suggest that endogenous PAF may contribute to LPS+hypoxia-induced intestinal hypoperfusion and necrosis.     

Shorter PFA-100® closure times in neonates than in adults: role of red cells, white cells, platelets and von Willebrand factor

In neonates, despite poor platelet function in various in vitro tests, closure times (CTs) in PFA-100^® measurements are shorter than in adults. Neonates have a higher polymeric von Willebrand factor (vWF). They also have a higher haematocrit and higher white blood cell count than adults, which may interfere with the evaluation of platelet and vWF function by means of the PFA-100 in neonates. To assess the role of different blood constituents on neonatal CTs, red blood cell, platelet and white blood cell counts in cord blood were modified. These modifications did not provide any evidence that the difference in number between adult and neonatal blood cells was responsible for shorter neonatal CTs. In further experiments, platelets and/or vWF were inhibited by means of abciximab and anti-vWF antibody, and mixing experiments with neonatal platelet-rich and platelet-poor plasma were performed. The results showed that short cord blood PFA-100 CTs were caused by a constituent of neonatal platelet-poor plasma, probably the neonatal high multimeric vWF. Conclusion: This study demonstrates that CTs in neonates are dependent on the same components, platelets and vWF, as in adults, making it likely that the PFA-100 can be used in neonates in the same way as in adults to investigate platelet and vWF function.     

Low von Willebrand factor in pediatric patients: Retrospective analysis of 293 cases informs diagnostic and therapeutic decision making

Low von Willebrand factor (VWF) poses diagnostic and therapeutic challenges for predicting bleed risk and need for empiric hemostatic therapy, particularly in children. Retrospective review identified 293 low VWF pediatric patients and investigated clinical and laboratory features. Low activity to antigen ratio was observed in 142 patients. When evaluation included VWF activity with gain‐of‐function glycoprotein Ib fragments (VWF:GPIbM) assay or VWF exon 28 sequencing, low VW was excluded in the majority (62%) of these patients. Application of a condensed bleeding assessment tool to quantify bleeding symptoms and use of the VWF:GPIbM assay may reduce the number of patients with bleed risk anxiety and unnecessary hemostatic management plans.     

Early elevation of plasma von Willebrand factor antigen in pediatric acute lung injury is associated with an increased risk of death and prolonged mechanical ventilation.

OBJECTIVE: Von Willebrand factor antigen (vWF-Ag) is a marker of pulmonary and systemic endothelial activation and injury. Adult studies indicate that patients with plasma vWF-Ag levels > or = 450% of control early in the course of acute lung injury (ALI) have an increased risk of death. The objective of this study was to evaluate whether vWF-Ag is elevated in the early phase of ALI in children and whether the magnitude of the increase was predictive of two important outcomes: mortality or duration of mechanical ventilation. DESIGN: Two-center, prospective observational study. SETTING: Two pediatric intensive care units: one in an academic university setting and one in a major community children's hospital. PATIENTS: After appropriate consent, plasma was collected from 48 pediatric patients on day 1 of ALI, 45 patients on day 2 of ALI, and four intubated controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Mean PaO2/FiO2 at the onset of ALI was 140 +/- 70, and mortality rate was 17%. vWF-Ag levels on day 1 of ALI were higher in patients compared with controls (287 +/- 183 vs. 87 +/- 84% of control [mean +/- SD], p < .05). Patients with vWF-Ag levels > or = 450% of control on day 1 of ALI had a markedly greater risk of death (odds ratio, 7.0; confidence interval, 1.31, 37.30; p < .05). Multivariate analysis revealed that elevated vWF-Ag level and either presence of multiple organ system failure or Pediatric Risk of Mortality III score independently predict increased risk of death. vWF-Ag levels on day 2 of ALI were significantly higher in patients who required prolonged mechanical ventilation (316 +/- 173 vs. 191 +/- 89% of control, p < .05). CONCLUSIONS: Early injury to the systemic and pulmonary endothelium, as measured by plasma vWF-Ag levels, is associated with an increased risk of death and prolonged mechanical ventilation in pediatric patients with ALI.