MICROVASCULAR
RESEARCH
Platelet
9,
22-28 (1975)
Aggregability and Vessel Contraction Microvascular Haemostasis
in
K.-E. ARFORSAND D. BERGQVIST~ Department
of Experimental
Medicine, Pharmacia AB, Uppsala, Sweden
Received March II,1974
Use of the dual-slit technique for on-line recording of blood how velocity and a film technique with time-lapse cinematography for the estimation of vessel contraction and haemostatic plug volume, makes it possible to determine the proportion of platelets which participate in the formation of an effective haemostatic plug in individual vessels of the microvasculature. Platelet aggregability is significantly higher in plugs formed at injuries on the arteriolar side of the microcirculation than at injuries on the venular side. The extent of vessel contraction is of no importance in microcirculatory haemostasis.
INTRODUCTION When a microvessel is transected with a sharp knife, bleeding starts at once. This bleeding will stop when a haemostatic plug has formed, sealing the transected vessel end (Zucker, 1947; Hugues, 1953). In the initial haemostatic mechanism, normal platelet reaction as well as intact coagulation and fibrinolytic systems are of great importance (Kjaerheim and Hovig, 1962; Bergqvist and Arfors, 1973, 1974a). In previous studies it has been shown that arterioles and venules differ in their haemostatic characteristics (Hugues, 1953; Arfors et al., 1972; Bergqvist, 1974; Bergqvist and Arfors, 1974b). Thus, the haemostatic plugs in proximal arteriolar segmentsgrow to a larger volume in a much shorter time than in other vesselsegments.The influence of microvesselcontraction on the haemostatic processis a controversial issue(Macfarlane, 1941; Zucker, 1947; Hugues, 1953; Cruz, 1965). In a study on the influence of blood flow velocity on experimental haemostatic plug formation, we used results from different investigations to make approximate calculations. These suggested that platelets participated more effectively in haemostatic plug formation in proximal arteriolar segments(Arfors and Bergqvist, 1974) than in venular segments.This led to investigation of the growth rate of haemostatic plugs in relation to platelet flow in individual vessels,as an estimate of the platelet activity in different vessel segments, At the same time it was possible to establish the significance of vessel contraction in microvascular haemostasis. MATERIALS
AND METHODS
Nine New Zealand white rabbits of both sexes(wt 2.4 + 0.3 kg) were studied. They were fed on a standard diet (Teknosan pellets, Ferrosan AB, Malmo, Sweden) and starved 12 hr before experiment. 1 Present address: Department of Surgery, Klrnsjukhuset, 541 00 Skovde, Sweden. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
22
HAEMOSTATIC
PLUGS
Ot
23
1lICROVASCULATURE
Haemostatic plug jhmation. This method is described in detail elsewhere (Arforh et al.. 1972; Bergqvist, 1974).In short, mesenteric microvessels40-60 ;tm in diameter
in urethane-anaesthetizedrabbits are transected, while observed under a Leitz Biomed CAPILLARY
ARTERIOLE
VENULE
ARTERIOLAR TRANSECT/ON
segment
segment
seynent
segnent
I
FIG. 1. Schematic diagram illustrating the definition used in this study of proximal and distal vessel segments. Arrows indicate the flow direction.
Intravital microscope. The time between transection and the first arrest of haemorrhage is measured and called the primary haemostatic plug formation time (PHT). When a vesselis transected there are two vesselsegments,the positions of which are shown in Fig. 1. Blood flow is reversedafter transection in distal arteriolar and proximal venular
FIG. 2. Ground glass screen with photodiode holder positioned over an arteriole (-45 pm) measuring the centerline velocity. At the end of the transected vessel an incomplete haemostatic plug can be seen.
24
ARFORS
AND
BERGQVIST
segments. The following number of vessels were studied: 14 proximal arteriolar segments, 12 distal arteriolar segments. 12 proximal venular segments, and I6 distal venular segments. VeZocit~ measurement. The technique to measure red blood cell velocity in the microvasculature has been described by Intaglietta et al. (1970). This method is based on the dual-slit technique of Wayland and Johnson (1967), who measured the delay between signals that originate from two photodetectors when an image of the blood
FIG. 3. A diagram of the ocular mounted ground glassscreen,front view (cf. Fig. 1) and side view. 1. Adjustable photodiode holder, 2. Chip carrying photodiodesfacing ocular. 3. Ocular x 10. stream is projected onto them. The detectors are fixed with a known separation in the direction of flow. While measuring flow in the mesentery, peristaltic movements sometimes make it necessary to rapidly readjust the position of the two photodetectors. We have, therefore, developed a simple photodiode holder mounted inside a ground glass screen (Figs. 2 and 3), on which the image from the mesentery is projected through an ocular (Arfors et aE., 1973). The detectors used were two photodiodes (SP 50, thin film substrate: Hafo, Stockholm, Sweden) mounted with a 1.0 mm center to center
HAEMOSTATIC
PLUGS
OF MICROVASCULATL’RE
35 i.
separation. The optical signal pattern of the passing red cells was continuously cross correlated (Hewlett-Packard Correlator 3721 A, Palo Alto, California) to determine
the transit time (t) of cells passing from one diode to the other. The distance between the diodes is known (s), and the velocity was computed on-line according to the formula s!t = L’.The resulting axial red blood cell velocity (c) was continuously recorded on a Hewlett-Packard Recorder (Mosley Autograph 680 M, Hewlett-Packard, California). Haemostaticplug w&me and vesselcontraction. This was measured using time-lapse photography (Beaulieu R 16, Kodachrome II daylight film, Hasselblad Timer, Reflex, Stockholm, Sweden) with an exposure every 5 sec.Analysis from the films was made with a single frame motion analyser (Motion analysis projector, S, Mark 111;John Hadland Ltd., Bovingdon, England). The systemwascalibrated by filming a micrometer slide through the same optical system. When calculating the volume, the haemostatic plug was approximated to the form of an ellipsoid, rotated around its long axis. The perpendicular short (a) and long (b) axes were measuredand the volume (v) calculated according to the formula: v = (47c/3)a2b.The maximum vessel contraction (C) was calculated as a percentage according to C = (D - Dmin)100/D, where D is the diameter before transection and Dminthe diameter at maximum contraction. Platelet counts. These were determined in duplicate on an Improved Neubauer Chamber, according to the method of Brecher and Cronkite (1950). Calculation of theproportion ofplateletsparticipating in theformation qf a haemostatic plug. The total number of platelets that passed(platelet flow) before arrest of bleeding in individual vesselswas obtained by multiplying the integrated blood flow velocity until haemostasisby the platelet count for the particular rabbit. For eachvesselsegment the continuously recorded values for centerline velocity were used to obtain volume flow rate (Q): Q = vIIR2/2, where R is the radius (Baker, 1972). The total number of platelets in the haemostatic plug was calculated by dividing the final plug volume (u) by the volume of individual platelets (5.5 pm3) in the rabbit microcirculation (Arfors e2al., 1974). Individual vesselplatelet aggregability as a percentagewas calculated as platelet number in final plug x 100 platelet flow RESULTS In Table 1 the maximum degree of vesselconstriction during haemostasisis shown. Both arteriolar segments show the same degree of contraction, as do both venular segments,the constriction being approximately three times as great in arterioles. This table also illustrates the linear correlation coefficients for the relationship between the degreeof contraction and the primary haemostatic plug formation time (PHT). There was no correlation in any of the segments. Forty-one percent of the platelets passing the proximal arteriolar orifice after vessel transection participate in the formation of an effective haemostatic plug. The corresponding figures for the other vessel segmentsare: 12% in distal arteriolar segments, 6 % in proximal venular segments,and 3 1% in distal venular segments.These data can also be seenin Fig. 4. Individual vesselplatelet aggregability is significantly higher in plugs where blood is flowing from the arteriolar side of the microcirculation (proximal
26
ARFORS
AND
BERGQVIST
TABLE THE
MAXIMUM DEGREE
I
OF VESSEL CONSTRICTION
DUKIKG
HAEMOSTASIS
Arterioles
Maximum degree of contraction in percent Linear correlation (v) between degree of contraction and PHT PHT” (set)
Venules
proximal segment
distal segment
16.0
18.2
proximal segment 5.8
distal segment 5.5
0.52 NP
0.46 NS
-0.11 NS
0.42 NS
75
458
439
303
a PHT = primary haemostatic plug formation time. b NS = not significant according to the Student’s t test.
FIG. 4. Bar-chart showing the individual platelet aggregability in the four vessel segments; 95% confidence limits are given. AP = proximal arteriolar segment; AD = distal arteriolar segment; VD = distal venular segment; VP = proximal venular segment.
arteriolar and distal venular segments), than where it is flowing from the venular side-proximal venular and distal arteriolar segments (see also Fig. I). DISCUSSION Several authors have described differences in the initial haemostatic processes in arterioles and venules (Apitz, 1942; Chen and Tsai, 1948; Hugues, 1953; Arfors et al., 1972; Bergqvist, 1974) although these differences have not been explained. Previous experimental results have been inconclusive. These physiological variations in haemostatic properties are not reflected morphologically, as judged by ultrastructural studies of haemostatic plugs from arterioles and venules in the rabbit mesentery (Kjaerheim and Hovig, 1962). However, several possible explanations exist (Bergqvist, 1973). Vessel constriction has been considered to be an important factor in haemostasis (Macfarlane, 1941; Apitz, 1942; Tocantins, 1947; Zucker, 1947; Cruz, 1965), but the finding in the distal arteriolar segment of a longer primary haemostatic plug formation
HAEMOSTATIC
PLUGS OF MICROVASCULATURE
27
time than in the proximal arteriolar segmentdespite the samedegreeof posttraumatic contraction in the two vessel segmentssuggeststhat contraction is not an important factor in microvascular haemostasis. The lack of correlation between the degree of contraction and the haemostatic plug formation time further supports this conclusion as does the similar haemostatic tissues between distal arteriolar segmentsand venules despite the much greater contraction in the former (Table 1). In experiments in which xylocaine was added to the superfusatehaemostatic plug formation time was unaltered despitethe abolition of vascular contraction (unpublished observations). It can therefore be concluded that vesselconstriction is of little importance in microcirculatory haemostatic plug formation in the rabbit mesentery. It is noteworthy that despite differences in methodology, Hugues (1953) also came to the conclusion that vascular constriction was not an important determinant of microvascular haemostasis. In an experimental mode1where arteriolar blood flow velocity was reduced, we were able to show that the difference in haemostasis between arterioles and venules could not be explained on the basis of different flow velocities (Arfors and Bergqvist, 1974). This view was supported by analysis of different flow velocity parameters after microvessel transection. Approximate calculations suggested that platelets passing the proximal arteriolar segment participated more actively in forming the haemostatic plug than in the other vessel segments. This finding prompted us to perform this investigation, using a method to measure the individual vessel platelet aggregability. The results are similar to those obtained by our approximate calculations, and suggest that the platelets coming from the arteriolar side of the microcirculation (proximal arteriolar and distal venular segments)are significantly more active than those coming from the venular side. The individual vesselplatelet aggregability is thus higher in the arteriolar blood. Differences in blood fibrinolytic activity and white cell behaviour are not responsible for the haemostatic differences (Bergqvist and Arfors, 1973, 1974a). In rabbits the difference in haemostasis between arterioles and venules tends to be diminished by continuous infusion of adenosine diphosphate (ADP) into the cranial mesenteric artery and by a preceding laser-induced intravascular injury upstream of the transection site of the microvessel (Bergqvist and Arfors, to be published). Several unpublished investigations have given us the impression that haemostatic plug formation may be mainly an ADP-induced platelet aggregation and one possibility is that cells (red cells and platelets) in statesof a high sheardisrupt more easily, releasing a higher concentration of platelet aggregating substances.In this way the cells contribute to a faster haemostasisthan would otherwise be the case.This is in agreement with claims made by Marr et al. (1965) and Johnson et al. (1966) emphasizing the important role of ADP in initial haemostasis-ADP probably released from the red blood cells. Red blood cells contain 90 % of the nucleotides of the blood and platelets the remaining 10% (Johnson et al., 1967). This view is also supported by Jorgensen and Borchgrevink (1964), based on histological studies of skin wounds made for bleeding time tests in normal humans and patients with various bleeding disorders. REFERENCES APITZ, K. (1942). Die Bedeutung der Gerinnung und Thrombose fiir die Blutstillung. Virch. Arch. Pathol. Anat. Physiol. 308, 540. ARFORS, K.-E., AND BERGQVIST, D. (1974). Influence of blood flow velocity on experimental haemostaticplug formation.Throm. Res. 4,447.
28
ARFORS
AND
BERGQVIST
AKFOKS,K.-E., BEKGQVIST, D., BYGDEMAN, S., MCKPJZIE, F. N., AND SvENsJO, E. (1972). The effect of inhibition of the platelet release reaction on platelet behaviour in vitro and in vivo. Scud J. Haematol. 9, 322. ARFORS,K.-E., BERGQVIST, D., INTAC;LI~.I~A, M., AND WEsTERCiREh, B. (1973a). Measurements 01 blood flow velocity in the microcirculation. L/ps. J. Med. Sri., in press. ARFORS, K.-E., COCKBURN, J., AND GROSS, J. (1974). Measurement of growth rate of laser-induced intravascular platelet aggregation. To be published. BAKEK, M. (1972). “Double-Slit Photometric Measurement of Velocity Profiles for Blood Flow in Microvessels and Capillary Tubes.” Thesis. California Institute ofTechnology, University microfilm no. 72-30791, Pasadena, California. BAUMGARTNER, H. R., GRIMM, L., AND ZBINDEN, G. (1973). Effect of generation of mural platelet thrombi on circulating blood in rabbit aorta. Experientia 29, 442. BERGQVIST, D. (1973). Haemostatic plug formation time and stability in the microvasculature of rabbit mesentery. Acra Univ. Ups. 167, Abstracts of Uppsala Dissertations from the faculty of Medicine. BERGQVIST, D. (1974). Haemostatic plug formation in the rabbit mesentery. A methodological study. Ups. J. Med. Sci. 79, 28. BERGQVIST,D., AND ARFORS, K.-E. (1973). Influence of platelet count on haemostatic plug formation and stability. An experimental study in rabbits with graded thrombocytopenia. Thromb. Diath. Haermorrh. 30,586. BERGQVIST, D., AND ARFORS, K.-E. (1974a). Influence of fibrinolysis and coagulation on haemostatic plug formation. An experimental study in rabbits. Thromb. Res. 4, 345. BERGQVIST, D., AND ARFORS, K.-E. (1974b). Growth rate and volume of haemostatic plugs in the mesentery of normal and thrombocytopenic rabbits. Thromb. Res. 4,77. BORCHGREVINK, CHR. (1960). A method for measuring platelet adhesiveness in vivo. Acta Med. Stand. 168, 157. BRECHER, G., AND CRONKITE, E. (1950). Morphology and enumeration of human blood platelets. J. Appl. Physiol. 3, 365. CHEN, 7. I., AND TSAI, C. (1948). The mechanism of haemostasis in peripheral vessels. J. Physiol. 107, 280. CRUZ, W. 0. (1965). The significance of a smooth muscle component in hemostasis. Proc. SOC.Exp. Biol. Med. 119, 876. HUGUES, J. (1953). Contribution a l’ttude des facteurs vasculaires et sanguins dans l’hemostase spontanee. Arch. Znt. Physiol. 41,565. INTAGLIETTA, M., TOMPKINS, W. R., AND RICHARDSON, D. R. (1970). Velocity measurements in the microvasculature of the cat omentum by on-line method. Microvasc. Res. 2,462. JOHNSON, S., v. HORN, D., PEDERSON,H., AND MARR, J. (1966). The function of platelets: a review. Transfusion 6,3. JOHNSON, S., FREDELL, L., SHEPARD, J., TEBO, T., CHANG, C., PEDERSON,H., AND VON HORN, D. (1967). Red blood cells, fibrin and platelets in hemostasis. In “Physiology of Hemostasis and Thrombosis” (Johnson, S., and Seegers, W., eds.), p. 44. Charles G. Thomas, Springfield, Illinois. JORGENSEN, L., AND BORCHGREVINK, CHR. (1964). The haemostatic mechanism in patients with haemorrhagic diseases. A histological study of wounds made from primary and secondary bleeding time tests. Acta Path. Microbial. Stand. 60,55. KJAERHEIM, A, AND HOVIG, T. (1962). The ultrastructure of haemostatic blood platelet plugs in rabbit mesenterium. Thromb. Diath. Haemorrh. 7, 1. MACFARLANE, R. G. (1941). Critical review: the mechanism of haemostasis. Quart. J. Med. N.S. 10,l. MARR, J., BARBORIAK, J. J., AND JOHNSON, S. (1965). Relationship of appearance of adenosine diphosphate, fibrin, formation and platelet aggregation in the haemostatic plug in vivo. Nature (London) 205,259. TOCANTINS, L. (1947). The mechanism of hemostasis. Ann. Surg. 125,292. WAYLAND, H., AND JOHNSON, P. (1967). Erythrocyte velocity measurement in microvessels by a two-slit photometric method. J. Appl. Physiol. 22,333. ZUCKER, M. B. (1947). Platelet agglutination and vasoconstriction as factors in spontaneous hemostasis in normal, thrombocytopenic, heparinized and hypoprothrombinemic rats. Amer. J. Physiol. 148, 275.
RESEARCH
Platelet
9,
22-28 (1975)
Aggregability and Vessel Contraction Microvascular Haemostasis
in
K.-E. ARFORSAND D. BERGQVIST~ Department
of Experimental
Medicine, Pharmacia AB, Uppsala, Sweden
Received March II,1974
Use of the dual-slit technique for on-line recording of blood how velocity and a film technique with time-lapse cinematography for the estimation of vessel contraction and haemostatic plug volume, makes it possible to determine the proportion of platelets which participate in the formation of an effective haemostatic plug in individual vessels of the microvasculature. Platelet aggregability is significantly higher in plugs formed at injuries on the arteriolar side of the microcirculation than at injuries on the venular side. The extent of vessel contraction is of no importance in microcirculatory haemostasis.
INTRODUCTION When a microvessel is transected with a sharp knife, bleeding starts at once. This bleeding will stop when a haemostatic plug has formed, sealing the transected vessel end (Zucker, 1947; Hugues, 1953). In the initial haemostatic mechanism, normal platelet reaction as well as intact coagulation and fibrinolytic systems are of great importance (Kjaerheim and Hovig, 1962; Bergqvist and Arfors, 1973, 1974a). In previous studies it has been shown that arterioles and venules differ in their haemostatic characteristics (Hugues, 1953; Arfors et al., 1972; Bergqvist, 1974; Bergqvist and Arfors, 1974b). Thus, the haemostatic plugs in proximal arteriolar segmentsgrow to a larger volume in a much shorter time than in other vesselsegments.The influence of microvesselcontraction on the haemostatic processis a controversial issue(Macfarlane, 1941; Zucker, 1947; Hugues, 1953; Cruz, 1965). In a study on the influence of blood flow velocity on experimental haemostatic plug formation, we used results from different investigations to make approximate calculations. These suggested that platelets participated more effectively in haemostatic plug formation in proximal arteriolar segments(Arfors and Bergqvist, 1974) than in venular segments.This led to investigation of the growth rate of haemostatic plugs in relation to platelet flow in individual vessels,as an estimate of the platelet activity in different vessel segments, At the same time it was possible to establish the significance of vessel contraction in microvascular haemostasis. MATERIALS
AND METHODS
Nine New Zealand white rabbits of both sexes(wt 2.4 + 0.3 kg) were studied. They were fed on a standard diet (Teknosan pellets, Ferrosan AB, Malmo, Sweden) and starved 12 hr before experiment. 1 Present address: Department of Surgery, Klrnsjukhuset, 541 00 Skovde, Sweden. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
22
HAEMOSTATIC
PLUGS
Ot
23
1lICROVASCULATURE
Haemostatic plug jhmation. This method is described in detail elsewhere (Arforh et al.. 1972; Bergqvist, 1974).In short, mesenteric microvessels40-60 ;tm in diameter
in urethane-anaesthetizedrabbits are transected, while observed under a Leitz Biomed CAPILLARY
ARTERIOLE
VENULE
ARTERIOLAR TRANSECT/ON
segment
segment
seynent
segnent
I
FIG. 1. Schematic diagram illustrating the definition used in this study of proximal and distal vessel segments. Arrows indicate the flow direction.
Intravital microscope. The time between transection and the first arrest of haemorrhage is measured and called the primary haemostatic plug formation time (PHT). When a vesselis transected there are two vesselsegments,the positions of which are shown in Fig. 1. Blood flow is reversedafter transection in distal arteriolar and proximal venular
FIG. 2. Ground glass screen with photodiode holder positioned over an arteriole (-45 pm) measuring the centerline velocity. At the end of the transected vessel an incomplete haemostatic plug can be seen.
24
ARFORS
AND
BERGQVIST
segments. The following number of vessels were studied: 14 proximal arteriolar segments, 12 distal arteriolar segments. 12 proximal venular segments, and I6 distal venular segments. VeZocit~ measurement. The technique to measure red blood cell velocity in the microvasculature has been described by Intaglietta et al. (1970). This method is based on the dual-slit technique of Wayland and Johnson (1967), who measured the delay between signals that originate from two photodetectors when an image of the blood
FIG. 3. A diagram of the ocular mounted ground glassscreen,front view (cf. Fig. 1) and side view. 1. Adjustable photodiode holder, 2. Chip carrying photodiodesfacing ocular. 3. Ocular x 10. stream is projected onto them. The detectors are fixed with a known separation in the direction of flow. While measuring flow in the mesentery, peristaltic movements sometimes make it necessary to rapidly readjust the position of the two photodetectors. We have, therefore, developed a simple photodiode holder mounted inside a ground glass screen (Figs. 2 and 3), on which the image from the mesentery is projected through an ocular (Arfors et aE., 1973). The detectors used were two photodiodes (SP 50, thin film substrate: Hafo, Stockholm, Sweden) mounted with a 1.0 mm center to center
HAEMOSTATIC
PLUGS
OF MICROVASCULATL’RE
35 i.
separation. The optical signal pattern of the passing red cells was continuously cross correlated (Hewlett-Packard Correlator 3721 A, Palo Alto, California) to determine
the transit time (t) of cells passing from one diode to the other. The distance between the diodes is known (s), and the velocity was computed on-line according to the formula s!t = L’.The resulting axial red blood cell velocity (c) was continuously recorded on a Hewlett-Packard Recorder (Mosley Autograph 680 M, Hewlett-Packard, California). Haemostaticplug w&me and vesselcontraction. This was measured using time-lapse photography (Beaulieu R 16, Kodachrome II daylight film, Hasselblad Timer, Reflex, Stockholm, Sweden) with an exposure every 5 sec.Analysis from the films was made with a single frame motion analyser (Motion analysis projector, S, Mark 111;John Hadland Ltd., Bovingdon, England). The systemwascalibrated by filming a micrometer slide through the same optical system. When calculating the volume, the haemostatic plug was approximated to the form of an ellipsoid, rotated around its long axis. The perpendicular short (a) and long (b) axes were measuredand the volume (v) calculated according to the formula: v = (47c/3)a2b.The maximum vessel contraction (C) was calculated as a percentage according to C = (D - Dmin)100/D, where D is the diameter before transection and Dminthe diameter at maximum contraction. Platelet counts. These were determined in duplicate on an Improved Neubauer Chamber, according to the method of Brecher and Cronkite (1950). Calculation of theproportion ofplateletsparticipating in theformation qf a haemostatic plug. The total number of platelets that passed(platelet flow) before arrest of bleeding in individual vesselswas obtained by multiplying the integrated blood flow velocity until haemostasisby the platelet count for the particular rabbit. For eachvesselsegment the continuously recorded values for centerline velocity were used to obtain volume flow rate (Q): Q = vIIR2/2, where R is the radius (Baker, 1972). The total number of platelets in the haemostatic plug was calculated by dividing the final plug volume (u) by the volume of individual platelets (5.5 pm3) in the rabbit microcirculation (Arfors e2al., 1974). Individual vesselplatelet aggregability as a percentagewas calculated as platelet number in final plug x 100 platelet flow RESULTS In Table 1 the maximum degree of vesselconstriction during haemostasisis shown. Both arteriolar segments show the same degree of contraction, as do both venular segments,the constriction being approximately three times as great in arterioles. This table also illustrates the linear correlation coefficients for the relationship between the degreeof contraction and the primary haemostatic plug formation time (PHT). There was no correlation in any of the segments. Forty-one percent of the platelets passing the proximal arteriolar orifice after vessel transection participate in the formation of an effective haemostatic plug. The corresponding figures for the other vessel segmentsare: 12% in distal arteriolar segments, 6 % in proximal venular segments,and 3 1% in distal venular segments.These data can also be seenin Fig. 4. Individual vesselplatelet aggregability is significantly higher in plugs where blood is flowing from the arteriolar side of the microcirculation (proximal
26
ARFORS
AND
BERGQVIST
TABLE THE
MAXIMUM DEGREE
I
OF VESSEL CONSTRICTION
DUKIKG
HAEMOSTASIS
Arterioles
Maximum degree of contraction in percent Linear correlation (v) between degree of contraction and PHT PHT” (set)
Venules
proximal segment
distal segment
16.0
18.2
proximal segment 5.8
distal segment 5.5
0.52 NP
0.46 NS
-0.11 NS
0.42 NS
75
458
439
303
a PHT = primary haemostatic plug formation time. b NS = not significant according to the Student’s t test.
FIG. 4. Bar-chart showing the individual platelet aggregability in the four vessel segments; 95% confidence limits are given. AP = proximal arteriolar segment; AD = distal arteriolar segment; VD = distal venular segment; VP = proximal venular segment.
arteriolar and distal venular segments), than where it is flowing from the venular side-proximal venular and distal arteriolar segments (see also Fig. I). DISCUSSION Several authors have described differences in the initial haemostatic processes in arterioles and venules (Apitz, 1942; Chen and Tsai, 1948; Hugues, 1953; Arfors et al., 1972; Bergqvist, 1974) although these differences have not been explained. Previous experimental results have been inconclusive. These physiological variations in haemostatic properties are not reflected morphologically, as judged by ultrastructural studies of haemostatic plugs from arterioles and venules in the rabbit mesentery (Kjaerheim and Hovig, 1962). However, several possible explanations exist (Bergqvist, 1973). Vessel constriction has been considered to be an important factor in haemostasis (Macfarlane, 1941; Apitz, 1942; Tocantins, 1947; Zucker, 1947; Cruz, 1965), but the finding in the distal arteriolar segment of a longer primary haemostatic plug formation
HAEMOSTATIC
PLUGS OF MICROVASCULATURE
27
time than in the proximal arteriolar segmentdespite the samedegreeof posttraumatic contraction in the two vessel segmentssuggeststhat contraction is not an important factor in microvascular haemostasis. The lack of correlation between the degree of contraction and the haemostatic plug formation time further supports this conclusion as does the similar haemostatic tissues between distal arteriolar segmentsand venules despite the much greater contraction in the former (Table 1). In experiments in which xylocaine was added to the superfusatehaemostatic plug formation time was unaltered despitethe abolition of vascular contraction (unpublished observations). It can therefore be concluded that vesselconstriction is of little importance in microcirculatory haemostatic plug formation in the rabbit mesentery. It is noteworthy that despite differences in methodology, Hugues (1953) also came to the conclusion that vascular constriction was not an important determinant of microvascular haemostasis. In an experimental mode1where arteriolar blood flow velocity was reduced, we were able to show that the difference in haemostasis between arterioles and venules could not be explained on the basis of different flow velocities (Arfors and Bergqvist, 1974). This view was supported by analysis of different flow velocity parameters after microvessel transection. Approximate calculations suggested that platelets passing the proximal arteriolar segment participated more actively in forming the haemostatic plug than in the other vessel segments. This finding prompted us to perform this investigation, using a method to measure the individual vessel platelet aggregability. The results are similar to those obtained by our approximate calculations, and suggest that the platelets coming from the arteriolar side of the microcirculation (proximal arteriolar and distal venular segments)are significantly more active than those coming from the venular side. The individual vesselplatelet aggregability is thus higher in the arteriolar blood. Differences in blood fibrinolytic activity and white cell behaviour are not responsible for the haemostatic differences (Bergqvist and Arfors, 1973, 1974a). In rabbits the difference in haemostasis between arterioles and venules tends to be diminished by continuous infusion of adenosine diphosphate (ADP) into the cranial mesenteric artery and by a preceding laser-induced intravascular injury upstream of the transection site of the microvessel (Bergqvist and Arfors, to be published). Several unpublished investigations have given us the impression that haemostatic plug formation may be mainly an ADP-induced platelet aggregation and one possibility is that cells (red cells and platelets) in statesof a high sheardisrupt more easily, releasing a higher concentration of platelet aggregating substances.In this way the cells contribute to a faster haemostasisthan would otherwise be the case.This is in agreement with claims made by Marr et al. (1965) and Johnson et al. (1966) emphasizing the important role of ADP in initial haemostasis-ADP probably released from the red blood cells. Red blood cells contain 90 % of the nucleotides of the blood and platelets the remaining 10% (Johnson et al., 1967). This view is also supported by Jorgensen and Borchgrevink (1964), based on histological studies of skin wounds made for bleeding time tests in normal humans and patients with various bleeding disorders. REFERENCES APITZ, K. (1942). Die Bedeutung der Gerinnung und Thrombose fiir die Blutstillung. Virch. Arch. Pathol. Anat. Physiol. 308, 540. ARFORS, K.-E., AND BERGQVIST, D. (1974). Influence of blood flow velocity on experimental haemostaticplug formation.Throm. Res. 4,447.
28
ARFORS
AND
BERGQVIST
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