Malonyldialdehyde formation, oxygen consumption, fatty acid composition in newborn platelets stimulated by thrombin.

The release reaction, the formation of malonyldialdehyde (MDA), the pattern of oxygen consumption, and the variation in fatty acid composition after addition of thrombin (1.67 U/ml) have been investigated in newborn platelets, comparing the obtained data with analogous values showed by adult platelets assumed as normal controls. Newborn platelets showed a release reaction 20% lower than that of controls. MDA formation, even in the presence of NEM and the burst in oxygen consumption, resulted to be similar in newborn and adult platelets (p greater than 0.3); the burst was also similar after the addition of thrombin (10 U/ml) in the presence of antimycin and aspirin. The ratio between formed MDA and oxygen consumption was 1:10 in adult platelets while it was 1:15 in those of newborns. The study of fatty acid composition demonstrated that in newborn platelets at rest, arachidonic acid is significantly in a higher concentration than in controls and that it decreases after stimulation with thrombin. It is concluded that the pathway of prostaglandins is normally stimulated by thrombin in newborn platelets.     

A circulating inhibitor of platelet aggregation in Bartter's syndrome.

Aggregation studies were performed on platelets from five patients with Bartter's syndrome. Epinephrine failed to induce aggregation in all five patients. Adenosine 5'-diphosphate (ADP) produced a single reversible phase of aggregation, and there was depressed sensitivity to collagen. Response to ristocetin was normal. There was a dose-related inhibition of ADP-induced platelet aggregation when plasma from the patients was addeded to normal platelet-rich plasma. This inhibition was diminished or absent when patients were receiving aspirin. Washed platelets from two patients who were no longer undergoing aspirin therapy, showed a normal response to epinephrine in normal platelet-poor plasma. Bleeding time was reduced from 23 minutes to 12 minutes in one patient while on aspirin therapy. These studies suggest that a circulating inhibitor of platelet aggregation, probably of prostaglandin origin, is present in the plasma of patients with Bartter's syndrome.     

Differences in thromboxane production between neonatal and adult platelets in response to arachidonic acid and epinephrine

In this study, we have investigated the possible role of the pro-aggregatory arachidonic acid (AA) metabolite thromboxane, in the impaired function of neonatal platelets. In platelet-rich plasma thromboxane production (measured by radioimmunoassay of thromboxane B2) was not different between neonates and adults when stimulated by thrombin (at 0.1 or 1.0 U/ml) or collagen (70 micrograms/ml) although neonatal platelets produced decreased thromboxane (TBX2) postepinephrine stimulation. In response to 1 U/ml thrombin, adult and neonatal platelet-rich plasmas produced mean values of 3.41 +/- 0.35 (SEM) and 3.11 +/- 0.49 pmol of TXB2/10(6) platelets, respectively. Production of TXB2 in response to 0.1 U/ml thrombin was not dissimilar between neonates (1.01 +/- 0.46 pmol) and adults (1.04 +/- 0.38 pmol). When collagen was used as the aggregating agent, TXB2 production was also not significantly different with values of 2.44 +/- 0.48 and 1.90 +/- 0.46 pmol/10(6) platelets produced by adult and neonatal platelet-rich plasma, respectively. In response to 200 microM epinephrine, adult platelets produced 1.03 +/- 0.39 pmol TXB2/10(6) platelets while neonatal platelet TXB2 production was significantly decreased (0.15 +/- 0.04; P less than 0.05). Thromboxane production in response to AA, however, was markedly elevated in neonatal platelet-rich plasma. When 200 and 400 microM concentrations of AA were used as the aggregating stimuli, neonatal platelet rich plasma produced 3.17 +/- 0.77 and 8.0 +/- 1.47 pmol TXB2/10(6) platelets, respectively. These values were significantly elevated P less than 0.02 and less than 0.005) when compared to mean values of 0.41 +/- 0.10 and 3.32 +/- 0.15 pmol in adult platelet-rich plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of amniotic fluid on platelet thromboxane production

We have studied the effect of amniotic fluid on thromboxane A2 (TXA2) production as an initial step in an evaluation of the role of this metabolite as the mediator of the pulmonary hypertension that accompanies perinatal aspiration. Term amniotic fluid enhanced platelet thromboxane B2 (TXB2) production in the presence of the aggregating agents thrombin and arachidonic acid, activity being confined to the lipid fraction. Compared with a baseline production of 1.4 +/- 0.45 pmol TXB2/10(6) platelets in response to thrombin (1 U/ml), unfractionated amniotic fluid or its lipid fraction enhanced TXB2 production to 2.87 +/- 0.53 and 2.81 +/- 0.62 pmol, respectively (P less than 0.01). Values for the aqueous extract were no different from buffer control values (1.14 +/- 0.5). No enhancement of platelet TXB2 production was observed in amniotic fluid obtained at 15 to 17 weeks. Similar activity was observed with either adult or neonatal platelets. This thromboxane enhancing property of amniotic fluid appears to be distinct from its thrombin generating property. Following perinatal aspiration, in situ production of thrombin and proaggregatory TXA2 could recruit more platelets, enhance local TXA2 production, and be responsible for the platelet thrombi that have been documented at autopsy in the pulmonary microcirculation in infants with perinatal aspiration syndrome.     

Effect of maternal low dose aspirin on neonatal platelet function

OBJECTIVE: To evaluate the effect of maternal low dose aspirin ingestion in platelet function of newborn. DESIGN: Prospective randomized placebo controlled study. METHODS: 25 neonates born to mothers receiving low dose aspirin and 25 matched neonates with no maternal exposure to aspirin were studied. 2 ml of EDTA and 4.5 ml of citrate blood was collected from umbilical vein using double clamped umbilical stump for hemogram, coagulation profile and platelet functions. RESULTS: The platelet counts (10(9)/l) of study and control groups were 186.4 +/- 22.76 (116-225) and 205.28 +/- 17.34 (176-225), respectively. There was no significant difference in coagulation parameters. Prothrombin time index (PTI) was 86.24 +/- 6.623 and 87 +/- 6.43, respectively in the study and control group while PTTK (sec) was 55.88 +/- 20.54 and 52.12 +/- 11.82 in study and control subjects, respectively. The platelet aggregation studies (platelet function) with various platelet agonists in study and control group did not show any significant difference. Clinically, none of the babies had bleeding. CONCLUSIONS: Use of low dose aspirin in pregnant women was found to be safe and had no adverse effects on platelet functions of newborn.     

Platelet function in the neonate

Impairment of platelet function is well recognized in the neonate. The abnormalities include a reduction in platelet factor 3 activity and availability, a reduction in the release of nonmetabolic storage pool ADP and ATP, and platelet factor 4 following stimulation, decreased adhesiveness, and impaired aggregation with ADP, epinephrine, collagen, and thrombin. Whether the cause of the platelet abnormality and the impairment in platelet secretion is due to a "storage pool deficiency" or an "aspirin-like defect" has been unclear. However, recent data suggests that the neonatal platelet possesses neither a significant deficiency in prostaglandin synthesis nor a significant decrease in storage pool adenine nucleotides. The abnormalities noted appear most likely to be due to a membrane-related phenomenon.     

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.     

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.     

Glutathione peroxidase and glutathione S-transferase activity of platelets

A low Se intake in dietetically treated patients with phenylketonuria (PKU) or maple syrup urine disease (MSUD) leads to a marked reduction of the platelet glutathione peroxidase activity (GSHPx). The mean value amounted to 2.0 U/10(11) platelets with t-butyl hydroperoxidase (t-BOOH) (2.2 U/10(11) with H2O2) in patients and 5.8 U/10(11) with t-BOOH (5.4 U/10(11) with H2O2) in the control children. After Se supplementation with yeast rich in Se (dose: 135 micrograms Se/m2) the GSHPx activities rapidly increased. They reached a plateau after 2-3 weeks and remained there during the following 15-20 weeks of supplementation. After the cessation of supplementation there was a slow decrease, the values reached a low plateau after 24 weeks. In addition platelet glutathione S-transferase (GSHTf) was estimated with 1-chloro-2,4-dinitrobenzene. No significant difference between the values in healthy and dietetically treated patients in a low or normal Se state was observed. GSHTf did not exhibit peroxidase activity and did not show a compensatory increase when Se dependent GSHPx activity was low. The patients do not reveal clinical signs of disturbed platelet function. GSHPx may act in platelets via lipoxygenase on the prostaglandin pathway. The physiologic consequence of altered arachidonate metabolism, when GSHPx is deficient in platelets, remains to be elucidated.     

Platelet factor 4 and beta thromboglobulin in Moya-Moya disease

Increased concentrations of PF4 and beta TG were found in two patients with Moya-Moya disease. Although the pathogenesis of these abnormalities is unknown, the increased concentration of these proteins may result from platelet activation in the abnormal vascular network, leading to platelet aggregation and secretion of these two platelet-derived proteins, or alternatively, the increased concentration of these proteins may be the result of platelet aggregation due to a defect in endothelial prostaglandin production.     

Comparison of high-dose and low-dose aspirin plus intravenous immunoglobulin in the treatment of Kawasaki syndrome

The efficacy of intravenous immunoglobulin (IVIG) in the treatment of Kawasaki syndrome (KS) has been unequivocally established, but questions remain concerning the proper dose of adjunctive aspirin therapy in the treatment of KS. The medical records of 72 children with KS were reviewed. All patients were treated with IVIG; 21 received 400 mg/kg/dose on 4 consecutive days and 51 received 2 g/kg as a single infusion. Seventy patients also received aspirin. Twenty-four of the 70 patients were started on high-dose aspirin (80-100 mg/kg/day) at the time of diagnosis. High-dose aspirin was given for a mean (+/- SE) duration of 6.1+/-0.9 days, then switched to low-dose aspirin (3-5 mg/kg/day). Forty-six of the 70 patients were started on low-dose aspirin at the time of diagnosis and remained on low-dose aspirin for the duration of treatment. Coronary artery abnormalities were present at the time of diagnosis in 12 of 72 patients (17%), including 6 of 6 of patients (100%) with atypical KS and 6 of 66 patients (9%) with typical KS. None of the remaining 60 patients developed coronary artery abnormalities after treatment with IVIG and aspirin. The mean duration of fever after initiation of therapy was 44+/-6 hours in patients treated with IVIG 400 mg/kg/dose on 4 consecutive days and 35+/-5 hours in patients treated with 2 g/kg as a single infusion (p=0.3). The mean duration of fever after the initiation of therapy was 47+/-8 hours in patients treated with high-dose aspirin compared to 34+/-5 hours in patients treated with low-dose aspirin (p=0.13). These preliminary results indicate there is no benefit to high-dose aspirin compared to low-dose aspirin in the treatment of children with KS.     

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。     

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.     

Hyporeactivity of neonatal platelets is not caused by preactivation during birth

Little information exists concerning platelet function in neonates due to the small blood volume. Most studies using conventional aggregation methods have shown a diminished response to various agonists. This is in contrast to the lack of a bleeding tendency and to a short bleeding time in healthy neonates. In previous work we have shown that in healthy term neonates even after an uncomplicated delivery signs of thrombin generation can be demonstrated. This activation of the clotting system may also lead to platelet activation in the neonate. We investigated by means of flow cytometry the expression of activation markers GMP 140 and GP 53 on the surface of neonatal platelets and GP53 intracellularly by means of a newly developed assay. The expression of GMP 140 and of GP 53 after thrombin stimulation was significantly higher on adult rather than neonatal platelets, while there was no difference between neonatal and adult platelets using GP 53 as intracellular marker. In none of the healthy term neonates after an uncomplicated delivery were activated platelets demonstrated. Conclusion Our data show that the decreased reactivity of neonatal platelets is not caused by preactivation during birth but rather represents a developmental phenomenon. Possibly the observed hyporeactivity of neonatal platelets to thrombin helps to prevent harmful effects of birth stress on the clotting system of neonates. Received: 14 October 1996 / Accepted in revised from: 1 April 1997     

In vitro comparison of the efficacy of cyclooxygenase inhibitors on the adult versus neonatal platelet

Neonates manifest more hemorrhagic tendencies when exposed to either acetylsalicylic acid (ASA) or indomethacin in comparison to adults. We therefore assessed the susceptibility of neonatal and adult platelets to the effects of these cyclooxygenase inhibitors in vitro. Baseline thromboxane B2 production in response to thrombin was similar in platelets from the adult and neonate. Following exposure to varying concentrations (0.5-100 microM) of either ASA or indomethacin, platelet thromboxane B2 was inhibited to a similar extent in both adult and neonatal platelets. Our study demonstrates that the enhanced tendency to bleeding observed in the neonate following exposure to ASA or indomethacin is not due to an enhanced susceptibility of the neonatal platelet enzyme to the effects of cyclooxygenase inhibition.     

Platelet aggregation in term and preterm newborns

The platelets of newborns have a hyporeactive period. This period, during which the platelet count is normal but their functions are deficient, is called transient platelet hyporeactivity of newborns. The platelet functions and their normalizing process of term and preterm neonates are investigated. Twenty term and 20 preterm (gestational age <37 weeks) newborns were enrolled in the study. Twenty-eight healthy children aged 2 months to 3 years old participated in the study as the control group. Healthy newborns were followed for 15 days after birth longitudinally in 3 periods: period 1 (0-4 days), period 2 (5-9 days), period 3 (10-15 days). Aggregation studies were performed from whole blood samples. Whole blood aggregation was measured by the impedance method. Transient hyporeactivity of platelets was found in term and preterm groups, and there was no difference between term and preterms. Platelets of newborns gained their normal functions at postnatal 10-14 days. The results show that hyporeactivity of platelets during the first 9 days of life is physiological and transient.     

The relationship of von Willebrand factor binding to activated platelets from healthy neonates and adults

von Willebrand Factor (VWF) is important in platelet adhesion and shear-dependent platelet activation. We performed flow cytometric analyses of VWF binding to and activation of platelets from healthy neonates, children, and adults. Platelets from cord blood (n = 38; gestational age: 36-42 wk; birth weight: 2.4-5.1 kg), neonatal venous blood (n = 19; d 2-3 of life), children (n = 15; age: 1.5-16.3 y), and adults (n = 22; age: 18-55 y) were studied. Binding of VWF was assessed using an antihuman VWF polyclonal antibody and a FITC-conjugated secondary antibody. Platelet activation was determined by the expression of CD62P, CD63, CD41, CD42b, activated GPIIb/IIIa (PAC-1), procoagulant surface (as reflected by annexin V binding), and microparticle formation. Although the mean percentage of VWF-positive platelets was not significantly higher in unstimulated platelets from 2- to 3-d-old neonates, their platelets were more activated than those from adults, and there was a positive correlation of VWF binding with platelet activation (CD62P: r = 0.74, p < 0.001; annexin V: r = 0.46, p < 0.05). In adults, after in vitro activation of platelets with thrombin and ADP, VWF binding to platelets increased and correlated significantly with CD62P expression (r = 0.71, p < 0.001). VWF binding to unstimulated neonatal platelets was, however, higher than that to in vitro-stimulated platelets from adults at the same level of expression of platelet activation markers. Further studies are required to assess the mechanism and significance of VWF binding to activated platelets in the neonatal period.     

PLATELET FUNCTIONS IN CHILDREN WITH CONGENITAL HEART DISEASE

Abstract. Goldschmidt, B. (Second Department of Paediatrics, Semmelweis Medical University, Budapest, Hungary). Platelet functions in children with congenital heart disease. Acta Paediat Scand, 63 271, 1974.–Examinations of platelet functions (bleeding time, capillary resistance, clot retraction, thrombocyte adhesiveness in vitro and in vivo, spontaneous platelet aggregation, ADP and collagen induced platelet aggregation) were performed in children with CHD. In the cases with acyanotic heart disease (30 cases) the platelet functions were normal. The majority of cases with cyanotic CHD (35 cases) are thrombocytopenic. In these cases protracted bleeding time, increased capillary fragility and reduced clot retraction may be 'observed. The pathologic symptoms are connected with the low number of platelets. In patients with cyanotic CHD the platelet adhesiveness shows in vitro and in vivo a statistically significant increase. In one third of cases, platelet aggregation is also increased in cyanotic patients. Following the administration of ADP and collagen the aggregation shows a normal course.     

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.     

Surface expression of major membrane glycoproteins on resting and TRAP-activated neonatal platelets.

Platelets of full-term newborns and those of healthy adult donors were compared for constitutive expression of surface glycoproteins (GP) Ia-IIa, GP Ib, GP IIb-IIIa, and GP IV and for their activation responses to an agonist by detection of surface expression of activation markers P-selectin and CD63. Resting neonatal platelets showed significantly lower expression of GP Ia-IIa, GP Ib, and GP IIb-IIIa. In contrast, the expression of GP IV was significantly higher compared with platelets of adults. The expression of activation markers P-selectin and CD63 was assessed after in vitro activating of platelets with 0-15 microM human thrombin receptor-activating peptide. At low concentrations of thrombin receptor-activating peptide, the extent of surface expression of activation markers did not differ significantly between adult and neonatal platelets. However, after activation with 15 microM thrombin receptor-activating peptide, the extent of surface expression of P-selectin and CD63 was significantly lower in neonatal platelets. Because of ethical reasons, our study was conducted on neonates with a moderate neonatal hyperbilirubinemia. The remote possibility that hyperbilirubinemia could influence the expression of platelet surface receptors and the reactivity of neonatal platelet cannot be excluded. The role of higher expression of GP IV on neonatal platelets, also seen in certain hematologic malignancies in adults, remains to be elucidated. The lower expression of platelet adhesive receptors and the limited ability to up-regulate granular glycoproteins may play a role in the impairment of function of neonatal platelets.