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Department of Human Physiology, School of Medicine, University of California, Davis, California 95616
Submitted 21 March 2003 ; accepted in final form 30 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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kinase; endothelium; rat; mesentery; inflammation; platelet activating factor
Because we were interested in direct comparison of the mechanisms, which increase permeability in cultured monolayers and in intact endothelial barriers, the initial aim of our experiments was to measure the effect of thrombin on the permeability of the walls of individually perfused venular microvessels in rat mesentery where the direct effect of thrombin on the permeability of the wall could be separated from hemodynamic changes. In preliminary experiments, we found that direct vascular exposure to thrombin did not increase permeability. We also showed that inhibition of the GTPase Rho A, one component of the signaling pathway linking the thrombin PAR-1 receptors to increased permeability in cultured endothelial cells, did not modulate changes in permeability in intact rat and mice microvessels (1). Finally, we noted that components of the Rho pathways and thrombin receptors were upregulated in other cell types after injury (11, 14). This observation laid the basis for the current experiments. We argued that the response to thrombin in microvascular endothelial cells might change after cells were "injured" by being isolated and cultured or by exposure to inflammatory conditions or injury in intact vessels. Thus the specific aim of the present experiments was to test the hypothesis that prior exposure of microvessels to inflammatory stimuli or injury by cannulation and perfusion modified the response of the endothelial cells to thrombin and possibly other directly acting acute inflammatory agents.
We designed experiments to test whether the endothelial cells in rat venular microvessels, which do not respond to the serum protease thrombin by increasing microvascular permeability when first cannulated, perfused, and exposed directly to thrombin, do respond to thrombin after the same microvessels are retested close to 24 h after being replaced in the animal. To do this, we have modified techniques using individual microvessels in rat mesentery, which are cannulated and perfused with solutions of known composition. With these modifications, the permeability response, measured as hydraulic conductivity, of single perfused venules after exposure to thrombin can be measured on two successive days. In particular, we describe experiments below on microvessels cannulated and perfused under aseptic conditions on day 1. The micropipette is then removed from the microvessels and blood flow allowed to resume. On day 2 the same vessel is located in the mesentery, recannulated and perfused to measure a new baseline permeability, and then exposed to thrombin. We (4, 5) previously used this approach in frog mesenteric microvessels to investigate both the acute response on the microvessels to VEGF (day 1) and the longer term effects of this exposure to increase permeability during the subsequent 24–72 h.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Each rat was anesthetized as above and placed on a heating pad to maintain normal body temperature. A midline surgical incision (2–3 cm) was made in the abdominal wall, and the mesentery was gently taken out from the abdominal cavity and spread over a quartz pillar for hydraulic conductivity (Lp) measurements. The upper surface of the mesentery was continuously superfused with Ringer solution maintained at 37°C during preparation and experimentation. Experiments were performed on straight nonbranched segments of venules typically 30–45 µm diameter. Before cannulation, all vessels selected for experiments had brisk blood flow and were free of leukocytes sticking or rolling on the vessel wall.
For 2-day experiments, rats were prepared as above on day 1 with the use of aseptic techniques developed in association with our institutional veterinarian in accordance with the Animal Use and Care Committee Policy Statement on Survival Surgery in Rodents. At the end of the day 1 protocol, the perfusion pipette was removed, blood flow through the venule was restored, a map of the local microvasculature was drawn, and the intact vessel in the rat mesentery returned to the rat peritoneal cavity. The abdominal wall was sutured closed and the animal allowed to recover from anesthesia and returned to the cage overnight. The next day, the animal was anesthetized and the incision opened, the gut spread, and the same vessel was located in the mesentery.
Measurement of Lp. All measurements were based on the modified Landis technique, which measures the volume flux of water across the wall of a vessel perfused via a glass micropipette after occlusion of the vessel. Assumptions and limitations of the technique have been evaluated in detail (16). The initial transcapillary water flow per unit area of the capillary wall (Jv/S)0 was measured at predetermined capillary pressures of 30–60 cmH2O. Venule Lp was calculated as the slope of the relation between (Jv/S)0 and applied hydraulic pressure. For most experiments, (Jv/S)0 was estimated from a single occlusion at one hydraulic pressure with the assumption that the net effective pressure determining fluid flow was equal to the applied hydraulic pressure minus 3 cmH2O, the approximate oncotic pressure contributed by the bovine serum albumin (BSA) in all perfusates.
Experimental protocols. For the single-day protocol, each vessel was initially perfused with a control solution containing 10 mg/ml BSA (catalog no. A0281, Sigma-Aldrich) in Ringer solution. Several occlusions during 20–40 min were used to establish a stable control Lp. Then the first pipette was replaced with a pipette containing test perfusate with PAF (10 nM). At the end of 
25-min exposure to PAF the pipette was removed and replaced with a pipette containing the control perfusate. About 40 min after the initial PAF exposure, the perfusate was replaced with a solution containing rat thrombin (10 U/ml) in BSA-Ringer solution. Occlusions were made about every 60 s to monitor changes in (Jv/S)0.
On the first day of the 2-day protocol, vessels were perfused with BSA-Ringer control solution alone, or BSA-Ringer control solution, followed by PAF as a test solution. After 60 min of total perfusion time, the perfusion pipette was removed and the animal recovered. On the second day of the 2-day protocol, the vessels were initially perfused with control BSA-Ringer solution for 30–70 min and (Jv/S)0 was monitored to establish a day 2 control Lp. The control perfusion pipette was then replaced with a test pipette containing rat thrombin (10 U/ml) in BSA-Ringer solution and (Jv/S)0 was monitored for up to 20 min to test the responsiveness to thrombin.
Solutions and reagents. All perfusates were mammalian Ringer solution additionally containing BSA (10 mg/ml). Mammalian Ringer solution was composed of (in mM) 132 NaCl, 4.6 KCl, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, 5.0 NaHCO3, and 20 HEPES. The ratio of acid-HEPES to Na-HEPES was adjusted to achieve a pH of 7.40–7.45. Two stock solutions were prepared in advance and diluted into the final BSA-Ringer solution immediately before use. 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) (catalog no. 511075, CalBiochem) was prepared at 1 mM in ethanol and stored no >2 wk. Thrombin from rat plasma (catalog no. T-5772, Sigma) was diluted to 200 U/ml in Ringer solution and frozen in small aliquots. Both were diluted to working concentrations on the day of use. Sodium nitroprusside was purchased from Sigma (catalog no. S-0501).
Labeling of endothelial clefts with silver. To enable measurement of cell areas, vessels were perfused with AgNO3 using methods previously described (12). Briefly, at the end of perfusion experiments, vessels were cannulated with a micropipette containing AgNO3 for 10 to 20 s. AgNO3 was prepared in a chloride-free Ringer solution with Na gluconate replacing Na chloride isoosmotically. Immediately before the pipette was placed on the tissue, the mesentery superfusate was switched to a chloride-free Ringer solution to prevent silver chloride precipitation. During AgNO3 perfusion, the superfusion with chloride-containing Ringer was resumed. On removal of the AgNO3 pipette, the vessel was recannulated with solution containing BSA (10 mg/ml) in normal Ringer solution. Vessels were occluded with perfusion pressure set at 50 cmH2O, and fixative (2% glutaraldehyde and 4% paraformaldehyde in phosphate-buffered saline) was dripped on the mesentery. After development of silver lines under a bright light, the vessels were imaged with the use of a laser scanning confocal microscope in transmission mode (Zeiss LSM510, x25 lens, 0.8 numerical aperture). Cell areas were measured on the captured images using analysis software from Zeiss.
Analysis and statistics. Lp measurements during the final 5–10 min of control periods were averaged to establish a single value for control Lp for each vessel. Peak Lp values were the single highest measurement recorded after treatment with an inflammatory mediator. Throughout, averaged values were reported as means ± SE. The indicated statistical tests were performed assuming significance for P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Response to thrombin seen 24 h after initial perfusion. Venules of a second group (n = 11) were investigated over a 24-h period. First, on day 1, we established a baseline Lp in the presence of BSA-Ringer over 15–25 min with a mean Lp of 0.9 ± 0.1 x 10–7 cm · s–1 · cmH2O–1, which was not different from that of group 1. Of these venules, six were also perfused with PAF (10 nM) on day 1 to demonstrate inflammatory responsiveness. A map of the microvessels in the mesentery was drawn, the pipette withdrawn, blood flow was allowed to resume, and the venule and mesentery were returned to the peritoneal cavity. On the day after initial perfusion (i.e., after 25–27 h), the mesentery was again exposed, and the same vessel was recannulated and perfused with BSA-Ringer for 30–90 min to establish a day 2 baseline Lp. The venules on day 2 initially had numerous leukocytes in the tissue surrounding the perfused vessel as well as adhering to the walls of the vessel. The initial Lp in all day 2 vessels was high and fell rapidly. This characteristic is shown in both representative vessels of Fig. 2. At the end of BSA-Ringer perfusion, there remained very few leukocytes adhering to the luminal wall and venule Lp reached stable values with a baseline of 4.7 ± 0.9 x 10–7 cm · s–1 · cmH2O–1. On recannulation with a perfusate containing thrombin (10 U/ml), each vessel responded with a transient increase in permeability, which reached a peak in 6 ± 1 min, and then began to return toward the day 2 baseline Lp (Fig. 2). The mean peak Lp in response to thrombin reached 24 ± 4.2 x 10–7 cm · s–1 · cmH2O–1 (Fig. 3B). The two subgroups (5 venules perfused on day 1 with BSA-Ringer only and 6 venules perfused additionally with PAF to test for initial responsiveness on day 1) were not different from one another in reponse to thrombin on day 2. Representative vessels from each subgroup are shown in Fig. 2. The increase in Lp during thrombin exposure was not the result of a nonspecific response to recannulation of the vessel on day 2 as recannulation and perfusion with BSA-Ringer alone, after establishing the stable day 2 baseline Lp, did not increase permeability.
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Change in venule diameter. One assumption in our experiments is that the change in venule response to thrombin reflects a phenotypic change in some or all of the endothelial cells forming the vessel wall. The most apparent change was an increase in vessel diameter on day 2 compared with values on day 1. At a pressure of 50 cmH2O, the mean diameter of day 1 venules was 39 ± 1 µm, whereas the mean diameter of venules on day 2 was 68 ± 3 µm (n = 11; P < 0.001, Wilcoxon matched pairs). A small part of this increase could have been due to changes in the compliance of the vessel's wall, but compliance does not account for most of the change because the diameter of vessels decreased <2 µm when the pressure was reduced by up to 20 cmH2O. Another possiblity was that part of the injury response was venular dilatation seen at 24 h as the measured increase in vessel diameter. We tested a group of vessels for capacity to dilate by perfusion with BSA-Ringer also containing the vasodilator sodium nitroprusside (10 µM). None of the vessels showed a measurable change in diameter (perfusion pressure 50 cmH2O) in up to 30-min perfusion with the vasodilator (n = 5, mean diameter 35 ± 3 µm). Alternatively, the 74% increase in diameter of the day 2 vessels could occur with either an increase in the number of endothelial cells forming the venule wall or an increase in the size of individual cells. We tested the latter idea by labeling the endothelial clefts with silver and measuring the sizes of individual cells. The mean cell area dramatically increased from 705 ± 24 µm2 (n = 32) in noninflamed vessels to 1,230 ± 44 µm2 (n = 28) in vessels examined after 24 h of inflammation (P < 0.001, Mann-Whitney U-test). The mean cell size increased 74%, thus conforming to the hypothesis that at 24 h the increase in venule diameter was accounted for by increased surface area of individual cells and suggesting that the number of cells did not increase (Fig. 4).
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Responsiveness to PAF not significantly increased. As a further test of the changes at 24 h after initial perfusion, we examined the responsiveness of venules to PAF both on day 1 and day 2 using a protocol similar to that used to check for thrombin-induced Lp response (Fig. 5). On day 1, venules (n = 6) were perfused with BSA-Ringer solution for 30–40 min to establish a baseline Lp of 1.2 ± 0.2 x 10–7 cm · s–1 · cmH2O–1. Each venule was then perfused with PAF (10 nM), and the mean peak Lp was 28 ± 9.7 x 10–7 cm · s–1 · cmH2O–1. After 25 min of PAF perfusion (total perfusion time on day 1 of 60–70 min for each venule), the perfusion pipette was removed and the animals were allowed to recover overnight. On day 2 (24–27 h later) venules were perfused with BSA-Ringer for 25–60 min to establish a baseline Lp of 6.0 ± 1.9 x 10–7 cm·s–1·cmH2O–1. The venules were then recannulated with PAF (10 nM), and the mean peak response was 48 ± 10 x 10–7 cm·s–1·cmH2O–1. Although the mean was somewhat higher on day 2 than on day 1, the paired comparison did not reach statistical significance (P = 0.094, Wilcoxon matched pairs). This absence of a significant change in response to inflammatory mediator PAF clearly contrasts the observations with thrombin in which venules change in 24 h from no response to a well-defined transient increase in Lp.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Changes in endothelial cell size and/or number. When we observed that the day 2 vessels were much larger than the control vessels, a key question was whether the increased diameter in response to thrombin was due to changes in endothelial cells already present in the vessel wall on day 1 or to growth of new endothelial cells formed in the wall by cell division. Our observation that the endothelial cells increased in size by 74%, sufficient to account for the measured a 1.7-fold increase in the diameter of the venules on day 2, suggests that the number of new endothelial cells formed by normal cell division was small and that the changed properties of the vessels wall are due to phenotypic change characterized in part by the enlarged endothelial cells. We note, however, that Ezaki and colleagues (8) have recently described increased endothelial cell size and number and increased microvessel diameters in tissues exposed to an infectious agent. Specifically, they examined tracheal microvascular remodeling in an experimental mouse model of chronic inflammation. At 4–7 days after infection with Mycoplasma pulmonis there was a significant increase in the diameter of arterioles, capillaries, and venules in the tracheal microvasculature. Venule diameter increased to 61 µm by day 7, an increase of 76% over noninflamed vessels. This was correlated with an increase in endothelial cell proliferation that also peaked at 7 days. Importantly, Ezaki et al. (8) also noted a minor population of enlarged endothelial cells, up to twice the size of normal endothelial cells. Thus the increased venular diameters and areas of individual endothelial cells measured in our present study may represent an early response similar to the early stages of the airway inflammation. If so, then we would predict that at longer times than 24 h, we may see increased endothelial proliferation.
Although leakage of Monastral blue was noted in the inflamed tracheal tissues, Ezaki et al. (8) were unable to positively correlate increased permeability using Evans blue-labeled albumin with characteristics of the remodeled microvasculature. This may require a more detailed investigation of permeability of individual vessels to water and solutes using the methods to measure both permeability and microvessel ultrastructure available in our laboratory (1). Bates (3) noted in his study of venular permeability at 24 h after VEGF perfusion that whereas Lp was greatly increased, the effective oncotic pressure of serum albumin was not decreased, implying that solute permeability was not likely changed. Therefore, we may be able in future experiments to examine separate consequences for large solute, small solute, and hydraulic pathways in response to chronic inflammation.
We also note that these investigations may provide new ways to understand differences between responses of endothelial cells in intact microvessels and the responses in culture to some inflammatory agents. For example, many of the cells in cultured endothelial monolayers have recently undergone mitosis, and some cells may still be dividing. Also, some of the conditions encountered during preparation for culture (scraping of endothelial cells to remove from the extracellular matrix and/or exposure to digestive enzymes) may inflict mild injury, sufficient to stimulate mechanisms similar to those due to an acute inflammatory response (including transient increase in cytoplasmic calcium). We cannot say that all cultured cells undergo these changes as they may depend on many additional factors (e.g., site of origin of the endothelial cells, the nature of the substrate on which they are grown, the number of passages before being exposed to the agents and others), but in the light of the long-standing difficulties of correlating responses in intact microvessels with responses in cultured endothelial cells, it is reasonable to test whether some of these mechanisms may bias the response of cultured endothelial cells toward a postinjury phenotype.
Leukocyte attachment. It is important to emphasize that the vessels studied on day 2 were perfused with the rats blood between the initial perfusion on day 1 and the recannulation on day 2. Thus the vessels were exposed to all circulating plasma components and inflammatory cells. All vessels cannulated and perfused in frog and rat mesenteric vessels have leukocytes attached when reexposed on day 2. Many but not all of the attached leukocytes are washed away rapidly (within 1–2 min) after cannulation. There were also migrated cells in the tissue. The initial Lp was high but fell rapidly during the vehicle perfusion and reached a stable value in most vessels by 40 min. It is possible that part of the high permeability seen 24 h after initial cannulation and perfusion is due to a leukocyte-mediated permeability response. This initially high day 2 Lp may be part of a chronic inflammatory state, and the decrease in Lp during vehicle perfusion may be associated with the washing away of leukocytes. However, in our present experiments we cannot attribute the fall in Lp to specific mechanisms. In some of the investigated vessels, the day 1 occlusion site or cannulation site was included within the vessel segment investigated on day 2 and we cannot exclude the possibility that a portion of the initially high Lp is an artifact of direct local damage from the day 1 procedure. Nonetheless, in all vessels, we eventually established the stable day 2 baseline Lp with a mean value about threefold higher than the day 1 baseline (see Fig. 2) during 40 min of vehicle perfusion from which to test the thrombin. The initially attached leukocytes have not been characterized in our study, and the number has not been quantified, but an important question is whether endothelial exposure to inflammatory conditions associated with attached or emigrated leukocytes in the 24 h before day 2 perfusion is necessary for the change in response to thrombin that we describe. Our methods provide several ways to investigate this question. One way would be to perfuse individual venules with artificial perfusates, with or without leukocytes and platelets, for periods up to 24 h. Another method would be to use antibodies to endothelial binding sites for leukocytes (intracellular adhesion molecules) to stimulate leukocyte-dependent responses in the endothelium. Repeated tests with thrombin during this period would not establish the time course of the changes in permeability and diameter before the 24-h test used in this experiment but would evaluate the contribution of leukocytes to the response.
Signaling pathways. Our methods are a direct extension of the approaches previously described from our laboratory, and subsequently used by Bates and colleagues (5), to investigate the acute and chronic effect of VEGF on permeability in individually perfused microvessels of frog mesentery. A similar approach will enable us to test the hypothesis that upregulation of thrombin receptors and/or signaling pathways occurs within 24 h of exposure of venules to an acute inflammatory agent. We note that in isolated human coronary arteries, exposure to interleukin-1
or tumor necrosis factor- results in upregulation of receptors PAR-2 and PAR-4, which modify the vasodilatory response to thrombin in these vessels (11). These receptors were identified using peptide agonists specific for the PAR receptors. As already noted, experiments by Vergnolle and colleagues (17) found that the accumulation of leukocytes in rat venules exposed to thrombin in the superfusate could not be reproduced by the specific PAR-1 activating peptide but were reproduced by stimulation of PAR-4 receptors. A role of PAR-4 receptors in direct activation of endothelial cells has not been investigated further. These elegant approaches may enable evaluation of changes in receptor expression in our experiments.
Bates et al. (3–5) have demonstrated that venular microvessels in frog mesentery, which responded with a transient increase in permeability after a brief initial exposure to VEGF (day 1), have a significantly elevated baseline permeability when measured at 24 and 48 h. Furthermore, if the transient response on day 1 was blocked by inhibiting the calcium influx into the endothelial cells through transient receptor potential-like calcium channels, the subsequent response was also inhibited (5). Bates and colleagues (3, 5) also showed that the vessels were larger on day 2, with an increase in vessel diameter of 48 ± 13% (3, 5). Part of this increase in diameter was accounted for by increased compliance of the vessel, but not all, suggesting an increase in endothelial cell size or number contributed to the increase in diameter. The action of thrombin in these frog mesenteric vessels was not studied, nor were the studies extended to mammalian vessels.
Important clues to further understanding mechanisms in the present studies are the experiments on frog microvessels to distinguish mechanisms that increased permeability on day 2 and mechanisms that increased diameter. Specifically, the increase in both diameter and compliance on day 2 in frog microvessels was attenuated by PD-98059, which blocks mitogen-activated protein kinase (MAPK) activation (5). In contrast, MAPK inhibition did not attenuate the increase in microvessel permeability. Thus it appears that separate signaling pathways modulate the increase in vessel diameter and increased microvessel permeability after VEGF. Furthermore, an increase in microvessel diameter and compliance was also measured on day 2 in microvessels exposed to ATP on day 1 (5). If a pathway involving MAPK regulates changes of rat mesenteric venule diameter, we are in a good position to extend our investigations to evaluate the contribution of this pathway to thrombin-dependent changes in permeability.
Our recent experiments (2) using inhibitors of Rho A and Rho A-dependent kinase demonstrated that rat mesenteric venules do not require these signaling molecules to increase permeability after stimulation by bradykinin and PAF. We were not able to test the hypothesis that thrombin activation did involve these pathways because normal rat venules did not respond to thrombin. We are now in position to directly test the hypothesis that a Rho A-dependent pathway modulates the thrombin response that we have described in the day 2 vessels. We note that there is evidence to support the hypothesis that Rho A pathways are upregulated in vascular smooth muscle cells after injury leading to increased contractility due to inhibition of myosin light chain phosphatase and possibly direct activation of myosin light chain kinase (14).
We cannot fully exclude possible indirect effects of thrombin in our in situ perfused microvessel model. It is conceivable that thrombin escapes from the vessel and interacts with either sensory nerves or mast cells in the tissue surrounding the perfused vessel during the day 2 perfusion (6, 7). Our finding that thrombin does not have an effect on the day 1 baseline vessels either after vehicle control perfusion or after stimulation with PAF speaks against these hypotheses. However, the day 2 basal Lp is somewhat higher than on day 1 and it is possible that thrombin more readily penetrates to the extravascular space under the day 2 conditions. These possibilities can be tested in future studies.
In conclusion, the observation that rat mesenteric venules change from a phenotype that fails to respond to thrombin to a phenotype with a robust response to thrombin within 24 h after an inflammatory stimulus not only demonstrates that the plasticity of endothelial phenotypes may be an important mechanism responsible for the long-term regulation of venular permeability but also provides a useful experimental model to address several poorly understood mechanisms of permeability modulation. Of particular interest is the possibility that endothelial cell injury may account at least in part for the change in venule response to thrombin. The same mechanism may play a key role in the high permeability of some endothelial cells in culture and their response to inflammatory agents and may also account for the formation of highly localized leakage sites in intact venules if these form at sites where there had been a previous inflammatory response.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-Thrombin-induced pulmonary vasoconstriction. J Appl Physiol 63: 1993–2000, 1987.This article has been cited by other articles:
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  G. P. van Nieuw Amerongen, R. J. P. Musters, E. C. Eringa, P. Sipkema, and V. W. M. van Hinsbergh Thrombin-induced endothelial barrier disruption in intact microvessels: role of RhoA/Rho kinase-myosin phosphatase axis Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1234 - C1241. [Abstract] [Full Text] [PDF]
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 F.-R. E. Curry Permeability measurements in an individually perfused capillary: the 'squid axon' of the microcirculation Exp Physiol, April 1, 2008; 93(4): 444 - 446. [Full Text] [PDF]
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 B. Troyanovsky, D. F. Alvarez, J. A. King, and K. L. Schaphorst Thrombin enhances the barrier function of rat microvascular endothelium in a PAR-1-dependent manner Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L266 - L275. [Abstract] [Full Text] [PDF]
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 U. M. Chandrasekharan, L. Yang, A. Walters, P. Howe, and P. E. DiCorleto Role of CL-100, a Dual Specificity Phosphatase, in Thrombin-induced Endothelial Cell Activation J. Biol. Chem., November 5, 2004; 279(45): 46678 - 46685. [Abstract] [Full Text] [PDF]
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), the vessel was perfused with 10 nM 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF; 
) to demonstrate responsiveness to inflammatory stimulus, reperfused with vehicle to allow recovery to baseline Lp, and tested with thrombin (10 U/ml; 
). There was no Lp response to thrombin perfusion.
different from day 1 thrombin, nonpaired.
