States in adherent platelet morphology and the processing of adsorbed protein on biomaterials

137

States in adherent platelet morphology and the processing of adsorbed protein on biomaterials LA, Feuerstein and JL Sheppard Departments Cana da

of Chemical

Engineering

and Pathology,

McMaster

University,

Hamilton,

Ontario

L&S 4L7,

This study evaluates the range of adherent platelet morphologies and their relationship to preadsorbed protein. Fluorescently labelled protein was used so changes in its distribution could be followed along with morphological states assessed with modulation-contrast microscopy. Our particular concern was with the quantitative relationship between the platelet and the fluorescent image. The findings from this study continue to support the idea that platelets do interact with adsorbed protein so that protein redistribution occurs and that thrombin accelerates this. Evidence is also presented to support platelet shrinkage, membrane vesicle formation and destruction as a result of thrombin. The shrinking of adherent platelets causes areas free of pre-adsorbed protein to be exposed. This process will be important determining the nature of the substrate available to cells contacting surfaces along with other adsorbed protein-related processes, e.g. reversible adsorption and post-adsorptive transitions. Keywords: Received

Platelets,

17 April

fibrinogen,

thrombin,

1992; revised 30 June 1992; accepted

The sequence of events when blood contacts a biomaterial is believed by many to be protein adsorption, platelet adhesion, thrombin generation (due to contact activation), and thrombus formation. There has been much work on each of these events individually. In particular, protein adsorption has been studied extensively demonstrating the dynamic nature of adsorbed protein, continuous adsorption and desorptionl, turnover and post-adsorptive transitions which are likely to be the result of changes in the conformation of adsorbed protein” 3. Since platelets adhere soon after protein adsorption is initiated, we asked if they could contribute to the local surface concentration of adsorbed protein. We have shown on glass that there is a redistribution of adsorbed protein when platelets are presenta. The work described here extends the earlier study to a polymeric material and introduces thrombin, added in controlled amounts, to the platelet-protein interaction process. Thrombin is likely to be present near polymeric surfaces in contact with whole blood. It may be produced following plasma Factor XII activation at solid surfaces5 or possibly through the prothrombinase complex on the plasma membranes of activated adherent platelets5! 6. This study looks more closely at the range of morphology of platelets as they interact with their substrate. As before4, fluorescently labelled protein was used so changes in its distribution could be followed. Our Correspondence

to Dr LA. Feuerstein.

0 1993 Butterworth-Heinemann 0142-9212/93/020137-11

Ltd

PMMA, glass 1 July 1992

particular concern was with the quantitative relationship between the platelet and the fluorescent image. Finally, we attempted to assess if the presence of adherent platelets created access to the substrate for additional fluid-phase protein.

MATERIALS

AND METHODS

The methods described provide details of a morphological study of platelets adherent to adsorbed fibrinogen on two different substrates over a 90 min time-span. Concurrent evaluations were also made of the protein layer below the cells. Thrombin and a calcium ionophore were introduced to the system and the effects analysed. Viability testing of the platelets and the protein were performed, along with a number of protein adsorption studies which could reveal differences in the protein surface concentrations amongst the experimental conditions. Platelet preparation Venous blood drawn from healthy adult donors was anticoagulated with 14% acid-citrate-dextrose (ACD). Following centrifugation, the platelet-rich plasma was removed and the platelet suspensions were prepared using the method of Mustard et al.‘. Using Tyrode’s solution containing calcium and magnesium, pH 7.35, Biomaterials

1993, Vol. 14 No. 2

Platelet

138

morphology

the red blood cells were washed three times at a 1:l (v/v) ratio. The final blood cell suspensions contained 3 X 10’ platelets/ml, the haematocrit of red cells equalled 40%, 3.5 g/l of bovine albumin [Fraction V, Sigma Chemical Co., St Louis, MO, USA) and apyrase in the Tyrode’s solution.

Protein-related

methods

Preparation of lz51 labelled protein Purified fibrinogen, Fb (Fibrinogen, Human Grade L, KabiVitrum, Stockholm, Sweden), prepared using the method of Lawrie et aL8, was radiolabelled with “‘1 (Na”‘I, Amersham Corp., Arlington Heights, IL, USA) using immobilized lactoperoxidaseg. Unincorporated lz51 was separated from labelled protein by gel filtration on a chromatography column (Bio-Gel AG l-X4, Bio-Rad Laboratories, Richmond, CA, USA). Free “‘1 was between 0.5 and 1.5% of the total radioactivity, determined by the trichloroacetic acid precipitation of labelled protein lo . The labelled Fb was used within 48 h of preparation.

Adsorption

studies of lz51 labelled protein

Poly(methy1 methacrylate) (PMMA, Cyro Canada Inc., Etobicoke, Ontario, Canada) and alkali-silicate glass coverslips (Catalogue no. 1.2545M, Fisher Scientific Ltd, Whitby, Ontario, Canada) were cut into 1 X 1 cm2 then ultrasonically cleaned with a 0.1% detergent solution (Liqui-Nox, Alconox Inc., New York, NY, USA) for 10 min at 37’C, followed by two deionized, distilled water ultrasonic rinses for 10 min at 37°C. All samples were stored overnight at 4°C in Tyrode’s buffer. Before adsorption, the samples were transferred to 1.5 ml polystyrene microtitre plate wells (Costar, Cambridge, MA, USA) and filled with degassed Tyrode’s buffer. After a 1 h equilibration period, the degassed buffer was removed from the sample well and replaced with the appropriate protein adsorption solution. Glass or PMMA was immersed in a 0.1 mg/ml Fb solution (2% “‘1-Fb) in Tyrode’s solution for 1 h at 21°C. The samples were dip-rinsed three times in fresh buffer then divided into three separate treatment groups (three samples/group) and a base case (three samples) as follows: 90 min of incubation at 37’C in Tyrode’s buffer with albumin (3.5 g/ml). 90 min of incubation at 37OC in Tyrode’s buffer with albumin and 0.025 units/ml thrombin (thrombin from human plasma, 3375 NIH units/mg protein, Sigma Chemical Co.). 90 min of incubation at 37’C in Tyrode’s buffer with albumin and 0.4,~~ calcium ionophore (A23187, Sigma Chemical Co.). 0 min base case (no incubation). The buffer system for each treatment group was replaced with a fresh equivalent volume of the appropriate buffer following 45 min of incubation. Treatment groups (l)-(3) were subjected to three additional dip-rinses following the 90 min incubation period, blotted dry and transferred to counting vials. Biomaterials

1993, Vol. 14 No. 2

and adsorbed

protein:

LA. Feuerstein and J.I. Sheward

Preparation of fluorescently

labelled proteins

Purified human Fb was conjugated with a fluorescent label, fluorescein isothiocyanate (FITC, Sigma Chemical Co.), using modified standard conjugation methods1’-13. The preparation of these conjugates involved the reaction of purified Fb with the fluorochrome. The range of reaction times was 1.5-2.0 h with a range of dye to protein ratio of 1.0-l-5 mg FITC:lOO mg protein, Purification of the conjugates was achieved by column chromatography (Sephadex G-25, Sigma Chemical Co.). To evaluate the conjugates with respect to binding to activated platelets, a biological test was used. Aggregation of platelets was tested in an aggregometer (Aggregation module 300B-5, Payton Associates Ltd, Scarborough, Ontario, Canada). The agonist was ADP, 5pM (Sigma Chemical Co.), with a concentration of added Fb, labelled or unlabelled, at a level where the Fb concentration was limiting the extent of aggregation, 12 ,ag/ml. A reduction in Fb concentration reduced the extent of aggregation with ADP, 5 PM. Labelled Fb with a molar fluorochrome to protein ratio, F:P, varying from 4.5:1 to 12:l was tested. An F:P ratio > 8:l resulted in a lower aggregation peak when compared to unlabelled Fb, whilst those < 8:l provided similar traces to that of the unlabelled Fb control. For the protein adsorption work to follow with FITC-labelled Fb, an F:P ratio of 5:l was used. The above indicated that unlabelled Fb and FITC-labelled Fb were equivalent with respect to support of platelet aggregation. This is consistent with both types of Fb binding to their receptor on the platelet membrane in an equivalent way. The conjugates were tested for clottability using 1 unit/ml of thrombin, and were found to be similar to unlabelled Fb > 98% clottable. To ensure that ‘251-labelled Fb and unlabelled Fb adsorbed in the same way to a solid substrate, a preferential adsorption test was performed14. The percentage of “‘1-Fb was varied from 0.1 to 100% in Tyrode’s buffer, having a final total concentration of 0.01 mg/ml of protein, The protein solutions were contacted with 1 X 1 cm2 PMMA samples [three samples/ solution) for 2 h at 2l”C, after which the samples were quickly dip-rinsed three times in fresh Tyrode’s buffer, blotted dry and transferred to counting vials. With preferential adsorption absent, the surface concentration of Fb should be independent of the percentage of ‘251-labelled Fb. A second preferential adsorption test comparing FITC-labelled Fb with ‘251-labelled Fb was performed in parallel with the above study. In this case, the FITClabelled Fb was substituted for the unlabelled Fb of the previous test. The second test was necessary to elucidate the effect of FITC-labelling on Fb, as ‘251-labelling of FITC-conjugated Fb would prove too disruptive to the Fb molecule, a direct preferential adsorption comparison with unlabelled Fb was not possible. The surface concentration data emerging from these two tests are summarized in Table 1. The trend towards slightly higher surface concentration values for the 100% “‘1-Fb may be due to the high specific activity and high counts associated with these samples’4. In all cases, the difference in surface concentrations between the mixtures containing the various percentages of 1251-labelled Fb is much smaller than would be expected for a denatured

Platelet

morphology

Table 1

and adsorbed

Tests for preferential

‘%fibrinogen

protein:

adsorption

/.A. Feuerstein

of fibrinogen

and J.I. Sheppard

to PMMA’

(%)

Amount adsorbed Native-Fb/“‘I-Fb Experiment

139

1

(pg/cm*)

mixtures

FITC-Fb/‘251-Fb mixtures

Experiment 2

Experiment

1

Experiment 2

0.1 1.0 25.0 50.0 100.0

0.614 0.578 0.631 0.632 0.720

0.517 0.497 0.505 0.545 0.625

0.589 0.540 0.530 0.523 0.579

0.619 0.539 0.533 0.532 0.668

Mean Std. Dev.

0.635 0.052

0.538 0.019

0.552 0.019

0.578 0.040

‘Fibrinoaen adsorotion 100% %fibrinogk.

to PMMA after 2 h at 21 “C from a series of solutions

protein, or if free iodide was present. An analysis of variance (ANOVA) performed on the series of surface concentrations for native Fb-“‘1-Fb mixtures and the FITC-labelled Fb-‘?-Fb mixtures together revealed that there was no significant difference between the two series of surface concentrations (P > 0.05). This indicated that the FITC-labelled Fb does not adsorb differently from native Fb.

Fixation and staining

for platelet

cytoskeleton

A fluorescent phallotoxin, rhodamine phalloidin (R415, Molecular Probes Inc., Eugene, OR, USA), was used to stain large and small F-actin filaments of thrombinstimulated platelets adherent for 90 min to FITC-labelled Fb-coated glass. All fixing and staining solutions were made in Tyrode’s buffer. After 90 min from the initial adhesion, a 5 min Tyrode’s rinse, followed by a 5 min 0.2% glutaraldehyde (E.M. grade, 25% glutaraldehyde, Marivac Limited, Halifax, Nova Scotia, Canada) rinse and 10 min of incubation without flow was performed. Non-specific staining was avoided by rinsing the cell adherent coverslips twice in 0.05 M glycine (Fisher Scientific, Fair Lawn, NJ, USA) before incubating at 4°C in the same buffer for 15 min’5*‘6. The cells were stained with 200~1 of rhodamine phalloidin at 2.5 units/ml for 20 min at 21°C while protected from light. The coverslips were rinsed in Tyrode’s buffer twice and glycerol mounted on to a glass slide. Photography with fluorescence and Hoffman modulation contrast (HMC) optics was performed the following day.

Flow studies The coverslip (24 X 60 mm), composed of alkali-silicate glass, was cleaned with 5% Liqui-Nox and rinsed with distilled water followed by deionized water. The PMMA coverslip (30 X 60 mm] was rinsed thoroughly with distilled water and followed by deionized water. The coverslips were mounted in a flow cell which was filled with Tyrode’s solution. Fibrinogen labelled with FITC in Tyrode’s solution displaced that buffer from the flow cell, as described previously4. The protein at a concentration of 0.1 mg/ml was allowed to adsorb for 1 hat 21°C. There followed a 15 min Tyrode’s buffer rinse at a flow rate of 0.2 ml/mix-i, which corresponds to a wall shear rate of steps were done at 37°C. 100 s-l. This and subsequent

at a final total concentration

of 0.01 mg/ml in Tyrode’s buffer, varying from 0.1 to

The platelet/red cell suspension was next drawn over the surface at a flow rate of 1.0 ml/min, equivalent to a wall shear rate of 500 s-l for 30 s. Tyrode’s solution with albumin, with or without thrombin, 0.025 units/ml, followed, first at a shear rate of 500 s-l for 5 min, then at a shear rate of 50 s-l for the remainder of 90 min. Thrombin was kept on ice until needed and was added just before the time of use: fresh thrombin solution was added after 45 min. A calcium ionophore was also tested on PMMA with adherent platelets. A concentration range from 0.2 to 0.8pM calcium ionophore was tested by including it in the Tyrode’s albumin rinse, as was performed with the thrombin experiments above. It was found that, after 90 min, the degree of platelet spreading was similar to that of the thrombin condition when a concentration of 0.4pM was employed. A simultaneous experiment comparing 0.025 units/ml thrombin and 0.4 FM calcium ionophore was performed on PMMA. Both fluorescent and HMC images of platelets adherent to FITC-labelled Fb were photographed 90 min after initial platelet adhesion.

Photographic

methods

Photographs of HMC and fluorescent images were taken of the adherent platelets and the fluorescently labelled protein, respectively. The Wild Leitz Photoautomat (Wild MPS46, Leica Canada Inc., Willowdale, Ontario, Canada) was used in conjunction with a photomicrographic unit and Leitz Laborlux S microscope (Leica Canada Inc.). For HMC photography, Kodak TMY 400 ASA black and white film was used, and for fluorescent photography Kodak TMZ 3200 ASA black and white film was used. The same HMC 100 X oil immersion objective (1.25 NA, 160/0.17, Modulation Optics Inc., Greenvale, NY, USA) was used for both types of photography. This allowed for dual records of the same microscopic field. The negatives were then superimposed to assess the relationship between the two images. Hoffman modulation contrast photography This optical system allowed visualization of the morphological structures of the adherent platelets. A test was performed to determine the effects of serial exposures to transmitted light. Serial exposures to such light did not noticeably enhance or inhibit the morphological sequence for control and thrombin-treated platelets. Three states Biomaterials

1993, Vol. 14 No. 2

Platelet

140

morphology

of platelet morphological development were defined as follows: Unspread platelet - this cell was raised upon the substrate and either discoid or dendritic with small pseudopodia. The average cell diameter at this stage of development was found to be 2-4pm. Spread platelet - this platelet was flat, round and has reached, or is near, its maximum diameter of 7-10 ,um. Pseudopodia were very short or absent and small round raised areas may be found near the centre of the cell. These may be in the form of a peak or a raised ring. Pancake platelet - almost completely circular, 3-5 ,um in diameter, this cell may contain a few round raised protuberances located at the periphery. The platelet had a mainly smooth unperturbed surface.

and adsorbed

protein:

Morphological

experiments

Quantitative morphological studies HMC optics were used to study platelet morphological changes over 80 min. Every 20 min, a field of adherent cells was photographed for each experimental condition (four conditions, PMMA and glass with and without thrombin). The mean percentage of each morphological type (unspread, spread and pancake) was recorded from 4 d of experimentation in a manner similar to that used by Goodman et z11.l~. Single field analysis of platelet morphology over time A single field of thrombin-stimulated platelets adherent to PMMA coated with FITC-labelled Fb was photographed every 15 min for 90 min using transmitted light. The resultant fluorescent image of the same area of cells was photographed at the end of the 90 min study. HMC and fluorescent counterparts on PMMA and Glass 1. Paired photographs of HMC and fluorescent images of platelets on FITC-labelled Fb-coated glass and Biomaterials

1993, Vol. 14 No. 2

and J.I. Sheppard

PMMA, in the absence of thrombin, were taken 90 min after platelet contact. Every 15 min for 90 min, a field, containing thrombinstimulated platelets adherent to PMMA coated with FITC-labelled Fb, was photographed for morphological and fluorescent images. Each field was photographed only once with both types of optics. Thrombin-stimulated platelets adherent to FITClabelled fibrinogen-coated glass for 90 min were prepared for rhodamine phalloidin staining, Photomicrographs of a single group of platelets were taken with HMC optics for cell morphology and fluorescent optics for both FITC and rhodamine-derived images.

RESULTS Protein adsorption

Fluorescent photography A preliminary experiment was performed involving fluorescent photography of the FITC-labelled Fb-coated glass and PMMA surfaces without platelets, in the presence or absence of thrombin or calcium ionophore. Observations were made periodically for 90 min. Visually, the FITC-labelled Fb remained unaltered after 90 min of exposure to the surface in the absence of adherent cells. Changes in the adsorbed FITC-labelled Fb beneath the adherent platelets were visualized with fluorescence optics. Photography, using fluorescence optics, was always performed by exposing a single field of cells for < 5 s to light before moving to a new area of cells. Thus, any microscopic fields which were exposed to light were excluded from further study to avoid the effects of photobleaching of FITC-labelled Fb and photoactivation of cells.

LA. Feuerstein

experiments

Our experiments with surface-bound FITC-labelled Fb and adherent platelets were up to 90 min long. After the initial adsorption of Fb and adhesion of platelets, the surfaces were exposed to Tyrode’s buffer with albumin with no added material, with thrombin added or with calcium ionophore added. In the absence of platelets, measurements of the surface concentration of Fb were made at the beginning, before any additions, base case and at the end of the 90 min period for the above conditions. The same relative results were found for PMMA and glass while surface concentrations on glass were approximately 25% of those on PMMA. Surface concentrations for all the 90 min cases were less than their respective base case surface concentrations. These results are summarized in Figure 1 and indicate the same reduction with buffer containing albumin alone and with buffer containing albumin plus calcium ionophore, and less of a reduction with buffer containing albumin plus thrombin than with albumin alone. In the case of incubation with Tyrode’s buffer containing albumin alone, > 85% of the reduction in surface concentration occurred within 15 min of contact (data not shown).

Morphological

experiments

Quantitative morphological studies These results are presented in the form of four sets of bar charts: glass with and without thrombin and PMMA with and without thrombin (Figure 2). Three linea? models of the following form were fit with all of the data for each of the three responses obtained: unspread platelets, spread platelets and pancake platelets. Each response was modelled for time, treatment (with or without thrombin) and surface type (glass or PMMA). Since more than two time points were available a (time)’ term was included. Also, to elucidate the response, interaction terms between time and treatment, time and surface and treatment and surface were included. The form of the model is: Percentage of unspread, spread or pancake cells = + At + Btr + Cs + D(t X tr) + E(t X s) + F(tr X s) PMWI + Gt” where t = time, tr = treatment, s = surface, A,B, C,D,E,F and G are parameters which were determined for each of the three responses.

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142

Platelet

morphology

and

adsorbed

protein: /.A. Feuerstein

and J.I. Sheppard

Figure 3 A single microscope field of thrombin stimulated platelets, using HMC optics, is displayed at different times after initial contact on FITC-labelled fibrinogen-coated PMMA, a-c. a, 15 min; b, 30 min; c, 90 min; d, the corresponding fluorescent image to the HMC image of c at 90 min. Scale bar = 10pm.

than their cellular counterparts. The areas of altered fluorescence at 90 min are similar in size to the cells’ maximum size, attained at an earlier stage of spreading. Measurements of the largest distances across the surfaces of three cells, isolated from this series of photographs, (Figure 3) were made (Table 2). A similar measurement was then taken for the fluorescent counterpart. These measurements quantitatively verify that the region of altered fluorescence can be larger than the size of the covering platelet after 90 min of surface contact. HMC and fluorescent image counterparts on PMMA and glass In tests over 90 min, with and without thrombin, platelets adherent to unlabelled Fb-coated PMMA and glass did not produce any fluorescent images and FITC-labelled Fb in the absence of platelets showed no change in the fluorescence patterns. Table 2 adherent Time from attachment

variation of thrombin-stimulated platelets to PMMA coated with FITC-labelled fibrinogen

Size

initial (min)

Largest Cell 1

;z 90

90

l

linear

distance

Cell 2

(pm) Cell 3

a.0 a.5

6.0 6.5 7.0 5.0 3.0 4.5 5.5 Largest linear distance of altered fluorescence image+ (pm) 6.5 a.0 6.5

*The designations cell 1, cell 2 and cell 3 refer to Figure 3. +This is a distance across the regions of non-uniform fluorescence, composed of darker and brighter portions, associated with cell 1. cell 2 and cell 3.

Biomaterials

1993. Vol. 14 No. 2

In the absence of thrombin, platelet morphology and fluorescence patterns on FITC-labelled Fb-coated PMMA or glass are shown in Figure 4 after 90 min of contact. The unstimulated platelets on the PMMA surface (Figure 4a) are unspread and the protein layer remains unchanged (Figure 4b), exhibiting uniform background fluorescence similar to an FITC-labelled Fb-coated surface in the absence of any cells. The unstimulated platelets on the glass substrate, on the other hand, are well spread (Figure 4~) and produce an area of redistribution of fluorescence similar in size to that of the platelet above (Figure 4d). A fine black region of protein depletion is also seen encompassing the cell boundary (cell 1 of Figure 4c and d, with a central crater produced an equal area of bright fluorescence while cell 2, revealed areas of fluorescent centralization corresponding to a raised portion of the platelet]. In the presence of thrombin, the time-dependen’t changes in adherent platelet morphology and corresponding changes in the pattern of fluorescence of FITClabelled Fb on PMMA, are shown in Figure 5. Matched pairs of photomicrographs of different fields for each of four times after initial platelet contact using HMC and fluorescence optics, are shown. The determination of a record of the time progression of fluorescence images for a single field is not feasible, due to photobleaching of the labelled protein and photoactivation of platelets. The progression in the platelet spreading sequence of Figure 5 is similar to that of Figure 3 where records over time, using HMC optics, were made of one field. The fluorescent images at the 15 min stage of development (Figure 5b) are represented by areas of fluorescent concentration, as well as areas of fluorescent depletion, which are located beneath corresponding platelets. By

Platelet

morphology

and adsorbed

protein:

LA. Feuerstein and J.I. Sheppard

Figure 4 Side by side HMC and fluorescent paired imagesof platelets: without thrombin, after 90 min, on FITC-labelled fibrinogencoated on a and b PMMA, and c and d glass, cell 1 has a crater and cell 2 has a raised centre. Scale bar = 10,um.

30 min, the local changes in fluorescence, concentration and depletion, were larger than the platelets covering them (Figures 5~41). The contrast between areas of labelled protein concentration and areas of depletion increased with time. Some of the fluorescent images below the pancake platelets exhibited a pale general fluorescence at the later stages of development which was noticeably larger than the cell to which it correlated (cell 1, Figure 5g,h). Other pancake platelets had areas of bright fluorescence and dark areas arranged at opposite poles (cell 2, Figure 5g,h). Thrombin-stimulated platelets adherent to FITClabelled Fb-coated glass, 90 min from initial adhesion, are shown in Figure 6. There are several contracted cells much smaller than the single spread platelet shown near the top (Figure 6a). Each platelet produced central areas of FITC-fluorescence concentration and depleted regions (Figure 6b). The corresponding rhodamine phalloidin image of the well spread cell (Figure 6c) produced a central fluorescent concentration, similar to that of the central FITC-fluorescence concentration. Note the peripheral concentration and linear arrangement of fluorescence. Platelets, in the more contracted stage of development, exhibit an even fluorescence within the body of the cell and a few bright dots of fluorescence surround the central image.

Sfimula tion of pla telets with a calcium ionophore A comparison was made between calcium ionophorestimulated and thrombin-stimulated platelets on PMMA 90 min after first platelet contact. Both of these conditions led to the formation of a large percentage of pancake platelets (data not shown]. In both cases, photomicrographs of the fluorescently labelled protein had areas of

fluorescent underneath

concentration and darker the corresponding cell.

areas

placed

DISCUSSION The experiments described are an extension of previous work4. They explore the relationship between the changing states of adherent platelet morphology and the distribution of pre-adsorbed Fb. Two materials were used, (PMMA] and glass. In some cases, a low level of thrombin was introduced immediately after platelet adhesion and maintained during the experiments. The data obtained were of three kinds: (I) photographic, from modulation contrast microscopy of adherent platelets, (2) quantitative, in the form of percentages of adherent platelet morphological types with time, and (3) photographic, from fluorescence microscopy of fluorescently labelled surface-bound protein. In the absence of thrombin, the range of morphological states observed and the Fb surface concentrations measured are consistent with previous worklal lg. There is a distinct difference between the ability of PMMA and glass to adsorb Fb and participate in its redistribution. Platelets adhere and spread in small (PMMA, 10%) to moderate (glass, 30%) numbers within 20 min; following that, there are only small changes in morphology up to 80 min. This is consistent with the results of others”* 20.21. However, glass adsorbs less protein and demonstrates more redistribution of that protein. These features may result from a relatively weak binding of Fb to glass when compared to PMMA or possibly from a difference in the conformation of adsorbed Fb between the two materials. Thrombin is an important agonist for platelets and can be made available near biomaterial surfaces in contact Biomaterials

1993, Vol. 14 No. 2

144

Platelet

morphology

and adsorbed

protein:

/.A. Feuerstein

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Figure 5 A series of paired HMC and fluorescent images of thrombin stimulated platelets adherent to FITC-labelled fibrinogencoated PMMA after a and b 15 min, c and d 30 min, e and f 60 min, g and h 90 min of spreading time (cell 1 is associated with fluorescenceconcentration and cell 2 with polarized alteration of fluorescence). These pairs are not from thesame microscopefield. Scale bar = 10pm.

with whole blood. It can be produced via plasma Factor XII at solid surface? or via the prothrombinase complex at the surface of activated plateletssV6. Although we do not know quantitatively what concentrations of thrombin can exist near our surfaces with whole blood, we have attempted to simulate the presence of thrombin with a low concentration, 0.025 units/ml, capable of stimulating platelets in the aggregomete?. The addition of thrombin produces continued changes in the distribution of morphological states for the 80 min observation time. Spread cells, > 85% for both surfaces, were present after 20 min. Both surfaces presented, after 80 min, a picture of > 85% of cells which had spread to a maximum area then contracted, pancake platelets. In addition, the presence of dark regions, indicating absence of fluorescence, was now found with PMMA; these were not found on PMMA in the absence of thrombin. We are not aware of any report of pancake platelets like those observed here. Their formation requires a means by which the diameter of spread platelets can be reduced over time. Also, the appearance of these cells without their organelles seen protruding from the centres of spread platelets makes them appear less functional. Biomaterials

1993. Vol. 14 No. 2

Two possibilities which could lead to the above condition are alteration in the ultrastructure of the platelet accompanied by disintegration of the cell focused at the cell extremities and direct shrinkage of the adherent platelets in the plane of the substrate, which would require a thickening of the cell in the direction perpendicular to the substrate. During the clotting of platelet-rich plasma, there is evidence that platelets can undergo major alterations of their granules, internal and external membranes and in time disintegrate. The presence and later disappearance of vesicular material coming from internal and external membranes has been observed external to the main body of the platelet’“. These phenomena occur after secretion of granular material from platelets24325. The mechanism for the destruction of platelets is not known. We also observed platelet fragments at the outer periphery of our pancake platelets and with samples stained for actin. These could have been formed as a response to thrombin in a manner similar to that described above. The common features between the clotting experiment and the formation of pancake platelets are the presence of thrombin and possibly fibrin. The clotting experimentz3, in recalcified

Platelet

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FiguIN 6 Photographs of a single microscope field of thromb linstim ulated platelets adherent to FITC-labelled fibrinogt 3ncoat :ed glass: a, HMC optics; b, fluorescence optics for FIT‘Cc, fluorescence optics for rhodami ine labe illed fibrinogen; phal lloidin staining of F-actin. Scale bar = 10pm.

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plasma, would have thrombin concentrations near 1 unit/ml which could lead to more rapid effects than those of the current experiments with a thrombin concentration of 0.025 units/ml. The shrinkage of adherent platelets could alternatively be through the retraction of pseudopods with partial reformation of the discoid shape of the unstimulated platelet. A thicker cell in a direction perpendicular to the surface could be a consequence of such a process. It was found that a low level of thrombin reduced the time necessary to achieve all morphological states and redistribution of adsorbed protein by approximately tenfold, It is likely that this was related to additional platelet stimulation and secretion. The protein substrate and secretions from already adherent platelets can provide a base activation and the thrombin adds to this. We have previously provided evidence which associates the glycoprotein IIb/IIIa complex with the redistribution of surface-bound fibrinogen4’ “, “. The ability of thrombin to increase the number of available glycoprotein IIb/IIIa complex unit? is probably the means by which the speed and degree of protein redistribution increases. The attachment of actin filaments to the glycoprotein IIb/IIIa complex molecules of thrombin stimulated platelets has been noted by others”’ 3D. The presence of centrally located concentrations of fluorescently labelled Fb in combination with a centrally positioned actin concentration has been demonstrated here. This further supports the connection between the glycoprotein IIb/IIIa complex and the redistribution of adsorbed Fb. The formation of fibrin may also result from the addition of thrombin to platelets adherent to surfacebound Fb. Measurements of Fb surface concentration after adsorption of Fb and again after 90 min of flowing buffer, with or without thrombin, were made in the absence of platelets. Without thrombin, the surface concentration of Fb reduces over time; in the presence of thrombin, there was about half as much reduction, Parallel visual observations of fluorescently labelled adsorbed Fb in the absence of platelets showed an absence of the redistribution seen with platelets. This protein surface concentration evidence indicated that adsorbed Fb was being altered over time in the presence of thrombin. Possibly, fibrinopeptide A is being cleaved and the resultant charge on the adsorbed molecule is changed so that it is more strongly bound. Since platelets arrive on the substrate before thrombin in our experiments, an important question is: Can thrombin access the adsorbed Fb on the abluminal side of adherent spread platelets? If thrombin has no or limited access, its effect is likely to be on the adherent platelet alone. Since platelets are adherent but not completely spread before the arrival of thrombin, at least some portion of the Fb on the abluminal side of platelets can be accessible to thrombin. An attempt was made to determine if platelet stimulation alone could reduce the time for development of the full spectrum of morphological states and redistribution of adsorbed protein. Calcium ionophore, which was shown to have no effect on adsorbed Fb, was used to stimulate adherent platelets. It was found, like thrombin, to increase the speed of change of cell shapes, the percentage of later forms at earlier times and the speed of redistribution of adsorbed Fb. Biomaterials

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Our current experiments are consistent with the idea that no action of thrombin on adsorbed Fb is needed for the redistribution of that protein; in that context, its action is as a stimulator on platelets. Although we could not block the action of thrombin on adsorbed Fb as it bound to platelets, the ability of calcium ionophore, a platelet stimulator, to produce morphological changes and Fb redistribution similar to that of thrombin strongly supports this statement. In our previous work, we showed that platelets could redistribute Fb and fibronectin adsorbed to glass’. It was also shown that this redistribution was likely to be associated with the movement of the glycoprotein IIb/ IIIa receptors of the platelet, demonstrated by others”. The current experiments indicate that the changes in adsorbed protein distribution occur over areas covered by spread platelets. In the presence of thrombin, the regions showing absence of protein later, after redistribution of protein and platelet shrinkage, become accessible to fluid-phase protein. The current work demonstrates that the amount of redistribution of Fb is considerably less with PMMA, a hydrophobic polymer than with glass in the absence of thrombin. However, the addition of a small concentration of thrombin makes possible an additional redistribution of adsorbed Fb on PMMA and causes PMMA and glass to appear equivalent with respect to the distribution of morphological forms over time and the redistribution of Fb. The findings from this study continue to support the idea that platelets do interact with adsorbed protein so protein redistribution occurs. This process will be important for determining the nature of the substrate available to cells contacting surfaces along with other processes, reversible adsorption’ and post-adsorptive transitions” 3.

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ACKNOWLEDGEMENTS

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This work was supported by grants from the Heart and Stroke Foundation of Ontario, the Natural Sciences and Engineering Research Council of Canada and The Ontario Centre for Materials Research. The authors wish to thank: Mr W.G. McClung for performing the protein adsorption experiments and the statistical analyses, and Mr T. Sutton for advice on statistical matters.

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Chan, B.M.C. and Brash, J.L., Adsorption of fibrinogen on glass: reversibility aspects, I. Colloid Inter. Sci. 1961, 182,217-225 Bohnert, J.L. and Horbett, T.A., Changes in adsorbed fibrinogen and albumin interactions with polymers indicated by decreases in detergent elutability, I. Coil. Znterf. Sci. 1966, 111,363-377 Chinn, J.A., Posso, S.E., Horbett, T.A. and Ratner, B.D., Post-adsorptive transitions in fibrinogen adsorbed to Biomer: changes in baboon platelet adhesion, antibody binding, and sodium dodecyl sulfate elutability, 1, Biomed. Mater. Res. 1991, 25, 535-555

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Second European Conference on Engineering and Medicine Biennial Conference of the European Society for Engineering and Medicine (ESEM)

Stuttgart, Germany, 25-28 April 1993 Conference Topics + Medical informatics l Medical challenges for biomedical engineering l Minimal invasive tools for diagnosis and therapy l Implants l l Sensor devices, signal processing and transmission l l Imaging techniques, processing and transmission l

l

The Scientific Committee will be chaired by Dr S Gamwell-Dawids of the Institute of Engineering Design, Technical University of Denmark. For further information please contact the conference organisers: Die Kongress-Partner, Eberhardt & Neumann Savignystrasse 30, D-6000 Frankfurt/M.l., Germany. Tel: + 49 69 746 101 Fax: +49 69 746 110

Biomaterials

1993, Vol. 14 No. 2