Dissecting sites important for complement regulatory activity in membrane cofactor protein (MCP; CD46).

Membrane cofactor protein (MCP; CD46), a widely distributed regulator of complement activation, is a cofactor for the factor I-mediated degradation of C3b and C4b deposited on host cells. MCP possesses four extracellular, contiguous complement control protein modules (CCPs) important for this inhibitory activity. The goal of the present study was to delineate functional sites within these modules. We employed multiple approaches including mutagenesis, epitope mapping, and comparisons to primate MCP to make the following observations. First, functional sites were located to each of the four CCPs. Second, some residues were important for both C3b and C4b interactions while others were specific for one or the other. Third, while a reduction in ligand binding was invariably accompanied by a parallel reduction in cofactor activity (CA), other mutants lost or had reduced CA but retained ligand binding. Fourth, two C4b-regulatory domains overlapped measles virus interactive regions, indicating that the hemagglutinin docks to a site important for complement inhibition. Fifth, several MCP regulatory areas corresponded to functionally critical, homologous positions in other CCP-bearing C3b/C4b-binding proteins. Based on these data and the recently derived crystal structure of repeats one and two, computer modeling was employed to predict MCP structure and examine active sites.

Conservation in decay accelerating factor (DAF) structure among primates.

The decay accelerating factor (DAF, CD55) protects self cells from activation of autologous complement on their surfaces. It functions to disable the C3 convertases, the central amplification enzymes of the cascade. Its active site(s) are contained within four approximately 60 amino acid long units, termed complement control protein repeats (CCPs), which are suspended above the cell surface on a 68 amino acid long serine/threonine (S/T)-rich cushion that derives from three exons. We previously proposed a molecular model of human DAF's four CCPs in which certain amino acids were postulated to be recognition sites for the interaction between DAF and the C3 convertases. In the current study, we characterized DAF in five non-human primates: the great apes, gorilla and common chimpanzee, and the Old World monkeys: hamadryas baboon, Rhesus macaque, and patas monkey. Amino acid homology to human DAF was approximately 98% for the two great apes and 83% for the three Old World monkeys. The above cited putative ligand interactive residues were found to be fully conserved in all of the non-human primates, although there were amino acid changes outside of these areas. In the chimpanzee, alternative splicing of the S/T region was found potentially to be the source of multiple protein isoforms in erythrocytes, whereas in the patas monkey, similar alternative splicing was observed but only one protein band was seen. Interestingly, a Rhesus macaque was found to exhibit a phenomenon paralleling the human Cromer Dr(a-) blood group, in which a 44-base pair deletion in CCP3 leads to a frameshift and early STOP codon.

Decay accelerating activity of complement receptor type 1 (CD35). Two active sites are required for dissociating C5 convertases.

The goal of this study was to identify the site(s) in CR1 that mediate the dissociation of the C3 and C5 convertases. To that end, truncated derivatives of CR1 whose extracellular part is composed of 30 tandem repeating modules, termed complement control protein repeats (CCPs), were generated. Site 1 (CCPs 1-3) alone mediated the decay acceleration of the classical and alternative pathway C3 convertases. Site 2 (CCPs 8-10 or the nearly identical CCPs 15-17) had one-fifth the activity of site 1. In contrast, for the C5 convertase, site 1 had only 0.5% of the decay accelerating activity, while site 2 had no detectable activity. Efficient C5 decay accelerating activity was detected in recombinants that carried both site 1 and site 2. The activity was reduced if the intervening repeats between site 1 and site 2 were deleted. The results indicate that, for the C5 convertases, decay accelerating activity is mediated primarily by site 1. A properly spaced site 2 has an important auxiliary role, which may involve its C3b binding capacity. Moreover, using homologous substitution mutagenesis, residues important in site 1 for dissociating activity were identified. Based on these results, we generated proteins one-fourth the size of CR1 but with enhanced decay accelerating activity for the C3 convertases.

The C3b/C4b receptor (CR1, CD35) on erythrocytes: methods for study of the polymorphisms.

This report is devoted to methodologies used in analyzing the C3b/C4b receptor (CR1, CD35) on erythrocytes (E), its soluble form, the CRI structural or allotype polymorphism, and CR1 density polymorphism. In primates E CR1 serves as the main system for processing and clearance of complement opsonized immune complexes (IC). CR1 copy numbers decrease with aging of E in normal individuals. Erythrocyte CR1 is also decreased in pathological conditions such as systemic lupus erythematosus (SLE), HIV infection, certain hemolytic anemias, and many other conditions featuring immune complexes. Consequently, CRI on E has an important physiological role in immune complex handling and has interesting alterations in disease.

Mapping epitopes for 20 monoclonal antibodies to CR1.

Complement receptor type one (CR1; CD35) binds and processes C3b and C4b opsonized immune complexes and regulates complement activation. We have characterized the epitopes of 13 previously reported and seven new MoAbs to human CR1. The MoAbs formed seven groups based on their reactivity with a panel of deletion forms of CR1. Seventeen of the MoAbs reacted with CR1 at more than one site, a consequence of its repetitive sequence. All five of the MoAbs recognizing epitopes in the nearly identical repeats 3, 10, and 17, as well as one MoAb which reacted with repeats 8 or 1/2 of 9 and 15 or 1/2 of 16, blocked cofactor activity for C3b. Knowledge of the repeats bearing the epitopes for these MoAbs should facilitate the further characterization of CR1.

Substitution of two amino acids confers C3b binding to the C4b binding site of CR1 (CD35). Analysis based on ligand binding by chimpanzee erythrocyte complement receptor.

The chimpanzee (Ch) E complement receptor type 1 (CR177) appears to be an alternatively spliced product of the Ch CR1 gene transcript. Its cDNA-derived amino acid sequence contains complement protein-repeating modules (CP) 1-6, 28, 29, and 30 in tandem and is 98.8% homologous to the corresponding regions of human (Hu) CR1. It differs from the C4b binding site of Hu CR1 only by two amino acids, Tyr for Ser37 in CP 1 and Asp for Gly79 in CP 2. However, in addition to binding C4b, Ch E binds C3b. As homologous substitution of one of these amino acids (Tyr for Ser37) was previously shown to not confer C3b binding, we reasoned that either single substitution of the other amino acid or a combination of the two amino acid changes would be required for C3b binding. To test this, using a truncated form of Hu CR1 that has a binding site only for C4b, we made these additional constructs. Single substitution of either amino acid did not affect the ligand binding or cofactor activity. However, the double substitution induced C3b binding and increased cofactor activity for C3b without changing the C4b-binding property. Of interest, these two amino acids are also found in the homologous positions of CP 9 and 16, which form part of the C3b binding site of Hu CR1. Thus, Ch E CR177, one-third of the size and with only a single ligand binding site, by acquiring key amino acid substitutions, binds C3b and C4b and functions analogous to Hu E CR1.

Identification of complement receptor type 1-related proteins on primate erythrocytes.

The purpose of this study was to characterize the structure and function of the immune adherence receptor (CR1, CD35, C3b/C4b receptor) of primates. Western blotting, immunoprecipitation, ELISA, and affinity chromatography with homologous C3b and C4b were utilized. The major cross-reactive E membrane protein of ten species of primates tested was lower in m.w. than was human CR1 and fell into two size groups of 55 to 75 and 130 to 165 kDa. There was 10- to 100-fold more CR1 per primate E than human E. Five species also expressed lesser quantities of a protein similar in m.w. (approximately 200 kDa) to human CR1. In contrast to E, the major cross-reactive protein on PBMC was similar in size to human CR1. Four species also expressed lesser amounts of a lower m.w. protein on their PBMC of the same M(r) as that found on their E. Affinity chromatography demonstrated that the approximately 200-kDa form, if present, was recovered with a similar efficiency to that of human CR1. Three patterns of binding, however, were identified among the lower m.w. proteins: 1) C3b > or = C4b; 2) C4b > C3b; and C3b only or predominantly. The fact that these E proteins cross-react with Ab to human CR1, bind homologous C3b and, in most cases, C4b, and for some species represent the only such protein expressed on their E identifies them as immune adherence receptors. The 70-kDa CR1 of the chimpanzee E seems to arise by alternative splicing of the mRNA encoding the 200-kDa protein. These data raise interesting questions relative to the evolution of CR1 in primates and provide a basis for analysis of structure-function relationships among these size forms of CR1.

Comparison of diltiazem at two dose levels with propranolol for treatment of stable angina pectoris.

Two dose levels of diltiazem with propranolol were compared in the management of chronic stable angina. Two groups of patients were treated for alternate periods of 4 weeks with each drug in a double-blind crossover with computer-assisted maximal treadmill tests and ambulatory ST-segment monitoring for evaluation of efficacy and safety. In 12 patients who received diltiazem, 180 mg/day, the time to development of angina increased from 5.9 +/- 0.7 minutes (+/- standard error of the mean) during placebo treatment to 8.3 +/- 0.8 minutes during diltiazem treatment and to 9.2 +/- 0.8 minutes with propranolol, 240 mg/day. Three patients became angina-free when they were treated with both drugs. Among 12 patients who received diltiazem, 360 mg/day, 1 patient became angina-free during treatment with both drugs and 1 became angina-free with diltiazem only. The mean exercise time increased from 5.8 +/- 0.7 minutes with placebo to 8.6 +/- 1.0 minutes with diltiazem, 360 mg/day, and to 8.2 +/- 0.6 minutes with propranolol, 240 mg/day. Analysis of variance showed no difference in efficacy between the 2 doses of diltiazem or between the 2 drugs. Ambulatory heart rate was reduced both during the day and at night with both drugs and significantly more with propranolol than with diltiazem treatment. Except for 1 patient in whom a rash developed when given diltiazem, 180 mg/day, and another who had both a rash and first-degree heart block with diltiazem, 360 mg/day, both drugs were well tolerated. Thus, diltiazem in a daily dose of 180 or 360 mg/day is as effective as propranolol for the treatment of chronic stable angina.

Changes of Q wave amplitude during exercise for the prediction of coronary artery disease.

We have examined the changes of Q wave amplitude during exercise in 156 patients with chest pain with a view to improving the accuracy of stress testing for the diagnosis of coronary artery disease. Coronary arteriography showed significant disease in 127 patients and normal arteries or minimal disease in 29. The Q wave amplitude was measured in lead CM5 from the computer-derived average of 25 consecutive beats immediately before and at the peak of maximal treadmill exercise. The amplitude was greater in the normal subjects at rest and increased with exercise, but the reverse occurred in those with coronary disease. Using the criterion of decrease or no change of Q wave amplitude during exercise as indicating a positive test, the discriminative capacity of Q wave changes was equivalent to that of ST segment depression and was maintained when patients with myocardial infarction were excluded. Using either an abnormal Q wave or ST segment response to exercise improved the test's sensitivity with a loss of specificity but no change of predictive value. In 42% of patients with coronary disease when both the Q wave and ST segment exercise responses were abnormal coronary disease was predicted with an accuracy of 91%. Analysis of subgroups of patients with coronary artery disease suggested a possible explanation for the observed changes in Q wave amplitude, measurement of which can improve the stress test's accuracy for predicting obstructive coronary artery disease.

Randomized double-blind comparison of gallopamil and propranolol in stable angina pectoris.

A new calcium ion antagonist, gallopamil, 150 mg/day, was compared with propranolol, 240 mg/day, in 20 patients with stable chronic angina. The patients were studied in a randomized, placebo-controlled, double-blind, crossover trial. Multistage treadmill exercise with computer-assisted electrocardiographic analysis was performed after 2 weeks of placebo therapy and at the end of each 4-week active treatment period. The mean (+/- standard error of the mean) exercise time to development of angina was 5.4 +/- 0.3 minutes with placebo; this increased to 9.4 +/- 0.7 minutes with propranolol (p less than 0.001) and 10.1 +/- 0.7 minutes with gallopamil (p less than 0.001 vs placebo; difference not significant vs propranolol). Both drugs significantly prolonged the time to development of 1 mm of ST depression. Five patients became free of angina during treadmill testing with gallopamil therapy and 2 with propranolol. Both drugs decreased the heart rate at rest; propranolol also decreased the maximal exercise heart rate, which was slightly increased with gallopamil. With the exception of 1 patient in whom raised liver enzymes developed, gallopamil was well tolerated. Thus, gallopamil is an effective antianginal agent that has few of the unwanted effects associated with other calcium channel-blocking drugs.

The role of a slow channel inhibitor, verapamil, in the management of hypertension.

The 'Oxford' system for continuous monitoring of the ambulatory blood pressure was used to assess the changes in blood pressure following therapy with the slow channel inhibitor, verapamil. Sixteen patients were studied on no therapy and following a minimum of 6 weeks therapy (dose range 120-160 mg t.d.s.). During each study patients underwent standardized physiological tests including tilt, isometric handgrip and dynamic bicycle exercise. Verapamil was demonstrated to produce a consistent reduction of blood pressure over most of the 24 h period studied but this was most marked during the day. Heart rate was also reduced. There was no postural hypotension and the absolute responses to dynamic and isometric exercise were reduced. The results demonstrate the antihypertensive efficacy of 'slow channel inhibitors' as represented by verapamil.

Evaluation of verapamil and high dose nifedipine in patients with chronic stable angina with objective methods.

The efficacy and safety of verapamil and nifedipine were objectively assessed in patients with chronic stable angina. Twenty four patients entered a double blind randomized cross over trial of nifedipine (10 mg thrice daily) and placebo. In this dosage nifedipine did not show any significant change in exercise duration and the variables obtained using computer assisted exercise testing when compared to placebo. The next stage consisted of another double blind randomized cross over trial comparing the effects of verapamil (120 mg thrice daily) and nifedipine (20 mg thrice daily) with an initial placebo run-in period in 32 patients. At this dose level nifedipine showed a definite and significant improvement in all the objective variables; however an increase in side effects was observed resulting in withdrawal of the drug in seven patients. A common problem was tachycardia precipitating angina after nifedipine ingestion. On the other hand verapamil produced a marked improvement in exercise tolerance and other variables as compared to placebo and nifedipine, was well tolerated and produced a mild bradycardia. This study clearly indicates that verapamil is distinctly superior to nifedipine in efficacy side effects and safety in patients with chronic stable angina. This may be attributable to the differential effect on heart rates induced by these drugs.

Nifedipine or verapamil as sole treatment of hypertension. An intraarterial study.

Intraarterial ambulatory blood pressures were recorded prior to and during therapy with two different calcium ion antagonists, nifedipine and verapamil, in two separate groups of patients. In the first group, nine patients were studied off therapy and following a minimum of 6 weeks of nifedipine treatment (dose range, 20 to 60 mg twice daily). A second group of 16 patients followed the identical protocol but were prescribed verapamil (120 to 160 mg, three times daily). During both studies, patients underwent standardized physiological tests including tilt, isometric handgrip, and dynamic bicycle exercise. Both verapamil and nifedipine caused a reduction in blood pressure over most of the 24 hours studied. Nifedipine did not affect heart rate whereas verapamil caused a reduction of approximately 10 bpm. Nifedipine and verapamil did not induce postural hypotension, and the absolute responses to dynamic and isometric exercise were reduced. These results show the efficacy of slow channel inhibitors in the management of essential hypertension.

Long-term effectiveness of verapamil in stable and unstable angina pectoris. One-year follow-up of patients treated in placebo-controlled double-blind randomized clinical trials.

The clinical responses to 12 months' treatment with verapamil were evaluated in 63 patients with stable and unstable angina pectoris in whom the effectiveness of verapamil had been established in short-term double-blind placebo-controlled randomized clinical trials. In 41 patients with effort-related angina, long-term responses were sustained for periods exceeding 1 year. Twenty patients were evaluated by clinical history and showed a sustained reduction in frequency of anginal attacks and consumption of nitroglycerin with verapamil compared with the initial placebo control periods; the magnitude of this benefit was similar to that observed during double-blind treatment with the drug. Twenty-one patients were evaluated by serial treadmill exercise testing and showed a sustained improvement in exercise duration after 4, 8, 16, 24, and 52 weeks of verapamil treatment; withdrawal of the drug resulted in a deterioration of exercise performance to levels similar to those seen before initiation of therapy. In 22 patients with unstable angina at rest, verapamil produced an amelioration of anginal symptoms that was sustained in most patients for longer than 1 year. However, these patients continued to have a high incidence of death and myocardial infarction in a frequency similar to that previously reported in large clinical studies using either combinations of verapamil and nitrates, nifedipine and propranolol, or propranolol and nitrates. Calcium-channel antagonists may decrease the number of patients requiring coronary artery bypass surgery for relief of refractory angina, but they do not appear to alter the natural history of the disease.

Rationale for the choice of calcium antagonists in chronic stable angina. An objective double-blind placebo-controlled comparison of nifedipine and verapamil.

The effectiveness and safety of verapamil, nifedipine, and placebo in patients with chronic stable angina pectoris were evaluated and compared in two double-blind randomized crossover trials. In the first study, nifedipine (10 mg 3 times daily) was compared with placebo in 24 patients with chronic effort-related angina pectoris; no significant differences in exercise performance were observed with nifedipine compared with placebo. In the second study, the effects of verapamil (120 mg 3 times daily), nifedipine (20 mg 3 times daily), and placebo were compared in 32 patients with chronic stable angina using a double-blind crossover study design. Compared with placebo, both nifedipine and verapamil prolonged exercise duration (5.7 +/- 0.3 minutes with placebo, 7.9 +/- 0.5 minutes with nifedipine [p less than 0.001], and 10.0 +/- 0.7 minutes with verapamil [p less than 0.001]), but the improvement with verapamil was greater than that seen with nifedipine (p less than 0.01). Seven patients had increasing angina with nifedipine, none did with verapamil; the exacerbation of angina during nifedipine therapy appeared related to our observation that, compared with placebo, patients receiving nifedipine had higher heart rates, while patients receiving verapamil had slower heart rates. This study indicates that, at the doses used, verapamil was more effective and better tolerated than nifedipine in patients with chronic stable angina pectoris.

Calcium channel blockade as primary therapy for stable angina pectoris. A double-blind placebo-controlled comparison of verapamil and propranolol.

The effectiveness and safety of the beta-adrenergic blocking agent propranolol and the calcium channel antagonist verapamil were compared in 22 patients with chronic stable angina pectoris using a double-blind randomized placebo-controlled crossover protocol. The double-blind phase was preceded by a 2 week single-blind placebo period, followed by randomization to either 4 weeks' therapy with verapamil, 360 mg/day, or propranolol, 240 mg/day, followed by crossover to the other drug. Both verapamil and propranolol increased exercise tolerance (5.5 +/- 0.4 minutes with placebo, 7.8 +/- 0.5 minutes with propranolol [p less than 0.001], and 9.1 +/- 0.5 minutes with verapamil [p less than 0.001]), but the increase with verapamil was significantly greater (p less than 0.01). Both drugs prolonged the exercise duration to 1 mm S-T depression (3.3 +/- 0.4 minutes with placebo, 5.7 +/- 0.5 minutes with propranolol [p less than 0.001] and 5.5 +/- 0.6 minutes with verapamil [p less than 0.001]); the degree of improvement was similar with both active drugs. Both drugs decreased the resting heart rate (76 +/- 3 beats/min with placebo, 56 +/- 2 beats/min with propranolol [p less than 0.001], and 71 +/- 3 beats/min with verapamil [p less than 0.01]), but the heart rate decreased more with propranolol than with verapamil (p less than 0.001). Neither drug produced significant adverse reactions. This study, along with 8 similar double-blind placebo-controlled randomized investigations which have compared verapamil with propranolol, indicate that verapamil is as effective and safe as propranolol in relieving symptoms and improving exercise tolerance in patients with chronic stable angina pectoris and may be considered a first-line therapeutic agent in patients with ischemic heart disease.