Fibrinolysis In Man Research Articles

Histone deacetylase inhibitors stimulate tissue-type plasminogen activator production in vascular endothelial cells

A reduced capacity for acute tissue-type plasminogen activator (t-PA) release is likely to be associated with an impaired endogenous defense against intravascular thrombosis. Efficient approaches to pharmacologically restore a defective t-PA release have been lacking, but recent observations suggest that histone deacetylase inhibitors (HDACis) enhance t-PA production in vitro. HDACis have diverse chemical structures and different HDAC-enzyme sub-class targeting. We here compared the effects of several clinically used HDACis on t-PA production in endothelial cells. Human umbilical vein endothelial cells were exposed to a panel of 11 different HDACis and t-PA mRNA and protein levels were quantified. All HDACis dose-dependently stimulated t-PA mRNA and protein expression with similar maximal efficacy but with different potencies. Already at low concentrations, the majority of inhibitors caused significant and sustained effects on t-PA production. In addition, selected HDACis were capable of normalizing t-PA production when suppressed by the inflammatory cytokine TNF-α. We conclude that HDACis targeting classical HDAC enzymes are powerful inducers of t-PA expression in cultured endothelial cells and could be promising candidates for pharmacological modulation of endogenous fibrinolysis in man.

Role of Histone Acetylation in the Stimulatory Effect of Valproic Acid on Vascular Endothelial Tissue-Type Plasminogen Activator Expression

AimsStimulated release of tissue-type plasminogen activator (t-PA) is pivotal for an intravascular fibrinolytic response and protects the circulation from occluding thrombosis. Hence, an impaired t-PA production is associated with increased risk for atherothrombotic events. A pharmacological means to stimulate the production of this enzyme may thus be desirable. We investigated if the anti-epileptic drug valproic acid (VPA) is capable of enhancing t-PA expression in vitro in vascular endothelial cells, and further examined if its histone deacetylase (HDAC)-inhibitory activity is of importance for regulating t-PA expression.Methods and ResultsHuman endothelial cells were exposed to valproic acid and t-PA mRNA and protein levels were quantified. Potential changes in histone acetylation status globally and at the t-PA promoter were examined by western blot and chromatin immunoprecipitation. Valproic acid dose-dependently stimulated t-PA mRNA and protein expression in endothelial cells reaching a 2–4-fold increase at clinically relevant concentrations and 10-fold increase at maximal concentrations. Transcription profiling analysis revealed that t-PA is selectively targeted by this agent. Augmented histone acetylation was detected at the t-PA transcription start site, and an attenuated VPA-response was observed with siRNA knock of HDAC3, HDAC5 and HDAC7.ConclusionsValproic acid induces t-PA expression in cultured endothelial cells, and this is associated with increased histone acetylation at the t-PA promoter. Given the apparent potency of valproic acid in stimulating t-PA expression in vitro this substance may be a candidate for pharmacological modulation of endogenous fibrinolysis in man.

Endothelin-1 does not contribute to the release of tissue plasminogen activator in vivo in man

Objectives: Endothelin-1 is a potent endothelium-derived vasoconstrictor peptide with autocrine and paracrine actions. Tissue plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI-1), are also released from the vascular endothelium and play a pivotal role in endogenous fibrinolysis. We, therefore, examined the effects of exogenous and endogenous endothelin-1 on t-PA and PAI-1 release in vivo in man. Design: Open investigative study. Setting: Clinical Research Centre, University of Edinburgh. Subjects: Fourteen healthy male volunteers. Interventions: Unilateral brachial artery infusions of endothelin-1 at 2.5 and 10 pmol/min, and the selective endothelin type B (ET B) receptor antagonist, BQ-788, at 1 nmol/min. Main outcome measures: Blood, flow and plasma fibrinolytic factors were measured in both forearms using venous occlusion plethysmography and venous blood samples withdrawn from the antecubital fossae. Results: Endothelin-1 caused a slow onset dose-dependent forearm vasoconstriction ( P<0.001) with a maximal reduction in blood flow of 40 ± 4% and 63 ± 3% at 2.5 and 10 pmol/min respectively. BQ-788 also caused a slow onset reduction in forearm blood flow ( P<0.001) reaching a maximum of 21 ± 3%. However, BQ-788 and endothelin-1 did not affect plasma concentrations of t-PA or PAI-1 in the venous effluent of the infused forearm. Conclusions: Despite sustaining significant vasoconstriction, neither endogenous nor exogenous endothelin-1 influences the release of t-PA or PAI-1 in the forearm vascular bed of man. This suggests that endothelin-1 does not provide a major contribution to the regulation of endogenous fibrinolysis in man.

375 The effect of ancrod on fibrin formation and fibrinolysis in man

375 The effect of ancrod on fibrin formation and fibrinolysis in man

Effects of the converting enzyme inhibitor captopril on blood coagulation and fibrinolysis in man.

Systematic blood coagulation analyses were conducted in 32 severely hypertensive patients treated with the angiotensin converting enzyme inhibitor captopril. Two hours after the first captopril dose, fibrin monomer complexes had already increased. This rise was even more distinct after 26 h and 1 week. Tests after 6 and 12 months of therapy showed a regression of fibrin monomer complexes to pretreatment values. In several patients with a marked increase in fibrin monomer complexes, the partial thromboplastin time (PTT) became shorter and antiplasmin activity increased. The most pronounced increase in fibrin monomer complexes was seen in patients with a rapid and excessive blood pressure reduction. The concentration of fibrin monomer complexes also rose in 15 healthy normotensive subjects, after a single oral dose of captopril (25 mg). Additionally, the PTT was shortened and antiplasmin significantly rose. An inhibition of fibrinolysis by captopril could be demonstrated by the effect on fibrin plates and thrombus weight after streptokinase. Out of 58 patients with severe hypertension and atherosclerosis treated with captopril, 7 patients suffered vascular complications during antihypertensive therapy: myocardial infarction (n = 2), coronary insufficiency (1), cerebral ischemia (1), renal insufficiency (3). These ischemic lesions may be partly explained by the alterations of coagulation and fibrinolysis under captopril therapy.

Natural inhibitors of fibrinolysis

I NTRAVASCULAR fibrin deposits or thrombus formation is a major pathophysiologic phenomenon in health and disease. The dissolution of such fibrin deposits or thrombi in vivo is achieved by plasmin formed from plasminogen by plasminogen activator that has been added extrinsically for the therapeutic purposes (urokinase and streptokinase) or endogenously generated in the vascular trees (blood activators or vascular activator). Regardless of the manner in which plasminogen is activated dissolution of fibrin is believed to be checked or retarded by natural inhibitors of fibrinolysis. Natural inhibitors of fibrinolysis inhibit the activation of plasminogen to plasmin or act upon the plasmin already formed. Although the presence versus absence of the former inhibitors in plasma has been a subject of debate, the presence of inhibitors of plasmin has been firmly established. The presence in tissues of inhibitors acting upon plasminogen activator is also known. There are synthetic inhibitors (epsilon-amino caproic acid, tranexamic acid, etc.) and bovine trypsin inhibitor (aprotinin) available for therapeutic purposes in the regulation of fibrinolysis. These extrinsic inhibitors are beyond the scope of this review and only endogenously occurring inhibitors of fibrinolysis in man will be discussed.

Molecular Mechanisms of Fibrinolysis in Man

Molecular Mechanisms of Fibrinolysis in Man

Blood coagulation and fibrinolysis in man after myocardial infarction compared with a representative population sample

Blood coagulation and fibrinolysis were studied in a group of 83 patients 3 months after their first myocardial infarction (MI). All patients were below 55 years of age (mean 49 years). A randomly selected subsample ( n = 76) of 55-year-old men from the general population was used as a control series. The MI group had shorter clotting time of recalcified plasma, shorter partial thromboplastin time (PTT) in silicone tubes but not in glass tubes and lower factor II-VII-X activity than the control series. No other significant differences were found. The possibilities of finding out the significance of blood coagulation and fibrinolysis for coronary thrombosis and MI are discussed.

Effects of adrenaline and alprenolol (Aptin�) on blood coagulation and fibrinolysis in man

The effects of intravenous infusions of adrenaline alone, and after alprenolol (Aptin®) pre-treatment, on whole blood clotting time, factor VIII, fibrinolysis, partial thromboplastin time (PTT), platelet count and fibrinogen have been studied in four healthy, fasting male subjects by double-blind technique using saline infusions as control. The adrenaline infusion caused increases in factor VIII levels, fibrinolysis and platelet counts and a decrease in whole blood clotting time. Alprenolol inhibited the effects of adrenaline on factor VIII and fibrinolysis in all subjects, and the effects on whole blood clotting time in all but one subject. The adrenaline-induced rise in platelet count was not significantly influenced by alprenolol pre-treatment. No effects were seen of the alprenolol infusion itself.

The kidney and fibrinolysis in man.

Wahrend Nierenoperationen vorgenommene Messungen ergaben, dass eine in der Funktion stark eingeschrankte Niere weniger Urokinase in den Urin ausscheidet, ohne dass jedoch die fibrinolytische Aktivitat im Venenblut zunimmt. Es wird daraus geschlossen, dass die Nieren keine erheblichen Mengen Plasminogen-aktivierender Stoffe an das Blut abgeben.

Streptokinase as a thrombolytic agent

E XTENSIVE clinical studies in manycenters have indicated the thrombolytic potential of various intravenously infused plasmin preparations. Interpretations of these clinical studies are complicated by (1) wide variations in the natural course of disease; (2) an antiinflammatory effect which may follow clinical use of the enzymes; and (3) inadequate definition and standardization of these plasmin preparations containing varying amounts of streptokinase, streptokinase-activated plasminogen as the activator complex, and streptokinaseactivated plasminogen as plasmin. In the present studies, forty-one experimental thrombi, 2 to 5 inches long, were induced in the superficial veins in the arms and legs of human volunteers by direct irritation of the intima with a dental broach or by chemical irritation with sodium morrhuate. The position and size of the thrombi were determined by clinical observation and by venograms. Various ~brinolytic systems were then produced by infusing streptokinase (SK) into a contralateral extremity at varying time intervals after formation of clots. Similar studies were also made on a patient with a fibrinolytic system due to circulating endogenous activator. Thus, objective comparison was made of the various methods to produce thrombolysis and to prevent reformation of the thrombi. Three different systems were established by varying the amount of SK infused : (1) moderate amounts of circulating plasmin with no measurable free SK (method P) ; (2) negligible plasmin with moderate amounts of free SK or activator (method SK) ; and (3) small amounts of both free SK and plasmin (method SK-P). Thrombolysis was also tested in a fourth system, moderate amounts of endogenous activator with negligible amounts of plasmin (method A). These systems were produced and utilized under rigidly controlled biochemical conditions, each system representing a different, sharply defined agent for the production of thrombolysis. Since circulating SK in human plasma could not be distinguished by assay from the activator complex of SK-plasminogen under these experimental conditions, the use of endogenous human activator also aided in the evaluation of the SK-activator complex as a thrombolytic agent. Figure 1 represents most of the important components of the human fibrinolytic system. The kinases from tissue, plasma or streptokinase appear to react stoichiometrically with a proenzyme in plasminogen to form an activator complex.1-3 This complex, designated “activator,” or the naturally occurring activator in urine, plasma or tissues, reacts catalytically with plasminogen (the proteolytic precursor) to form a proteolytic enzyme, plasmin.*,” The plasmin in turn acts upon fibrin, fibrinogen and other proteins to produce soluble split products of these native proteins. In order to produce pathologic fibrinolysis in man, this proteolytic system must be uninhibited. Normally, there may be interference with the system at three levels. Streptokinase may be neutralized 1:’ _ a specific antibody and by less specific inhtbltorsfi Activator may be neutralized by inhibitors found primarily in the alpha-2 globulin fraction of

Butter, corn oil and fibrinolysis in rats.

We have recently reported a dietary method for the production of thrombosis and infarcts in rats and have suggested that part of the mechanism of thrombosis in these animals was interference by the diet with some hematologic factor, possibly fibrinolysis. In the study reported herein, we have developed a method for studying fibrinolysis in rats in vitro, utilizing an electronic device called the Thrombelastograph. Results of this study correlate well with the previously demonstirated thrombogenic properties of 2 of the diets. Rats on a "butter-thiouracil-cholesterol-bile salt" diet (known to produce thrombi and infarcts) have prolonged clotlysis times, as compared with rats on a similar diet (and not thus far associated with thrombi and infarcts) in which corn oil is substituted for butter. Although the in vitro test is performed under highly artificial conditions, they are carefully standardized and the fact that the results correlate well with previously demonstrated thrombogenic properties of the diet would suggest that it has practical value for the study of thrombogenic effects of various diets in experimental animals. It is possible that it will prove to be even more useful in the study of fibrinolysis in man.

Intravenous protein-free pyrogen; a powerful fibrinolytic agent in man.

Administration of a variety of nonfibrinolytic, nonenzymatic substances induces fibrinolysis in man. The most potent materials of this type known at present are purified protein-free pyrogens. Their action after intravenous injection on the human fibrinolytic system, on some of its components, and on the clotting system of 67 individuals has been studied. The possible therapeutic implications are discussed of the marked fibrinolysis.