Platelet adhesion and spreading on protein-coated surfaces: variations in behavior in washed cells, PRP, and whole blood

Platelet attachment and spreading were monitored on glass and various protein coated glass, under shear with washed platelets, platelet rich plasma (PRP) and whole blood, using fluorescence Optimas imaging system and software. Results showed that the platelet adhesion and spreading were sensitive to the nature of precoated proteins and the type of medium used for introducing platelet suspension for the study. In general, the cell adhesion and spreading were higher with fibrinogen (Fg), fibronectin (Fn), von Willebrand Factor (vWF), and collagen precoated surfaces. In the presence of albumin on the surface, however, platelets could not attach and spread fully when using washed cells. But, the surface attachment and spreading of the cells were higher on albumin substrates on exposure to PRP or whole blood. This may be due to the replacement of precoated albumin by other plasma proteins, like Fg to facilitate the platelet-surface attachment. The composition of this layer determines the extent of platelet activation and the adhesive strength between platelets and polymer surface. These results indicate that multiple adhesion receptors can mediate platelet adhesion and spread to matrix proteins immobilized on surfaces. Further, these studies combined with some of our earlier observations and suggestions propose the need for developing in vitro tests that resemble in vivo conditions.     

壳聚糖与丙烯酸甲酯接枝用水合肼改性及其吸附性能的研究

本文通过对壳聚糖(chitosan)用H_2O_2-Fe(Ⅱ)作引发剂,用丙烯酸甲酯接枝及用水合肼对其进行改性。接枝和改性后产物对金属离子吸附性明显增加,尤其是提高了Au、Pd的分离富集效果。改性的产物,吸附容量Au为496mg/g干树脂。Pd为580mg/g干树脂。     

An FT-Raman spectroscopic investigation of dentin and collagen surfaces modified by 2-hydroxyethylmethacrylate

Although 2-hydroxyethylmethacrylate (HEMA) is commonly used for adhesive bonding to dentin, its role in promoting adhesion is not completely understood. Here, we use FT-Raman spectroscopy to elucidate further the nature of the interaction of HEMA with dentin. Ground dentin was exposed to 2.5% (w/w) nitric acid, washed, dried in air, and treated with HEMA. The samples were then sequentially washed with distilled water, with FT-Raman spectra being obtained after different wash times. Hydroxyapatite and bovine type I collagen were similarly treated with HEMA except for the acid exposure. The FT-Raman spectra of these samples were also recorded. The spectra of HEMA-treated water-washed dentin and collagen revealed the following changes: (1) The band intensities of HEMA absorbed on dentin and collagen decreased with increasing wash times (2) the nu(C=O) and nu(CCO) modes of HEMA at 1718 and 607 cm-1, respectively, either disappeared or decreased after extensive washing; (3) the nu (C=C) (1640 cm-1) and delta (=CH2), (1403 cm-1) bands exhibited minor variations in band position and relative intensity. These results demonstrate that HEMA interacts with dentin both physically and chemically. The chemical interaction can be interpreted by either hydrogen bonding or the formation of a new bond to the ester group of HEMA.     

Biomimetic peptide surfaces that regulate adhesion, spreading, cytoskeletal organization, and mineralization of the matrix deposited by osteoblast-like cells

In an effort to regulate mammalian cell behavior in contact with solid material surfaces, we have functionalized surfaces with different ratios of both the putative cell binding (-Arg-Gly-Asp-) domain and a consensus heparan-binding domain. The peptide sequences -Arg-Gly-Asp- (-RGD-) and -Phe-His-Arg-Arg-Ile-Lys-Ala- (-FHRRIKA-) or mixtures of the two in the ratios of 75:25 (mimetic peptide surface I), 25:75 (mimetic peptide surface II), and 50:50 (mimetic peptide surface III) were immobilized on model surfaces using a heterobifunctional cross-linker to link the peptide(s) to amine-functionalized quartz surfaces. Contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to confirm the chemistry, thickness of the overlayers, and surface density of immobilized peptides ( approximately 4-6 pmol/cm2). The degree of rat calvaria osteoblast-like cell spreading, focal contact formation, cytoskeletal organization, proliferation, and mineralization of the extracellular matrix (ECM) on model biomaterial surfaces was examined. Mimetic peptide surface II (MPS II) and MPS III supported the highest degree of cell spreading (p < 0.05), following 4 h of incubation, compared to MPS I, homogeneous -RGD-, and homogeneous -FHRRIKA- grafted surfaces. Furthermore, MPS I, MPS II, MPS III, and homogeneous -RGD- surfaces promoted the formation of focal contacts and stress fibers by attached bone cells. The strength of bone cell detachment following 30 min of incubation was significantly higher (p < 0.05) on MPS II surfaces compared to homogeneous -RGD- and -FHRRIKA-. However, the degree of cell proliferation on the peptide surfaces were not significantly different from each other (p > 0.1). Following 24 d in culture, the areas of mineralized ECM formed on MPS II and MPS III surfaces were significantly (p < 0.05) larger than those of other surfaces. These results demonstrate that utilizing peptide sequences incorporating both cell- and heparin-adhesive motifs can enhance the degree of cell surface interactions and influence the long-term formation of mineralized ECM in vitro.     

Platelet endothelial cell adhesion molecule-1 is phosphorylatable by c-Src, binds Src-Src homology 2 domain, and exhibits immunoreceptor tyrosine-based activation motif-like properties

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is 130-kDa member of the immunoglobulin gene superfamily that localizes to cell-cell borders of confluent endothelial cells and has been shown to play a role in the control of endothelial sheet migration and leukocyte transmigration through the endothelium. The cytoplasmic tail plays an important role in the modulation of PECAM-1 function. Mutation of tyrosine 663 or 686 in the cytoplasmic tail reduces phosphorylation and mutation of 686 is associated with a reduction in PECAM-1-mediated inhibition of cell migration (1). We have previously noted that these two tyrosine residues are surrounded by consensus sequences for Src homology 2 (SH2) domain binding (1, 2), and the experiments presented explore the potential for PECAM-1-Src and PECAM-1-SH2 domain interactions. PECAM-1 is more highly phosphorylated in endothelial cells overexpressing c-Src, and in in vitro kinase assays, c-Src can phosphorylate a glutathione S-transferase (GST)-PECAM cytoplasmic tail fusion protein. The phosphorylated fusion protein associates with the bead-bound c-Src. This association appears to be mediated by Src-SH2 domain, because PECAM-1 can be precipitated by a GST-Src-SH2 affinity matrix. The binding to the GST-Src-SH2 affinity matrix correlates directly with the level of PECAM-1 phosphorylation, because more PECAM-1 is precipitated from c-Src overexpressors and from wild-type rather than Tyr663 --> Phe and Tyr686 --> Phe mutant PECAM-1 expressors. Yet unidentified phosphoproteins can also be coimmunoprecipitated with wild-type but not mutant PECAM-1. Finally, we note the similarity of the PECAM-1 cytoplasmic domain sequence to the immunoreceptor tyrosine-based activation motif. Our data begin to delineate how tyrosines 663 and 686 may play a role in mediating PECAM-1 signal transduction.     

六氰合铁酸铁/全氟磺酸 聚四氟乙烯共聚物复合膜修饰玻碳电极差分脉冲伏安法测定铅和镉

利用电聚合的方法将普鲁士蓝修饰到玻碳电极表面,然后修饰上全氟磺酸 聚四氟乙烯共聚物(Nafion)膜制成修饰电极。利用差分脉冲伏安法（DPV）对Pb2+和Cd2+在该修饰电极上的电化学行为进行了研究,建立了差分脉冲伏安法灵敏测定Cd2+和Pb2+的新方法。对富集电位、富集时间以及Nafion用量等实验条件进行了优化。在01 mol/L pH 45的NaAc HAc缓冲液中,在-11 V处搅拌富集450 s,用DPV分别测定-048 V和-073 V处的氧化峰电流。溶出峰电流与Pb2+和Cd2+的浓度分别在5×10-8~5×10-5 mol/L和2×10-8~2×10-5 mol/L范围内呈良好的线性关系,相关系数分别为0995和0992。检出限分别为5×10-9 mol/L (Pb2+)和2×10-9 mol/L(Cd2+) (S/N=3)。方法用于水样中Cd2+和Pb2+的测定,测定值与原子吸收光谱法的结果相一致,相对标准偏差为21%~38%。