Robert J. Gould

Disintegrins: a family of integrin inhibitory proteins from viper venoms.

Abstract Disintegrins represent a new class of low molecular weight, RGD-containing, cysteine-rich peptides isolated from the venom of various snakes. They interact with the β 1 and β 3 families of integrins and their potency is at least 500–2000 times higher than short RGDX peptides. Analysis of the amino acid sequences of 14 different disintegrins suggests that the RGD sequence, in the spatial configuration determined by the appropriate pairing of the cysteine residues, functions as a cell recognition site. However, certain nonconserved amino acids appear to modify the activity of disintegrins, their specificity for various receptors, and their ability to compete specifically with various ligands.

A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy

Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks. Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance. Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families. Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids. Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration.

Expression and secretion of biologically active echistatin in Saccharomyces cerevisiae

A synthetic gene coding for a platelet aggregation inhibitor, echistatin (ECS), was inserted into a Saccharomyces cerevisiae expression vector utilizing the alpha-mating factor pre-pro leader sequence and galactose-inducible promoter, GAL10. Cleavage of the pre-pro leader sequence in vivo results in the secretion of a properly processed recombinant ECS with the native N-terminal glutamic acid residue. Recombinant ECS was recovered from yeast supernatants and purified by reverse phase high performance liquid chromatography. Recombinant ECS expressed and purified from yeast was identical to native ECS in its ability to inhibit platelet aggregation.

Antiplatelet Effects of MK‐852, a Platelet Fibrinogen Receptor Antagonist, in Healthy Volunteers

MK‐852, a cyclic heptapeptide, is a potent platelet fibrinogen receptor antagonist When administered to normal healthy male subjects by 1 ‐ and 4‐hour constant rate intravenous infusions, it provides a generally well‐tolerated and reversible means of inhibition of platelet function. At infusion rates of 1 μg/kg/min for 1 hour and 0.44 μg/kg/min for 4 hours, respectively, MK‐852 extended baseline bleeding time by greater than 2.2‐fold and 2.6‐fold, inhibited ADP‐induced platelet aggregation by 76% and 69%, and inhibited collagen‐induced platelet aggregation by 65% and 67%, respectively. The pharmacokinetics of MK‐852 include an elimination half‐life of approximately 2 hours, total clearance of about 150ml/min, and volume of distribution of about 18 liters. Examination of the relationship between MK‐852 whole‐blood concentration in vitro and inhibition of platelet aggregation showed an EC50 of about 55 ng/ml and a Hill coefficient of 1.55. The infusions were generally well tolerated, with no study drug‐related changes in blood counts or biochemical profiles.

High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin

A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.

Effects of inhibition of α-CGRP receptors on cardiac and peripheral vascular dynamics in conscious dogs with chronic heart failure

Whether endogenous calcitonin gene-related peptide (CGRP) plays a role in heart failure is unclear. Seven dogs were instrumented with left ventricular (LV) pressure gauges, pacers, coronary occluder and aortic, atrial, and coronary sinus catheters. Hemodynamic recordings and response to &agr;-CGRP challenge were obtained for baseline in the conscious state. Rapid pacing (240 beats/min) was then initiated. The coronary artery was occluded for 90 minutes followed by reperfusion after 2 weeks of pacing. After 6 weeks of pacing, LV pressure (−11 ± 6%), LV dP/dt (−53 ± 5%), and mean arterial pressure (−15 ± 4%) decreased (P < 0.01), while left atrial pressure (+19 ± 3 mm Hg from 7 ± 1 mm Hg) and heart rate (+53 ± 16%) increased (P < 0.01). Infusion of the &agr;-CGRP receptor antagonist &agr;-CGRP[8-37] (30 &mgr;g/kg/min, iv), which blocked the exogenous &agr;-CGRP challenge, did not affect any of these indices. Regional blood flow, as measured by the microsphere technique, in the nonischemic myocardium, as well as cerebral and renal vasculatures were unaltered during the infusion of &agr;-CGRP[8-37]. Plasma concentrations of CGRP from both arterial and coronary sinus samples were unchanged after 6 weeks of pacing as compared with control. Thus, we conclude that endogenous &agr;-CGRP does not appear to play a major role in the regulation of cardiac and peripheral vascular dynamics in the late stage of heart failure.

Nonpeptide glycoprotein IIB/IIIA inhibitors : 18. Indole alpha-sulfonamide acids are potent inhibitors of platelet aggregation

Abstract The structure-activity relationship (SAR) of a series of orally active glycoprotein IIb/IIIa antagonists containing an alkyl or aryl sulfonamide grafted onto an indole core is described.

Regulated production of recombinant echistatin by yeast

SummaryThe α-mating factor pre-pro-leader sequence under the regulation of theGAL10 promoter was used to direct the secretion of echistatin by recombinant yeasts. Optimization of the culture medium and host strain increased the productivity of shake flask cultures twenty-fold to 8 mg/L. In fermentors, the production of echistatin was greater than 40 mg/L.

Proaggregatory effect of epinephrine on rabbit platelets inhibited by ticlopidine

Ticlopidine is a potent inhibitor of ADP-induced aggregation of rabbit platelets ex vivo. In vivo, however, multiple agonists play a role in platelet activation. In this study, we examined the effect of epinephrine on the antiplatelet action of ticlopidine in rabbit platelets. Epinephrine reversed the inhibitory effect of drug on ADP-induced platelet aggregation. The potentiating effect of epinephrine was mediated through alpha 2-adrenergic receptors, was reversed by pretreatment with the Na+/H+ exchange inhibitor dimethylamiloride, and was mimicked by agents that increased intracellular sodium or pH. Ticlopidine had no effect on resting intracellular pH, an indication that the effect of epinephrine was not compensating for a drug-induced intracellular acidification. While this potentiation was also found to be inhibited by aspirin, it did not involve enhanced release of thromboxane A2. Our results demonstrate that epinephrine can overcome the inhibitory effect of ticlopidine on ADP-induced aggregation through a mechanism involving activation of Na+/H+ exchange and through an as yet unidentified mechanism sensitive to aspirin.

Inhibition of Osteoclastic Bone Resorption in Vivo by Echistatin, an “Arginyl-Glycyl-Aspartyl” (RGD)-Containing Protein

Osteoclastic bone resorption requires the formation of a tightly sealed compartment between the osteoclast and the mineralized bone matrix. This compartment functions as an extracellular lysosome which contains proteolytic enzymes and acids. Vitronectin receptors (VnR, integrin alpha v beta 3) displayed on the osteoclast cell surface may play a role in the attachment of osteoclasts to the resorption surface. VnR are known to bind to arginyl-glycyl-aspartyl (RGD)-containing matrix proteins and it has recently been reported that soluble peptides containing RGD sequences can block osteoclast attachment to bone and inhibit bone resorption in vitro. In this study echistatin, a naturally-occurring protein containing an RGD-sequence motif, was shown to completely inhibit osteoclast-mediated bone resorption in vivo. Echistatin or smaller derivative peptides may prove useful in the treatment of disorders characterized by excess bone resorption, such as osteoporosis and metastatic bone disease.