Calf Skin Collagen Research Articles

Isolation and characterisation of acid-solubilised collagen from the skin of Nile tilapia (Oreochromis niloticus)

Acid-solubilised collagen (ASC) was extracted from the skin of Nile tilapia (Oreochromis niloticus) and characterisation was studied. The results indicated that the yield of ASC was 39.4% on the basis of dry weight. This ASC was rich in glycine (35.6%). The amount of imino acids, proline and hydroxyproline, in ASC was 210 residues per 1000 residues. The ultraviolet (UV) absorption spectrum of ASC showed that the distinct absorption was at 220 nm. ASC showed transition curve at maximum temperature (Tmax) of 32.0 °C in 0.05 M acetic acid, about 12 °C lower than that of calf skin collagen. Maximum solubility (0.75 mg/ml) in 0.5 M acetic acid was observed at pH 3. Solubility reached the minimum at pH 7. A sharp decrease in solubility was observed in 2% (w/v) NaCl or above. Biochemical studies indicated that ASC was composed of the α1α2α3 heterotrimers.

Robust Pore Size Analysis of Filamentous Networks from 3D Confocal Microscopy

We describe a robust method for determining morphological properties of filamentous biopolymer networks, such as collagen or other connective tissue matrices, from confocal microscopy image stacks. Morphological properties including pore size distributions and percolation thresholds are important for transport processes, e.g. particle diffusion or cell migration through the extracellular matrix. The method is applied to fluorescently labeled fiber networks prepared from rat tail tendon and calf skin collagen, at concentrations of 1.2, 1.6 and 2.4 mg/ml. The collagen fibers form an entangled and branched network. The medial axes, or skeletons, representing the collagen fibers are extracted from the image stack by threshold intensity segmentation and distance-ordered homotopic thinning. The size of the fluid pores as defined by the radii of largest spheres that fit into the cavities between the collagen fibers is derived from Euclidean distance daps and maximal covering radius transforms of the fluid phase. The size of the largest sphere that can traverse the fluid phase between the collagen fibers across the entire probe, called the percolation threshold, was computed for both horizontal and vertical directions. We demonstrate that by representing the fibers as the medial axis the derived morphological network properties are both robust against changes of the value of the segmentation threshold intensity and robust to problems associated with the point-spread function of the imaging system. We also provide empirical support for a recent claim that the percolation threshold of a fiber network equals the fiber diameter for which the Euler index of the networks becomes zero.

황다랑어 복부 껍질을 이용한 콜라겐의 제조 및 물리화학적 특성 해석

Processing of collagen from yellowfin tuna (Thunnus albacares) abdominal skins was optimized by response surface methodology and central composite design. The values of independent variables at optimal conditions were NaOH concentration: 0.5 N, NaOH treatment time: 36.2 hr, pepsin concentration: 1:4.9 ratio (0.245%, w/v), and digestion time: 48.1 hr, respectively. The collagen content estimated under optimal conditions was 33.1%, and the actual experimental collagen content was 32.3%. Physicochemical properties of collagen from yellowfin tuna abdominal skin were investigated by amino acids analysis, SDS-PAGE, FT-IR, viscosity and denaturation temperature. Amino acids content of the collagen was 21.0%. SDS-PAGE pattern of the collagen showed two different <TEX>$\alpha$</TEX>-chain (<TEX>${\alpha}_1$</TEX>- and <TEX>${\alpha}_2$</TEX>- chain), <TEX>$\beta$</TEX>-component and <TEX>$\gamma$</TEX>-component. The spectrum of FT-IR of the collagen showed wavenumber at 3,434, 1,650, 1,542 and <TEX>$1,235\;cm^{-1}$</TEX> representing the regions of amide A, I, II and III, respectively. Relative viscosity of the collagen decreased continuously on heating up to <TEX>$32^{\circ}C$</TEX>, and the rate of decrease was retarded in the temperature range of <TEX>$35-50^{\circ}C$</TEX>. Denaturation temperature (Td) of the collagen solution (0.06%, w/v) was <TEX>$31^{\circ}C$</TEX> and was lower than calf skin collagen (<TEX>$35^{\circ}C$</TEX>).

Robust Pore Size Analysis of Filamentous Networks from Three-Dimensional Confocal Microscopy

We describe a robust method for determining morphological properties of filamentous biopolymer networks, such as collagen or other connective tissue matrices, from confocal microscopy image stacks. Morphological properties including pore size distributions and percolation thresholds are important for transport processes, e.g., particle diffusion or cell migration through the extracellular matrix. The method is applied to fluorescently labeled fiber networks prepared from rat-tail tendon and calf-skin collagen, at concentrations of 1.2, 1.6, and 2.4mg/ml. The collagen fibers form an entangled and branched network. The medial axes, or skeletons, representing the collagen fibers are extracted from the image stack by threshold intensity segmentation and distance-ordered homotopic thinning. The size of the fluid pores as defined by the radii of largest spheres that fit into the cavities between the collagen fibers is derived from Euclidean distance maps and maximal covering radius transforms of the fluid phase. The size of the largest sphere that can traverse the fluid phase between the collagen fibers across the entire probe, called the percolation threshold, was computed for both horizontal and vertical directions. We demonstrate that by representing the fibers as the medial axis the derived morphological network properties are both robust against changes of the value of the segmentation threshold intensity and robust to problems associated with the point-spread function of the imaging system. We also provide empirical support for a recent claim that the percolation threshold of a fiber network is close to the fiber diameter for which the Euler index of the networks becomes zero.

Fluorescence of putative chromophores in Skh‐1 and citrate‐soluble calf skin collagens

Fluorescence of Skh-1 hairless mouse and calf skin acid-soluble type I collagens are envelopes of several bands putatively due to tyrosine (excitation/emission peak at 275/300 nm), dihydroxyphenylalanine (dopa; 280/325 nm), tyrosine aggregate (285/360 nm), dityrosine 325/400 nm), and advanced glycation end (AGE) product (370/450 nm), respectively. As these fluorophores can be markers of pathological conditions, we wish to present further evidence for or against these assignments. We analysed intact type I mouse collagens and AGE by conventional fluorescence spectroscopy, synchronous spectroscopy, high-performance liquid chromatography (HPLC), and borate quenching. The relative amount of each fluorophore depends on the collagen sample. Acid- or enzyme-hydrolysis results in loss of fluorescence intensity at 350-400 nm, and enhanced emission 400-500 nm which could be reproduced by controlled dopa oxidation. Borate buffer quenches fluorescence at lambda>400 nm from intact collagens, dityrosine, dopa, and oxidized dopa but does not quench tyrosine or AGE fluorescence. Collagen fluorescence depends on its source and previous history. The data indicate that collagen fluorescence is derived from tyrosine, dopa, interacting tyrosine residues, covalent dityrosine, and dopa oxidation product(s), but not AGE.

Nitroxides are more efficient inhibitors of oxidative damage to calf skin collagen than antioxidant vitamins

Reactive oxygen species generated upon UV-A exposure appear to play a major role in dermal connective tissue transformations including degradation of skin collagen. Here we investigate on oxidative damage to collagen achieved by exposure to (i) UV-A irradiation and to (ii) AAPH-derived radicals and on its possible prevention using synthetic and natural antioxidants. Oxidative damage was identified through SDS-PAGE, circular dichroism spectroscopy and quantification of protein carbonyl residues. Collagen (2 mg/ml) exposed to UV-A and to AAPH-derived radicals was degraded in a time- and dose-dependent manner. Upon UV-A exposure, maximum damage was observable at 730 kJ/m 2 UV-A, found to be equivalent to roughly 2 h of sunshine, while exposure to 5 mM AAPH for 2 h at 50 °C lead to maximum collagen degradation. In both cases, dose-dependent protection was achieved by incubation with μM concentrations of nitroxide radicals, where the extent of protection was shown to be dictated by their structural differences whereas the vitamins E and C proved less efficient inhibitors of collagen damage. These results suggest that nitroxide radicals may be able to prevent oxidative injury to dermal tissues in vivo alternatively to commonly used natural antioxidants.

Two novel monoclonal antibodies to VWFA3 inhibit VWF-collagen and VWF-platelet interactions.

The interaction of collagen-von Willebrand factor (VWF)-GPIb is essential for platelet adhesion, especially under high shear conditions. VWF, which acts as a bridge between platelets and exposed subendothelium, interacts with collagen through its A3 domain, which is a new target for the antithrombotic agent. To develop functional blockers that specifically inhibit VWF-dependent adhesion of platelets to collagen under high shear stress. To develop murine antihuman VWF A3 monoclonal antibodies (mAbs) by standard hybridoma technology, and characterize their abilities to block interactions between VWF A3 and collagen as well as platelet function. Thirty anti-VWF-A3 mAbs were obtained. Among them, two mAbs, designated as SZ-123 and SZ-125, were found to inhibit VWF-collagen type III interaction. SZ-123 and SZ-125 inhibited the binding of purified human VWF (1.5 or 3 mug mL(-1)) to human placenta collagen type III (IC(50) = 0.07 +/- 0.02 and 0.15 +/- 0.03 mug mL(-1), respectively) or to calf skin collagen type III (IC(50) = 0.48 +/- 0.06 and 0.51 +/- 0.07 mug mL(-1), respectively) coated on plates. Under flow shear condition (1000 s(-1)), SZ-123 and SZ-125 inhibited platelet adhesion on human placenta collagen- or calf skin collagen-coated surfaces. Both mAbs also inhibited platelet aggregation induced by ristocetin, botrocetin or bovine plasma. SZ-123 and SZ-125 inhibited VWF-collagen and VWF-platelet interactions.

On the mechanism of cerebral accumulation of cholestanol in patients with cerebrotendinous xanthomatosis

The most serious consequence of sterol 27-hydroxylase deficiency in humans [cerebrotendinous xanthomatosis (CTX)] is the development of cholestanol-containing brain xanthomas. The cholestanol in the brain may be derived from the circulation or from 7alpha-hydroxylated intermediates in bile acid synthesis, present at 50- to 250-fold increased levels in plasma. Here, we demonstrate a transfer of 7alpha-hydroxy-4-cholesten-3-one across cultured porcine brain endothelial cells (a model for the blood-brain barrier) that is approximately 100-fold more efficient than the transfer of cholestanol. Furthermore, there was an efficient conversion of 7alpha-hydroxy-4-cholesten-3-one to cholestanol in cultured neuronal and glial cells as well as in monocyte-derived macrophages of human origin. It is concluded that the continuous intracellular production of cholestanol from a bile acid precursor capable of rapidly passing biomembranes, including the blood-brain barrier, is likely to be of major importance for the accumulation of cholestanol in patients with CTX. Such a mechanism also fits well with the observation that treatment with chenodeoxycholic acid, which normalizes the level of the bile acid precursor, results in a reduction of cholestanol-containing xanthomas even in the brain.

In vitro growth and differentiated activities of human periodontal ligament fibroblasts cultured on salmon collagen gel

Marine-derived collagen is expected to be a much safer alternative to calf collagen, which in medical applications carries the risk of bovine spongiform encephalopathy. In this study, acid-soluble collagen was extracted from salmon skin and crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide during fibril formation to produce a crosslinked salmon collagen (SC) gel. The growth rates and the differentiated functions of human periodontal ligament fibroblasts (HPdLFs) cultured on the SC gel were investigated. Growth was faster on the SC gel than on porcine collagen (PC) gel. In addition, the HPdLFs cultured on the SC gel exhibited higher alkaline phosphatase (ALP) activity than those cultured on the PC gel. Quantitative RT-PCR revealed higher mRNA expression of type I collagen, ALP, and osteocalcin in the HPdLFs cultured on the SC gel. HPdLFs had a flat shape on the SC gel and a spindle shape on the PC gel, as revealed by observation with scanning electron microscopy and immunostaining with cytoskeletal protein and vinculin. The results showed that HPdLFs could grow and show highly differentiated activity on the SC gel as well as on the PC gel.

New Index for the Stability of a Type I Collagen Affected by Hydrophobic Environment

Effects of hydrophobic environment adjusted by various alcohols on the structural stability of calfskin collagen (CSC) were studied to elucidate the nature of collagen-monomer interaction in adhesion. The stability of CSC in aqueous alcohol solutions was represented by its denaturation temperature, Td, measured by DSC. The hydrophobicity of the alcohol solutions was quantified with their specific dielectric constants, epsilon(r), calculated from their concentrations. The effect of each alcohol to stabilize or destabilize CSC was evaluated by the initial slope of each Td vs. epsilon(r) plot, denoted as -(dTd/d epsilon(r))ini and termed as stabilization power. Results showed that a hydrophobic environment with a smaller epsilon(r) lowered the stabilization power. Stabilization power ranged from -3 (strong destabilization) for phenol (epsilon(r) =12) to +0.3 (weak stabilization) for glycerol (epsilon(r)=47). In view of the encouraging results obtained in this study, the new index was therefore helpful in predicting the effects of new dental materials of known epsilon(r) values on the stability of dentinal collagen.

Isolation and partial characterization of pepsin-soluble collagen from the skin of grass carp ( Ctenopharyngodon idella)

Pepsin-soluble collagen was extracted from the skin of grass carp ( Ctenopharyngodon idella) with a yield of 46.6%, on a dry weight basis. Electrophoretic patterns showed that the collagen contained α1 and α2 chains, similar to those of calf skin collagen. The imino acid content of the collagen from grass carp skin was much lower than those of mammalian’s collagens, as also were the transition temperature and denaturation temperature which were only 24.6 °C and 28.4 °C respectively. Peptide maps of the collagen digested by trypsin and V8 protease showed different peptide fragments from those of calf skin collagen and other fish skin collagens, suggesting differences in amino acid sequences and collagen conformation. In addition, the lyophilized collagen sponge from grass carp skin had a uniform and regular network structure, just like calf skin collagen sponge. These results suggest that grass carp skin has potential for use as a supplementary source of collagen.

Production and Characterization of Novel Neutralizing mAbs Against vWF-A3 Domain, an Inhibitor of Binding of vWF to Collagen and Influence of Interaction between vWF and Platelet GPIbα.

The interaction of collagen-vWF-GPIb is the first instance of the adherence, aggregation and activation of platelets and also is a new target of antithrombotic agent. At sufficiently elevated shear forces, platelets bind to collagen through a multistep process, involving both vWF-independent and soluble large vWF-dependent interactions. The vWF-A3 domain mediates vWF binding to collagen, after which vWF-platelet interactions occure via the vWF-A1 domain (platelet GPIb/IX/V receptor recognization ). Therefore, production of novel neutralizaing anti-vWF-A3 mAbs blocking binding of vWF to collagen is new antithrombotic target of preventing arterial thrombosis.AIM: To produce novel neutralizing anti-vWF-A3 mAbs and study its effect on binding of vWF to collagen.METHODS: Balb/c mices were immunized by purified recombinant vWF-A3( rvWF A3). After fusion, screening with rvWF-A3 and cloning. Hybridomas for inhibition of vWF binding to collagen were selected with ELISA. The McAbs were purified from murine ascites on a protein G-Sepharose 4B column. The recognition of mAbs with rvWF-A3 and reduced human vWF was studied by Western-blot. Inhibition of binding of purified human vWF (3μg/ml for calf skin collagen III-coated plates; 1.5ug/ml for human placenta collagen III-coated plates) or diluted normal plasma ( human and Rhesus monkey plasma :1/20 on human collagen III and 1/10 on calf skin collagen III; Beagle dog plasma : 1/10 on human collagen III and 1/5 on calf skin collagen III) to collagen by anti-vWF-A3 McAbs was studied with collagen binding test . Platelets aggregation was tested with the inducers of ristocetin (1.25mg/ml), collagen (2μg/ml), ADP(2μM).RESULTS: A group of 30 anti-vWF-A3 mAbs was obtained, from which 2 clones were selected and identified as inhibitory. They were designated 1B9 and 3D2. Both of them are IgG1. It was shown that 1B9 and 3D2 could react specifically with human vWF and rvWF-A3 respectively, while neither of them reacted with rvWF-A1 and rvWF-A2. Western-blot showed that 1B9 and 3D2 can recognize a band at the 27 KDa of rvWF-A3 and 2 bands at 250 KDa and 170KDa of reduced human vWF. 1B9 and 3D2 dose dependently inhibited the binding of purified human vWF to human type III collagen (IC50= 0.055 ± 0.008 and 0.15 ± 0.004μg/ml, respectively) and to calf skin collagen III (IC50= 0.48 ± 0.06 and 0.51 ± 0.07 μug/ml, respectively). Furthermore, 1B9 and 3D2 showed similar inhibitory potency against the binding of human, Rhesus monkey, and Beagle dog plasma vWF to these collagens. Platelets aggregation tests showed that 1B9 and 3D2 dose dependently inhibited platelets aggregation induced by ritocetin (IC50=4.54 ± 0.62 and 8.21 ± 1.02 μg/ml, respectively), but they failed to inhibit platelets aggregation induced with collagen or ADP.COUNCLUSION: These data demonstrate that 1B9 and 3D2, as novel neutralizing mAbs against vWF-A3 domain, are potent inhibitors of vWF-dependent platelets adhesion to collagen and at the same time, they influence the interaction of binding of vWF to platelets. Therefore, They may have therapeutic potential as antithrombotic agent.

Temperature dependence of collagen fluorescence

Dermal collagens have several fluorescent moieties in the UV and visible spectral regions that may serve as molecular probes of collagen. We studied the temperature-dependence of a commercial calf skin collagen and acid-extracted Skh-1 hairless mouse collagen at temperatures from 9 degrees C to 60 degrees C for excitation/emission wavelengths 270/305 nm (tyrosine), 270/360 nm (excimer-like aggregated species), 325/400 nm (dityrosine) and 370/450 nm (glycation adduct). L-tyrosine (1 x 10(-5) M in 0.5 M HOAc) acted as a "reference compound" devoid of any collagen structural effects. In general, the fluorescence efficiency of these fluorophores decreases with increasing temperature. Assuming that rate constant for fluorescence deactivation has the form k(d)(T) = k(d) degrees exp (-DeltaE/RT), an Arrhenius plot of log[(1/Phi) - 1] vs. 1/T affords a straight line whose (negative) slope is proportional to the activation energy, DeltaE, of the radiationless process(es) that compete with fluorescence. Because it is difficult to accurately measure Phi(f) for collagen-bound fluorophores, we derived an approximate formula for an activation parameter, DeltaE*, evaluated from an Arrhenius-like plot of log 1/I(N)vs. 1/T, (1/I(N)vs. is the reciprocal normalized fluorescence intensity). Tyrosine in dilute solution affords a linear Arrhenius plot in both of the above cases. Using the known value of Phi(f) = 0.21 for free tyrosine at room temperature, we determined that DeltaE* is accurate to approximately 25% in the present instance. Collagen curves are non-linear, but they are quasi-linear below approximately 20 degrees C, where the helical form predominates. Values of DeltaE* determined from the data at T < 20 degrees C ranged from 6.2-8.4 kJ mol(-1) (1.5-2.0 kcal mol(-1)) for mouse collagen and 10.3-11.4 kJ mol(-1) (2.5-2.7 kcal mol(-1)) for calf skin collagen, consistent with collisional deactivation of the fluorescent state via thermally enhanced molecular vibrations and rotations. Above 20 degrees C, log 1/I(N)vs. 1/T plots from Skh-1 hairless mouse collagen are concave-downward, suggesting that fluorescence deactivation from the denatured coil has a significant temperature-independent component. For calf skin collagen, these plots are concave-upward, suggesting an increase in activation energy above Tm. These results suggest that collagen backbone and supramolecular structure can influence the temperature dependence of the bound fluorophores, indicating the future possibility of using activation data as a probe of supramolecular structure and conformation.

Fish Skin Type I Collagen

ABSTRACT The acid soluble collagens (ASC) and pepsin soluble col-lagens (PSC) of albacore tuna and silver-line grunt skin, by-products from frozen fish fillet industry, had been extracted, purified and characterized. Analysis of the collagens by SDS-PAGE indicated the absence of a disulfide bond, which is a characteristic of type I collagen. They also contained different subunits, a, β and ?. At least two different a chains, a1 and a2, were presented. The proportion of ? trimer in ASC was higher than in PSC. This caused reduced solubility of ASC, whereas, PSC were lower molecular weight and had fewer crosslinks, which resulted in increased solubility. Collagen water solubility was lower than in 0.5 M acetic acid, but the solubility increased as the temperature increased. Denaturation temperature (Td) of silver-line grunt collagen was slightly higher than albacore tuna collagen, but about 7–9°C lower than calfskin collagen.

Biochemical and Enzymatic Characterization of Human Kallikrein 5 (hK5), a Novel Serine Protease Potentially Involved in Cancer Progression

Human kallikrein 5 (KLK5) is a member of the human kallikrein gene family of serine proteases. Preliminary results indicate that the protein, hK5, may be a potential serological marker for breast and ovarian cancer. Other studies implicate hK5 with skin desquamation and skin diseases. To gain further insights on hK5 physiological functions, we studied its substrate specificity, the regulation of its activity by various inhibitors, and identified candidate physiological substrates. After producing and purifying recombinant hK5 in yeast, we determined the k(cat)/K(m) ratio of the fluorogenic substrates Gly-Pro-Arg-AMC and Gly-Pro-Lys-AMC, and showed that it has trypsin-like activity with strong preference for Arg over Lys in the P1 position. The serpins alpha(2)-antiplasmin and antithrombin were able to inhibit hK5 with an inhibition constant (k(+2)/K(i)) of 1.0 x 10(-) (2)and 4.2 x 10(-4) m(-1) min(-1), respectively. No inhibition was observed with the serpins alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, although alpha(2)-macroglobulin partially inhibited hK5 at high concentrations. We also demonstrated that hK5 can efficiently digest the extracellular matrix components, collagens type I, II, III, and IV, fibronectin, and laminin. Furthermore, our results suggest that hK5 can potentially release (a) angiostatin 4.5 from plasminogen, (b) "cystatin-like domain 3" from low molecular weight kininogen, and (c) fibrinopeptide B and peptide beta15-42 from the Bbeta chain of fibrinogen. hK5 could also play a role in the regulation of the binding of plasminogen activator inhibitor 1 to vitronectin. Our findings suggest that hK5 may be implicated in tumor progression, particularly in invasion and angiogenesis, and may represent a novel therapeutic target.

Isolation and characterization of collagen from bigeye snapper (Priacanthus macracanthus) skin

AbstractAcid‐solubilized collagen (ASC) and pepsin‐solubilized collagen (PSC) were isolated from the skin of bigeye snapper (Priacanthus macracanthus) with yields of 64 and 11 g kg−1 wet weight, respectively. Both ASC and PSC were characterized as type I collagens with no disulfide bonds. Peptide maps of ASC and PSC digested by V8 protease and lysyl endopeptidase showed some differences in peptide patterns and were totally different from those of calf skin collagen. The maximum solubility was observed at pH 4 and 5 for ASC and PSC, respectively. A sharp decrease in solubility of both collagens in acetic acid was found with NaCl concentration above 30 g l−1. Thermal transitions of ASC and PSC in deionized water were observed with Tmax of 30.37 and 30.87 °C, respectively, and were lowered in the presence of acetic acid (0.05 mol kg−1 solution). Therefore, ASC was a major fraction in bigeye snapper skin and it exhibited some different characteristics to PSC. Copyright © 2005 Society of Chemical Industry

Isolation and characterisation of acid and pepsin-solubilised collagens from the skin of Brownstripe red snapper ( Lutjanus vitta)

Acid-solubilised collagen (ASC) and pepsin-solubilised collagen (PSC) were successfully extracted from the skin of Brownstripe red snapper ( Lutjanus vitta) with yields of 9% and 4.7%, respectively, on the basis of wet weight. Both ASC and PSC consisted of two different α chains (α1 and α2), and were characterised to be type I with no disulfide bond. PSC had a lower content of high molecular weight cross-links, than did ASC. Peptide maps of ASC and PSC hydrolysed by V8 protease and lysyl endopeptidase showed some differences in peptide patterns between the two fractions and were totally different from those of calf skin collagen type I, suggesting differences in amino acid sequences and collagen conformation. Maximum solubility in 0.5 M acetic acid was observed at pH 3 and pH 4 for ASC and PSC, respectively. A sharp decrease in solubility was observed in the presence of NaCl, above 2% and 3%, (w/v) for ASC and PSC, respectively. T max values of both collagen fractions were similar and shifted to a lower value in the presence of acetic acid, suggesting some changes in the collagen structure caused by acid induction.

αIIbβ3antagonism vs. antiadhesive treatment to prevent platelet interactions with vascular subendothelium

Platelets adhering to blood vessels promote coagulation and inflammation, and release growth factors that trigger smooth muscle cell activation. We have therefore studied the pharmacological modification of platelet deposition quantitatively by comparing adhesion of flowing platelets to various subendothelial ligands in the absence or presence of an antialpha(IIb)beta(3) antagonist with the effects of antiadhesive treatment consisting of von Willebrand factor (VWF) and fibronectin neutralization or of the combined inhibition of platelet adhesion and aggregation. In vitro, perfusion of anticoagulated human blood over calf skin collagen reiterated that alpha(IIb)beta(3) antagonism prevents platelet aggregation, but not adhesion per se: single platelets strongly bound to collagen at wall shear rates of both 1300 and 2700 s(-1), largely VWF-independent. When perfused over a human umbilical vein endothelial cell-derived extracellular matrix, single alpha(IIb)beta(3)-antagonized platelets primarily adhered to matrix-bound VWF when perfused at 2700 s(-1), but at 1300 s(-1) they also adhered significantly to fibronectin. During perfusion of anticoagulated rabbit blood over de-endothelialized rabbit aorta at a wall shear rate of 1100 s(-1), alpha(IIb)beta(3) antagonism even increased the absolute numbers of adhering platelets and VWF neutralization redirected alpha(IIb)beta(3)-antagonized platelets towards other vascular ligands. Finally, in vivo, following photochemically induced blood vessel injury in mice, alpha(IIb)beta(3) antagonism inhibited platelet-rich thrombus formation, but platelet adhesion was only significantly inhibited when associated with fibronectin neutralization. In conclusion, antiadhesive platelet treatment more potently interferes with platelet deposition on injured blood vessels than alpha(IIb)beta(3) antagonism, but abrogating platelet adhesion can only be achieved by carefully selected antiplatelet drug combinations.

Characterisation of acid-soluble collagen from skin and bone of bigeye snapper ( Priacanthus tayenus)

The compositions and some properties of acid-soluble collagens (ASC) of the skin and bone of bigeye snapper, ( Priacanthus tayenus) were investigated. The collagens were extracted with the yields of 10.94% and 1.59% on the basis of wet weight, from skin and bone, respectively. Similar electrophoretic patterns of collagens from the skin and bone were observed. Both collagens comprised two different α chains, α1 and α2 and were classified as type I collagen. However, peptide maps of collagen from the skin and bone of bigeye snapper, digested by V8 protease and lysyl endopeptidase, revealed differences between collagens from skin and bone, and both were completely different from those of calf skin collagen. Collagen rehydrated in acetic acid had lower T max and enthalpy than those rehydrated in deionised water, suggesting a conformational change caused by acid. Collagens form the skin and bone had the highest solubility at pH 2 and 5, respectively. No changes in solubility were observed in the presence of NaCl up to 3% (w/v). However, a sharp decrease in solubility was found with NaCl above 3% (w/v).

Biochemical properties of black drum and sheepshead seabream skin collagen.

Acid-soluble collagen (ASC) and pepsin solubilized collagen (PSC) were isolated from the skins of black drum (Pogonias cromis) and sheepshead seabream (Archosargus probatocephalus) harvested in the Gulf of Mexico coastal waters. The yields of ASCs on dry basis from black drum and sheepshead were estimated at 2.3 and 2.6%, and the yields of PSCs were 15.8 and 29.3%, respectively. Analyses of molecular weight profile, amino acid composition, and secondary structure showed that the skin collagens from both species were typical type-I collagen. The molecular mass of alpha(1) and alpha(2) subunits, as determined by SDS-PAGE using Tris-Acetate gels, was 127 kDa and 116 kDa, respectively. The amino acid composition of ASC and PSC for both species was closer to calf skin ASC than to cod skin ASC. Thermal denaturation temperatures, measured by melting point using circular dichroism, gave the following values: black drum ASC, 34.2 degrees C; sheepshead ASC, 34.0 degrees C; black drum PSC, 35.8 degrees C; sheepshead PSC, 34.3 degrees C. The literature value for the heat stability of calf skin collagen is 36.3 degrees C. The potentials of collagens from black drum and sheepshead skins in the functional food, healthcare, and pharmaceutical industries are discussed.