Platelet Plug Formation in an Extracorporeal Unit A. B. C H A N D L E R , M.D.,
A N D M. S. H U T S O N ,
B.S.
Department of Pathology, Medical College of Georgia, Eugene Talmadge Memorial Hospital, Augusta, Georgia 30902
ABSTRACT
I N HEMOSTASIS and thrombosis, platelets tions of venous pressure, plugs of aggrereact to vascular injury by adhesion to the gated platelets that formed in the unit vessel wall and by aggregation to form were stable enough to stop flow in the hemostatic plugs or i n t r a v a s c u l a r absence of fibrin. T h e absence of fibrin thrombi. 9 T h e initial response of platelets was confirmed by electron microscopy, is generally considered to be independent of fibrin coagulation, but after aggregaMaterials and Methods tion occurs, fibrin may form around and T h e extracorporeal unit for platelet between the platelet aggregates to make p i u g formation consisted of a stainless the mass more stable.2-9 This study de- s t e e l needle threaded into silicone rubber scribes observations on the formation of tubing that was constricted by a brass clip platelet plugs from flowing venous blood to form a stenosis (Fig. 1). T h e procedure in an extracorporeal microunit modeled for plug formation was carried out on after the experimental animal work of venous blood of healthy men and women Didisheim and associates3-4 and of Evans w n o had not taken any medication for the and Gordon. 5 Under controlled condi- previous 10 days. T h e assembly of the unit and the procedure are described in Received November 19, 1974; accepted for publi-
cation December 20, 1974.
flow
Supported in part by Grants HL-3973 and HL15158 from the United States Public Health Service. Address reprint requests to Dr. Chandler. 101
detail below. Briefly, b l o o d was a l l o w e d to . •
.
at a
steady pressure from an antecu-
.. J ., . .. . . t h r o u g h a collection line a n d the a t t a c h e d e x t r a c o r p o r e a l unit until flow
bltal v e l n
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Chandler, A. B., and Hutson, M. S.: Platelet plug formation in an extracorporeal unit. Am J Clin Pathol 64: 101-107, 1975. Platelet plugs were formed in an extracorporeal unit from flowing venous blood and studied by electron microscopy. T h e unit consisted of a stainless steel needle threaded into a section of silicone rubber tubing that was constricted to form a slit-like stenosis equivalent in cross-sectional area to an arteriole 100 fi in diameter. Blood was allowed to flow at a steady pressure from an antecubital vein through a collection line and the attached unit until bleeding was stopped by the formation of a platelet plug at the stenosis. Electron microscopy of the plugs showed closely packed aggregated platelets. No fibrin was detected. T h e formation of a stable plug in the absence of fibrin was considered a measure of the capacity of platelets in hemostasis and thrombosis to aggregate and resist the force of the blood current. (Key words: Platelet aggregation; Platelet plug; Hemostasis; Thrombosis.)
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CHANDLER AND HUTSON
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was arrested. T h e unit containing the platelet plug was immediately removed and placed in fixative for preparation for electron microscopic examination. Assembly of Extracorporeal Unit A 15-mm. length of 0.30 mm. ID x 0.64 mm. OD silicone tubing* was cut across with a sharp surgical blade so that the ends were smooth. T h e tubing was threaded for 2 mm. over the shaft of a 27-gauge '/6-in. blunt-cut, deburred, stainless steel n e e d l e f that h a d been siliconized. A brace of glass tubing 10 mm. long and 0.9 mm. ID x 1.2 mm. OD$ was threaded over the silicone tubing until it rested against the hub of the needle. T h e exposed silicone tubing was compressed 2 mm. from the end with fine forceps and slipped into the slot of a brass clip. T h e clip was made by cutting grooves * Silastic medical-grade silicone elastomer tubing, Dow Corning, Midland, Michigan. t Becton-Dickinson, Rutherford, New Jersey. t Unopette 5878, Becton-Dickinson.
to a depth of 2 mm. into the edge of a 0.007-in.-thick brass sheet§ with a 0.012in.-thick circular jeweler's saw,H then trimming around the slot, 1.5 mm. on three sides, with small sharp scissors to make a square plate with one slot. Calibration of the Unit T h e assembled unit was adapted to the tip of a 50-ml. glass buret** that was filled to the 0 mark with sterile distilled water. Care was taken to remove air bubbles from the water, which was then allowed to flow through the unit. If necessary, clips were filed out slighdy o r the tubing was adjusted in the slot to modify flow rate. When a flow rate of 25 drops/min. (± 1 drop) was achieved, the unit was acceptable for use. The size of the slit-like stenosis as determined by measurement of a paraffin wax cast of the site was 15 x 520 ix. T h e cross-sectional area of § Small Parts, Inc., Miami, Florida. f Ewing Brothers, Atlanta, Georgia. ** Kimble 17030, Owens-Illinois, Toledo, Ohio.
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FIG. 1. Collection line and extracorporeal unit. A venipuncture needle shown on the left is attached to one end of the collection line, which consists of silicone rubber tubing that has nylon connectors inserted into each end. After venipuncture and filling of the line, the extracorporeal unit for platelet plug formation shown on the right is attached to the other connector. T h e unit consists of a blunt-cut needle threaded into a silicone tubing that is constricted by a brass clip near the end to create a stenosis. A glass tube is slipped over the silicone tubing for support. T h e inset shows a close-up view of the slit-like stenosis produced by the clip.
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103
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the stenosis was 7,800 sq. /JL, which equalled attached clip was immediately dropped into fixative, 15 to 30 seconds after the that of an arteriole 100 /A in diameter. unit had been occluded. Collection Line Preparation of Specimens for Electron T h e collection line consisted of a 9-cm. Microscopy length of 1.98 mm. ID x 3.18 mm. OD T h e trimmed tubing with the brass clip silicone tubing that had 4-cm.-long nylon in place was fixed for 30 minutes under connectorsff inserted into each end. A 11 p.s.i. vacuum in cold 3 % glutaralsterile 19-gauge thin-wall siliconized venidehyde buffered at pH 7.2 with 0.1 M puncture n e e d l e ^ was attached to the phosphate buffer. T h e specimen was connector on one end. postfixed in cold phosphate-buffered 1% osmium tetroxide for 1 hour. After deProcedure hydration in a series of graded concentraT h e subject was seated on an adjustable tions of ethyl alcohol, the unit was imstool alongside a narrow table with the mersed in propylene oxide and the brass arm resting outward and downward clip was removed. At this stage the proacross a wood block sloping at an angle of pylene oxide caused the silicone tubing to 12 degrees with the horizontal. A blood swell so that the contents could be slipped pressure cuff was placed about the arm, out. T h e specimen was then embedded in attached to a mercury sphygmomanome- epoxy resin. Thick sections were cut until ter, and inflated to a pressure of 30 mm. the platelet plug was identified, and the Hg. After aseptic preparation of the skin block was trimmed. Thin sections were puncture site, and after cuff pressure had cut, stained sequentially with uranyl acebeen stable at 30 mm. Hg for 1 minute, a tate and lead citrate, and examined with a venipuncture was made into an antecubi- Phillips EM 200 electron microscope. tal vein. Three to 4 ml. of blood were first allowed to flow through the line to flush Results out any contaminating tissue thromboplastin. While blood was still flowing, the Rate of Occlusion extracorporeal unit was quickly connected to the line and a stopwatch started. Drops As soon as the extracorporeal unit was of blood flowing out of the unit were attached to the collection line, flow out of collected at 0.2-minute intervals by lighdy the unit was so gready reduced that touching the end of the tube to the edge successive drops of blood could be blotted of a strip of filter paper. When flow onto a strip of filter paper. As occlusion of ceased, the collection was discontinued, the stenosis took place, the volume of and the unit was removed from the blood leaving the unit progressively dicollection line. minished, as illustrated by the tapered T h e unit was placed on a glass block pattern of the drop size on the paper that had a narrow slot to accommodate strips in Figure 2. Occasionally, after flow the clip, and the tubing was cut 1 mm. on had decreased almost to the point of either side of the clip with a sharp surgical cessation, a resurgence of flow occurred, blade. T h e portion of the tubing with the only to be followed by complete occlusion and arrest of flow. T h e time elapsed from t t Supplied through the courtesy of Cutter the start of flow to the last visible drop Laboratories, Inc., Berkeley, California. picked u p by the filter paper was ret t Monoject 200, 1 !4 in. A bevel, Sherwood Medi- corded as the occlusion time. cal Industries, Inc., Deland, Florida.
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A J. C.P. — Vol. 64
aggregants added to platelet-rich plasma in vitro was demonstrated by aggregometer studies. §§ Morphologic Studies
T h e rate of occlusion was determined for 8 healthy adult subjects on two successive occasions at intervals of 1 to 20 days. Occlusion times ranged from 0.6 to 3.6 minutes in the first series and from 0.8 to 2.6 minutes in the second series. T h e mean ± 1 S.D. was 1.73 ± 0.9 for the first series and 1.70 ± 0.6 for the second series. In subsequent studies, these and other healthy subjects periodically did not produce a sharp end point, and bleeding of progressively reduced volume persisted until the procedure was discontinued at 5 minutes. In one apparently healthy adult subject in whom occlusive platelet plugs consistently failed to form, a markedly decreased response of the platelets to i'
Electron microscopy of the occlusive white bodies, or plugs, showed them to be composed of closely packed aggregated platelets (Fig. 3). Most of the aggregated platelets were altered by changes in shape. §§ Kindly performed by Dr. C. L. Lutcher.
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FIG. 2. Record of occlusion rate. As occlusion of the stenosis in the extracorporeal unit proceeds, drops of blood collected every 0.2 minutes on strips of filter paper progressively decrease in size. In this instance, the occlusion time of 1.8 minutes is recorded as the last visible smallest drop on the filter paper strip on the right.
T h e drops of blood leaving the unit were sequentially collected on glass slides instead of filter paper in one experiment and covered by a cover slip. T h e wet preparations were immediately examined under the microscope. Rouleaux of erythrocytes were evident in the second or third d r o p and persisted until flow ceased. A few small, loosely aggregated, clusters of 3 to 4 platelets were identified in the last 2 to 3 drops before arrest of flow, along with rare small clumps of densely aggregated platelets, estimated to be 25 to 50 ^i in diameter. In another experiment, flow was interrupted at 1.0 min., just after flow noticeably diminished, and epoxyembedded thick sections of the region of stenosis were prepared. Occasional small aggregates of platelets were in the lumen as well as along the peripheral zone, where they had contacted the inner wall of the tubing. T h e aggregates were on both sides of the stenosis, predominantly on the proximal side nearest the vein, but were sparse in the narrowest parts of the stenotic channel. Examination of the site of stenosis under the dissecting microscope after flow had stopped showed that a small opaque white body had formed a plug on the proximal side. T h e white body had a flattened bar shape that corresponded to the configuration of the stenotic zone. In those instances when bleeding failed to stop before the procedure was discontinued, a small plug that partly occluded the stenosis was found.
July 1975
105
PLATELET PLUG FORMATION
Discussion T h e platelet plugs that were produced in the extracorporeal unit are equivalent in structure to hemostatic plugs or occlusive thrombotic plugs. 11 They were composed almost entirely of closely packed aggregated platelets, and lacked any evidence of fibrin. T h e early stages of hemostasis or mural thrombosis probably depend in large part on the capacity of platelets to aggregate and resist the shearing force of the blood current. Subsequent formation of fibrin a r o u n d platelet aggregates contributes to their stabilization. 2,9 T h e cohesiveness of the aggregated platelets and the stability of the plugs that formed in the unit were measured by the
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FlG. 3. Electron micrograph of platelet plug. T h e plug is composed of closely that appear flattened in this plane of section, which is parallel to the line Pseudopods project from the platelets to interlock with each other. A long containing glycogen particles (arrow) lines the surface of the plug that contacted of the extracorporeal unit, x27,600.
••;•-_•"' packed aggregated platelets of flow through the unit. pseudopod of one platelet the inner wall of the tubing
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Pseudopods of the platelets interlocked with each other in a complex mosaic arrangement within the aggregates, but were most prominent on the surface of the plug that bordered the lumen of the tube. Frequently, the platelets on the external surface of the plug that had been in contact with the wall of the silicone tubing were flattened and elongated. Occasional leukocytes, mainly neutrophilic granulocytes, were observed between aggregates in the plug or along the border. Erythrocytes were rarely detected within the plug. In the zone away from the plug, erythrocytes often were in rouleaux formation. Fibrin was not identified, either within the plug or on its surface.
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that platelet aggregation is the important event that leads to the formation of the occlusive platelet plug that arrests flow. Didisheim, 3 who studied experimental thrombosis and hemostatic plug formation in a Teflon arteriovenous shunt, considered the formation of a platelet aggregate of primary importance in these fundamentally similar processes. He further suggested that in hemostasis and thrombosis the blood vessel acts primarily as a tube, and that its vital components may play contributory but not necessarily essential roles in these reactions. T h e observations recorded in the present study and in related studies of other investigators 5,10 lend support to this concept. In histologic studies of platelet plugs formed in small vessels of skin wounds removed 15 to 20 minutes after the arrest of bleeding, Zucker 11 observed that occlusive platelet thrombi could resist blood pressure and arrest flow in arterioles as large as 55 fi in diameter, a size that would be fairly comparable to that of the stenosis in the extracorporeal unit. Fibrin-erythrocyte clots that formed in the wound were thought to be a secondary, but important, reinforcement to the platelet thrombi plugging the small vessels. Jorgensen and Borchgrevink 7 performed a similar study and noted the consistent formation of a thin fibrin "perimetric" net around the distal part of the platelet plug protruding out of the vessel. They also concluded that fibrin formed in association with the extravasated blood in the skin wound. Electron microscopic studies of hemostatic plugs produced in the experimental animal have demonstrated the presence of fibrin on the surface of platelet plugs, but not inside them, in specimens fixed immediately 6 or within 5 minutes 8 after arrest of bleeding. In the present study, the plugs were fixed for electron micro-
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rate at which flow was arrested while venous pressure remained constant. Preliminary experiments had shown that once occlusion occurred, the plug remained in place even if the cuff pressure was raised from 30 to 40 mm. Hg for a period of 30 seconds immediately following occlusion. T h e failure consistently to arrest flow by occlusive plugs in one subject who was shown to have markedly reduced platelet aggregation in vitro in an aggregometer further suggests that the extracorporeal procedure for plug formation is an estimate of platelet cohesiveness. In occasional instances, instability of the developing plug was reflected by a "washout" near the time of occlusion; renewed bleeding that followed was then rearrested by the formation of a stable plug. T h e size of the stenosis was critical for stable plug formation. Washouts and failure to form plugs were frequently observed in initial studies when a larger stenosis was used. T h e mechanism of formation of the platelet plugs may be related to several factors. In sections studied by light and electron microscopy, foci of aggregated platelets observed at the site of the inner wall of the silicone tubing suggest that the plugs could have started by adhesion of platelets to the silicone. Loose embolic aggregates in the lumen and in wet preparations of the blood leaving the unit before occlusion may have originated by fragmentation from the wall or by aggregation of platelets in the flowing blood. T h e zones adjacent to the stenotic slit are probably regions of turbulent flow that promote aggregation. Brice and associates 1 have shown that a slit configuration creates more resistance to flow than a circular lumen of the same area. Slowing of flow and the associated rouleaux of erythrocytes that occurred might have further enhanced aggregation. Regardless of the initiating stimulus, it seems clear
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PLATELET PLUG FORMATION
scopic examination less than 30 seconds after bleeding had stopped. Although it would seem likely that, in time, some fibrin might have formed in the plasma around the platelet plugs, none was necessary to stop the bleeding.
6.
7.
References 8.
9.
10.
11.
catheters (abstr). Proc Int Soc T h r o m b Haemostasis 356, 1972 French JE, MacFarlane RG, Sanders AG: T h e structure of haemostatic plugs and experimental thrombi in small arteries. Br J Exp Pathol 45:467-474, 1964 Jorgensen L, Borchgrevink CF: T h e platelet plug in normal persons. 1. T h e histological appearance of the plug 15 to 20 minutes and 24 hours after the bleeding and its role in the capillary haemostasis. Acta Pathol Microbiol Scand 57:40-56, 1963 Kjaerheim A, Hovig T : T h e ultrastructure of haemostatic blood platelet plugs in rabbit mesenterium. T h r o m b Diath H a e m o r r h 7 : 1 - 1 5 , 1962 Mustard JF: T h e relationship between the structure of a thrombus or a hemostatic plug and the mechanisms involved in its formation. T h r o m b Diath Haemorrh suppl 28:57-64, 1968 Rowntree LG, Shionoya T: Studies in experimental extracorporeal thrombosis. I. A method for the direct observation of extracorporeal thrombus formation. J Exp Med 46:7-12, 1927 Zucker HD: Platelet thrombosis in human hemostasis. A histologic study of skin wounds in normal and purpuric individuals. Blood 4:631-645, 1949
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1. Brice JG, Dowsett DJ, Lowe RD: T h e effect of constriction on carotid blood-flow and pressure gradient. Lancet 1:84-85, 1964 2. Chandler AB: T h e platelet in thrombus formation, T h e Platelet. Edited by KM Brinkhous, RW Shermer, FK Mostofi. Baltimore, Williams and Wilkins, 1971, p p 183-197 3. Didisheim P: Microscopically typical thrombi and hemostatic plugs in Teflon arteriovenous shunts, Dynamics of Thrombus Formation and Dissolution. Edited by SA Johnson, MM Guest. Philadelphia, J.B. Lippincott, 1969, pp 64-71 4. Didisheim P, Pavlovsky M, Kobayashi I: Factors affecting hemostatic plug formation in an extracorporeal model. Ann NY Acad Sci 201:307-315, 1972 5. Evans RJ, Gordon J L : Quantitative experimental studies of thrombus formation in arterial
107
A N D M. S. H U T S O N ,
B.S.
Department of Pathology, Medical College of Georgia, Eugene Talmadge Memorial Hospital, Augusta, Georgia 30902
ABSTRACT
I N HEMOSTASIS and thrombosis, platelets tions of venous pressure, plugs of aggrereact to vascular injury by adhesion to the gated platelets that formed in the unit vessel wall and by aggregation to form were stable enough to stop flow in the hemostatic plugs or i n t r a v a s c u l a r absence of fibrin. T h e absence of fibrin thrombi. 9 T h e initial response of platelets was confirmed by electron microscopy, is generally considered to be independent of fibrin coagulation, but after aggregaMaterials and Methods tion occurs, fibrin may form around and T h e extracorporeal unit for platelet between the platelet aggregates to make p i u g formation consisted of a stainless the mass more stable.2-9 This study de- s t e e l needle threaded into silicone rubber scribes observations on the formation of tubing that was constricted by a brass clip platelet plugs from flowing venous blood to form a stenosis (Fig. 1). T h e procedure in an extracorporeal microunit modeled for plug formation was carried out on after the experimental animal work of venous blood of healthy men and women Didisheim and associates3-4 and of Evans w n o had not taken any medication for the and Gordon. 5 Under controlled condi- previous 10 days. T h e assembly of the unit and the procedure are described in Received November 19, 1974; accepted for publi-
cation December 20, 1974.
flow
Supported in part by Grants HL-3973 and HL15158 from the United States Public Health Service. Address reprint requests to Dr. Chandler. 101
detail below. Briefly, b l o o d was a l l o w e d to . •
.
at a
steady pressure from an antecu-
.. J ., . .. . . t h r o u g h a collection line a n d the a t t a c h e d e x t r a c o r p o r e a l unit until flow
bltal v e l n
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Chandler, A. B., and Hutson, M. S.: Platelet plug formation in an extracorporeal unit. Am J Clin Pathol 64: 101-107, 1975. Platelet plugs were formed in an extracorporeal unit from flowing venous blood and studied by electron microscopy. T h e unit consisted of a stainless steel needle threaded into a section of silicone rubber tubing that was constricted to form a slit-like stenosis equivalent in cross-sectional area to an arteriole 100 fi in diameter. Blood was allowed to flow at a steady pressure from an antecubital vein through a collection line and the attached unit until bleeding was stopped by the formation of a platelet plug at the stenosis. Electron microscopy of the plugs showed closely packed aggregated platelets. No fibrin was detected. T h e formation of a stable plug in the absence of fibrin was considered a measure of the capacity of platelets in hemostasis and thrombosis to aggregate and resist the force of the blood current. (Key words: Platelet aggregation; Platelet plug; Hemostasis; Thrombosis.)
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CHANDLER AND HUTSON
A.J.C.P. — Vol.64
was arrested. T h e unit containing the platelet plug was immediately removed and placed in fixative for preparation for electron microscopic examination. Assembly of Extracorporeal Unit A 15-mm. length of 0.30 mm. ID x 0.64 mm. OD silicone tubing* was cut across with a sharp surgical blade so that the ends were smooth. T h e tubing was threaded for 2 mm. over the shaft of a 27-gauge '/6-in. blunt-cut, deburred, stainless steel n e e d l e f that h a d been siliconized. A brace of glass tubing 10 mm. long and 0.9 mm. ID x 1.2 mm. OD$ was threaded over the silicone tubing until it rested against the hub of the needle. T h e exposed silicone tubing was compressed 2 mm. from the end with fine forceps and slipped into the slot of a brass clip. T h e clip was made by cutting grooves * Silastic medical-grade silicone elastomer tubing, Dow Corning, Midland, Michigan. t Becton-Dickinson, Rutherford, New Jersey. t Unopette 5878, Becton-Dickinson.
to a depth of 2 mm. into the edge of a 0.007-in.-thick brass sheet§ with a 0.012in.-thick circular jeweler's saw,H then trimming around the slot, 1.5 mm. on three sides, with small sharp scissors to make a square plate with one slot. Calibration of the Unit T h e assembled unit was adapted to the tip of a 50-ml. glass buret** that was filled to the 0 mark with sterile distilled water. Care was taken to remove air bubbles from the water, which was then allowed to flow through the unit. If necessary, clips were filed out slighdy o r the tubing was adjusted in the slot to modify flow rate. When a flow rate of 25 drops/min. (± 1 drop) was achieved, the unit was acceptable for use. The size of the slit-like stenosis as determined by measurement of a paraffin wax cast of the site was 15 x 520 ix. T h e cross-sectional area of § Small Parts, Inc., Miami, Florida. f Ewing Brothers, Atlanta, Georgia. ** Kimble 17030, Owens-Illinois, Toledo, Ohio.
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FIG. 1. Collection line and extracorporeal unit. A venipuncture needle shown on the left is attached to one end of the collection line, which consists of silicone rubber tubing that has nylon connectors inserted into each end. After venipuncture and filling of the line, the extracorporeal unit for platelet plug formation shown on the right is attached to the other connector. T h e unit consists of a blunt-cut needle threaded into a silicone tubing that is constricted by a brass clip near the end to create a stenosis. A glass tube is slipped over the silicone tubing for support. T h e inset shows a close-up view of the slit-like stenosis produced by the clip.
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103
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the stenosis was 7,800 sq. /JL, which equalled attached clip was immediately dropped into fixative, 15 to 30 seconds after the that of an arteriole 100 /A in diameter. unit had been occluded. Collection Line Preparation of Specimens for Electron T h e collection line consisted of a 9-cm. Microscopy length of 1.98 mm. ID x 3.18 mm. OD T h e trimmed tubing with the brass clip silicone tubing that had 4-cm.-long nylon in place was fixed for 30 minutes under connectorsff inserted into each end. A 11 p.s.i. vacuum in cold 3 % glutaralsterile 19-gauge thin-wall siliconized venidehyde buffered at pH 7.2 with 0.1 M puncture n e e d l e ^ was attached to the phosphate buffer. T h e specimen was connector on one end. postfixed in cold phosphate-buffered 1% osmium tetroxide for 1 hour. After deProcedure hydration in a series of graded concentraT h e subject was seated on an adjustable tions of ethyl alcohol, the unit was imstool alongside a narrow table with the mersed in propylene oxide and the brass arm resting outward and downward clip was removed. At this stage the proacross a wood block sloping at an angle of pylene oxide caused the silicone tubing to 12 degrees with the horizontal. A blood swell so that the contents could be slipped pressure cuff was placed about the arm, out. T h e specimen was then embedded in attached to a mercury sphygmomanome- epoxy resin. Thick sections were cut until ter, and inflated to a pressure of 30 mm. the platelet plug was identified, and the Hg. After aseptic preparation of the skin block was trimmed. Thin sections were puncture site, and after cuff pressure had cut, stained sequentially with uranyl acebeen stable at 30 mm. Hg for 1 minute, a tate and lead citrate, and examined with a venipuncture was made into an antecubi- Phillips EM 200 electron microscope. tal vein. Three to 4 ml. of blood were first allowed to flow through the line to flush Results out any contaminating tissue thromboplastin. While blood was still flowing, the Rate of Occlusion extracorporeal unit was quickly connected to the line and a stopwatch started. Drops As soon as the extracorporeal unit was of blood flowing out of the unit were attached to the collection line, flow out of collected at 0.2-minute intervals by lighdy the unit was so gready reduced that touching the end of the tube to the edge successive drops of blood could be blotted of a strip of filter paper. When flow onto a strip of filter paper. As occlusion of ceased, the collection was discontinued, the stenosis took place, the volume of and the unit was removed from the blood leaving the unit progressively dicollection line. minished, as illustrated by the tapered T h e unit was placed on a glass block pattern of the drop size on the paper that had a narrow slot to accommodate strips in Figure 2. Occasionally, after flow the clip, and the tubing was cut 1 mm. on had decreased almost to the point of either side of the clip with a sharp surgical cessation, a resurgence of flow occurred, blade. T h e portion of the tubing with the only to be followed by complete occlusion and arrest of flow. T h e time elapsed from t t Supplied through the courtesy of Cutter the start of flow to the last visible drop Laboratories, Inc., Berkeley, California. picked u p by the filter paper was ret t Monoject 200, 1 !4 in. A bevel, Sherwood Medi- corded as the occlusion time. cal Industries, Inc., Deland, Florida.
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aggregants added to platelet-rich plasma in vitro was demonstrated by aggregometer studies. §§ Morphologic Studies
T h e rate of occlusion was determined for 8 healthy adult subjects on two successive occasions at intervals of 1 to 20 days. Occlusion times ranged from 0.6 to 3.6 minutes in the first series and from 0.8 to 2.6 minutes in the second series. T h e mean ± 1 S.D. was 1.73 ± 0.9 for the first series and 1.70 ± 0.6 for the second series. In subsequent studies, these and other healthy subjects periodically did not produce a sharp end point, and bleeding of progressively reduced volume persisted until the procedure was discontinued at 5 minutes. In one apparently healthy adult subject in whom occlusive platelet plugs consistently failed to form, a markedly decreased response of the platelets to i'
Electron microscopy of the occlusive white bodies, or plugs, showed them to be composed of closely packed aggregated platelets (Fig. 3). Most of the aggregated platelets were altered by changes in shape. §§ Kindly performed by Dr. C. L. Lutcher.
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FIG. 2. Record of occlusion rate. As occlusion of the stenosis in the extracorporeal unit proceeds, drops of blood collected every 0.2 minutes on strips of filter paper progressively decrease in size. In this instance, the occlusion time of 1.8 minutes is recorded as the last visible smallest drop on the filter paper strip on the right.
T h e drops of blood leaving the unit were sequentially collected on glass slides instead of filter paper in one experiment and covered by a cover slip. T h e wet preparations were immediately examined under the microscope. Rouleaux of erythrocytes were evident in the second or third d r o p and persisted until flow ceased. A few small, loosely aggregated, clusters of 3 to 4 platelets were identified in the last 2 to 3 drops before arrest of flow, along with rare small clumps of densely aggregated platelets, estimated to be 25 to 50 ^i in diameter. In another experiment, flow was interrupted at 1.0 min., just after flow noticeably diminished, and epoxyembedded thick sections of the region of stenosis were prepared. Occasional small aggregates of platelets were in the lumen as well as along the peripheral zone, where they had contacted the inner wall of the tubing. T h e aggregates were on both sides of the stenosis, predominantly on the proximal side nearest the vein, but were sparse in the narrowest parts of the stenotic channel. Examination of the site of stenosis under the dissecting microscope after flow had stopped showed that a small opaque white body had formed a plug on the proximal side. T h e white body had a flattened bar shape that corresponded to the configuration of the stenotic zone. In those instances when bleeding failed to stop before the procedure was discontinued, a small plug that partly occluded the stenosis was found.
July 1975
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PLATELET PLUG FORMATION
Discussion T h e platelet plugs that were produced in the extracorporeal unit are equivalent in structure to hemostatic plugs or occlusive thrombotic plugs. 11 They were composed almost entirely of closely packed aggregated platelets, and lacked any evidence of fibrin. T h e early stages of hemostasis or mural thrombosis probably depend in large part on the capacity of platelets to aggregate and resist the shearing force of the blood current. Subsequent formation of fibrin a r o u n d platelet aggregates contributes to their stabilization. 2,9 T h e cohesiveness of the aggregated platelets and the stability of the plugs that formed in the unit were measured by the
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FlG. 3. Electron micrograph of platelet plug. T h e plug is composed of closely that appear flattened in this plane of section, which is parallel to the line Pseudopods project from the platelets to interlock with each other. A long containing glycogen particles (arrow) lines the surface of the plug that contacted of the extracorporeal unit, x27,600.
••;•-_•"' packed aggregated platelets of flow through the unit. pseudopod of one platelet the inner wall of the tubing
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Pseudopods of the platelets interlocked with each other in a complex mosaic arrangement within the aggregates, but were most prominent on the surface of the plug that bordered the lumen of the tube. Frequently, the platelets on the external surface of the plug that had been in contact with the wall of the silicone tubing were flattened and elongated. Occasional leukocytes, mainly neutrophilic granulocytes, were observed between aggregates in the plug or along the border. Erythrocytes were rarely detected within the plug. In the zone away from the plug, erythrocytes often were in rouleaux formation. Fibrin was not identified, either within the plug or on its surface.
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that platelet aggregation is the important event that leads to the formation of the occlusive platelet plug that arrests flow. Didisheim, 3 who studied experimental thrombosis and hemostatic plug formation in a Teflon arteriovenous shunt, considered the formation of a platelet aggregate of primary importance in these fundamentally similar processes. He further suggested that in hemostasis and thrombosis the blood vessel acts primarily as a tube, and that its vital components may play contributory but not necessarily essential roles in these reactions. T h e observations recorded in the present study and in related studies of other investigators 5,10 lend support to this concept. In histologic studies of platelet plugs formed in small vessels of skin wounds removed 15 to 20 minutes after the arrest of bleeding, Zucker 11 observed that occlusive platelet thrombi could resist blood pressure and arrest flow in arterioles as large as 55 fi in diameter, a size that would be fairly comparable to that of the stenosis in the extracorporeal unit. Fibrin-erythrocyte clots that formed in the wound were thought to be a secondary, but important, reinforcement to the platelet thrombi plugging the small vessels. Jorgensen and Borchgrevink 7 performed a similar study and noted the consistent formation of a thin fibrin "perimetric" net around the distal part of the platelet plug protruding out of the vessel. They also concluded that fibrin formed in association with the extravasated blood in the skin wound. Electron microscopic studies of hemostatic plugs produced in the experimental animal have demonstrated the presence of fibrin on the surface of platelet plugs, but not inside them, in specimens fixed immediately 6 or within 5 minutes 8 after arrest of bleeding. In the present study, the plugs were fixed for electron micro-
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rate at which flow was arrested while venous pressure remained constant. Preliminary experiments had shown that once occlusion occurred, the plug remained in place even if the cuff pressure was raised from 30 to 40 mm. Hg for a period of 30 seconds immediately following occlusion. T h e failure consistently to arrest flow by occlusive plugs in one subject who was shown to have markedly reduced platelet aggregation in vitro in an aggregometer further suggests that the extracorporeal procedure for plug formation is an estimate of platelet cohesiveness. In occasional instances, instability of the developing plug was reflected by a "washout" near the time of occlusion; renewed bleeding that followed was then rearrested by the formation of a stable plug. T h e size of the stenosis was critical for stable plug formation. Washouts and failure to form plugs were frequently observed in initial studies when a larger stenosis was used. T h e mechanism of formation of the platelet plugs may be related to several factors. In sections studied by light and electron microscopy, foci of aggregated platelets observed at the site of the inner wall of the silicone tubing suggest that the plugs could have started by adhesion of platelets to the silicone. Loose embolic aggregates in the lumen and in wet preparations of the blood leaving the unit before occlusion may have originated by fragmentation from the wall or by aggregation of platelets in the flowing blood. T h e zones adjacent to the stenotic slit are probably regions of turbulent flow that promote aggregation. Brice and associates 1 have shown that a slit configuration creates more resistance to flow than a circular lumen of the same area. Slowing of flow and the associated rouleaux of erythrocytes that occurred might have further enhanced aggregation. Regardless of the initiating stimulus, it seems clear
A.J.C.P. — Vol. 64
July 1975
PLATELET PLUG FORMATION
scopic examination less than 30 seconds after bleeding had stopped. Although it would seem likely that, in time, some fibrin might have formed in the plasma around the platelet plugs, none was necessary to stop the bleeding.
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7.
References 8.
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catheters (abstr). Proc Int Soc T h r o m b Haemostasis 356, 1972 French JE, MacFarlane RG, Sanders AG: T h e structure of haemostatic plugs and experimental thrombi in small arteries. Br J Exp Pathol 45:467-474, 1964 Jorgensen L, Borchgrevink CF: T h e platelet plug in normal persons. 1. T h e histological appearance of the plug 15 to 20 minutes and 24 hours after the bleeding and its role in the capillary haemostasis. Acta Pathol Microbiol Scand 57:40-56, 1963 Kjaerheim A, Hovig T : T h e ultrastructure of haemostatic blood platelet plugs in rabbit mesenterium. T h r o m b Diath H a e m o r r h 7 : 1 - 1 5 , 1962 Mustard JF: T h e relationship between the structure of a thrombus or a hemostatic plug and the mechanisms involved in its formation. T h r o m b Diath Haemorrh suppl 28:57-64, 1968 Rowntree LG, Shionoya T: Studies in experimental extracorporeal thrombosis. I. A method for the direct observation of extracorporeal thrombus formation. J Exp Med 46:7-12, 1927 Zucker HD: Platelet thrombosis in human hemostasis. A histologic study of skin wounds in normal and purpuric individuals. Blood 4:631-645, 1949
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1. Brice JG, Dowsett DJ, Lowe RD: T h e effect of constriction on carotid blood-flow and pressure gradient. Lancet 1:84-85, 1964 2. Chandler AB: T h e platelet in thrombus formation, T h e Platelet. Edited by KM Brinkhous, RW Shermer, FK Mostofi. Baltimore, Williams and Wilkins, 1971, p p 183-197 3. Didisheim P: Microscopically typical thrombi and hemostatic plugs in Teflon arteriovenous shunts, Dynamics of Thrombus Formation and Dissolution. Edited by SA Johnson, MM Guest. Philadelphia, J.B. Lippincott, 1969, pp 64-71 4. Didisheim P, Pavlovsky M, Kobayashi I: Factors affecting hemostatic plug formation in an extracorporeal model. Ann NY Acad Sci 201:307-315, 1972 5. Evans RJ, Gordon J L : Quantitative experimental studies of thrombus formation in arterial
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