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It has been shown that NO and prostacyclin (prostaglandin I(2)) from cultured endothelium synergistically inhibit blood platelet aggregation in vitro. However, it is unknown whether this synergism is also effective in the inhibition of thromboembolism in vivo and, if it is, whether it differs between vessel types. Therefore, the effect of endogenous NO and prostacyclin, in combination or alone, on thromboembolism was studied in an in vivo model. Thromboembolism was induced by micropipette puncture of rabbit mesenteric arterioles and venules (diameter 18 to 40 micrometer). In addition, the influence of wall shear rate was analyzed. In arterioles, the combined inhibition of NO synthase (N(G)-nitro-L-arginine [L-NA] 0.1 mmol/L; local superfusion) and of cyclooxygenase (aspirin [ASA] 100 mg/kg IV) resulted in a pronounced, significant prolongation of embolization duration (median >600 seconds) compared with control (median 153 seconds) or treatment with either L-NA (234 seconds) or ASA (314 seconds). This combined effect of L-NA+ASA was greater than the sum of the individual effects of L-NA and ASA. In contrast, in venules L-NA+ASA had no additional effect on embolization duration (209 seconds) compared with the effect of L-NA alone (230 seconds); ASA alone had no effect (122 seconds; control 72 seconds). Interestingly, only in the L-NA+ASA arterioles did embolization correlate positively with wall shear rate (r(s)=0.687; P=0.028). In conclusion, this study indicates that in arterioles, but not in venules, endogenous NO and prostaglandins synergistically counteract ongoing thromboembolism after vessel wall injury and that the combination of endogenous NO and prostaglandins appears to protect against enhancement of arteriolar thromboembolism by wall shear rate.

Because nitric oxide (NO) inhibits aggregation and adhesion of blood platelets, NO may play a role in platelet-vessel wall interactions. Therefore, the purpose of this study was to investigate the involvement of endogenous NO in thromboembolic processes, as induced by wall puncture, in rabbit mesenteric arterioles and venules (diameters 20 to 43 microm). In venules, inhibition of NO synthase by superfusion of the mesentery with N omega-nitro-L-arginine (L-NA; 0.1 mmol/L) significantly increased the duration of embolization (from 50 seconds to 511 seconds) and the number of emboli produced (from 2 to 11 emboli per vessel), while the median period of time needed to produce an embolus was not influenced. On the contrary, in arterioles, L-NA had no significant effect on embolization (duration of embolization: 426 seconds in the control and 382 seconds in the L-NA group, with 20 and 12 emboli per vessel, respectively). Addition to the L-NA superfusate of L-arginine (L-ARG; 1 mmol/L), the active precursor for endogenous NO synthesis, resulted in a complete reversal of the L-NA effects in venules, while addition of the inactive D-arginine (D-ARG; 1 mmol/L) had no effect. Addition of L-ARG and D-ARG had no significant effect in arterioles. Addition to the L-NA superfusate of the exogenous NO donor sodium nitroprusside (0.1 micromol/L) also resulted in reversal of the L-NA effects in venules, while in arterioles, it slightly but significantly decreased embolization duration. The differences in effect of L-NA on embolization between arterioles and venules were not caused by differences in fluid dynamic conditions. It is concluded that the role of endogenous NO in inhibiting thromboembolic processes is more important in venules than in arterioles.

The involvement of prostaglandins in thromboembolic processes, as induced by wall puncture, was studied in rabbit mesenteric arterioles and venules using intravital videomicroscopy. Inhibition of prostaglandin formation with aspirin (100 mg/kg, i.v.) significantly increased in arterioles duration of embolization (from 91 to 200 s) and number of emboli produced (from 4 to 8.5 per vessel), while rate of embolus production was not influenced. In venules, aspirin only influenced embolization rate (a significant decrease from one embolus/14 s to one/23 s). Specific blockade of TXA2-receptors by sulotroban (30 mg/kg, i.v.) only influenced the arteriolar reaction: it significantly decreased embolization duration (from 560 to 218 s) and number of emboli produced (from 23 to 10 emboli per vessel), without affecting embolization rate. These findings indicate that both platelet activating and inhibiting prostaglandins play a more important role in thromboembolism in arterioles than in venules; this suggests a difference in prostaglandin synthetic capacity between arteriolar and venular endothelium.

Platelet distribution was investigated in 21 venules (V) and 10 arterioles (A) of the rabbit mesentery (vessel diam 15-33 microns). Circulating platelets were labeled in vivo with the dye acridine red and observed with fluorescence video microscopy. Only platelets flowing in a thin (5-7 microns) optical section located about the median plane of the vessel were used. The relative position of each platelet, i.e., the distance of its centroid to the left vessel wall divided by the local vessel diameter, was determined. In addition, in 10 venules leukocyte margination was inhibited by intravenous injection of dextran sulfate (500,000 mol wt; 30 mg/kg body wt). The number of platelets per unit volume (i.e., platelet density) relative to the mean density was significantly higher in the vessel center of V (1.04) than of A (0.55; P less than 0.005). In contrast, near the wall this density was significantly higher in A compared with V. Mean values were as follows: at radial position (R) = 0.9-1.0, 0.30 in A and 0.11 in V (P greater than 0.05); at R = 0.8-0.9, 1.63 in A and 0.84 in V (P less than 0.002); at R = 0.7-0.8, 1.60 in A and 1.36 in V (P greater than 0.05); at R = 0.6-0.7, 1.16 in A and 1.60 in V (P less than 0.02); and at R = 0.5-0.6, 0.92 in A and 1.36 in V (P less than 0.02). These differences in platelet distribution between arterioles and venules are not caused by the presence of leukocyte margination in venules.

Spontaneous arteriolar vasomotion and its relation to arterial pressure reduction was studied in the rabbit tenuissimus muscle using intravital microscopy. Vasomotion was observed in all transverse arterioles and their first-order side branches. Vasomotion frequency ranged from 5 to 32 cycles per minute (median: 25 cpm). The relative vasomotion amplitude in transverse arterioles varied from 0.06 to 0.44 (median: 0.22). Vasomotion was generally of the on-off type in first-order side branches. A gradual reduction in arterial pressure as achieved by aortic occlusion resulted in an abrupt disappearance of vasomotion. Vasomotion disappeared between 19 and 59 mm Hg (median: 30.4 mm Hg). No differences were found between the pressures at which vasomotion ceased in transverse arterioles and first-order side branches. With a further reduction of arterial pressure transverse arterioles attained a maximal diameter of 98-265% (median: 119%) of the maximal diameter during vasomotion. After release of occlusion vasomotion reappeared much earlier in the first-order side branches than in their feeding transverse arterioles. It is concluded that although vasomotion influences capillary perfusion, it does not play a major role in the adaptation of vascular resistance following a reduction in arterial pressure.

Velocity profiles, as determined in vivo in rabbit mesenteric arterioles with fluorescently labeled platelets as natural flow markers, were used to calculate least estimates of the actual wall shear rate in these microvessels (17-32 micron diam). The fit of the velocity data points described the profile as close to the wall as 0.5 micron. To satisfy the no-slip condition, a thin layer of fluid with a steep velocity gradient near the wall was assumed. Least estimates of wall shear rate, as calculated from the fitted platelet-velocity profiles and using the mean velocity gradient in this layer of fluid, ranged from 472 to 4,712 s-1 with a median value of 1,700 s-1. Red blood cell center-line velocities varied between 1.3 and 14.4 mm/s (median 3.4). The wall shear rates were at least 1.46-3.94 (median 2.12) times higher than expected on the basis of a parabolic velocity distribution but with the same volume flow in the vessel. Considerable spatial differences in wall shear rate might exist even within a short segment of a vessel.

The walls of rabbit mesenteric arterioles and venules (diameter 20 to 40 microns) were punctured with glass micropipets (tip diameter 6 to 8 microns). Thromboembolic reactions resulting from this standardized, small mechanical vessel wall injury could be quantified in vivo with the use of intravital video-microscopy. Following induction of the injury thrombus growth started immediately (less than 0.1 s). Bleeding times were short, on the average less than 2 s, and did not differ between arterioles and venules. The duration of the embolization process was significantly longer in arterioles than in venules (median 101 and 17 s, respectively), and more emboli were produced in arterioles than in venules (median 6 and 1, respectively). Arteriolar thrombi were more effective in plugging the punctured holes than venular thrombi. The differences in thromboembolic reaction between arterioles and venules, as found in the present study, can probably not be explained by fluid dynamic factors.

Changes in vasomotion parameters and their consequences for local arteriolar resistance were studied in transverse arterioles and their first order side branches in the tenuissimus muscle of 10 young urethane anesthetized rabbits during stepwise reduction of arterial pressure, using intravital microscopy. To assess the influence of vasomotion on mean local arteriolar resistance, the effective vascular diameter, as a measure of mean flow carrying capacity, was calculated. The contribution of vasomotion to the mean local resistance is limited in transverse arterioles, but important in first order side branches, dominating the flow fluctuations in the downstream capillaries. During pressure reduction, an over-all increase in vasomotion cycle length and amplitude was found in both transverse arterioles and first order side branches, concomitant with an increase in effective arteriolar diameter and a decrease in local blood flow and reduced velocity, as a measure of wall shear rate. Flow autoregulation was observed in 70% of the arterioles. The changes in cycle length and amplitude showed only limited correlations with local blood flow, reduced velocity, arterial pressure and effective arteriolar diameter. This indicates that it is unlikely that only one of these variables is responsible for the changes in the vasomotion parameters.