A novel engineered subtilisin BPN' lacking a low-barrier hydrogen bond in the catalytic triad.

The low-barrier hydrogen bond (LBHB) between the Asp and His residues of the catalytic triad in a serine protease was perturbed via the D32C mutation in subtilisin BPN' (Bacillus protease N'). This mutant enzyme catalyzes the hydrolysis of N-Suc-Ala-Ala-Pro-Phe-SBzl with a k(cat)/K(m) value that is only 8-fold reduced from that of the wild-type (WT) enzyme. The value of k(cat)/K(m) for the corresponding p-nitroanilide (pNA) substrate is only 50-fold lower than that of the WT enzyme (DeltaDeltaG++ = 2.2 kcal/mol). The pK(a) controlling the ascending limb of the pH versus k(cat)/K(m) profile is lowered from 7.01 (WT) to 6.53 (D32C), implying that any hydrogen bond replacing that between Asp32 and His64 of the WT enzyme most likely involves the neutral thiol rather than the thiolate form of Cys32. It is shown by viscosity variation that the reaction of WT subtilisin with N-Suc-Ala-Ala-Pro-Phe-SBzl is 50% (sucrose) to 100% (glycerol) diffusion-controlled, while that of the D32C construct is 29% (sucrose) to 76% (glycerol) diffusion-controlled. The low-field NMR resonance of 18 ppm that has been assigned to a proton shared by Asp32 and His64, and is considered diagnostic of a LBHB in the WT enzyme, is not present in D32C subtilisin. Thus, the LBHB is not an inherent requirement for substantial rate enhancement for subtilisin.

Cardiac receptor physiology and its application to clinical imaging: present and future.

Both gamma imaging and positron emission tomography (PET) imaging of cell surface receptors have become possible through the development of agonists and antagonists with high specific radioactivity and high specificity for the receptors. An understanding of the physiology of the cardiac receptor system is essential to comprehending receptor imaging. The complexity of the physiologic information developed over the past decade has been compounded by the concomitant discovery of additional receptor subtypes. The following is a review of a select group of cardiac receptors and their regulation-namely, adrenergic, muscarinic-cholinergic, adenosine, and angiotensin I and II receptors. The role of imaging regional receptor localization and function in providing new insights into cardiac pathology and therapeutic avenues is explored.

Early patency of the infarct-related artery after myocardial infarction preserves diastolic filling.

A patent infarct-related artery (IRA) following myocardial infarction has been associated with lower mortality, increased systolic function, decreased left ventricular remodeling, and electrical stability. The purpose of this study was to determine whether coronary artery patency early after myocardial infarction is associated with greater early diastolic filling than a closed artery. Radionuclide ventriculograms were performed at a central laboratory on 167 patients who received alteplase for an acute myocardial infarction and had infarct artery patency determined by cardiac catheterization. The peak early filling rate (PEFR) was assessed by 4 different methods: (1) PEFR (EDV/s)--normalized to the end-diastolic volume; (2) PEFR (SV/s)--normalized to the stroke volume; (3) PEFR (ml/s/m(2))--an absolute diastolic filling rate; and (4) PEFR (PER)--normalized to the peak ejection rate. Patients with a closed IRA (n = 16, Thrombolysis In Myocardial Infarction [TIMI] 0 or 1 flow) and patients with an open IRA (n = 151, TIMI 2 or 3 flow) had similar ages, ejection fractions, and cardiac volumes. However, among patients with an occluded IRA, the PEFR was decreased by 12% to 18% by the 4 measures of diastolic filling (3 of 4 methods, p <0.05). PEFR (EDV/s) was 1.69 +/- 0.9 in the occluded group versus 2.06 +/- 0.4 EDV/s in the open artery group (p = 0.005). By multivariate analysis, IRA patency was an independent predictor of the PEFR by all 4 methods. Early coronary artery patency after an acute myocardial infarction preserves diastolic filling. Improved diastolic function may in part explain part of the long-term benefits of a patent IRA after thrombolytic therapy when there is no documented improvement in the ejection fraction.

Semirational design of a potent, artificial agonist of fibroblast growth factor receptors.

Fibroblast growth factors (FGFs) are being investigated in human clinical trials as treatments for angina, claudication, and stroke. We designed a molecule structurally unrelated to all FGFs, which potently mimicked basic FGF activity, by combining domains that (1) bind FGF receptors (2) bind heparin, and (3) mediate dimerization. A 26-residue peptide identified by phage display specifically bound FGF receptor (FGFR) 1c extracellular domain but had no homology with FGFs. When fused with the c-jun leucine zipper domain, which binds heparin and forms homodimers, the polypeptide specifically reproduced the mitogenic and morphogenic activities of basic FGF with similar potency (EC50 = 240 pM). The polypeptide required interaction with heparin for activity, demonstrating the importance of heparin for FGFR activation even with designed ligands structurally unrelated to FGF. Our results demonstrate the feasibility of engineering potent artificial agonists for the receptor tyrosine kinases, and have important implications for the design of nonpeptidic ligands for FGF receptors. Furthermore, artificial FGFR agonists may be useful alternatives to FGF in the treatment of ischemic vascular disease.

The time course of haemodynamic, autonomic and skeletal muscle metabolic abnormalities following first extensive myocardial infarction in man.

We investigated the time course of genesis of skeletal muscle dysfunction and sympatho-vagal imbalance after myocardial infarction. We studied 22 normal controls, 22 patients with >6 months stable chronic heart failure and 10 patients after a first massive myocardial infarction at 1-3 weeks (the "early" period), 6-8 weeks ("mid") and 6-9 months ("late") following their infarct. Four patients developed overt heart failure. Forearm muscle metabolism was studied using (31)P magnetic resonance spectroscopy (MRS). Sympatho-vagal balance was assessed by heart rate variability and radiolabelled norepinephrine kinetics. Increased norepinephrine spillover (0.55+/-0.02 v 0.27+/-0.04 mg/min/m(2); P<0.01) and decreased heart rate variability were confined to those post-myocardial infarction patients who subsequently developed heart failure. Resting cardiac output was normal in all the post-myocardial infarction patients, although the response of cardiac output to supine bicycle exercise at the "mid" study point was less in the group who subsequently developed heart failure (9+/-1 v 41+/-8 %; P<0.005). In the MRS studies, there were no detectable differences between those who did or did not develop heart failure. The initial rate of ATP turnover, calculated from initial-exercise changes in pH and phosphocreatine (PCr), was increased in established chronic heart failure, but in the post-myocardial infarction patients a numerically similar increase reached statistical significance only in the early group (19+/-3 v 11+/-1 mM/min; P<0.005). The apparent maximum rate of oxidative ATP synthesis, calculated from post-exercise PCr recovery kinetics, was lower than control in the late post-myocardial infarction and established chronic heart failure groups 34+/-5 v 55+/-4 mM/min; P<0.03 and 38+/-3 v 55+/-4 mM/min; P<0.003, respectively). Skeletal muscle metabolism and autonomic function become abnormal after an extensive myocardial infarction. While skeletal muscle abnormalities are relatively slow to develop and unrelated to the degree of failure, excessive neurohormonal activation and impaired cardiac output response to exercise seem from an early stage to characterize patients who subsequently develop chronic heart failure.

Urokinase receptor antagonists: discovery and application to in vivo models of tumor growth.

Urokinase receptor antagonists based on the growth factor domains of both human and murine urokinase which show sub-nanomolar affinities for their homologous receptors have been expressed as recombinant proteins. Further modification of these molecules by preparing fusions with the constant region of human IgG has led to molecules with high affinities and long in vivo half-lives. Smaller peptidic inhibitors have been obtained by a combination of bacteriophage display and peptide analog synthesis. All of these molecules inhibit the binding of the growth factor domain of uPA to the uPA receptor and enhance binding of the uPA receptor to vitronectin. Protein uPA receptor antagonists were tested in an in vivo tumor model using the human breast carcinoma MDAmb231 in immunodeficient mice. Both human and murine receptor antagonists showed significant inhibition of primary tumor growth, demonstrating that in vivo, both tumor and stromal cell uPA receptor dependent plasminogen activation can modulate tumor growth.

Effect of endurance exercise training on heart rate variability at rest in healthy young and older men.

Heart rate variability (HRV) (SD of the RR interval), an index of parasympathetic tone, was measured at rest and during exercise in 13 healthy older men (age 60 to 82 years) and 11 healthy young men (age 24 to 32 years) before and after 6 months of aerobic exercise training. Before exercise training, the older subjects had a 47% lower HRV at rest compared with the young subjects (31 +/- 5 ms vs 58 +/- 4 ms, p = 0.0002). During peak exercise, the older subjects had less parasympathetic withdrawal than the young subjects (-45% vs -84%, p = 0.0001). Six months of intensive aerobic exercise training increased maximum oxygen consumption by 21% in the older group and 17% in the young group (analysis of variance: overall training effect, p = 0.0001; training effect in young vs old, p = NS). Training decreased the heart rate at rest in both the older (-9 beats/min) and the young groups (-5 beats/min, before vs after, p = 0.0001). Exercise training increased HRV at rest (p = 0.009) by 68% in the older subjects (31 +/- 5 ms to 52 +/- 8 ms) and by 17% in the young subjects (58 +/- 4 ms to 68 +/- 6 ms). Exercise training increases parasympathetic tone at rest in both the healthy older and young men, which may contribute to the reduction in mortality associated with regular exercise.

Fate of gelatin-resorcinol-formaldehyde/glutaraldeyde adhesive on femoral vessel morphology.

Several clinical reports have demonstrated that gelatin-resorcinol-formaldehyde/glutaraldehyde (GRFG) glue can be useful in the repair of acute aortic dissection; however, the cellular and extracellular events that follow GRFG application, as well as the mechanisms responsible for the long-term strength and adhesive properties of GRFG, remain unclear. Accordingly, the present study examined the long-term effects of GRFG adhesive application on femoral vessel extracellular structure and composition. The left and right femoral artery and vein were sterilely exposed in adult rats, and GRFG (2 mL) was applied between and around one pair of vessels. An equivalent amount of sterile saline was applied to the contralateral vessels to serve as an intrinsic control. At either 1 (n = 6) or 2 (n = 6) months postoperatively, the lower extremities were perfusion fixed and harvested to preserve the native anatomy and cytoarchitecture of the femoral region. Gross examination of the specimens revealed no evidence of necrosis or wound breakdown. Tissue blocks (4 microm) were then sectioned perpendicular to the treated vessel region and subjected to histomorphometric analysis using computer-assisted microscopy. The perivascular capsule area, relative content of fibrillar collagen, and number of nucleated cells within the interstitial space were computed. At 1 and 2 months following the application of GRFG adhesive, perivascular capsular size increased by 42 and 221%, respectively. Perivascular interstitial collagen content increased by 21% at 1 month and by 50% at 2 months. The nucleated cell number increased by 107% at 1 month and by 166% at 2 months. This cellular infiltrate appeared to be of fibroblastic morphology. Thus, a potential contributory mechanism to the long-term strength and adhesive capacities of GRFG adhesive may be extracellular remodeling and not the intrinsic properties of GRFG glue itself.

Abnormalities in exercising skeletal muscle in congestive heart failure can be explained in terms of decreased mitochondrial ATP synthesis, reduced metabolic efficiency, and increased glycogenolysis.

OBJECTIVE: To distinguish between the effects of reduced oxidative capacity and reduced metabolic efficiency on skeletal muscle bioenergetics during exercise in patients with congestive heart failure. DESIGN AND PATIENTS: Patients were studied by 31P magnetic resonance spectroscopy during aerobic exercise and recovery, and results compared with controls. RESULTS: In flexor digitorum superficialis muscle (26 patients) there was a 30% decrease in oxidative capacity compared with control (mean (SE) 36 (2) v 51 (4) mM/min) and also a 40% decrease in "effective muscle mass" (5 (1) v 9 (1) arbitrary units), probably at least partly the result of reduced metabolic efficiency. Both contribute to increased phosphocreatine depletion and intracellular acidosis during exercise. However, an increased concentration of ADP (an important mitochondrial regulator) during exercise permitted near-normal rates of oxidative ATP synthesis. Results were similar in gastrocnemius muscle (20 patients), with a 30% decrease in maximum oxidative capacity (29 (4) v 39 (3) mM/min) and a 65% decrease in effective muscle mass (5 (1) v 13 (2) arbitrary units). Exercise training improved maximum oxidative capacity in both muscles, and in gastrocnemius effective muscle mass also. CONCLUSIONS: Skeletal muscle exercise abnormalities in patients with congestive heart failure results more from decreased metabolic efficiency than from the abnormalities in mitochondrial oxidation. Both decreased efficiency and defective mitochondrial oxidation result in an increased activation of glycogen phosphorylase, and may be improved by exercise training.

Effects of endurance training on the circadian rhythm of fibrinolysis in men and women.

This randomized study compared the fibrinolytic circadian rhythm of healthy older men and older women (average age 66 +/- 5), before and after 6 months of endurance training versus stretching controls. Compared with men, women at baseline had similar rhythms for tissue plasminogen activator (t-PA) activity and plasminogen activator inhibitor 1 (PAI-1) activity, but lower levels of total t-PA antigen. In men (N = 16), endurance training increased VO2max 15% (P < 0.001), while decreasing PAI-1 activity 37% (P = 0.034) and total t-PA antigen 18% (P = 0.0003) between midnight and 6 a.m., but did not affect t-PA activity. In women (N = 9), endurance training increased VO2max 18% (P = 0.003), and increased t-PA activity 20% (P = 0.027) and total t-PA antigen 55% (P = 0.007) between 10 p.m. and 4 a.m., but had no effect on PAI-1 activity. After endurance training there were no significant differences in the fibrinolytic circadian rhythm of men versus women. Six months of nonaerobic stretching had no effect on VO2max or fibrinolysis in men (N = 11) or women (N = 8). This study indicates that potentially favorable changes occur in fibrinolytic factors after endurance training in older men and older women.

Developmental differences in myocyte contractile response after cardioplegic arrest.

Although developmental differences in left ventricular function after cardioplegic arrest and rewarming have been postulated, whether differences exist at the level of the myocyte remains unexplored. This project tested the hypothesis that there is a differential effect of hypothermic hyperkalemic cardioplegic arrest with subsequent rewarming on contractile function of immature compared with adult ventricular myocytes. Myocytes were isolated from the left ventricular free wall of five immature and five adult rabbits and incubated for 2 hours in hyperkalemic modified Ringer's solution at 4 degrees C (cardioplegia) or for 2 hours in cell culture medium at 37 degrees C (normothermia). Myocytes were resuspended ("rewarmed") in 37 degrees C cell culture medium after the incubation protocol. Normothermic baseline contractile performance was lower in immature, compared with adult, myocytes. Specifically, myocyte shortening velocity was 62 +/- 4 microm/sec in immature and 112 +/-6 microm/sec in adult myocytes (p < 0.01). After cardioplegia and rewarming, immature myocyte contractile function was unchanged, whereas adult myocyte contractile function was significantly diminished. For example, myocyte shortening velocity was 65 +/- 4 microm/sec in immature and 58 +/- 3 microm/sec in adult myocytes (p < 0.01 versus normothermic). Myocyte surface area, which reflects myocyte volume, was increased after cardioplegia and rewarming in adults (3582 +/- 55 versus 3316 +/- 46 microm2, p < 0.01), but remained unchanged in immature myocytes (2212 +/- 27 versus 2285 +/- 28 microm2, P = not significant). These unique findings demonstrate a preservation of myocyte contractile function and volume regulation in immature myocytes after cardioplegic arrest and rewarming. Thus this study directly demonstrates that developmental differences exist in myocyte responses to hypothermic hyperkalemic cardioplegic arrest with subsequent rewarming.

The circulatory regulation of TPA and UPA secretion, clearance, and inhibition during exercise and during the infusion of isoproterenol and phenylephrine.

BACKGROUND: Exercise to exhaustion and infusions of isoproterenol and phenylephrine were used to study interactions between plasminogen activator regulation and the control of regional blood flow in 10 healthy males. METHODS AND RESULTS: Experimental measurements of cardiac output, heart rate, tissue plasminogen activator (TPA), urokinase plasminogen activator (UPA), plasminogen activator inhibitor (PAI-1), C1-inhibitor, and TPA/C1-inhibitor complex during the infusions and exercise were used to develop a comprehensive fluid-phase model of the circulatory regulation of fibrinolysis. alpha- and beta-adrenergic agonists increased TPA and UPA in plasma by different mechanisms: Phenylephrine decreased hepatic blood flow and thus clearance while isoproterenol stimulated increased secretion of TPA and UPA. Exercise to exhaustion increased TPA and UPA through a combination of increased secretion and decreased clearance. The time course of UPA and TPA release were similar, but the magnitude of their secretion responses differed. In vivo, C1-inhibitor bound to TPA at a rate of 553 mol-1.s-1. C1-inhibitor contributed equally with PAI-1 to TPA inhibition when active PAI-1 levels were low (20 to 50 pmol/L) but was less important when active PAI-1 levels were high. CONCLUSIONS: We conclude that secretion, inhibition, clearance, and regional blood flow effects must all be taken into account when evaluating changes in plasminogen activator levels.

Selective uptake of radiolabeled annexin V on acute porcine left atrial thrombi.

BACKGROUND: Annexin V is a human phospholipid binding protein that binds to activated platelets in vitro. We sought to determine the potential of this agent for imaging intracardiac thrombi in swine. METHODS AND RESULTS: Left atrial thrombi were formed by crush injury. In initial nonimaging experiments using intravenous 125I-labeled human annexin V, the mean thrombus/whole blood ratio was 13.4 +/- 4.8 for the entire thrombus using well counting of resected specimens (n = 8). Using intravenously injected 99mTc-labeled human annexin V, the left atrial thrombus/blood ratio by well counting was similar (14.2 +/- 10.6 for the entire thrombus and 26.2 +/- 14.9 for the peak section) (n = 12). The ratio for a control protein, 125I-ovalbumin, was only 1.0 +/- 0.2. 99mTc tomographic imaging was positive (n = 10) or equivocal (n = 2) in all experiments with but negative in 10 controls without left atrial thrombi. By region-of-interest analysis of the tomographic images, the mean left atrial appendage/blood ratio at 2 hours in animals with a thrombus was 3.90 +/- 1.12 compared with 0.84 +/- 0.10 in closed chest controls and 1.01 +/- 0.23 in open chest controls (P < .001). CONCLUSIONS: We conclude that 99mTc-labeled human annexin V detects acute left atrial thrombi in vivo in swine. The combination of a new thrombus detection agent, annexin V, with a 99mTc label may allow in vivo imaging of thrombi in humans.

Beta-adrenergic effects on left ventricular filling: influence of aging and exercise training.

Reduced heart rate and contractile responses to beta-agonist stimulation characterize normal cardiac aging, but whether diastolic responses also decline with aging has not been determined in humans. Diastolic filling responses to isoproterenol were determined in 13 older (60-82 yr) and 11 young (24-32 yr) healthy men before and after endurance training. Filling rates were expressed in three ways: 1) normalized to end-diastolic volume per second, 2) normalized to stroke volume per second, and 3) as absolute milliliters of blood (ml.s-1.m-2). Peak early filling rates by all methods were reduced at rest and all isoproterenol doses with aging (all P < 0.0001 for old vs. young), whereas peak atrial filling rates were increased with aging. During isoproterenol, both peak early and peak atrial filling rates increased significantly (all P < 0.01); the increase in filling rates with isoproterenol was not different with aging (all NS for old vs. young x dose). Endurance training did not augment diastolic filling responses to isoproterenol. Although diastolic filling rates at rest are markedly altered by aging, diastolic filling responses to isoproterenol are not reduced with aging. Thus the age-associated declines in heart rate, ejection fraction, and cardiac output responses to beta-adrenergic stimulation with isoproterenol do not extend to diastolic filling responses.

Imaging arterial thrombosis: comparison of technetium-99m-labeled monoclonal antifibrin antibodies and indium-111-platelets.

UNLABELLED: Imaging with the 99mTc-T2G1s monoclonal antifibrin antibody fragment (Fab') has demonstrated promise in the noninvasive detection of venous thrombi in humans. The purpose of this study was to determine whether chronic arterial thrombi can also be detected by antifibrin antibody imaging. METHODS: Eighteen subjects with chronic arterial thrombi were studied with planar and tomographic imaging at 0 to 24 hr postinjection of 99mTc-labeled T2G1s monoclonal antifibrin antibody fragment. Imaging with 111In-labeled platelets was also performed. Images were visually graded by two observers as 0, 1, 2 or 3 (no, faint, moderate or marked) uptake, and quantitative analysis of tomographic images was done in 13 subjects. RESULTS: On visual analysis of planar images, 44% (8 of 18) of antifibrin patient studies were 1.0 or more and 66% (10 of 18) were judged negative compared with 94% (15 of 16) of platelet patient studies judged 1.0 or more and 6% (1 of 16) judged as negative (p < 0.01). Visual analysis of tomographic images was similar, with 61% (11 of 18) of antifibrin studies graded 1.0 or more compared with 100% (17 of 17) of platelet studies (p < 0.01). The tomographic target-to-background ratio was higher with platelets than with antifibrin antibody (2.5 +/- 1.4 versus 1.8 +/- 1.0, p < 0.05). CONCLUSION: In the large-vessel chronic arterial thrombi studied, the results of 99mTc-labeled monoclonal T2G1s antifibrin Fab' imaging were positive in only one-half of the patients studied, significantly less than the findings with platelet imaging, which were positive in all subjects. The higher rate of positive images with labeled platelets than with labeled antifibrin antibodies may be largely due to thrombus age, with continued platelet deposition but little active fibrin deposition.

Evaluation of annexin V as a platelet-directed thrombus targeting agent.

Annexin V is a human phospholipid binding protein (M(r) 36,000) that binds with high affinity to activated platelets in vitro. We studied the biodistribution and thrombus binding of annexin V in rabbit and swine models of fully occlusive arterial thrombi formed 1-2 h prior to injection of annexin V. Iodinated annexin V was cleared from blood in a rapid early phase (t1/2 = 6.4 min, 76% of radioactivity) and a slower late phase (t1/2 = 71 min, 24% of radioactivity). Organ uptake was highest in the kidney and spleen and lowest in heart and skeletal muscle. Thrombus/blood uptake ratios were (mean +/- SEM): 6.39 +/- 1.80 for rabbit iliac artery, 6.97 +/- 1.45 for swine carotid artery, and 7.68 +/- 1.70 for swine femoral artery (all p values < 0.01 versus control artery); a control protein, ovalbumin, showed an uptake ratio of 0.59 +/- 0.08 in swine femoral artery thrombi. These results indicate that annexin V is useful as an agent for selective targeting of platelet-containing thrombi.

Laboratory evaluation of fibrinolysis in patients with a history of myocardial infarction.

The aims of this study were to determine whether elevated plasminogen activator inhibitor type 1 (PAI-1) activity after myocardial infarction reflects baseline PAI-1 or represents an acute-phase response secondary to the infarction, and to determine how tissue plasminogen activator (t-PA) activity and total t-PA antigen levels in healthy control subjects differ from those in patients after myocardial infarction. Compared with healthy control subjects, patients studied 1-3 months after infarction had elevated levels of PAI-1 activity and fibrinogen but normal levels of C-reactive protein and von Willebrand factor antigen, whereas patients with a noncardiac acute-phase response showed elevation of all four proteins. Elevated PAI-1 activity in the absence of elevations in other acute-phase proteins suggests an intrinsic increase in PAI-1 secretion in the post-myocardial infarction group. In addition, when compared with healthy control subjects, post-myocardial infarction patients had higher levels of total t-PA antigen (bound and free t-PA) but lower t-PA activity and a lower percentage of active t-PA. Overall, survivors of myocardial infarction have reduced t-PA activity and increased PAI-1 activity that is not due to a prolonged acute-phase response.