Dissociation of heparin-dependent thrombin and factor Xa inhibitory activities of antithrombin-III by mutations in the reactive site.

Antithrombin-III (AT-III) is a heparin-dependent inhibitor of thrombin and Factor Xa, two serine proteases that are crucial for blood coagulation. In order to assess whether it would be possible to target AT-III only towards Factor Xa, we replaced parts of the reactive site, or P region, of AT-III by sequences present in prothrombin, a substrate of Factor Xa in the coagulation cascade. We show that replacement of the P3 to P3' region generates the hypothesized phenotype. In fact, point mutation of the P1' site from Ser (present in AT-III) to Ile (present in prothrombin) is sufficient to dissociate heparin-dependent thrombin and Factor Xa inhibitory activities. Interestingly, a combined mutation at P3 and P3' brings about the same dissociation. We show that besides Ile, other amino acids at P1' can lead to the dissociation in inhibitory activity. Amino acids with small side chains (Gly, Ser, Ala, and Thr) have only a marginal effect on the inhibitory activity against either protease. However, larger residues at the P1' position abolish the heparin-dependent anti-thrombin activity, whereas the heparin-dependent anti-Factor Xa activity is not at all or only moderately affected. These results can be rationalized by a comparison of the x-ray structure and a three-dimensional model of the S1' binding pockets of thrombin and Factor Xa, respectively. It appears that the S1' pocket of Factor Xa leaves much more space for the P1' residue of AT-III than the S1' pocket of thrombin.

Regional cerebral and extracranial blood flow measurements in acutely and chronically implanted cats: hydrogen clearance technique.

A technique is described for measuring regional blood flow concomitantly in the brain and in extracranial tissues of the cat. Hydrogen clearance using the tissue polarographic electrode appears to be a useful technique for intermittent measurements of cerebral blood flow (CBF) in relatively small areas. H2 was administered by inhalation for 10 min. Both chronic and acutely implanted electrodes were placed at different depths in the cat brain, on the surface of the cortex, and in extracranial tissues. Clearance rates in gray matter of 75 to 119 ml/min/100 g tissue have been obtained and of 11 to 14 ml/min/100 g tissue in white matter. Clearance curves have invariably been monoexponential in character in white matter and biexponential in gray matter. Successful recordings of H2 clearance curves were obtained from both chronically (up to 5 months) and acutely implanted electrodes. A new type of electrode is described. The "paperclip" electrode is placed at the surface of the cortex, has a reactive surface much greater than that of needle electrodes, thus limiting the possible variations due to vascularization differences from one local area to the next, and induces no damage to the brain tissue. To test the reliability of the technique, blood flow was measured during hypercapnia and progressive exsanguination. All electrodes indicated increased rCBF following 5-7% CO2 inhalation. A marked decrease in blood flow was seen with peripheral electrodes during exsanguination, whereas it was necessary to lower arterial blood pressure by more than 60% of the baseline value to record decreased flow in brain tissues. The constancy of response from electrodes and the lack of obvious tissue damage on dissection of the brain renders the method an adequate one. It provides highly focal recording of both CBF and extracranial flow in chronically implanted animals.

An automated superfusion technique for measuring circular muscle contraction. Effects of serotonin on intra- and extracranial arteries.

A new in vitro technique is described and its advantages are demonstrated: "true" circular contraction is measured; arteries are in cascade, permitting comparison of intra- and extracranial arteries from the same animal; the mechanical influences on contractility are reduced; long-lasting experiments of up to 50 h can be performed; the procedure is fully automated. The maximal effect of an agonist was not only markedly modified during the first 2--4 h after fixing the arteries in the apparatus but also slightly during the next 3--4 h. After the initial stabilization period, the arterial response to an agonist remained highly reproducible for 24--48 h (standard deviation not exceeding 7%). This was demonstrated in all the types of arteries tested. With serotonin as agonist, there was a significant difference in --log ED50 values for intracranial arteries (basilar 8.70, middle cerebral 8.72) vs. extracranial (lateral nasal 8.15; facial 8.14) and peripheral (branch of saphenous 8.14) arteries.