Intraventricular and brachial artery thrombosis in nephrotic syndrome

PURPOSE: To investigate the influence of hyperthermia up to 45 degrees C on fibrinolysis with recombinant tissue-type plasminogen activator (rt-PA). METHODS: Standardized fibrin clots were incubated in a water bath for 5 hr with either rt-PA (test group) or 0.9% sodium chloride (control group) and blood plasma at temperatures of 30-45 degrees C. Concentrations of D-dimer and time to complete clot lysis were measured. RESULTS: The activity of fibrinolysis with rt-PA rose with increasing temperature: time to lysis approximately halved from 30 degrees C to 40 degrees C and the concentration of D-dimer tripled. In the control group clot size did not change. CONCLUSIONS: Activity of rt-PA-induced fibrinolysis rises distinctly with higher temperatures. Since even healthy subjects show a physiologic decline in body temperature in the extremities, in patients with occlusive arterial disease decreased activity of fibrinolysis with rt-PA can be expected. Controlled hyperthermia may improve fibrinolysis with rt-PA and should be investigated in vivo.     

Recombinant lys-plasminogen given before, but not after, recombinant tissue-type plasminogen activator markedly improves coronary thrombolysis in dogs: relationship of thrombolytic efficacy with parameters of fibrinolysis

Recombinant tissue-type plasminogen activator (rt-PA) administration rapidly restores blood flow in thrombosed coronary arteries, but coronary arteries often reocclude after initial thrombolysis. This occurs because of the short half-life of rt-PA and rapid increase in plasminogen activator inhibitor (PAI-1) and alpha2-antiplasmin levels in plasma. We hypothesized that administration of lys-plasminogen, which binds to fibrin with 10 times greater affinity and results in a loose fibrin structure (as compared with native glu-plasminogen), before rt-PA would enhance the thrombolytic efficacy of rt-PA and modulate parameters of fibrinolysis. To examine this hypothesis, dogs with electrically induced stable thrombus in the left anterior descending coronary artery (LAD) were treated with saline (group A, n = 9) or lys-plasminogen (group B, 2 mg/kg, n = 5), followed 10 min later by rt-PA (1 mg/kg in 20 min). Four other dogs with occlusive LAD thrombus were first given rt-PA, followed by lys-plasminogen (2 mg/kg) 50 min later (group C). Lys-plasminogen given before rt-PA restored flow in all dogs in 14 +/- 4 min (vs. 22 +/- 9 min in group A, p < 0.05), continuing > 2 h (vs. 41 +/- 15 min in group A, p < 0.02). Lys-plasminogen given after rt-PA did not potentiate the effect of rt-PA. Plasma t-PA antigen concentrations were highest in group B dogs at 2 h after rt-PA infusion. PAI-1 and alpha2-antiplasmin plasma levels were suppressed in all dogs receiving lys-plasminogen whether it was given before or after rt-PA. Therefore, lys-plasminogen given before rt-PA markedly potentiates the effect of rt-PA and alters the parameters of fibrinolysis. In contrast, lys-plasminogen given after rt-PA does not influence the thrombolytic effect of rt-PA, whereas it suppresses PAI-1 and alpha2-antiplasmin levels in plasma. This study also suggests that binding of plasminogen to the clot is more important than the plasma levels of PAI-1 and alpha2-antiplasmin.     

Molecular basis of fibrinolysis, as relevant for thrombolytic therapy

The fibrinolytic system comprises an inactive proenzyme, plasminogen, that is converted by plasminogen activators to the active enzyme, plasmin, that degrades fibrin. Two physiological plasminogen activators have been identified: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Plasminogen activation for clot lysis is regulated by specific molecular interactions between tissue-type plasminogen activator (t-PA), plasminogen and fibrin, whereby the lysine-binding sites of the plasminogen molecule play a crucial role by mediating its binding to fibrin, and by controlling the inhibition rate of plasmin by alpha 2-antiplasmin. The recognition that thrombosis within the infarct related coronary artery plays a major role in the pathogenesis of acute myocardial infarction and the observation that early administration of thrombolytic agents results in recanalization of occluded coronary arteries, have provided the basis for the development of thrombolytic therapy in acute myocardial infarction. The elucidation of the biochemical mechanism of fibrin-specific plasminogen activation has fueled the hope that specific and efficacious thrombolytic agents might become available. Comparative studies between the non-fibrin-selective streptokinase and fibrin-selective recombinant t-PA (rt-PA) have shown a difference in efficacy for early coronary artery recanalization, whereas the GUSTO trial has established that clinical benefit in patients with acute myocardial infarction is indeed correlated with the rapidity and frequency of sustained recanalization and that effective thrombolysis requires adequate anticoagulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mast cell as site of tissue-type plasminogen activator expression and fibrinolysis

Recent data suggest that mast cells (MC) and their products (heparin, proteases) are involved in the regulation of coagulation and fibrino(geno)lysis. The key enzyme of fibrinolysis, plasmin, derives from its inactive progenitor, plasminogen, through catalytic action of plasminogen activators (PAs). In most cell systems, however, PAs are neutralized by plasminogen activator inhibitors (PAIs). We report that human tissue MC as well as the MC line HMC-1 constitutively produce, express, and release tissue-type plasminogen activator (tPA) without producing inhibitory PAIs. As assessed by Northern blotting, highly enriched lung MC (>98% pure) as well as HMC-1 expressed tPA mRNA, but did not express mRNA for PAI-1, PAI-2, or PAI-3. The tPA protein was detectable in MC-conditioned medium by Western blotting and immunoassay, and the MC agonist stem cell factor (c-Kit ligand) was found to promote the release of tPA from MC. In addition, MC-conditioned medium induced fibrin-independent plasmin generation as well as clot lysis in vitro. These observations raise the possibility that MC play an important role in endogenous fibrinolysis.     

Distribution of CCR5-delta 32 gene deletion across the Russian part of Eurasia

We describe the cases of two premature infants carrying a central venous line who developed on the fourth day of life a catheter related intracardiac thrombus. Although they were clinically asymptomatic we opted for thrombolytic treatment considering the potential risks of this situation. Treatment with recombinant tissue plasminogen activator (rt-PA) was performed for one day in the first case and for three days in the second one, in association with fresh frozen plasma. Thrombus dissolution occurred in both patients and no adverse effects were noted. In our experience tissue plasminogen activator was a therapy acceptably safe and effective inducing clot lysis in very low birthweight neonates into critical situations.     

Thrombolytic and pharmacokinetic properties of a recombinant chimeric plasminogen activator consisting of a fibrin fragment D-dimer specific humanized monoclonal antibody and a truncated single-chain urokinase

A recombinant chimeric plasminogen activator consisting of a humanized monoclonal antibody specific for cross-linked human fibrin (MA-15C5Hu) and a 32 kDa single-chain urokinase-type plasminogen activator (scu-PA-32k) comprising amino acids Leu144-Leu411, MA-15C5Hu/scu-PA-32k, was previously found to have a 12-fold higher fibrinolytic potency than recombinant scu-PA-32k towards a human plasma clot in a human plasma milieu in vitro (Vandamme et al., Eur J Biochem 1992; 205: 139-46). Therefore, the thrombolytic and pharmacokinetic properties of MA-15C5Hu/scu-PA-32k were compared with those of recombinant single-chain urokinase-type plasminogen activator (scu-PA) in 3 different venous thrombosis models in vivo. In hamsters with a pulmonary embolus consisting of a human plasma clot, the thrombolytic potency (% lysis per dose in mg/kg administered) of MA-15C5Hu/scu-PA-32k was 23-fold higher than that of scu-PA (p less than 0.0005). In rabbits with a jugular vein clot prepared from human plasma, the thrombolytic potency of MA-15C5Hu/scu-PA-32k was 11-fold higher than that of scu-PA (p = 0.012). In baboons with an autologous whole blood clot in the femoral vein, the chimera had a 5-fold higher thrombolytic potency than scu-PA. In all three animal species, the clearance of the chimera was 10- to 27-fold reduced as compared to scu-PA. The specific thrombolytic activity (% lysis per micrograms/ml steady-state plasma u-PA antigen) was increased up to 7-fold with MA-15C5Hu/scu-PA-32k as compared with scu-PA, which is indicative of targeting of the chimera to the clot. No fibrinogen breakdown or alpha 2-antiplasmin depletion was observed during thrombolysis with the chimera. Thus, MA-15C5Hu/scu-PA-32k constitutes a recombinant chimeric plasminogen activator with a significantly enhanced thrombolytic potency in 3 different animal models of venous thrombosis.     

Comparison of the in vitro fibrinolytic activities of low and high molecular weight single-chain urokinase-type plasminogen activator

The fibrinolytic activity of low molecular weight (LMW) single-chain urokinase-type plasminogen activator (scu-PA) lacking the epidermal growth factor domain and the kringle domain was compared with the activity of high molecular weight (HMW) scu-PA. LMW scu-PA was 1-5 times less active than HMW scu-PA in a fibrin plate method, in a purified fibrin clot lysis assay and in a plasma clot lysis assay. Time course experiments in a chromogenic plasminogen activator assay suggested that LMW scu-PA was less sensitive to activation by plasmin than HMW scu-PA. This was confirmed in a scu-PA activation test, which showed that at a concentration of 40 IU/ml LMW scu-PA required a three-fold higher plasmin concentration for 50% activation in 20 min than did HMW scu-PA. Kinetic experiments in the presence of 0.1 M NaCl showed non-standard Michaelis-Menten kinetics for the activation by plasmin of both HMW and LMW scu-PA. In contrast, standard kinetics was observed at 0.15 M NaCl, showing a 2.6-fold lower catalytic efficiency for LMW scu-PA than for HMW scu-PA. It is concluded that the plasmin activation of LMW scu-PA is about three times slower than the activation of HMW scu-PA. This explains, at least partially, the lower fibrinolytic activity of LMW scu-PA in comparison with HMW scu-PA.     

The absence of a procoagulant effect after TNK-t-PA: a comparison with streptokinase and rt-PA

A limitation of current fibrinolytic drugs is the procoagulant activity induced by their administration. TNK is a mutant of tissue plasminogen activator (t-PA) with high fibrin specificity, resistance to plasminogen activator inhibitor-1 and slow plasma clearance, which is administered in a single intravenous bolus. In this study we investigated the procoagulant effect of TNK-t-PA compared to streptokinase, rt-PA or no thrombolysis. Twenty-nine patients with acute myocardial infarction, treated within 6 hours of symptom onset with 1.5 MU streptokinase over 1 hour (n = 12), 100 mg rt-PA in 1.5 hours (n = 12) or 30-40 mg TNK-t-PA in 15 s (n = 5), were studied and compared to 7 patients with contraindications to thrombolysis (control group). All patients received a similar i.v. heparin regimen for at least 24 hours. Blood samples were drawn before the start of treatment (time 0) and after 2 hours. Thrombin formation was assessed as plasma concentrations of thrombin-antithrombin complex (TAT). The four patient groups did not differ significantly in age, sex, time to treatment, infarct location, and TAT values at time 0 (mean value +/- standard error of the mean 9 +/- 2 micrograms/l). Mean TAT levels at 2 hours were 26 +/- 6 micrograms/l in streptokinase treated patients (p = 0.005 vs time 0), 21 +/- 4 micrograms/l in rt-PA treated patients (p < 0.05 vs time 0), 5 +/- 0.6 micrograms/l in TNK treated patients, and 4 +/- 0.4 micrograms/l in controls (NS vs time 0 for TNK and controls). In conclusion, our data suggest that, in patients with acute myocardial infarction, bolus TNK-t-PA, unlike streptokinase or rt-PA infusions, is devoid of procoagulant effects, evaluated 2 hours after its administration.     

Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice

BACKGROUND: Platelet-rich arterial thrombi are resistant to lysis by plasminogen activators. However, the mechanisms underlying thrombolysis resistance are poorly defined. Plasminogen activator inhibitor-1 (PAI-1), which is present in plasma, platelets, and vascular endothelium, may be an important determinant of the resistance of arterial thrombi to lysis. However, in vitro studies examining the regulation of platelet-rich clot lysis by PAI-1 have yielded inconsistent results. METHODS AND RESULTS: We developed a murine arterial injury model and applied it to wild-type (PAI-1 [+/+]) and PAI-1-deficient (PAI-1 [-/-]) animals. FeCl3 was used to induce carotid artery thrombosis. Thrombi consisted predominantly of dense platelet aggregates, consistent with the histology of thrombi in large-animal arterial injury models and human acute coronary syndromes. To examine the role of PAI-1 in regulating endogenous clearance of platelet-rich arterial thrombi, thrombi were induced in 22 PAI-1 (+/+) mice 14 PAI-1 (-/-) mice. Twenty-four hours later, the amount of residual thrombus was determined by histological analysis of multiple transverse sections of each artery. Residual thrombus was detected in 55 of 85 sections (64.7%) obtained from PAI-1 (+/+) mice compared with 19 of 56 sections (33.9%) from PAI-1 (-/-) mice (P=.009). Computer-assisted planimetry analysis revealed that mean thrombus cross-sectional area was 0.033+/-0.0271 mm2 in PAI-1 (+/+) mice versus 0.016+/-0.015 mm2 in PAI-1 (-/-) mice (P=.048). CONCLUSIONS: PAI-1 is an important determinant of thrombolysis at sites of arterial injury. Application of this model to other genetically altered mice should prove useful for studying the molecular determinants of arterial thrombosis and thrombolysis.     

Occluded peripheral arteries and bypass grafts: lytic stagnation as an end point for pulse-spray pharmacomechanical thrombolysis

Angiographic demonstration of luminal narrowing during pulse-spray pharmacomechanical thrombolysis (PSPMT) may reflect residual lysable clot, organized thrombus, platelet-rich clot, atherosclerosis, neointimal hyperplasia, or functional narrowing. The authors evaluated the efficacy and safety of lytic stagnation (initial rapid lysis followed by insubstantial further lysis with additional treatment) as an end point for PSPMT. Lytic stagnation was evaluated with serial angiography in 16 arterial and five bypass graft occlusions. Substantial lysis occurred after administration of mean doses of 512,000 units +/- 182,000 of urokinase or 5.3 mg +/- 2.0 of tissue-type plasminogen activator. Additional treatment with either of those agents produced minimal or no further change in the appearance of residual disease. Recanalization was successful in all patients after angioplasty. Distal emboli were noted in four cases, in three of which angioplasty of large intraluminal filling defects had been performed. The authors conclude that lytic stagnation is a reliable and safe end point for PSPMT in the absence of large intraluminal filling defects.     

Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently identified fibrinolysis inhibitor in plasma, that when converted to an enzyme potently attenuates fibrinolysis. It is activated by relatively high concentrations of thrombin that exceed the thrombin concentration required for fibrin formation. These high concentrations of thrombin are generated by the intrinsic pathway via activation of factor XI by thrombin. The down regulation of fibrinolysis by TAFI can be measured in a clot lysis assay. When the clot lysis times of healthy individuals were determined, large inter-individual differences were observed. To determine if differences in concentration of TAFI explain the variation in clot lysis between individuals, specific assays were developed for the measurement of TAFI antigen and activity in plasma. In normal plasma, there was a dose-dependent relationship between TAFI antigen and TAFI activity. There was also a correlation between clot lysis time and plasma TAFI antigen, indicating that the amount of TAFI that is activated during the clot lysis assay, is dependent on the concentration of TAFI. In the plasmas of 20 healthy individuals, clot lysis times, TAFI antigen and TAFI activity were determined. Both TAFI antigen and TAFI activity showed a significant correlation with the clot lysis time. No correlation between TAFI antigen and clot lysis time was found when the clot lysis time was determined in the presence of an antibody blocking the factor XI feedback loop. These results indicate that plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation.     

A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor

TAFI (thrombin-activable fibrinolysis inhibitor) is a recently described plasma zymogen that, when exposed to the thrombin-thrombomodulin complex, is converted by proteolysis at Arg92 to a basic carboxypeptidase that inhibits fibrinolysis (TAFIa). The studies described here were undertaken to elucidate the molecular basis for the inhibition of fibrinolysis. When TAFIa is included in a clot undergoing fibrinolysis induced by tissue plasminogen activator and plasminogen, the time to achieve lysis is prolonged, and free arginine and lysine are released over time. In addition, TAFIa prevents a 2.5-fold increase in the rate constant for plasminogen activation which occurs when fibrin is modified by plasmin in the early course of fibrin degradation. The effect is specific for the Glu- form of plasminogen. TAFIa prevents or at least attenuates positive feedback expressed through Lys-plasminogen formation during the process of fibrinolysis initiated by tissue plasminogen activator and plasminogen. TAFIa also inhibits plasmin activity in a clot and prolongs fibrinolysis initiated with plasmin. We conclude that TAFIa suppresses fibrinolysis by removing COOH-terminal lysine and arginine residues from fibrin, thereby reducing its cofactor functions in both plasminogen activation and the positive feedback conversion of Glu-plasminogen to Lys-plasminogen. At relatively elevated concentrations, it also directly inhibits plasmin.     

Tissue plasminogen activator, plasminogen activator inhibitor-1, and fibrin as indexes of clinical course in cardiac allograft recipients. An immunocytochemical study

BACKGROUND: Tissue-type plasminogen activator (TPA) is the principal activator of plasminogen. Since hemostasis in the microcirculation of allografts is a well-recognized complication of transplantation, we asked (1) whether the distribution and amount of cellular TPA in biopsies of transplanted human hearts are associated with fibrin deposits in and around the microcirculation, (2) whether such changes involve the physiological inhibitors of TPA and plasmin, and (3) whether the presence of these activators and inhibitors of fibrinolysis in tissue is correlated with clinical outcome. METHODS AND RESULTS: We immunocytochemically quantified the presence of fibrin, plasmin, TPA, and the TPA inhibitor PAI-1 in 938 biopsies from 68 consecutive cardiac allografts over a 54-month period. The localization, distribution, and quantification of TPA in arteriolar smooth muscle cells revealed that 35 of the 68 allografts maintained vascular TPA reactivity consistent with time-zero biopsies of autologous donor hearts: this was designated as the normal TPA group. In contrast, 33 of the 68 allografts significantly lost vascular TPA reactivity compared with time-zero biopsies of autologous donor hearts: this was designated as the depleted TPA group. Analysis of sequential biopsies from both groups during 54 months revealed that the mean cumulative quantitative TPA value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.9 +/- 0.02 (P = .0001), and the mean cumulative quantitative fibrin value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.5 +/- 0.05 (P = .0001). Biopsies of allografts in the depleted TPA group contained endothelial reactivity for TPA-PAI-1 complexes, whereas biopsies from the normal TPA group did not. Plasmin-associated molecules were rarely identified in biopsies of the normal TPA group but were present in the depleted TPA group, and the fibrin-to-plasmin ratio in the normal TPA group always was less than the fibrin-to-plasmin ratio in biopsies from the depleted TPA group. Analysis of demographic and risk factors revealed no significant differences between patients in the normal and depleted TPA groups, but none of the 35 patients in the normal TPA group died or were retransplanted, and 13 of the 33 patients in the depleted TPA group died or required retransplantation (P = .0001). CONCLUSIONS: Time-zero hearts (n = 68) and 34 of 38 stable allografts contained immunocytochemically detectable TPA only in vascular smooth muscle cells. Twenty-nine of 30 patients with normal TPA in their time-zero biopsies who subsequently developed a poor clinical outcome were found to have depleted TPA in biopsies evaluated during their first postoperative month and remained depleted throughout the study. Of 33 patients with depleted TPA, 39% died or required retransplantation. Depleted arteriolar TPA associated significantly with vascular and interstitial deposits of fibrin, plasmin, and endothelial TPA-PAI-1 complexes. These findings indicate that hemostatic and fibrinolytic pathways are activated in falling allografts, and they reveal evidence of depleted TPA before clinical or histopathological signs of failure. Patients with such allografts were found to be at high risk of death independently of other widely used clinical/laboratory parameters of prediction.     

Intracoronary infusion of E6010 has more potent thrombolytic activity than tissue plasminogen activator (t-PA) in dogs: a higher plasma level of E6010 than t-PA causes potent thrombolytic activity

We examined the thrombolytic properties of a novel modified human tissue plasminogen activator (PA) (E6010), in which cysteine 84 is replaced by serine, and which has a prolonged biologic half-life (t1/2). We compared the thrombolytic efficacy of continuous intracoronary (i.c.) infusion of E6010 with that of recombinant human tissue PA (rt-PA) in a canine model with copper coil-induced 1-h-old coronary artery thrombi and also compared the relation between thrombolytic efficacy and plasma clearance represented by pharmacokinetic parameters of i.c.-infused E6010 and rt-PA. Sixty-minute E6010 and rt-PA i.c. infusions were compared. The thrombolytic effects of i.c.-infused E6010 and rt-PA, represented by time to reperfusion (TR), reperfusion rate at 60 min (RR), and reocclusion rates at 60 min after reperfusion (OR) were as follows. E6010: Dose 0.06, 0.15, 0.3 (mg/kg/h); TR 25 +/- 10, 15 +/- 10, 13 +/- 5 (min); RR 100, 100, 100 (%); and OR 0, 0, 17 (%), respectively. Recombinant t-PA: Dose 0.06, 0.15, 0.3 (mg/kg/h); TR 47 +/- 12, 18 +/- 17, 14 +/- 4 (min); RR 50, 75, 100 (%); and OR 100, 33, 33 (%), respectively. These findings indicate that E6010 has more potent thrombolytic activity than rt-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

A steady-state template model that describes the kinetics of fibrin-stimulated [Glu1]- and [Lys78]plasminogen activation by native tissue-type plasminogen activator and variants that lack either the finger or kringle-2 domain

The kinetics of activation of both [Glu1]- and [Lys78]Plg(S741C-fluorescein by native (recombinant) tissue-type plasminogen activator and its deletion variants lacking either the finger or kringle-2 domain were measured by fluorescence within fully polymerized fibrin clots. The kinetics conform to the Michaelis-Menten equation at any fixed fibrin concentration so long as the plasminogen concentration is expressed as either the free or fibrin-bound, but not the total. The apparent kcat and Km values both vary systematically with the concentration of fibrin. Competition kinetics disclosed an active site-dependent interaction between t-Pa and [Glu1]Plg(S741C-fluorescein) in the presence, but not the absence, of fibrin. A steady-state template model having the rate equation v/[A]o = kcat(app).[Plg]/(Km(app) + [Plg]) was derived and used to interpret the data. The model indicates that catalytic efficiency is determined by the stability of the ternary activator-fibrin-plasminogen complex rather than the binding of the activator or plasminogen to fibrin. This implies that efforts to improve the enzymatic properties of t-PA might be more fruitfully directed at enhancing the stability of the ternary complex rather than fibrin binding.     

Properties of chimeric (tissue-type/urokinase-type) plasminogen activators obtained by fusion at the plasmin cleavage site

Two hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 x 10(6) and 1.0 x 10(6) t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 microgram/ml for K2tu-PA and 1 microgram/ml for FK2tu-PA, as compared to 0.5 microgram/ml and > 2 micrograms/ml for t-PA and scu-PA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasminogen deficiency causes severe thrombosis but is compatible with development and reproduction

Plasminogen (Plg)-deficient mice were generated to define the physiological roles of this key fibrinolytic protein and its proteolytic derivatives, plasmin and angiostatin, in development, hemostasis, and reproduction. Plg-/- mice complete embryonic development, survive to adulthood, and are fertile. There is no evidence of fetal loss of Plg-/- mice based on the Mendelian pattern of transmission of the mutant Plg allele. Furthermore, embryonic development continues to term in the absence of endogenous, sibling-derived, or maternal Plg. However, Plg-/- mice are predisposed to severe thrombosis, and young animals developed multiple spontaneous thrombotic lesions in liver, stomach, colon, rectum, lung, pancreas, and other tissues. Fibrin deposition in the liver was a uniform finding in 5- to 21-week-old mice, and ulcerated lesions in the gastrointestinal tract and rectal tissue were common. A remarkable finding, considering the well-established linkage between plasmin and the proteolytic activation of plasminogen activators, was that the level of active urokinase-type plasminogen activator in urine was unaffected in Plg-/- mice. Therefore, Plg plays a pivotal role in fibrinolysis and hemostasis but is not essential for urokinase proenzyme activation, development, or growth to sexual maturity.     

Alpha 2-antiplasmin causes thrombi to resist fibrinolysis induced by tissue plasminogen activator in experimental pulmonary embolism

BACKGROUND: In patients with pulmonary embolism, thrombi resist fibrinolysis induced by plasminogen activators. Because the molecular basis of this thrombus resistance is poorly understood, we used a potent inhibitor to examine the potential role of alpha 2-antiplasmin (alpha 2AP) in experimental pulmonary embolism. METHODS AND RESULTS: Lysis of experimental pulmonary emboli was measured 4 hours after embolization in anesthetized ferrets. All animals received heparin (100 U/kg). Five experimental groups were studied: (1) no recombinant tissue plasminogen activator (rTPA); (2) rTPA at 1 mg/kg; (3) rTPA at 2 mg/kg; (4) rTPA at 1 mg/kg plus a control monoclonal antibody (MAb); and (5) rTPA at 1 mg/kg plus an alpha 2AP inhibitor (MAb 77A3). In comparison with ferrets receiving no rTPA (15.6 +/- 10.5% lysis, mean +/- SD), rTPA-treated groups showed significantly greater lysis (P < .01). Animals treated with rTPA and alpha 2AP inhibitor (56.2 +/- 4.7% lysis) showed significantly greater lysis than all other treatment groups, including ferrets treated with the same dose of rTPA alone (38.5 +/- 6.3%, P < .01), with twice the rTPA dose alone (45.0 +/- 6.5%, P < .05), or with a control MAb (35.2 +/- 4.6%, P < .01). The combination of rTPA treatment and alpha 2AP inhibition caused no consumption of fibrinogen. CONCLUSIONS: Inhibition of alpha 2AP significantly amplified the lysis of experimental pulmonary emboli by rTPA without increasing fibrinogen consumption. These results suggest that alpha 2AP may play an important role in thrombus resistance in patients with venous thromboembolism.     

Sixty-minute alteplase protocol: a new accelerated recombinant tissue-type plasminogen activator regimen for thrombolysis in acute myocardial infarction

OBJECTIVES: Our aim was to design and evaluate a new and easily administered recombinant tissue-type plasminogen activator (rt-PA) regimen for thrombolysis in acute myocardial infarction (AMI) based on established pharmacokinetic data that improve the reperfusion success rate. BACKGROUND: Rapid restoration of Thrombolysis in Myocardial Infarction (TIMI) grade 3 flow is a primary predictor of mortality after thrombolysis in AMI. However, TIMI grade 3 patency rates 90 min into thrombolysis of only 50% to 60% indicate an obvious need for improved thrombolytic regimens. METHODS: Pharmacokinetic simulations were performed to design a new rt-PA regimen. We aimed for a plateau tissue-type plasminogen activator (t-PA) plasma level similar to that of the first plateau of the Neuhaus regimen. These aims were achieved with a 20-mg rt-PA intravenous (i.v.) bolus followed by an 80-mg i.v. infusion over 60 min (regimen A). This regimen was tested in a consecutive comparative trial in 80 patients versus 2.25 10(6) IU of streptokinase/60 min (B), and 70 mg (C) or 100 mg (D) of rt-PA over 90 min. Subsequently, a confirmation trial of regimen A in 254 consecutive patients was performed with angiographic assessment by independent investigators of patency at 90 min. RESULTS: The comparative phase of the trial yielded, respectively, TIMI grade 3 and total patency (TIMI grades 2 and 3) of 80% and 85% (regimen A), 35% and 50% (B), 50% and 55% (C) and 60% and 70% (D). In the confirmation phase of the trial, regimen A yielded 81.1% TIMI grade 3 and 87.0% total patency. At follow-up angiography 7 (4.1%) of 169 vessels had reoccluded. In-hospital mortality rate was 1.2%. Nadir levels of fibrinogen, plasminogen and alpha2-antiplasmin were 3.6 +/- 0.8 mg/ml, 60 +/- 21% and 42 +/- 16%, respectively (mean +/- SD). Fifty-seven patients (22.4%) suffered from bleeding; 3.5% needed blood transfusions. CONCLUSIONS: The 60-min alteplase thrombolysis in AMI protocol achieved a TIMI grade 3 patency rate of 81.1% at 90 min with no indication of an increased bleeding hazard; it was associated with a 1.2% overall mortality rate. These results are substantially better than those reported from all currently utilized regimens. Head to head comparison with established thrombolytic regimens in a large-scale randomized trial is warranted.     

Reward magnitude, but not time of day, influences the trial-spacing effect in autoshaping with rats

An important regulator of the initiation of blood coagulation is the plasma glycoprotein, tissue factor pathway inhibitor (TFPI). TFPI inhibits factor Xa and factor VIIa/tissue factor complex, thereby dampens the proteolytic cascade of the tissue factor pathway. Plasma clot lysis is primarily mediated by the fibrinolytic enzyme, plasmin, which is generated through limited proteolysis of plasminogen by endogenous or exogenously administered plasminogen activators. In this study, the interaction of plasmin with recombinant E. coli-derived TFPI (rTFPI) was examined. Plasmin was found to cause a time and concentration dependent proteolysis of rTFPI, resulting in the decrease of anti-factor Xa (measured by chromogenic substrate assay) and anticoagulant (measured by tissue factor-induced clotting assay) activities. Amino-terminal sequencing of the proteolytic fragments revealed that plasmin cleaved rTFPI at K86-T87, R107-G108, R199-A200, K249-G250, and K256-R257. Western blot analysis showed that proteolysis of exogenously added rTFPI also occurred in plasma supplemented with urokinase, and this is accompanied by decrease of anticoagulant activity. These changes were abolished by addition of aprotinin, an inhibitor of plasmin. These data indicate that TFPI is susceptible to proteolysis when plasma fibrinolytic system is activated. The results taken together suggest that plasmin degradation of TFPI may contribute to rethrombosis after thrombolysis, and may contribute to the variability of the efficacy of TFPI in various thrombolysis/reocclusion studies reported previously.