A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly.

Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.

Antihypertensive and natriuretic effects of CGS 30440, a dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase 24.11.

Dual angiotensin-converting enzyme (ACE)/neutral endopeptidase (NEP) inhibitors, by decreasing angiotensin-II production and by preventing the degradation of atrial natriuretic peptide (ANP), may be useful for the treatment of hypertension and congestive heart failure. The thiol dipeptide CGS 30440 (prodrug of CGS 30008, IC50: ACE/NEP = 19/2 nM) administered to rats (10 mg/kg p.o.) inhibited lung tissue ACE activity by 98% and 61% at 1 and 24 hr (P < .001) and inhibited the angiotensin-I pressor response by 75 to 90% for more than 6 hr. Renal tissue NEP activity was reduced by 80% at 1 hr and 73% at 24 hr (P < .001). In rats supplemented with exogenous ANP, CGS 30440 (1 mg/kg p.o.) elevated the concentration of circulating ANP (133%, P < .025) for 4 hr and increased the excretion of urine (300%, P < .001), sodium (194%, P < .025) and cyclic GMP (238%, P < .005). CGS 30440 (10 mg/kg p.o.) administered to hypertensive rats with aortic ligation between the renal arteries (mean arterial blood pressure, 209 +/- 4 mm Hg) produced a 48 mm Hg blood pressure reduction (P < .001) within 4 hr. CGS 30440 given to cynomolgus monkeys at 2 mg/kg inhibited plasma ACE activity by 96% within 1 hr (P < .001), and this inhibition was maintained for 7 and 21 days in monkeys receiving the compound orally at 2.5 mg/kg b.i.d. These studies demonstrate that CGS 30440 is an orally active agent which produces tissue ACE and NEP inhibition in rats and plasma ACE inhibition in primates and suggest that the compound may be useful in the treatment of hypertension and congestive heart failure.

Effects of cross-linking on clearance of circulating alpha-fibrin monomer and its complexes.

Preceding studies showed that fibrin-monomer of the type lacking only fibrinopeptide A (alpha-fibrin) cleared from the circulation more rapidly than a tighter aggregating form of monomer that lacks both fibrinopeptides A and B (alpha beta-fibrin). The rapid clearance of alpha-fibrin may be related to the high dissociability of the soluble complexes that it forms with fibrinogen in blood. In this study, we use cross-linking by factor XIIIa to suppress dissociation of fibrin complexes and examine the effect of the cross-linking on the circulatory half-life of the fibrin. Incubation of alpha-fibrin-monomer/fibrinogen solutions with factor XIIIa before injection in rabbits increased the circulatory half-life of the fibrin (range, 2 to 16 hours) in proportion to the percentage conversion of monomer to cross-linked dimers and small oligomers. Electrophoretic analyses of plasma samples confirmed that, compared with non-cross-linked monomer, cross-linked dimers and small oligomers were long lived and, further, were not degraded. The inhibition of clearance through cross-linking occurs only under conditions that produce partial cross-linking of the fibrin. An opposite effect occurs when cross-linking is allowed to approach completion, as a result of polymerization of the fibrin into large fibers that disappear almost immediately after injection. Cross-linking elicited in vivo with injected factor XIIIa has an inhibitory effect on clearance of injected monomer similar to the effect produced by partial cross-linking in vitro. It is proposed that the prolonged survival of cross-linked dimers and small oligomers in the circulation provides a partial explanation for the frequent prevalence of cross-linked rather than non-cross-linked complexes in blood of human subjects with vascular disease.

Antihypertensive effects of CGS 19281A, an inhibitor of phenylethanolamine-N-methyltransferase.

In experimental hypertension, phenylethanolamine-N-methyltransferase (PNMT) activity and adrenaline levels are elevated in brainstem centers involved in cardiovascular regulation. Known PNMT inhibitors used to lower blood pressure have invariably shown alpha adrenergic activity as a side effect. A search for new inhibitors disclosed that CGS 19281A (4,9-dihydro-7-methoxy-3H-pyrido[3,4b]indole) inhibits PNMT (IC50, 2.7 x 10(-6) M) without interacting with the alpha-1 or alpha-2 adrenergic receptors. CGS 19281A (20 mg/kg i.v.) reduced PNMT activity (60% decrease; P less than 0.001) and adrenaline concentration (38%; P less than .025) in the brainstem of normal rats. In conscious deoxycorticosterone acetate-salt rats and spontaneously hypertensive rats, CGS 19281A (20 mg/kg i.v.) decreased blood pressure (50 mm Hg; P less than .001) and heart rate (26-36%; P less than .001) for 3 hr. Elevated brainstem adrenaline levels in deoxycorticosterone acetate-salt rats were decreased by CGS 19281A (42%; P less than .005) whereas i.c.v. administration of CGS 19281A (845 nmol/rat) to conscious spontaneously hypertensive rats decreased blood pressure (20 mm Hg; P less than .010) and heart rate (84 beats/min; P less than .001) as well. Therefore, CGS 19281A may be useful for the study of the function of PNMT and adrenaline in the central nervous system.

Malignant hypertension, fibrinoid deposition, and fibrinogen electrophoresis.

Electrophoretic profiles of the molecular weight distributions of fibrinogen derivatives in blood provide a tool for combined assessment of coagulation and fibrinolysis in the course of vascular disease. Profiles obtained in studies on an experimental model of hypertension and in humans with occlusive vascular disease are discussed. In the experimental studies elevations in the level of cross-linked dimers provided a more reliable means for predicting development of malignant hypertension than did many other criteria, especially near the outset when blood pressure changed to similar degrees in rats with malignant and benign hypertension. Similarly, we find that levels of dimeric and occasionally trimeric forms of fibrinogen are more prominently elevated than degraded forms of fibrinogen in patients with occlusive vascular disease.

Elevated arterial cyclic AMP levels during the development of one-kidney, one-clip and DOCA hypertension in rats.

A marked increase in the cyclic AMP content and concentration of the thoracic aorta was detected during the rise in blood pressure produced by one-kidney, one-clip and DOCA hypertension. This alteration was accompanied by the development of vascular hypertrophy and preceded the abnormal increase in DNA content observed in the arterial system during the more chronic stages of the disease. These experiments suggest the participation of cyclic AMP in the development of hypertension vascular growth.

Increased DNA replication in the arterial adventitia after aortic ligation.

After microsurgical dissection of the thoracic aorta of normotensive rats, biochemical and morphological comparisons were performed between the intima-media and adventitia. The DNA content, wet weight, and dry defatted weight of the adventitia were half that of the intima-media. Collagen was the main component of the adventitia (collagen greater than nonfibrous protein greater than elastin) whereas elastin was the main protein in the intima-media (elastin greater than nonfibrous protein greater than collagen), and the results correlated with morphological observations. Hypertension induced by aortic ligation between the renal arteries resulted in rapid elevations in circulating humoral factors and blood pressure. A sixfold increase in DNA synthesis was observed in the adventitia (p less than 0.001), resulting in a significant increase in DNA content as early as 6 days after aortic ligation (75% increase; p less than 0.001). Increased DNA replication was accompanied by elevations in nonfibrous protein and elastin contents. Autoradiograms showed labeled adventitial fibroblasts throughout the thickness of the adventitia and along the entire length of the aorta and smaller vessels. DNA synthesis and content and labeled smooth muscle cells were increased in the intima-media. These studies indicate that the adventitia participates in the development of vascular hypertrophy and arterial disease produced by aortic ligation.

Elevated plasma catecholamines and adrenal phenylethanolamine N-methyl transferase in experimental renal hypertension.

The participation of plasma catecholamine alterations in the development of renal hypertension is uncertain. Therefore, plasma catecholamines and phenylethanolamine N-methyl transferase (PNMT) activity in the adrenal gland were studied in rats with aortic ligation between the renal arteries. Blood pressure reached a plateau after 12 days (mean arterial pressure (MAP): 194 +/- 3 mmHg; P less than 0.001) and its elevation was accompanied by a biphasic elevation in plasma adrenaline. The first elevation (4-fold above control levels; P less than 0.001) occurred at 24 h after aortic ligation and lasted for 4 days. The second elevation commenced on day 6, reached its zenith at day 9 (16-fold increase; P less than 0.005) and lasted for 6 days. The first elevation was associated with the highest levels of plasma renin activity (PRA) (34-fold increase; P less than 0.001) and glucocorticoids (74% increase; P less than 0.001) but plasma noradrenaline, plasma dopamine and adrenal PNMT activity were minimally affected. However, a statistically significant increase in PNMT activity preceded and accompanied the second adrenaline elevation. Despite falling PRA and glucocorticoid levels, marked increases in plasma noradrenaline (5-fold increase; P less than 0.001) and plasma dopamine (2.5-fold increase; P less than 0.010) were observed. These experiments identify an early activation of the sympatho-adrenal axis in renal hypertension. Apparently there is a rapid release of the adrenaline pool followed by an elevation in PNMT activity. The results suggest that the sympatho-neuronal axis is also activated leading to increases in both plasma noradrenaline and dopamine levels.

Elevated arterial cyclic AMP levels during the development of spontaneous hypertension in rats.

Vascular cyclic AMP alterations were studied during the initiation of vascular hypertrophy and hyperplasia in spontaneously hypertensive rats (SHR). The onset of hypertension at 6 weeks of age coexisted with a three-fold elevation in the aortic content and concentration of cyclic AMP, whereas aortic DNA and protein contents were identical to those of WKY controls. A similar cyclic AMP elevation was present in 12-week-old SHR when vascular hypertrophy and hyperplasia were already established. These experiments suggest the participation of cyclic AMP in the process of hypertensive vascular growth.

Kinetic characterization of a saturable pathway for rapid clearance of circulating fibrin monomer.

The mechanism of clearance of circulating fibrin monomer was investigated in rabbits through (1) study of decay in plasma concentrations of 125I-labeled monomers with variant fibrinopeptide content and (2) concurrent analysis of decay of the monomers relative to coinjected 131I-fibrinogen. Under the conditions employed, essentially all of the fibrin became distributed in a soluble form in plasma and decayed independently of the coinjected fibrinogen. Among the species of fibrin studied, monomer lacking fibrinopeptide A alone (alpha-fibrin) underwent very rapid clearance by a saturable mechanism that was not evident in relatively sluggish clearance of monomer lacking either fibrinopeptide B alone (beta-fibrin) or both fibrinopeptides A and B (alpha beta-fibrin). Decay of alpha-fibrin conformed with a kinetic mechanism involving first-order permeation of the fibrin into extravascular space at a rate equivalent to that of permeation of fibrinogen; unlike fibrinogen, however, the alpha-fibrin underwent immediate absorption in parallel with permeation (t1/2 = 2.6 hours) at doses below an apparent saturating level of 3 mg/kg. At doses near the absorptive limit, the uptake accompanying permeation diminished as in a second-order kinetic mechanism, and at very high doses the plasma decay of the alpha-fibrin approached that of fibrinogen. The beta- and alpha beta-fibrins also permeated extravascular space in parallel with fibrinogen, but absorption proceeded sluggishly (t1/2 = 11 and 16 hours, respectively) at low doses and did not change with increasing dose. The uniquely rapid and saturable clearance of alpha-fibrin is suggested to involve uptake through the fibrin aggregation site that is blocked by fibrinopeptide A in fibrinogen and beta-fibrin and by tight binding to fibrinogen in soluble complexes formed by alpha beta-fibrin. A corollary of this hypothesis is that rapid uptake depends on dissociability of fibrin complexes for access to the aggregation site, a mechanism that is just the converse of uptake through aggregation.

Acute arterial fibrinoid deposition and ischaemic parenchymal damage of the kidney. Pathogenic factors in the development of malignant hypertension.

The development and evolution of hypertensive vascular lesions affecting the arterial supply of (a) the kidney and (b) organs other than the kidney were studied in rats developing either malignant (MHY) or benign (BHY) hypertension 3, 6, 9 and 12 days after aortic ligation between the renal arteries. Vascular disease evolved into two distinct patterns which suggested acute renal damage to be the determinant for the development of either the malignant or benign form of hypertension. Three days after aortic ligation MHY and BHY animals showed widespread fibrinoid deposition in vascular territories above the aortic ligature. However, in MHYs these lesions were much more severe and, in the kidney, they were accompanied by the development of focal parenchymal atrophy, microinfarcts and hyalin droplet degeneration of cells of the Bowman capsule. The degree of renal damage correlated with elevations in blood urea nitrogen (BUN) and plasma creatinine; however, there was no correlation with rises in blood pressure, plasma renin activity (PRA), aldosterone or corticosterone which were similarly elevated in 3-day MHY and 3-day BHY animals. Between 6 and 12 days a marked clearance of fibrinoid took place in all organ beds of BHYs, but in the non-renal vasculature of MHY animals fibrinoid remained prominent and served as the central core for necrotising arterial lesions. In the kidney of MHYs some reduction in the fibrinoid content was observed, but the parenchymal damage perpetuating from the earlier stages had exacerbated leading to collagen deposition and a marked increase in the collagen concentration of the renal cortex. These features were accompanied by further elevations in PRA and corticosteroids and a progressive deterioration of renal function. By contrast, in 12-day BHY animals, despite sustained hypertension, PRA and corticosteroids were falling from their previously higher levels and normal renal function was maintained. These studies warrant inference that extensive parenchymal damage of the kidney due in part to severe arterial fibrinoid deposition is one of the initial events in the development of malignant hypertension.

Fibrinopeptide B in fibrin assembly and metabolism: physiologic significance in delayed release of the peptide.

The delayed release of peptide B that accelerates towards the end of fibrin formation unmasks accessory (b-) epitopes for monomer interaction. Ultracentrifuge and chromatographic analysis of the composition and dissociation of soluble complexes formed by monomers in fibrinogen solution indicate that the b-epitope augments aggregation by acting cooperatively with the a-epitope to reinforce rather than cross-bridge oligomer assembly. Monomer/fibrinogen association by coordinated interactions through both epitopes is strengthened by an additional order of magnitude over associations (10(7) and 1.6 X 10(6) M-1) through the a- and b-epitopes individually, without affecting oligomer thickness. It is suggested that the delayed release of B has purpose in allowing early complexes to dissociate for (1) rapid equilibration across interstitial fluids, and for (2) rapid uptake by phagocytic cells which depend on access to the a-epitope for monomer absorption. In late stages of coagulation, stabilization of oligomer assembly imparted by the b-epitope blocks both equilibration of fibrin concentrations and phagocytic clearance of the fibrin to localize deposition.

Cascade immunoelectrophoresis: combined electrophoretic and solid-phase processing of immunoreactive protein by zonal immobilization.

A new approach to immunochemical analysis of complex mixtures of proteins has been devised through development of a system for (1) sequentially absorbing, desorbing, and cascading antibodies in a profile indicative of both the quantity and electrophoretic characteristics of the antigen, and (2) for carrying out multiple tests of antigenic determinants at sub-picomole levels regardless of the solubility or precipitin forming characteristics of the immunoreactive protein. The procedure employs zonal immobilization in which a novel, aldehyde-rich gel (glyoxyl agarose) is used interchangeably as an inert support for electrophoresis and as an immobilizing matrix to fix the protein for analysis by solid-phase techniques. Proteins may be separated on the gel as with ordinary agarose, and then driven to combine covalently with the gel upon exposure to NaCNBH3. After removing NaCNBH3, the distribution of specified antigens is then established by exposing the gel to antibody an profiling the pattern of antibody uptake by a cross-electrophoretic technique in which the absorbed antibody is (1) desorbed with dodecyl sulfate, then (2) transferred through a gel containing potassium ion to halt migration of the detergent, and (3) displayed by immunoprecipitation with anti-IgG antibodies. To cascade the process further, the IgG may be immobilized and used as a surrogate antigen to bind additional antibody protein. In addition to enhancing sensitivity, the cross-electrophoretic measurement of uptake of antibody by immobilized antigen eliminates dependence on direct immunoprecipitation of the antigen, and may accordingly be of particular advantage with non-precipitin forming antigens and monoclonal antibodies.

Fibrinopeptide B and aggregation of fibrinogen.

Removal of fibrinopeptide B from human fibrinogen by reaction with the procoagulant enzyme from copperhead snake venom below 25 degrees C resulted in tight aggregation of the fibrinogen, which, in turn, progressively blocked a concomitant but sluggish release of fibrinopeptide A by the enzyme. When the clots obtained at less than 25 degrees C were warmed, they dissociated into soluble aggregates and monomers. Release of fibrinopeptide A then resumed, and a secondary coagulation followed. The aggregation induced by release of fibrinopeptide B itself involves a plasmin-susceptible segment located just distal to B in the B beta chain of fibrinogen, a segment previously shown to be of little importance in the aggregation induced by release of fibrinopeptide A.