Protein–platelet and platelet–leukocyte interaction at materials in contact with humanbloodHåkan Nygren, Magnus Braide, and Christin Karlsson Citation: Journal of Vacuum Science & Technology A 13, 2613 (1995); doi: 10.1116/1.579459 View online: http://dx.doi.org/10.1116/1.579459 View Table of Contents: http://scitation.aip.org/content/avs/journal/jvsta/13/5?ver=pdfcov Published by the AVS: Science & Technology of Materials, Interfaces, and Processing Articles you may be interested in Ultrasonic method to define human serum blood total protein and protein fractions J. Acoust. Soc. Am. 123, 3224 (2008); 10.1121/1.2933433 The effects of plasma-processing conditions on the morphology of adherent human blood platelets J. Appl. Phys. 103, 093302 (2008); 10.1063/1.2908200 Effects of Electron Beam and Microwave Irradiation on Human Blood Proteins AIP Conf. Proc. 899, 815 (2007); 10.1063/1.2733556 Dielectric Characterization of Leukocytes from Human Blood AIP Conf. Proc. 854, 170 (2006); 10.1063/1.2356437 Reduced adhesion of human blood platelets to polyethylene tubing by microplasma surface modification J. Appl. Phys. 96, 4539 (2004); 10.1063/1.1786668

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Protein–platelet and platelet–leukocyte interaction at materials in contactwith human blood

Hakan Nygren, Magnus Braide, and Christin KarlssonDepartment of Anatomy and Cell Biology, University of Go¨teborg, Goteborg, Sweden

~Received 28 September 1994; accepted 12 December 1994!

The adhesion and activation of platelets and leukocytes at blood–material interfaces was studied byfluorescence microscopy and photometry using specific anti-CD antibodies, antiplasma proteinantibodies, and the calcium probe Fura-2. Hydrophilic glass or methylized, hydrophobic glass wasprepared and capillary blood was placed as droplets on the surface in a humified chamber. Theadsorption of plasma proteins was monitored with FITC-labeled antibodies directed againstalbumin, IgG, fibrinogen, fibronectin, the v. Willebrand factor, prothrombin/thrombin, andcomplement factor C3c. The adhesion of platelets was shown by anti-CD 61 antibodies, specific forthis cell type. Adhesion of leukocytes was measured by staining their DNA with acridine orange.Adhering platelets were found after 15 s of blood–material contact on both surfaces. The number ofadhering platelets rapidly decreased at the hydrophilic surface, but remained high for more than 8min at the hydrophobic surface. Fibrinogen was the dominating protein at the material surface,whereas fibronectin and the v. Willebrand factor were found at the cell surfaces. Platelet-derivedmicrovesicles were found after 4 and 8 min of blood–material contact. These microvesicles showedintense staining with anti-C3c antibodies. Significant numbers of leukocytes~PMN cells! were seenafter 2 h of blood–material contact. In other experiments, granulocytes were isolated and incubatedwith Fura-2. The supernatant of hirudin-treated blood, exposed to hydrophilic or hydrophobic glasssurfaces, was added to the cells and the fluorescence was recorded after emission at 340 and 380 nm.A rapid peak was seen, indicating calcium influx into the cytoplasm. The activating substance wasremoved from the supernatant by filtering it through a 0.1–0.45mm Millipore filter. The bloodsamples were taken from patients undergoing treatment with extracorporeal circulation. The sampleswere incubated with monoclonal antibodies against surface antigens CD-11b, 16, 35, 61 and 62. Thefluorescence was measured in a flow cytofluorometer. The PMN cells were shown to be activatedrapidly after the onset of oxygenator circulation. ©1995 American Vacuum Society.

I. INTRODUCTION

Almost any medical device introduced into the humanbody will interact initially with blood. Plasma proteins, beingpresent in high concentrations, will be the first componentspresent at the blood–material interface. The adsorption ofproteins onto a clean surface is a rapid process and masstransport may well become the rate limiting step.1,2 Thismeans that a foreign surface introduced into blood will becovered with a monolayer of albumin within approximately0.0004 s. The initially formed layer will also contain 10% ofimmunoglobulin and less than 1% of fibrinogen if only con-centrations and diffusion coefficients are considered. Thisinitially formed layer is not stable but is exchanged uponprolonged exposure to blood.3 The kinetics of the exchangereaction will differ between surfaces4,5 but the general rule isthat large and less soluble proteins will be selected on thesurface.6 This may be expressed by the Bro¨nsted partition asC1/C25exp(2lA/kT) whereA is the molecular area andlrelates to molecular properties, e.g., to hydrophobicity.6

Fibrinogen, fibronectin, vitronectin, and the v. Willebrandfactor, acting as adhesive proteins for platelets, are oftenfound to be enriched at the surface of foreign materials.7

Several different studies have shown that adsorption of fi-brinogen and platelet activation on surfaces are importantparts of the nonself recognition of foreign materials7,8 andmay lead to an inflammatory reaction in the surrounding

tissue.9,10 The question asked in the present study is, How isinformation transferred from fibrinogen adsorbed at a blood–material interface to cells surrounding the site of exposure?The answer was sought along three lines of experimentalmodels: exposure of capillary blood to material surfaces andspecific labeling of proteins and cells reacting with the sur-face; incubation of blood with material surfaces followed byexposure of a pure preparation of Fura-2 loaded PMN cellsto the supernatant of the incubate, and measuring calciumflow into the cytoplasm; investigation by flow cytofluorom-etry of blood cells from patients undergoing treatment withextracorporeal circulation, and measuring the integrin ex-pression of platelets and PMN cells.

II. MATERIAL AND METHODS

A. Exposure of capillary blood

Capillary blood was placed in drops on glass slides~15315 mm!, either clean washed in acid ethanol~hydro-philic glasses! or methylized with hexamethyldisilazane bydeposition from gas phase: 0.1 mbar; 40 °C~hydrophobicglass!. The contact time was from 15 s to 2 h incubation timein a humified chamber at 37 °C.

The blood, which coagulated after approximately 4 min,was washed off with Dulbeccos’ phosphate-buffered salineand the glass was incubated with FITC-labeled antibodiesdirected against platelet Gp IIIa~CD 61!, albumin, IgG, fi-

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brinogen, fibronectin, the v. Willebrand factor, prothrombin/thrombin, and the complement factor C3c~DAKO, Patt,Copenhagen, Denmark! or was stained with acridine orange0.1%, which is a specific stain for DNA-containing cells, i.e.,leukocytes in blood. The glass slides were examined under afluorescence microscope; the fluorescence intensity was re-corded as 1/exposure time and pictures were taken as colorslides. The pictures were scanned, digitized, and printed asblack and white prints. The number of cells per image field~43104 mm2! adhering to the surface was counted at thepictures.

B. Exposure of PMN cells to whole blood plasma,interacted with material surfaces

1. Cell preparation

Human neutrophils~PMN cells! were obtained fromhealthy donors and isolated from peripheral blood byvenepuncture and the vacutainer system was anticoagulatedwith ethylenediaminetetraacetic acid~EDTA!. The PMNcells were isolated by a one-step Percoll technique,11 sus-pended and recalcified in Hanks’ balanced salt solution~HBSS! at apH of 7.4 containing 0.5% bovine serum albu-min ~BSA! ~Sigma Chemical Co., St. Louis, MO! until load-ing.

2. Fura-2 loading

The PMN cells~23106 cells/ml! in a HBSS buffer wereloaded with the calcium indicator Fura-2/AM~MolecularProbes, Inc., Eugene, OR!. The probe was added from a 0.5mM stock solution of Fura-2/AM dissolved in dry dimethyl-sulphoxide. The cells were incubated with 1mM Fura-2/AMat 37 °C for 30 min in a HBSS buffer together with 0.05%pluronic acid~Calbiochem, San Diego, CA!. Pluronic acid, anonionic dispersing agent, was added to make solubilizedFura-2 soluble in physiological media.12 After loading, thecells were washed three times by centrifugation followed byresuspension and were kept in a HBSS buffer until use. Mi-croscopic examination of the cells showed homogeneousfluorescence during this period.

3. Whole blood interaction with material surfaces invitro

Glass test tubes~Assistent, Germany! with a total volumeof 0.65 ml were treated to get a hydrophilic or hydrophobicsurface as described earlier. The glass test tubes were thenfilled with a 0.9% saline solution.

Venepuncture was performed upon the same healthy hu-man donor as previous. The blood was collected in polyeth-ylene syringes containing saline with hirudin~Sigma Chemi-cal Co.! and human serum albumin~HSA! to preventadsorption of hirudin into the syringe itself. The final con-centration of hirudin in the filled syringes was 50 units/mland this allowed the blood to remain liquid in the tubes for 2h or longer.7 The blood was used for experiments within 1min and syringed into the glass tubes under saline to avoidair–blood contact; noncontact was a prerequisite for repro-

ducible results. After 10 min the tube was centrifugated andhuman platelet-poor plasma~PPP! was received in the super-natant of the incubate.

4. Measurements of cytosolic free Ca 2 1

The PMN cells~23106 cells/ml! loaded with the calciumindicator Fura-2/AM were added to a cuvette in a Perkin-Elmer spectrofluorometer LS-50. Measurements were madeat 37 °C while continuously stirring the cell suspension. Cal-cium fluxes in PMN cells were detected by a change from380 nm in its unbound state to 340 nm when bound to cal-cium that occurs in the excitation spectrum of Fura-2 andemitted light was read at 510 nm.13 The supernatant of thehirudin-treated blood, incubated with hydrophilic or hydro-phobic glass surfaces, was added to the cell preparation at afinal dilution of 1/100 and the fluorescence was recorded. Insome experiments the supernatant of the hirudin-treatedblood was filtering through a Millipore filter with a pore size0.1–0.45mm ~Millipore Corp.! and then added to the cellpreparation. As a biological control of the system we usedthe chemotactic peptide for leukocytes, N-formyl-Met-Leu-Phe, fMLP~Sigma Chemical Co.! as a stimulus, with a finalconcentration of 1027 M fMLP in the cuvette. After the mea-surements, an identical reference recording was made withunloaded cells to compensate for medium autofluorescence.

C. Cell surface antigens in patients duringextracorporal circulation

Consecutive blood samples were drawn from five patientsat defined stages of coronary bypass operations:~1! beforesurgery;~2! after 1 h of surgery; ~3! after cooling and 15 minof oxygenator circulation;~4! after 45 min of extracorporalcirculation; ~5! after blood warming before disconnection;and ~6! 30 min after disconnection. Blood samples weredrawn into syringes, prefilled with hirudin~50 units/ml!,cooled, incubated with antibodies for 30 min and fixed withformaldehyde. In the FACSCAN evacuations that followed,the PMN cells were discriminated by size and granularityand measured by mean fluorescence in relationship to con-trols with nonrelated antibodies. In each patient the obtainedvalues were normalized to the values obtained before theoperation. Results were expressed as the mean of all patients~n55! for each antibody and stage of surgery. Monoclonalantibodies directed against CD-11b, CD-16, CD-35, CD-61,and CD-62 were performed in order to evaluate the occur-rence of thrombocyte–granulocyte aggregates and the activa-tion state of the thrombocyte in any such aggregates.14

III. RESULTS

The kinetics of platelet adhesion from native blood ontohydrophilic and hydrophobic glass surfaces is shown in Fig.1. As can be seen, high numbers~250–300 cells/image field!of platelets are seen after 15 s of blood–material contact. Atthe hydrophilic surface, the number of platelets decreasesrapidly down to a level of about 10% of the initial number.At the hydrophobic surface, some fluctuations in the number

2614 Nygren, Braide, and Karlsson: Protein–platelet and platelet–leukocyte interaction at material–blood interfaces 2614

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of adhering cells can be seen, but the level remains high, andis a factor of 10 higher than at the hydrophilic surface from 1through 8 min of blood–material contact.

The kinetics of leukocyte adhesion from native blood ontohydrophilic and hydrophobic glass is shown in Fig. 2. Only afew scattered cells can be seen on either surface from 2through 32 min of blood–material contact. After 64 min thenumber of cells increases significantly at the hydrophobicsurface and after 128 min, a significant increase is seen onboth surfaces, with a higher number of adhering PMN cellsat the hydrophobic surface.

The presence of plasma proteins at the surfaces is shownin Figs. 3 and 4. The surface concentration of plasma pro-teins measured as the fluorescence intensity~Fig. 3! showsthe surface concentration of fibrinogen is twofold higher thanthat of albumin and IgG. Fibrinogen was the only adhesionprotein that was found adsorbed onto the material surface@Figs. 4~a! and 4~b!#. An increase of the fluorescence inten-sity was seen in connection with platelets sticking to thesurface. The cell-related antifibrinogen fluorescence wasstronger on the hydrophilic surface than on the hydrophobic@Figs. 4~a! and 4~b!#. Labeled anti-v Willebrand factor anti-bodies were found localized at the cell surface of adheringplatelets@Figs. 4~c! and 4~d!#. The fluorescence intensity was

stable with time at the hydrophic surface and decreased rap-idly at the hydrophilic surface parallel with the decrease ofthe number of adhering CD-611 cells ~Fig. 1!. Labeled an-tifibronectin antibodies showed activity only at the cell sur-faces of scattered adhering platelets~not shown!.

Antigenic C3c was found localized at microvesicles after4–8 min of blood–material contact@Fig. 4~e!#. The numberof vesicles decreased at the hydrophilic surface during 4–8min of incubation and increased significantly on the hydro-phobic surface during the same period of blood contact.

Activation of Fura-2 loaded PMN cells was investigatedby measuring the fluorescence emitted~Fig. 5!. The valuespresented represent the mean of six individual measurementswith the mean values of the corresponding references sub-tracted.

The chemotactic peptide fMLP induces a fast, transient,and three-phasic rise in fluorescence which then slowly de-creases, showing an initial rise in cytosolic free Ca21 con-centrations. The supernatant of hirudin-treated blood ex-posed to hydrophilic glass surfaces was added to the PMNcells and the fluorescence was recorded. A rapid two-phasepeak was seen, although not as high as that for the fMLPstimuli, indicating calcium influx into the cytosol. The super-natant from the hydrophobic glass surface interaction withhirudin-treated blood shows only a weak increase in fluores-cence, indicating a low calcium concentration leaking intothe cytosol.

In the experiments with the the supernatant of the hirudin-treated blood in contact first with a hydrophilic surface, thenfiltering through the Millipore filter with a pore size 0.1mmand then added to the cell preparation, a low fluorescencesignal is seen, indicating that the activating substance wasremoved from the supernatant by filtering.

After the measurements, addition of digitonin~100mg/ml;Sigma Chemical! to the cuvette resulted in an increase influorescence~data not shown!. This was interpreted as thepresence of vital permeabilized cells, with calcium leakinginto the cytosol. The FACSCAN measurements~Fig. 6!showed an upregulation of the IgG receptor~CD-16! andcomplementary receptors 1~CD-35! and 3~CD-11b! startingat the onset of extracorporal circulation and remaining after

FIG. 1. A plot of the number of CD-611 cells adhering to hydrophilic~h:acid washed, OH! and hydrophobic~j: methylized, CH3! glass surfaces vstime of blood–material contact~in seconds!.

FIG. 2. A plot of the number of acridine orange1~DNA-containing! cellsadhering to hydrophilic~h: OH! and hydrophobic~j: CH3! glass surfacesvs time of blood–material contact~in seconds!.

FIG. 3. Surface concentration of albumin, fibrinogen, and IgG on hydro-philic ~OH! and hydrophobic~CH3! glass measured as fluorescence intensity~1/exposure time3 1000!. Background intensity~equal for all categories!has been subtracted from data.

2615 Nygren, Braide, and Karlsson: Protein–platelet and platelet–leukocyte interaction at material–blood interfaces 2615

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FIG. 4. Immunofluorescence micrographs showing the distribution of FITC-labeled antibodies on hydrophilic and hydrophobic glass surfaces representingone-half of an image field.~a! Antifibrinogen antibodies. Hydrophilic glass after 15 s of blood–material contact. Fluorescence is found evenly distributed onthe surface with a higher intensity at the surfaces of adhering platelets.~b! Antifibrinogen antibodies. Hydrophobic glass after 15 s of blood–material contact.Fluorescence is found evenly distributed on the surface with a higher intensity at the surfaces of adhering platelets. Note that some cells appear as black holesdue to a negative surface reaction and shielding of the underlying fibrinogen.~c! Anti-v. Willebrand factor antibodies. Hydrophilic glass after 15 s ofblood–material contact. Fluorescence is found localized at the surface of adhering platelets.~d! Anti-v. Willebrand factor antibodies. Hydrophobic glass after15 s of blood–material contact. Fluorescence is found localized at the surface of adhering platelets.~e! Anti-C3c antibodies. Hydrophilic glass after 240 s ofblood–material contact. Fluorescence is found localized at platelet surfaces and on microvesicles adhering to the surface. The number of microvesicles ishigher than the number of adhering CD-611 cells ~see Fig. 1!.

2616 Nygren, Braide, and Karlsson: Protein–platelet and platelet–leukocyte interaction at material–blood interfaces 2616

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disconnection. The time histories of the three receptors werealmost identical~Fig. 6!, suggesting that the underlyingmechanisms for upregulation were closely connected.

The presence of platelet-associated antigens CD-61 andCD-62 ~P selectin! on granulocytes was ascribed to adheringplatelets. The number of adhering platelets increased duringsurgery. Oxygenator circulation induced a decrease of non-activated platelets~CD-61! and a concomitant increase ofactivated platelets~CD-62! adhering to the granulocytes.There was no apparent correlation in time between the ex-pression of the platelet antigens and the granulocyte surfacereceptors.

IV. DISCUSSION

The flux of cells towards a material surface is governedby parameters similar to those for proteins, namely, size andconcentration. Erythrocytes with a concentration'63109

cells/ml and 6–8mm in size and platelets~43108 cells/ml; 3mm! collide most frequently with a surface introduced intothe blood. Leukocytes~63106 cells/ml; 15mm! representless than 0.1% of the total flux of cells towards a blood–material interface. The number of cells found adhering to thesurface will, of course, represent a fraction of this flow withthe property of expressing receptors for adhesion proteins.The finding that platelets are the dominating cells adhering toforeign surfaces during the first hours of blood–material con-tact is thus not a surprising one, but still one not often men-tioned in the literature.

The pattern of surface-adsorbed protein strongly suggeststhat the platelets adhere to surface-immobilized fibrinogensince other adhesive proteins were found localized at the cellsurface and not on the material. Platelet adhesion to surface-immobilized fibrinogen has been shown to induce thyrosinephosphorylation, cause reorganization of the cytoskeleton re-sulting in cell spreading, but doesnot induce secretion ofplatelet vesicles.15 Fibrinogen is thus suggested to be the

substrate for platelet adhesion to the surfaces, in accordancewith the result of other studies.16 Candidates for platelet ac-tivation of secretion are the v. Willebrand factor that hasbeen shown to affect platelet aggregation17 and thrombinwhich has been shown previously to be activated on hydro-philic but not on hydrophobic glass surfaces.5 Thrombin iswell known as a potent activator of platelet secretion15,18andmay increase the binding of fibrinogen to the platelets19 asseen on the hydrophilic surface of Fig. 4~b!. In the presentstudy, we failed to demonstrate prothrombin/thrombin on thecell surfaces that may be due to lack of sensitivity of theimmunoassay, but our data suggests that the v. Willebrandfactor is the cause of secretory activation of platelets at thematerials used.

The results of the present study indicate that activatedplatelets may leave the material surface, probably by stickingto fibrin formed in the coagulating blood. Platelet-derivedmicrovesicles stick to the surfaces in large numbers whereasonly small numbers of intact cells can be seen. That plateletshave really left the surface and have not changed only theirintegrin expression was verified by scanning electron micros-copy ~not shown!. The platelet-derived microvesicles havepreviously been shown to bind coagulation factors V andVIII, 20 and other coagulation enzymes,21,22but have not pre-

FIG. 6. Flow cytofluorometric~FACSCAN! measurements of the surfacereceptors on leukocytes in connection with cardiopulmonary bypass~CPB!and thoracic surgery~coronary bypass!. Upregulation of the IgG receptor~CD-16! and complementary receptors 1~CD-35! and 3~CD-11b! started atthe onset of CPB and remained after disconnection from the CPB circuit.The presence of thrombocyte-associated antigens~CD-61 and CD-62! ongranulocytes was ascribed to adhering platelets. The number of adheringplatelets increased during surgery. CPB induced a decrease in nonactivatedplatelets~CD-61! and a concomitant increase in activated platelets~CD-62!adhering to the granulocytes.

FIG. 5. Changes in intracellular calcium concentration measured as fluores-cence intensity of the calcium probe Fura-2 after stimulation of humangranulocytes at timet50. Plasma was obtained from blood, anticoagulatedwith hirudin and incubated in either hydrophilic~OH! or hydrophobic~CH3!glass tubes. Microfiltration of the plasma after incubation in a hydrophilictube abolished the subsequent calcium response of the white cells.

2617 Nygren, Braide, and Karlsson: Protein–platelet and platelet–leukocyte interaction at material–blood interfaces 2617

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viously been shown to dress complement factors.Platelet-derived microvesicles are strong candidates as the

morphological substrate of the PMN cell-activating, filter-removable substance that can be transferred from blood aftercontact with surfaces~Fig. 5!. Platelet activation of PMNcells has previously been discussed as a result of interactionwith the P selectin~CD-62!,23,24 which has also been sug-gested as the mechanism behind the clearance ofmicrovesicles.25 The results of the present study also indicatea possible role for complement factor C3 on microvesicles inthe activation of white cells. This pathway is strongly sup-ported by our clinical data, showing the expression ofcomplementary receptors in patients during treatment withextracorporeal circulation~see Fig. 6!. This is in accordancewith other studies, reporting upregulation of the complementreceptor CD-11b on neutrophils after contact with activatedplatelets.26

V. CONCLUSIONS

Platelets are the primary reactive cells in the nonself rec-ognition of foreign materials.

Platelets adhere to fibrinogen, which is the most abundantplasma protein during the initial blood–material contact.

Activated platelets secrete microvesicles which dressserine proteases and activated platelets can mediate activa-tion of PMN cells.

1R. R. Gorman, G. E. Stoner, and A. Catlin, J. Phys. Chem.75, 2103~1971!.2P. Wojcieshowskij, P. Ten Hove, and J. L. Brash, J. Colloid Interface Sci.111, 455 ~1986!.

3L. Vroman and A. L. Adams, J. Biomed. Mater. Res.3, 43 ~1969!.4L. Vroman, A. L. Adams, and M. Kling, Blood55, 156 ~1980!.5H. Nygren, J. H. Elam, and M. Stenberg, Acta Physiol. Scand.133, 573~1988!.6H. Nygren, S. Alaeddine, I. Lundstro¨m, and K.-E. Magnusson, Biophys.Chem.49, 263 ~1994!.7J. H. Elam and H. Nygren, Biomater.13, 3 ~1992!.8R. C. Woodman and L. A. Harker, Blood76, 1680~1990!.9L. Tang and J. W. Eaton, J. Exp. Med.178, 2147~1993!.10J. H. Elam and M. Elam, Biomater.14, 861 ~1993!.11M. Braide and L. M. Bjursten, J. Immunol. Methods93, 183 ~1986!.12M. Poenie, J. Alderton, R. Steinhardt, and R. Y. Tsien, Science233, 886

~1986!.13G. Grynkiewicz, M. Poenie, and R. Y. Tsien, J. Biol. Chem.260, 3440

~1985!.14C. S. Rinder, J. L. Bonan, H. M. Rinder, J. Mathew, R. Hines, and B. R.Smith, Blood79, 1201~1992!.

15B. Haimovich, L. Lipfert, J. S. Brugge, and S. J. Shattil, J. Biol. Chem.268, 15868~1993!.

16H. Nagai, M. Hande, Y. Kawai, K. Watanabe, and Y. Ikede, Thromb. Res.71, 467 ~1993!.

17J. R. O’Brien and G. P. Salmon, Blood70, 1354~1987!.18C. Taparelli, R. Mettesnick, and N. S. Cook, Trends Pharmacol. Sci.14,426 ~1993!.

19A. S. Huzoor, N. Kundu, and R. Kornhauser, Life Sci.53, 1967~1993!.20P. J. Sims, E. M. Faioni, T. Wiedmer, and S. J. Shattil, J. Biol. Chem.263,18205~1988!.

21G. E. Gilbert, P. J. Sims, T. Wiedmer, B. Furie, B. C. Furie, and S. J.Shattil, J. Biol. Chem.266, 17261~1991!.

22J. D. Wencel-Drake, M. G. Dieter, and S. C. T. Lam, Blood82, 1197~1993!.

23R. P. McEver, J. Cell Biochem.45, 156 ~1991!.24D. E Lorant, M. K. Topham, R. E Whatley, R. P. McEver, T. M McIntyre,S. M Prescott, and G. A. Zimmerman, J. Clin. Invest.92, 559 ~1993!.

25W. Jy, L. Horstman, W. Mao, and Y. Ahn, Blood82Supp. 1, 281a~1993!.26E. Morrison, P. M. Guyre, E. Larsen, Blood82 Supp. 1, 157a~1993!.

2618 Nygren, Braide, and Karlsson: Protein–platelet and platelet–leukocyte interaction at material–blood interfaces 2618

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