The effect of fluid shear and co-adsorbed proteins on the stability of immobilized fibrinogen and subsequent platelet interactions.

The conformation adopted by the plasma protein fibrinogen upon its adsorption onto synthetic surfaces has been implicated to play an important role in determining the blood compatibility of biomaterials. It has recently been shown that adsorbed fibrinogen undergoes biologically significant conformational changes with increasing residence time on the surface of selected biomaterials. The purpose of this study was to examine the effects of co-adsorbed proteins and shear forces on such time-dependent functional changes in fibrinogen adsorbed onto polyethylene (PE), polytetrafluoroethylene (PTFE), and silicone rubber (SR). Fibrinogen was adsorbed onto these materials for 1 min and then allowed to 'reside' on these surfaces for up to 2 h prior to assessing its biological activity. Changes in fibrinogen reactivity were determined by measuring the adhesion of 51Cr-labeled platelets and the ability of blood plasma to displace previously adsorbed fibrinogen. The magnitude of platelet adhesion to substrates adsorbed with pure fibrinogen increased in the presence of shear, compared with static conditions; at the lowest shear rate of 200 s(-1), samples exhibited a 20-fold increase in adhered platelet levels. In contrast, at a higher shear rate of 1000 s(-1), the three polymers supported minimal levels of platelet attachment. Surfaces pre-adsorbed with 10% plasma did not promote a significant increase in the number of adherent platelets with increasing shear when compared with the pure fibrinogen-coated substrates. The presence of shear also significantly altered the materials' ability to retain fibrinogen. Under static conditions, the amount of fibrinogen retained following incubation in blood plasma increased on all materials with increasing fibrinogen residence time. However, the materials varied distinctly in their ability to retain adsorbed fibrinogen with increasing fibrinogen residence time, shear rate, and nature of the co-adsorbed proteins. Thus, the results from this study indicate that fluid shear, residence time of the adsorbed protein, nature of the co-adsorbed proteins, and surface chemistry of the material all play important roles in influencing platelet-surface interactions and that they act in a complex manner to influence the biocompatibility of a material.

Effects of fibrinogen residence time and shear rate on the morphology and procoagulant activity of human platelets adherent to polymeric biomaterials.

Fibrinogen readily adsorbs to the surface of biomaterials and, because of its demonstrated ability to support platelet adhesion and aggregation, plays a role in thrombotic events associated with the implantation of synthetic materials in the human body. Thus, understanding the factors influencing the interactions of fibrinogen with biomaterials, and how platelet responses are affected, is crucial for the development of synthetic materials exhibiting improved blood compatibility. In this study, the effects of fibrinogen residence time and shear rate on the procoagulant activity of adherent platelets, along with their morphologic status, as deduced from scanning electron microscopy, were investigated. To examine whether adherent platelets promoted the generation of thrombin, polymeric materials (polytetrafluoroethylene, polyethylene, and silicone rubber) preadsorbed with fibrinogen were exposed to platelet suspensions at different wall shear rates and then incubated with clotting factors for 5 minutes under static conditions. The amount of thrombin generated per platelet was calculated from the optical density of the color developed by adding substrate S-2238. Scanning electron microscopy images of the platelets revealed that the platelets exhibited different morphologies, depending on the shear rate and residence time of the adsorbed fibrinogen. Platelets ranged from their normal discoid shape observed primarily under static conditions, to that of fully spread platelets. Results from this study show that platelets, in the presence of shear forces, undergo activation on exposure to surfaces on which adsorbed fibrinogen has resided for short residence times rather than long residence times. Interestingly, studies examining the procoagulant responses of such adherent platelets demonstrated that the platelets attached to the fibrinogen coated materials did not promote significant thrombin generation. Such low prothrombinase activity of adherent platelets suggests that adsorbed fibrinogen, while capable of supporting platelet adhesion and spreading on biomaterials, does not necessarily enhance the procoagulant activity of adherent platelets.

Particle diameter influences adhesion under flow.

The diameter of circulating cells that may adhere to the vascular endothelium spans an order of magnitude from approximately 2 microm (e.g., platelets) to approximately 20 microm (e.g., a metastatic cell). Although mathematical models indicate that the adhesion exhibited by a cell will be a function of cell diameter, there have been few experimental investigations into the role of cell diameter in adhesion. Thus, in this study, we coated 5-, 10-, 15-, and 20-microm-diameter microspheres with the recombinant P-selectin glycoprotein ligand-1 construct 19.ek.Fc. We compared the adhesion of the 19.ek.Fc microspheres to P-selectin under in vitro flow conditions. We found that 1) at relatively high shear, the rate of attachment of the 19.ek.Fc microspheres decreased with increasing microsphere diameter whereas, at a lower shear, the rate of attachment was not affected by the microsphere diameter; 2) the shear stress required to set in motion a firmly adherent 19.ek.Fc microsphere decreased with increasing microsphere diameter; and 3) the rolling velocity of the 19.ek.Fc microspheres increased with increasing microsphere diameter. These results suggest that attachment, rolling, and firm adhesion are functions of particle diameter and provide experimental proof for theoretical models that indicate a role for cell diameter in adhesion.

Differential expression of a ligand induced binding site (LIBS) by GPIIb-IIIa ligand recognition peptides and parenteral antagonists.

The glycoprotein (GP) IIb-IIIa complex is an attractive anti-platelet target for the prevention of thrombotic events associated with coronary artery disease. Although GPIIb-IIIa antagonists inhibit GPIIb-IIIa binding to its ligands, the interactions have not been fully clarified, particularly with respect to their ability to induce structural changes in the complex that lead to exposure of neoantigenic epitopes or ligand-induced binding sites (LIBS). In this study we used the anti-LIBS monoclonal antibody (mAb) D3 to further define the activation states of purified active and inactive GPIIb-IIIa. We also compared the data obtained in the purified system to that observed with intact human platelets. Active GPIIb-IIIa expressed significantly greater high-affinity D3 LIBS sites compared to the inactive form. In addition, the ligand recognition peptides RGDS and H12 caused increased expression of the D3 epitope, with RGDS eliciting a much more potent response. The response of the purified GPIIb-IIIa to these peptides paralleled that observed with human platelets. To explore whether the platelet antagonists abciximab, eptifibatide and tirofiban induced expression of the D3 LIBS site, a modified competitive ELISA was developed. Our data indicate that the use of purified GPIIb-IIIa with this ELISA system provides a reproducible approach for exploring the interactions between GPIIb-IIIa and its antagonists. Whereas abciximab caused no detectable increase in the expression of the D3 epitope on purified GPIIb-IIIa, eptifibatide, tirofiban, RGDS, and H12 induced differential expression of the high-affinity LIBS. Studies with intact platelets suggested that abciximab blocked the binding of the D3 and LIBS6 mAbs, and that the pre bound anti-LIBS D3 sterically hindered abciximab binding.

Residence-time dependent changes in fibrinogen adsorbed to polymeric biomaterials.

It has generally been accepted that biomaterials adsorbing the least amount of the plasma protein fibrinogen following exposure to blood will support less platelet adhesion and therefore exhibit less thrombogenicity. Several studies suggest, however, that the conformation or orientation of immobilized fibrinogen rather than the total amount adsorbed plays an important role in determining the blood compatibility of biomaterials. The purpose of this study was to investigate time-dependent functional changes in fibrinogen adsorbed to polytetrafluoroethylene (PTFE), polyethylene (PE), and silicone rubber (SR). Fibrinogen was adsorbed to these materials for 1 min and then allowed to 'reside" on the surfaces for up to 2 h prior to assessing its biological activity. Changes in fibrinogen reactivity were determined by measuring the adhesion of 51Cr-labeled platelets, the binding of a monoclonal antibody (mAb) directed against an important functional region of the fibrinogen molecule (the gamma-chain dodecapeptide sequence 400-411), and the ability of blood plasma to displace previously adsorbed fibrinogen. Platelet adhesion differed among the polymeric materials studied, and PTFE and PE samples exhibited a small decrease in adhesion with increasing fibrinogen residence time. Platelet adhesion to SR was the least among all materials studied and showed no variation with residence time. When using PTFE and SR as substrates, mAb recognition of adsorbed fibrinogen did not change with residence time whereas that on PE decreased slightly. The mAb binding was least to fibrinogen adsorbed to SR, which is in agreement with the platelet adhesion results. Finally, the ability of plasma to displace previously adsorbed fibrinogen decreased dramatically with increasing residence time on all materials. These in vitro studies support the hypothesis that fibrinogen undergoes biologically significant conformational changes upon adsorption to polymeric biomaterials, a phenomenon that may contribute to the hemocompatibility of the materials following implantation in the body.

Tissue factor expression by rat osteosarcoma cells adherent to tissue culture polystyrene and selected orthopedic biomaterials.

Tissue factor (TF), a transmembrane glycoprotein expressed by numerous cell types, plays a critical role in the initiation of blood coagulation at sites of vascular injury. Activated products of the coagulation cascade may then enhance the inflammatory responses associated with wound healing. In the present investigation the ability of rat osteosarcoma (ROS) cells to express TF activity was examined following their growth on tissue-culture polystyrene (TCPS) and selected orthopedic biomaterials (titanium and zirconium alloys, and stainless steel). ROS cells exhibited significant TF activity as evidenced by the conversion of Factor X to Factor Xa in the presence of TF, Factor VIIa, and Ca2+. Factor Xa concentrations ranged from 1.0 fM per cell at 10 min to 6.0 fM per cell after 60 min. Additionally, ROS cells stimulated with calcium ionophore (A23187) exhibited approximately twice the activity of non-stimulated cells when grown on TCPS but not on the metallic substrates. ROS cells (stimulated or unstimulated) adherent to the zirconium alloy generated lower amounts of Factor Xa compared to those bound to the other alloys and unstimulated cells grown on TCPS. These results indicate that ROS cells cultured on these synthetic surfaces differentially express procoagulant activity and that cells grown on TCPS, but not the metallic alloys, exhibit increased TF activity in response to stimulation by calcium ionophore. This procoagulant activity may potentiate subsequent inflammatory responses associated with the use of orthopedic biomaterials and thereby influence the tissue compatibility of the implant.

Vascular cell attachment and procoagulant activity on metal alloys.

The attachment and growth of vascular smooth muscle cells on biomaterials used as components of devices implanted in the vascular space may influence the biocompatibility of such materials. The nature of the materials may affect the attachment and/or the activation of these cells' procoagulant responses. Therefore, the main objective of this study was to measure the strength of adhesion of these vascular cells to potential biomaterials (titanium, zirconium alloys, and stainless steel) by exposing them to a range of shear stresses (50-300 dyn cm(-2)) in a parallel plate flow chamber. The procoagulant responses of the cells were evaluated by measuring the tissue factor (TF) activity promoted by the different materials under flow conditions. The materials supported distinctly different levels of initial cell adhesion in static culture. However, the fraction of adherent cells did not decline significantly with incrementally increasing shear stress within the range tested. TF expression, as measured by factor Xa (FXa) production. was material-dependent. For example, cells cultured on Ti1313 exhibited more FXa production (13.2 nM 10(-5) cells) than Ti1313(DH) (8.5 nM 10(-5) cells) or stainless steel (2 nM 10(-5) cells). Thus, our studies indicate that the level of adhesion, strength of attachment and the expression of procoagulant activity of adherent vascular cells depend strongly on the nature of the underlying biomaterial.

A computational analysis of FXa generation by TF:FVIIa on the surface of rat vascular smooth muscle cells.

A computational model was developed to investigate the contribution of classical mass transport and flow parameters to factor X (FX) activation by the tissue factor-factor VIIa complex (TF:VIIa) on one wall of a parallel-plate flow chamber. The computational results were compared to previously obtained experimental data for the generation of factor Xa (FXa) by TF:VIIa on the surface of cultured rat vascular smooth muscle cells. In this study, the complete steady-state convection-diffusion equation was solved using the commercial software package, FLUENT (Fluent Inc., Lebanon, New Hampshire). A user-defined subroutine interfaced with FLUENT implemented the surface reaction which was modeled using classical Michaelis-Menten reaction kinetics. The numerical solutions were obtained for 12 cases which used combinations of three wall shear rates and four reaction rates. The numerically obtained fluxes for a given reaction rate displayed a wall shear rate dependence which ranged from classical kinetic reaction control (no dependence) to pure diffusional control (maximum dependence). The experimental data, however, were not represented by numerical data generated using a single reaction rate. The three numerically obtained fluxes which corresponded most closely to the experimental fluxes were determined using three different Vmax values. This finding supports the hypothesis that there may be a direct effect of flow on the TF:VIIa complex or the cell membrane.

Hemocompatibility studies of surface-treated polyurethane-based chronic indwelling catheters.

The objectives of this research were to evaluate and compare the interactions of several polyurethane-based central venous catheter materials with blood. Specifically, measurements of fibrinogen adsorption, platelet adhesion, kallikrein generation, and fibrinopeptide A (FPA) release were performed. The catheter materials examined in this study included: platinum-cured, 50 shore A durometer, barium sulfate-filled, silicone (SI); Tecoflex EG85A-B20 polyurethane (PU); PU catheters whose outer surface had been impregnated with ion beam-deposited silver atoms (AgI and AgII); PU catheters coated with a hydrophilic, polyacrylic acid polymer (UC); PU catheters coated with an air-cured PTFE emulsion (CS); and PU catheters coated with an aminofunctional dimethylsiloxane copolymer (JG). The time course of fibrinogen adsorption from plasma to the SI, JG, PU, and CS materials was similar, with CS exhibiting the least amount of adsorbed fibrinogen after 1 h (65 +/- 4.7 ng cm-2) and PU the greatest (144 +/- 16.5 ng cm-2). After 90 min of contact, AgI and AgII exhibited the greatest number of adherent platelets, levels that were approximately two to three times higher than those on the other catheter materials. With the exception of UC and PU, which caused kallikrein generation levels approximately half that of the positive (glass) control, little kallikrein formation was observed for any of the materials relative to the negative control. Finally, FPA generation was greatest using the SI, CS, and PU materials, with the latter causing the production of almost four times the amount of FPA as the negative control. This preliminary assessment of the hemocompatibility of the various catheters suggests that the surface treatments did not adversely affect their interactions with blood components; further investigations of these materials are therefore warranted in order to completely characterize their behavior prior to use in clinical situations.

Initial hemocompatibility studies of titanium and zirconium alloys: prekallikrein activation, fibrinogen adsorption, and their correlation with surface electrochemical properties.

Two novel metal alloys, Ti-13Nb-13Zr and Zr-2.5Nb, have been engineered for applications in orthopedic implants because of their favorable mechanical properties, corrosion resistance, and compatibility with bone and tissue. These alloys also have the ability to form a hard, abrasion-resistant, ceramic surface layer upon oxidative heat treatment (diffusion hardening, DH). Previous studies have indicated that these and other ceramics cause limited hemolysis and exhibit remarkable structural integrity after extended exposure to physiological environments. Such observations suggest that DH Ti-13Nb-13Zr and ZrO2/Zr-2.5Nb could be used successfully as components in blood-contacting devices. Materials intended for such applications must possess properties that do not elicit adverse physiological responses, such as the initiation of the coagulation cascade or thrombus formation. In the present study measurements of prekallikrein activation, fibrinogen adsorption from diluted human plasma, and the strength of fibrinogen attachment as judged by residence-time experiments were performed to evaluate the potential hemocompatibility of these materials. The results of the prekallikrein activation and fibrinogen-retention studies correlated well with two electrochemical properties of the alloys, the open circuit potential and reciprocal polarization resistance. The results indicate that both the original and treated Ti and Zr alloys activate prekallikrein and adsorb as well as retain fibrinogen in amounts similar to other materials used as components of blood-contacting devices. On the basis of these studies, these alloys appear to be promising candidates for cardiovascular applications and merit further investigation.

A numerical analysis of factor X activation in the presence of tissue factor--factor VIIa complex in a flow reactor.

A mathematical model has been developed to investigate previously obtained experimental findings relating to the activation of factor X by surface-bound tissue factor--factor VIIa (TF:VIIa) in a tubular flow reactor. In those experiments, factor X was perfused through a microcapillary tube over a range of flow (shear) conditions and the activated product, factor Xa, was measured at the outlet of the tube using a chromogenic assay. In the present study, the steady-state convection-diffusion equation with Michaelis-Menten kinetics used to describe the reaction at the wall was numerically integrated using an implicit method based on linear systems of ordinary differential equations. The results from the numerical analysis indicated that shear rate directly affects both Km and Vmax. Values of Km decreased from 151 to 16 nM as the shear rate increased from 25 to 2400 sec-1. Additionally, there was a twofold increase in Vmax from 1.4 to 3.0 pmol/cm2/min as the shear rate increased from 25 to 300 sec-1. These findings are in contrast with classical enzyme behavior and imply a direct effect of fluid flow on the kinetics of factor X activation.

Immunological comparisons of integrin alpha IIb beta 3 (GPIIb-IIIa) expressed on platelets and human erythroleukemia cells: evidence for cell specific differences.

Platelet glycoprotein IIb-IIIa (GPIIb-IIIa, alpha IIb beta 3) is expressed on the cell surface of the human erythroleukemia (HEL) cell line. Previous studies have demonstrated differences in GPIIb-IIIa ligand binding properties of HEL cells when compared to platelets. Although the mRNA sequences for GPIIb and GPIIIa are identical in platelets and HEL cells, cell specific differences in the conformation states of the GPIIb-IIIa complex may exist and may explain in part the contrasting functional properties. Two monoclonal antibodies (mAbs), an anti-GPIIb mAb C3 and an anti-GPIIIa mAb D3, were used to determine whether differences in GPIIb-IIIa conformational states could be measured. Initial studies in a purified system showed that the mAbs' binding to isolated GPIIb-IIIa conformers was increased to the active GPIIb-IIIa and to dissociated receptor subunits when compared to the inactive form. Furthermore, soluble active GPIIb-IIIa was a much better inhibitor of D3 binding to the immobilized receptor compared to soluble inactive GPIIb-IIIa. Extending these studies with intact cells, we detected at least two classes of binding sites for each mAb on each cell type. Differences in Bmax and in the relative affinities of the mAbs were identified and may represent subpopulations of GPIIb-IIIa conformations. Total HEL cell and platelet GPIIb-IIIa was determined in our binding assays using a radiolabeled GPIIb-IIIa complex specific mAb, 10E5. HEL cells express approximately five times more GPIIb-IIIa on a per cell basis. The percent of total GPIIb-IIIa that represented each class of mAb binding sites was determined. In summary, the relative differences in GPIIb-IIIa conformation found on platelets and HEL cells may be related to cell-specific ligand binding properties and activation states of the receptor.

Mathematical Analysis of Bleeding Time Data in Patients with Platelet Disorders and von Willebrand's Disease.

In a previous study the volume of blood obtained from bleeding time incisions was measured every 30 s from normal subjects (n = 15), patients with thrombasthenia (TSA, n=4), idiopathic thrombocytopenic purpura (ITP, n=4), von Willebrand's disease ((v)WD, n=3), Bernard-Soulier syndrome (BSS, n = 2), and δ and αδ-storage pool deficiencies (SPD, n=4 and 5, respectively) and the experimental results analyzed by empirical curve-fitting of the data. In the present investigation, a mathematical model based on blood flow physiology was developed to describe the rate of blood loss over time from these same patients as a function of two parameters, α, which describes the magnitude of vessel contraction following transection, and ß, the rate of vessel dilation to its nominal diameter. For the normal controls a third parameter, δ, was used to describe the rate of vessel closure due to the formation of a hemostatic plug. Optimal values for these parameters for the normal subjects and each patient group were determined by least-squared fitting of the experimental bleeding time data. For all subjects, values for the magnitude of vessel contraction were similar (α=0.65±0.02). However, values for ß were reduced in both TSA (ß=0.22±0.04) and (v)WD (ß = 0.30±0.03) and were increased relative to normal controls (ß=0.39±0.03) in BSS (ß=0.50±0.01) and both δSPD (ß=0.50±0.07) and αδSPD (ß=0.50±0.05). The initial rate of blood loss was also significantly greater in patients with BSS, ITP, δ-SPD, and αδSPD than in the normal subjects, as determined by a one-way analysis of variance. These results suggest that: (1) the initial contraction of severed blood vessels does not appear to be mediated by any plasma or platelet compounds absent in the various bleeding disorders considered in this study; and (2) the increased initial bleeding observed in SPD may reflect the absence of vasoactive agents, such as ADP or serotonin, released from platelet dense granules following platelet activation. These conclusions are consistent with those reported previously on the same patients and indicate that mathematical modeling of bleeding time measurements, based on assumptions of vascular and platelet reactivity, can provide insights into the complex series of events occurring at sites of vessel injury.

Platelet size distribution measurements as indicators of shear stress-induced platelet aggregation.

The mechanisms underlying shear stress-induced platelet aggregation (SIPA) were investigated by measuring changes in the platelet size distributions resulting from the exposure of human platelet-rich plasma (PRP) to well-defined shear stresses in a modified viscometer. Exposure of PRP to a shear stress of 100 dyne/cm2 for 1 min at 37 degrees C resulted in the loss of single platelets, an overall shift in the distribution to larger particle sizes, and the generation of platelet fragments. Treatment of PRP prior to shearing with a monoclonal antibody directed against platelet glycoprotein (GP) IIb-IIIa (integrin alpha IIb beta 3) at a concentration that completely inhibited ADP-induced platelet aggregation also inhibited SIPA. Furthermore, incubation of PRP with a recombinant fragment of von Willebrand factor (vWF) that abolishes ristocetin-induced platelet agglutination significantly inhibited but did not eliminate SIPA. Pretreatment of PRP with the tetrapeptides RGDS or RGDV, which constitute the GP IIb-IIIa peptide recognition sequences on fibrinogen and vWF, almost completely blocked platelet aggregation at 100 dyne/cm2, whereas the negative control peptide RGES had no discernible effect. Finally, incubation of PRP with a monoclonal antibody directed against the platelet vitronectin receptor (integrin alpha v beta 3) did not affect SIPA. These results indicate that both GP IIb-IIIa and GP Ib, the latter through its interaction with vWF, are required for SIPA at 100 dyne/cm2; that the interaction of GP IIb-IIIa with its adhesive ligands under shear stress can be inhibited by RGD-containing peptides; and that the vitronectin receptor on platelets, which shares the same beta 3 subunit as GP IIb-IIIa, plays no role in SIPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in fibrinogen adsorbed to segmented polyurethanes and hydroxyethylmethacrylate-ethylmethacrylate copolymers.

Fibrinogen adsorption from blood to biomaterials may regulate platelet adhesion and thrombus formation because of fibrinogen's central role in the coagulation cascade and its ability to bind specifically to the platelet membrane glycoprotein (GP) IIb-IIIa. Adsorption of fibrinogen from blood plasma to many materials exhibits a maximum with respect to plasma dilution and exposure time (the Vroman effect). In this study fibrinogen adsorption to several polymers was examined to ascertain the influence of controlled changes in surface chemistry on the Vroman effect. The materials included hydroxyethylmethacrylate-ethylmethacrylate (HEMA/EMA) copolymers, Biomer, and a series of segmented polyurethanes (PEUs), two of which contained fluorinated chain extenders. Each material exhibited maximal adsorption of fibrinogen at intermediate plasma concentrations. Little effect of soft-segment type or molecular weight was observed and no significant differences in fibrinogen adsorption to the fluorinated PEUs were seen. Changes in the strength of fibrinogen attachment to these materials with time after adsorption were also assessed. Fibrinogen adsorbed for 1 min was displaced more readily by blood plasma than that adsorbed for 1 h, regardless of the material. The more hydrophobic polymers exhibited greater retention of adsorbed fibrinogen. In addition, the fraction of fibrinogen retained by polyethylene depended on the amount of fibrinogen adsorbed to the surface, being greatest when the surface loading was the least. These studies indicate that spreading or transition of adsorbed fibrinogen molecules from a weakly to tightly bound state is a general consequence of protein adsorption to solid surfaces.

Measurement of fibrinogen adsorption from blood plasma using 125I-fibrinogen and a direct ELISA technique.

A direct enzyme-linked immunosorbent assay (ELISA), using a polyclonal anti-fibrinogen conjugated to horseradish peroxidase, was used to detect fibrinogen adsorption from blood plasma to ten different materials. Adsorption was also measured with [125I]-fibrinogen. The materials studied included glass, Biomer, Immulon I, and a series of hydroxyethylmethacrylate (HEMA) and ethylmethacrylate (EMA) co-polymers. For all the materials studied, the results from the ELISA technique closely paralleled those obtained using [125I]-fibrinogen. The cross-reactivity of the antibody with proteins other than fibrinogen was generally small. Both experimental methods detected the presence of a maximum in fibrinogen adsorption (as a function of the plasma dilution) to the more hydrophobic materials. For all but two HEMA/EMA co-polymers, a linear correlation between the ELISA and [125I]-fibronogen measurements was indicated by inspection of cross plots as well as by a statistical test.

The effects of temperature and buffer on fibrinogen adsorption from blood plasma to glass.

[125I]-Fibrinogen was used to measure the adsorption of fibrinogen from baboon plasma to two types of glass (Pyrex and a borosilicate glass) at 25 and 37 degrees C using two different buffers to dilute the plasma, the first being citrate-phosphate buffered saline (CPBSz) and the second isotonic Tris-saline (TRIS), both pH 7.4. In addition, the effects of hydration conditions, rinsing techniques, and glass-cleaning treatments on fibrinogen adsorption were evaluated. The data reveal that lower temperatures and the use of TRIS to dilute the plasma significantly enhance fibrinogen adsorption to both types of glass. As has been observed in the past, fibrinogen adsorption peaked at intermediate plasma concentrations on both Pyrex and borosilicate glass (the so-called Vroman effect), but almost twice as much fibrinogen adsorbed to glass when TRIS was used to dilute the plasma instead of CPBSz. Moreover, up to five times as much fibrinogen adsorbed to both types of glass at 25 degrees C compared with 37 degrees C. No effects of the rinsing technique or glass-cleaning treatment were observed.

The effects of surface chemistry and coagulation factors on fibrinogen adsorption from plasma.

Fibrinogen adsorption from plasma exhibits an unusual displacement phenomenon (the Vroman effect) in that decreases in adsorption occur after longer contact time or as the plasma concentration increases. Fibrinogen adsorption and the displacement effect were found to depend markedly on the nature of the surface, the time and temperature of adsorption, and to a lesser extent on certain contact activation factors. Displacement still occurred from plasmas lacking kininogens, factor IX, and prekallikrein, and from plasma that had been treated with BaSO4 to remove factors II, VII, IX, and X. Furthermore, displacement was also observed from fibrinogen solutions to which either albumin or hemoglobin had been added. In addition, competitive adsorption of binary protein mixtures was also shown to depend strongly on surface type. It therefore appears that fibrinogen adsorption from plasma is subject to similar if not identical competitive processes that occur in simpler protein mixtures. The final adsorption then reflects the influence of all the proteins in plasma, each competing for the limited number of adsorption sites according to the fundamental physical properties of surface activity and mass concentration.