Increased plasma fibrinolysis and tissue-type plasminogen activator/tissue-type plasminogen activator inhibitor ratios after ethanol withdrawal in chronic alcoholics.

The effects of alcohol withdrawal on fibrinolysis were studied in 10 middle-aged male chronic alcoholics institutionalized for withdrawal therapy. All patients were sampled on admission [day 1 (D1)] and 21 days after alcohol withdrawal [day 22 (D22)]. The overall plasma fibrinolytic capacity (OFC) was assayed by measuring the ability of patient plasma to generate D-dimers from a standardized fibrin clot, and tissue-type plasminogen activator (t-PA) and t-PA inhibitor (PAI-1) levels were assayed together with serum cholesterol, triglyceride and cholesterol fractions. At D22, the OFC significantly increased in seven patients [D1 = 10 +/- 0.7 microg/h (mean +/- SD), D22 = 17 +/- 7.4 microg/h; P < 0.01], while t-PA and PAI-1 levels decreased in all patients but two (t-PA: D1 = 16.6 +/- 5 ng/ml, D22 = 10.2 +/- 3.8 ng/ml; P < 0.001; and PAI-1: D1 = 46 +/- 39 ng/ml, D22 = 21 +/- 28 ng/ml; P < 0.01). This study clearly demonstrates an increase in overall fibrinolytic activity after alcohol withdrawal, which is mainly due to a decrease in PAI-1 levels. These changes induced by alcohol abstinence might provide clear benefit by reducing the risk of thromboembolic events and particularly of stroke associated with elevated PAI-1 levels described in heavy drinkers.     

Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1.

Fibrinolysis is regulated in part by the interaction between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1, a serine protease inhibitor of the serpin family). It is known from our earlier work that deletion of a loop of amino acids (residues 296-302) from the serine protease domain of t-PA suppresses the interaction between the two proteins without altering the reactivity of t-PA towards its substrate, plasminogen. To define more precisely the role of individual residues within this loop, we have used site-directed mutagenesis to replace Lys-296, Arg-298, and Arg-299 with negatively charged glutamic residues. Replacement of all three positively charged amino acids generates a variant of t-PA that associates inefficiently with PAI-1 and is highly resistant to inhibition by the serpin. Two t-PAs with point mutations (Arg-298----Glu and Arg-299----Glu) are partially resistant to inhibition by PAI-1 and associate with the serpin at intermediate rates. Other point mutations (Lys-296----Glu, His-297----Glu, and Pro-301----Gly) do not detectably affect the interaction of t-PA with PAI-1. None of these substitutions has a significant effect on the rate of catalysis by t-PA or on the affinity of the enzyme for its substrate, plasminogen. On the basis of these results, we propose a model in which positively charged residues located in a surface loop near the active site of t-PA form ionic bonds with complementary negatively charged residues C-terminal to the reactive center of PAI-1.     

Changes in plasminogen activator inhibitor 1 and tissue-type plasminogen activator during exercise in patients with coronary artery disease

As depressed fibrinolysis is implicated in the pathogenesis of coronary artery disease, we have studied the activation of fibrinolysis during maximal, symptom-limited exercise in a group of 68 men. After exercise they were divided, according to their coronary angiography and exercise 201Tl emission computed tomography results, into three groups. Group 1: persons with normal exercise 201Tl emission computed tomography results and no underlying diseases who served as controls; group 2: patients with coronary artery disease without exercise-induced myocardial ischemia, and group 3: patients with coronary artery disease with transient, exercise-induced myocardial ischemia. Before and at peak exercise we measured the plasminogen activator activity (PAA) in the euglobulin fraction of plasma by an amidolytic method and the concentrations of tissue plasminogen activator (t-PA), activator-inhibitor complex - plasminogen activator inhibitor 1 (PAI-1) complexed with t-PA - and total PAI-1 by enzyme immunoassay. The concentration of free PAI-1 in plasma was calculated by subtraction of the concentration of activator-inhibitor complex from that of total PAI-1. Under basal conditions, group 3 had significantly higher free and total PAI-1 levels than group 1. There were no statistically significant differences between the three groups in PAA, t-PA, and activator-inhibitor complex levels. At peak exercise, group 1 showed the highest release of t-PA accompanied with highest increases in PAA as well as in activator-inhibitor complex, the proportion of released t-PA antigen not bound to PAI-1 being highest in group 1. Free PAI-1 decreased significantly, but there were no differences between individual groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular advances in plasminogen activator inhibitor 1 interaction with thrombin and tissue-type plasminogen activator

Plasminogen activator inhibitor 1 (PAI-1) is a glycoprotein that controls the activity of the key enzymes of the fibrinolytic system, the serine proteases tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Inhibition is accomplished by rapid formation of inactive, equimolar PAI-1/PA complexes. The physiological importance of PAI-1 for the fibrinolytic system has been underscored by the observation that in humans, a homozygous defect results in hemorrhagic episodes. In addition to its function in surveillance of the integrity of clots, PAI-1 efficiently inhibits the serine protease thrombin in vitro, provided that either the high molecular weight glycosaminoglycan heparin or the glycoprotein vitronectin is present. These cofactors accelerate the rate of thrombin inhibition by PAI-1 by more than two orders of magnitude. Inhibition of thrombin by PAI-1 proceeds according to a "suicide substrate mechanism," typified by a branched reaction pathway, leading either to stable PAI-1/thrombin complexes or to degradation of the inhibitor and recycling of enzyme. The cofactors heparin and vitronectin, although increasing inhibition through different mechanisms, essentially promote PAI-1 degradation by thrombin. In view of the multitude of functions attributed to thrombin, the authors propose that the relevance of thrombin inhibition by PAI-1 is to restrict its mitogenic activity, rather than to affect its coagulation function in plasma. (Trends Cardiovasc Med 1997;7:47-51). ? 1997, Elsevier Science Inc.     

Reactive hyperemia and tissue-type plasminogen activator release in hypertensive men.

A relationship may exist between endothelial-mediated vasodilation and tissue-type plasminogen activator (t-PA) release. However, the existing evidence is mainly based upon exogenous agonist administration, and needs testing under more physiological conditions. We evaluated the link between t-PA, the key fibrinolytic factor in man, and forearm reactive hyperemia, a model of endogenous endothelial-mediated vasodilation, in 13 uncomplicated hypertensive subjects and six elderly hypertensive patients with atherosclerotic peripheral vascular disease and hypercholesterolemia (i.e a group in whom post-ischemic hyperemia was probably defective because of dysfunctional endothelium). To characterize further the phenomenon, 29 additional uncomplicated hypertensive patients underwent intra-arterial drug infusions. Study variables were forearm blood flow (strain-gauge plethysmography), arterial and venous concentrations of t-PA mass concentrations, and calculated net release (forearm plasma flow x veno-arterial differences). Reactive hyperemia was induced by inflating a cuff midway between systolic and diastolic pressure for 10 min; blood and forearm blood flow were sampled before and after cuff release. Post-ischemic t-PA release increased in uncomplicated hypertensives, and did not change in hypercholesterolemic atherosclerotic patients in whom post-ischemic vasodilation was negligible. Local adenosine (n = 9), acetylcholine (n = 12) and bradykinin (n = 8) vasodilated similarly, but only bradykinin increased t-PA release. Thus, reactive hyperemia stimulates t-PA release, and that relationship is altered when endothelium is dysfunctional. Release of t-PA is independent of forearm vasodilatation, adenosine or biological products of muscarinic stimulation and may, perhaps, be related to the activity of the endogenous kininogen/kinin system.     

Biochemical determinants of clearance of tissue-type plasminogen activator from the circulation

Biochemical modification of tissue-type plasminogen activator (t-PA) designed to alter pharmacokinetics and pharmacodynamics offers promise for development of pharmaceuticals particularly suitable for treatment of specific disorders and for induction of coronary thrombolysis by intramuscular as well as intravenous administration. Accordingly, to identify biochemical determinants of clearance of t-PA from the circulation, we injected rabbits intravenously with three different preparations of t-PA synthesized from the same human gene and expressed in Chinese hamster ovary cells cultured under disparate conditions. Influences of glycosylation on clearance were defined by experiments with enzymatically treated t-PA in which clearance was assessed with concomitant administration of selected neoglycoproteins that compete with t-PA for specific glycoprotein receptors. The role of an intact active catalytic site, as reflected by differences in clearance with and without prior treatment of t-PA with the protease inhibitor PPACK, was defined also. Results indicate that clearance is altered by inhibition of the active site and that the nature and extent of glycosylation--not evident simply by analysis of peptide structure--influence clearance as well. These findings suggest that mannose/N-acetylglucosamine-specific glycoprotein receptors expressed on hepatic reticuloendothelial cells participate in clearance of t-PA from the circulation but that galactose-specific glycoprotein receptors probably do not. The observations may explain differences in clearance seen with different preparations of t-PA that have been seen in clinical pilot studies and may identify biochemical determinants of clearance amenable to modification for development of agents with potentially desirable, specific biological properties.     

PAF-acether-induced release of tissue-type plasminogen activator from vessel walls

Platelet-activating factor (PAF-acether; 1-0-octadecyl-2-acetyl-sn- glyceryl-3-phosphorylcholine) induced the release of plasminogen activator in rat, both in vivo and in perfused hind legs. The released plasminogen activator was shown by immunologic and functional criteria to be tissue-type plasminogen activator (t-PA). Release of t-PA by PAF- acether could be inhibited by phospholipase inhibitors and by lipoxygenase inhibitors, but not by cyclooxygenase inhibitors. It is suggested that PAF-acether induces the release of t-PA from vascular endothelial cells by the (calcium-dependent) activation of a phospholipase-lipoxygenase pathway.     

Tissue plasminogen activator release and plasminogen activator inhibitor levels in coronary artery disease

Unstable angina and Q wave myocardial infarction are associated with intraluminal coronary thrombosis, a process to which impaired fibrinolysis may contribute. The authors examined the extrinsic fibrinolytic system, including tissue plasminogen activator antigen, plasminogen activator inhibitor activity and antigen, and euglobulin clot lysis time before and after venous occlusion in 56 patients undergoing coronary angiography for chest pain syndromes and in 16 healthy controls. Fibrinolysis variables were similar (with greater than 95% confidence) in the patients with thrombus-associated coronary syndromes as compared with those with chest pain syndromes not due to coronary thrombosis. These fibrinolytic variables were also similar to those in patients without coronary artery disease and in healthy controls. Their data suggest that defective fibrinolysis is not involved, at least systemically, in the pathogenesis of thrombus-associated coronary artery syndromes.     

Baseline fibrinolytic state and the risk of future venous thrombosis. A prospective study of endogenous tissue-type plasminogen activator and plasminogen activator inhibitor.

BACKGROUND. Although isolated abnormalities of plasminogen activation and inhibition have been reported among selected patients with venous thrombosis, it is unclear whether these deficiencies of fibrinolysis are important risk factors for thromboembolic disease. METHODS AND RESULTS. To evaluate whether baseline levels of endogenous tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) predict the future occurrence of venous thrombosis, levels of these proteins were measured in prospectively collected plasma samples from 55 participants in the Physicians' Health Study who later developed deep venous thrombosis or pulmonary embolism and from an equal number of age- and smoking-matched control subjects who remained free of vascular disease during a mean follow-up period of 60.2 months. Overall, there were no statistically significant differences between case patients and control subjects in baseline levels of PAI-1 (50.5 versus 59.5 ng/ml, p = 0.26), t-PA (13.4 versus 13.3 ng/ml, p = 0.94), or PAI-1:t-PA ratio (6.84 versus 6.58, p = 0.82). No evidence of a threshold effect or trend was seen when these data were analyzed by increasing quartiles of PAI-1 (p = 0.73), t-PA (p = 0.62), or PAI-1:t-PA ratio (p = 0.93). These results were unchanged after multivariate analysis that simultaneously controlled for other baseline cardiovascular risk factors. CONCLUSIONS. In contrast to previous uncontrolled case series and smaller retrospective studies, these prospective data provide strong evidence that baseline fibrinolytic state, as measured by t-PA and PAI-1, does not predict the occurrence of future venous thrombosis.     

Origins of the specificity of tissue-type plasminogen activator.

The role of subsite interactions in defining the stringent substrate specificity of tissue-type plasminogen activator (t-PA) has been examined by using an fd phage library that displayed random hexapeptide sequences and contained 2 x 10(8) independent recombinants. Forty-four individual hexapeptides were isolated and identified as improved substrates for t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide that contained the primary sequence of the actual cleavage site in plasminogen. These results suggest that small peptides can mimic determinants that mediate specific proteolysis, emphasize the importance of subsite interactions in determining protease specificity, and have important implications for the evolution of protease cascades.     

Circulating tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in proliferative diabetic retinopathy: a pilot study

Several haemostatic abnormalities are associated with proliferative diabetic retinopathy. While abnormalities in plasma fibrinolytic activity have been described in diabetic retinopathy, platelets (a rich source of plasminogen activator inhibitor type 1, PAI-1) have received little attention. As a result, little is known about the fibrinolytic potential of circulating whole blood in diabetic retinopathy. The concentrations of tissue-type plasminogen activator (t-PA) and of its fast-acting inhibitor. PAI-1 were measured in plasma from eight patients with type 1 diabetes complicated by proliferative retinopathy, and from eight patients with type 1 diabetes and background or no retinopathy, matched for age, sex and duration of diabetes. The concentration of PAI-1 in platelets was also measured. The ratio of t-PTA to PAI-1 in plasma was significantly higher in patients with proliverative retinopathy than in those without (0.66 vs. 0.37, p < 0.02). The average quantitiy of PAI-1 per patelet was significantly lower in the group with proliferative retinopathy (0.33 vs. 0.50 ng/10⁶ platelets, p < 0.02). These data suggest that among patients with type 1 diabetes, total circulating fibrinolytic potential is higher in those with proliferative retinopathy. Received: 14 October 1998 / Accepted in revised form: 30 September 1999     

Acetylcholine-mediated vasodilatation and tissue-type plasminogen activator release in normal and hypertensive men

Muscarinic agents release tissue plasminogen activator (t-PA) in the forearm circulation of normal subjects, but no information exists about their effect in those hypertensive patients in whom the response to endothelial-mediated vasodilators is blunted. Acetylcholine, an endothelium-dependent vasodilator and a muscarinic agonist that releases t-PA from in-vitro systems, and sodium nitroprusside, an endothelium-independent vasodilator, were infused into the brachial artery at rates calculated to cause a similar degree of vasodilatation. The study was performed in five elderly, smoking hypertensive patients in whom the clustering of detrimental factors for endothelial function permitted prediction of defective endothelial-mediated vasorelaxation, and five young, normotensive, nonsmoking male volunteers. Forearm blood flow was assessed by venous plethysmography; t-PA and plasminogen activator inhibitor 1 (PAI-1) antigen values were expressed as flow-dependent (net release, the product of venoarterial concentration gradient and forearm blood flow) or independent (absolute and fractional concentration gradients) indices. In patients, acetylcholine did not change flow and net release and concentration gradients of t-PA, suggesting that vasodilatation as such, possibly by increasing fluid shear stress, may induce t-PA release in human forearm. In normal subjects, acetylcholine and sodium nitroprusside increased t-PA antigen net release at the highest infusion rate, an effect attributable to forearm hyperperfusion, since absolute and fractional gradients did not change significantly. PAI-1 antigen did not change during either infusion in both controls and patients, indicating the absence of an endothelial pool to be mobilized acutely.     

The dynamic changes of plasma tissue-type plasminogen activator level and the activity of its inhibitor during coronary vasospasm

This study aimed to examine the dynamic changes of the fibrinolytic system during coronary vasospasm. Tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI) and fibrinopeptide A (FPA) levels were measured in the great cardiac venous and arterial blood of 9 patients with clinically and angiographically proven vasospastic angina and 11 controls. Before ergonovine provocation, although there was no difference between the above 2 groups in t-PA levels in the aorta or the great cardiac vein, the PAI level in patients with variant angina was lower than in the controls both in the aorta (4.2 +/- 3.5 IU/ml vs 10.9 +/- 5.2 IU/ml) and in the great cardiac vein (2.3 +/- 2.9 IU/ml vs. 11.9 +/- 4.9 IU/ml). During ergonovine-induced coronary vasospasm in patients with variant angina, the t-PA level in the great cardiac vein significantly increased from 3.4 +/- 0.7 ng/ml to 4.4 +/- 0.5 ng/ml (p less than 0.05), but it did not change in the aorta. The maximal dose of ergonovine (0.4 mg) induced mild diffuse coronary vasoconstriction in the controls, and this diffuse coronary vasoconstriction induced a reduction of PAI levels in the great cardiac vein from 11.9 +/- 4.9 IU/ml to 9.5 +/- 4.8 IU/ml (p less than 0.05). FPA levels in the great cardiac vein did not change during ergonovine-induced coronary vasospasm in either group. Thus, the coronary vasospasm induced the release of t-PA from endothelial cells of coronary vessels and resulted in the reduction in the PAI activity in the great cardiac vein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet and vascular function during coronary thrombolysis with tissue-type plasminogen activator

Platelet activation may limit the response to tissue-type plasminogen activator (t-PA) during coronary thrombolysis in humans. As an index of platelet activation, we assessed thromboxane A2 biosynthesis during coronary thrombolysis with intravenous t-PA in patients with acute myocardial infarction. Urinary 2,3-dinor-thromboxane B2, a metabolite of thromboxane A2, was increased to a peak of 3,327 +/- 511 pg/mg creatinine (n = 12) following administration of intravenous t-PA and remained elevated for 48 hours. This increase was abolished by pretreatment with aspirin 325 mg orally (n = 6), indicating de novo biosynthesis of thromboxane A2 rather than washout of preformed metabolites during reperfusion. Prostacyclin (PGI2) biosynthesis, determined by excretion of 2,3-dinor-6-keto-PGF1 alpha, also increased after t-PA administration. However, this increase was less pronounced in patients who reperfused (28 +/- 3.3 ng.hr/mg creatinine) than in patients who failed to reperfuse (118 +/- 30 ng.hr/mg creatinine, p less than 0.05). These data provide evidence of platelet activation during coronary thrombolysis with t-PA. In patients who reperfuse, the reduction in PGI2 biosynthesis may be a marker of reperfusion injury to the vasculature and may further amplify platelet activation.     

Preserved tissue-type plasminogen activator release and endothelium-dependent vasodilation in postmenopausal women with NIDDM

We have recently shown that the net release of tissue-type plasminogen activator (t-PA) antigen can be rapidly enhanced by the muscarinic receptor stimulation in healthy males. Since diabetes mellitus has been associated with endothelial dysfunction, the aim of the present study was to compare the endothelium-derived local net release of t-PA with vasodilation in response to muscarinic receptor stimulation by metacholine (Mch) and fluid shear stress in a group of postmenopausal women with non-insulin-dependent diabetes mellitus (NIDDM), and to elucidate the influence of estrogen on this process. Six postmenopausal women with NIDDM were in randomized order exposed to step-wise intra-arterial infusions of Mch (0.1-0. 8-4.0 microg/min) and nitroprusside (SNP; 0.5-2.5-10.0 microg/min). Forearm blood flow (FBF) was assessed by plethysmography. The infusions with Mch and SNP were repeated during simultaneous intra-arterial infusion of 17-beta estradiol (E; 20 ng/min). During placebo infusion, FBF increased significantly in response to Mch and SNP (p<0.001), but no differences between Mch and SNP were found. In parallel to the blood flow increase in response to Mch stimulation, the t-PA net release was increased over 30 times (p<0.001). Estrogen did not produce any change in blood flow or net release of t-PA at baseline or in response to either drug (Mch or SNP). The present study demonstrates a preserved endothelium-dependent vasodilation and stimulated tissue-type plasminogen activator release in NIDDM postmenopausal women in response to Mch stimulation. Acute intra-arterial infusion of 17-beta estradiol did not affect the vasodilation or the t-PA net release.