K. Serizawa

Relationship between serotonergic measures in periphery and the brain of mouse

Circadian rhythm and the relationship between the concentration of serotonin (5HT) and related substances (5-hydroxyindoleacetic acid; 5HIAA and tryptophan; Trp) in mouse brain, stomach and blood have been studied. All factors underwent circadian changes in the brain and blood. 5HT and 5HIAA levels in the stomach showed no circadian fluctuation. The concentrations of 5HT in the brain and blood did not correlate. Significant correlations were found between other serotonergic parameters analyzed in brain, stomach and blood. A significant negative correlation was observed between brain 5HIAA and blood 5HIAA. The concentration of tryptophan in the brain was correlated with the plasma total tryptophan level. There was fairly significant correlation (p less than 0.06) between brain serotonin and plasma tryptophan levels. The brain serotonin and tryptophan levels were strongly correlated (R = 0.410, p less than 0.03). Significant negative correlation was found between serotonin in the blood and serotonin in the stomach as well as between its level in the brain and in the stomach. The significance of these findings and their relationship to the use of peripheral serotonergic system as a model of neurons are discussed.

Correlation between serotonergic measures in cerebrospinal fluid and blood of subhuman primate

The relationship between the concentration of serotonin (5-hydroxytryptamine; 5-HT), its precursor; tryptophan (Trp) and the main metabolite 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid (CSF) and blood of monkey have been studied. 5-HT, Trp and 5-HIAA underwent circadian changes in both CSF and blood. Significant correlations were found between 5-HT, 5-HIAA and Trp in CSF and blood. The significance of these findings and their relationship to the use of peripheral serotonergic system as a functional model of the central nervous system are discussed.

Impaired fibrinolytic activity induced by ingestion of butter : effect of increased plasma lipids on the fibrinolytic activity

To investigate the effects of the increased plasma lipid level on fibrinolysis, we measured the levels of fibrinolytic components in serially obtained plasma samples from healthy volunteers after the intake of different amounts of butter. Plasma triglyceride level increased significantly after butter intake compared to the control group. Eight hours after the intake of 100g of butter, plasminogen activator inhibitor 1 (PAI-1) level in plasma was significantly higher and euglobulin clot lysis time was significantly prolonged compared to those of the control group. There was no effect on plasma tissue plasminogen activator level. These results suggest that the temporary increase in plasma triglyceride level induced high PAI-1 level, resulting in impaired fibrinolytic activity. The effect of temporary hyperlipidemia on platelet function was also analyzed and revealed that the response of platelets to ADP and collagen was lower in the butter intake group compared to those of the control.

The potential role of platelet PAI-1 in t-PA mediated clot lysis of platelet rich plasma

The potential role of platelets in platelet rich plasma clot lysis induced by tissue plasminogen activator (t-PA) was investigated. At the various concentrations of both single chain t-PA (sct-PA) and two chain t-PA (tct-PA) (1.5nM, 3nM, and 6nM), we compared the t-PA mediated lysis time of platelet rich plasma clot (PRP-clot) with that of platelet poor plasma clot (PPP-clot). At the concentrations ranged from 1.5 to 6 nM of both types of t-PA, the clot lysis time of PRP-clot was longer than that of PPP-clot. This elongation was more significant in the tct-PA induced clot lysis than that in the sct-PA induced clot lysis. At the concentration of 3nM of tct-PA, the lysis time of PRP-clot was longer by a factor of 30% in comparison with that of PPP-clot. When the release and the aggregation of platelets were blocked by prostaglandin E1 (PGE1) and theophylline in this experiment, the lysis time of PRP-clot was essentially the same as that of PPP-clot. We then measured the antigen levels of total PAI-1 and t-PA-PAI-1 complex in the lyzed solutions of PRP-clot and PPP-clot to analyse the possible effect of plasminogen activator inhibitor-1 (PAI-1) present in platelets. Most of PAI-1 in a PPP-clot lyzed sample existed as t-PA-PAI-1 complex. In the lyzed solution of PRP-clot, however, the antigen levels of both total PAI-1 and t-PA-PAI-1 complex were significantly higher than those in PPP-clot, and larger amounts of PAI-1 existed as free PAI-1 which possesses activity. These data suggest that at least certain amounts of PAI-1 in platelets exist as an active form and inhibits t-PA activity resulting in the prolongation of the clot lysis time. Activation of platelets, therefore, seems to play an important role in the platelet rich plasma clot lysis induced by t-PA.

Heparin and heparan sulfate enhancement of the inhibitory activity of plasminogen activator inhibitor type 1 toward urokinase type plasminogen activator

To study effects of glycosaminoglycan on the interaction between two chain urokinase type plasminogen activator (tcu-PA) (EC 3.4.21.31) and plasminogen activator inhibitor type 1 (PAI-1) the second order rate constant (k1) between high molecular weight tcu-PA and active recombinant prokaryotic PAI-1 (rpPAI-1) was determined employing a continuous method using chromogenic substrate S-2444 either in the presence or absence of various kinds of glycosaminoglycans. k1 was (5.9 +/- 1.6).10(6)/mol per s in the absence of effector molecule, and following addition of heparin (1.0 U/ml) k1 was enhanced to (3.22 +/- 0.73).10(7). A significant enhancement of k1 was also obtained by heparan sulfate (1.87 +/- 0.25).10(7). Dermatan sulfate or chondroitin sulfate did not show a significant effect on k1 although a slight decrease was obtained by mono-dextran sulfate (4.2 +/- 1.2).10(6). The intrinsic fluorescence of rpPAI-1 was shown to be slightly increased following addition of heparin (1.49 +/- 0.22%, n = 6), suggesting that heparin may enhance the inhibitory activity of PAI-1 toward tcu-PA both by a template mechanism and by a modification of PAI-1 structure.

Effect of acute alcohol ingestion on the fibrinolytic activity

Effects of alcohol consumption on the function of fibrinolytic system were studied. The group which consisted of 12 male volunteers consumed standardised amounts of whisky. 3 blood samples for the assessment of euglobulin clot lysis time, t-PA, urokinase, PAI-1 and its complex with t-PA were taken: 1 before, 1 taken 45 min after and 1 on the next morning after drinking. No change was noted in those parameters 45 min after the consumption of ethanol. Alcohol ingestion had a specific tendency to decrease fibrinolytic activity on the next day after drinking. Therefore, acetaldehyde, the main metabolite of ethanol, rather than ethanol itself may be responsible for the enhancement of PAI-1 secretion by endothelial cells. These results show that the fibrinolytic system does not account for commonly accepted protection from coronary heart disease mediated by moderate consumption of alcohol.

Electric-foot-shock induced the supression of fibrinolytic activity in rats

Electric-foot-shock was given to rats to initiate constant mental stress and its effect on fibrinolytic activity was analyzed. After the termination of electric-foot-shock which was given for an hour, euglobulin clot lysis time in the stressed group significantly prolonged than those in the control group. tissue plasminogen activator activity was also significantly lower in the stressed group. These effects lasted at least for an hour and returned to the control values 24 hours after the stress. Whole blood serotonin levels, which mainly show serotonin contents in platelets, were higher in the stressed group. A negative correlation between whole blood serotonin and tPA activity in the stressed group was obtained. These results suggest that prolonged mental stress impairs fibrinolysis by decreasing tPA activity with a concomitant increase of serotonin contents in platelets.

The Role of Platelet PAI-1 in the Platelet Rich Plasma Clot Lysis

Plasminogen activator inhibitor-1 (PAI-1) is physiologically the most important inhibitor of tissue plasminogen activator(t-PA) in plasma(1). It has been suggested that more than 80 to 90% of PAI-1 is stored in the platelet α-granules(2,3). It is not clear, however, whether PAI-1 released from platelet plays an important role in the clot lysis.

The Cleavage of a Serine Protease Inhibitor at the Reactive Center by Its Target Protease: Analysis of the Substrate-Like Form of a Serpin

Many enzymes involved in coagulation and fibrinolysis have a serine residue at their active site and they are therefore defined as serine proteases. In plasma the activity of such proteases are controlled by specific inhibitors belonging to the serine protease inhibitor (SERPIN) family that forms stoichiometric tight complexes with the target proteases. The inhibitors of the SERPIN family are thought to have a common tertiary structure[1] with a reactive center located on an exposed loop denoted “the strained loop”, situated near the carboxyl terminus of the inhibitor molecule[2–4]. These inhibitors interact with their target proteases by providing a so called “bait” residue (P1 residue), that mimics the normal substrate of the target proteases[2,3,5]. The complex is supposed to exist either as Michaelis type, tetrahedral type[6,7] or acylintermediate[8,9], although which stage is the stable state is not clear. Recently, we discovered that the SERPIN plasminogen activator inhibitor 1 (PAI-1) can be cleaved by plasminogen activators at its reactive site and that treatment with low concentrations of sodium dodecyl sulphate (SDS) converts active PAI-1 to a form that is cleaved like a substrate[10]. Since the conformation of the cleavable PAI-1 is completely different from its native form we defined it as the substrate-like form of PAI-1. The conversion of the inhibitor to a substrate like form is caused by a conformational change without alteration of the amino acid sequence and is of interest in order to study the mechanism of the interaction between serine protease and SERPIN. In this manuscript we demonstrate the cleavage of PAI-1 and discuss the possible mechanism by which a SERPIN is cleaved by its target protease.

Effect of Calcium Ion on the Euglobulin Clot Lysis Time: Significant shortening by physiological concentration of calcium ion

Euglobulin clot lysis time (ECLT) assay has been considered to be useful to assess the systemic fibrinolytic activity [1] [2]. ECLT is respected to show mainly the activity of tissue plasminogen activator (tPA), since antibody against tPA significantly prolonged ECLT. We have recently shown, however, that ECLT is primarily determined by the amounts of plasminogen activator inhibitor type 1 (PAI-1) due to the fact that ECLT inversely correlates with the concentration of PAI-1 whereas it did not have any meaningful correlation with tPA antigen level [3]. Taken together these results suggest that the activity of tPA either in plasma or in the euglobulin fraction is controlled by the balance of the concentrations of tPA and PAI-1 and that ECLT reflects the amounts of the active form of tPA which is not bound to PAI-l. We also recently showed that the addition of kaolin to ECLT (kaolin activated ECLT), in which the contact phase of both the coagulation and fibrinolysis is fully activated, significantly shortened regular ECLT, suggesting that intrinsic fibrinolytic pathway is not mainly involved in regular ECLT [3]. In this paper we report another pathway to enhance ECLT which is induced by the addition of physiological concentration of calcium ion. We believe it important because Ca2+ has been considered to work only to prolong fibrinolysis.