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Influence of shear rate on platelet aggregation in cerebral microvessels
MICROVASCULARRESEARCH 23, 311-315 (1982)
Influence of Shear Rate on Platelet Aggregation in Cerebral Microvessels 1 WILLIAM I. ROSENBLUM 2 AND FAROUK EL-SABBAN Department of Pathology (Neuropathology), Medical College of Virginia, Richmond, Virginia 23298 Received April 2, 1981 The aggregation of platelets in injured cerebral arterioles was delayed as the wall shear rate rose, over a range of 0.2 to 1.4 s e c - ~. The shear rates were calculated from centerline velocities determined with the two-slit technique and a cross correlator. The velocity range was 2 to 13 m m / s e c . The correlation between aggregation latency and shear rate was 0.44, P < 0.02, and 0.55, P < 0.05, in each of two studies. The correlation between aggregation latency and centerline velocity was slightly less and was slightly less significant than the correlation between shear rate and aggregation latency.
The physical factors affecting platelet adherence to and aggregation on blood vessel walls have been investigated by several groups using in vitro test systems (1,2). These factors and their interaction are highly complex, and include the velocity or shear rate of blood and the numbers of platelets delivered to the wall per unit time (1,2). To our knowledge, in vivo studies of the relationship between shear rate or velocity and platelet aggregation have only been reported by Begent and Born (3) who observed microvessels of the hamster cheek pouch and used the velocity of platelet emboli as the measure of blood flow velocity within the microvessel. We have measured the time required to induce recognizable aggregation within small arterioles on the cerebral surface (pial arterioles) and we have measured red blood cell (RBC) velocity within the same vessels just before onset of aggregation. From the velocity data we have calculated the shear rate at the vessel wall for each observed aggregation. Such calculations of shear rate cannot be made from the data presented by Begent and Born. Our velocity and shear rate values, together with their relationship to the ease with which platelet aggregates could be produced, are presented below. METHODS Aggregate induction. Platelet aggregates were induced in pial arterioles of male ICR mice as previously described (4,5). Briefly stated tracheotomy and craniotomy are performed in mice anesthetized with urethane, and the pial arterioles are observed with a Leitz Ultropak microscope equipped with a 22 x Supported by Public Health Service Grant HL-23560. 2 Reprint requests to Dr. R o s e n b l u m , Box 17, M C V Station, Richmond, VA. 23298. 311 0026-2862/82/03031!-05502.00/0 Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
312
ROSENBLUM AND EL-SABBAN
objective and dipping cone. As described in earlier publications, platelet aggregates are produced by injuring endothelium, to which they adhere (6). The injury is produced (6) by exposing the microscopic field to light from an appropriately filtered mercury lamp, after injecting the mice intravenously with 0.2 ml of 2% sodium fluorescein per 25 g body wt. The filters were a Leitz KG-1 and a Leitz dichroic reflecting filter which together exclude virtually all wavelengths above 800 and below 350 lambda. In addition a Leitz BG-12 exciter filter is used with peak transmission at 400 lambda. The fluorescein is excited at 475 lambda. Neither the light alone nor the fluorescein damages the endothelium or induces aggregation, but the combination produces aggregates which are readily recognized because they fluoresce (7). The time from the onset of the noxious stimulus (light plus dye) to the recognition of the first aggregate is recorded with a stopwatch. Measurement of RBC velocity. This is done with a two-slit velocimeter and cross correlator manufactured by IPM (San Diego, Calif.). The theoretical bases underlying velocity measurement with the two-slit technique and the use of the correlator have been thoroughly documented by others (8-11). The recording of RBC velocity from pial arterioles was performed with the aid of a TV microscope permitting us to align the photodiodes of the velocimeter longitudinally along the midline of the vessel. This and the measurement of RBC velocity were performed wh.en the vessel was illuminated by the same mercury lamp and regulated dc power supply as was used to induce platelet aggregation. However, a light-green filter was inserted into the light path rather than the BG-12 blue filter used during the induction of platelet aggregation. Immediately after the measurement of RBC velocity, the blue filter was reinserted, the fluorescein was injected via the tail vein, and the stopwatch was started in order to measure the time elapsing before the combination of light plus dye produced aggregation. The RBC velocity at the vessel center was converted to shear rate at the wall by the well-known formula, shear rate = (8 x velocity)/(1.6 x diameter). The diameters were measured at the time of the velocity measurement, utilizing an ocular micrometer. All vessels had an internal diameter between 20 and 50 Ixm. RESULTS Two studies are reported. In the first study 30 mice were used, one pial arteriole per mouse. In the second, 14 animals and 14 arterioles were used but 5 min before measuring RBC velocity and inducing aggregation these animals were injected subcutaneously with varying amounts of vasopressin and norepinephrine in order to raise their blood pressures and hence to increase their RBC velocities. The purpose of the second study was to increase the range of velocity values over which our observations of platelet aggregation were made. The data from study 1 are shown in Fig. 1. There was a significant positive correlation between shear rate and the number of seconds required to initiate aggregation. There was also a significant correlation (y = 8.4x + 8, r = 0.39, P = 0.03) between velocity (x) and time (y) required to initiate aggregation in this study. In study 2, there was again a significant linear correlation (y -- 18x + 23, r = 0.55, P = 0.04) between shear rate (x) and the number of seconds (y) required to initiate aggregation, and a less significant relationship between
313
SHEAR RATE AND PLATELET AGGREGATION
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IO-3xSHEAR RATE AT WALL (sec - I ) FIG. 1. The data show a linear relationship between shear rate at the wall and the time required for aggregates to appear in cerebral arterioles damaged by a noxious stimulus.
velocity (x) and aggregation time (y) (y = 1.9x + 25, r = 0.48, P = 0.08). The mean velocity in study 1 was 6 _+ 2 mm/sec and in study 2 was 7 + 3 mm/sec (M _+ SD). Thus we were only marginally successful in increasing the velocities in group 2, probably because after the initial rise in blood pressure which followed the injection of the pressor drugs, the pressure rapidly returned to basal levels. DISCUSSION The interpretation of our data is complex because of the complex nature of the phenomena themselves. Perhaps the most important practical point is, that with this particular method of inducing platelet aggregation there is a slight but definite direct relationship between shear rate or RBC velocity, and the time required to initiate an adherent platelet aggregate. Consequently, one must display caution when comparing aggregation in differently treated groups of animals lest changes in aggregation time be mistakenly ascribed to effects on platelets or vessel wall when really caused by systematic differences in shear rates or velocities. A similar conclusion can be drawn from the in vivo studies of Begent and Born (3) in which iontophoretic application of ADP was used to induce platelet aggregates in venules of the hamster cheek pouch, and the growth rate
314
ROSENBLUM AND EL-SABBAN
of aggregates increased as velocity increased from 0.0 to 0.4 mm/sec. Above 0.4 mm/sec the growth rate sharply declined, and above 2.5 mm/sec no aggregates could be produced. Our own data fail to show such a biphasic relationship between the ease with which aggregation was induced and either a rising flow velocity or a rising shear rate. Indeed, a linear relationship was found between the time required to produce aggregation and the velocity or shear rate. This may be because all the velocities in our study were many times greater than 0.4 mm/sec and our arteriolar diameters were smaller than the 40- to 70-~m diameters of the venules observed by Begent and Born, so that the shear rates in our studies are very much higher than those in their studies. The absence of extremely low velocities and shear rates from our study may thus account for our failure to find a velocity range in which aggregation was favored rather than discouraged by increasing velocities within that range. Nevertheless both their work and our own suggest that it would be beneficial to have measurements of velocity and shear in studies of platelet aggregation in the microcirculation. The fact that Begent and Born failed to induce aggregates at velocities greater than 3 mm/sec while we can produce them at velocities of 10 mm/sec and more, probably reflects different methodologies used to initiate aggregation and the much greater endothelial injury induced by our technique (3, 6). Indeed the question of endothelial damage creates one of the complexities in the interpretation of our results. We have previously documented endothelial defects and exposed basement membrane (6) caused by our technique of producing aggregation. Continuing electron microscopic studies indicate lesser amounts of damage as well and the sequence of morphologic changes leading to aggregation still requires clarification. It is possible that a substance(s) released from injured vessels is important in initiating aggregation and is simply diluted at higher velocities, thereby accounting for the greater delay in aggregation at increasing velocities and shear rates. However, current concepts emphasize the antiaggregating substances such as prostacyclin which are released by vessel walls (12) and thus it seems unlikely that a more rapid carrying away of released agents would increase the latency of aggregation. Moreover, the technique of Begent and Born (3) is not thought to injure vessels, yet they also observe a decreasing tendency toward aggregation as velocities rise above 0.4 mm/sec. It seems most likely that our data, like theirs, are due at least in part to the fact that as velocity and shear rate increases, it becomes more difficult for platelets to adhere to a vessel wall or to each other. At the same time, in the study of Begent and Born, where no injury is said to occur, and where a chemical that causes aggregation is being locally deposited in the vascular lumen, it is possible that reduced aggregation at higher velocities, was a simple reflection of dissipation of the drug. When describing results such as ours or those of Begent and Born, the question occurs as to whether shear rate or RBC velocity is the more appropriate parameter against which to look at aggregation. In vessels of less than 100-~m diameter, blood becomes increasingly heterogeneous, so that some workers may feel that terms like shear rate and viscosity lose their meaning. However, studies of pressure and flow in microvessels indicate that (13) the microvascular bed can be treated as if blood were a homogeneous fluid, provided a correction is made for the true hematocrit. Moreover, our data show a better correlation between shear rate and aggregation latency, than between velocity and latency.
SHEAR RATE AND PLATELET AGGREGATION
315
T h i s s u g g e s t s t h a t s h e a r f o r c e s a n d n o t j u s t flow v e l o c i t y a r e i m p o r t a n t in t h e d e t e r m i n a t i o n o f a g g r e g a t i o n l a t e n c y , a n d t h a t s h e a r r a t e at t h e w a l l is a u s e f u l p a r a m e t e r in t h e c o n t e x t o f o u r s t u d i e s . Finally, our studies indicated that a linear relationship between velocity or s h e a r r a t e , a n d a g g r e g a t i o n l a t e n c y , e x i s t e d in a p o p u l a t i o n o f m i c e i n j e c t e d w i t h p r e s s o r d r u g s . T h e s l o p e o f this line w a s l o w e r t h a n t h a t o f t h e line o b t a i n e d w i t h u n t r e a t e d m i c e , b u t w a s n o t significantly different. It is p o s s i b l e t h a t t h e d r u g s t h e m s e l v e s a l t e r e d a g g r e g a t i o n in s o m e w a y , b u t t h e f a c t t h a t a l i n e a r relationship existed between shear rate or velocity and latency, does not seem r e a s o n a b l y a s c r i b e d to t h e d r u g s in v i e w o f the l i n e a r r e l a t i o n s h i p a l s o e x i s t i n g f o r u n t r e a t e d m i c e . T h i s is p a r t i c u l a r l y so, s i n c e t h e d r u g - t r e a t e d m i c e s h o w e d a closer relationship between shear rate and latency, than between velocity and l a t e n c y , a p o i n t w h i c h o n c e again e m p h a s i z e s the role o f p h y s i c a l f a c t o r s in m o d i f y i n g a g g r e g a t i o n in the m i c r o c i r c u l a t i o n . REFERENCES 1. TURITTO, V. T., WEISS, H. J., AND BAUr~GARTNER,H. R. (1980). The effect of shear rate on platelet interaction with subendothelium exposed to citrated human blood. Microvasc. Res. 19, 352-365. 2. DOSNE,A. M., MERVlLLE,C., DROUET,L., ANTONINI,G., GUIFFANT,G., ANDQUEMODA,D. (1977). Importance of transport mechanisms in circulating blood for platelet deposition on arterial subendothelium. Microvasc. Res. 14, 45-52. 3. BEGENT, N., AND BORN, G. V. R. (1970). Growth rate in vivo of platelet thrombi, produced by iontophoresis of ADP, as a function of mean blood flow velocity. Nature 227, 926-930. 4. ROSENBLUM,W. I., AND ZWEIFACH,B. W. (1963). Cerebral microcirculation in the mouse brain. Arch. Neurol. 9, 414-423. 5. ROSENBLUM, W. I. (1976). Pial arteriolar responses in the mouse brain, revisited. Stroke 7, 283-287. 6. ROSENBLUM,W. I., AND EL-SABBAN,F. (1977). Platelet aggregation in the cerebral microcirculation. Effect of aspirin and other agents. Circ. Res. 40, 320-328. 7. ROSENBLUM, W. 1. (1978). Fluorescence induced in platelet aggregates as a guide to luminal contours in the presence of platelet aggregation. Microvasc. Res. 15, 103-106. 8. WAYLANO,H., AND JOHNSON, P. C. (1967). Erythrocyte velocity measurement in microvessels by a two-slit photometric method. J. Appl. Physiol. 22, 333-337. 9. BAKER, M., AND WAVLAND,H. (1974). On-line volumetric flow rate and velocity profile measurement for blood in microvessels. Microvasc. Res. 7, 131-143. 10. TOMPKINS,W, R., MONTX,R., ANOINTAGLIETTA,M. (1974). Velocity measurements by self-tracking correlator. Res. Sci. Inst. 45, 647-649. 11. LIPOWSKV,H., ANDZWEIFACH,B. W. (1978), Application of the "two-slit" photometric technique to measurement of microvascular volumetric flow rates. Microvasc. Res. 15, 93-101. 12. MONCADA,S., ANDVANE,J. R. (1979). Arachidonic acid metabolites and the interactions between platelets and blood vessel walls. N. Engl. J. Med. 300, 1142-1147. 13. LlvowsKv, H. H., USAMI,S., ANDCmEN, S. (1980). In vivo measurements of"apparent viscosity" and the microvessel hematocrit in the mesentery of the cat. Microvasc. Res. 19, 297-319.
Influence of Shear Rate on Platelet Aggregation in Cerebral Microvessels 1 WILLIAM I. ROSENBLUM 2 AND FAROUK EL-SABBAN Department of Pathology (Neuropathology), Medical College of Virginia, Richmond, Virginia 23298 Received April 2, 1981 The aggregation of platelets in injured cerebral arterioles was delayed as the wall shear rate rose, over a range of 0.2 to 1.4 s e c - ~. The shear rates were calculated from centerline velocities determined with the two-slit technique and a cross correlator. The velocity range was 2 to 13 m m / s e c . The correlation between aggregation latency and shear rate was 0.44, P < 0.02, and 0.55, P < 0.05, in each of two studies. The correlation between aggregation latency and centerline velocity was slightly less and was slightly less significant than the correlation between shear rate and aggregation latency.
The physical factors affecting platelet adherence to and aggregation on blood vessel walls have been investigated by several groups using in vitro test systems (1,2). These factors and their interaction are highly complex, and include the velocity or shear rate of blood and the numbers of platelets delivered to the wall per unit time (1,2). To our knowledge, in vivo studies of the relationship between shear rate or velocity and platelet aggregation have only been reported by Begent and Born (3) who observed microvessels of the hamster cheek pouch and used the velocity of platelet emboli as the measure of blood flow velocity within the microvessel. We have measured the time required to induce recognizable aggregation within small arterioles on the cerebral surface (pial arterioles) and we have measured red blood cell (RBC) velocity within the same vessels just before onset of aggregation. From the velocity data we have calculated the shear rate at the vessel wall for each observed aggregation. Such calculations of shear rate cannot be made from the data presented by Begent and Born. Our velocity and shear rate values, together with their relationship to the ease with which platelet aggregates could be produced, are presented below. METHODS Aggregate induction. Platelet aggregates were induced in pial arterioles of male ICR mice as previously described (4,5). Briefly stated tracheotomy and craniotomy are performed in mice anesthetized with urethane, and the pial arterioles are observed with a Leitz Ultropak microscope equipped with a 22 x Supported by Public Health Service Grant HL-23560. 2 Reprint requests to Dr. R o s e n b l u m , Box 17, M C V Station, Richmond, VA. 23298. 311 0026-2862/82/03031!-05502.00/0 Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
312
ROSENBLUM AND EL-SABBAN
objective and dipping cone. As described in earlier publications, platelet aggregates are produced by injuring endothelium, to which they adhere (6). The injury is produced (6) by exposing the microscopic field to light from an appropriately filtered mercury lamp, after injecting the mice intravenously with 0.2 ml of 2% sodium fluorescein per 25 g body wt. The filters were a Leitz KG-1 and a Leitz dichroic reflecting filter which together exclude virtually all wavelengths above 800 and below 350 lambda. In addition a Leitz BG-12 exciter filter is used with peak transmission at 400 lambda. The fluorescein is excited at 475 lambda. Neither the light alone nor the fluorescein damages the endothelium or induces aggregation, but the combination produces aggregates which are readily recognized because they fluoresce (7). The time from the onset of the noxious stimulus (light plus dye) to the recognition of the first aggregate is recorded with a stopwatch. Measurement of RBC velocity. This is done with a two-slit velocimeter and cross correlator manufactured by IPM (San Diego, Calif.). The theoretical bases underlying velocity measurement with the two-slit technique and the use of the correlator have been thoroughly documented by others (8-11). The recording of RBC velocity from pial arterioles was performed with the aid of a TV microscope permitting us to align the photodiodes of the velocimeter longitudinally along the midline of the vessel. This and the measurement of RBC velocity were performed wh.en the vessel was illuminated by the same mercury lamp and regulated dc power supply as was used to induce platelet aggregation. However, a light-green filter was inserted into the light path rather than the BG-12 blue filter used during the induction of platelet aggregation. Immediately after the measurement of RBC velocity, the blue filter was reinserted, the fluorescein was injected via the tail vein, and the stopwatch was started in order to measure the time elapsing before the combination of light plus dye produced aggregation. The RBC velocity at the vessel center was converted to shear rate at the wall by the well-known formula, shear rate = (8 x velocity)/(1.6 x diameter). The diameters were measured at the time of the velocity measurement, utilizing an ocular micrometer. All vessels had an internal diameter between 20 and 50 Ixm. RESULTS Two studies are reported. In the first study 30 mice were used, one pial arteriole per mouse. In the second, 14 animals and 14 arterioles were used but 5 min before measuring RBC velocity and inducing aggregation these animals were injected subcutaneously with varying amounts of vasopressin and norepinephrine in order to raise their blood pressures and hence to increase their RBC velocities. The purpose of the second study was to increase the range of velocity values over which our observations of platelet aggregation were made. The data from study 1 are shown in Fig. 1. There was a significant positive correlation between shear rate and the number of seconds required to initiate aggregation. There was also a significant correlation (y = 8.4x + 8, r = 0.39, P = 0.03) between velocity (x) and time (y) required to initiate aggregation in this study. In study 2, there was again a significant linear correlation (y -- 18x + 23, r = 0.55, P = 0.04) between shear rate (x) and the number of seconds (y) required to initiate aggregation, and a less significant relationship between
313
SHEAR RATE AND PLATELET AGGREGATION
200180" y'69x
+14
r-0.44 160
•
p-.OI6
-
Z 0
<~ 1400 bJ nO
0 ,,:[
120-
bJ
_~ IO0Z m
go
o
so
(g) e-, Z o
60
,.o,
•
•
(/)
ee
•
•
40
20
I
i
I
.2
.4
.6
~ .
!
I
!
1.0
1,2
1.4
IO-3xSHEAR RATE AT WALL (sec - I ) FIG. 1. The data show a linear relationship between shear rate at the wall and the time required for aggregates to appear in cerebral arterioles damaged by a noxious stimulus.
velocity (x) and aggregation time (y) (y = 1.9x + 25, r = 0.48, P = 0.08). The mean velocity in study 1 was 6 _+ 2 mm/sec and in study 2 was 7 + 3 mm/sec (M _+ SD). Thus we were only marginally successful in increasing the velocities in group 2, probably because after the initial rise in blood pressure which followed the injection of the pressor drugs, the pressure rapidly returned to basal levels. DISCUSSION The interpretation of our data is complex because of the complex nature of the phenomena themselves. Perhaps the most important practical point is, that with this particular method of inducing platelet aggregation there is a slight but definite direct relationship between shear rate or RBC velocity, and the time required to initiate an adherent platelet aggregate. Consequently, one must display caution when comparing aggregation in differently treated groups of animals lest changes in aggregation time be mistakenly ascribed to effects on platelets or vessel wall when really caused by systematic differences in shear rates or velocities. A similar conclusion can be drawn from the in vivo studies of Begent and Born (3) in which iontophoretic application of ADP was used to induce platelet aggregates in venules of the hamster cheek pouch, and the growth rate
314
ROSENBLUM AND EL-SABBAN
of aggregates increased as velocity increased from 0.0 to 0.4 mm/sec. Above 0.4 mm/sec the growth rate sharply declined, and above 2.5 mm/sec no aggregates could be produced. Our own data fail to show such a biphasic relationship between the ease with which aggregation was induced and either a rising flow velocity or a rising shear rate. Indeed, a linear relationship was found between the time required to produce aggregation and the velocity or shear rate. This may be because all the velocities in our study were many times greater than 0.4 mm/sec and our arteriolar diameters were smaller than the 40- to 70-~m diameters of the venules observed by Begent and Born, so that the shear rates in our studies are very much higher than those in their studies. The absence of extremely low velocities and shear rates from our study may thus account for our failure to find a velocity range in which aggregation was favored rather than discouraged by increasing velocities within that range. Nevertheless both their work and our own suggest that it would be beneficial to have measurements of velocity and shear in studies of platelet aggregation in the microcirculation. The fact that Begent and Born failed to induce aggregates at velocities greater than 3 mm/sec while we can produce them at velocities of 10 mm/sec and more, probably reflects different methodologies used to initiate aggregation and the much greater endothelial injury induced by our technique (3, 6). Indeed the question of endothelial damage creates one of the complexities in the interpretation of our results. We have previously documented endothelial defects and exposed basement membrane (6) caused by our technique of producing aggregation. Continuing electron microscopic studies indicate lesser amounts of damage as well and the sequence of morphologic changes leading to aggregation still requires clarification. It is possible that a substance(s) released from injured vessels is important in initiating aggregation and is simply diluted at higher velocities, thereby accounting for the greater delay in aggregation at increasing velocities and shear rates. However, current concepts emphasize the antiaggregating substances such as prostacyclin which are released by vessel walls (12) and thus it seems unlikely that a more rapid carrying away of released agents would increase the latency of aggregation. Moreover, the technique of Begent and Born (3) is not thought to injure vessels, yet they also observe a decreasing tendency toward aggregation as velocities rise above 0.4 mm/sec. It seems most likely that our data, like theirs, are due at least in part to the fact that as velocity and shear rate increases, it becomes more difficult for platelets to adhere to a vessel wall or to each other. At the same time, in the study of Begent and Born, where no injury is said to occur, and where a chemical that causes aggregation is being locally deposited in the vascular lumen, it is possible that reduced aggregation at higher velocities, was a simple reflection of dissipation of the drug. When describing results such as ours or those of Begent and Born, the question occurs as to whether shear rate or RBC velocity is the more appropriate parameter against which to look at aggregation. In vessels of less than 100-~m diameter, blood becomes increasingly heterogeneous, so that some workers may feel that terms like shear rate and viscosity lose their meaning. However, studies of pressure and flow in microvessels indicate that (13) the microvascular bed can be treated as if blood were a homogeneous fluid, provided a correction is made for the true hematocrit. Moreover, our data show a better correlation between shear rate and aggregation latency, than between velocity and latency.
SHEAR RATE AND PLATELET AGGREGATION
315
T h i s s u g g e s t s t h a t s h e a r f o r c e s a n d n o t j u s t flow v e l o c i t y a r e i m p o r t a n t in t h e d e t e r m i n a t i o n o f a g g r e g a t i o n l a t e n c y , a n d t h a t s h e a r r a t e at t h e w a l l is a u s e f u l p a r a m e t e r in t h e c o n t e x t o f o u r s t u d i e s . Finally, our studies indicated that a linear relationship between velocity or s h e a r r a t e , a n d a g g r e g a t i o n l a t e n c y , e x i s t e d in a p o p u l a t i o n o f m i c e i n j e c t e d w i t h p r e s s o r d r u g s . T h e s l o p e o f this line w a s l o w e r t h a n t h a t o f t h e line o b t a i n e d w i t h u n t r e a t e d m i c e , b u t w a s n o t significantly different. It is p o s s i b l e t h a t t h e d r u g s t h e m s e l v e s a l t e r e d a g g r e g a t i o n in s o m e w a y , b u t t h e f a c t t h a t a l i n e a r relationship existed between shear rate or velocity and latency, does not seem r e a s o n a b l y a s c r i b e d to t h e d r u g s in v i e w o f the l i n e a r r e l a t i o n s h i p a l s o e x i s t i n g f o r u n t r e a t e d m i c e . T h i s is p a r t i c u l a r l y so, s i n c e t h e d r u g - t r e a t e d m i c e s h o w e d a closer relationship between shear rate and latency, than between velocity and l a t e n c y , a p o i n t w h i c h o n c e again e m p h a s i z e s the role o f p h y s i c a l f a c t o r s in m o d i f y i n g a g g r e g a t i o n in the m i c r o c i r c u l a t i o n . REFERENCES 1. TURITTO, V. T., WEISS, H. J., AND BAUr~GARTNER,H. R. (1980). The effect of shear rate on platelet interaction with subendothelium exposed to citrated human blood. Microvasc. Res. 19, 352-365. 2. DOSNE,A. M., MERVlLLE,C., DROUET,L., ANTONINI,G., GUIFFANT,G., ANDQUEMODA,D. (1977). Importance of transport mechanisms in circulating blood for platelet deposition on arterial subendothelium. Microvasc. Res. 14, 45-52. 3. BEGENT, N., AND BORN, G. V. R. (1970). Growth rate in vivo of platelet thrombi, produced by iontophoresis of ADP, as a function of mean blood flow velocity. Nature 227, 926-930. 4. ROSENBLUM,W. I., AND ZWEIFACH,B. W. (1963). Cerebral microcirculation in the mouse brain. Arch. Neurol. 9, 414-423. 5. ROSENBLUM, W. I. (1976). Pial arteriolar responses in the mouse brain, revisited. Stroke 7, 283-287. 6. ROSENBLUM,W. I., AND EL-SABBAN,F. (1977). Platelet aggregation in the cerebral microcirculation. Effect of aspirin and other agents. Circ. Res. 40, 320-328. 7. ROSENBLUM, W. 1. (1978). Fluorescence induced in platelet aggregates as a guide to luminal contours in the presence of platelet aggregation. Microvasc. Res. 15, 103-106. 8. WAYLANO,H., AND JOHNSON, P. C. (1967). Erythrocyte velocity measurement in microvessels by a two-slit photometric method. J. Appl. Physiol. 22, 333-337. 9. BAKER, M., AND WAVLAND,H. (1974). On-line volumetric flow rate and velocity profile measurement for blood in microvessels. Microvasc. Res. 7, 131-143. 10. TOMPKINS,W, R., MONTX,R., ANOINTAGLIETTA,M. (1974). Velocity measurements by self-tracking correlator. Res. Sci. Inst. 45, 647-649. 11. LIPOWSKV,H., ANDZWEIFACH,B. W. (1978), Application of the "two-slit" photometric technique to measurement of microvascular volumetric flow rates. Microvasc. Res. 15, 93-101. 12. MONCADA,S., ANDVANE,J. R. (1979). Arachidonic acid metabolites and the interactions between platelets and blood vessel walls. N. Engl. J. Med. 300, 1142-1147. 13. LlvowsKv, H. H., USAMI,S., ANDCmEN, S. (1980). In vivo measurements of"apparent viscosity" and the microvessel hematocrit in the mesentery of the cat. Microvasc. Res. 19, 297-319.