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STABILITY OF THE THROMBUS-RESISTANT GRAPHITE-BENZALKONIUM-HEPARIN SURFACE IN AN ANTI-HEPARIN ENVIRONMENT
STABILITY OF THE THROMBUS-RESISTANT GRAPHITE-BENZALKONIUM-HEPARIN SURFACE I N A N ANTI-HEPARIN ENVIRONMENT James D. Whiffen,
M.D.,
Vincent L. Gott, M.D.*
William Madison,
P. Young,
M.D.,
and
Wis.
F
OLLOWING the development of the thrombus-resistant graphite-benzalkoniumheparin (GBH) surface for use on intravascular prostheses, 1 ' 2 · 3 the ques tion was raised as to the in vivo fate of this surface after the intravascular infusion of protamine sulfate or hexadimethrine bromide (Polybrene). Heparinization,' with the eventual intravenous administration of an anti-heparin agent, is necessary for placement of intracardiac prostheses with the use of the pump oxygenator, and it is occasionally necessary to administer an antiheparin compound to heparinized patients with vascular prostheses. If intravascular infusion of an anti-heparin substance would immediately neutralize the heparin on the surface of the prosthesis, the thrombus-resisting property might be lost. The present study was undertaken to determine the in vivo stability of the thrombus-resisting property of the GBH surface in an anti-heparin environment. METHOD
The placement of plastic rings (Pig. 1) in the canine thoracic vena cava has been shown to be a severe in vivo clotting test. 2 ' 3 · 4 In these previous studies, plain plastic and, even, untreated graphite- or silicone-coated plastic rings contained large amounts of thrombus within 2 hours after placement in the vena cava. GBH-coated plastic rings were found to be completely free of thrombus formation at 2 hours (and also at 2 months). In the present study, GBH-coated rings were placed in the thoracic vena cava after which an antiheparin substance was infused into the vena cava directly upstream to the prosthesis. If the thrombus-resisting property of the surface was neutralized, severe thrombus formation would be expected to take place within 2 hours. Rigid rings of polycarbonate (Lexan) were made by injection molding. They were 9 mm. long with a 7 mm. internal diameter and a 0.5 mm. wall, From the Department of Surgery and Cardiovascular Laboratories, University of Wis consin Medical School, Madison, Wis. Supported in part by National Institutes of Health Grant H-4162. Received for publication March 16, 1964. •Markle Scholar in Medical Sciences. 317
3]8
W H I F F E N , YOUNG, GOTT
J. Thoracic and Cardiovas. Surg.
and had streamlined leading and trailing edges. After cleansing and pretreatment of the plastic surface with ethanol, a durable coat of graphite was obtained by dipping the rings in a Dag 154 colloidal graphite solution,* diluted with two parts 95 per cent ethanol and one part benzene. While Dag 154 will not coat all plastic materials well, it will give an excellent graphite coat to both polycarbonate and methylmethacrylate. The rings were placed in a forced draft oven at 130° F. for 48 hours to remove all diluting solvents. The GBH coat was then completed by soaking the rings in aqueous benzalkonium chloride (1:1000) for 24 hours, rinsing them with saline to remove excess benzalkonium, and then exposing them to a dilute heparin solution (0.63 mg. of heparin per cubic centimeter of saline) for one hour.
Vig. 1.—The polycarbonate rings used in this study showing their size and design. The rings on the right have been coated with graphite.
Control studies were performed on graphite-coated rings that were (1) plain or untreated, (2) treated with benzalkonium chloride alone, (3) treated with heparin alone, and (4) treated with benzalkonium chloride and heparin. The concentrations and exposure times of the benzalkonium chloride and heparin solutions used in the preparation of the control rings were the same as described for the test rings. Forty healthy dogs, weighing between 7 and 10 kilograms, were used in this study. They were anesthetized with intravenous sodium pentobarbitol (26.4 mg. per kilogram) and respired by a mechanical respirator. Exposure of the right atrium and superior and inferior venae cavae was made through the right fifth intercostal space under aseptic conditions. The azygos vein was ligated and a purse-string suture placed in the right atrial wall. Tapes were then placed about the venae cavae as far from the atrium as possible. To simulate clinical conditions, some animals were given heparin 1.0 or 2.0 mg. per kilogram while others did not receive this anticoagulant (Figs. 2, 3, and 4). During a short period of inflow stasis, a ring was placed in either the *Dag 154 colloidal graphite—Acheson Colloids Co., Port Huron, Michigan.
Vol. 48, No. 2 August. 1964
S T A B I L I T Y OF THROMBUS-RESISTANT SURFACE
319
superior or inferior vena eava through an atriotomy inside the purse-string suture. 2 Although there was little discrepancy in the ring and vena cava diam eters with the use of dogs in this weight range, a fabric wrap was placed tightly around the vena cava and ring to prevent the formation of a cul-de-sac between the ring and the vein wall. When a ring was placed in the superior vena cava the right external jugular vein was exposed and used as the antiheparin infusion site. The left common femoral vein was used in a similar manner after placement of a ring in the inferior vena cava. No difference in clotting tendencies has been noted in the two venae cavae. No vessel was used twice. Glass clotting times were determined for all animals and all were within normal limits. The concentrations of both the protamine sulfate solution and the hexadimethrine bromide solution used in this study were 10 mg. per cubic centimeter. The anti-heparin solutions were infused at a rate of 10 mg. or 1.0 c.c. per minute. CONTROL STUDIES Graphite Treatment Untreated Benzalkonium Heparin Benzalkonium + Heparin
Thrombus in Ring Lumen At Two Hours
0 0 OOOG Ό O0 O0 •
•
•
•
•
•
•
•
•
Fig. 2.—The thrombus formation (red venous thrombus) in graphite-coated plastic vena cava rings, after various treatments of the graphite surface, is shown diagrammatically. Only the graphite-benzalkonium-heparin rings were free of thrombus formation after 2 hours in the vena cava. None of these animals received heparin or an anti-heparin agent.
Commonly used neutralizing doses for 1.0 mg. of heparin are 1.5 mg. of protamine sulfate and 0.7 to 1.1 mg. of hexadimethrine bromide. 5 ' e We used higher anti-heparin doses so that an excess would be available to the heparin on the graphite surface. In very high concentrations both protamine sulfate and hexadimethrine bromide can act as anticoagulants themselves. This does not occur in the dose ratios used in this study. 7 This was confirmed by deter mining Lee-White clotting times 5 minutes after the infusion of the antiheparin agents as all were normal and essentially the same (± 2.0 min.) as the control clotting times. RESULTS
Control studies are depicted diagrammatically in Fig. 2. Both untreated graphite and graphite-benzalkonium treated rings showed complete occlusion of four rings and partial occlusion of the fifth ring 2 hours after placement
W H I F F E N , YOUNG, GOTT
320
J. Thoracic and Cardiovas. Surg.
in the thoracic vena cava. The graphite-heparin treated rings also showed a severe amount of thrombus formation present, while the graphite-benzalkonium-heparin treated rings were completely free of thrombus at 2 hours. Figs. 3 and 4 depict the absence of thrombus formation in the graphitebenzalkonium-heparin treated rings placed in the vena cava of both heparinized and unheparinized dogs 2 hours after infusion of various doses of anti-heparin compounds directly upstream from the ring. It can be seen that neither protamine sulfate nor hexadimethrine bromide infusions, even in excess, disturbed the in vivo thrombus-resisting property of the graphitebenzalkonium-heparin surface. PROTAMINE INFUSION Heparin Dose Protamine Infusion Thrombus in Ring Lumen (Mg.Ailo) Up Stream (Mg./Kilo) At Two Hours 1
2
2
4
None
3
ooooo ooooo ooooo
Pig. 3.—The absence of thrombus formation in GBH-coated plastic vena cava rings, 2 hours after the intravenous infusion of protamine sulfate directly upstream to the rings, is shown. The animals received from zero to 2.0 mg. of heparin per kilogram before ring placement and protamine neutralization.
HEXADIMETHRINE INFUSION Heparin Dose (Mg./kilo.)
Hexadimethrine Infusion Thrombus in Ring Lumen UpStream (Mg./Kilo.) At Two Hours . 1. 5
OOOOO
Fig. 4.—The absence of thrombus formation in GBH-coated plastic vena cava rings, 2 hours after heparinization and neutralization with hexadimethrine bromide, is depicted.
DISCUSSION
The GBH surface, when used on intravascular prostheses, is superior to a silicone surface in resisting thrombus formation. 1 It is believed that heparin adsorption and its presence on the surface of the graphite is at least the initial factor responsible for the thrombus-resisting properties of the surface.1· 2 · 3 It has been shown that the presence of a cationic surface active agent, such as benzalkonium chloride, is a critical factor in the binding or adsorption of the heparin to the graphite surface. 2,3 Preliminary studies in this laboratory with the use of C14 benzalkonium have shown the presence of this cation on the GBH surface even after a 2 month exposure to the venous blood stream. When solutions of heparin and benzalkonium chloride are mixed, a white
Vol. 48, No. 2 August, 1964
S T A B I L I T Y O F THKOMBUS-RESISTANT SURFACE
321 "
Λ ί
precipitate forms, presumably due to the attraction of the strong negative charge of the sulfonic and sulfate groups of the heparin to the strong positive charge of the quaternary nitrogen of the benzalkonium. 3 Thus far it has also been observed that the thrombus-resisting property of the GBH surface persists for at least 6 months in the vena cava (cava rings), 9 months in the mitral valve area (valve housings), and 2y2 years in the pulmonary artery (valve housings and grafts). Whether the exogenous heparin which is initially applied to the surface remains or is replaced by endogenous heparin, or other heparin-like mucopolysaccharides or proteins, is unknown. Certainly the latter seems more plausible. It is of interest that the present study has shown that while intravenous infusion of an anti-heparin agent, such as protamine sulfate or hexadimethrine bromide, will neutralize heparin circulating in the blood stream, it will not neutralize the heparin on the graphite-benzalkoniumheparin surface. If other mucopolysaccharides or proteins are adsorbed or substituted onto the surface after placement in the vascular stream and are the cause of the persistence of the thrombus-resisting properties of the sur face, these too are not neutralized by protamine or hexadimethrine. Initially the GBH surface has at least one chemical similarity to vascular endothelium in that it contains an anticoagulant acid mueopolysaccharide (heparin). The cement or ground substance and tissue mast cells of normal vascular endothelium are rich in acid mucopolysaccharides. 8 " 13 The specific components include chrondroitin sulfates A and B, hyaluronic acid, and heparitin sulfate. 8 - 9 Gore and Larkey 8 have shown that the mueopolysaccha ride extract of human aorta (0.5 per cent by weight) displayed anticoagulant properties which were neutralizable with protamine and when injected intra venously into rats stimulated the production of lipid clearing factor. They identified the active mueopolysaccharide as chondroitin sulfate B (a chemical relative of heparin with known anticoagulant activity 14 ) and estimated the anticoagulant potency of human aortic tissue as about 100 mg. of heparin per kilogram of tissue. McGovern 12 has demonstrated the in vivo surface anti coagulant effect of endothelial cement substance as it spreads out over in jured endothelium after trauma. Vascular endothelium, even when moderately traumatized, is resistant to neutralization of the anticoagulant mucopolysaccharides in its wall and on its surface by intravenous anti-heparin agents. Mersereau 15 has shown in rats and dogs that the local application of an anti-heparin agent to the exter nal surface of a traumatized vein caused thrombosis of the vein, while, for the most part, intravenous infusion of an anti-heparin agent through a trau matized vein did not. He concluded that the external application brought about thrombosis of the injured vessels by neutralizing an anticoagulant pro duced locally in the vessel wall while neutralization did not occur with intra venous administration of the anti-heparin agent. The present study suggests that the initial heparin layer of the graphitebenzalkonium-heparin surface is apparently resistant to circulating antiheparin agents just as is the heparin-like mucopolysaccharides on normal vascular endothelial surfaces.
322
W H I F F E N , YOUNG, GOTT
J. Thoracic and Cardiovas. Surg.
The plastic surfaces of both our experimental canine and human prosthetic heart valves are now coated with GBH. We have observed no immediate or long-term deterioration in the thrombus-resisting property of the coated valve surfaces due to the administration of the anti-heparin agent at the completion of heart-lung bypass. SUMMARY
The effect of the intravascular infusion of anti-heparin substances, protamine sulfate and hexadimethrine bromide, directly upstream to the thrombus-resistant graphite-benzalkonium-heparin surface have been studied in the dog. The thrombus-resisting property of the surface was found to per sist in this anti-heparin environment. A similarity to the thrombus resisting property of normal vascular endothelium is suggested. The authors wish to thank Dr. Charles W. Crumpton, Director of the Cardiovascular Laboratory, Mr. Francis McMahan, and Mr. Dennis Copper for their assistance in this study. REFERENCES 1. Gott, V. L., Whiff en, J . D., Dutton, R. C. : Heparin Bonding on Colloidal Graphite Surfaces, Science 142: 1297, 1963. 2. Whiff en, J . D., Dutton, E. C , Young, W. P., and Gott, V. L.: Heparin Application to Graphite Coated Intravascular Prostheses, Surgery 56: 404, 1964. 3. Whiff en, J . D., and Gott, V. L. : Effect of Various Surface Active Agents on Heparin Binding and Clot Formation on Graphite Surfaces, Proc. Soc. Exper. Biol. & Med. 116: 314, 1964. 4. Gott, V. L., Koepke, D. E., Daggett, R. L., Zarnstorff, W., and Young, W. P . : The Coating of Intravascular Plastic Prostheses With Colloidal Graphite, Surgerv 50: 382, 1961. 5. Rothnie, N. G., and Kinmonth, J . B.: Bleeding After Perfusion for Open Heart Surgery, Brit. M. J . 1: 73, 1960. 6. Weiss, W. A., Gilman, J. S., Catenacci, A. J., and Osterberg, A. E.: Heparin Neutra lization With Polybrene Administration I. V., J. A. M. A. 166: 603, 1958. 7. Keats, A. S., Cooley, D. A., and Telford, J . : Relative Anti-heparin Potency of Poly brene and Protamine in Patients Undergoing Extracorpcreal Circulation, ,T. THORACIC & CARDIOVAS. SURG. 38: 362
1959.
8. Gore, I., and Larkev, B. J. : Functional Activity of Aortic Mucopolvsaccharides, J. Lab. & Clin. Me*d. 56: 839, 1960. 9. Meyer, K., and Rapport, M. M.: The Mucopolvsaccharides of the Ground Substance of Connective Tissue, Science 113: 596, 1951. 10. Fisher, E. R.: Tissue Mast Cells, J. A. M. A. 173: 171, 1960. 11. Lovelock, ,T. E., and Porterfield, J. S.: Blood Coagulation: Its Prolongation in Vessels W i t h Negatively Charged Surfaces, N a t u r e 167: 39, 1951. 12. McGovern, V. J. : Reactions to Injury of Vascular Endothelium With Special Refer ence to the Problem of Thrombosis, J . Exper. Path. & Bact. 69: 283, 1955. 13. Kirk, J . E. : Anticoagulant Activity of Human Arterial Mucopolysaccharides, Circu lation 20: 975, 1959. 14. Grossman, B. J., and Dorfman, A.: In Vivo Comparison of the Antithrombic Action of Heparin and Chondroitinsulfuric Acid-B, Pediatrics 20: 506, 1957. 15. Mersereau, M. A.: Effect of Anti-heparin Agents on Venous Thrombosis Following \ r ascular Injury, Circulation Res. 11: 765, 1962.
M.D.,
Vincent L. Gott, M.D.*
William Madison,
P. Young,
M.D.,
and
Wis.
F
OLLOWING the development of the thrombus-resistant graphite-benzalkoniumheparin (GBH) surface for use on intravascular prostheses, 1 ' 2 · 3 the ques tion was raised as to the in vivo fate of this surface after the intravascular infusion of protamine sulfate or hexadimethrine bromide (Polybrene). Heparinization,' with the eventual intravenous administration of an anti-heparin agent, is necessary for placement of intracardiac prostheses with the use of the pump oxygenator, and it is occasionally necessary to administer an antiheparin compound to heparinized patients with vascular prostheses. If intravascular infusion of an anti-heparin substance would immediately neutralize the heparin on the surface of the prosthesis, the thrombus-resisting property might be lost. The present study was undertaken to determine the in vivo stability of the thrombus-resisting property of the GBH surface in an anti-heparin environment. METHOD
The placement of plastic rings (Pig. 1) in the canine thoracic vena cava has been shown to be a severe in vivo clotting test. 2 ' 3 · 4 In these previous studies, plain plastic and, even, untreated graphite- or silicone-coated plastic rings contained large amounts of thrombus within 2 hours after placement in the vena cava. GBH-coated plastic rings were found to be completely free of thrombus formation at 2 hours (and also at 2 months). In the present study, GBH-coated rings were placed in the thoracic vena cava after which an antiheparin substance was infused into the vena cava directly upstream to the prosthesis. If the thrombus-resisting property of the surface was neutralized, severe thrombus formation would be expected to take place within 2 hours. Rigid rings of polycarbonate (Lexan) were made by injection molding. They were 9 mm. long with a 7 mm. internal diameter and a 0.5 mm. wall, From the Department of Surgery and Cardiovascular Laboratories, University of Wis consin Medical School, Madison, Wis. Supported in part by National Institutes of Health Grant H-4162. Received for publication March 16, 1964. •Markle Scholar in Medical Sciences. 317
3]8
W H I F F E N , YOUNG, GOTT
J. Thoracic and Cardiovas. Surg.
and had streamlined leading and trailing edges. After cleansing and pretreatment of the plastic surface with ethanol, a durable coat of graphite was obtained by dipping the rings in a Dag 154 colloidal graphite solution,* diluted with two parts 95 per cent ethanol and one part benzene. While Dag 154 will not coat all plastic materials well, it will give an excellent graphite coat to both polycarbonate and methylmethacrylate. The rings were placed in a forced draft oven at 130° F. for 48 hours to remove all diluting solvents. The GBH coat was then completed by soaking the rings in aqueous benzalkonium chloride (1:1000) for 24 hours, rinsing them with saline to remove excess benzalkonium, and then exposing them to a dilute heparin solution (0.63 mg. of heparin per cubic centimeter of saline) for one hour.
Vig. 1.—The polycarbonate rings used in this study showing their size and design. The rings on the right have been coated with graphite.
Control studies were performed on graphite-coated rings that were (1) plain or untreated, (2) treated with benzalkonium chloride alone, (3) treated with heparin alone, and (4) treated with benzalkonium chloride and heparin. The concentrations and exposure times of the benzalkonium chloride and heparin solutions used in the preparation of the control rings were the same as described for the test rings. Forty healthy dogs, weighing between 7 and 10 kilograms, were used in this study. They were anesthetized with intravenous sodium pentobarbitol (26.4 mg. per kilogram) and respired by a mechanical respirator. Exposure of the right atrium and superior and inferior venae cavae was made through the right fifth intercostal space under aseptic conditions. The azygos vein was ligated and a purse-string suture placed in the right atrial wall. Tapes were then placed about the venae cavae as far from the atrium as possible. To simulate clinical conditions, some animals were given heparin 1.0 or 2.0 mg. per kilogram while others did not receive this anticoagulant (Figs. 2, 3, and 4). During a short period of inflow stasis, a ring was placed in either the *Dag 154 colloidal graphite—Acheson Colloids Co., Port Huron, Michigan.
Vol. 48, No. 2 August. 1964
S T A B I L I T Y OF THROMBUS-RESISTANT SURFACE
319
superior or inferior vena eava through an atriotomy inside the purse-string suture. 2 Although there was little discrepancy in the ring and vena cava diam eters with the use of dogs in this weight range, a fabric wrap was placed tightly around the vena cava and ring to prevent the formation of a cul-de-sac between the ring and the vein wall. When a ring was placed in the superior vena cava the right external jugular vein was exposed and used as the antiheparin infusion site. The left common femoral vein was used in a similar manner after placement of a ring in the inferior vena cava. No difference in clotting tendencies has been noted in the two venae cavae. No vessel was used twice. Glass clotting times were determined for all animals and all were within normal limits. The concentrations of both the protamine sulfate solution and the hexadimethrine bromide solution used in this study were 10 mg. per cubic centimeter. The anti-heparin solutions were infused at a rate of 10 mg. or 1.0 c.c. per minute. CONTROL STUDIES Graphite Treatment Untreated Benzalkonium Heparin Benzalkonium + Heparin
Thrombus in Ring Lumen At Two Hours
0 0 OOOG Ό O0 O0 •
•
•
•
•
•
•
•
•
Fig. 2.—The thrombus formation (red venous thrombus) in graphite-coated plastic vena cava rings, after various treatments of the graphite surface, is shown diagrammatically. Only the graphite-benzalkonium-heparin rings were free of thrombus formation after 2 hours in the vena cava. None of these animals received heparin or an anti-heparin agent.
Commonly used neutralizing doses for 1.0 mg. of heparin are 1.5 mg. of protamine sulfate and 0.7 to 1.1 mg. of hexadimethrine bromide. 5 ' e We used higher anti-heparin doses so that an excess would be available to the heparin on the graphite surface. In very high concentrations both protamine sulfate and hexadimethrine bromide can act as anticoagulants themselves. This does not occur in the dose ratios used in this study. 7 This was confirmed by deter mining Lee-White clotting times 5 minutes after the infusion of the antiheparin agents as all were normal and essentially the same (± 2.0 min.) as the control clotting times. RESULTS
Control studies are depicted diagrammatically in Fig. 2. Both untreated graphite and graphite-benzalkonium treated rings showed complete occlusion of four rings and partial occlusion of the fifth ring 2 hours after placement
W H I F F E N , YOUNG, GOTT
320
J. Thoracic and Cardiovas. Surg.
in the thoracic vena cava. The graphite-heparin treated rings also showed a severe amount of thrombus formation present, while the graphite-benzalkonium-heparin treated rings were completely free of thrombus at 2 hours. Figs. 3 and 4 depict the absence of thrombus formation in the graphitebenzalkonium-heparin treated rings placed in the vena cava of both heparinized and unheparinized dogs 2 hours after infusion of various doses of anti-heparin compounds directly upstream from the ring. It can be seen that neither protamine sulfate nor hexadimethrine bromide infusions, even in excess, disturbed the in vivo thrombus-resisting property of the graphitebenzalkonium-heparin surface. PROTAMINE INFUSION Heparin Dose Protamine Infusion Thrombus in Ring Lumen (Mg.Ailo) Up Stream (Mg./Kilo) At Two Hours 1
2
2
4
None
3
ooooo ooooo ooooo
Pig. 3.—The absence of thrombus formation in GBH-coated plastic vena cava rings, 2 hours after the intravenous infusion of protamine sulfate directly upstream to the rings, is shown. The animals received from zero to 2.0 mg. of heparin per kilogram before ring placement and protamine neutralization.
HEXADIMETHRINE INFUSION Heparin Dose (Mg./kilo.)
Hexadimethrine Infusion Thrombus in Ring Lumen UpStream (Mg./Kilo.) At Two Hours . 1. 5
OOOOO
Fig. 4.—The absence of thrombus formation in GBH-coated plastic vena cava rings, 2 hours after heparinization and neutralization with hexadimethrine bromide, is depicted.
DISCUSSION
The GBH surface, when used on intravascular prostheses, is superior to a silicone surface in resisting thrombus formation. 1 It is believed that heparin adsorption and its presence on the surface of the graphite is at least the initial factor responsible for the thrombus-resisting properties of the surface.1· 2 · 3 It has been shown that the presence of a cationic surface active agent, such as benzalkonium chloride, is a critical factor in the binding or adsorption of the heparin to the graphite surface. 2,3 Preliminary studies in this laboratory with the use of C14 benzalkonium have shown the presence of this cation on the GBH surface even after a 2 month exposure to the venous blood stream. When solutions of heparin and benzalkonium chloride are mixed, a white
Vol. 48, No. 2 August, 1964
S T A B I L I T Y O F THKOMBUS-RESISTANT SURFACE
321 "
Λ ί
precipitate forms, presumably due to the attraction of the strong negative charge of the sulfonic and sulfate groups of the heparin to the strong positive charge of the quaternary nitrogen of the benzalkonium. 3 Thus far it has also been observed that the thrombus-resisting property of the GBH surface persists for at least 6 months in the vena cava (cava rings), 9 months in the mitral valve area (valve housings), and 2y2 years in the pulmonary artery (valve housings and grafts). Whether the exogenous heparin which is initially applied to the surface remains or is replaced by endogenous heparin, or other heparin-like mucopolysaccharides or proteins, is unknown. Certainly the latter seems more plausible. It is of interest that the present study has shown that while intravenous infusion of an anti-heparin agent, such as protamine sulfate or hexadimethrine bromide, will neutralize heparin circulating in the blood stream, it will not neutralize the heparin on the graphite-benzalkoniumheparin surface. If other mucopolysaccharides or proteins are adsorbed or substituted onto the surface after placement in the vascular stream and are the cause of the persistence of the thrombus-resisting properties of the sur face, these too are not neutralized by protamine or hexadimethrine. Initially the GBH surface has at least one chemical similarity to vascular endothelium in that it contains an anticoagulant acid mueopolysaccharide (heparin). The cement or ground substance and tissue mast cells of normal vascular endothelium are rich in acid mucopolysaccharides. 8 " 13 The specific components include chrondroitin sulfates A and B, hyaluronic acid, and heparitin sulfate. 8 - 9 Gore and Larkey 8 have shown that the mueopolysaccha ride extract of human aorta (0.5 per cent by weight) displayed anticoagulant properties which were neutralizable with protamine and when injected intra venously into rats stimulated the production of lipid clearing factor. They identified the active mueopolysaccharide as chondroitin sulfate B (a chemical relative of heparin with known anticoagulant activity 14 ) and estimated the anticoagulant potency of human aortic tissue as about 100 mg. of heparin per kilogram of tissue. McGovern 12 has demonstrated the in vivo surface anti coagulant effect of endothelial cement substance as it spreads out over in jured endothelium after trauma. Vascular endothelium, even when moderately traumatized, is resistant to neutralization of the anticoagulant mucopolysaccharides in its wall and on its surface by intravenous anti-heparin agents. Mersereau 15 has shown in rats and dogs that the local application of an anti-heparin agent to the exter nal surface of a traumatized vein caused thrombosis of the vein, while, for the most part, intravenous infusion of an anti-heparin agent through a trau matized vein did not. He concluded that the external application brought about thrombosis of the injured vessels by neutralizing an anticoagulant pro duced locally in the vessel wall while neutralization did not occur with intra venous administration of the anti-heparin agent. The present study suggests that the initial heparin layer of the graphitebenzalkonium-heparin surface is apparently resistant to circulating antiheparin agents just as is the heparin-like mucopolysaccharides on normal vascular endothelial surfaces.
322
W H I F F E N , YOUNG, GOTT
J. Thoracic and Cardiovas. Surg.
The plastic surfaces of both our experimental canine and human prosthetic heart valves are now coated with GBH. We have observed no immediate or long-term deterioration in the thrombus-resisting property of the coated valve surfaces due to the administration of the anti-heparin agent at the completion of heart-lung bypass. SUMMARY
The effect of the intravascular infusion of anti-heparin substances, protamine sulfate and hexadimethrine bromide, directly upstream to the thrombus-resistant graphite-benzalkonium-heparin surface have been studied in the dog. The thrombus-resisting property of the surface was found to per sist in this anti-heparin environment. A similarity to the thrombus resisting property of normal vascular endothelium is suggested. The authors wish to thank Dr. Charles W. Crumpton, Director of the Cardiovascular Laboratory, Mr. Francis McMahan, and Mr. Dennis Copper for their assistance in this study. REFERENCES 1. Gott, V. L., Whiff en, J . D., Dutton, R. C. : Heparin Bonding on Colloidal Graphite Surfaces, Science 142: 1297, 1963. 2. Whiff en, J . D., Dutton, E. C , Young, W. P., and Gott, V. L.: Heparin Application to Graphite Coated Intravascular Prostheses, Surgery 56: 404, 1964. 3. Whiff en, J . D., and Gott, V. L. : Effect of Various Surface Active Agents on Heparin Binding and Clot Formation on Graphite Surfaces, Proc. Soc. Exper. Biol. & Med. 116: 314, 1964. 4. Gott, V. L., Koepke, D. E., Daggett, R. L., Zarnstorff, W., and Young, W. P . : The Coating of Intravascular Plastic Prostheses With Colloidal Graphite, Surgerv 50: 382, 1961. 5. Rothnie, N. G., and Kinmonth, J . B.: Bleeding After Perfusion for Open Heart Surgery, Brit. M. J . 1: 73, 1960. 6. Weiss, W. A., Gilman, J. S., Catenacci, A. J., and Osterberg, A. E.: Heparin Neutra lization With Polybrene Administration I. V., J. A. M. A. 166: 603, 1958. 7. Keats, A. S., Cooley, D. A., and Telford, J . : Relative Anti-heparin Potency of Poly brene and Protamine in Patients Undergoing Extracorpcreal Circulation, ,T. THORACIC & CARDIOVAS. SURG. 38: 362
1959.
8. Gore, I., and Larkev, B. J. : Functional Activity of Aortic Mucopolvsaccharides, J. Lab. & Clin. Me*d. 56: 839, 1960. 9. Meyer, K., and Rapport, M. M.: The Mucopolvsaccharides of the Ground Substance of Connective Tissue, Science 113: 596, 1951. 10. Fisher, E. R.: Tissue Mast Cells, J. A. M. A. 173: 171, 1960. 11. Lovelock, ,T. E., and Porterfield, J. S.: Blood Coagulation: Its Prolongation in Vessels W i t h Negatively Charged Surfaces, N a t u r e 167: 39, 1951. 12. McGovern, V. J. : Reactions to Injury of Vascular Endothelium With Special Refer ence to the Problem of Thrombosis, J . Exper. Path. & Bact. 69: 283, 1955. 13. Kirk, J . E. : Anticoagulant Activity of Human Arterial Mucopolysaccharides, Circu lation 20: 975, 1959. 14. Grossman, B. J., and Dorfman, A.: In Vivo Comparison of the Antithrombic Action of Heparin and Chondroitinsulfuric Acid-B, Pediatrics 20: 506, 1957. 15. Mersereau, M. A.: Effect of Anti-heparin Agents on Venous Thrombosis Following \ r ascular Injury, Circulation Res. 11: 765, 1962.