Ghislain Defreyn

A THROMBOXANE SYNTHETASE INHIBITOR REORIENTS ENDOPEROXIDE METABOLISM IN WHOLE BLOOD TOWARDS PROSTACYCLIN AND PROSTAGLANDIN E2

During incubation of citrated blood at 37 degrees C the levels of 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and prostaglandin E2 (PGE2) remain constant, but rise markedly within one minute after the addition of collagen, particularly when thromboxane synthetase is blocked. The amount of 6-keto PGF1 alpha formed is dose-dependent for both collagen and the thromboxane synthetase inhibitor (UK-37,248). Moreover, the number of platelets will determine the extent of the 6-keto PGF1 alpha jump, that does not occur when blood is drawn after aspirin ingestion. The production of 6-keto PGF1 alpha in function of time is composed of a fast platelet-related (intercept) and a slower probably leukocyte-dependent contribution (slope). In the absence of UK-37,248 the intercept is 115 +/- 85 pg/ml, the slope is 12.9 +/- 7.7 pg/min/ml whereas in the presence of the thromboxane synthetase inhibitor they are 411 +/- 177 pg/ml and 56.2 +/- 25 pg/min/ml respectively. The present findings indicate that a thromboxane synthetase inhibitor, by not only reducing thromboxane A2 production but also enhancing prostacyclin generation with blood is exposed to thrombogenic stimuli such as collagen, should be superior to aspirin as an antithrombotic agent, although possible interference by enhanced PGE2 production should be taken into account.

Resolution of the ATP-Mg-dependent phosphorylase phosphatase from liver into a two protein component system

The inacti~ti~~ of a ho~one-sen~tive lipase from rat adipose tissue with ATP in the presence of Mg* has been described fl] and the resolution of an ATP-Mg-dependent system for the degradation of proteins in rabbit reticulocytes into two protein components reported [2]. An inhibition of phosphorylase phosphatase by ATP has been described in some enzyme preparations ]3-7] whereas other preparations of pho~ho~~se phosphatase in bovine adrenal cortex [8,9], atian skeletal muscle f 1 0] and dog liver [6] are activated by ATP-Mg, From these observations one may conclude that ATF-Mg could play an important role in the regulation of these enzyme systems. The ATP-Mg-dependent phosphorylase phosphatase present in liver cytosol constitutes an important fraction of the total phosphorylase phosphatase activity [6]; its activity is controlled by heat stable protein inhibitors, one of which is activated by cyclic AMPdependent protein kinase ill]. We describe here that this phosphorylase phosphatase activity results from the interaction of at least two different thermolabile fractions which can be separated by DEAEeel&lose ~~omatography. part of this work has been reported in [ 121.

A deinhibitor protein neutralizing the effect of the protein inhibitors on dog liver phosphorylase phosphatase

The level of active phosphorylase is the result of a balance between the activities of phosphorylase kinase and of phosphorylase phosphatase. In recent years, attention has been increasingly focused on the enzymes responsible for the dephosphorylation of phosphoproteins. Among them phosphorylase phosphatase and the protein inhibitors [l-5] of this enzyme have been studied in some detail. Several types of phosphorylase phosphatase have been extracted from dog liver [6] . Two of these are present in the cytosol: one type of enzyme (mol. wt. 77 OOO2 15 000) is spontaneously active in vitro, but is presumed to be inhibited in vivo owing to the presence of small amounts of free ATP; the other is an ATPMg-dependent phosphorylase phosphatase (mol. wt 138 000) expected to be fully active in vlvo. A third and still different phosphorylase phosphatase (mol. wt 5 1 000) is associated with particulate glycogen; it is active as such and presumably also active in vivo. Protein kinase-dependent (inhibitor-l) and -independent (inhibitor-2) proteins extracted from dog liver [3,4] inhibit the ATP-Mg-dependent phosphorylase phosphatase from the cytosol as well as the spontaneously active enzyme associated with particulate glycogen [7]. The ATP-inhibited phosphatase extracted from the cytosol is not affected by either protein inhibitor. Now we have isolated from the glycogen pellet a

The Effect of Clinical Prostacyclin Infusions in Advanced Arterial Disease on Platelet Function and Plasma 6-keto PGF1α Levels

Summary. We have infused synthetic prostacyclin (PGI2) continuously for approximately 72 h at the maximum tolerated dose (ranging from 5 to 60 ng/kg/min) into nine patients with advanced arterial disease. Prior to the infusion seven out of nine patients had spontaneous platelet aggregation and five out of six patients tested had an abnormal circulating platelet aggregate ratio. During the infusion only one patient still had spontaneous aggregation and all the abnormal circulating platelet aggregate ratios returned to the normal range. However, none of the patients showed any suppression of ADP induced aggregation. The level of exogenous PGI2 required in vitro prior to the infusion to completely inhibit ADP induced aggregation was 5–10 ng/ml in three of the four patients tested. Ten healthy adults showed complete inhibition with 1 ng/ml of PGI2. It appears that the platelets of some patients with arterial disease are more resistant to the anti‐aggregating properties of PGI2.

Multiple molecular forms of phosphorylase phosphatase associated with particulate glycogen and extracted from the cytosol of dog liver

The glycogen pellet of dog liver extracts contains a phosphorylase phosphatase which has characteristics different from those of the phosphatases extracted from the cytosol. The phosphatase associated with glycogen is characterized by a M, of 51,000, a half maximal inhibition at 0.3 mM ATP (Hill coefficient : 2) and a Ki for Mg2+ of 1 mM. Treatment with urea or mercaptoethanol of the phosphatase associated with glycogen does not influence the activity, the Mr or the half maximal inhibition by ATP, but a decrease of the Hill coefficient for ATP is observed. A similar treatment of the phosphatases extracted from the high speed supernatant results in a decrease of the Mr of the spontaneously active form from 215,000 to 43,000, without an effect on the Ki for ATP (7 micronM), but accompanied by an increase in activity. The ATP-Mg dependent form of the phosphatase from the high speed supernatant (Mr : 138,000 ; Ka for ATP in the presence of 0.1 mM Mg2+ : 0.3 micronM), is denatured by urea or mercaptoethanol. The phosphatase associated with particulate glycogen cannot be found in the supernatant, nor the phosphorylase phosphatases present in the supernatant in the glycogen pellet. When all the glycogen is mobilized (starvation, glucagon) the phosphatase specifically associated with glycogen cannot be found as such in the cytosol. No activation of synthase beta can be detected neither with the phosphatases extracted from the cytosol nor with the enzyme released from the glycogen pellet.

Low prostacyclin synthetase activity of fetal rat aorta. Progressive increase after birth

Aortae from fetal or 3 weeks old rats produced very small amounts of PGI2, prostacyclin. This production increased from 4 weeks on, reaching adult values at about ten weeks. This maturation seemed to be predominantly determined by a change in the PGI2 synthetase system, rather than in arachidonic acid availability, phospholipase or cyclo-oxygenase activity. The anti-oxidant ascorbic acid stimulated prostacyclin production more strongly in adult than in young rat aortae. This finding suggests that the lower production of PGI2 by young tissues is not due to an enhanced inhibition of prostacyclin synthetase by lipid peroxides.