THROMBOSIS RESEARCH 15 ; 157-167 Pergamon Press Ltd .1979 . Printed in Great Britain
PLATELET AND PLASMA COAGULATION COMPATIBILITY OF HEPARINIZED AND SULPHATED SURFACES R . Larsson***, J-C . Eriksson**, H . Lagergren* and P . Olsson* *Department of Experimental Surgery, Thoracic Clinics, Karolinska Sjukhuset, **Department of Physical Chemistry, Royal Institute of Technology and ***Aminkemi AB, Stockholm, Sweden (Received 18 .12 .1978 ; in revised form 16 .2 .1979 . Accepted by Editor E . W . Salzman)
ABSTRACT The degrees of platelet adhesion and activation of the plasma coagulation system ware measured separately on unmodified polyethylene, sulphated polyethylene and different modifications of heparinized surfaces stabilized with glutardialdehyde . Thranbus formation on the unmodified surfaces was shown to involve activation of both the platelets and the coagulation system . Despite minimal platelet adhesion on the sulphated surface and the host stable heparinized me, surface-induced plasma coagulation was demonstrated . Two slightly different modifications of the heparinglutar surface proved to inhibit platelet adhesion and surfaceinduced plasma coagulation .
lwrRDDO MON In a recent Task Force Report on Bicmmaterials from NIH (1), it is concluded that both the lack of fundamental knowledge of tissue-material interaction and adequate test procedures are two of the main obstacles for the development of blood-oampai-ihlcp artificial materials . For some years to have studied a glutardialdehyde-stabilized heparin surface, which proved to be highly thranboresistant (2, 3, 4, 5) . The platelet cammp,.tibility of this heparin surface is well docurented (3, 4, 6), and this particular property of artificial surfaces has been regarded as the crucial one for achieving non-thro bogaiicity (7) . A platelet-compatible surface, however, may still be thranbogenic by activating enzymes in the coagulation caqoade . In order to ascertain whether such a situation exists, different modifications of the heparinized surfaces were compared with sulphated and unmodified polyethylene in test models, which permitted separate evaluation of events related to the platelets and to the coagulation system . 157
158
Vol .15,No .1/2
HEPARINIZED SURFACES
ie:5r SMACES Polyethylene (low density) tubings (A/S Surgined, Oelstykke, Denmark) length 90 cm, inner diameter 3 .8 mm, volume 10 ml and inner surface area 107 ant Were used . After cleaning in an ultrasonic bath containing 70 % ethanol for 60 minutes, the tubings were modified and characterized as described below . One sulphated surface with oovalently bound sulphate groups and three modifications of the heparin-glutar surface with different rates of initial release of heparin were investigated . Pbeth_ylene PE The polymer showed only minute amounts of oxygen on the surface, as 3anonstrated by electron spectroscopy for chemical analysis (ESCA) (Table I) . TABLE I Chemical surface analysis by means of ESCA of unmodified PE, sulphated and heparin-glutar I surfaces . Relative peak intensities, corrected for differences in cross sections, are listed for S 2p, 0 1s, C 1s, N ls-transitions . Intensities I/I Transition
Energy Unmodified
S C N 0
2p is is is
is
169 .2 285 .0 400 .3 582 .8
c0 .001 1 '0 .003 '0 .026
Sulphated 0 .04 1 0 .24
Heparin-glutar I 0 .01 1 0 .05 0 .17
SuijMted polye tJ ene The sulphated surface was prepared by incubating PEtubings with concentrated sulphuric acid containing potassium pennanganate (2 g/1) for two minutes at roam temperature and then rinsing in ultrAp+re water . Increased oxygen and sulphur signals were recorded by FSCA (Table I) . The electron binding energy of the sulphur signal indicates that sulphate ions are localized on the surface (8) . Judging by scanning electron microscopy at a magnification of 30 000 there were no morphological changes induced by the sulphation procedure . Both the sulphated and the heparin-glutar surfaces exhibited rapid wetting kinetics in contrast to the unuLodified PEsurface . Heparin~luutar I surface PE-tubings were treated sequentially with water solutions ofhexadecylanIne hydrocloride (C16-amine), heparin (130 Wing, Vitrum, Stockholm, Sweden), C15-amine and glutardialdehyde as previously described (9) . By this procedure a stabilized amine-heparin complex is precipitated onto the surface . The size of the precipitated amine-heparin canplex particles cannot be controlled with this technique and scanning electron microscopy has shown major variations in the grain size (Fig . 1 A) . The results of ESCA-analysis were similar to those obtained on the sulphated surface, although the intensity of the sulphur signal was swat leer (Table I) . The heparin-glutar I surface was also investigated with the aid of Auger electron spectroscopy, which gave essentially the sane result as those obtained by ESCA (10) . Beparin-!Ilutar II A surface Sulphated PE-tubings were heparinized by means of adsorption ofcolloidal amine-heparin particles by sequential treatments with the following solutions : Solution -1- (S 1) : 115 mg of heparin dissolved
Vol .15,No .1/2
HEPARTNIZED SURFACES
159
in 250 ml of ultrapure water at 600C and the final pH adjusted to 3 with HC1 . 250 mg of C16 -amine dissolved in 50 ml water at 50°C and pH adjusted to 3 . The heparin solution was added to the amine solution during vigorous mixing . By using an excess of amine, the heparin molecules are surrounded by a double layer of cationic surfactants and aggregate irreversibily to stable colloidal particles with a positive net charge, on their surfaces . With the aid of quasielastic light scattering the particle size was assessed at about 80 nn . Solution 2 (S .2) : 22 mg of heparin were dissolved in 300 ml and the final pH adjustedto
3.
The sulphated PE-tubings were first exposed to S 1 at 600C for 2 minutes and then, after rinsing in water, they were exposed to S 2 at 60°C for 5 minutes and thereafter rinsed in water again . This sequence, which includes adsorption of the positive particles to the negatively charged surface and reversal of the positivly charged adsorbate with heparin, was repeated three times . The final stabilization treatrent with glutar ciial dehyde was carried out as described previously (9) . In comparison with the heparin-glutar I surface, this technique yields a much smoother surface (Fig . 1 B) . Heparin-glutar II B surface Sulphated PE-tubings were treated in the same way as II A above, with the exception of 23 mg heparin and 50 mg C16-amine being used to prepare S 1 . Judging from the light scattering properties, these particles were considerably smaller (< 50 non) than in the case of S 1 for the II A preparation . Auger electron sp troscopy revealed similar srnx.tra for the heparin-glutar surfaces I, II A and II B (to be published separately) . METHODS Heparin _concentration inylasna was determined by using a factor Xe inhibition test (CoatestR, Rabi Diagnostica, Stockholm, Sweden) . The standard plasma was the same as that used for the experiments and the incubation times ware longer (4 and 7 min) than recamended in the test kit . By these measures the detection limit could be set as low as 0 .02 IU/ml . The heparin surface concentration of the tubings was determined by dissolving the unstabilized amine-heparin Lt.,vlex in plasna . Five 1111 plasma portions
were rotated at 6 (11), then rinsed for one hour . The amount of heparin
r .p .m . for four hours in tubings formed as Chandler loops in saline and again exposed to another 5 ml plasma portion heparin surface concentration was calculated from the total recovered in the eluates .
The degree of heparin desorption from the glutardiai dehyde-stabilized surface was determined in the same way by consecutive rotation of 4 portions of one ml plasma for one hour each . Platelet adhesion in vitro was determined by measuring the concentration of adenosinetriphosphate (ATP) on the surfaces after contact with freshly drawn heparinized blood (150 ml in 20 ml 5 .5 % glucose solution and 0 .3 ml heparin, 5000 IU/ml Vitnm, Stockholm, Sweden, resulting in a heparin concentration of 10 IU/ml) . Five ml portions were rotated in the Chandler loops for one hour . After rinsing with saline, the surface concentration of ATP was determined with a bioluminiscence technique previously described in detail (12) .
16o
HEPARINIZED SURFACES
Vol .15,No .1/2
Platelet adhesion in vivo The uptake of platelets on the surface of the tubings insertedas arteriovenous shunts in dogs was measured by using autologous S-Cr-labelled platelets . After removal, the tubings were rinsed in 2 x 20 ml of saline and the surface concentration of 51Cr-activity was determined . The platelet specific 51Cr-activity determined in a pre-experimental arterial blood sample was used to calculate the number of surface-attachedd platelets/ant, as previously described (3) . Surface concentration of fibrinogen in vitro The radi .oimnuioassay of fibrino---------------peptide A (EPA) according to Nossel et al ., (13) was employed as a method for measuring surface adsorption of fibrinogen . The tubings were rotated for me hour with heparinized human plasma . After rinsing in 3 X 10 ml of saline, the surfaces were exposed for one hour to 5 ml of DefibraseR, (2 B units/ml , Pentaphaan, Basel, Switzerland) . The procedure was repeated in order to ensure the complete release of FPA from the fibrinogen molecules adsorbed to the surface . The amount of FPA in the eluates was determined by using a kit for radioinn noassay (linro AS, Stockholm, Sweden) . The surface concentration of fibrinogen was calculated from the recovered amounts of FPA . Surface_induced coagulation The concentration of FPA in dog plasma was measured radioinnamolog twtm y, using a rabbit antibody against human FPA . A particular rabbit produced an antibody, which bound radio-labelled canine EPA to the same extent as radiolabelled human FPA (the former was generously supplied by Dr . George Wilner, Columbia University, New York, U .S .A .) . The t racer was 12 5J-labelled desaminotyrosol EPA (D= AB) . Animal en~eriurents Mongrel dogs (17 to 26 kg), anesthetized with intravenous sodiun peitothal, tracheally intubated and ventilated with a mixture of oxygen and dinitrous oxide in a volume csuttrolled respirator (Mivab, Stockholm, Sweden), were used . one carotid artery was calculated with a heparin-glutar I catheter to the level of the aortic arch for monitoring blood pressure with a strain gauge and Grass Polygraph (Grass Instrument Co ., Qunicy, Mass ., U .S .A .) . Samples for FPA detemdnations were taken from this catheter before and after the experiments . The femoral artery and vein were dissected bilaterlly at a length of five can . In one group of experiments comprising six animals, PE, sulphated PE, heparinglutar 1, II A and II B tubings ware tested for one hour . In each animal, two PE-tubings were compared with one or two of the modified tubings . Fifteen an long heparin-glutar I tubings were inserted in each femoral artery and vein . The test tubings were connected to these tubings with the aid of ncn-heparinized polymer sleeves . The middle section (30) was squeezed between two circular plates (Fig 2) to give a resistance resulting in a flow of water of 80 ml/min at a pressure of 70 mm H20 . The blood flow through the shunts was cxzntinously measured electro agnetically (Nycotro n AS, Dramren, Norway) with the flow probe placed on the artery . Before complete occlusion or after ate hour, the shunts were disconnected an the venous side and blood was sampled from the tubings for FPA analysis . After removal, the blood and rinsing solutions (2 x 20 ml saline) were poured through a fabric for detection of thrombi . The squeezed section of each tubing was cut into pieces for determination of 51Cr-activity, i .e . platelet adhesion . In a send group comprising four animals, heparin-glutar I and II A tubings were tested to ascertain if these surfaces would initiate Plasma Coagulation for six hours . In order to avoid thrachs formation at connection sites with other tubings, the test tubings were now inserted directly into the vessels .
161
HEPARINIZED SURFACES
Vol . 1 5 ,No .1/2
Samples for FPA determination were taken 2, 4, 5 and 6 hairs fran the venous side of the shunts by disconnecting the tubings on that side . Statistics All results are given as mean values ± S .D . Individual t-statistics with the significant level set at p
PLATELET AND PLASMA COAGULATION COMPATIBILITY OF HEPARINIZED AND SULPHATED SURFACES R . Larsson***, J-C . Eriksson**, H . Lagergren* and P . Olsson* *Department of Experimental Surgery, Thoracic Clinics, Karolinska Sjukhuset, **Department of Physical Chemistry, Royal Institute of Technology and ***Aminkemi AB, Stockholm, Sweden (Received 18 .12 .1978 ; in revised form 16 .2 .1979 . Accepted by Editor E . W . Salzman)
ABSTRACT The degrees of platelet adhesion and activation of the plasma coagulation system ware measured separately on unmodified polyethylene, sulphated polyethylene and different modifications of heparinized surfaces stabilized with glutardialdehyde . Thranbus formation on the unmodified surfaces was shown to involve activation of both the platelets and the coagulation system . Despite minimal platelet adhesion on the sulphated surface and the host stable heparinized me, surface-induced plasma coagulation was demonstrated . Two slightly different modifications of the heparinglutar surface proved to inhibit platelet adhesion and surfaceinduced plasma coagulation .
lwrRDDO MON In a recent Task Force Report on Bicmmaterials from NIH (1), it is concluded that both the lack of fundamental knowledge of tissue-material interaction and adequate test procedures are two of the main obstacles for the development of blood-oampai-ihlcp artificial materials . For some years to have studied a glutardialdehyde-stabilized heparin surface, which proved to be highly thranboresistant (2, 3, 4, 5) . The platelet cammp,.tibility of this heparin surface is well docurented (3, 4, 6), and this particular property of artificial surfaces has been regarded as the crucial one for achieving non-thro bogaiicity (7) . A platelet-compatible surface, however, may still be thranbogenic by activating enzymes in the coagulation caqoade . In order to ascertain whether such a situation exists, different modifications of the heparinized surfaces were compared with sulphated and unmodified polyethylene in test models, which permitted separate evaluation of events related to the platelets and to the coagulation system . 157
158
Vol .15,No .1/2
HEPARINIZED SURFACES
ie:5r SMACES Polyethylene (low density) tubings (A/S Surgined, Oelstykke, Denmark) length 90 cm, inner diameter 3 .8 mm, volume 10 ml and inner surface area 107 ant Were used . After cleaning in an ultrasonic bath containing 70 % ethanol for 60 minutes, the tubings were modified and characterized as described below . One sulphated surface with oovalently bound sulphate groups and three modifications of the heparin-glutar surface with different rates of initial release of heparin were investigated . Pbeth_ylene PE The polymer showed only minute amounts of oxygen on the surface, as 3anonstrated by electron spectroscopy for chemical analysis (ESCA) (Table I) . TABLE I Chemical surface analysis by means of ESCA of unmodified PE, sulphated and heparin-glutar I surfaces . Relative peak intensities, corrected for differences in cross sections, are listed for S 2p, 0 1s, C 1s, N ls-transitions . Intensities I/I Transition
Energy Unmodified
S C N 0
2p is is is
is
169 .2 285 .0 400 .3 582 .8
c0 .001 1 '0 .003 '0 .026
Sulphated 0 .04 1 0 .24
Heparin-glutar I 0 .01 1 0 .05 0 .17
SuijMted polye tJ ene The sulphated surface was prepared by incubating PEtubings with concentrated sulphuric acid containing potassium pennanganate (2 g/1) for two minutes at roam temperature and then rinsing in ultrAp+re water . Increased oxygen and sulphur signals were recorded by FSCA (Table I) . The electron binding energy of the sulphur signal indicates that sulphate ions are localized on the surface (8) . Judging by scanning electron microscopy at a magnification of 30 000 there were no morphological changes induced by the sulphation procedure . Both the sulphated and the heparin-glutar surfaces exhibited rapid wetting kinetics in contrast to the unuLodified PEsurface . Heparin~luutar I surface PE-tubings were treated sequentially with water solutions ofhexadecylanIne hydrocloride (C16-amine), heparin (130 Wing, Vitrum, Stockholm, Sweden), C15-amine and glutardialdehyde as previously described (9) . By this procedure a stabilized amine-heparin complex is precipitated onto the surface . The size of the precipitated amine-heparin canplex particles cannot be controlled with this technique and scanning electron microscopy has shown major variations in the grain size (Fig . 1 A) . The results of ESCA-analysis were similar to those obtained on the sulphated surface, although the intensity of the sulphur signal was swat leer (Table I) . The heparin-glutar I surface was also investigated with the aid of Auger electron spectroscopy, which gave essentially the sane result as those obtained by ESCA (10) . Beparin-!Ilutar II A surface Sulphated PE-tubings were heparinized by means of adsorption ofcolloidal amine-heparin particles by sequential treatments with the following solutions : Solution -1- (S 1) : 115 mg of heparin dissolved
Vol .15,No .1/2
HEPARTNIZED SURFACES
159
in 250 ml of ultrapure water at 600C and the final pH adjusted to 3 with HC1 . 250 mg of C16 -amine dissolved in 50 ml water at 50°C and pH adjusted to 3 . The heparin solution was added to the amine solution during vigorous mixing . By using an excess of amine, the heparin molecules are surrounded by a double layer of cationic surfactants and aggregate irreversibily to stable colloidal particles with a positive net charge, on their surfaces . With the aid of quasielastic light scattering the particle size was assessed at about 80 nn . Solution 2 (S .2) : 22 mg of heparin were dissolved in 300 ml and the final pH adjustedto
3.
The sulphated PE-tubings were first exposed to S 1 at 600C for 2 minutes and then, after rinsing in water, they were exposed to S 2 at 60°C for 5 minutes and thereafter rinsed in water again . This sequence, which includes adsorption of the positive particles to the negatively charged surface and reversal of the positivly charged adsorbate with heparin, was repeated three times . The final stabilization treatrent with glutar ciial dehyde was carried out as described previously (9) . In comparison with the heparin-glutar I surface, this technique yields a much smoother surface (Fig . 1 B) . Heparin-glutar II B surface Sulphated PE-tubings were treated in the same way as II A above, with the exception of 23 mg heparin and 50 mg C16-amine being used to prepare S 1 . Judging from the light scattering properties, these particles were considerably smaller (< 50 non) than in the case of S 1 for the II A preparation . Auger electron sp troscopy revealed similar srnx.tra for the heparin-glutar surfaces I, II A and II B (to be published separately) . METHODS Heparin _concentration inylasna was determined by using a factor Xe inhibition test (CoatestR, Rabi Diagnostica, Stockholm, Sweden) . The standard plasma was the same as that used for the experiments and the incubation times ware longer (4 and 7 min) than recamended in the test kit . By these measures the detection limit could be set as low as 0 .02 IU/ml . The heparin surface concentration of the tubings was determined by dissolving the unstabilized amine-heparin Lt.,vlex in plasna . Five 1111 plasma portions
were rotated at 6 (11), then rinsed for one hour . The amount of heparin
r .p .m . for four hours in tubings formed as Chandler loops in saline and again exposed to another 5 ml plasma portion heparin surface concentration was calculated from the total recovered in the eluates .
The degree of heparin desorption from the glutardiai dehyde-stabilized surface was determined in the same way by consecutive rotation of 4 portions of one ml plasma for one hour each . Platelet adhesion in vitro was determined by measuring the concentration of adenosinetriphosphate (ATP) on the surfaces after contact with freshly drawn heparinized blood (150 ml in 20 ml 5 .5 % glucose solution and 0 .3 ml heparin, 5000 IU/ml Vitnm, Stockholm, Sweden, resulting in a heparin concentration of 10 IU/ml) . Five ml portions were rotated in the Chandler loops for one hour . After rinsing with saline, the surface concentration of ATP was determined with a bioluminiscence technique previously described in detail (12) .
16o
HEPARINIZED SURFACES
Vol .15,No .1/2
Platelet adhesion in vivo The uptake of platelets on the surface of the tubings insertedas arteriovenous shunts in dogs was measured by using autologous S-Cr-labelled platelets . After removal, the tubings were rinsed in 2 x 20 ml of saline and the surface concentration of 51Cr-activity was determined . The platelet specific 51Cr-activity determined in a pre-experimental arterial blood sample was used to calculate the number of surface-attachedd platelets/ant, as previously described (3) . Surface concentration of fibrinogen in vitro The radi .oimnuioassay of fibrino---------------peptide A (EPA) according to Nossel et al ., (13) was employed as a method for measuring surface adsorption of fibrinogen . The tubings were rotated for me hour with heparinized human plasma . After rinsing in 3 X 10 ml of saline, the surfaces were exposed for one hour to 5 ml of DefibraseR, (2 B units/ml , Pentaphaan, Basel, Switzerland) . The procedure was repeated in order to ensure the complete release of FPA from the fibrinogen molecules adsorbed to the surface . The amount of FPA in the eluates was determined by using a kit for radioinn noassay (linro AS, Stockholm, Sweden) . The surface concentration of fibrinogen was calculated from the recovered amounts of FPA . Surface_induced coagulation The concentration of FPA in dog plasma was measured radioinnamolog twtm y, using a rabbit antibody against human FPA . A particular rabbit produced an antibody, which bound radio-labelled canine EPA to the same extent as radiolabelled human FPA (the former was generously supplied by Dr . George Wilner, Columbia University, New York, U .S .A .) . The t racer was 12 5J-labelled desaminotyrosol EPA (D= AB) . Animal en~eriurents Mongrel dogs (17 to 26 kg), anesthetized with intravenous sodiun peitothal, tracheally intubated and ventilated with a mixture of oxygen and dinitrous oxide in a volume csuttrolled respirator (Mivab, Stockholm, Sweden), were used . one carotid artery was calculated with a heparin-glutar I catheter to the level of the aortic arch for monitoring blood pressure with a strain gauge and Grass Polygraph (Grass Instrument Co ., Qunicy, Mass ., U .S .A .) . Samples for FPA detemdnations were taken from this catheter before and after the experiments . The femoral artery and vein were dissected bilaterlly at a length of five can . In one group of experiments comprising six animals, PE, sulphated PE, heparinglutar 1, II A and II B tubings ware tested for one hour . In each animal, two PE-tubings were compared with one or two of the modified tubings . Fifteen an long heparin-glutar I tubings were inserted in each femoral artery and vein . The test tubings were connected to these tubings with the aid of ncn-heparinized polymer sleeves . The middle section (30) was squeezed between two circular plates (Fig 2) to give a resistance resulting in a flow of water of 80 ml/min at a pressure of 70 mm H20 . The blood flow through the shunts was cxzntinously measured electro agnetically (Nycotro n AS, Dramren, Norway) with the flow probe placed on the artery . Before complete occlusion or after ate hour, the shunts were disconnected an the venous side and blood was sampled from the tubings for FPA analysis . After removal, the blood and rinsing solutions (2 x 20 ml saline) were poured through a fabric for detection of thrombi . The squeezed section of each tubing was cut into pieces for determination of 51Cr-activity, i .e . platelet adhesion . In a send group comprising four animals, heparin-glutar I and II A tubings were tested to ascertain if these surfaces would initiate Plasma Coagulation for six hours . In order to avoid thrachs formation at connection sites with other tubings, the test tubings were now inserted directly into the vessels .
161
HEPARINIZED SURFACES
Vol . 1 5 ,No .1/2
Samples for FPA determination were taken 2, 4, 5 and 6 hairs fran the venous side of the shunts by disconnecting the tubings on that side . Statistics All results are given as mean values ± S .D . Individual t-statistics with the significant level set at p
