Laser-induced Thrombus Formation Research Articles

New NO donors with antithrombotic and vasodilating activities, Part 28. N-(1-cyanoalkyl)-N-hydroxyureas.

Nineteen N-(1-cyanoalkyl)-N-hydroxyureas comprising aliphatic (3a-i, 4a, b, and 5a) and aromatic (3j-n, 4c, 5b) compounds were prepared, fourteen of them for the first time, and tested for antithrombotic (p.o. administration to rats, 60 mg/kg) effects. The N-(1-cyanocyclohexyl)-N-hydroxy-N'-phenylurea (3j) was most potent and inhibited laser-induced (35 mW, 50 ms) thrombus formation in arterioles by 21% and that in venules by 15%. The compounds form nitric oxide in vitro by the addition of a Fe3(+)-porphyrin complex and an oxygen donor. Moreover, the most active compound 3j in vivo exhibits the highest NO formation in vitro. Furthermore, it was shown that the cyano group is essential for the desired activities and NO formation. These results suggest that the title compounds act as NO donors.

Experimental Studies on the Interaction of LW 10082 (Aprosulphate) with Other Antithrombotic Agents in Laser-Induced Thrombosis

The aim of this study was to investigate the possible interactions of LW 10082 with several other antithrombotic agents. The antithrombotic effects of all agents were studied in a thrombosis model in which rat mesenteric venules (diameter 20–30 μm) were injured by well-defined argon laser lesions. The number of laser injuries which were needed to induce a thrombus that intermittently occluded the vessel was used to quantify the results. LW 10082 was injected s.c. 2 h before testing. The minimal effective dose which significantly inhibited thrombus formation was 5 mg/kg−1. The minimal effective dose of Daltroban (orally 2 h before testing) was 10 mg/kg−1. At 30 min after i.v. injection of 0.1 mg/kg−1 Molsidomin, a significant antithrombotic activity was present. Ticlopidine inhibited thrombus formation if administered orally for 3 days at a dose of 2×10 mg/kg−1. A 5 mg/kg−1 dose of LW 10082 given together with Molsidomin produced a strong additive effect. The combination of LW 10082 and Daltroban also had a significant additive effect. There was a slight but not significant additive effect between LW 10082 and Ticlopidine. In conclusion, LW 10082 and some other antithrombotic agents show additive effects in the prevention of laser-induced thrombus formation in the rat microcirculation. The clinical use of such combinations seems possible in patients at high risk of thrombotic vascular occlusion.

Effects of Polysulfonate Derivative (GL 522-Y-1) on Coagulation in vitro and Thrombosis in vivo

We have investigated the influence of a polysulfonate derivative – GL 522-Y-1 – on platelet-induced thrombin generation time, platelet adhesion to siliconized glass, platelet aggregation induced by collagen and ADP, on aPTT, PT and TT in vitro and studied its antithrombotic effect in an animal model of thrombosis in vivo. In vitro, GL 522-Y-1 caused inhibition of ADP- and collagen-induced aggregation. In a dose-dependent manner this compound inhibited PITT, aPTT, and PT. GL 522-Y-1 did not prolong thrombin time. GL 522-Y-1 inhibited in vivo the laser-induced thrombus formation after intravenous and oral administration. On the basis of its unique antithrombotic properties, GL 522-Y-1 seems to open a new pathway in the field of antithrombotics.

Thrombotic Tendency of Pial Vessels in SHRSP/Izm, Studies with He-Ne Laser-induced Thrombus Formation Technique

Thrombotic Tendency of Pial Vessels in SHRSP/Izm, Studies with He-Ne Laser-induced Thrombus Formation Technique

Antithrombotic effects of aspirin and LMWH in a laser-induced model of arterials and venous thrombosis

Antiplatelet drug aspirin and anticoagulant low molecular weight heparin (LMWH) were compared as arterial and venous antithrombotic preparations in the rat experimental model of the laser induced thrombus formation. A method to induce microthrombi in small mesenteric vessel (15–25 μm) has been developed to investigate antithrombotic drugs and to study platelet reactions. Mesenteric injuries are induced in the vascular system of Wistar rats with an argon laser. The laser beam induced formation of the vessel wall injury with damage of endothelial cells. Thrombus was formed within seconds after laser injury and grew rapidly. The aggregate can be swept away by the flow and a new thrombus was formed again. This embolization began within the minute following the laser flash. Thrombus formation and embolization were repetitive phenoma. Aspirin (100 mg/kg) and LMWH (1 mg/kg) are approximately the same as to decrease the number of emboli detached from the thrombus and the duration of embolization; both in venules and in arterioles. This results suggest reflexion about the role of platelets in venous thrombosis induced by laser beam.

Arterial antithrombotic effect of piyav1t, the novel pharmacological preparation from the medicinal leech, and of its components, prostanoids and enzyme destabilase

Abstract Piyavit, the novel pharmacological preparation from the medicinal leech (Hirudo medicinalis) contained the leech saliva, produces the potent arterial antithrombotic effect examined on experimental of Laser Induced Thrombus formation. Administrated orally into rats or injected subcutaneously as water extract, non-diluted or in 1600 times diluted, it inhibits statistically significant comparing with control platelet thrombus stimulated by laser beams. Its components, prostanoid fraction and purified enzyme destabilase, endo-ε-(γ-Glu)-Lys-isopeptidase, also inhibit thrombus formation in statistically different manner, comparing with control. All the tested preparations inhibit platelet aggregation induced by ADP. The dependence of arterial antithrombotic effect on the leech prostanoid is discussed.

Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

To assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded. In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i.v. injection inhibits risocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombogenic properties of ultra-low-dose of acetylsalicylic acid in a vessel model of laser-induced thrombus formation

Various studies have demonstrated the antithrombotic effect of high dose acetylsalicylic acid (ASA) and more reports show the similar efficiency of the much better tolerated low dose ASA. Recently, in vitro studies hinted the potent thrombotic properties of ultra-low-dose ASA (ULD-ASA, < 1 mg/day) showing a significant decrease in bleeding time( 1,2). This effect was, at least in part, related to the inhibition of endothelial cyclooxygenase -and not to platelet oneleading to a decrease of prostacyclm release (3). To investigate the effect of ASA in vivo, we used an intravital microscopic technique, allowing to evaluate (anti)-thromboembolic events at previously determined locations of the microvasculature. Thrombus formation was induced by Argon-laser shot. The instrumental test set-up was completed with a video-system. The kinetic feature of thrombus formation was estimated in terms of number of laser shots required to induce endothelium injury leading to thrombosis, number of emboli removed by blood stream and duration of embolization. The reliability and reproductibility of our laser-induced thrombus model have been previously tested in rat mesenteric arterioles and veinules and we showed that ASA used at current doses (50-200 mgkg) decreased the number of emboli and the duration of embolization (4). The aim of this preliminary study was the investigation of the effects of ULD-ASA in vivo. In so doing, we evaluated ULD-ASA in our experimental laser-induced thrombus model. This might represent a further step in the understanding of the action of ASA according to the range of concentration used.

New NO-donors with antithrombotic and vasodilating activities, VI: thiazole-2-nitrosimines.

24 new thiazole-2-nitrosimines were prepared and described by means of spectroscopical methods (NMR, IR, MS, UV). At pH 7 in cell free systems as well as in platelet rich plasma the compounds are stable against hydrolysis and do not react with the platelet glutathione. The chemical stability is underlined by the mass spectra: M+. is of high intensity and sometimes even forms the base peak (e.g. 8a). Thermal elimination of N2 is of minor importance. The =N-NO bond in solution is susceptible to cleavage by visible light. The metabolite so formed is able to inhibit the platelet aggregation induced by collagen (Born-test). Five compounds exhibit this activity in concentrations below 10 mumol/L (IC50). This is due to the release of a NO species, as could be demonstrated by the stimulation of soluble guanylate cyclase in a cell free system (e.g. 8a, KM = 72 mumol/L). In vivo the nitrosimines show antithrombotic properties. Two h after a single oral dose of 8g (60 mg/kg) a 57% inhibition of the laser induced thrombus formation in the mesenteric arterioles of rats is observed. After 8 h a 43% inhibition still is seen.

Antithrombotic effect of argatroban on the pial vessels of the rat: a study with He-Ne laser-induced thrombus formation.

The antithrombotic effect of the synthetic thrombin inhibitor (2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfon yl)-L-arginyl]-2-piperidinecarboxylic acid monohydrate (argatroban) was investigated in cerebral vessels of the rat. An occlusive thrombus was formed in pial vessels using a He-Ne laser in a closed cranial window technique. Argatroban retarded the formation of thrombi in a dose-dependent manner. The antithrombotic effect of a single intravenous dose of argatroban at 0.5 mg/kg was diminished after 30 min in arterioles and after 50 min in venules, respectively. The antithrombotic activity was maintained, however, by continuous intravenous infusion (2 mg/kg/h).

Thrombotic Tendency of Spontaneously Hypertensive Rats Measured by the He-Ne Laser-induced Thrombus Formation Method

This experiment measured the thrombotic tendency and platelet aggregability of a spontaneously hypertensive rat (SHR) and its control, Wistar Kyoto (WKY), using the He-Ne laser-induced thrombus formation method and the whole blood aggregation method, respectively. The two findings of this experiment were the number of irradiations required to induce occlusive thrombus formation was smaller in SHR than in WKY and platelet aggregability in SHR was higher than in WKY. These results suggest that higher thrombotic tendency may be partly due to the higher platelet aggregability in SHR.

Effect of antibiotics on laser-induced thrombus formation in rat mesenteric arterioles.

Antibiotics were tested in a thrombosis model, in which thrombi are produced in small rat mesenteric arterioles. An interference contrast system based on a Leitz Orthoplan microscope, was used to visualize thrombus formation. Vascular lesions were produced with a Coherent CR-2 supergraphite ion laser (argon laser) in arterioles of 25-35 microns diameter. Cefmenoxim, cefotaxim (i.v.) and cephalexin orally at doses of 20 and 40 mg/kg and gentamycin 10 and 20 mg/kg (i.v.) had a marked and significant antithrombotic effect. Even more effective were cefoperazon and lamoxactam 20 and 40 mg/kg (i.v.) and tobramycin 10 and 20 mg/kg (i.v.). Azlocillin, cefoxitin, mezlocillin or spectinomycin (20 mg/kg i.v.) and penicillin or piperacillin (50 mg/kg i.v.) also showed a significant antithrombotic effect, which, however, disappeared on doubling of the applied dose.

Effect of physical training on thrombotic tendency in rats: decrease in thrombotic tendency measured by the He-Ne laser-induced thrombus formation method.

The effect of physical training on thrombotic tendency was assessed in rats. Exercise was done on a flat treadmill for 30 min at a rate of 1,400 m/h (submaximal speed), 5 times a week for either 1.5 or 3 months. The thrombotic tendency was measured by the He-Ne laser-induced thrombus formation method in microvessels of mesentery, i.e. measurement of the number of laser irradiations necessary to induce stasis of blood flow by occlusive thrombus formation. An increase in the number of irradiations necessary to induce occlusive thrombus formation was observed in arterioles, but not in venules after physical training for 1.5 and 3 months.

Effect of low-molecular-weight heparins on laser-induced thrombus formation in rat mesenteric vessels.

Laser-induced thrombi have been frequently produced in rat mesenteric vessels to investigate the effect of antithrombotic drugs. We have tested low molecular heparins in comparison to unfractionated heparin. The investigations were carried out on male Wistar rats weighing 200-300 g; the animals were anesthetized with pentobarbital sodium (Nembutal) 60 mg/kg i.p. Vascular lesions were induced with a Coherent CR-2 supergraphite ion laser (argon laser) mounted on a Leitz Orthoplan microscope. An intestinal loop was spread on a self-constructed object stage, mounted on the microscope table. The laser beam was directed through the optical path of the microscope on small mesenteric vessels in the fat-free portion of the mesentery. For the evaluation of thrombus formation the number of laser injuries needed to induce a defined thrombus was used. All low-molecular-weight heparins (preparation B, Braun; BR-Z-0601, Braun; CY 216, Choay; Fragmin, Kabi; low molecular heparin, Sandoz and Org 10172, Organon) or unfractionated heparin (Liquemin; Hoffmann-La Roche) showed a significant and dose-dependent antithrombotic effect after subcutaneous injection, if venules had been damaged. The effect lasted for 48 h, partly for over 48 h. If thrombus formation was studied after intravenous injection into arterioles as well as into venules 8 h after injection, no antithrombotic effect could be demonstrated under the influence of CY 216 or unfractionated heparin.

Surprising effects of the sequential administration of pentoxifylline and low dose acetylsalicylic acid on thrombus formation's

The effect of the combined oral administration of pentoxifyl-line (pof) and low dose acetylsalicylic acid (ASA) was evaluated with the help of the laser-induced thrombosis in rat mesenteric arterioles. Laser-induced thrombosis is inhibited in a dose-dependent way by both drugs. The administration of ASA, either simulataneously with or 1 hour prior to pof, does not show any effects in the laser model. On the contrary, the administration of pof followed 1 h later by ASA not only exhibited a significant effect but also produced a supraadditive inhibition of the laser-induced thrombus formation. Specific investigations concerning the time interval between the administration of both drugs determined that a significant effect can be achieved only after an interval of 30 to 90 minutes (principle of HWA 5112). The striking results could also be shown in diseased animals after sequential chronic administration of pof 1 h prior to ASA. HWA 5112 exhibits significant effects on laser-induced thrombus formation in the following chronic animal models: 1. adjuvant arthritic rats, 10→1 mg/kg for 21 days; 2. spontaneously hypertensive stroke-prone rats, 10→1 mg/kg three times within 24 h; 3. cholesterol-induced atherosclerosis in rabbits, 10→1 mg/kg for 14 days. The reported data clearly demonstrate that the sequential drug administration of first pentoxifylline followed 30 to 90 min later by ASA exhibits a supraadditive antithrombotic effect.

Inlfuence of ADP, blood flow velocity, and vessel diameter on the laser-induced thrombus formation

The laser-induced thrombus model in rat mesenteric arterioles and venules represents a reliable and reproducible in vivo method. It is suitable for basic investigations concerning factors involved in thrombus formation as well as for testing antithrombotic effects of drugs. The laser-induced thrombus formation depends significantly on the presence of ADP, as ADP-utilizing enzymes inhibit thrombosis in the animal model. The instrumental test set-up consists of a 4 W Argon laser (Spectra Physics, Mountain View, CA, USA), a ray adaptation and adjusting device (BTG, Munich, FRG), a microscope (ICM 405, Zeiss, Oberkochen, FRG), and a video system (Sony, Japan). RBC velocity data were recorded with the help of a modified dual-slit technique (acc. to Wayland and Johnson). Results were expressed as number of laser injuries necessary to produce a defined thrombus (minimum size: 1/4 of the vessel diameter) under constant conditions (effective capacity: 30 mW, exposure time: 1/5 sec). The number of laser lesions necessary to induce a defined thrombus decreased with an increase in arteriole diameter (10 to 20 μm) but increased again in larger arterioles and small arteries (>25μm). On the arteriolar side there are significant correlation coefficients between vessel diameter and RBC velocity (r = 0.69), vessel diameter and No. of laser injuries (r = 0.70), and RBC velocity and No. of laser injuries (r = 0.71). Due to relative low flow conditions in the venules, the number of laser injuries required to induce a defined thrombus does not significantly depend on the vessel diameter.

Effects of propentofylline (HWA 285) on laser-induced thrombus formation in healthy and diseased rat mesenteric arterioles.

The antithrombotic effects of the xanthine derivative propentofylline (HWA 285) were investigated in the laser-induced thrombus model in healthy and diseased rat mesenteric arterioles. Laser-induced platelet thrombi were documented by the help of an intravital microscope and a video system. The statistical comparison of drug-treated and untreated animals, in respect to the number of laser shots necessary to induce a defined thrombus, served as a quantitative measure for a potential antithrombotic drug effect. Propentofylline and acetylsalicylic acid, in single dosages of 10 and 30 mg/kg orally, significantly increased the number of laser shots required to produce a defined thrombus. Moreover, the drug decreased the laser-induced thrombus formation in a chronic experiment in adjuvant arthritic rats. After oral administration of 30 mg/kg for 21 days, propentofylline and pentoxifylline reduced significantly the thrombus formation, whereas acetylsalicylic acid exerted no effect.

ANTITHROMBOTIC AND THROMBOLYTIC EFFECTS OF LIDOCAINE AND RELATED COMPOUNDS ON LASER-INDUCED MICROVASCULAR INJURY

In addition to an amply documented local anaesthetic effect, lidocaine has been demonstrated to reduce the adhesion of leucocytes to blood vessel walls (1). This observation, together with further experimental (2) and clinical (3) findings, has indicated that an antithrombotic effect is attributable to lidocaine.The purpose of the present investigation was to study whether topically or intravenously (i. v.) applied amide-type local anaesthetics, e. g. lidocaine and bupivacaine, would influence microvascular thrombosis, and whether they would restitute microcirculation after laser-induced thrombus formation in the hamster cheek pouch. It was further intended to record whether the major lidocaine metabolite, mono-ethyl-glycinexylidide, and tocainide (an antiarrhythmic compound, structurally related to lidocaine but suitable for oral administration) produced microvascular effects similar to that of lidocaine.The investigation was carried out in fore series: two for the purpose of recording the formation of a thrombus using laser microbeam irradiation subsequent to topical or i. v. application of the local anaesthetic (Series I and II); and the other to study the circulatory restitution effects on topical or i. v. application of the compounds onto already formed thrombi (Series II and IV).

Laser-Induced Thrombus Formation and Vascular Reactivity in the Microcirculation of the Spontaneously Hypertensive Rat

Thrombi formation resulting from laser-induced injury to mesenteric circulation has been studied in spontaneously hypertensive (SH) and normotensive rats. The thrombotic reaction was enhanced in both venules and arterioles of the SH rats. In the arterioles of the hypertensive rats, the vasoconstriction which develops downstream from the thrombus is greatly intensified and the time to form microvascular haemostatic plugs significantly decreased in the arterioles of these animals. These phenomena have been explained on the basis of an increased reactivity of the vascular smooth muscle of the SH rats to vasoconstrictor substances liberated from platelets at the site of injury.