The effect of menstrual cycle on platelet aggregation in reproductive-age women

Our aim was to assess the change in platelet activity along the menstrual cycle. We conducted a prospective observational study. The study group included 16 healthy women with regular menstrual cycles, which were compared to a control group of 14 healthy males. Exclusion criteria were age <18 years or >45 years, use of oral contraceptives or any other forms of hormonal therapy and medical disorders or medications that might affect platelet aggregation. Blood samples were taken from each of the women at four different phases of the menstrual cycle: day 1 ± 1, day 7 ± 1, day 14 ± 1, and day 21 ± 1. A single blood sample was taken from the males. Platelet aggregation was assessed in whole blood samples using the Multiplate? analyzer with three different agonists (ADP, arachidonic acid (AA), and thrombin-receptor activating peptide (TRAP)). Platelet aggregation for each of the women at each of the phases of the menstrual cycle was expressed as the percentage change from the day 1 ± 1 value. A total of 390 aggregation assays were performed. The mean aggregation activity was significantly higher in females compared with males, irrespective of the agonist used. For the TRAP and the ADP agonists, the relative platelet activity decreased along the menstrual cycle from day 1 towards day 21 and from day 7 towards day 21, respectively, although differences reached statistical significance only for day 21 (?12.4% ± 3.2%, P < 0.05 for TRAP, and ?9.5% ± 3.9%, P < 0.05 for ADP). When using AA to induce platelet aggregation, the relative platelet activity was highest around the time of ovulation (11.0% ± 4.7%) and was significantly lower on day 21 (?8.5% ± 6.7%, P < 0.05). In conclusion, platelet aggregation activity is higher in females compared with males. The association between the phase of the menstrual cycle and platelet activity appears to vary with the type of agonist, but platelet aggregation is consistently lowest in the mid-luteal phase irrespective of the agonist used.     

Effects of pH and concentration of sodium citrate anticoagulant on platelet aggregation measured by light transmission aggregometry induced by adenosine diphosphate

The 2013 ISTH-SSC guidelines for the standardization of light transmission aggregometry (LTA) were largely based on expert consensus, as studies directly comparing LTA methodologies were lacking. We experimentally tested the cogency of ISTH-SSC recommendations pertaining to use of anticoagulant, in particular whether: (1) buffered citrate (BC) is preferable to unbuffered citrate (C); (2) the two recommended concentrations of sodium citrate (109 and 129 mM) are equivalent in terms of platelet aggregation (PA). Blood from 16 healthy volunteers was collected into BC and C (109 and 129 mM). PA was measured by LTA in platelet-rich plasma (PRP) stimulated by adenosine diphosphate (ADP) (2 μM) immediately after PRP preparation and up to 4 hr after blood collection; pH and platelet counts in PRP were measured in parallel. pH in PRP increased with time up to about 8 for all anticoagulants, although it was lower in BC than in C at all times. In BC, PA was lower at 45 min, but equivalent at all other times. PA was higher and more stable in sodium citrate 109 mM than in 129 mM at all times. The extent of PA did not change for up to 2 hr after blood collection, and subsequently dramatically decreased. In contrast with ISTH-SSC recommendations, (1) BC does not show advantages compared to C; (2) 109 mM citrate is preferable to 129 mM, because it better supports PA; and (3) LTA studies should be completed within 2 hr of blood collection, instead of the recommended 4 hr.     

Effect of oral contraceptives and ovarian cycle on platelet function

In past decades, numerous epidemiological and clinical studies in women taking oral contraceptives revealed the impact of sex steroids on coagulation factors and the incidence of venous thrombosis. To date, only scarce data regarding the impact of oral contraceptives on platelet function are available. The aim of this study was to further elucidate the impact of sex steroids on platelet function. We conducted an observational study in young women using different types and dosages of monophasic oral contraceptives (OCs) compared to women not taking OCs. During the follicular phase, the mean closure time (CT) in Col/Epi was 168.0?±?64.9?s compared to 131.5?±?28.9?s during the luteal phase (p?=?0.012). In Col/Epi cartridges, no difference was detected between women taking second/third generation OCs and low-dose OCs (145.2?±?44.3 vs. 169.4?±?63.5, p?=?0.34). In contrast, mean Col/Epi values of women using anti-androgen-containing OCs were less (110.3?±?15.6?s) than in both other OC groups (p?=?0.03 for both comparisons). The same holds for Col/Epi values from women during the follicular- and luteal phases compared to women using anti-androgen-containing OCs (p?=?0.0002, p?=?0.013). Significant correlations between progesterone and platelet function in women not using OCs (p?=?0.02) could be found. In conclusion, the results of the study show that platelet function might be modulated by OCs and the female cycle. As for OCs, the main factor seems to be the progestagen. During the female cycle, the main impact on platelet function might be mediated by progesterone.     

Pre-analytical effects of pneumatic tube transport on impedance platelet aggregometry

Point-of-care platelet monitoring is increasingly used in cardiac patients treated with antiplatelet agents. The validity of a new assay needs to be evaluated not only for reproducible data in clinical samples, but also for other pre-analytical conditions that may influence measurements. The aim of this study was to evaluate the influence of a pneumatic tube system (PTS) for specimen transport on impedance platelet aggregometry. We evaluated 50 consecutive patients scheduled for coronary artery bypass surgery under oral therapy with 100 mg/d acetylsalicylic acid (aspirin). In each patient, three blood samples for platelet function analysis were taken before induction of anesthesia. The first sample was measured in the operating room (OR) area and designated as the reference value. The second sample was again measured by the same operator in the OR area using a random PTS transport. The third sample was sent to the central laboratory by PTS where it was measured by a local technician. Platelet function was assessed in whole blood by impedance aggregometry with a Multiplate? analyzer using thrombin-related activation peptide (TRAP test) and arachidonic acid (ASPI test) (Dynabite GmbH, Munich, Germany). Mean ± SD for TRAP test was 1009 ± 196 in the reference measurement. Bias ± 95% limit of agreement between the reference measurement and a second measurement for TRAP test were 126 ± 284 (n = 25) for untransported and 181 ± 316 (n = 25) for PTS transported samples. In the reference measurements, 48/50 (96%) of TRAP values were within the normal range. After PTS transport, 35/50 (70%) of TRAP measurements in the central laboratory were within the normal range (p < 0.001). Mean ± SD for ASPI test was 175 ± 137. Bias ± 95% limit of agreement for ASPI test were 12 ± 109 (n = 25) for untransported and 68 ± 250 (n = 25) for PTS transported samples. In the reference measurements, 7/50 (14%) ASPI values were above the cut-off level and defined as reduced aspirin responsiveness. After PTS transport, only 1/50 (2%) of the patients showed reduced aspirin responsiveness in the central laboratory measurements (p = 0.031). In conclusion, PTS transport had a significant influence on platelet function testing by the Multiplate^? analyzer. Significantly fewer test results indicated normal platelet function in TRAP test and reduced aspirin responsiveness in ASPI test after PTS transport. Therefore, clinical decisions regarding platelet function and aspirin responsiveness should not be based on blood specimens transported by a PTS system.     

Investigation of the contribution of an underlying platelet defect in women with unexplained heavy menstrual bleeding

Heavy menstrual bleeding (HMB) is often undiagnosed in women and can cause discomfort and distress. A haemostatic cause for excessive bleeding is often not routinely investigated and can lead to hysterectomy at an early age. A prospective cohort study was carried out to determine whether certain patients with unexplained HMB have an underlying platelet function defect (PFD). The Genotyping and Phenotyping of Platelets (GAPP) study recruited 175 women with HMB and 44 unrelated volunteers from 25 Haemophilia Centres across the UK, and a tertiary gynaecology service. Bleeding history was assessed using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT). Platelet count, platelet size, haemoglobin and mean corpuscular volume were measured in whole blood using the Sysmex XN-1000 Haematology Analyzer. Platelet function testing using lumiaggregometry and flow cytometry was performed in patients included in this study. A PFD was identified in 47% (82/175) of patients with HMB. Cutaneous bleeding was the most frequent additional bleeding symptom (89% in PFD and 83% with no PFD). Whole blood platelet count was significantly lower (P < 0.0001) between the PFD group and no PFD group. The prevalence of anaemia did not differ between patients and healthy volunteers. Clinical evaluation alone is insufficient to determine presence of an underlying PFD in patients with HMB. Platelet function tests may be considered and clinical guidelines may include them in their algorithms. An appropriate diagnosis and subsequent tailored management of HMB may prevent unnecessary surgery and help manage future haemostatic challenges.     

Effects of interleukin-2 administration on platelet function in cancer patients

Platelet function in 16 patients with metastatlc renal cell carcinoma and melanoma was studied sequentially over the first 96 hr of treatment with moderate and highdose inter-leukin-2 (IL-2). During the first 96 hr of therapy, an increased ex vivo platelet maximal aggregation (MA) response to ADP, epinephrine, and arachidonlc acld was paralleled by a decrease In the peripheral platelet count. Plasma speclmens from patients receiving the moderate dose schedule showed a significant IL-2 induced secretory response of the platelet α-granule components β-thromboglobulin (BTG) and platelet factor 4 (PF4) and the eicosanoid thromboxane B₂ (TBX₂) as measured by RIA. The increase in TXB₂ was highly correlated with MA when analyzed by bivariate regression analysls, whereas the addition of PF4 to TXB₂ in a multiple regression analysls further Increased their correlation to MA. The observed decrease In peripheral platelet count correlated significantly with MA and PF4 secretion. High-dose IL-2-treated patlents showed a statistically significant increase in the percentage of large platelets exceeding 12 fl in diameter and platelet responsiveness to hypotonic shock. These observations suggest that IL-2 therapy results in a reduced peripheral platelet pool, with an increased proportion of the remaining pool of platelets larger, more viable, and actlvated. © 1994 Wiley-Liss, Inc.     

Effects of pathogen reduction systems on platelet microRNAs,mRNAs, activation,and function

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen?+?ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin?+?UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p?<?0.05) and an impaired platelet aggregation response to ADP (p?<?0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.     

Urinary vasodilator and vasoconstrictor angiotensins during menstrual cycle, pregnancy, and lactation

Since normal human pregnancy is characterized by normotension in the face of an increased renin-angiotensin-aldosterone system (RAAS), we evaluated the temporal pattern of urinary excretion of a novel vasodilator within this system, angiotensin-(1–7) (Ang-[1–7]), during the menstrual cycle, pregnancy, and lactation. The urinary profiles of Ang I, Ang II, human chorionic gonadotropin, 17β-estradiol, and progesterone were also determined. During the menstrual cycle, urinary Ang-(1–7) and Ang II remained stable (mean cycle value: 94.6±11.3 and 11.4±1.1 pmol/g of creatinine, respectively) in nine females. In 10 normal pregnant women, urinary Ang-(1–7) and Ang II increased throughout gestation, averaging 1499.8±310 and 224.4±58 pmol/g of creatinine, respectively (p<0.05) at wk 35 and falling during lactation to 394.0±95 and 65.7±20 pmol/g of creatinine (p<0.05), respectively. The Ang-(1–7)/Ang II ratio was unchanged in the different reproductive periods. During the menstrual cycle, Ang II and Ang-(1–7) correlated with 17β-estradiol and progesterone using multivariate analysis (r=0.31, p<0.001) and r=0.28, p<0.02, respectively). During gestation, 17β-estradiol and progesterone correlated with urinary Ang-(1–7) (r=0.48, p<0.001 and r=0.47, p<0.001, respectively) and Ang II (r=0.24, p<0.03 and r=0.25, p<0.03, respectively); by multiple regression, only Ang-(1–7) correlated with both steroids (r=0.49, p<0.001). The progressive rise of Ang-(1–7) throughout gestation, probably modulated by estrogen and progesterone, suggests a physiologic counterregulation within the RAAS.     

Diagnostic evaluation of platelet function disorders

Platelet function disorders are inherited and acquired conditions that represent a common cause of bleeding. Their clinical findings are generally similar to von Willebrand disease. It is often challenging to diagnose common platelet function disorders due to heterogeneity in their features, uncertainties about their pathogenesis and genetic cause, variability in the procedures used to assess platelet function in diagnostic laboratories and the lack of diagnostic criterion. Some inherited platelet function disorders have been established to increase risks for bleeding and bleeding scores. However, bleeding history assessment tools are not validated for use in diagnosing platelet function disorders. Standardized tests that assess aggregation function, dense granule deficiency and dense granule secretion are useful for diagnosing common platelet function disorders, in addition to some rare conditions. Guidelines have emerged to improve and standardize the laboratory tests for diagnosing platelet function disorders, including how to interpret aggregometry findings. Nonetheless, there is need to further evaluate the features, pathogenesis and genetic cause of many platelet function disorders, including the inherited conditions that impair granule secretion.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

阿加曲班对急性脑梗死患者血小板功能的影响

目的探讨阿加曲班抗凝治疗对急性脑梗死患者血小板功能的影响及其可能的作用机制。方法将急性脑梗死患者60例分为阿加曲班组(30例)和对照组(30例)。2组均给予脑梗死基础治疗。在基础治疗上,阿加曲班组第1、2天给予阿加曲班120 mg/d,48 h持续静脉泵入,第3～7天给予阿加曲班20 mg,2次/d,抗凝治疗结束后加用氯吡格雷75 mg/d;对照组入院后即给予氯吡格雷75 mg/d,持续应用。测定2组治疗前后次日晨、48 h、第7、14天的血小板聚集率(PA),并观察治疗前后血小板计数、活化部分凝血酶时间的变化。结果阿加曲班组治疗后48 h、第7天的PA明显高于治疗前,治疗后第14天的PA明显低于治疗前(P<0.01)。对照组氯吡格雷治疗后次日晨、第7、14天PA明显低于治疗前(P<0.01)。阿加曲班组治疗前同治疗后次日晨、第7天PA差值与对照组治疗前同治疗后次日晨、第7天PA差值比较,差异有统计学意义(P<0.01)。结论急性脑梗死患者应用阿加曲班抗凝治疗时二磷酸腺苷诱导的PA增加。     

国产和进口氯吡格雷对不稳定性心绞痛患者血小板功能的影响

目的　观察国产氯吡格雷和进口氯吡格雷对不稳定性心绞痛 (UAP)患者血小板功能的影响 ,比较两药抗血小板作用的优劣及其安全性。方法　 4 0例UAP患者随机分为 2组 ,其中国产氯吡格雷组 2 0例 ,进口氯吡格雷组 2 0例。另选健康对照组 10例。两治疗组分别于服用氯吡格雷前、服用氯吡格雷 30 0mg 2h后及服用氯吡格雷 75mg1次 d 1周后抽血查血小板聚集率及血小板活化指标。结果　UAP患者血小板聚集率及血小板活化状态较健康对照组明显增高。治疗前国产和进口氯吡格雷组的血小板聚集率及血小板活化指标无显著差异。治疗后两治疗组之间无显著差异。结论　血小板的活化在UAP的发生、发展过程中起着重要的作用。国产和进口氯吡格雷均有良好的抗血小板作用 ,两者抗血小板聚集和活化的作用相似 ,且无明显的不良反应。     

雷帕霉素洗脱支架对冠心病患者介入术后血小板功能的影响

目的　比较雷帕霉素洗脱支架(CypherTM)与Bxsonic支架对冠心病不稳定性心绞痛患者血小板功能的影响。方法　选择6 2例冠心病不稳定性心绞痛患者,30例置入CypherTM 支架(CypherTM支架组) ,32例置入Bxsonic支架(Bxsonic支架组)。分别于术前、术后即刻、术后6h取冠状静脉窦血测定血小板α颗粒膜蛋白14 0 (GMP14 0 )浓度以及血小板表面GMP14 0的分子数和血小板最大聚集率(MPAR) ;分别于术前、术后2 4h、7天、1个月取肘静脉血检测上述指标。结果　冠状窦血样显示CypherTM支架组血浆GMP14 0、MPAR和血小板表面GMP14 0表达水平在术前和术后即刻与Bxsonic支架组无明显差异,血浆GMP14 0、MPAR和血小板表面GMP14 0分子数在6h明显低于Bxsonic支架组(P <0 .0 5 ) ;外周静脉血样显示GMP14 0、MPAR和血小板表面GMP14 0分子数在2 4h、7天时CypherTM支架组高于Bxsonic支架组(P <0 .0 5 ) ,1个月时CypherTM支架组血小板表面GMP14 0分子数仍然高于Bxsonic支架组(P <0 .0 5 )。结论　CypherTM支架对冠心病患者介入术后血小板功能的影响与Bxsonic支架不同,血小板活化时间较长,应注意坚持1个月以上的抗血小板治疗。     

Clopidogrel anti-platelet effect: an evaluation by optical aggregometry, impedance aggregometry, and the platelet function analyzer (PFA-100)

Platelet aggregation inhibition by clopidogrel may be suboptimal in 4-30% of patients. Traditionally, optical aggregometry is used to assess clopidogrel's anti-platelet effects by inhibition of ADP-induced aggregation in platelet rich plasma. Red blood cells are an important source of ADP and, thus, are known to modulate platelet function. Because the whole blood aggregation by impedance method assesses platelet function in a physiological milieu, we compared clopidogrel response by this method with the optical method in platelet rich plasma (PRP) and the Platelet Function Analyzer (PFA-100). Platelet function studies were performed in 17 healthy subjects at baseline and after 10 days of clopidogrel intake (75 mg/day). Optical and impedance aggregometry were performed after addition of ADP (10 and 20 microM) and collagen (1 and 2 microg/mL). For PFA-100 analysis, whole blood closure time was measured in collagen-coated cartridges with ADP and epinephrine. All subjects except one showed a decrease in ADP-induced aggregation using both aggregation methods. However, ADP-induced platelet aggregation was significantly inhibited when assessed in whole blood as compared to the optical method (71+/- 34% vs. 34.2+/- 23%, p = 0.0002); this suggests that whole blood aggregometry is more sensitive in the detection of clopidogrel effect in the presence of red cells, which are known to modulate platelet function. The PFA-100 ADP closure time was slightly prolonged above the reference interval in only 5/17 (29%) subjects, suggesting that this instrument is not able to detect clopidogrel effect. We conclude that whole blood aggregation appears to be more sensitive in detecting clopidogrel effect compared with the platelet rich plasma method; the PFA-100 was unable to detect clopidogrel effect in the majority of the subjects.     

Calcitonin immunostaining in monkey uterus during menstrual cycle and early pregnancy

Calcitonin has been shown to be a progesterone-regulated potential marker of the receptive endometrium in the rat and human. The present study was undertaken to immunohistochemically investigate the changes in calcitonin in the monkey uterus during the menstrual cycle and periimplantation period. Calcitonin immunostaining was primarily localized in the glandular epithelium on d 16, 20, and 25 of the menstrual cycle. During early pregnancy, calcitonin immunostaining was strongly observed in the glandular epithelium only on d 9 of pregnancy, the day before implantation. Since the high level of calcitonin immunostaining in the glandular epithelium during the luteal phase of the menstrual cycle and periimplantation period matched the high level of maternal progesterone during this period, the expression of calcitonin in monkey endometrium may be under the regulation of maternal progesterone.     

A new platelet parameter,the mean platelet component,can demonstrate abnormal platelet function and structure in myelodysplasia

Summary The mean platelet component (MPC) is a new platelet parameter generated by the Bayer ADVIA^TM 120 full blood count analyser as part of the routine complete blood count (CBC) test cycle. We report a case of myelodysplasia with bleeding complications and abnormal template bleeding time in whom low mean platelet component parameters were associated with partial platelet granule deficiency, demonstrated by transmission electron microscopy. We suggest that the mean platelet component is an inexpensive and rapid test to screen for platelet dysfunction related to ultrastructural abnormalities in myelodysplasia.     

Blockade of menstrual cycle by thyroidectomy in Japanese monkeys (Macaca fuscata fuscata)

To examine the role of thyroid hormones in the seasonal breeding cycle in Japanese monkeys (Macaca fuscata fuscata), sexually mature females were thyroidectomized (n=6) in early December, during the midbreeding season, or they received sham operations (n=4). They were housed indoors individually, and blood samples were collected two to three times a week to monitor gonadotropin and gonadal steroid hormone secretions. Control monkeys exhibited ovulatory cycles during the breeding season. The mean dates of onset and end of the ovulatory cycles were October 22±13 d and February 25±14 d, respectively. These dates coincided well with those of our colonies under captivity. By contrast, three of the six thyroidectomized monkeys terminated ovulatory cycles immediately after operations; the remaining three monkeys ovulated only once or twice after thyroid removal. The mean dates of onset and end of the ovulatory cycles of thyroidectomized monkeys were October 18±4 d and December 31±4 d, respectively. This was a significantly earlier termination of the ovulatory cycles than in controls. Mean concentrations of plasma thyroxine of control monkeys were maintained throughout the experimental period, whereas plasma thyroxine concentrations of thyroidectomized monkeys decreased abruptly to undetectable levels. Thyroidectomized monkeys exhibited significantly higher levels of plasma prolactin (PRL) than controls. Moreover, even in control monkeys, plasma PRL increased during the transition out of the breeding season. These results suggest that thyroid hormones play an important role in the regulation of ovulatory cycles in Japanese monkeys, directory or indirectly, possibly by mediating the changes of PRL secretion.     

Thrombocytopenia model with minimal manipulation of blood cells allowing whole blood assessment of platelet function

In vitro models of thrombocytopenia are useful research tools. Previously published models have shortcomings altering properties of platelets and other blood components. The aim of the present study was to develop a whole blood method to induce thrombocytopenia with minimal manipulation, and to describe platelet function in induced thrombocytopenia in individuals with healthy platelets. Hirudin anticoagulated blood was obtained from 20 healthy volunteers. One part of the blood was gently centrifuged at 130g for 15 minutes. The platelet-rich plasma was replaced with phosphate-buffered saline to establish thrombocytopenia. Various levels of thrombocytopenia were achieved by combining different volumes of baseline whole blood and thrombocytopenic blood. Platelet counts were measured by flow cytometry (Navios, Beckman Coulter) and routine haematological analyser (Sysmex XE-5000). Platelet function was analysed by impedance aggregometry (Multiplate® Analyzer, Roche) and by flow cytometry (Navios, Beckman Coulter) using collagen, adenosine diphosphate, thrombin receptor activating peptide-6 and ristocetin as agonists. Median baseline platelet count was 227×10⁹/l. The in vitro model yielded median platelet counts at 51×10⁹/l (range 26–93×10⁹/l). We observed minor, yet significant, changes in platelet size and maturity from baseline to modelled thrombocytopenia. In the thrombocytopenic samples, significant and positive linear associations were found between platelet count and platelet aggregation across all agonists (all p-values<0.001). Platelet function assessed by flow cytometry showed minimal alterations in the thrombocytopenic samples. A new whole blood-based model of thrombocytopenia was established and validated. This new model serves as a useful future tool, particularly to explore platelet function in patients with thrombocytopenia.     

Hydrogen peroxide,an inhibitor of platelet function: Effect on adenine nucleotide metabolism,and the release reaction

The in vitro effects of H₂O₂ on platelet adenine nucleotide metabolism and on the ADP-induced platelet release reaction were examined. All studies were performed on human platelet-rich plasma (PRP) preincubated with (³ H)-adenine. Within 3 min of incubation with PRP, H₂O₂ (100–500 μM) caused an irreversible reduction in the (³ H)-ATP level with a concomitant increase in (³ H)-IMP and (³ H)-inosine and hypoxanthine levels. The adenylate energy charge (AEC) initially decreased while the ATP level fell. The AEC, however, returned to levels slightly lower than the control during further incubation. No leakage of ATP and ADP to plasma was observed. The fall in the steady-state levels of (³ H)-ATP increased with increase of the H₂O₂ concentration (decrease of 8.7–40% at H₂O₂ concentrations from 5 to 600 μM). H₂O₂ pretreatment of PRP caused absence of ADP-induced biphasic aggregation, partial inhibition of the primary wave, and complete inhibition of release of platelet nonmetabolic ATP and ADP. Our in vitro findings support the view that part of the inhibitory effect of H₂O₂ may be related to the lowering of metabolic ATP levels in platelets.