Competitive Adsorption of Proteins During Exposure of Human

Chapter 11

Competitive Adsorption of Proteins During Exposure of Human Blood Plasma to Glass and Polyethylene P. Turbill, T. Beugeling, and A. A. Poot

Downloaded by UNIV LAVAL on April 24, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch011

Department of Chemical Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, Netherlands

Fibrinogen adsorption to glass from various human blood plasmas has been measured as a function of time. The plasmas were 11 single donor plasmas, pooled plasma, a single donor HWMK-deficient plasma and HMWK-deficient plasma, which had been reconstituted with HWMK. For adsorption times between 1 min and 1 h more fibrinogen adsorbed from HWMK-deficient plasma compared to the amounts of fibrinogen which adsorbed from the other plasmas. This result supports the conclusion of several authors that HWMK is involved in the displacement of fibrinogen, initially adsorbed from normal human plasma to glass. Glass surfaces, preexposed to solutions of plasma and subsequently exposed to 1:1 diluted plasma, give rise to a relatively high adsorption of HMWK which is independent of the plasma concentration of the precoating solution. This result demonstrates that HMWK displaces preadsorbed plasma proteins from the glass surface. The experiments with polyethylene as a substrate reveal that HDL displaces preadsorbed plasma proteins from the polyethylene surface. Moreover, evidence is presented that substantial amounts of albumin and fibrinogen, adsorbed from 1:1,000 diluted plasma to glass and polyethylene, are displaced from the material surfaces by proteins different from HMWK and HDL, when these surfaces are subsequently exposed to 1:1 diluted plasma.

Several studies strongly suggest that fibrinogen, initially adsorbed from blood plasma to glass or a glass-like surface, is subsequently displaced from the surface by H M W K (high molecular weight kininogen) (7-6). These studies include experiments in which the time dependent adsorption of fibrinogen from single donor HMWK-deficient plasma had been compared with the adsorption of fibrinogen from normal single donor plasma or from pooled plasma. The amount of adsorbed fibrinogen may, however, depend on the plasma composition. In

0097-6156/95/0602-0150$12.00/0 © 1995 American Chemical Society Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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order to get more insight into the real differences between normal donor plasmas and HMWK-deficient plasma with respect to fibrinogen adsorption, we determined the adsorption of fibrinogen to glass from 11 donor plasmas and pooled plasma as a function of time. The 11 donors did not belong to the group of 15 donors, who donated blood for the preparation of pooled plasma. In addition, we measured fibrinogen adsorption from single donor HMWK-deficient plasma and the same plasma, which had been reconstituted with H M W K . The displacement of adsorbed plasma proteins by H M W K was also studied in another way. Glass surfaces were first preexposed to solutions with different concentrations of plasma, resulting in protein layers having different protein compositions. The precoated glass surfaces were subsequently exposed to 1:1 diluted plasma. Thereafter the amounts of H M W K in the newly formed protein layers were determined and compared with the amounts of this protein adsorbed to the preexposed glass surfaces. Measurements of albumin and fibrinogen adsorption were also included in this study. Similar experiments were carried out with polyethylene instead of glass, but in this study the adsorption of H D L (high density lipoprotein) was investigated because it has been reported that H D L preferentially adsorbs to hydrophobic polymers such as polyethylene (6,7). Materials and Methods Test device. Protein adsorption from plasma solutions to the material surfaces was studied by means of a two step enzyme-immunoassay (6,8). In this enzymeimmunoassay (EIA) a special test device is used (Fig.l). The device consists of a stainless steel bottom plate (13 x 9.5 x 0.2 cm) provided with nine screw pins, and a Teflon upper part (13 x 9.5 x 1 cm) containing 24 cylindrical holes with a diameter of 10 mm as well as holes for the screw pins. At the bottom side, each of the 24 holes has a stepped recess (15.5 mm ID, depth 2.0 mm) in which a silicone sealing ring (10.77 ID x 2.62 mm; Eriks, Alkmaar, The Netherlands) is placed. Either a polymer sheet or two glass plates (in order to prevent breakage) can be placed between the bottom plate and the sealing rings in the Teflon upper part. After the components have been pressed together by means of wing nuts, a 24 wells test device is formed which allows the adsorption as well as the detection of proteins adsorbed to the surface of a polymer or glass. The test surface area and the maximum content of each well are 0.9 cm and 800 pL respectively. 2

Material surfaces. Glass plates were obtained from Corning, New York, U S A (hard glass, type 7059, thickness 2mm). Polyethylene sheet (low density polyethylene, thickness 0.05 mm) was obtained from T A L A S , Zwolle, The Netherlands. Glass plates and polyethylene sheets were cleaned as described by Poot et al (6). Plasmas and HMW kininogen. Pooled normal human plasma was obtained from 15 healthy male donors. From each donor 100 mL of venous blood was collected via a 1.5 mm needle and 'Silastic' Medical-Grade tubing (length 15 cm, 3/16 in ID) into two polypropylene centrifuge tubes (50 ml each), containing

Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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Figure 1. Test device for studying protein adsorption with the aid of an enzyme-immunoassay. (1) Stainless steel bottom plate, (2) Teflon upper part, (3) silicone rubber sealing ring.

Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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anticoagulant. The anticoagulant was 130 m M trisodium citrate and the anticoagulant to blood ratio was 1:9 (v/v). The tubes were centrifuged for 15 min at 1,570 g and the remaining plasmas were centrifuged for 15 min at 3,000 g. Thereafter the plasmas were pooled in a polypropylene beaker of 1,000 mL and the pooled plasma was transferred into polypropylene vessels of 2.2. mL. Vessels with plasma were kept at -30°C. Just before use, plasma was thawed in a water bath of + 3 7 ° C . Serial plasma dilutions were made with phosphate buffered saline, p H 7.4 (PBS) (NPBI, Emmer-Compascuum, The Netherlands), and put into polypropylene vessels of 2.2 mL before (diluted) plasma was transferred into these vessels. The 11 single donor plasmas were prepared from buffycoats which were obtained from the Blood Bank Twente-Achterhoek (Enschede, The Netherlands). These buffycoats had been prepared from citrated/dextran A blood collected in P V C blood bags. The buffycoats were centrifuged in polypropylene tubes for 15 min at 1,570 g, and the remaining plasmas were centrifuged for 15 min at 3,000 g. The single donor plasmas were put into polypropylene vessels of 2.2 mL and kept at -30°C. Just before use, the plasma was thawed in a water bath of + 3 7 ° C . Congenitally HMWK-deficient plasma was obtained from George King Biomaterials, Overland Park, K S , U S A , and kept at -30°C. Just before use, the plasma was thawed in a water bath of + 3 7 ° C . Purified native (single chain) H M W K (0.6 mg.mL" ) was kindly provided by Dr B . N . Bouma (Department of Hematology, University Hospital, Utrecht, The Netherlands). The purified H M W K has been extensively characterized (P). This protein was also kept at -30°C until use. HMWK-deficient plasma, reconstituted with H M W K , had a H M W K concentration of 70 ixg.mV . 1

1

Protein adsorption and E I A . A description of protein adsorption experiments and the subsequent enzyme-immunoassay (EIA) of adsorbed proteins with the aid of the test device has been given by Poot et al (

Competitive Adsorption of Proteins During Exposure of Human

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