Mast cell derived heparin activates the contact system: a link to kinin generation in allergic reactions

Contact activation occurs when plasma comes in contact with negatively charged man-made surfaces but no substance that initiates contact activation in vivo has been identified. We have isolated a mast cell heparin proteoglycan (MC-HepPG) from a Furth mouse mastocytoma-derived cell line that is analogous to human tissue-type mast cell HepPG. This material and other glycosaminoglycans (GAGs) were tested for their ability to accelerate the reciprocal activation of factor XII and prekallikrein and the autoactivation of factor XII. Quantitative analysis showed the MC-HepPG to be as active as dextran sulfate on a weight basis; hog intestine heparin, dermatan sulfate. keratan polysulfate and chondroitin sulfate C were less active, other sulfated polysaccharides were essentially inactive. Incubation of MC-HepPG in 1:4 diluted plasma resulted in complete cleavage of high molecular weight kininogen in a factor Xll-dependent reaction. All of the MC-HepPG dependent reactions described above were inhibited by preincubation of MC-HepPG with heparinase I and II but not by pretreatment with heparitinase. chondroitinase ABC or the serine protease inhibitor aPMSF thus indicating that heparin proteoglycan is indeed acting as an initiating ‘surface’. We analysed the proteoglycan preparation by HPLC gel filtration. Fractions spanning a molecular weight range of > 400000–8000 were active initiators. Comparison of the chromatograms obtained before and after cleavage of GAG side chains from the protein core suggested that dissociated GAGs in the MW range 69000–17000 are the most active species rather than the complete proteoglycan. MC-HepPG GAGs therefore represent a physiologic macromolecule with activity comparable to non-physiological surfaces in a purified system and with the capability to induce activation of the contact system in diluted plasma. Its ability to promote kinin generation links cellular and humoral inflammatory responses in the perivasculature and provides a possible explanation for the elevated kinin levels observed after allergen exposure.     

Use of liquid heparin for blood gas sampling in pediatric intensive care unit: A comparative study of effects of varying volumes of heparin on blood gas parameters

Background and Aims:

Pre-analytical errors in sample collection affect the reliability of blood gas (BG) analysis. Amount of liquid heparin as anticoagulant in samples for BG can affect results by its dilutional direct binding and compositional effects. The aim of this study was to examine the effect of varying amounts of heparin in blood samples on results.

Materials and Methods:

The prospective study was conducted on 15 children at a pediatric intensive care unit (PICU). Three different heparinized syringes were used containing minimal, 60 IU and 120 IU of heparin. A total volume of 1 ml blood in each syringe was taken and was analyzed by blood gas analyzer. Statistical analysis used related samples Friedman''s test and Wilcoxon signed ranks test for paired comparisons. The observed bias was also compared with the desirable bias according to specifications by Ricos et al.

Results:

There was a significant difference (P < 0.05) in values of pH, pCO₂, HCO₃⁻, Hb and Na⁺ in the three syringes. The pCO₂, HCO₃⁻ and Na⁺ levels decreased with the increasing amount of heparin. The observed percentage bias was more than desirable percentage bias specifications for pCO₂, HCO₃⁻, Hb, Na⁺, K⁺ and Cl⁻ levels.

Conclusions:

Syringes with minimal liquid heparin are most appropriate for studying BG parameters as they have the least effect on these parameters. There is a need to standardize the procedure of syringe preparation for BG analysis. Further studies are needed to compare minimal amounts of heparin with commercially available dry balanced heparin syringes.     

Improved blood compatibility and decreased VSMC proliferation of surface-modified metal grafted with sulfonated PEG or heparin

Although the technique of coronary stenting has remarkably improved long-term results in recent years, (sub)acute thrombosis and late restenosis still remain problems to be solved. Metallic surfaces were regarded as thrombogenic, due to their positive surface charges, and stenosis resulted from the activation and proliferation of vascular smooth muscle cells (VSMCs). In this study, a unique surface modification method for metallic surfaces was studied using a self-assembled monolayer (SAM) technique. The method included the deposition of thin gold layers, the chemisorption of disulfides containing functional groups, and the subsequent coupling of PEG derivatives or heparin utilizing the functional groups of the disulfides. All the reactions were confirmed by ATR-FTIR and XPS. The surface modified with sulfonated PEG (Au-S-PEG-SO₃) or heparinized PEG (Au-S-PEG-Hep) exhibited decreased static contact angles and therefore increased hydrophilicity to a great extent, which resulted from the coupling of PEG and the ionic groups attached. In vitro fibrinogen adsorption and platelet adhesion onto the Au-S-PEG-SO₃ or Au-S-PEG-Hep surfaces decreased to a great extent, indicating enhanced blood compatibility. This decreased interaction of the modified surfaces should be attributed to the non-adhesive property of PEG and the synergistic effect of sulfonated PEG. The effect of the surface modification on the adhesion and proliferation of VSMCs was also investigated. The modified Au-S-PEG-SO₃ or Au-S-PEG-Hep surfaces also exhibited decreased adhesion of VSMCs, while the deposited gold layer itself was effective. The enhanced blood compatibility and the decreased adhesion of VSMCs on the modified metallic surfaces may help to decrease thrombus formation and suppress restenosis. It would therefore be very useful to apply these modified surfaces to stents for improved functions. A long-term in vivo study using animal models is currently under way.     

Action of heparin and heparin-precipitated fraction of human blood plasma on antibody-producing cells in vitro

The effect of heparin and heparin-precipitated fraction of human blood plasma (HPF) on the ability of spleen cells of mice immunized with sheep's red blood cells to form plaques in vitro was studied. Heparin and HPF were found to inhibit plaque formation as a result of interaction with lymphocytes. It is postulated that the possible point of application of the action of heparin and HPF may be surface cell membranes of the antibody-forming cells.Laboratory of Clinical Immunology, Institute of Clinical and Experimental Medicine, Academy of Medical Sciences of the USSR, Siberian Branch, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 57–59, January, 1976.     

Effects of adenosine on ex vivo filtragometry platelet aggregation in relation to plasma levels

The aim of the present study was to investigate the concentration effect of adenosine on unstimulated platelet aggregation in humans. Adenosine infusion was given intravenously to 12 volunteers in the antecubital vein with infusion rates increasing from 20 to 100 μg kg^?1 min^?1. Filtragometry measurements were obtained from the contralateral antecubital vein before and during 100 μg kg^?1 min^?1 or during maximal tolerable infusion rate. In another set of experiments with 10 volunteers, basal filtragometry measurements were obtained before and after infusion of various concentrations of adenosine into the filtragometer test unit. With intravenous infusion aggregation time tended to increase from 333±42 to 418±8 s (mean±SEM) and increased the venous plasma adenosine concentration from 0.42±0.09 μM to 1.52±0.38 μM . Adenosine infusion into the filtragometer tubing system dose-dependently inhibited aggregation (P<0.05). Adenosine was rapidly eliminated with a half-life of adenosine in the filtragometry tubing system calculated to be about 6 s. These data extend our knowledge from an in vitroto an ex vivo situation that adenosine dose-dependently has a platelet antiaggregatory effect.     

低分子肝素对URSA患者外周血免疫细胞的影响

目的探讨低分子肝素对不明原因复发性流产（URSA）患者外周血免疫细胞的影响。方法研究组包括28例URSA患者,予低分子肝素（Fraxiparine 0.4-0.6ml/d或Enoxaparin 0.4-0.6ml/d iH）治疗至妊娠12w,于治疗前、3w后采用流式细胞术检测患者外周血中淋巴细胞亚群及血常规;15例未接受低分子肝素治疗的URSA患者为对照组,于首次就诊（血HCG确认妊娠）及2~3w后（流产清宫前）检测以上项目。结果研究组保胎成功率为86%,明显高于对照组33%（P〈0.05）;使用低分子肝素后USRA患者外周血中总T细胞数比例为（66.9±5.6）%,较使用前（61.2±7.4）%升高（P〈0.05）;NK细胞比例为（16.6±4.4）%,较使用前（19.4±7.3%）下降（P〈0.05）,中性粒细胞比例为（67.1±7.9）%,红细胞压积为（36.1±3.9）%,较使用前（70.7±7.8%）、（33.8±7.8%）均下降（P〈0.05）。结论低分子肝素对URSA有显著治疗效果,其作用机理除抗凝外,还可能直接降调URSA外周血中的NK细胞及中性粒细胞。     

慢性ITP患者血小板膜表面GPⅣ、GPⅤ特异性自身抗体的测定

目的：检测慢性ITP患者血小板膜表面GPⅣ、GPⅤ特异性自身抗体。方法：应用改良直接MAIPA(单克隆抗体俘获血小板抗原技术)检测血小板膜糖蛋白(GPⅣ、GPⅤ)特异性自身抗体。结果：慢性ITP患者血小板洗脱液中GPⅣ特异性和GPⅤ特异性自身抗体的阳性率分别为6．3％和4．8％。结论：血小板膜GPⅣ、GPⅤ分子是慢性ITP自身抗体的靶抗原，但多与GPⅡb／Ⅲa或GPⅠb自身抗原共存。     

Determination of Optimal Conditions for the Immobilization of Cells in a Cell Capture Enzyme Immunoassay (CC-EIA) by a Simple Giemsa Assay

To determine the optimal conditions for the immobilization of cells in a cell capture enzyme immunoassay (CC-EIA), the most suitable diluent, and the optimal pH, temperature and period of incubation were examined using WI-38, a human embryonic lung fibroblast cell line. For the evaluation, we devised a simple Giemsa assay method, in which immobilized cells on a microplate were stained with Giemsa solution, the stained dye was eluted with ethanol after washing the plate, and the optimal density (O.D.) was measured at wavelength 620 nm. the optimal conditions for the immobilization were determined to be treatment with 5% formalin in phosphate-buffered saline (PBS) (pH 7.2) for 15 minutes at room temperature, which were confirmed to be suitable for the measurement of cell associated collagen by CC-EIA. Additionally, we found that the simple Giemsa staining method was also useful for evaluating the number of immobilized cells on the microplate after CC-EIA.     

Prediction of inflammatory responses induced by biomaterials in contact with human blood using protein fingerprint from plasma

Inappropriate complement activation is often responsible for incompatibility reactions that occur when biomaterials are used. Complement activation is therefore a criterion included in legislation regarding biomaterials testing. However, no consensus is yet available regarding appropriate complement-activation-related test parameters. We examined protein adsorption in plasma and complement activation/cytokine release in whole blood incubated with well-characterized polymers. Strong correlations were found between the ratio of C4 to its inhibitor C4BP and generation of 10 (mainly pro-inflammatory) cytokines, including IL-17, IFN-γ, and IL-6. The levels of complement activation products correlated weakly (C3a) or not at all (C5a, sC5b-9), confirming their poor predictive values. We have demonstrated a direct correlation between downstream biological effects and the proteins initially adhering to an artificial surface after contact with blood. Consequently, we propose the C4/C4BP ratio as a robust, predictor of biocompatibility with superior specificity and sensitivity over the current gold standard.     

Anticoagulant activity and platelet aggregation in blood plasma from rats after intranasal administration of adenosine triphosphate and its complex with heparin

Fourfold intranasal administration of ATP was followed by an increase in anticoagulant activity and decrease in platelet aggregation in blood plasma of healthy rats and, particularly, of animals with suppressed function of the anticoagulant system. These changes decrease the risk of the prethrombotic state. Complex of ATP and high-molecular-weight heparin decreased platelet aggregation only in the blood from healthy animals, but increased anticoagulant potential of plasma hemostasis in prethrombotic animals. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 9, pp. 244–246, September, 2008     

Effect of heparin on activity of oxidative enzyme systems of liver microsomes in healthy rats and rats with experimental glomerulonephritis

To study the effect of heparin on activity of the oxidative enzyme systems of the liver microsomes of healthy rats and of rats with glomerulonephritis the hexobarbital test was carried out, the content of cytochrome P₄₅₀ in the liver and the relative weight of the liver were determined, and a histological and histochemical investigation of the liver was undertaken. Heparin was found to stimulate the detoxicating function of the liver, disturbed in experimental glomerulonephritis.Central Postgraduate Medical Institute, Moscow. N. I. Pirogov Second Moscow State Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 4, pp. 412–414, April, 1978.     

Influence of heparin coating on in vitro bacterial adherence to poly(vinyl chloride) segments

End-point attached, covalently bound heparin has been shown to be effective in preventing activation of the coagulation cascade by biomaterials. Data concerning its possible influence on bacterial attachment and resistance to biomaterial-associated infection are, so far, lacking. In the present work, the in vitro adherence of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli, one isolate of each species, to plain poly(vinyl chloride) (plain PVC) and heparin coated poly(vinyl chloride) (EPA-PVC) segments was compared. Also, the influence of precoating the segments with human normal plasma for 2 h was studied. ³⁵S-Methionine was used to radiolabel bacteria. The segments were exposed to bacterial suspensions of approximately 10⁷ colony forming units (CFU) per milliliter at 37°C for 0.5-6 h. Following repeated washing in phosphate buffered saline (PBS), radioactivity associated with the segments was measured. Plain PVC as compared to EPA-PVC bound significantly more cells of all three tested species. Plasma precoating significantly decreased adherence of the tested species to plain PVC but did not affect the binding to EPA-PVC. However, after precoating with human plasma, EPA-PVC compared to plain PVC showed a higher binding of S. aureus which might possibly be due to bridging effects of fibronectin or other plasma proteins, interacting with S. aureus.     

增殖诱导配体在自身免疫病患者外周血中的表达

为检测增殖诱导配体(APRIL)在自身免疫病(SLE和RA)患者中的表达情况以及它与B细胞刺激因子(BAFF)表达、疾病预后、抗-dsDNA抗体之间的相关性。收集58名自身免疫病患者以及20名健康对照的外周血,用实时定量RT-PCR方法检测PBMC中APRIL和BAFF mRNA的表达,用ELISA方法检测血浆中APRIL和BAFF蛋白水平。同时,检测血浆中IgG、IgA、IgM、BF和抗-dsDNA抗体水平。结果同对照组相比,APRIL mRNA水平在自身免疫病患者中显著升高,与BAFF mRNA表达水平有相关性。与疾病初发组和治疗后缓解组相比,治疗后来缓解组患者APRIL mRNA表达显著升高。疾病组外周循环中BAFF蛋白显著升高,APRIL蛋白未见升高,APRIL和BAFF之间未见显著意义相关性,A-PRIL蛋白与抗-dsDNA和疾病活动度之间未见显著相关。检测APRIL mRNA含量是判断自身免疫病患者治疗情况和疾病预后的有用指标。循环中APRIL和BAFF蛋白的产生可能受到不同机制调节,在自身免疫病的发生、发展以及转归中所起的作用也不同,为研究APRIL的产生机制、表达水平和自身免疫病程度、早期诊断、预后等的关系打下基础。     

Markers of platelet activation in plasma of patients suffering from persistent allergic rhinitis with or without asthma symptoms

BACKGROUND: Increased circulating levels of platelet release products are detected in various types of inflammation. It has been demonstrated that allergen challenge promotes platelet activation and leads to the release of chemokines such as platelet factor 4 (PF-4) and beta-thromboglobulin (beta-TG). OBJECTIVE: The aim of this study was to determine whether or not circulating platelets get into an activated state during allergic inflammatory reactions induced by long-term natural exposure to allergens. METHODS: Plasma levels of PF-4 and beta-TG (established markers of in vivo platelet activation) were determined by ELISA method in symptomatic patients with allergy to house dust mites, suffering from persistent allergic rhinitis (PAR) in the absence of asthma symptoms (12 men and 8 women; mean age 22 years) and from PAR with mild asthma (10 men and 6 women; mean age 23 years), as well as in healthy controls (10 men and 10 women; mean age 23 years). RESULTS: No significant differences were found between PAR patients with or without symptoms of asthma and healthy non-atopic subjects with respect to plasma levels of PF-4 and beta-TG as well as platelet count. CONCLUSIONS: It seems that patients undergoing continuous natural exposure to sensitizing allergens, with subsequent PAR alone or with concomitant mild asthma, have no altered platelet activity in vivo, as reflected by plasma levels of the chemokines. These findings, in conjunction with earlier data, indicate that differences may exist in platelet activity, including releasability of platelet products between patients with distinct clinical manifestation of atopy.     

Influence of intraperitoneal and systemic application of taurolidine and taurolidine/heparin during laparoscopy on intraperitoneal and subcutaneous tumour growth in rats

Background: Recent clinical and experimental studies investigated the problem and possible pathomechanisms of port-site metastases after laparoscopic resection of malignant tumours. A generally accepted approach to prevent these tumour implantations does not exist so far. Methods: After subcutaneous and intraperitoneal injection of 10⁴ cells of colon adenocarcinoma (DHD/K12/TRb) the influences of either taurolidine or taurolidine/heparin on intraperitoneal and subcutaneous tumour growth were investigated in 105 rats undergoing laparoscopy with carbon dioxide. The animals were then randomised into seven groups. A pneumoperitoneum was established using carbon dioxide for 30 min (8 mmHg). Three incisions were used: median for the insufflation needle, and a right and left approach in the lower abdomen for trocars. To investigate the intraperitoneal (local) influence of either taurolidine and heparin on tumour growth the substances were instilled intraperitoneally. Systemic effects were expected when the substances were applied intravenously (iv). Synergistic influences were tested when both application forms were combined. The number and the weight of tumours as well as the incidence of abdominal wall and port-site metastases were determined four weeks after intervention. Blood was taken to evaluate the influences of taurolidine and heparin on systemic immunologic reactions: seven days before laparoscopy, two hours, two days, seven days, and four weeks after operation, and the peripheral lymphocytes were determined. Results: Intraperitoneal (ip) tumour weight in rats receiving taurolidine (median 7 mg) and taurolidine/heparin (0 mg) intraperitoneally was significantly reduced when compared to the control group (52 mg) (P=0.001). There was no difference of subcutaneus tumour growth among the groups (P=0.4). Trocar recurrences were decreased when taurolidine was applied ip (3/15), ipiv (4/15), and ip in combination with heparin (4/15) in comparison to the control group (10/15). Immediately after intervention treated and untreated groups showed a peripheral lymphopenia. Conclusions: The intraperitoneal therapy with taurolidine and the combination with heparin inhibits the intraperitoneal tumour growth and trocar recurrences. Neither the intraperitoneal nor the systemic application or the combination of taurolidine and heparin did reduce the subcutaneous tumour growth. The intervention caused a lymphopenia which was compensated on day two. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immobilization of heparin/poly-l-lysine nanoparticles on dopamine-coated surface to create a heparin density gradient for selective direction of platelet and vascular cells behavior

Restenosis, thrombosis formation and delayed endothelium regeneration continue to be problematic for coronary artery stent therapy. To improve the hemocompatibility of the cardiovascular implants and selectively direct vascular cell behavior, a novel kind of heparin/poly-l-lysine (Hep/PLL) nanoparticle was developed and immobilized on a dopamine-coated surface. The stability and structural characteristics of the nanoparticles changed with the Hep:PLL concentration ratio. A Hep density gradient was created on a surface by immobilizing nanoparticles with various Hep:PLL ratios on a dopamine-coated surface. Antithrombin III binding quantity was significantly enhanced, and in plasma the APTT and TT times as coagulation tests were prolonged, depending on the Hep density. A low Hep density is sufficient to prevent platelet adhesion and activation. The sensitivity of vascular cells to the Hep density is very different: high Hep density inhibits the growth of all vascular cells, while low Hep density could selectively inhibit smooth muscle cell hyperplasia but promote endothelial progenitor cells and endothelial cell proliferation. These observations provide important guidance for modification of surface heparinization. We suggest that this method will provide a potential means to construct a suitable platform on a stent surface for selective direction of vascular cell behavior with low side effects.