Effects of age,gender and menstrual cycle on platelet function assessed by impedance aggregometry

Platelets are needed to prevent or arrest bleeding and aggregate at the site of injury upon vascular damage. Platelets express receptors for estrogens which might affect the function of the platelets and their hemostatic ability. The aim was to identify possible differences in platelet function related to age, gender, and phases of the menstrual cycle by use of impedance aggregometry with Multiplate. In the first part of the study, platelet function was assessed in 60 healthy individuals (30 men and 30 women) in each of three age groups (20–25, 40–45, and 60–65 years). In the second part of the study, the platelet function was analyzed on four occasions during the menstrual cycle in women without oral contraceptives (OCs) (n = 17) and compared to 19 women on OCs and 18 men of similar age (20–40 years). For the women on OCs, aggregation was analyzed once during the tablet-free week and once late during the period with OCs. The men were sampled once. Women of younger age (<45 years) had significantly higher agonist-induced aggregation response than both men and post-menopausal women (60–65 years). The agonist-induced aggregation response did not differ between phases of the menstrual cycle or OC use. The results suggest that estradiol and/or progesterone affect spontaneous aggregation since it was found to be lowest in the mid-luteal phase. Spontaneous aggregation was significantly lower in women on OCs than in both men and women without OCs. Our findings indicate that fertile age is associated with higher aggregation response capacity of the platelets, possibly to prevent excessive bleeding during menstruation, but this response capacity is not altered during the menstrual cycle or by use of OCs.     

Does the menstrual cycle affect anorectal physiology?

It has been shown that during the luteal phase of the menstrual cycle women excrete hard stools and have delayed transit, while at the time of menses, stools are looser and more frequent. The relationship between the menstrual cycle and anorectal physiology, and whether the timing of manometry testing should be standardized within the menstrual cycle has not been previously investigated. In this study, the anorectal responses to balloon distension during days 1–4 (menses), 8–10 (follicular phase), 18–20 (luteal phase) and 24–28 (premenstrual) of the menstrual cycle in 20 healthy female volunteers (ages 18–39 years) was measured. Stools were significantly looser during menses [2.32 (2.1–2.5); mean (95% CI)] compared with the luteal phase [1.98 (1.8–2.2):F(3,30) = 3.1;P<0.04). However, there were no changes in rectal sensitivity, distension-induced rectal motility, rectal compliance, or the volumes required to induce the initial and sustained internal aral sphincter relaxations. In conclusion, although stools become looser at the time of menses, anorectal responses to balloon distension are unaltered. This suggests that the changes in bowel habit associated with the menstrual cycle are unrelated to changes in anorectal physiology. Furthermore, routine manometric testing can be performed at any time during the menstrual cycle without fear of conflicting results.     

半胱氨酰-精氨酰-甘氨酰-天冬氨酰-色氨酰-青霉胺对血小板聚集和血栓形成的抑制作用

目的探讨自行合成的半胱氨酰-精氨酰-甘氨酰-天冬氨酰-色氨酰-青霉胺(Cys-Arg-Gly-Asp-Trp-Pen,W2003;即Arg-Gly-Mp肽类血小板抑制剂)抗人血小板聚集和动物体内预防血栓形成的活性,并与以前报道的N~a-乙酰-精氨酰-甘氨酰-天冬氨酰-苯乙胺(AcArg-Gly-Asp- NHCH_2CH_2C_6H_5,W2001)和N~a-乙酰-精氨酸-甘氨酸-天冬氨酸-对甲氧基苯乙胺(N~a-Ac-Arg-Gly-Asp-p-methoxyl phenylethylamine,W2002)比较。方法用比浊法和血小板计效的方法测定二磷酸腺苷(ADP)诱导的血小板聚集;用放射性配体分析法测定W2003竞争性抑制[~(125)I]纤维蛋白原(FGN)与血小板糖蛋白(GP)Ⅱb/Ⅲa的结合;用20?Cl_3刺激Wistar大鼠的颈总动脉产生动脉血栓动物模型,以被刺激动脉段形成的血栓湿重和干重评价该化合物预防血栓形成的作用。结果W2003有明显的抑制ADP诱导血小板聚集的活性.各浓度点(150,100,50,10,1μmol/L)间与生理盐水对照组比较差异均非常显著(P<0.01);抑制[~(125)I]FGN与血小板结合的IC50值为(31.5±7.0)μmol/L;W2003与以前报道的W2001和W2002比较.预防血栓形成作用的强弱顺序为:W2003>W2001>W2002,与生理盐水比较差异显著(P<0.01)。结论W2003通过抑制FGN与GPⅡb/Ⅲa的结合而发挥抗血小板聚集和预防血栓形成的生物活性。     

Inhibitory effect of aqueous extracts of some herbs on human platelet aggregation in vitro

Effect of aqueous extract of several herbs on human platelet aggregation in vitro was investigated. Out of 28 herbs/nutriceuticals investigated, camomile, nettle alfalfa, garlic and onion exhibited most significant anti-platelet activity (≥45% inhibition). Aqueous extracts of alfalfa, fresh nettle, and camomile inhibited ADP induced-platelet aggregation by 73, 65 and 60%, respectively, compared with control (P?<?0.05). Camomile and alfalfa inhibited collagen-induced platelet aggregation by 84 and 65%, respectively, but nettle could not inhibit collagen-induced aggregation. In contrast, nettle was the most potent inhibitor (66%) of whole blood aggregation induced by collagen, followed by alfalfa (52%), and camomile (30%) compared with control (P?<?0.05). None of these three herbs however could inhibit arachidonic acid or thrombin induced platelet aggregation. Camomile and alfalfa strongly inhibited thromboxane B₂ synthesis induced by ADP or collagen, but nettle had no effect. Alfalfa and nettle increased cGMP levels in platelets by 50 and 35%, respectively, compared with the control (1.85?±?0.23?nM) (P?<?0.005). All these data indicate that camomile, nettle and alfalfa have potent anti-platelet properties, and their inhibitory actions are mediated via different mechanisms.     

The effect of endogenous oestradiol levels on protein S concentration during a menstrual cycle and after GnRH analogues and gonadotropin therapy

Summary. We studied two groups of females to investigate the effect of endogenous oestradiol levels on total and free protein S (tPS, fPS) plasma concentrations. One group (group I) consisted of 12 healthy volunteers who were studied throughout one menstrual cycle; the other group (group II) consisted of 16 young women who were treated with GnRH analogues and gonadotropins before undergoing in vitro fertilization. Neither tPS nor fPS varied significantly with respect to the physiological changes of oestradiol or to the very low and high levels of oestradiol, achieved after GnRH analogues suppression and gonadotropin stimulation. These results indicate that endogenous oestradiol does not affect PS concentration.     

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.     

The effect of doubling the dose of clopidogrel on platelet aggregation in patients with clopidogrel resistance

Antiplatelet drugs play a pivotal role in the management of patients with acute coronary syndrome. Clopidogrel is an important antiplatelet drug commonly used in the treatment of those patients. However, the variability in patient response to clopidogrel is worrisome because a decreased response may lead to unfavorable clinical outcomes. Thus, it is crucial to study possible ways to overcome this problem. In the current study, we measured platelet aggregation in vitro to identify patients with clopidogrel resistance. Those patients were given double the standard maintenance dose of clopidogrel and platelet aggregation was reassessed. It was found that there was a significant increase in clopidogrel response (p < 0.001). Therefore, we recommend that using double the standard maintenance dose of clopidogrel should be considered in those patients.     

不同负荷剂量氯吡格雷对血小板聚集率的影响

目的比较两种剂量氯吡格雷的起效时间及安全性,为急性冠状动脉（冠脉）综合征患者用药方案提供依据。方法60例急性冠脉综合征使用不同负荷剂量氯吡格雷患者随机分为A组（300mg）和B组（600mg）,均予氯吡格雷75mg／d后续治疗。以腺苷二磷酸（ADP）5μmol／L及20μmol／L作为诱导剂检测服药前及服药后2h和6h的血小板聚集率,并检测服药前及服药后第3天的血自细胞及血小板计数。结果在ADP20μmoL／L诱导的血小板聚集检测中,两组均显示服药后6h比服药后2h达到更高的血小板聚集抑制水平[A组（29．75±12．11）％比（43．63±14．31）％,P〈0．05;B组（28．86±10．24）％比（34．86±10．84）％,P〈0．05]。B组与A组相比,在服药后2h即起到更加明显的血小板聚集抑制作用[（34．86±10．84）％比（43．63±14．31）％,P〈0．05]。服药后3d内所有人选患者均无出血、自细胞减少及血小板减少等事件发生。结论氯吡格雷600mg作为负荷剂量较之300mg可以更快地达到较高水平的血小板抑制作用,且两者安全性相似。     

The effect of ex vivo anticoagulants on whole blood platelet aggregation

Pre- and intraoperative platelet function monitoring is increasingly recommended in order to detect risk factors for bleeding and to target coagulation management. The ideal anticoagulant for accurate platelet aggregometry remains controversial. The aim of this experimental trial was to compare platelet aggregability in whole blood stored in citrate, heparin and direct thrombin inhibitors. Whole blood was drawn from 11 healthy adult volunteers who had not taken any medication in the previous 14 days. Blood was stored in trisodium citrate, unfractionated heparin, melagatran, lepirudin and argatroban. Platelet aggregation was performed using the impedance aggregometer Multiplate® (Dynabyte, Munich, Germany) with adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP), collagen, arachidonic acid and ristocetin as agonists. Samples were analysed immediately after blood sampling (baseline), as well as 30 and 120 min afterwards. At baseline there were no significant differences in aggregability between samples containing direct thrombin inhibitors and heparin. In contrast, aggregation in response to all agonists except for ristocetin was significantly impaired in citrated blood. During storage the response to arachidonic acid and collagen was maintained by direct thrombin inhibitors and heparin, whereas ADP-, TRAP- and ristocetin-induced aggregation varied considerably over time in all ex vivo anticoagulants tested. Pre-analytical procedures should be standardized because storage duration and anticoagulants significantly affect platelet aggregability in whole blood. For point-of-care monitoring with immediate analysis after blood withdrawal all tested direct thrombin inhibitors as well as unfractionated heparin can be used as anticoagulants whereas citrate is not recommended.     

Exome-chip meta-analysis identifies association between variation in ANKRD26 and platelet aggregation

Previous genome-wide association studies (GWAS) have identified several variants associated with platelet function phenotypes; however, the proportion of variance explained by the identified variants is mostly small. Rare coding variants, particularly those with high potential for impact on protein structure/function, may have substantial impact on phenotype but are difficult to detect by GWAS. The main purpose of this study was to identify low frequency or rare variants associated with platelet function using genotype data from the Illumina HumanExome Bead Chip. Three family-based cohorts of European ancestry, including ~4,000 total subjects, comprised the discovery cohort and two independent cohorts, one of European and one of African American ancestry, were used for replication. Optical aggregometry in platelet-rich plasma was performed in all the discovery cohorts in response to adenosine diphosphate (ADP), epinephrine, and collagen. Meta-analyses were performed using both gene-based and single nucleotide variant association methods. The gene-based meta-analysis identified a significant association (P = 7.13 × 10^–7) between rare genetic variants in ANKRD26 and ADP-induced platelet aggregation. One of the ANKRD26 SNVs - rs191015656, encoding a threonine to isoleucine substitution predicted to alter protein structure/function, was replicated in Europeans. Aggregation increases of ~20–50% were observed in heterozygotes in all cohorts. Novel genetic signals in ABCG1 and HCP5 were also associated with platelet aggregation to ADP in meta-analyses, although only results for HCP5 could be replicated. The SNV in HCP5 intersects epigenetic signatures in CD41+ megakaryocytes suggesting a new functional role in platelet biology for HCP5. This is the first study to use gene-based association methods from SNV array genotypes to identify rare variants related to platelet function. The molecular mechanisms and pathophysiological relevance for the identified genetic associations requires further study.     

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.     

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.     

In vivo effect of losartan on platelet aggregation in patients with hypertension

The angiotensin II receptor, losartan, has been found to inhibit platelet aggregability to some extent in in vitro experiments. There have been conflicting results about the in vivo effects of losartan. We sought to clarify the in vivo effect of losartan on platelet aggregation. Forty patients with grade I essential hypertension were treated with losartan for 3 weeks. Platelet aggregation tests with adenosine diphosphate (ADP) and ristocetin were analyzed and compared before and at the end of the study. Losartan effectively decreased systolic (SBP) and diastolic (DBP) blood pressure. Mean SBP before and after treatment was 159.6 ± 12.8 and 149.2 ± 17.3mmHg, respectively. Mean DBP decreased from 93.7 ± 8.2 to 87.7 ± 10.3mmHg after treatment. The results of the platelet aggregation tests with ADP and ristocetin were not significantly different when both rate and amplitude of maximal aggregation were included. Peak platelet aggregation with ADP regarding the lowest light transmission in the aggregometer was 59.8% ± 24.3% before and 58.3% ± 18.1% after the treatment. The same variables with ristocetin were 66.8% ± 21.6% and 60.8% ± 23.3%, respectively. In vivo effects of losartan on platelet aggregation with ADP and ristocetin were insignificant.     

The effect of radiographic contrast agents on bleeding time and platelet aggregation

The purpose of this study was to determine whether the use of relatively small amounts of radio-graphic contrast agents may impair hemostasis. Before and after injection of contrast agents, we measured the template bleeding time, platelet count in whole blood and platelet-rich plasma, platelet aggregation induced by epinephrine and adenosine diphosphate (ADP), and percent disaggregation of ADP-induced platelet aggregates in platelet-rich plasma of 18 patients. Ten patients received 50–100 ml of sodium iothalamate (66.8%) for intravenous urography and 8 patients received 125–200 ml of a combination of meglumine diatrizoate (66%) and sodium diatrizoate (10%) for coronary an-giography and left ventriculography. Neither group demonstrated a statistically significant mean difference (p >0.05) for any parameter measured before and after administration of the contrast agents. However, 2 patients who received diatrizoate had lengthening of their bleeding times to greater than twice the baseline value without any change in in vitro platelet aggregation. Physicians should be aware of this potential cause of bleeding which could be of particular danger to patients undergoing cardiopulmonary bypass with its inherent hemostatic defects.     

'Spontaneous' platelet aggregation in whole blood in diabetic patients with and without microvascular disease.

Consistent abnormalities of agonist-induced platelet aggregation, in either whole blood or platelet rich plasma, have not been demonstrated in diabetic patients without microvascular disease. In the present study platelet aggregation in the absence of exogenous agonists ('spontaneous' aggregation) was compared between 22 non-diabetic subjects and 23 Type 1 diabetic patients with (n = 12) and without (n = 11) microvascular disease. 'Spontaneous' aggregation was determined by measuring the percentage fall in single platelet number in aliquots of whole blood shaken for 60 min. Diabetic patients without microvascular disease had fewer single platelets remaining (greater aggregation) than non-diabetic subjects at all time-points (69.7 +/- 6.6 vs 82.3 +/- 7.3% at 60 min p less than 0.001), but more platelets remaining than in diabetic patients with microvascular disease at all time-points (69.7 +/- 6.6 vs 61.0 +/- 7.8% at 60 min p less than 0.02). No significant correlations were observed between platelet aggregation and plasma glucose, blood cell counts, or glycated haemoglobin levels. The study suggests that platelet abnormalities antedate the appearance of microvascular disease in diabetic patients.     

Alcohol consumption and platelet activation and aggregation among women and men: the Framingham Offspring Study

BACKGROUND: Alcohol intake has been associated with lower platelet activity; however, few large-scale studies have included women, and to our knowledge, the relationship of alcohol intake with measures of platelet activation has not been studied. METHODS: We performed a cross-sectional analysis of adults free of cardiovascular disease enrolled in the Framingham Offspring Study. Study physicians assessed alcohol consumption with a standardized questionnaire. We measured platelet activation in response to 1 and 5 microm of adenosine diphosphate (ADP) with a P-selectin assay among 1037 participants and platelet aggregability in response to ADP, epinephrine, and collagen among 2013 participants. RESULTS: Alcohol consumption was inversely associated with P-selectin expression in response to 1 microm ADP (p = 0.007) and 5 microm ADP (p = 0.02) among men but not women. Alcohol consumption was also inversely associated with platelet aggregation induced by ADP among both women (p = 0.04) and men (p trend = 0.008) and by epinephrine among men (p = 0.03) CONCLUSIONS: Alcohol consumption is inversely associated with both platelet activation and aggregation, particularly in men. Additional research is needed to determine whether these findings contribute to the contrasting associations of alcohol consumption with risk of thrombotic and hemorrhagic cardiovascular events.     

The effect of desmopressin on platelet aggregation defect in systemic amyloidosis: a preliminary report

Abstract: Systemic amyloidosis may often be complicated with haemorrhagic tendency. The causes of this manifestation are factor deficiencies, hyperfibrinolysis and vasculopathy. In order to investigate the role of platelets, if any, we performed platelet aggregation tests with different aggregants in 10 patients with systemic amyloidosis due to familial Mediterranean fever and 10 healthy controls. Platelet aggregation was defective with different aggregants (ADP, epinephrine, collagen) in patients compared with controls. Platelet aggregation tests repeated after desmopressin (DDAVP) administration were normalized. These findings may suggest a role of a platelet aggregation defect in haemorrhagic diathesis complicating systemic amyloidosis. DDAVP may benefit patients with this disease in case of bleeding and before surgical interventions.