Protein C inhibitor (plasminogen activator inhibitor-3) and the risk of venous thrombosis

Protein C inhibitor (PCI), also known as plasminogen activator inhibitor-3, is a serine proteinase inhibitor that can inhibit enzymes in blood coagulation, fibrinolysis and fertility. The role of PCI in regulating the blood coagulation mechanism is not known, as it can inhibit both procoagulant (thrombin, factor Xa, factor XIa) and anticoagulant (activated protein C, thrombin-thrombomodulin, urokinase) enzymes. To determine the relevance of this inhibitor in thrombosis, PCI levels were assessed in the Leiden Thrombophilia Study, a case-control study of venous thrombosis in 473 patients with a first deep-vein thrombosis and 474 age- and sex-matched control subjects. PCI levels above the 95th percentile of the controls (136.1%) increased the risk 1.6-fold compared with PCI levels below the 95th percentile (95% confidence interval 0.9-2.8). There was a gradual increase in risk of thrombosis with further increasing levels of PCI. Adjustment for a number of possible confounders led to a reduction of the risk estimates associated with PCI. However, it is unclear whether adjustment for such factors in the risk models is justified. These results indicate that high levels of PCI may constitute a mild risk factor for venous thrombosis.     

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.     

Blood cells participate in the fibrinolytic response to tissue-type plasminogen activator

Exercise and venous occlusion stimulate fibrinolysis. In addition to increased concentrations of tissue-type plasminogen activator (tPA) and increased plasmin-mediated fibrinolysis in plasma, these stimuli produce additional cellular-phase activity in blood that is of the same magnitude as the plasma response. To determine whether tPA alone, rather than other consequences of these stimuli, is responsible for the cellular response, the in vitro effects of tPA on whole blood, plasma, and calculated cellular-phase (whole blood minus plasma) activities were determined by solid-phase radiofibrin assay on venous blood from ten normal subjects (seven men, three women). At tPA concentrations encompassing physiological and therapeutic levels (5 to 100 ng/mL; 0.7 to 14 IU/mL), increments in whole blood were consistently in excess of those in companion plasma and represented increased cellular-phase activity equivalent in magnitude to the well-known increase in plasma activity. Fibrinolytic activity produced by 10 to 20 ng tPA/mL (1.4 to 2.8 IU/mL) was consistently detected in whole blood and plasma by 60 minutes, with higher concentrations (100 ng or 14 IU tPA/mL) detectable after a five-minute assay in all subjects. Thus, tPA alone, without invoking fibrinolytic factors extraneous to blood or other effects of exercise or venous occlusion, accounts for both cellular and plasma responses to these stimuli. The considerable cellular response, the mechanism of which remains to be elucidated, may constitute a determinant of individual therapeutic responsiveness to tPA.     

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.     

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     

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.     

Time dependent release of tissue-type plasminogen activator and plasminogen activator inhibitor into the circulation of pigs during shock.

To investigate short-term activation and inhibition of fibrinolysis during shock, we studied plasma levels of tissue-type plasminogen activator (t-PA) and t-PA inhibition capacity (PAI) in anaesthetized pigs. t-PA in euglobulin fractions of plasma was measured by the conversion of plasminogen to plasmin in the presence of fibrin split products. Plasmin thus generated was measured in a chromogenic substrate assay. PAI was measured as plasma inhibition capacity for human melanoma t-PA. Controls (n = 8) had constant t-PA and PAI for 6 h. Lipopolysaccharide from Salmonella abortus equi in four different doses (n = 9 - 11), or live Escherichia coli (n = 3) induced a transient t-PA increase with peak values at 2 h. PAI decreased to 50% at 2 h and increased to 250% at 6 h. Phorbol myristate acetate (n = 7) induced no change of t-PA or PAI. Dextran sulphate (n = 4) produced a t-PA rise at 30 min, followed by a rapid decline. Endotoxin was an appropriate stimulus for activation and inhibition of fibrinolysis whereas phorbol ester failed to elicit this response.     

Thrombolytic properties of human tissue-type plasminogen activator, single-chain urokinase-type plasminogen activator, and synergistic combinations in venous thrombosis models in dogs and rabbits

Thrombolysis with single and combined four-hour intravenous (IV) infusions of recombinant tissue-type plasminogen activator (rt-PA), recombinant single-chain urokinase-type plasminogen activator of 54,000 molecular weight (mol wt) (rscu-PA), and rscu-PA-32 kD, an rscu-PA derivative of 32,000 mol wt was studied in a femoral vein thrombosis model in the dog and in a jugular vein thrombosis model in the rabbit. In both species, the dose-response curves were linear, and no systemic activation of the fibrinolytic system or fibrinogen breakdown was observed. The steady-state levels of rt-PA-, rscu-PA-, and rscu-PA-32 kD-related antigens in plasma were proportional to the infusion rates. In the dog model, 25% lysis was obtained with 0.11 mg/kg rt-PA, 0.8 mg/kg rscu-PA, and 0.37 mg/kg rscu-PA-32 kD. Combinations of rt-PA and rscu-PA were 2.6 times more active (P less than .005) than anticipated on the basis of their pharmacologic additive effects, whereas combinations of rt-PA and rscu-PA-32 kD were 2.7 times more active (P less than .05). In the rabbit model, 25% lysis was obtained with 0.24 mg/kg rt-PA, 0.75 mg/kg rscu-PA, and 1.25 mg/kg rscu-PA-32 kD. Combinations of rt-PA and rscu-PA have a fivefold synergistic interaction, but surprisingly no synergism was observed between rt-PA and rscu-PA-32 kD. This study shows that synergism between rt-PA and rscu-PA occurs both in rabbits and dogs in a relatively narrow concentration range that allows a fractional reduction of the total equipotent dose by a factor of 2.5-fold to fivefold. Combination therapy is not associated with systemic fibrinolytic activation. This range of synergistic interaction, although limited, may be useful in devising the best thrombolytic therapy for patients with thromboembolic disease.     

Prevention of coronary thrombosis with subthrombolytic doses of tissue-type plasminogen activator

To determine whether tissue-type plasminogen activator (t-PA) may prevent coronary thrombosis or accelerate the lysis of clot formed under conditions in which increased concentration of the activator is present before thrombosis, clot lysis studies were undertaken in vitro and in vivo. In vitro, exogenous t-PA (6 to 100,000 ng/ml) accelerated the lysis of clot in a dose-dependent fashion when the clot was formed either from whole plasma or from euglobulin fractions (n = 316 determinations). Adding t-PA before clot formation shortened the time to lysis by at least threefold with euglobulin fractions and by at least 10-fold with whole plasma clots, which is consistent with the presence of inhibitors of fibrinolysis in whole plasma and with the binding of t-PA to nascent fibrin. In an intact dog preparation of coronary thrombosis (n = 25), occlusive thrombus formation was prevented when t-PA was present in subthrombolytic concentrations (430 to 1200 ng/ml, n = 5). Occlusive thrombus formation occurred after only discontinuation of the t-PA infusion and clearance of t-PA. Lower concentrations of t-PA (147 to 427 ng/ml, n = 6) significantly delayed occlusion (26 +/- 6.5 vs 7.8 +/- 2.8 min for controls). In animals with t-PA concentrations of less than 140 ng/ml (n = 4), the time to occlusion was unaltered (7.7 +/- 4.5 min). The present study demonstrates that t-PA present before clot formation inhibits thrombosis or accelerates thrombolysis depending on concentration, and that subthrombolytic doses of t-PA can prevent thrombus formation in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Treatment of prosthetic tricuspid valve thrombosis with recombinant tissue-type plasminogen activator

Recombinant tissue-type plasminogen activator (rt-PA) was admministeredintravenously to a 64-years old female with thrombotic malfunctionof a Björk–Shiley prosthetic tricuspid valve. 150mg of single-chain rt-PA was infused over 8 hours followed byan additional dose of 50 mg over the next 8 hours. At the endof the first infusion, restoration of normal valve functionwas demonstrated by fluoroscopic and echo-Doppler examinations.Mild systemic activation of the fibrinolytic system occurred,with a decrease of fibrinogen and ₂-antiplasmin to 53% and 33%,respectively, of the preinfusion value at the nadir.     

Treatment of prosthetic tricuspid valve thrombosis with recombinant tissue-type plasminogen activator

Recombinant tissue-type plasminogen activator (rt-PA) was admministeredintravenously to a 64-years old female with thrombotic malfunctionof a Björk–Shiley prosthetic tricuspid valve. 150mg of single-chain rt-PA was infused over 8 hours followed byan additional dose of 50 mg over the next 8 hours. At the endof the first infusion, restoration of normal valve functionwas demonstrated by fluoroscopic and echo-Doppler examinations.Mild systemic activation of the fibrinolytic system occurred,with a decrease of fibrinogen and ₂-antiplasmin to 53% and 33%,respectively, of the preinfusion value at the nadir.     

Expression of tissue-type and urokinase-type plasminogen activator activities in chronic venous leg ulcers.

BACKGROUND: Chronic wounds have been shown to exhibit elevated levels of several classes of proteinases. Plasminogen activators (PAs) are proteinases which play a major role in the biological processes involved in wound healing and abnormalities in PAs may play a role in the pathology associated with chronic wounds. Here, we investigated the expression of tPA and uPA activities in chronic venous ulcer biopsies. PATIENTS AND METHODS: In 22 patients with chronic venous leg ulcers, punch biopsies were taken from the ulcer base, ulcer margin and uninvolved skin from the thigh of the affected limb and PA activities were assessed using in situ histological zymography. RESULTS: tPA is the main PA activity in uninvolved skin but was reduced in ulcer margin skin and venous leg ulcer tissue compared to normal skin. uPA activity appeared throughout the ulcer margin skin but was at low levels in normal skin. Ulcer base tissue appeared to exhibit a plasminogen-independent proteinase activity not seen in normal or ulcer margin skin. CONCLUSION: PA activities are altered in and around chronic venous leg ulcers and their distribution suggests that blood vessels in CVI may be damaged and that the tissue is in an inflamed state.     

Increased concentration of the fast-acting plasminogen activator inhibitor in plasma associated with familial venous thrombosis

We have earlier demonstrated that in a family with a tendency to recurrent venous thrombosis the release of tissue plasminogen activator (t-PA) activity in blood after stimulation was abnormally low. This observation could be related either to an impaired release of t-PA into the blood stream or to a masking of the released t-PA by a high concentration of PA inhibitor(s). In order to distinguish between these two possibilities the family was reinvestigated using various newer techniques, including an ELISA for t-PA, an assay for quantitation of the fast-acting PA inhibitor and SDS polyacrylamide gel electrophoresis followed by fibrin-enzymography. Hereby the family members were demonstrated to have a high concentration in plasma of the PA inhibitor. After stimulation the release of t-PA into the blood was normal, the t-PA activity, however, was immediately inactivated by complex formation with the fast-acting PA inhibitor.     

Urokinase, tissue-type plasminogen activator and plasminogen activator inhibitor-1 expression in severely stenosed and occluded vein grafts with thrombosis.

Intimal hyperplasia and subsequent thrombotic occlusions limit the success of vascular reconstructive procedures. Plasminogen activation in situ may be an important factor affecting re-stenosis of the graft. Tissue specimens from eight patients with failing or failed infra-inguinal vein bypasses and three specimens from normal veins were harvested to study urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) by in situ hybridization and immunohistochemistry. The possible presence of thrombi was monitored by platelet and fibrin-specific stainings. In occluded grafts, platelet endothelial cell adhesion molecule (PECAM-1) antibody stained the thrombi but not the endothelial area, indicating the absence of endothelium. Platelet glycoprotein (GP) IIb/IIIa co-localized with PECAM-1 and, furthermore, GP IIb/IIIa staining was positive on the vein walls with thrombi and to some extent in the grafts without thrombi. PAI-1 and u-PA were uniformly upregulated in intimal thickening in grafts without thrombus. In organized thrombi, enhanced u-PA, t-PA and PAI-1 reactivity was detected in the ingrowing subendothelium. In non-occluded grafts with small thrombi, u-PA expression was enriched beneath microthrombi co-localizing with the graft wall injury, while PAI-1 was scattered in the (sub)endothelium. We conclude that fibrinolytic system is upregulated at sites of graft stenosis, and local proteolytic degradation of the graft wall associates with thrombus formation.     

A prospective, randomized trial comparing combination half-dose tissue-type plasminogen activator and streptokinase with full-dose tissue-type plasminogen activator. Kentucky Acute Myocardial Infarction Trial (KAMIT) Group

BACKGROUND. The potential benefits of combination thrombolytic agents in the treatment of myocardial infarction remain uncertain. In a small pilot study, we demonstrated that combining half-dose tissue-type plasminogen activator (t-PA) with streptokinase (SK) achieved a high rate of infarct vessel patency and a low rate of reocclusion at half the cost of full-dose t-PA. METHODS AND RESULTS. We designed a prospective trial in which 216 patients were randomized within 6 hours of myocardial infarction to receive either the combination of half-dose (50 mg) t-PA with streptokinase (1.5 MU) during 1 hour or to the conventional dose of t-PA (100 mg) during 3 hours. Acute patency was determined by angiography at 90 minutes, and angioplasty was reserved for failed thrombolysis. Heparin and aspirin regimens were maintained until follow-up catheterization at day 7. Acute patency was significantly greater after t-PA/SK (79%) than with t-PA alone (64%, p less than 0.05). After angioplasty for failed thrombolysis, acute patency increased to 96% in both groups. Marked depletion of serum fibrinogen levels occurred after t-PA/SK compared with t-PA alone at 4 hours (37 +/- 36 versus 199 +/- 66 mg/dl, p less than 0.0001) and persisted 24 hours after therapy (153 +/- 66 versus 252 +/- 75 mg/dl, p less than 0.0001). Reocclusion (3% versus 10%, p = 0.06), reinfarction (0% versus 4%, p less than 0.05), and need for emergency bypass surgery (1% versus 6%, p = 0.05) tended to be less in the t-PA/SK group. Greater myocardial salvage was apparent in the t-PA/SK group as assessed by infarct zone function at day 7 (-1.9 SD/chord versus -2.3 SD/chord after t-PA alone, p less than 0.05). In-hospital mortality (6% versus 4%) and serious bleeding (12% versus 11%) were similar between the two groups. CONCLUSIONS. These results suggest that a less expensive regimen of half-dose t-PA with SK yields superior 90-minute patency and left ventricular function and a trend toward reduced reocclusion compared with the conventional dose of t-PA.     

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)