Interaction of plasma proteins with heparinized gel particles studied by high-resolution two-dimensional gel electrophoresis.

In order to further the understanding of protein-surface interactions in the coagulation system, we have chosen to study plasma protein adsorption onto heparin-immobilized surfaces. Heparin-binding proteins are abundant in plasma: a search of amino acid sequences revealed that many plasma proteins have possible heparin binding sites. Plasma protein adsorption to the heparinized surfaces is monitored by a novel technique in which the solution depletion of proteins is analytically determined using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). This method enables simultaneous, quantitative detection of the majority of plasma proteins before, during, and after their adsorption onto high surface area adsorbents. Using computerized densitometry of silver-stained 2-D PAGE gels, the amount of each protein can be determined from the integrated optical density of each protein "spot." Kinetics of adsorption and adsorption isotherms of four important heparin binding proteins, antithrombin III (ATIII), complement factor C3 (C3), apolipoprotein AI (Apo-AI) and apolipoprotein AIV (Apo-AIV) are reported in this paper. From the adsorption isotherms, the apparent binding constants of each protein-immobilized heparin complex, Ka, were calculated. The surface binding constants were of the same order of magnitude as the respective solution binding constants in the literature. The surface binding constants followed the same order as the respective solution binding constants: Ka (ATIII) greater than Ka (Apo-AIV) greater than Ka (C3) greater than Ka (Apo-AI), indicating that protein binding to the immobilized heparin used is not essentially different from solution binding.     

Interactions of thermally denatured fibrinogen on polyethylene with plasma proteins and platelets.

During the investigation of fibrin deposition onto hydrophobic polymers in contact with human blood, a model was developed in which fibrinogen was denatured and irreversibly coated onto a polyethylene surface by heating to 70 degrees C for 10 min. The denatured fibrinogen-polyethylene surface is resistant to fluid wall shear rates of up to 550 s-1 and the fibrinogen does not desorb in the presence of plasma proteins. Compared to uncoated polyethylene, little albumin or fibrinogen adsorbs to heat-denatured fibrinogen. Thrombin binds to the denatured fibrinogen-coated polyethylene with low affinity and also acts on the surface-bound denatured fibrinogen and cleaves fibrinopeptide A (FPA) quantitatively. Washed, 51Cr-labeled platelets do not adhere to the thermally denatured fibrinogen at either low or high shear rates compared to surfaces coated with undenatured fibrinogen (p < 0.01). These observations support the role of the D domain of fibrinogen in platelet adhesion because this is the region that is denatured by heating. In contrast, the E domain of fibrinogen is not altered by heating to 70 degrees C and hence remains susceptible to thrombin and/or plasmin cleavage. The characteristics of this surface are such that it can be used to develop fibrin-coated surfaces for use in studies of thrombus formation on artificial surfaces.     

The influence of plasma proteins and platelets on oxygen radical production and F-actin distribution in neutrophils adhering to polymer surfaces

It is well known that blood cell interactions with artificial surfaces might have deleterious effects on host tissue, however, the mechanisms involved are far from understood. In this study, neutrophil-platelet interaction on uncoated or protein-coated polymer surfaces was investigated. Cell spreading, reorganization of actin filaments and release of oxygen metabolites (measured as luminol-amplified chemiluminescence) were used as criteria for cell activation on positively charged, hydrophilic 1,2-diaminocyclohexane, and negatively charged, hydrophobic hexamethylene-disiloxane. The model surfaces were made by radio frequency plasma discharge polymerization. Neutrophil contact with the uncoated polymers induced a prolonged generation of oxygen radicals. Precoating of the polymer surfaces with human serum albumin (HSA) or fibrinogen, markedly reduced neutrophil activation, whereas coating with human immunoglobulin G (IgG), a well-known opsonin, resulted in significantly higher levels of cell activation. Consequently, protein coating overruled the activating effects of the polymer surfaces. The presence of unstimulated or thrombin-stimulated platelets markedly increased the reactivity of neutrophils against fibrinogen- and IgG-coated surfaces. However, neutrophils remained relatively unreactive in the presence of platelets on HSA-treated surfaces. Comparison of the different types of surfaces used, reveals a correlation between the degree of cell spreading, reorganization of the actin cytoskeleton and the amount of oxygen radicals produced. Our results suggest that the acute inflammatory reaction on a biomaterial surface is highly dependent on the nature and composition of the first adsorbed protein layer and the extent of platelet activation.     

Exposure of fibrinogen-adherent platelets to plasma proteins: a new method for studying protein interactions with platelets

To understand the effects of mediators in coagulating blood at biomaterial surfaces, it is important to use methods that resemble the normal sequence of events in wound healing around implants. The initial adhesion of platelets from whole blood onto material surfaces is mediated by the fibrinogen receptor glycoprotein IIb/IIIa, as shown in a previous study (Broberg et al., J Lab Clin Med 2002; 139:163-172). In this study, isolated platelets were adhered to fibrinogen and exposed to IgG, von Willebrand factor, or thrombin. The response was detected as the number of adherent platelets, the spreading of platelets, the exposure of CD62P (P-selectin), and the release of platelet factor 4 (PF4), ADP, and ATP. These results were compared to the response of platelets adhering to surfaces coated with the same proteins. Fibrinogen-adherent platelets exposed to thrombin generated the significantly highest exposure of CD62P and release of PF4, ADP, and ATP. When platelets were adhered to different protein coatings, von Willebrand factor generated the most CD62P exposure, IgG generated the most PF4 release, and thrombin generated the highest concentration of ADP. These results indicate that protein interactions with platelets may generate different results, depending on the mode of protein exposure.     

Methacrylate polymer layers bearing poly(ethylene oxide) and phosphorylcholine side chains as non-fouling surfaces: in vitro interactions with plasma proteins and platelets

Two methacrylate monomers, oligo(ethylene glycol) methyl ether methacrylate (OEGMA; MW = 300 g mol⁻¹, poly(ethylene glycol) (PEG) side chains of average length n = 4.5) and 2-methacryloyloxyethyl phosphorylcholine (MPC; MW = 295 g mol⁻¹), were grafted from silicon wafer surfaces via surface-initiated atom transfer radical polymerization. The grafted surfaces were used as model PEG and phosphorylcholine surface systems to allow comparison of the effectiveness of these two motifs in the prevention of plasma protein adsorption and platelet adhesion. It was found that at high graft density fibrinogen adsorption from plasma on the poly(MPC) and poly(OEGMA) surfaces for a given graft chain length was comparable and extremely low. At low graft density, poly(OEGMA) was slightly more effective than poly(MPC) in resisting fibrinogen adsorption from plasma. Flowing whole blood experiments showed that at low graft density the poly(OEGMA) surfaces were more resistant to fibrinogen adsorption and platelet adhesion than the poly(MPC) surfaces. At high graft density, both the poly(MPC) and poly(OEGMA) surfaces were highly resistant to fibrinogen and platelets. Immunoblots of proteins eluted from the surfaces after contact with human plasma were probed with antibodies against a range of proteins, including the contact phase clotting factors, fibrinogen, albumin, complement C3, IgG, vitronectin and apolipoprotein A-I. The blot responses were weak on the poly(MPC) and poly(OEGMA) surfaces at low graft density and zero at high graft density, again indicating strongly protein resistant properties for these surfaces. Since the side chains of the poly(OEGMA) are about 50% greater in size than those of poly(MPC), the difference in protein resistance between the poly(MPC) and poly(OEGMA) surfaces at low graft density may be due to the difference in surface coverage of the two graft types.     

Interaction of thrombin with mammalian platelets.

In this study we have explored the interaction of thrombin with platelets from human and rat. Compared to human platelets, rat platelets suspended in plasma required a higher concentration of thrombin for aggregation. This difference in sensitivity to thrombin was maintained when platelets were washed and suspended in buffer. Platelets from both mammals bound thrombin and showed a similar number of binding sites. However, the apparent dissociation constant of thrombin binding for rat platelets was approximately 15-fold higher than that for human. Thus, the decreased aggregation response of rat platelets may be due to a reduced binding of thrombin to its receptor. It is known that the thrombin receptor is located on the platelet surface. Gel electrophoresis of platelets followed by specific staining as well as fluorography showed significant differences in the surface glycoproteins of human and rat platelets. Human platelets showed labeled components corresponding to 210,000 and 160,000 daltons, whereas rat platelets showed glycoproteins with molecular weights of 240,000 and 190,000. A 135,000-dalton component was present in platelets from both sources. These results suggest that either or both glycoproteins of 210,000 and 160,000 daltons may be involved in the interaction of thrombin with human platelets.     

Synthesis and characterization of heparinized polyurethanes using plasma glow discharge

Polyurethanes (PU) were synthesized from 4,4'-diphenylmethane diisocyanate and polytetramethylene glycol, and subsequently with ethylene diamine as a chain extender. The PU film was exposed to oxygen plasma glow discharge to produce peroxides on the surfaces. These peroxides were then used as catalysts for the copolymerization of acrylic acid (AA) and methyl acrylate (MA) in order to prepare carboxyl group-introduced PU (PU-C). Heparin-immobilized PU was prepared using the coupling reaction of PU-C with polyethylene oxide (PEO) followed by the reaction of grafted PEO with heparin. The surface-modified PUs were then characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis (ESCA), and a contact angle goniometer. The concentration of carboxylic acid groups on the PU surfaces could be controlled within the range of 0.47-1.68 micromol cm(-2) by the copolymerization of AA and MA. The amounts of heparin coupled to terminus amino groups on PU-6 and PU-33 were 1.30 and 1.16 microg cm(-2), respectively. The water contact angle of the PU was decreased by AA grafting, and further decreased by PEO grafting and heparin immobilization, showing an increased hydrophilicity of the modified PUs. A 3% loss from the originally bound heparin appeared within several hours and thereafter almost no heparin was released when heparin-immobilized PUs were immersed in a physiological solution for 100 h, indicating the covalent immobilization of heparin on the surfaces.     

Interaction of polystyrene/poly(gamma-benzyl L-glutamate) and poly(methyl methacrylate)/poly(gamma-benzyl L-glutamate) block copolymers with plasma proteins and platelets

A-B-type block copolymers, consisting of polystyrene (PST) or poly(methyl methacrylate) (PMMA) forming segment (A) and poly(gamma-benzyl L-glutamate) (P[Glu(OBzl)]) segment (B), were synthesized and the thrombus formation on these block copolymer films was investigated in relation to the adsorption of plasma proteins and the activation of platelets. The relative amount of thrombus formation was higher on homopolymers than on block copolymers. The amount of thrombus formation became less, with decreasing content of P[Glu(OBzl)] in the PST block copolymers and with increasing content of P[Glu(OBzl)] in the PMMA block copolymers. Adsorption of bovine serum albumin(BSA), bovine gamma-globulin (B gamma G) and bovine plasma fibrinogen(BPF) onto polymer films was also investigated. More proteins were adsorbed and denatured when adsorbed onto PST and PMMA than onto block copolymers. With increasing content of P[Glu(OBzl)] in the PST block copolymers, the degree of denaturation of adsorbed proteins increased, while the amount of protein adsorption was unaffected. Conversely, with increasing content of P[Glu(OBzl)] in the PMMA block copolymers, the degree of denaturation of adsorbed proteins decreased, while similarly the amount of protein adsorption was unaffected. Adhesion of platelets from platelet suspension (WP) to polymer films coated with one of the plasma proteins showed that the activation of adhered platelets was suppressed when there was a lower degree of denaturation of coated proteins. In the same experiments using platelet-rich plasma(PRP), neither the number of platelets adhered nor the degree of activation of the adhered platelets was correlated with the composition of the polymer films.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen radical production in neutrophils interacting with platelets and surface-immobilized plasma proteins: role of tyrosine phosphorylation

The interaction between neutrophil granulocytes and platelets is considered to play an important role in the inflammatory process induced by an implanted foreign material. However, the cellular mechanisms involved remain incompletely understood. We used a luminol-dependent chemiluminescence (CL) technique to analyze the generation of reactive oxygen species (ROS) in human neutrophils interacting with different plasma protein-coated surfaces in the presence or absence of unstimulated or stimulated platelets. The role of tyrosine phosphorylation in the regulation of NADPH oxidase activity was evaluated with quantitative fluorescence microscopy and the specific tyrosine kinase inhibitor genistein. We found that the ROS-production is 2 to 3 times higher in neutrophils on immunoglobulin G (IgG)-coated surfaces than in cells interacting with albumin- or fibrinogen-coated surfaces. Incubation with superoxide dismutase and catalase revealed that about 45% of the ROS was released extracellularly on IgG surfaces whereas corresponding values were 90% and 85% in neutrophils interacting with albumin and fibrinogen, respectively. The presence of platelets markedly increased the extracellular generation of ROS, mainly in neutrophils interacting with IgG- or fibrinogen-coated surfaces whereas the intracellular production was only modestly affected. Quantitative fluorescence microscopy of neutrophils stained with FITC-conjugated anti-phosphotyrosine antibodies showed a correlation between tyrosine phosphorylation, cell spreading, and ROS production. Platelets markedly amplified the anti-phosphotyrosine staining on both fibrinogen- and IgG-coated surfaces whereas the low level of tyrosine phosphorylation in neutrophils on albumin-coated surfaces was not further elevated by platelets. Furthermore, the tyrosine kinase inhibitor genistein inhibited both extra- and intracellular ROS production in neutrophils regardless of the presence of platelets. We demonstrate that plasma protein coating and the presence of platelets are crucial for the inflammatory response of adhering neutrophils and that the oxidative response correlates with the extent of tyrosine phosphorylation of proteins in focal contacts.     

Interaction of rat platelets with Listeria monocytogenes.

Listeria monocytogenes induced aggregation of rat platelets in vitro and stimulated the nonlytic release of [3H]serotonin. Listeria-induced platelet aggregation and serotonin release required the presence of intact Listeria, was maximal at a 1:1 Listeria/platelet ratio, required a plasma cofactor, and was not inhibited by indomethacin, acetylsalicylic acid, or apyrase. Aggregation either of platelets in platelet-rich plasma with adenosine diphosphate or of washed platelets with thrombin resulted in the release of a listericidin from the platelets; however, direct interaction of L. monocytogenes with platelet-rich plasma did not kill Listeria. The ability of rats to clear an intravenous challenge of L. monocytogenes (0.005 50% lethal dose), as determined by the recovery of viable L. monocytogenes from the spleen and liver, was unaffected by prior treatment with antiplatelet serum.     

Lysed platelets shorten the activated coagulation time (ACT) of heparinized blood

Aliquots of heparinized whole blood were incubated at 37 degrees C with preparations of lysed platelets or buffer. In the presence of lysed platelets, the ACT decreased to 127 seconds (+/- 41, n = 9) versus 248 seconds (+/- 43, n = 9) in the controls (P = 0.0002). The shortening of the ACT by lysed platelets followed a typical dose-response relationship. Fractionation of lysed platelet preparations revealed that the procoagulant membrane fraction, not the cytosol fraction containing PF4, was responsible for this effect of lysed platelets on the ACT. These findings demonstrate that the ACT can be artifactually low in the presence of heparin, indicating a potential problem in the use of the ACT for monitoring of heparin neutralization.     

Assay of Factor XII clotting activity in heparinized plasma

Clotting tests based upon the activated partial thromboplastin time are disturbed by heparin. This brief communication shows that Factor XII clotting activity in plasma samples heparinized in vitro and in vivo can be measured accurately by means of an aPTT-test using congenitally deficient substrate plasma when the plasma dilution buffer contains hexadimethrine bromide (Polybrene), 15 mg/L. This method of neutralizing heparin obviates more complicated procedures such as heparin adsorption to anion exchange resins.     

Adsorption of plasma proteins and adhesion of platelets onto novel polyetherurethaneureas--relationship between denaturation of adsorbed proteins and platelet adhesion

Novel polyetherurethaneureas which have been synthesized by the present authors were chosen for the substrate polymers, on which adhesion of platelets was investigated. The number of adhered platelets and the amount of serotonin released from platelets adhered on the polymers and the protein-coated polymers were determined by radioisotope method. Both of them were enhanced with increasing content of urea linkages in the polyetherurethaneureas. The platelet adhesion was discussed in terms of the denaturation of plasma proteins upon adsorption, which was determined by Fourier-transform infrared spectroscopy. With increasing degree of protein denaturation, the platelet adhesion and the serotonin release were enhanced. This relationship was particularly evident in the case of albumin adsorption. It was shown that the surface properties of substrate polymers affect the protein adsorption, which in turn influences the adhesion of platelets.     

Importance of a biofouling-resistant phospholipid polymer to create a heparinized blood-compatible surface

Heparinization is believed to be one of the methods to suppress thrombus formation on blood-contacting surfaces. However, this study hypothesizes that heparinization alone might not be sufficient to provide a blood-compatible surface; that is, a surface property that resists biofouling is necessary to obtain an effective heparin-modified surface. 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers with 2-aminoethyl methacrylate (AEMA) were synthesized to immobilize heparin through ionic bonding. The primary amino groups of AEMA were considered to be the polymer surface because the zeta-potential of the surface was positive when the mole fraction of the AEMA units was above 0.2. The antithrombogenic character of the polymer surface modified with heparin was evaluated by both Lee-White and microsphere column methods. The coagulation period of human whole blood in the absence of anticoagulant in glass tubing coated with the MPC polymer was longer than that in the original glass tube. Cell adhesion was completely inhibited on the MPC polymer surface after contact with human whole blood without anticoagulant. However, many adherent blood cells were observed on poly(2-ethylhexyl methacrylate-co-AEMA) (no MPC unit) even after heparinization. These results strongly indicate that the MPC polymer is a useful substrate where the heparin works well and that the heparin-immobilized MPC polymer has superior blood compatibility to the simple MPC polymer.     

Importance of a biofouling-resistant phospholipid polymer to create a heparinized blood-compatible surface

Heparinization is believed to be one of the methods to suppress thrombus formation on blood-contacting surfaces. However, this study hypothesizes that heparinization alone might not be sufficient to provide a blood-compatible surface; that is, a surface property that resists biofouling is necessary to obtain an effective heparin-modified surface. 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers with 2-aminoethyl methacrylate (AEMA) were synthesized to immobilize heparin through ionic bonding. The primary amino groups of AEMA were considered to be the polymer surface because the ζ-potential of the surface was positive when the mole fraction of the AEMA units was above 0.2. The antithrombogenic character of the polymer surface modified with heparin was evaluated by both Lee-White and microsphere column methods. The coagulation period of human whole blood in the absence of anticoagulant in glass tubing coated with the MPC polymer was longer than that in the original glass tube. Cell adhesion was completely inhibited on the MPC polymer surface after contact with human whole blood without anticoagulant. However, many adherent blood cells were observed on poly(2-ethylhexyl methacrylate-co-AEMA) (no MPC unit) even after heparinization. These results strongly indicate that the MPC polymer is a useful substrate where the heparin works well and that the heparin-immobilized MPC polymer has superior blood compatibility to the simple MPC polymer.     

Interaction of Yersinia enterocolitica and Y. pseudotuberculosis with platelets.

Yersinia enterocolitica is a bacterium capable of growth at 4 degrees C in donated blood and has been responsible for many deaths following transfusion. Interaction of Y. enterocolitica with blood cells is of interest in understanding the mechanisms of survival and growth in blood. The closely related organism Y. pseudotuberculosis is known to invade platelets and cause platelet aggregation by a mechanism that involves expression of the chromosomal inv gene. Yersinia isolates were made to express green fluorescent protein (GFP) and their interaction with platelets was studied by flow cytometry, enterocolitica did not cause platelet aggregation or activation, not even when grown at 22 degrees C to maximise inv expression. Attachment of Y. enterocolitica O:9 to platelets occurred with virulence plasmid-bearing (pYV+) strains grown at 37 degrees C but not with pYV- strains nor with strains grown at 22 degrees C. Y. pseudotuberculosis containing inv did cause platelet activation and aggregation when grown at 22 degrees C, as has been shown before, but also showed enhanced attachment to platelets when grown at 37 degrees C. Electron microscopy studies confirmed that inv-expressing Y. pseudotuberculosis invaded platelets but Y. enterocolitica attached only to the outer surface of platelets. Interaction of Y. enterocolitica O:9 with platelets provided a modest protection against bacterial killing by human serum. Interaction of Y. enterocolitica O:9 with platelets does not lead to platelet invasion or activation, and is mediated through plasmid-coded factors, not inv.