Platelet Adhesion Studies Research Articles

Platelet Aggregation and Adhesion during Dietary Copper Deficiency in Rats

The role of dietary copper deficiency in platelet-to-endothelial cell adhesion and in platelet-to-platelet aggregation was studied in vitro. Platelets were obtained from male, weanling Sprague-Dawley rats fed purified diets which were either copper-adequate (CuA, 6.3 micrograms copper/g of diet) or copper-deficient (CuD, 0.3 microgram/g of diet) for 4 weeks. The platelet adhesion study was performed by adding CuA or CuD platelets either suspended in homologous plasma or in Tyrode buffer salt solution (TBSS) to cultured rate endothelial cells. After a one hour incubation at 37 degrees C non-adhered platelets were removed and counted in a microcytometer. Platelet aggregation in platelet rich plasma (PRP) samples was induced by adding ADP (2 x 10(-4)M) and measured in a turbidometric aggregometer. The content of von Willebrand factor (vWF) in platelets and in plasma and the content of fibrinogen in platelets was determined. Platelet adhesion to rat endothelial cells was significantly lower for platelets from CuD rats than for platelets from CuA rats. ADP induced platelet aggregation from CuD rats was significantly higher than platelet aggregation from CuA rats. The content of vWF in platelets and in plasma from CuD rats was significantly lower than in platelets and plasma from CuA rats. However, the amount of fibrinogen in platelets from ++CuD rats was about 4-fold higher than that in platelets from CuA rats while the plasma fibrinogen was lower in CuD rats than in CuA rats. These studies illustrate that copper deficiency diminishes platelet adhesion to endothelial cells but increases platelet aggregability. The results suggest that these physiological alterations may be the result of decreased platelet vWF and increased platelet fibrinogen during dietary copper deficiency.

The integrin alpha IIb beta 3 contains distinct and interacting binding sites for snake-venom RGD (Arg-Gly-Asp) proteins. Evidence that the receptor-binding characteristics of snake-venom RGD proteins are related to the amino acid environment flanking the sequence RGD.

We have previously demonstrated [Lu, Williams, Deadman, Salmon, Kakkar, Wilkinson, Baruch, Authi and Rahman (1994) Biochem. J. 304, 929-936] the preferential antagonism of the interactions of the integrin alpha IIb beta 3 on activated platelets with three immobilized glycoprotein ligands (fibrinogen, fibronectin and von Willebrand factor) by a selected panel of snake-venom RGD (Arg-Gly-Asp)-containing proteins including the disintegrins kistrin and elegantin, and the neurotoxin variant dendroaspin. Kistrin and dendroaspin, although structurally unrelated, contain similar amino acids flanking the tripeptide RGD and behaved as identical antagonists preferentially inhibiting platelet adhesion to immobilized fibrinogen as opposed to fibronectin. In contrast, elegantin, which shares extensive sequence similarity with kistrin but has different amino acids around the tripeptide RGD, preferentially inhibited platelet adhesion to immobilized fibronectin as opposed to fibrinogen. To develop further insights into the mechanisms underlying the preferential antagonism shown by the venom proteins in the adhesion studies, we, in the present study, sought to determine the binding properties of kistrin, elegantin and dendroaspin to the alpha IIb beta 3 complex by radioligand kinetic and competition studies. In direct binding experiments, both kistrin and dendroaspin were observed to bind to a single class of binding site on ADP-activated platelets with apparent equilibrium dissociation constant (Kdapp) values of 42 +/- 2 nM and 21 +/- 6 nM respectively. In competition studies, dendroaspin blocked the binding of 125I-labelled kistrin to ADP-activated platelets in a simple competitive manner, with an apparent equilibrium inhibition constant (Kiapp) of 143 +/- 14 nM, from which an indirect Kdapp = 22 nM for dendroaspin was determined. This result suggests that kistrin and dendroaspin bind to the same site on the integrin alpha IIb beta 3 consistent with their similar inhibitory properties. In contrast, elegantin recognized two classes of binding sites on the alpha IIb beta 3 complex with Kdapp values of 10.5 +/- 0.8 nM and 175 +/- 10 nM, and, unlike dendroaspin, did not inhibit the binding of 125I-labelled kistrin to ADP-activated platelets. However, in reciprocal experiments both kistrin and dendroaspin inhibited the binding of 125I-elegantin to ADP-activated platelets in a non-competitive manner, with Kiapp values of 34 +/- 3 nM and 21 +/- 2 nM respectively. Thus elegantin appears to interact with distinct but interacting sites on the alpha IIb beta 3 complex from the binding site of kistrin and dendroaspin, consistent with its distinctive inhibitory preferences as shown in platelet adhesion studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Thrombogenic properties of untreated and poly(ethylene oxide)-modified polymeric matrices useful for preparing intraarterial ion-selective electrodes.

In vitro platelet adhesion studies are used to compare the thrombogenic properties of various polymer matrices useful for preparing implantable ion-selective membrane electrodes. Conventional plasticized poly(vinyl chloride) and alternate polyurethane materials (Tecoflex, Pellethane) doped with proton- (tridodecylamine) and potassium-selective (valinomycin) ionophores are shown to be potentially thrombogenic. Incorporation of high molecular weight block copolymers of poly(ethylene oxide) and poly(propylene oxide) (e.g., Pluronic F108 and Tetronic 1508) within ion-selective membranes reduces platelet adhesion. A more marked decrease in platelet adhesion is, however, observed when the Tecoflex-based membranes are coated with a thin photo-cross-linked layer of poly(ethylene oxide). Such surface-modified membranes are shown to retain potentiometric ion response properties (i.e., selectivity, response times, response slopes, etc.) essentially equivalent to untreated membranes.

Polyethylene Surface Sulfonation: Surface Characterization and Platelet Adhesion Studies

A 1,3-propane sultone (C3H6SO3) solution was employed to incorporate sulfur-containing functionalities onto ammonia (NH3) and allylamine (CH2=CHCH2NH2) plasma treated low density polyethylene (LDPE). ESCA results indicated that nitrogen and sulfur concentrations in the surface region decreased with time for both the sulfonated ammonia and allylamine plasma treated LDPE due to migration of the hydrophilic nitrogen- and sulfur-containing functional groups into the bulk of the material and/or the loss of low molecular weight fragments to the atmosphere. Due to extensive post-plasma-treatment oxidation in the 1,3-propane sultone reagent and possible post-plasma-treatment oxidation with atmospheric oxygen before sulfonation, the O/C ratios of both sulfonated materials increased significantly after the sulfonation reaction. The increase in oxygen concentration after sulfonation reaction for both sulfonated materials suggested the occurrence of post-plasma-treatment oxidation with atmospheric oxygen on the modified surfaces. There was no difference in the air and octane contact angles for the sulfonated ammonia plasma treated LDPE when measured immediately after the sulfonation and 25 days later. The contact angles on the sulfonated allylamine plasma polymers, however, decreased 25 days after the sulfonation due to a slightly larger extent of the post-plasma-treatment oxidation. In vitro canine platelet adhesion results suggested the sulfonated ammonia plasma treated LDPE was more platelet activating than the control sample. The potential blood compatibility of the sulfonated allylamine plasma polymers was not assessed due to the poor adhesion between the sulfonated plasma polymer and LDPE substrate during the platelet adhesion experiment.

Surface characterization and platelet adhesion studies of plasma-sulphonated polyethylene

Canine platelet adhesion on sulphur dioxide and allyl phenyl sulphone plasma-treated low density polyethylene (LDPE) was studied. Both 5 W plasma excitation energy-treated surfaces showed the highest S:C atomic ratio by ESCA analysis. Surface hydrophilicity increased drastically after plasma treatment. However, surface hydrophilicity decreased with time. This might be due to the diffusion of the hydrophilic functional groups into the bulk of the material and/or the migration of low molecular weight hydrocarbon species into the surface region. The allyl phenyl sulphone plasma-treated LDPE was less platelet-activating in terms of platelet adhesion and spreading and was independent of the different plasma excitation energy levels used. Sulphur dioxide plasma-treated LDPE showed a higher-level platelet activation than the untreated LDPE. Among the sulphur dioxide plasma-treated LDPE samples, the sample prepared using the 5 W excitation energy was less platelet-activating than the samples treated by higher plasma excitation energies.

Surface Characterization and Platelet Adhesion Studies of Plasma-Carboxylated Polyethylene

Carboxyl groups were incorporated onto low-density polyethylene (LDPE) by carbon dioxide (CO 2) plasma modification, ammonia (NH 3) plasma modification with subsequent β-propiolactone (C 3H 4O 2) chemical grafting, and allylamine (CH 2=CHCH 2NH 2) plasma polymerization with subsequent β-propiolactone chemical grafting. ESCA and contact-angle experiments indicated that the hydrophilicity of the carbon dioxide plasma treated LDPE decreased with time due to the diffusion of the hydrophilic oxygen-containing functional groups away from the surface regions and/or the loss of the low molecular weight polar fragments. In contrast, the surface hydrophilicity of the β-propiolactone carboxylated ammonia and allylamine plasma treated LDPE determined by contact angle experiments was stable with time, but ESCA experiments showed that surface oxygen atomic concentration increased with time because of extensive post-plasma-treatment oxidation in air. The surface nitrogen atomic concentrations of both kinds of β-propiolactone carboxylated materials decreased with time due to diffusion of the nitrogen-containing functional groups into the bulk of the materials and/or the loss of the low molecular weight fragments to the environment. The blood compatibility of the carboxylated materials was examined by in vitro canine platelet adhesion as assessed by SEM. All three types of the plasma modified materials were more platelet reactive than the control LDPE base which suggests these materials are thrombogenic.

Type I von Willebrand disease, subtype 'platelet low': decreased platelet adhesion can be explained by low synthesis of von Willebrand factor in endothelial cells.

Endothelial cells (EC) were isolated from the umbilical vein of a newborn girl with type I 'platelet low' von Willebrand disease (I vWD) and endothelial localization and release and the ability of subendothelial von Willebrand factor (vWF) to support platelet adhesion were compared with those of normal EC. vWF was detectable by immunofluorescence in Weibel-Palade bodies, but the number of Weibel-Palade bodies positive for vWF was lower than in control EC. Patient EC released into the medium significantly smaller amount of vWF, both constitutively and after their stimulation. The vWF content of the extracellular matrix of patient EC was 38% that of control EC matrix. Platelet adhesion studies were performed under flow conditions with umbilical arteries and EC matrices of cultured EC. Using normal citrated whole blood as perfusate, platelet adhesion was lower in the umbilical artery of the patient (9 +/- 1% v 35 +/- 4% for the control) and in her EC matrix (7 +/- 1% v 21 +/- 2% of control). When patient EC matrix was perfused with vWF-deficient reconstituted blood, adhesion was 17 +/- 3% v 32 +/- 3% for control EC matrix; preincubation of patient EC matrix with 1 U/ml vWF increased the adhesion to 30 +/- 6%. These data establish that low contents of vWF in EC and subendothelium are important characteristics of type I vWD 'platelet low', and that such characteristics correlate with low platelet adhesion to the subendothelium.

Blood compatibility of SPUU-PEO-heparin graft copolymers.

Biological responses to heparinized segmented polyurethaneurea (SPUU-PEO-Heparin) were evaluated in vitro and ex vivo. In vitro assays involved plasma protein adsorption, platelet adhesion, and release reaction studies. In addition, an ex vivo rabbit arterio-artery (A-A) shunt experiment was also performed to measure occlusion times of the heparinized surfaces. All SPUU-PEO-Heparin surfaces demonstrated less protein adsorption than Biomer and protein adsorption patterns similar to SPUU-PEO surfaces. Platelet adhesion and release studies demonstrated that both SPUU-PEO-Heparin and SPUU-PEO surfaces adsorbed less platelets and inhibited platelet release, as compared to Biomer. These findings correlated with reduction in protein adsorption observed for the modified surfaces. In low flow rate ex-vivo A-A shunt experiments, all heparinized surfaces prolonged occlusion time longer than controls. However, SPUU-PEO surfaces did not prolong occlusion time when compared to Biomer, although these surfaces suppressed protein adsorption and platelet interaction in vitro. The improved blood compatibility of SPUU-PEO-Heparin surfaces attest to the usefulness of this approach in improving the blood compatibility of blood contacting surfaces.

Platelet microvesicles adhere to subendothelium and promote adhesion of platelets

A role in hemostasis has been suggested for platelet membrane microvesicles (mv). The objectives of the studies reported here include functional analysis of platelet mv in models developed for study of platelet adhesion, as well as investigation of possible interactions between mv and intact platelets in these same adhesion models. Microvesicles were prepared from washed platelet concentrates by repeated freezing and thawing. Adhesion to subendothelium was measured quantitatively by radiolabelling mv with 111-In, and morphologically by scanning electron microscopy. Platelet mv adhered to subendothelium quantitatively over time. Using a modified Baumgartner chamber, we found adhesion of mv to subendothelium significantly increased with increasing shear rates. With this same model we found that prior exposure of subendothelium to mv greatly increased subsequent adhesion of platelets to the same everted vessel, compared to platelet adhesion in the absence of mv. All of these experiments were conducted with mv suspended in ACD/saline, indicating that plasma components are not essential for adhesion of mv. Our studies show that platelet mv adhere to subendothelium in much the same way as do platelets, and support the concept of a hemostatic role for mv in that they appear to increase platelet adhesion.

Effect of Fabrication, Sterilization and Mediators - Blood Compatibility of Polyurethanes

The possible changes in the surface and physical properties of polyether urethane urea (PEUU) implants, including their interaction with blood, due to the different preparation methods, sterilization techniques and long term storage in different environmental conditions have been investigated by conducting the studies of mechanical properties, contact angle, platelet adhesion and protein adsorption. Considerable variations in the mechanical properties have been observed for the PEUU grafts stored in different conditions. Changes in platelet adhesion and albumin adsorption have also been observed in the case of samples that underwent different sterilization methods. The effect of mediators like bromelain, an enzyme present in pineapple juice, on albumin adsorption and platelet adhesion on PEUU surfaces have been investigated. It seems the presence of pineapple juice increases the adsorption of albumin and reduces the adhesion of platelets on PEUU surfaces.

Vessel wall proteins adhesive for platelets

Platelet adhesion to the vessel wall is the first step in the development of a haemostatic plug or thrombus. In vitro studies of platelet adhesion in flowing blood have become possible due to the development of suitable perfusion chambers. With the use of such chambers, adhesion to subendothelium and to isolated connective tissue components from the vessel wall has been realized. Such studies have begun to shed light on the molecular basis of the adhesion process. Von Willebrand factor (vWF), fibronectin, and collagen types I and III have been shown to be the most important adhesive proteins. The functional domains involved in vWF and fibronectin are being identified and the platelet receptors for these ligands have been established. A complicated picture of multiple mutually supportive ligand-receptor interactions has emerged. These insights are critical for the development of thrombo-resistant biomaterials.

Platelet adhesion to exposed endothelial cell extracellular matrixes is influenced by the method of preparation.

The relative thrombogenicity of extracellular matrixes (ECMs) produced by cultured human umbilical endothelial cells (ECs) was studied under flow conditions. ECMs were prepared using a number of physical and chemical methods, and their reactivity toward platelets was morphometrically evaluated. von Willebrand factor (vWF), fibronectin (FN), and 13-hydroxy-9-cis,11-trans-octadecadienoic acid (13-HODE) were also determined. We found that platelet adhesion to ECMs differed significantly, both quantitatively and qualitatively, with the method of ECM preparation. Mechanically prepared ECM exposed a less thrombogenic surface compared with ECM prepared by chemical methods (platelet-covered surface of 20% and 50%, respectively). Evaluation of the ECM components vWF, FN, and 13-HODE showed significant changes, both in their concentrations and distribution patterns, depending on the method of ECM preparation. The decrease measured in the levels of ECM-associated vWF (from 108 to 9.2 ng/10(4) cells) and the minor changes observed in the distribution pattern of subendothelial FN did not appear to be sufficient to explain the altered platelet adhesion observed in our model. This suggests that the amount of 13-HODE probably associated to the remaining ECs present in the mechanically exposed ECM could be one factor that specifically contributed to the nonthrombogenic state of these preparations. We conclude that the degree of ECM reactivity toward platelets is dependent on the method of ECM preparation and that this is related to the removal of specific EC/ECM components that modulate their thromboresistant/thrombogenic properties. This fact should be taken into account when ECMs produced by cultured ECs are used in platelet adhesion studies.

Comparison between Human Umbilical Artery and Rabbit Abdominal Aorta as Substrata for Platelet Adhesion and Platelet Thrombus Formation under Flow Conditions

Rabbit abdominal aortas and human umbilical arteries are currently used as substrata for the study of platelet adhesion and aggregate formation under flow conditions. Using immunohistochemical and ultrastructural methods, we have analyzed both vessel surfaces. The reactivity towards platelets of the subendothelium (SE) exposed on these vessels after mechanical or enzymatic digestion (alpha-chymotrypsin) was morphometrically quantified and the nature of the interaction studied in the electron microscope. After mechanical damage, the ultrastructural study of rabbit aortas showed a clearly defined internal elastic lamina (IEL). In contrast, umbilical vessels lacked a consistent IEL and masses of amorphous material often located deeper in the media were the main constitutents of the SE. Immunohistochemical labeling of the von Willebrand factor bound to both types of vessel differed considerably. Quantification of platelet interactions after perfusion of citrated blood showed qualitative differences between mechanically damaged rabbit or human vessels. Enzymatic digestion produced a more thrombogenic surface on rabbit aortas (p less than 0.01 vs. nondigested), but decreased their reactivity towards platelets on umbilical arteries (p less than 0.01 vs. nondigested). The ultrastructural study of the interacting platelets revealed that aggregates, when present, were found on the extracellular matrix underlying endothelial cells of rabbit aortas, but interacting with fibrillar structures probably derived from cell elements of the media in the case of umbilical arteries. These findings indicate that rabbit aortas and umbilical arteries possess structural characteristics that result in different thrombogenic properties with respect to circulating platelets.

Permeability of PEUU membranes: their modification towards blood compatibility.

An attempt was made to develop haemodialysis membranes using polyether urethane urea synthesised in our laboratory. It was observed that the processing parameters such as precipitation medium, precipitation temperature etc. can influence the porosity of the membrane and subsequently the permeability property. It was also noted that the permeability of the dried membrane was negligible even though it was kept in distilled water overnight before use. The effect of pH on permeability through the membrane was studied by dialysis experiment using mixtures of various components such as urea, creatinine, uric acid, inulin, albumin, NaCl and KCl at various pH. Standard cellulose acetate (CA) membrane was used for comparison. Membranes were also prepared using biomer solution by precipitating in distilled water at room temperature and the monomer, 2-hydroxy ethyl methacrylate (HEMA) was grafted onto it by glow discharge technique. It was found that the permeability was increased by HEMA grafting with some loss of tensile strength and strain. A comparative study of fibrinogen adsorption during dialysis and adsorption by direct exposure of samples to a mixture containing urea, uric acid, creatinine, dextran, fibrinogen and electrolytes like sodium and potassium ions was also done with 125I labelled fibrinogen. Platelet adhesion studies indicated that the number of adhered platelets was less on the HEMA grafted samples which may enhance blood compatibility. Finally, the membranes were subjected to different sterilization processes possible under wet conditions such as glutaraldehyde treatment and autoclaving. The contact angle, permeability, mechanical property and platelet adhesion studies indicated that the sterilization method can affect the performance of the membrane.

Changes in protein adsorption on polycarbonate due to L-ascorbic acid

When a synthetic material is introduced into blood, plasma proteins are rapidly adsorbed on to its surface, followed by the attachment of formed elements of blood, leading to thrombus formation. Previous research 8 reported that vitamin C (L-ascorbic acid) prolongs the clotting time due to the formation of vitamin C-calcium complexes, which reduce the availability of Ca 2+ ions for the clotting mechanism in in vitro conditions. Contact angle and platelet adhesion studies have indicated that vitamin C modiRes the surface-protein interaction and surface-platelet binding at the interface. In this paper an increased thickness of protein layer deposited on the polycarbonate substrate has been observed in the presence of vitamin C (~ 50 Å) using ellipsometric measurements. Further polyacrylamide gel electrophoresis (PAG) and infrared attenuated total reflection spectroscopy (IR-ATR) techniques have provided a better understanding of the interfacial phenomena. It seems, that the adsorption of albumin is increased, relatively, in the presence of vitamin C, when compared with that of fibrinogen and γ-globulin from an equal amount of protein mixture.

Surface modification of polyvinyl chloride towards blood compatibility

The surface modification of medical grade polyvinylchloride (pvc) from various sources with the ionic bonding of polyelectrolyte is investigated by exposing thepvc sheets to 1% zephiran chloride for 10 min and then to 50 mg% solution of polyelectrolyte for 10 min. Surface energy and platelet adhesion studies were carried out to demonstrate the suitability of our improved surface towards blood compatibility. Relative changes due toγ-irradiation are also discussed.

Radiation-induced albuminated surfaces—Their modifications toward blood compatibility

An effort is made here to bind albumin on the polyetherurethane surfaces using Co 60 γ-irradiation. The main objective is to develop a procedure suitable for albuminating the surfaces and to correlate such surface modifications with blood compatibility using contact angle technique and platelet adhesion studies, toward long term vascular graft applications under in vivo conditions.

Lipid adsorption/absorption on polycarbonate surfaces — an understanding

Thrombus formation on a foreign surface is a complicated process, involving many factors. The adhesion and aggregation of platelets play an important role in the initial events of thrombus formation on such surfaces. In this work, adsorption/absorption studies using saturated fatty acids were carried out on polycarbonate surface to evaluate how such surfaces may affect blood-polymer interaction. The surface change was investigated by measuring the interfacial energies as derived from advancing contact angle technique. The plasma recalicification time and platelet adhesion studies were also carried out to further develop an understanding.

Glutaraldehyde proteinated surfaces: blood compatibility

This paper reports our attempts to crosslink low molecular weight proteins namely trypsin and insulin using glutaraldehyde on polycarbonate surface and to evaluate how such surfaces may affect the blood compatibility of the polymer, by studying the interfacial energies of the modified polymer surface using advancing contact angle technique. The plasma recalcification time and platelet adhesion studies were also carried out. It has been observed that such low molecular weight proteins retard clotting.

Variations in glutaraldehyde-proteinated surfaces

An attempt is made here to crosslink different proteins using glutaraldehyde on polycarbonate surface and to evaluate how such surfaces may effect the blood compatibility of the polymer, by investigating the interfacial energies of the modified polymer surfaces using contact angle techniques. The plasma recalcification time and platelet adhesion studies were also carried out to develop an understanding further. It has been observed that the increased molecular weight of proteins seems to encourage thrombosis.