The establishment and validation of efficient assays for anti-IIa and anti-Xa activities of heparin sodium and heparin calcium

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0–102.8% and 101.6–102.8%, respectively), good intermediate precision (1.9–2.1% and 2.4–4.2%, respectively) and good reproducibility (4.0–4.8% and 3.6–6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.     

Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell line (JAR) to a human uterine epithelial cell line (RL95)

In vitro studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. In order to investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were used to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay was developed to determine if binding of JAR cells to RL95 cells was heparan sulfate–dependent. Labeled, single cell suspensions of JAR cells attached to confluent monolayers of RL95 cells in a dose- and time-dependent manner. Heparin-like glycosaminoglycans and JAR cell proteoglycans competitively inhibited JAR cell adhesion to RL95 cells by 50% or more. A panel of chemically modified heparins were used to demonstrate that O-sulfation and amino group substitution were critical for inhibition of cell-cell adhesion. Treatment with chlorate, an inhibitor of A ATP-sulfurylase, resulted in a 56% reduction in cell-cell binding compared to untreated controls. Heparinase and chondroitinase ABC markedly inhibited JARRL95 binding, while chondroitinase AC had no significant effect. These observations indicated that HSPGs as well as dermatan sulfate–containing proteoglycans participated in cell-cell binding. Collectively, these results indicate that initial binding interactions between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAGs) with heparin-like properties (i.e., heparan sulfate and dermatan sulfate). These observations are consistent with an important role for HS and heparin-like GAGs as well as their corresponding binding sites in early stages of human trophoblast-uterine epithelial cell binding.     

Prostaglandin F in monkey uterine fluid during the menstrual cycle and following steroid treatment

Prostaglandin F levels were determined in monkey uterine fluid collected daily from a silicone tubing collection system surgically installed in adult female rhesus monkeys. Sampling was obtained from intact and ovariectomized — hormone treated monkeys. During the primate menstrual cycle, uterine fluid prostaglandin F levels showed a cyclic pattern in concentration with highest values recorded during the 18–20th day of the cycle. Estrogen treatment to the ovariectomized female elicited a striking and marked increase in uterine fluid prostaglandin F levels while progesterone treatment had little effect. These results suggest that the presence of uterine fluid prostaglandin F is estrogen dependent and may be causely related to the control of menstrual cyclicity.     

Heparin oligosaccharide sequence and size essential for inhibition of pulmonary artery smooth muscle cell proliferation

Heparin has a wide range of important biological activities including inhibition of pulmonary artery smooth muscle cell proliferation. To determine the minimum size of the heparin glycosaminoglycan chain essential for antiproliferative activity, porcine intestinal mucosal heparin was partially depolymerized with heparinase and fractionated to give oligosaccharides of different sizes. The structure of these oligosaccharides was fully characterized by 1D and 2D 1H NMR spectroscopy. These oligosaccharides were assayed for antiproliferative effects on cultured bovine pulmonary artery smooth muscle cells (PASMCs). The tetrasaccharide (4-mer) exhibited no heparin-like activity. Decasaccharides (10-mers) and dodecasaccharides (12-mers) displayed a reduced level of activity when compared to full-length heparin. Little effect on activity was observed in deca- and dodecasaccharides with one less 2-O-sulfo group. The 14-, 16-, and 18-mers showed comparable growth-inhibition effects on PAMSC as porcine intestinal mucosal heparin. These data suggest that a 14-mer is the minimum size of oligosaccharide that is essential for full heparin-like antiproliferative activity. Since the 14- to 18-mers have no 3-O-sulfo groups in their glucosamine residues, their full activity confirms that these 3-O-sulfonated glucosamine residues, which are required for heparin's anticoagulant activity, are not an essential requirement for antiproliferative activity.     

Two-dimensional gel electrophoresis of endometrial protein in human uterine fluids: qualitative and quantitative analysis

By combining two-dimensional gel electrophoresis, protein staining and a sensitive computer-assisted gel scanning system, it was possible to examine human uterine fluid (n = 56) qualitatively and quantitatively for the presence of endometrial proteins. The protein concentration of uterine fluids ranged from 0.1 to 12.0 mg/ml with early secretory phase samples (n = 15) having significantly less protein (0.72 +/- 0.2 SEM mg/ml p less than or equal to 0.05) than the proliferative phase (n = 57) samples (1.58 +/- .29 SEM mg/ml). Whole blood contamination of uterine fluid, as measured by hemoglobin content, averaged 6.2 +/- 0.88% throughout the menstrual cycle. Human uterine fluids collected throughout the menstrual cycle were found to contain serum and up to 24 other proteins in addition to those previously described (MacLaughlin and Richardson, 1983). These proteins represent approximately 1% of the total protein in the gels and exhibit isoelectric points from 4.5 to 7.0 and molecular weights in the 26,000 to 60,000 range. These proteins are absent from human serum, which exhibits an identical pattern whether obtained in the proliferative or secretory phase of the menstrual cycle. These secreted endometrial proteins now become the standard against which to compare proteins identified in vitro using organ, gland and cell culture techniques and to characterize proteins that are regulated by steroid hormones in vivo.     

Anticoagulantly active heparin-like molecules from cultured fibroblasts

Heparin-like molecules, isolated from cultured rat and human skin fibroblasts, accelerated the inactivation of purified human thrombin via purified human antithrombin. Only 15% of the biologic activity of the complex carbohydrates derived from human skin fibroblasts was expressed when the heparin-binding domain an antithrombin was chemically modified at the Trp 49 residue. Human skin fibroblasts were metabolically labeled with [35S]Na2SO4, and radiolabeled heparin-like molecules were isolated utilizing ion-exchange chromatography and fractionated into two separate populations employing immobilized antithrombin. The species which bound with high avidity to the affinity matrix represented about 12% of the radiolabeled heparin-like molecules, accounted for almost 95% of the initial anticoagulant activity, and exhibited a specific activity of 3.98 USP units/10(6) 35S-cpm.     

Implantation-associated proteinase in mouse uterine fluid

Proteinase activity was detected in mouse uterine fluid by means of a new casein-substrate assay. The activity was found to be generally low in diestrous and proestrous stages of the estrous cycle and was more variable in proestrous. During pregnancy, activity was very low on day 1 (counting the plug date as day 0). By day 3, proteinase activity (expressed as Pronase equivalents/mg protein of uterine fluid) increased more than 100-fold, and then declined on day 4. Peak activity thus coincides with initiation of embryo implantation, which occurs on day 3 of pregnancy in the strain tested. The results provide direct biochemical support for previous indirect bioassay indications of the presence in uterine fluid of a proteolytic factor of uterine origin. The quantitative changes observed here are also consistent with previous bioassay observations and with the hypothesis that the uterine proteinase may mediate initial attachment of the blastocyst to the uterine wall. These and other data are used to formulate a 2-stage hypothesis of implantation, according to which uterine and trophoblast proteinases act sequentially to cause attachment and invasion, respectively.     

Cellular characteristics of nipple aspiration fluid during the menstrual cycle in healthy premenopausal women

Cellular characteristics of nipple aspiration fluid during the menstrual cycle in healthy premenopausal women Fifteen healthy premenopausal female volunteers underwent weekly nipple aspiration of ductal fluid from both breasts during two menstrual cycles to investigate the variability of the cellular profile of the ductal fluid. Ductal fluid was successfully obtained using breast massage and nipple-areolar suction from 247/280 (89%) breasts. 83% of samples available for cytological analysis were cellular and 30% of cellular aspirates contained ductal epithelial cells identified using standard morphological criteria. No significant variation in cell number or cell type was identified during the menstrual cycle. All samples tested had an 'H' score of zero for oestrogen receptor. Seven out of 14 women expressed the proliferation marker Mcm-2 in the cells of at least one of the specimens, with no evidence of a menstrual cycle influence on expression. In conclusion, the cellular profile of breast ductal fluid did not vary consistently during the menstrual cycle, permitting future breast cancer screening studies incorporating serial nipple aspirations to be performed independent of the phase of the cycle.     

Anti-inflammatory properties of a heparin-like glycosaminoglycan with reduced anti-coagulant activity isolated from a marine shrimp

The anti-inflammatory properties of a heparin-like compound from the shrimp Litopenaeus vannamei are related. Besides reducing significantly (p<0.001) the influx of inflammatory cells to injury site in a model of acute inflammation, shrimp heparin-like compound was able to reduce the matrix metalloproteinase (MMPs) activity in the peritoneal lavage of inflamed animals. Moreover, this compound also reduced almost 90% the activity of MMP-9 secreted by human activated leukocytes. Negligible anti-coagulant activities in aPPT assay and a poor bleeding potential make this compound a better alternative than mammalian heparin as a possible anti-inflammatory drug.     

Hormonal control of endometrial glycogen metabolism in the Macaca arctoides

Endometrial biopsies obtained throughout the menstrual cycle of the Macaca arctoides show the glycogen content paralleling the serum progesterone fluctuations which occur during the menstrual cycle. Secretory phase samples contained a three-fold higher concentration of glycogen when compared to follicular phase tissue. Changes in the activity levels of the glycogen metabolizing enzymes, glycogen phosphorylase and glycogen synthetase, during various stages of the menstrual cycle are in accord with the concept that the post-ovulatory increase in endometrial metabolism is a function of progesterone influence on this tissue. Endometrial glycogen synthetase activity remains low during the early proliferative phase of the cycle and becomes significantly elevated (two-to three-fold) during the early secretory phase of the cycle. Glycogen phosphorylase shows a similar cyclicity later in the luteal phase, reaching maximal activity between the seventeenth to nineteenth day of the cycle and remaining elevated through the twenty-sixth day of the cycle. The coincident nature of the rise in peripheral progesterone to increases in uterine glycogen metabolism suggest that progesterone may be the prime modulator of uterine endometrial metabolism during the post-ovulatory phase.     

超选择子宫动脉栓塞治疗子宫肌瘤

目的探讨超选择子宫动脉栓塞治疗子宫肌瘤的方法及疗效。方法子宫肌瘤65例采用Seldinger技术经皮股动脉插管至双侧髂内动脉,造影了解肿瘤的血供来源后,超选至两侧子宫动脉,缓慢注入平阳霉素碘油乳剂或PVA微粒栓塞治疗。结果患者症状缓解率为93．2％,月经增多,痛经,尿频、尿急,贫血等1-3个月内恢复正常。并发症：除栓塞综合征外,可见阴道不规则流血,腰腿痛,可自行恢复。治疗后3-6个月肌瘤缩小率在30％-68％之间。中短期疗效较稳定,末见复发。治疗前后性激素水平变化无明显差异。结论子宫动脉栓塞术治疗子宫肌瘤操作简单安全,疗效好。超选择子宫动脉插管其分支栓塞,并发症少,具有较大的临床应用价值。     

The effect of locally administered PGF2α on the contractility of the nonpregnant human uterus in vivo

The effect of locally administered prostaglandin F_2α on the sensitivity and reactivity of the nonpregnant human uterus during the menstrual cycle was studied. An increase in uterine contractility in response to as little as 1.0 μg PGF_2α could be observed in all patients during both the early and late portions of the menstrual cycle, but at the time of ovulation a marked decrease in sensitivity was noted. Endogenous prostaglandin normally occurs in the secretory endometrium in levels compatible with the amount of exogenous prostaglandin which elicited increased uterine activity. These findings support the hypothesis that PGF_2α plays an important physiological role in the cyclical regulation of uterine motility during the human menstrual cycle.     

Uterine infusion of conceptus fragments changes the protein profile from cyclic mares

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare''s uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.     

Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins

Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF165, the major isoform of VEGF, whereas 2-O-desulfated heparin and 6-O-desulfated heparin have weak but significant affinity (Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., and Kimata, K. (2004) J. Biol. Chem. 279, 12346-12354). In this study, we first examined the effect of heparin and modified heparins (completely desulfated N-sulfated heparin, 2-O-desulfated heparin, and 6-O-desulfated heparin) on VEGF165-dependent mitogenic activity and tube formation on type I collagen gels of human umbilical vein endothelial cells. Both were enhanced by heparin, but not by modified heparins, suggesting that both the 2-O-sulfate group of hexuronic acid and the 6-O-sulfation group of N-sulfoglucosamine in heparin/heparan sulfate are necessary for VEGF165 activity. We then examined the activation of VEGF receptor (VEGFR) to understand the mechanism. We have made several new findings; 1) heparin yielded a 1.7-fold enhancement of VEGF165-induced phosphorylation of VEGFR-2; 2) depletion of cell surface heparan sulfate by heparinase/heparitinase treatment and preferential reduction of trisulfated disaccharide units of cell surface HS by sodium chlorate treatment resulted in the reduction of such phosphorylation, suggesting the involvement of a heparin-like domain in the phosphorylation of VEGFR-2; and 3) VEGF121, an isoform without the exon 7-encoded region, which has no capacity to bind to heparin, did not show these effects. It is therefore likely that a heparin-like domain of heparan sulfate/heparin forms a complex with VEGF165 and VEGFR-2 via the exon 7-encoded region, thereby enhancing VEGF165-dependent signaling.     

未绝经宫颈癌患者肿瘤组织中环氧合酶2的表达及其临床意义

目的探讨环氧合酶2(COX-2)在宫颈癌组织中的表达与月经周期的关系及其临床意义。方法选取47例未绝经宫颈癌手术病人,病史结合子宫内膜HE染色观察判断患者所处月经周期时段,采用免疫组织化学SP法检测宫颈癌组织中COX-2及增殖细胞核抗原(PCNA)的表达,计算机图像分析COX-2免疫染色密度值及PCNA标记指数(LI)。结果宫颈癌组织中COX-2表达阳性者增生期表达强于分泌期(P<0.05),但阳性率无显著性差异(P>0.05),COX-2阳性癌组织中PCNALI高于阴性者,差异有显著性(P<0.01)。有淋巴结转移者COX-2的表达高于无转移者(P<0.05),宫颈癌组织中COX-2的表达与FIGO临床分期、组织分化程度无显著相关性(P>0.05)。结论未绝经宫颈癌患者肿瘤组织中COX-2的表达在增生期较分泌期明显增加,并参与调节肿瘤的生长、转移。     

不稳定型心绞痛患者血小板计数对低分子肝素抗凝有效性的影响

目的:通过检测抗Xa因子活性,探讨不稳定型心绞痛患者血小板计数(PLT)对低分子肝素抗凝有效性的影响。方法:入选不稳定型心绞痛患者63例,分为两组,A组(n=24):PLT≥240×109/L,B组(n=39):PLT240×109/L。首先比较两组患者之间一般情况有无差异,而后在两组患者皮下注射那曲肝素前及注射后8 h两个时间点分别进行抗Xa因子活性检测并记录结果,分析两组患者抗Xa因子活性水平并比较活性低于0.5 IU·mL-1患者所占比例。结果:两组患者之间一般情况无显著性差异,抗Xa因子活性治疗前后差异在两组中均有统计学意义,A组治疗后抗Xa因子活性均值低于B组,抗Xa因子0.5 IU·mL-1的患者比率高于B组,组间比较差异显著。结论:在低分子肝素抗凝治疗中,血小板计数≥240×109/L的患者抗Xa因子活性较低,提示不稳定型心绞痛患者在应用低分子肝素后的抗凝力度不足与血小板计数偏高相关。     

The effect of locally administered PGE2 on the contractility of the nonpregnant human uterus in vivo.

The effect of locally administered prostaglandin E2 on the sensitivity and reactivity of the nonpregnant human uterus during the menstrual cycle was studied in seven women. An increase in uterine contractility in response to as little as 0.25 mug PGE2 could be observed during both the mid-proliferative and mid-secretory phases of the menstrual cycle, but around ovulation a marked decrease in sensitivity to PGE2 was noted. An inhibition of uterine motility was observed during menstruation in response to 30--40 mug PGE2. Endogenous E prostaglandin normally occurs in the secretory endometrium in levels comparable to the amount of exogenous PGE2 which elicited increased or decreased uterine activity in this study. These findings suggest that PGE2 may play an important role in the cyclical regulation of uterine motility during the menstrual cycle.     

Capacitation of bovine spermatozoa by oviduct fluid

Oviduct fluid collected from chronically cannulated oviducts of heifers was evaluated for its effect on capacitation of bovine sperm in vitro. Capacitation was determined by the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine (LC). After incubation of sperm with 0-25% (v/v) estrual oviduct fluid (collected +/- 1 day from estrus) for 4 h, addition of LC (100 micrograms/ml) for an additional 0.25 h resulted in an increasing percentage of acrosome-reacted sperm as the concentration of oviduct fluid increased. Sperm incubated 4 h with 25% estrual oviduct fluid fertilized more oocytes than sperm incubated in medium alone (p less than 0.05) but was not different from sperm incubated with 10 micrograms/ml heparin (p greater than 0.05). Glucose inhibited the ability of LC to induce ARs in sperm incubated 4 h with heparin or estrual oviduct fluid. Incubation of sperm with 25% oviduct fluid collected at various days over the estrous cycle demonstrated that peak capacitating activity was found at estrus but was also present +/- 1 day from estrus. The active capacitating factor in oviduct fluid was found to be heat stable. In addition, when extraction procedures were applied in sequential order, oviduct fluid capacitating activity was resistant to protease digestion, precipitable by ethanol, size-excluded by Sephadex G-25, and destroyed by nitrous acid. These results suggest that a heparin-like glycosaminoglycan from the oviduct is a potential in vivo capacitating agent in the bovine.     

Effect of insemination dose and site on uterine inflammatory response of mares

It is unclear whether AI of mares deep into the uterine horn causes more or less inflammation of the endometrium than conventional AI. Thus, we compared uterine inflammatory reactions of mares inseminated with two different doses of frozen-thawed semen into the tip of the uterine horn (UH) ipsilateral to the preovulatory follicle with those of mares inseminated into the uterine body (UB). Thirty-two mares were assigned to one of four groups (eight mares/group): UB20=AI into UB, 20 x 10(6)sperm/0.5 mL; UB200=AI into UB, 200 x 10(6)sperm/0.5 mL; UH20=AI into UH, 20 x 10(6)sperm/0.5 mL; UH200=AI into UH, 200 x 10(6)sperm/0.5 mL, and inseminated 24 h after hCG administration. Before and 24 h after AI, they were examined with ultrasonography for the presence of intrauterine fluid. At 24 h, uterine fluid samples were obtained first by absorbing fluid into a tampon and then by uterine lavage. Uterine fluid was examined for polymorphonuclear leukocytes (PMN) and bacteriology, and frozen for lysozyme and TIC (trypsin-inhibitor capacity) assays. Only three mares conceived, one in each of the following groups: UB200, UH20, and UH200. Mares in the UH20 group accumulated less intrauterine fluid (p<0.05) than those in the other groups, which had similar amounts. No significant differences in PMN numbers were detected in either tampon or lavage fluid. Enzyme levels between groups did not differ statistically, except for TIC, which was lowest in the UH200 group. Thus, deep uterine horn AI caused no greater inflammation or irritation than uterine body AI in normal mares 24 h after insemination.     

不稳定型心绞痛患者血小板计数对低分子肝素抗凝有效性的影响

目的：通过检测抗Xa因子活性,探讨不稳定型心绞痛患者血小板计数（PLT）对低分子肝素抗凝有效性的影响。方法：入选不稳定型心绞痛患者63例,分为两组,A组（n=24）：PLT≥240×10^9／L,B组（n=39）：PLT〈240×10^9／L。首先比较两组患者之间一般情况有无差异,而后在两组患者皮下注射那曲肝素前及注射后8h两个时间点分别进行抗Xa因子活性检测并记录结果,分析两组患者抗Xa因子活性水平并比较活性低于0．5IU·mL^-1患者所占比例。结果：两组患者之间一般情况无显著性差异,抗Xa因子活性治疗前后差异在两组中均有统计学意义,A组治疗后抗Xa因子活性均值低于B组,抗Xa因子〈0．5IU·mL-1的患者比率高于B组,组间比较差异显著。结论：在低分子肝素抗凝治疗中,血小板计数≥240×10^9／L的惠者抗Xa因子活性较低,提示不稳定型心绞痛患者在应用低分子肝素后的抗凝力度不足与血小板计数偏高相关。