S35972, a direct-acting thrombin inhibitor with high oral bioavailability and antithrombotic efficacy.

OBJECTIVES: Dabigatran etexilate is the first oral thrombin inhibitor to demonstrate superior efficacy to warfarin for stroke prevention in patients with atrial fibrillation. This study describes the in vitro, ex vivo anticoagulant and in vivo antithrombotic effects of an oral thrombin inhibitor, S35972, in comparison with dabigatran etexilate. METHODS: Enzyme assays with thrombin and related serine proteases were performed. Clotting times, including activated partial thromboplastin time (APTT) and thrombin time (TT), were measured in vitro in different species and ex vivo in dogs and rats to determine pharmacologic bioavailabilities. The formation of occlusive venous and arterial thrombi in the rat vena cava and aorta was induced with stasis plus thromboplastin or ferrous chloride, respectively. RESULTS: S35972 inhibited human thrombin with an IC(50) of 3.7 nm, and did not inhibit other serine proteases. The anticoagulant activities of S35972 in vitro were comparable in dog and human plasmas, and the sensitivity of the clotting times to S35972 was TT > APTT > prothrombin time. In the fasted dog, oral administration of 3 mg kg(-1) S35972 increased TT rapidly and for at least 8 h, and its pharmacologic bioavailability was 75.4% ± 0.1%. In the rat venous thrombosis model, 3 mg kg(-1) oral S35972 or dabigatran etexilate significantly decreased the thrombus weight. In the rat aortic thrombosis model, oral S35972 at 10mg kg(-1) significantly decreased thrombus weight, by approximately 50%, whereas, at this dose, no effect was obtained with dabigatran etexilate. CONCLUSIONS: S35972 is a non-prodrug thrombin inhibitor with high selectivity, oral bioavailability, and antithrombotic efficacy.

Inactivation of plasminogen activator inhibitor-1 accelerates thrombolysis of a platelet-rich thrombus in rat mesenteric arterioles.

To investigate the role of active plasminogen activator inhibitor 1 (PAI-1) in the evolution of a microthrombus generated in the arteriolar microcirculation, the monoclonal antibody, 33H1F7, which transforms active PAI-1 to a tissue type plasminogen activator (t-PA) substrate, was evaluated in an arteriolar thrombosis model in the rat mesentery. Arterioles (200-300 um) were stimulated electrically to create an endothelial lesion; ADP was then perfused for 2 min to induce the formation of a platelet-rich thrombus which lysed spontaneously in 140 +/- 24 s. Two successive ADP superfusions produced comparable thrombi which lysed in comparable times. Different doses of 33H1F7 were infused to rats for 30 min and the dose which inactivates rapidly and totally active rat PAI-1 (300 microg/kg/min) was selected to be tested on the thrombosis model. Infusion of 33H I F7 beginning 10 min before the ADP application significantly reduced the lysis time in comparison to the control (123 +/- 30 s versus 169 +/- 33 s, P < 0.05, paired Student's t-test) and the cumulative thrombus area during the lysis period was decreased by 56 +/- 7%. These results demonstrate that inactivation of PAI-1 is able to accelerate lysis of a platelet-rich clot in a mesenteric arteriole of the rat. Thus active PAI-1 most likely participates to the resistance to thrombolysis in the arteriolar microcirculation and its inactivation may shorten ischemic periods after microvascular obstruction such as e.g. during cerebral stroke.

Off-line analysis of red blood cell velocity in renal arterioles.

Videomicroscopic methods with off-line analysis of microcirculatory parameters by multifunctional computer-assisted image analysis systems have significant advantages for in vivo microvascular research. A limitation of these methods is, however, that red blood cell velocities (V(RBC)) exceeding 2 mm/s cannot be measured using standard video framing rates. In the present study, a high-speed video camera, recording up to 600 frames per second, was incorporated in the set-up, and V(RBC) was measured off-line with the line-shift-diagram method. The aim of this study was to test the reproducibility and validity of the method using a high-speed video camera and to evaluate its applicability in vivo. V(RBC) were measured in arterioles of the split hydronephrotic kidney. The intra- and interindividual variability was small for V(RBC) below 40 mm/s. The validity of the method was tested using the mass conservation principle and found to be at least as good as that of the dual-slit photometric technique. The present approach extends the application of videomicroscopy coupled to image analysis systems to the analysis of high V(RBC).

Activation of thromboxane receptors and the induction of vasomotion in the hamster cheek pouch microcirculation.

1. The present study was designed to investigate a possible role of thromboxane A2 (TXA2) on arteriolar vasomotion (spontaneous rhythmic variations of the vessel diameter). Therefore the microcirculatory effects of the thromboxane-receptor (TP-receptor) agonist, U 46619, as well as the effects of the TP-receptor antagonists S 17733 and Bay U3405 were evaluated in the hamster cheek pouch microcirculation. For comparison some effects of angiotensin II were also investigated. 2. For microcirculatory measurements, the cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. The TV monitor display was used to obtain arteriolar internal diameter measurements by means of an image shearing device. 3. Superfusion (0.1 nM to 1 microM) or bolus application (1 pmol to 10 nmol) of U 46619 concentration- or dose-dependently decreased the arteriolar diameter and induced vasomotion in arterioles with a mean initial diameter of 24+/-2 microm. Both the vasoconstriction and the vasomotion induced by U 46619 were inhibited by the TP-receptor antagonists S 17733 (100 mg kg(-1), i.v.) and Bay U3405 (10 mg kg(-1), i.v.). 4. Bolus applications of angiotensin II (0.1 pmol to 1 nmol) induced transient vasoconstriction followed by vasodilatation in the cheek pouch arterioles. The dilatation but not the constriction, was sensitive to treatment with the NO-synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG; 100 microM). Angiotensin II did not induce vasomotion in control conditions or in the presence of L-NOARG. 5. Bolus application of phenylephrine (10 pmol) induced vasoconstriction but no vasomotion in previously quiescent hamster cheek pouch arterioles. 6. These results indicate that activation of TP-receptors causes vasomotion in the hamster cheek pouch arterioles. These spontaneous rhythmic variations in arteriolar diameter are not observed with equipotent doses of angiotensin II and phenylephrine. Thus, the vasoconstriction by itself cannot explain the occurrence of vasomotion observed with the TP-receptor agonist.

Mechanism of improvement in pulmonary gas exchange during growth in awake piglets.

Previous studies have shown a lower arterial PO2 (PaO2) in infants and young animals than in adults. To investigate the mechanism of this impairment of gas exchange we studied 13 piglets from 12 to 65 days of age. Two days after instrumentation we measured the distribution of ventilation-perfusion ratios (VA/Q) by use of the multiple inert gas technique on awake animals. We showed that PaO2 is lower in young animals, increasing from 72 +/- 11.5 Torr before 2 wk to 102 Torr at 2 mo. This hypoxemia is due to an enlarged alveolar-arterial O2 pressure difference that significantly decreases with age. This impairment in gas exchange is not due to shunting (0.6 +/- 1.3%). Mean dead space (36 +/- 11%) was not related to age. Mean modes of perfusion and ventilation did not differ significantly between age groups. However, the dispersion of perfusion as expressed by its logSD decreased significantly with age, whereas dispersion of ventilation remained constant. Furthermore, in the young animals only, a significant difference was evidenced between measured alveolar-arterial PO2 gradient and the value predicted by the inert gas model. We therefore conclude that the impairment of gas exchange in piglets is due to two mechanisms: VA/Q mismatch and diffusion limitation for O2.

Improvement of gas exchange during inhibition of hypoxic pulmonary vasoconstriction by nitrendipine in piglets.

To determine how nitrendipine (N), a calcium antagonist, interferes with haemodynamics and gas exchange during hypoxia, we studied 12 piglets (4-6 weeks, 4.3-9.0 kg) anaesthetized with pentobarbital (20 mg.kg-1 i.p.). Haemodynamics, blood gases and multiple inert gas elimination were measured at the end of three consecutive 30 min periods of spontaneous breathing: room air (RA), 11-12% FIO2 (H) and 11-12% FIO2 with a randomly selected infusion of N (3 micrograms.kg-1.min-1) (HN) or the carrier solution (HP) (6 animals in each group). Pulmonary vascular resistance (Rpv) doubled from 8.7 +/- 2.8 mmHg.min-1.1-1 in RA to 19.5 +/- 10.0 (mean +/- SD) in H while PaO2 fell from 83 +/- 8.3 mmHg to 28.7 +/- 5.2. With N, Rpv fell back to room air value: 8.7 +/- 2.0 (p less than 0.02; comparison H and HN), while PaO2 rose from 29.3 +/- 6.3 mmHg in H to 46.1 +/- 6.1 HN (p less than 0.001) and PaCO2 fell from 33.8 +/- 10 to 23.9 +/- 3.7 mmHg. There was a small non-significant rise in VE. Haemodynamics and blood gases of the placebo group were not statistically different in H and HP. No extrapulmonary shunting was evidenced during any experimental period. The perfusion to lung zones with VA/Q lower than 0.005 rose from 1.1 +/- 2.1% in RA to 8.9 +/- 5.7% in H, but no further increase was obtained with N: 5.1 +/- 2.5%. Overall VA/Q matching did not deteriorate with N during hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic sodium cyanate treatment induces "hypoxia-like" effects in rats.

Three weeks of sodium cyanate (NaCNO) intraperitoneal treatment in rats (n = 15) induced high hemoglobin O2 affinity, i.e., low PO2 at 50% hemoglobin saturation (P50), 20.5 +/- 1.4 Torr, in comparison with the mean control values, 34.5 +/- 1.6 Torr (n = 15). NaCNO rats showed a reduction in mean body weight, 376 +/- 27 g, in comparison with controls, 423 +/- 23 g (P less than 0.001). Despite arterial O2 partial pressure (PaO2) within normal limits NaCNO-treated rats had a higher systolic right ventricular pressure (SRVP), 33.7 +/- 3.1 Torr, in comparison with control value, 29.0 +/- 2.5 Torr (P less than 0.001). Right ventricle weights were significantly increased (P less than 0.001). After 60 min of an hypoxic challenge (fractional concentration of inspired O2 = 0.10) NaCNO-treated rats increased SRVP of only 7 +/- 4% compared with 46 +/- 9% in the control animals. Inducing high hemoglobin affinity in rats (n = 10; 6 wk NaCNO treatment) resulted in increases in hematocrit ratio and hemoglobin concentration (P less than 0.001). The characteristics of the red blood cell (RBC) itself changed; values of mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration being significantly increased (P less than 0.001) when compared with mean control values. The count of nucleated RBC's appeared to be significantly higher from the 2nd wk of NaCNO treatment. Chronic NaCNO treatment was demonstrated to exert "hypoxia-like" effects since it induced prevention of normal growth, polycythemia, pulmonary hypertension, right ventricular hypertrophy, and blunted pulmonary pressor response to acute hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological effects of high-P50 erythrocyte transfusion on piglets.

Rightward shifts of the O2 dissociation curve (ODC) were experimentally obtained in lysed and resealed erythrocytes following encapsulation of inositol hexaphosphate (IHP). This continuous lysing and resealing procedure led to in vitro P50 (Po2 at 50% hemoglobin saturation) increases up to 80 Torr (pH, 7.40; Pco2, 40 Torr; temp, 37 degrees C) for both human and pig erythrocytes. The Hill number of the transformed blood decreased when IHP was fixed on the hemoglobin, but the sigmoid shape of the ODC was maintained. The O2 hemoglobin binding capacity and the mean corpuscular hemoglobin content were found unchanged by the experimental procedure in human and pig erythrocytes. Isovolumic exchange transfusion of high-P50 erythrocytes in anesthetized and ambient air-ventilated piglets (n = 6) led to substantial in vivo P50 increases (range, 8-19 Torr). The rightward shift of the ODC was concomitant with an increase of the arterial Po2 and of the arteriovenous O2 content difference, 19 and 59% respectively above their control values. The mixed-venous Po2 (PVO2) remained unchanged. The cardiac output was shown to be inversely related to the P50 value. In spite of the O2-transport reduction (37%), O2 consumption was maintained due to enhanced O2 extraction.