Capillary Perfusion System for Quantitative Evaluation of Protein

Chapter 33

Capillary Perfusion System for Quantitative Evaluation of Protein Adsorption and Platelet Adhesion to Artificial Surfaces Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 29, 2018 | https://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch033

Jean-Pierre Cazenave and Juliette Mulvihill Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Institut National de la Santé et de la Recherche Médicale U.311, 10 rue Spielmann, 67085 Strasbourg Cédex, France

A capillary perfusion system has been developed for in vitro quantitation of protein adsorption and platelet accumulation on artificial surfaces under controlled hydrodynamic conditions (0-4,000s ). Using washed platelet suspensions or anticoagulated whole blood, platelet deposition and/or protein adsorption is followed by radioisotopic techniques, platelet deposition in whole blood being measured by surface phase radioimmunoassay with a monoclonal antibody against human platelet membrane glycoprotein Ilb-IIIa. Applications include studies of the influence of preadsorbed proteins on platelet accumulation on artificial surfaces and screening of biomaterials for short term hemocompatibility. -1

Under normal conditions, the luminal surface of the endothelium lining of the cardiovascular system is non thrombogenic and does not allow platelet and leucocyte adhesion (1,2). In contrast, exposure of an artificial surface to blood leads to basic reactions which may culminate in thrombus formation (3 ) · When flowing blood enters into contact with an artificial surface, a series of events is initiated : rapid adsorption of a layer of plasma proteins at the interface, platelet adhesion to the protein layer and conformational changes of adsorbed contact factors leading to activation of the coagulation system to form thrombin and fibrin. Hemodynamic and rheological conditions play an important role in the localisation, size, structure and turnover of the resulting thrombus. In high flow regions, it is composed mainly of platelets and fibrin. The adsorption of a layer of plasma proteins is the first event which occurs when blood is exposed to an artificial surface {^). As a result, a platelet never sees or adheres to abare surface. The nature of the adsorbed protein layer, which depends on the relative concentrations and mobilities of the proteins in plasma and on their affinity for the surface, will condition the subsequent plateletsurface interaction (5) · Protein adsorption to foreign surfaces has 0097-6156/87/0343-0537$06.00/0 © 1987 American Chemical Society

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

PROTEINS AT INTERFACES

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been studied experimentally by measuring the adsorption isotherms of single p u r i f i e d proteins (6,7) known to be involved i n thrombosis (fibrinogen, f i b r i n , contact factor XII, high molecular weight kininogen, von M l l e b r a n d factor, thrombin), inflammatory and immunological reactions (immunoglobulins, immune complexes, complement chemotactic fragments C5a and C3a) and c e l l adhesion (albumin, f i b r o n e c t i n , collagen). Adsorption of a protein at an interface may be followed by denaturation, conformational change or b i o l o g i c a l a c t i v a t i o n . Furthermore, adsorption from blood, which i s a complex mixture of proteins, i s complicated by competition between proteins, desorption and. s t r u c t u r a l modifications of the protein layer with time and rheological factors (6,8). The development of thromboresistant biomaterials i s important i n a l l situations where an a r t i f i c i a l surface i s exposed to blood, f o r example i n cardiovascular implants (heart valves, shunts, vascular grafts and catheters), extracorporeal c i r c u l a t i o n systems, blood f i l t e r s and blood storage containers. In order to predict the short term c l i n i c a l performance of biomaterials, we have developed an i n v i t r o technique for the determination of protein adsorption and p l a t e l e t adhesion to a r t i f i c i a l surfaces which s a t i s f i e s the following requirements : absence of an a i r - s o l u t i o n i n t e r f a c e , an essential condition to avoid a c t i v a t i o n of proteins or p l a t e l e t s by a i r contact; well characterised hydrodynamic conditions ( P o i s e u i l l e laminar flow) with wall shear rates corresponding to those encountered i n the cardiovascular system (0-4,000 s"'') (9); using radioisotopic methods, p o s s i b i l i t y of the simultaneous study of p l a t e l e t adhesion and the adsorption of one or more plasma proteins; quantitation of p l a t e l e t accumulation either with washed radiolabeled p l a t e l e t s or by surface phase radioimmunoassay using a monoclonal antibody directed s p e c i f i c a l l y against the membrane glycoprotein complex I l b - I I I a , thus enabling experiments to be performed with whole blood. Experimental Procedures C a p i l l a r y Perfusion System. The i n v i t r o perfusion system (Figure 1 ) consists of two p l a s t i c syringes mounted i n p a r a l l e l on a syringe pump (Precidor 5003, Infors, Easel, Switzerland) and leading to a pair of glass c a p i l l a r y tubes (Microcaps, Drummond S c i e n t i f i c Co., USA). P r i o r to use, c a p i l l a r i e s are cleaned by immersion for one hour i n sulphcchromic acid, followed by extensive r i n s i n g i n d i s t i l l e d water and oven drying at 10C°C. The c a p i l l a r y i n t e r n a l diameter i s constant to within 2%. Capi] l a r i e s of i n t e r n a l diameter 0.56 mm are used for perfusion at wall shear rates from 800 to 4.,000 s and c a p i l l a r i e s of i n t e r n a l diameter 0.80 mm for perfusion at shear rates less than 800 s~ . A three way stop-cock at the j o i n t between each syringe and i t s connecting c a p i l l a r y leads v i a a p e r i s t a l t i c pump (Minipuls 2, HP4-, Gilson, France) to a reservoir containing r i n s i n g buffer. The entire system i s maintained at 37.0 ± 0.2°C under a thermostated hood (ITH I, Infors, Basel, Switzerland). Using radiolabeled proteins, p l a t e l e t s or a n t i - p l a t e l e t antibody, protein adsorption or p l a t e l e t accumulation, i s measured according to the r a d i o a c t i v i t y deposited on c a p i l l a r y segments of known i n t e r n a l surface area, determined by radioactive counting i n a well-type gamma counter (1282 Compugamma, LKB, Sweden). Blood c o l l e c t i o n at the c a p i l l a r y exit enables quantitative determination of



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Capillary Perfusion System

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Figure 1 : Schematic diagram cf the in vitro capillary perfusion system. S : 50 ml plastic syringe containing protein or antibody solution, antiecagulated whole blood or washed platelet suspension; Ρ : piston driven by syringe pump; SC : 3-way stop-cock; J : joint in silicone tubing; C : glass capillary (0.80 or 0.56 mm i.d.); F : direction of blood flow; Τ : plastic tube for blood collection; R : rinsirg buffer from a reservoir at 37°C, flow controlled by a peristaltic pump. The entire apparatus is enclosed in a thermostatec hood at 37°C.


540



factors indicating activation of p l a t e l e t s (platelet factor 4 , 3 tbromboglobulin) or the coagulation system (fibrinopeptide A ) . Morphological examination of the c a p i l l a r y surface i s also possible by scanning electron microscopy ( 1 0 ) « P u r i f i c a t i o n and Radiolabeling of Proteins. Human serum albumin i s a s t e r i l e solution (175 g/1 ) prepared by the Cohn method of ethanol fractionation at the Centre Regional de Transfusion Sanguine de Strasbourg. Fibrinogen is p u r i f i e d from human plasma by the technique of Kekwick ( 1_1_). Human fibronectin i s p u r i f i e d by a f f i n i t y chromatography using g e l a t i n immobilised on Sepharose 4B, according to Miekka ( 1_2 ). Von Willebrand factor (vWF) i s isolated by gel f i l t r a t i o n of human plasma cryoprecipitate and characterised according to the von Willebrand antigen content, determined by I m m u n o e l e c t r o p h o r e s i s , and t h e r i s t o c e t i n cofactor a c t i v i t y , measured i n agglutination tests with washed human p l a t e l e t s (13,). The purity of plasms proteins, as v e r i f i e d by electrophoresis i n SDS-polyacrylamide gel (1^), i s always greater than 98 %. Bovine collagen (type I, insoluble, Sigma Chemicals, St Louis, USA) i s a 0.25 % suspension i n 0.522 M acetic a c i d , pH 2.8, prepared by the method of Cazenave (13). Purified human plasma proteins are radiolabeled with 'Îodine (Commissariat à l E n e r g i e Atomique, Saclay, France) by the iodogen technique ( 1 5 ) « f

Production, P u r i f i c a t i o n and Radiolabeling of Monoclonal Anti-human P l a t e l e t Antibody 6C9» The monoclonal antibody 6C9 directed s p e c i f i c a l l y against the membrane glycoprotein complex l i b - I l i a of human p l a t e l e t s i s a development of the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service i n Amsterdam. As described i n d e t a i l in previous publications (16), antibody i s produced i n BALB/C mice, p u r i f i e d by ammonium sulphate p r e c i p i t a t i o n and ion exchange chromatography and characterised by serological and immunochemical a n a l y s i s . Radiolabeling with ^Îodine j performed using the iodogen technique. s

1

Blood C o l l e c t i o n and Preparation of Washed Îndium Labeled Human P l a t e l e t s . Blood i s collected from healthy donors by venipuncture using a large diameter (18/10) needle. Washed human p l a t e l e t s are prepared by the method of Cazenave (13.) $ a modification of the technique of Mustard (17), and resuspended at 300,000/mm^ i n Tyrode's buffer containing 2 mM C a , 1 mM M g , 0.35 % human serum albumin, 0.1 % glucose and 20 y l / m l apyrase (1.3), pH 7.30. Radiolabeling of p l a t e l e t s i s carried out by incubation with Indium-oxine (0.25 g/ml) for 15 min at 3 7 ° C , according to Eber ( IS). Under these conditions, labeling y i e l d i s of the order of 90 % and p l a t e l e t aggregation with ADP, thrombin or collagen i s not modified. Washed red blood c e l l s from the same donor are prepared as described by Cazenave ( 1 9 ) · Immediately before perfusion experiments, packed erythrocytes are added to the suspension of washed Indium labeled human p l a t e l e t s at a volume r a t i o corresponding to a 40 % hematocrit. For perfusion studies with heparin (10 U/ml, Roche, France) anticoagulated whole blood, the stoppered tubes are kept at room temperature u n t i l required for perfusion, within a maximum delay of two hours. 2 +

Protein Adsorption.

Adsorption of

2 +

human plasma proteins

to glass



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capillary surfaces has been studied under static conditions az ambient temperature. Purified plasma proteins are diluted in rinsing buffer (Tyrode*s buffer without calcium or magnesium, pH 7.30). The capillaries are f i r s t f i l l e d with rinsing buffer, the protein solution is introduced at low flow rate (150 s ) by displacement of at least four capillary volumes and the tubes are then stoppered and left for one hour, the time required to attain adsorption equilibrium. Using radiolabeled proteins for the measurement of adsorption isotherms, rinsing (10 min at 1,000 s ) is followed by radioactive counting. Precoating of glass capillary surfaces with plasma proteins or bovine collagen for subsequent platelet adhesion studies is performed in the same manner as the determination of adsorption isotherms, the bulk solution concentration of plasma protein being chosen to l i e in the plateau region of the Langmuir-type adsorption curve. Bovine collagen is used as a 0.25 % suspension in 0.522 M acetic acid, pH 2.8. In the case of plasma proteins, the precoated tubes, rinsed and buffer f i l l e d , are left stoppered at ambient temperature until perfusion, within no longer than 18 hours. Collagen precoated capillaries are emptied to allow polymerisation of the collagen on contact with air (1_9) and may be stored at 4°C for up to 7 days. Care is taken at a l l times during protein adsorption or blood perfusion experiments to avoid the formation of air bubbles or an air-liquid interface, to prevent denaturation or activation of proteins or platelets on air contact. Perfusion of Whole Blood or Washed Platelets Suspensions. Capillaries are perfused with anticoagulated whole blood or with a suspension of washed 'Îndium labeled human platelets in "yrode salbumin buffer in the presence of a 4-0 % hematocrit. The blood or platelet suspension is introduced by displacement of rinsing buffer. Perfusion is continued for a fixed time interval, e.g. 2 or 5 min, at a steady flow rate corresponding to a predetermined wall shear rate, e.g. 50, 800, 2,000 or 4,000 s" . Rinsing with buffer (5 min at 1,000 s~ ) is followed by radioactive counting in the case of Îndium labeled platelets or by radioimmunoassay with ^Îodine labeled anti-human platelet antibody in experiments with whole blood. The perfusate is collected in plastic tubes. Platelet count is determined before and after perfusion, while standard immunoenzymatic (ELISA) techniques with commercial kits (Asserachrom, Stago, France) are used to measure the production of fibrinopeptide A (FPA), platelet factor 4 (PF4) or B-thromboglobulin ($TG). f

1

1

Quantitation of Platelet Deposition by Radioimmunoassay with Anti-human Platelet Antibody 6C9. In perfusion experiments with unlabeled platelets in anticoagulated whole blood, platelet deposition is quantitated by means of a surface phase radioimmunoassay using the monoclonal antibody 6C9, directed against the membrane glycoprotein complex Ilb-IIIa of human platelets. An aliquot of purified 'Îodine labeled antibody is diluted in rinsing buffer containing 0.1 % human serum albumin and introduced into the capillary tubes at low flow rate (150 s-1 ) by displacement of at least four volumes of buffer. The tubes are stoppered and incubation is continued under static conditions at 37°C for 30 min, the time required for immunoadsorption to reach a plateau value. Background antibody adsorption is determined using capillaries perfused with platelet poor plasma. After rinsing (5 min at


542



2

1,000 s" ), radioactive counting yield ε the ^ *Iodine radioactivity due to immunoadsorbed antibody for capillary segments of known surface area. Calibration of the radioimmunoassay is performed by means of cjlguble isotope experiments with ''indium labeled platelets and ^'Icdine labeled antibody. Capillaries are perfused with blocd containing autologous washed ''Îndium labeled platelets and a linear correlation is obtained between adherent ''indium-platelets and immunoadsorbed -'Iodine-antibody. For experiments using unlabeled platelets in whole blood, platelet adhesion is then calculated according to the relation: 2

adherent platelets/mm = ( 1 2

1 2 $

1 2

I - " I ) x slope /S A

1

E

(1)

c

12

where % is the measured Îodine radioactivity, ^ I g the background antibody adsorption, S the surface area and slope^ the slope of^the linear correlation for radioimmunoassay ( ' In-platelets versus Î-antibody), corrected for physical decay. A

Results and Discussion Flow Properties of Perfusion System. Under conditions of Poiseuille type laminar flou in capillary tubes, the Reynold's number (R ) is given by the relation : e

R

e

=

P
r

^

ζ

where Ρ is the volumetric mass, r the tube radius,^ ζ the fluid viscosity and < ν > the average fluid velocity (flow rate/r ). The onset of turbulent flow occurs at Reynold's numbers in excess of 2,500 (9) · In our system, at maximum shear stress (4,000 s"'' ), corresponding to a flow rate of 4· 14 ml/min in capillaries of internal diameter 0.56 mm, the Reynold's number calculated from Equation 2 is 80. This figure lies well below the limit of turbulent flow. Furthermore, experiments with tracer dyes have shown no visible evidence of turbulence at wall shear rates in the range 50-4,000 s , either within the capillaries or at entrance or exit points. The length of the inlet region (L) at the entrance of the capillary is estimated as (9) ï 2

( 3 )

L = 0.07 R r e

which in our case is never greater than : L = 0.07

= 0.16

2

χ 80 χ 2.8 χ 10" cm.

Since the minimum capillary length used in our system is 4 cm, the inlet region where the fluid flow is not of Poiseuille type represents 4 % or less of the total flow path studied. P. Ώ entrance effect would be reflected in increased protein or platelet deposition under dynamic conditions. Using radiolabeled platelets, when capillaries are cut into 1.0 cm segments for radioactive counting, there is no increase in



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radioactivity of the f i r s t segment other than that which may be attributed to the known variation of platelet accumulation with tube length (see following paragraph). Sensitivity in these measurements is sufficient to ensure less than 1 % error in radioactive counting of capillary segments. The velocity profile in the perfusion system is therefore considered to be parabolic and wall shear rates (γ) are calculated according to the relation : γ = U< ν > r

(4)

In platelet perfusion experiments, platelet accumulation on reactive adhesive surfaces such as collagen coated glass shows a steady decrease with distance from the capillary inlet, as previously observed in similar flow systems (20,21 ). This axial dependence does not, in our system, appear to follow a simple mathematical law as a function of distance from the capillary entrance (22). Platelet deposition is thus calculated as the mean deposition over the entire length ( ^ 10 cm) of a single capillary and in comparative studies capillaries of equal length and internal diameter are considered. Static Adsorption of Plasma Proteins on Glass. Initial studies of the interaction of proteins with a r t i f i c i a l surfaces concerned the highly simplified situation of static adsorption on glass from solutions of purified radiolabeled human plasma proteins. Albumin was chosen as a major plasma protein known for i t s non thrombogenic properties (5,6). Fibrinogen and fibronectin, on the contrary, are major proteins of plasma which enhance platelet and cellular adhesion (4,5.7,23-25). Static adsorption isotherms on glass were determined at ambient temperature for purified -"Iodine labeled human albumin, fibrinogen and fibronectin in Tyrode s buffer without calcium or magnesium, pH 7.30. Langmuir type adsorption was observed, the plateau region being attained for solution concentrations of 3.0 - 5.0 mg/ml (albumin), 0.51.0 mg/ml (fibrinogen) and 0.2 - 0.4· mg/ml (fibronectin). Plateau surface concentrations of protein (Table I) correspond approximately to monolayers of fibrinogen (26,27) and fibronectin (28) and to a bilayer of albumin (29). Affinity constants suggest an order of affinity for the glass surface : fibronectin > fibrinogen > albumin. f

Washed Human Platelet Suspensions. The capillary perfusion system has proved to be of particular value for the study cf the influence of a preadsorbed protein layer on subsequent platelet accumulation on a r t i f i c i a l surfaces. Platelet deposition was measured for varying wall shear rates (150, 500 or 1,000 s"*' ) and perfusion times (2, 5 or 10 min) on glass capillaries preadsorbed with purified human albumin, fibrinogen or fibronectin, using a suspension of washed Indium labeled human platelets in the presence of a 4-0 % hematocrit. Bulk solution concentrations of plasma protein for preadsorption were chosen to l i e in the plateau region of the previously determined adsorption isotherms. Results showed an increase in platelet accumulation with perfusion time on a l l three surfaces. On fibrinogen and fibronectin coated glass, platelet deposition increased with wall shear rate up to 1,000 s , indicating that under these conditions adhesion was largely controlled by transport to the interface (30). Consistent with the


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Table I. Static Adsorption Isotherms on Glass Capillary Tubes for Purified Human Plasma Proteins


Protein albumin fibrinogen fibronectin

H u g / cm )

Κ x 10

2

G, 40 o, 40 o, 25

9

(1/mole)

100 174 313

Adsorption 1 hour, static conditions, 22°C. Γ: Surface protein concentration in plateau region ; corresponding solution concentrations : albumin 3.0 - 5.0 mg/ml, fibrinogen 0.5 1.0 mg/nl, fibronectin 0.2 - 0.4 mg/ml. K: Affinity constant.

passive nature of albumin surfaces, accumulation of platelets on albumin coated glass was lower than on the other surfaces and decreased at high shear rate (1,000 ), where i t would appear that adhesion was no longer governed by transport phenomena but rather by surface phase biochemical reactions. At low wall shear rate (150 s ), platelet deposition was greater on fibronectin than on fibrinogen. Conversely, at high wall shear rate (1,000 s" ), platelet accumulation was greatest on fibrinogen. According to these studies, an albumin coated a r t i f i c i a l surface has low reactivity with respect to platelets, while a fibronectin coated surface shows greater reactivity under hemodynamic conditions corresponding to those of the venous circulation and a fibrinogen coated surface enhanced reactivity under arterial conditions ( 9 ) · In further experiments using washed human platelet suspensions, platelet accumulation on albumin ana fibrinogen coated glass was compared with accumulation on glass capillaries coated with type I bovine collagen for a perfusion time of 5 min and wall shear rates ranging from 50 to 4,000 s~ '. Surface reactivity followed the general order : albumin < fibrinogen < collagen (Table II). On the highly reactive collagen surface, platelet deposition increased continuously with wall shear rate up to 4,000 s '', indicating transport controlled adhesion, whereas on the passive albumin surface, peak deposition was observed at 800 s"'' and. lowest values at 4,000 s , where the rate of adhesion would be limited by biochemical reactions at the interface. The fibrinogen surface showed intermediate behaviour, v/ith a plateau level of platelet accumulation from 800 to 4,000 s . Results are thus in accord with the known high platelet reactivity of collagen, this protein being an important constituent of subendothelium, where i t is considered to play the major role in platelet adhesion and subsequent thrombus formation (3]_) · Platelet adhesion to subendothelium at high wall shear rate has been shown to be mediated by vWF (32). Preliminary results in the capillary perfusion system, using glass capillaries uncoated or coated with purified human albumin or bovine collagen, showed an increase in platelet deposition in the presence of vWF on the albumin surface at low (50 s" ) and high (2,000 s~ ) wall shear rate, but on the glass and 1

-

1

1


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Table II. Accumulation of Washed Human Platelets Labeled with Indium on Glass Capillary Surfaces Precoated with Purified Human Albumin, Human Fibrinogen or Bovine Collagen 1

Wall shear rate (s" )

2

Adherent platelets/mm


1

albumin 50 800 2,000 4,000

11,000 22,500 11,000 7,000

Protein coating fibrinogen

collagen 16,500 151,500 306,000 425,500

6,000 53,000 78,000 56,000

Preadsorption of protein coating 1 hour, static conditions, 22°C. Perfusion 5 min at 37°C, platelets 180,000/mm^ in Tyrode s-albumin buffer, hematocrit 40 %. Results are mean of four independent experiments. 1

collagen surfaces only at high shear rate (Table III). Experiments were of two types : vWF was added to the platelet suspension (final concentration 5 ug/ml) immediately before perfusion or the capillaries were preadsorbed with vWF (5 ug/ml) before perfusion. Control values were obtained by substituting buffer solution containing no vWF. The enhancement of platelet adhesion by vWFwas best seen on the more passive albumin surface, where the concentration of adherent platelets after perfusion for 2 min at 2,000 s increased by a factor of 1.87 when vWF was added to the platelet suspension and by a factor of 4 when vWF was already present on the surface in the form of a preadsorbed layer. Anticoagulated Whole Blood. In perfusion experiments using anticoagulated whole blood, platelet accumulation on protein coated glass capillary surfaces is determined by radioimmunoassay with a monoclonal antibody directed specifically against the membrane glycoprotein complex Ilb-IIIa of human platelets. The validity of this technique has been discussed in detail elsewhere ( 1_0). Briefly, since the glycoprotein complex Ilb-IIIa exists only in platelets and endothelial cells, when applied to the study of blood interaction in vitro with a r t i f i c i a l surfaces, the radioimmunoassay may be considered to give a specific measurement of platelet deposition, regardless of the presence of other blood cells. Calibration by the double isotope method results in, a linear correlation between adherent platelets ( I n d i u m labeled) and immunoadsorbed antibody (^Îodine labeled). Linearity i s conserved up to at least 7.5 x 10-* platelets/mm and fer a given preparation of purified, radiolabeled antibody the slope of the straight line relation, corrected for physical decay, remains constant to within 10 % in separate calibration experiments. Thus antigenantibody specificity is not destroyed by platelet adhesion. Nor does this specificity appear to be influenced by differing morphology of adherent platelets, since the slope of the linear relation is not 2



546


affected by the nature of the adhesive surface, e.g. glass coated with albumin, fibrinogen or collagen. Background antibody adsorption i n the absence of platelets i s low, of the order of 10 % of values for surfaces bearing p l a t e l e t s . As applied to the quantitation of p l a t e l e t accumulation i n whole blood, this technique offers the advantage of avoiding the lengthy procedure of p l a t e l e t separation, washing and radiolabeling, together with the possible selection of a particular population of p l a t e l e t s during this preparation. Table I I I . Accumulation of Washed Human Platelets Labeled with ^''indium i n Presence and Absence of vWF on Glass Capillary Surfaces Uncoated or Precoated with Purified Human Albumin or Bovine Collagen 1

Wall shear rate (s" )

Adherent

platelets : r a t i o vWF/control buffer

1

uncoated

Protein coating albumin

collagen

A.

50 2,000

0.98 1.33

1.18 1.87

0.94 1.49

B.

50 2,000

0.82 1.33

1.43 4.08

1.07 1.35

Preadsorption of protein coating 1 hour, s t a t i c conditions, 22°C. Perfusion 2 min at 37°C, platelets 180,000/mnK i n Tyrode s-albumin buffer, hematocrit 40 %. Results are mean of three independent experiments. A. vWF added to washed p l a t e l e t suspension ( f i n a l concentration 5 yg/ml). B. Preadsorption of vWF (5 yg/ml) 1 hour, s t a t i c conditions, 22°C. 1

Typical results f o r a series of measurements of p l a t e l e t accumulation by surface phase radioimmunoassay are presented i n Table IV. Glass c a p i l l a r i e s precoated with p u r i f i e d human albumin or fibrinogen or bovine collagen were perfused with heparin anticoagulated (10 U/ml) whole blood at varying wall shear rates (50-4,000 s~"^). As compared to equivalent experiments with washed p l a t e l e t suspensions (Table I I ) , p l a t e l e t deposition was reduced on a l l surfaces. This diminution of surface r e a c t i v i t y , undoubtedly related to albumin passivation from plasma, was p a r t i c u l a r l y important on albumin precoated glass, possibly due to favorable conditions for albumin adsorption by extension of the existing protein layer. In other respects, p l a t e l e t deposition i n whole blood followed trends similar to those observed i n washed p l a t e l e t suspensions. The order of surface r e a c t i v i t y was unchanged : albumin < fibrinogen < collagen. With r i s i n g wall shear rate, p l a t e l e t accumulation on collagen showed a continuous increase up to 4,000 s"'', as compared to a leveling out of values from 800 to 4f000 s~ on the less reactive albumin and fibrinogen surfaces. 1


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Table IV. Platelet Accumulation from Heparin Anticoagulated Whole Blood on Glass Capillary Surfaces Precoated with Purified Human Albumin, Human Fibrinogen or Bovine Collagen. Quantitation of Platelet Deposition by Radioimmunoassay with ^^'Iodine Labeled Monoclone 1 Antibody 6C-9 against Human Platelet Glycoprotein Complex Ilb-IIIa

Wall shear rate (s- )

Adherent platelets/mm'

1

Protein coating fibrinogen

albumin 50 800 2,000 4,000

90 150 170 100

4,500 27,000 25,500 31,000

collagen 16,000 39,000 43,500 79,000

Preadsorption of protein coating 1 hour, static conditions, 22°C. Perfusion 5 min at 37°C, heparin anticoagulated (10 U/ml) whole blood, platelets 173,000 ± 15,000/mm (χ ± SFM, n=4). Results are mean of four independent experiments. 3

Adaptation of Perfusion System to theStudy of Biomaterials in Catheter Form. Although originally developed for use with glass capillaries, the perfusion system is readily adapted to the study of protein adsorption and platelet accumulation on polymeric biomaterials in catheter form. As an example of this type of investigation, catheters produced from mixtures of polyvinylchlcride (PVC) with silicone based copolymers (Hospal, Meyzieu, France) were compared with pure silicone catheters. Using suspensions of washed Indium labeled human platelets in the presence of a 4.0 % hematocrit, platelet deposition and release of 3TG were determined for perfusion at flow rates corresponding to a wall shear rate of 500 s '' (Table V). Consistent with the weak thrombogenicity of silicone polymers (3,33), platelet accumulation and 6TG secretion were lower on pure silicone than on the mixed PVC/silicone surfaces. A similar series of experiments concerned a blend of a terpolymer of PVC with a cationic elastomer, containing or not ionically bound heparin. Perfusion with washed platelet suspensions, under conditions excluding thrombin generation from plasma, demonstrated the activation of platelets by heparin (34)> as reflected in a parallel increase in platelet deposition and gTG secretion (Table V). The perfusion system thus offers a simple means of in vitro screening of biomaterials for short term interactions with platelets and plasma proteins. -

American Chemical Society Library 1155 16th St., N.W. Brash and Horbett; Proteins at Interfaces Washington, 200 36 ACS Symposium Series; American ChemicalO.C Society: Washington, DC, 1987.

548


Table V. Platelet Interaction with Biomaterials in Catheter Form


Biomaterial

Adherent platelets/mm

A. Silicone PVC/silicone (1) PVC/silicone (2) PVC/silicone (3)

3TG secretion (ng/ml)

3,200 4,000 3,900 3,600

130 170 200 180

3C0 500

80 130

B. PVC/EC PVC/EC/H

Perfusion 5 min at 500 s , 22°C, washed Indium labeled human platelets 180,0C0/mm^ in Tyrode s-albumin buffer, hematocrit 4-0 %. Results are mean of two independent experiments. PVC/silicone (1), (2) and (3) : mixed PVC/silicone polymers containing respectively 15.9, 30.5 and 26.0 % silicone. PVC/EC : blend 50 % PVC, 50 % cationic elastomer. PVC/EC/H : blend 45 % PVC, 45 % cationic elastomer, 10 % ionically bound heparin. 1

Clinical Application ; Surface Passivation by Albumin Adsorption. Passivation of a r t i f i c i a l surfaces by preadsorption of human serum albumin is a technique frequently employed to improve the hemocompatibility of biomaterials used in clinical practice (3,35). The efficiency of this method is well illustrated by a study in our laboratory relating to therapeutic plasmapheresis (36). In the treatment of patients showing abnormal or excess immunoglobulins or immune complexes, plasmapheresis was hindered by blockage of the extracorporeal circulation system by platelet thrombi which formed in the filters and joints and in 44 % of cases led to premature termination of the plasmapheresis session. Perfusion tests in vitro indicated greatly reduced platelet accumulation on the PVC tubing of the extracorporeal system after preadsorption with purified human albumin. It was therefore decided to passivate the plasmapheresis circuits by preadsorption with 4 % human serum albumin, then to introduce the patient's blood by displacement of the albumin solution. Following adoption of this technique, blockage of the extracorporeal circulation system occured in only 5 % of cases treated. Conclusions In summary, the capillary perfusion system represents a simple in vitro technique for the quantitation of protein adsorption and platelet accumulation on a r t i f i c i a l surfaces under well defined hydrodynamic conditions (0-4,000 s~ ). Radioisotopic methods enable simultaneous study of platelet deposition and adsorption of one or more plasma proteins. Experiments may be performed using washed platelet suspensions or anticoagulated whole blood, platelet deposition being measured in the latter case by surface phase radioimmunoassay with a 1


33. CAZENAVE AND MULVIHILL


549


monoclonal antibody against the human platelet glycoprotein complex Ilb-IIIa, Activation of platelets or the coagulation system may be followed by determination of released PF4, 3TG or FPA. The system is of particular value for the study of tre influence of preadsorbed proteins on platelet accumulation on artificial surfaces, the screening of biomaterials for short term hercocompatibility and the evaluation of techniques such as albumin passivation designed to reduce the throncogenicity of systems used in clinical practice. A cknowledgments We gratefully acknowledge the assistance of Mrs. A. SutterBay, Mrs. J. Launay, Mrs. J. Eterhardt and Dr. S. Eemmendinger and the excellent secretarial assistance of Mrs. C. Helbourg and Mrs. M. Voyat. Monoclonal antibody 6C9 was a generous gift of Dr. H.G. Huisman (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam). Polymeric biomaterials were kindly supplied by Dr. C. Pusineri (Hospal, Meyzieu, France). This work was supported by a grant from CNRS GRECO n°48 "Polymères Hémocompatibles". J.-P. Cazenave is Directeur de Recherche, INSERM. Literature Cited 1. Zetter, B.R. Diabetes 1981, 30 Suppl. 2, 24-28. 2. Cazenave, J.-P.; Klein-Soyer, C.; Beretz, A. Inter. Angio. 1984, 3, 27-32. 3. Salzman, E.W.; Merrill, E.W. In Hemostasis and Thrombosis: Basic Principles and Clinical Practice ; Colman, R.W.; Hirsh, J.; Marder, V.J.; Salzman, E.W., Eds.; J.B. Lippincott Co.: Philadelphia, Toronto, 1982; pp 931-943. 4. Vroman, L.; Adams, A.L.; Klings, M.; Fischer, G.C.; Munoz, P.C.; Sclensky, R.P. Ann. N.Y. Acad. Sci. 1977, 283, 65-76. 5. Packham, M.A.; Evans, G.; Glynn, M.F.; Mustard, J.F. J. Lab. Clin. Med. 1969, 73, 686-697. 6. Brash, J.L. In Interaction of the Blood with Natural and Artificial Surfaces; Salzman, E.W., Ed.; Marcel Dekker Inc. : New York and Basel, 1981; pp 37-60. 7. Mosher, D.F. In Interaction of the Blood with Natural and Artificial Surfaces; Salzman, E.W., Ed.; Marcel Dekker Inc. : New York and Basel, 1981; pp 85-101. 8. Mulvihill, J.N.; Cazenave, J.-P.; Maennel, G.; Schmitt, A. ESAO Proceedings, 1982, pp 287-290. 9. Goldsmith, H.L.; Turitto, V.T. Thromb. Haemostas. 1986, 55, 415435. 10. Mulvihill, J.N.; Huisman, H.G.; Cazenave, J.-P.; van Mourik, J.Α.; van Aken, W.G. Thromb. Haemostas., submitted for publication. 11. Kekwick, R.A.; Mackay, M.E.; Nance, M.H.; Record, B.R. Biochem. J. 1955, 60, 671-683. 12. Miekka, S.I.; Ingham, K.C.; Menache, D. Thromb. Res. 1982, 27, 1-14. 13. Cazenave, J.-P.; Hemmendinger, S.; Beretz, Α.; Sutter-Bay, Α.; Launay J. Ann. Biol. Clin. 1983, 41, 167-179. 14. Laemmli, U.K. Nature 1970, 227, 680-685.



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15. Regoeczi, E. Iodine-labeled Plasma Proteins; CRC Press : Boca Raton, Florida, 1984. 16. Modderman, P.; Huisman, H.G.; von dem Borne, A.E.G.Kr. Thromb. Hemostas. 1985, 54, 197. 17. Mustard, J.F.; Perry, D.W.; Ardlie, N.G.; Packham, M.A. Brit. J. Haematol. 1972, 22, 193-204. 18. Eber, M.; Cazenave; J.-P.; Grob, J.C.; Abecassis, J.; Methlin G. In Blood Cells in Nuclear Medicine, Part I. Cell Kinetics and Bio-distribution; Hardeman, M.R.; Najean, Y., Eds.; Martinus Nijhoff Publishers : Boston, 1984; pp 29-43. 19. Cazenave, J.-P.; Blondowska, D.; Richardson, M.; Kinlough-Rathbone, R.L.; Packham, M.A.; Mustard, J.F. J. Lab. Clin. Med. 1979, 93, 60-70. 20. Sakariassen, K.S.; Baumgartner, H.S. Thromb. Haemostas. 1985, 54, 109. 21. Adams, G.A.; Feuerstein, I.A. Thromb. Haemostas. 1984, 52, 45-49. 22. Mulvihill, J.N. Ph.D. Thesis, Université Louis Pasteur, Strasbourg, France, 1984. 23. Park, K.; Mosher, D.; Cooper, S.L. J. Biomed. Mater. Res. 1986, 20, 589-612. 24. Grinnell, F.; Phan, T.V. Thromb. Pes. 1985, 39, 165-171. 25. Grinnell, F.; Feld, M.; Minter, R. Cell 1980, 19, 517-525. 26. Fowler, W.E.; Erickson, P.P. J. Mol. Biol. 1979, 134, 241-249. 27. Estis, L.F.; Haschemeyer, R.E. Proc. Natl. Acad. Sci. USA 1980, 77, 3139-3143. 28. Tooney, N.M.; Mosesson, M.W.; Amrani, D.L.; Hainfeld, J.F.; Wall, J.S. J. Cell. Biol. 1983, 97, 1686-1692. 29. De Baillou, N. Ph.D. Thesis, Université Louis Pasteur, Strasbourg, France, 1983; p 116. 30. Turitto, V.T.; Baumgartner, H.R. Methods in Hematology : Measurements of Platelet Function ; Parker, L.A.; Zinmermann, T.S., Eds.; Churchill Livingstone : Edinburgh, 1983; Vol. 8, pp 46-63. 31. Packham, M.A.; Mustard, J.F. In Progress in Hemostasis and Thrombosis; Spaet, T.H., Ed.; Grune and Stratton : Orlardo, Florida, 1984; Vol. 7, pp 211-288. 32. Sakariassen, K.S.; Bolhuis, P.Α.; Sixma, J.J. Nature 1979, 279, 636-638. 33. Pitlick, F.A. In Interaction of the Blood with Natural and Artificial Surfaces; Salzman, E.W., Ed.; Marcel Dekker Inc. : New York and Basel, 1981; pp 185-199. 34. Salzman, E.W.; Rosenberg, R.D.; Smith, M.A.; Lindon, J.N.; Faureau, L. J. Clin. Invest. 1980, 65, 64-73. 35. Sigot-Luizard, M.F.; Domurado, D.; Sigot, M.; Guidoin, R.; Gosselin, C.; Marois, M.; Girard, J.F.; King, M.; Badour, B. J. Biomed. Mater. Res. 1984, 18, 895-909. 36. Mulvihill, J.N.; Cazenave, J.-P. Bull. Soc. Chim. France 1985, 4, 551-554. RECEIVED January 13, 1987


Capillary Perfusion System for Quantitative Evaluation of Protein

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