Previous studies have shown that overall fibrinolytic activity in blood follows a diurnal rhythm with a peak in the morning and a trough in the evening. The purpose of this study was to determine which fibrinolytic factor(s) was responsible for this diurnal rhythm. Resting and postvenous occlusion tissue-type plasminogen activator (t-PA) activity, resting t-PA antigen, and resting plasminogen activator inhibitor 1 (PAI-1) activity were measured in the morning and evening in 33 healthy men (mean age, 31 years) and in 15 patients (mean age, 57 years) with previous myocardial infarction or unstable angina. PAI-1 activity and t-PA antigen were significantly higher (p <0.01) in the morning compared with the evening in controls and patients. In contrast, resting t-PA activity was significantly lower in the morning (p <0.01) in both groups and was inversely correlated with PAI-1 activity (r= -0.57, p < 0.0001). Postvenous occlusion t-PA activity and t-PA capacity were not significantly different between morning and evening in either group. Because t-PA antigen levels and PAI-1 activity were highest in the morning, the variation in t-PA activity was probably not due to decreased secretion of t-PA but instead to changes in the secretion of PAI-1. Our findings indicate that diurnal variations in PAI-1 activity may reduce fibrinolytic activity in the morning in healthy individuals and in patients with coronary artery disease. (Circulation 1989;79:101-106) I ncidence of acute myocardial infarction follows a circadian rhythm with a peak in the morning and a trough in the evening. Muller et all found a threefold increase in the frequency of myocardial infarction at 9 AM compared with 11 PM. A similar diurnal variation in the time of onset has been found for a number of other disorders associated with arterial thrombosis, including sudden cardiac death, angina at rest, and stroke.2-4A number of different factors may play a role in the circadian variation of arterial thrombosis. Heart rate, arterial blood pressure, and catecholamine levels have been shown to peak in the morning hours, indicating that coronary vasoconstriction may be increased in the morning as well.5-7Another approach has been to study factors that may increase the risk of initiating thrombus formation. Platelet aggregability has been found to increase in the morning after arising.8,9 A concurrent increase in From plasma epinephrine and norepinephrine were also found. These studies have concentrated on factors that may increase the risk of vasoconstriction or thrombus initiation. Another possible explanation would be alterations in blood components that depress the rate of thrombus removal. In vivo, newly forming thrombi are removed by the fibrinolytic system. Fibrinolysis is initiated by tissue-type plasminogen activator (t-PA), an enzyme secreted by endothelial cells that, in the presence of fibrin, converts the proenzyme plasminogen into its active form, plasmin.10 In turn, plasmin proteolytically degrades the fibrin that holds the thrombus together. Th...

A comparison of the physical-chemical properties of human, bovine, and horse antithrombin III has been made. These three plasma proteins are strong inhibitors of bovine factor Xa and form a 1:1 molar complex with this coagulation enzyme. Human, bovine, and horse antithrombin III are glycoproteins containing hexose, hexosamine, and neuraminic acid. The total carbohydrate was 9, 12, and 16% for human, bovine, and horse antithrombin III, respectively. These proteins have a similar amino acid composition, although some monor variations were noted. Each antithrombin III is composed of a single polypeptide chain with an amino-terminal histidine residue. Of the first 17 amino-terminal residues, only three differences were noted between the three proteins. These occur in position 2 which is occupied by Gly, Arg, and Trp in human, bovine, and horse, respectively; position 6 which has a deletion in human antithrombin III; and position 8 where Ile in human and horse antithrombin III has been replaced by Val in the bovine preparation. The remainder of the first 17 residues is the same in all three proteins. The molecular weights for the bovine and horse preparation were 56 600 and 52 500, respectively, as determined by sedimentation equilibrium in the presence of guanidine hydrochloride. Some immunological cross-reactivity was also observed between the three different proteins.

Factor IXa and factor Xabeta are serine proteases which participate in the middle phase of blood coagulation. These two enzymes are inhibited by antithrombin III by the formation of an enzyme-inhibitor complex containing 1 mol of enzyme and 1 mol of antithrombin III. The complex was readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and loss of coagulant or esterase activity at increasing concentrations of inhibitor. The inactivation of factor IXa by antithrombin III was relatively slow, but the reaction was greatly accelerated by the addition of heparin.

Human and bovine antithrombin II/III have been isolated by a simple procedure essentially using only affinity chromatography on heparin-agarose and polyethylene glycol precipitation. An additional ammonium sulphate step removes polyethylene glycol and trace contaminants. The final preparation is homogeneous by SDS-, disc- and agar slide electrophoresis. End group analysis of human antithrombin II/III shows histidine as the N-terminal amino acid. The pure preparation showed both progressive antithrombin activity and heparin cofactor activity.

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