Hypoxia induces differential expression of the integrin receptors alpha(vbeta3) and alpha(vbeta5) in cultured human endothelial cells.

The integrins alpha(vbeta3) and alpha(vbeta5) have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits beta(3) and beta(5). beta(3) and beta(5) messenger RNA (mRNA), protein levels, and alpha(v)beta(3) function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O(2)) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of alpha(V) and beta(3), peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, beta(5) mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in alpha(v)beta(3) protein levels, measured by blotting with LM609, evident by 24 h. alpha(v)beta(5) protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an alpha(v)beta(3) mediated process. These results indicate that hypoxia can increase expression of alpha(v)beta(3) in HUVECs, and that hypoxic regulation of alpha(v)beta(3) may be an important regulator of angiogenesis.

Inhibition of neointima formation by a nonpeptide alpha(v)beta(3) integrin receptor antagonist in a rabbit cuff model.

This study was performed to determine whether a highly selective nonpeptide alpha(v)beta(3) antagonist (SH306) would prove effective in inhibiting neointima formation in a rabbit cuff model. The animals were dosed with SH306, 5 mg/kg i.v., followed by 10 mg/kg s. c., 3 times daily for 3 days, or with vehicle (10% DMAC). Rabbits were sacrificed and perfused on days 1, 3, and 21; the vessels were paraffin embedded. A reduction in the intima/media (I/M) of the SH306-treated rabbits, as compared with the vehicle-treated control group, was noted (0.20 vs 0.36 [n = 4]). A significant increase in the area of the media was observed in the SH306-treated group versus the control group (0.20 vs 0.13). No difference was observed in cell proliferation between SH306 and vehicle after 1-day and 3-day dosing. Thrombi were found in 43% of the control vessels and in only 14% of the drug-treated vessels. No anticoagulant was used during the surgical procedure. No increase in inhibition of GPIIb/IIIa was observed in SH306-treated animals, as compared with the vehicle control group. We conclude that selective inhibition of alpha(v)beta(3) reduced neointima formation in a rabbit model at 3 weeks.

Orally active isoxazoline glycoprotein IIb/IIIa antagonists with extended duration of action.

Modification of the alpha-carbamate substituent of isoxazoline GPIIb/IIIa (alphaIIb beta3) antagonist DMP 754 (7) led to a series of alpha-sulfonamide and alpha-sulfamide diaminopropionate isoxazolinylacetamides which were found to be potent inhibitors of in vitro platelet aggregation. Aryl- and heteroaryl-alpha-sulfonamide groups, in conjunction with (5R)-isoxazoline (2S)-diaminopropionate stereochemistry, were found to impart a pronounced duration of antiplatelet effect in dogs, potentially due to high affinity for unactivated platelets. Isoxazolylsulfonamide 34b (DMP 802), a highly selective GPIIb/IIIa antagonist, demonstrated a prolonged duration of action after iv and po dosing and high affinity for resting and activated platelets. The prolonged antiplatelet profile of DMP 802 in dogs and the high affinity of DMP 802 for human platelets may be predictive of clinical utility as a once-daily antiplatelet agent.

Alphavbeta3 integrin binding affinity and specificity of SM256 in various species.

This study was undertaken to define the alphavbeta3 binding affinity and specificity of the low-molecular-weight nonpeptide integrin antagonist, SM256. SM256 demonstrated high potency (IC50, 0.057+/-0.030 nM) in inhibiting vitronectin binding to purified human alphavbeta3 receptors. Additionally, SM256 inhibited alphavbeta3-mediated human umbilical vein endothelial cell (HUVEC) or 293/beta3 (beta3-transfected cell line) adhesion to fibrinogen with IC50 values of 0.0054+/-0.0058 and 0.0023+/-0.0012 microM, respectively. SM256 demonstrated a relatively high degree of specificity for human alphavbeta3-mediated functions as compared with other human integrins including alphavbeta5 (IC50, 0.92+/-0.69 microM), alphaIIbbeta3 (IC50, 0.72+/-0.07 microM), alpha4/beta1 (IC50, >100 microM) and alpha5/beta1 (IC50, 2.3+/-2.1 microM). SM256 demonstrated different degree of species specificity in blocking alphavbeta3-mediated cellular adhesion with relatively higher affinity to dog (IC50, 0.005+/-0.002 microM), rabbit (IC50, 0.021+/-0.01 microM), mouse (IC50, 0.035+/-0.01 microM), and pig (IC50, 0.41+/-0.24 microM) endothelial or smooth-muscle cell alphavbeta3-mediated adhesion. Additionally, SM256 demonstrated high degree of alphavbeta3 specificity as compared with alphavbeta5, alpha5beta1, or alphaIIbbeta3-mediated binding in these species. SM256 is a potent alphavbeta3, antagonist with high affinity and specificity for alphavbeta3-mediated functions. Additionally, a comparable alphavbeta3 affinity for SM256 was demonstrated with endothelial cells obtained from various species (dog, mouse, rabbit, and pig) as compared with that from human.

Prothrombin activation in rabbits.

The suitability of rabbit prothrombin activation fragment F 1.2 as a marker for the activation of the coagulation system was tested. Monoclonal antibodies to rabbit F 1.2 were raised, and a competitive F 1.2 ELISA was developed. Within the detection limit of the ELISA, no increase in rabbit F 1.2 was detected upon recalcification of plasma, whereas human F 1.2 increased 1500-fold. The apparent lack of F 1.2 formation in rabbit serum was confirmed by immunoblotting analysis of endogenous and biotin-labeled prothrombin. Meizothrombin and the B-chain of thrombin were the only prothrombin fragments detectable. In contrast, labeled human prothrombin formed, in addition, prethrombin 2 and F 1.2 in both human and rabbit serum. In contrast, rabbit F 1.2 formation could be demonstrated using purified rabbit prothrombin and factor Xa. These observations raise the possibility that rabbit prothrombin is less susceptible than the human counterpart to factor Xa cleavage at the 271/272 peptide bond. Thus, the primary structure of rabbit prothrombin was deduced by cDNA sequencing. While the 320/321 Xa cleavage site giving rise to meizothrombin was identical in rabbit and human prothrombin, the flanking region of the 271/272 Xa sensitive site contained a six amino acid deletion in the rabbit sequence. Taken together, these observations suggest that the observed differences between human and rabbit prothrombin activation may be due to different susceptibilities of the two Xa cleavage sites rather than plasma or serum cofactor(s).

Disposition and exposure of the fibrinogen receptor antagonist XV459 on alphaIIBbeta3 binding sites in the guinea pig.

The disposition of XV459, a potent, selective GP IIb/IIIa antagonist, has been examined following intravenous administration of XP280, the benzenesulphonate salt, and 3H-SA202, the trifluroacetic acid salt, to male guinea pigs. A liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for XV459 quantitation in guinea pig plasma with an LLOQ of 0.1 ng/mL. Intravenous infusions (30 min) of XP280 at doses of 0.5 and 2.0 microg/kg were administered to guinea pigs which were sequentially sacrificed at 0.5, 1, 1.5, 4, 8, 12, 24, 48 and 72 h postinitiation of infusion. Maximum total (unbound and GP IIb/IIIa displaced) XV459 plasma concentration of approximately 3.5 microg/mL was obtained at the 2.0 microg/kg dose. Pooling individual concentration-time data yielded a systemic clearance of 1.42 mL/min/kg, Vss of 0.24 L/kg, and a terminal half-life of 2.8 h in the guinea pig at the 0.5 microg/kg dose. The 2.0 microg/kg dose yielded XV459 exposure that was less than proportional to the previous dose. Similar behaviour has been observed in human trials. Cumulative (up to 72 h) urinary and faecal recovery of total radioactivity was 66.4 and 11.2%, respectively. The time course of spleen, marrow and whole blood radioactivity profiles was similar, suggesting that XV459 was not preferentially sequestered on non-plasma GP IIb/IIIa binding sites. Tissue to blood ratios of 20.7 and 8.3 for the spleen and bone marrow, respectively, indicate that increased (relative to blood) exposure was evident for sites containing the GP IIb/IIIa receptor. In vitro studies confirmed the similarity of XV459 binding to both resting and activated platelets in the guinea pig and humans. Given the comparability of dissociation rate constants and IC50s based on in vitro platelet aggregation, human dosimetry estimates should assume similar partitioning of radiolabelled XV459 as in the guinea pig. These results suggest that the guinea pig may indeed be an appropriate animal model for pharmacokinetic and distribution studies with DMP754; in conjunction with recent pharmacological findings with GP IIb/IIIa antagonists, our results suggest that the guinea pig may be the rodent species of choice for preclinical studies with some other GP IIb/IIIa antagonists.

Thrombin and human plasma kallikrein inhibition during simulated extracorporeal circulation block platelet and neutrophil activation.

Cardiopulmonary bypass causes hemorrhagic complications, and initiates a chemical and cellular inflammatory response. Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, complement, and contact systems. Despite the fact that cardiopulmonary bypass is carried out in the presence of high doses of heparin, there is significant activation of both platelets and neutrophils. Thrombin is protected on cell and fibrin surfaces from antithrombin, even in the presence of high doses of heparin (approximately 5 U/ml). We therefore studied the effect of a small (Mr = 497), highly effective (Ki = 41 pM), reversible tripeptide inhibitor of thrombin, DUP 714 (1 microM), in a well characterized model of simulated extracorporeal circulation. In the absence of DUP 714, platelet counts decreased by 75% 5 min after the start of extracorporeal bypass and increased to 48% at 120 min of recirculation. DUP 714 significantly preserved platelet counts, decreased plasma levels of platelet beta-thromboglobulin levels, but did not prevent a decrease in sensitivity of platelets to adenosine diphosphate. Kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively from 0.32 U/ml to 0.67 U/ml and from 4.45 U/ml to 7.25 U/ml, respectively, during 120 min of recirculation without DUP 714. Addition of DUP 714 significantly inhibited kallikrein-C1-inhibitor complex formation but did not affect C1-C1-inhibitor complexes. In the absence of DUP 714, human neutrophil elastase levels rose from a baseline of 0.01 +/- 0.00 microg/ml to 1.18 +/- 0.21 microg/ml during 120 min of recirculation. Human neutrophil elastase release at 120 min was significantly inhibited in the presence of DUP 714 to 37% of the value with heparin alone. These results indicated that addition of this novel thrombin (and kallikrein) inhibitor to heparin preserved platelet counts, decreased platelet secretion, and provided the additional benefit of partially blocking neutrophil activation during simulated extracorporeal circulation.

Antiplatelet efficacy of XV459, a novel nonpeptide platelet GPIIb/IIIa antagonist: comparative platelet binding profiles with c7E3.

Recent advances in the development of i.v. platelet glycoprotein alphaIIb/beta3 integrin (GPIIb/IIIa) antagonists led to the development of either a class of small-molecular-weight antagonists with a short to ultra-short duration of antiplatelet effects (Integrelin, Tirofiban, DMP728) or a very long-acting antagonist (ReoPro). Thus the present study was undertaken to characterize the antiplatelet efficacy of a small-molecule GPIIb/IIIa antagonist, DMP754/XV459, and to determine its platelet GPIIb/IIIa receptor binding profiles. DMP754, upon its conversion with esterases to its free acid form XV459, and XV459 itself, demonstrated high potency (IC50 = 0.030-0.060 microM) in inhibiting human platelet aggregation induced by ADP (100 microM), thrombin receptor agonist peptide (10 microM) or collagen (20 microgram/ml) in citrate or heparin. Maximal platelet aggregation inhibition was achieved at 50 to >/=80% receptor occupancy, depending on the agonist used. Both XV459 and c7E3 bind with high affinity to either activated human platelets (Kd = 0.0008 and 0.0091 microM, respectively) or unactivated human platelets (Kd = 0.0025 and 0.0092 microM, respectively). XV459 demonstrated tight association with human, baboon and (to a lesser extent) canine platelets (t1/2 of dissociation = 7 +/- 0, 8 +/- 1 and 1.4 +/- 0.1 minutes, respectively). Both c7E3 and XV459 associate tightly with slower dissociation rates to unactivated human platelets. XV459 represents a potent antiplatelet agent in inhibiting platelet aggregation along with offering high affinity and a relatively slow dissociation rate from human platelet GPIIb/IIIa receptors that might allow for once-a-day p.o. dosage.

Oral antiplatelet efficacy and specificity of a novel nonpeptide platelet GPIIb/IIIa receptor antagonist, DMP 802.

This study was undertaken to define the platelet glycoprotein alphaIIb beta3 integrin (GPII/IIIa) affinity, specificity, and oral antiplatelet efficacy of DMP 802, a small-molecule nonpeptide antiplatelet agent. Platelet GPIIb/IIIa integrin binding affinity and specificity for DMP 802 were determined by using binding and adhesion assays with cells from various species, including human. DMP 802 demonstrated a potent antiplatelet efficacy [median inhibitory concentration (IC50), 0.029 +/- 0.0042 microM] in inhibiting human platelet aggregation induced by 10 microM adenosine diphosphate (ADP), as assessed by light-transmittance aggregometry. DMP 802 inhibited 125I-fibrinogen binding to activated (ADP, epinephrine, and arachidonic acid at 100 microM each) gel purified human platelets with an IC50 of 0.012 +/- 0.003 microM. DMP 802 demonstrated tight association with unactivated human, baboon, or canine platelets (t(1/2) of dissociation, 32 +/- 2, 32 +/- 13, and 11 +/- 1 min, respectively). DMP 802 binds with high affinity to both unactivated and activated human platelets (Kd = 0.61 +/- 0.17, 0.57 +/- 0.21 nM, respectively). DMP 802 demonstrated species specificity in inhibiting platelet aggregation with IC50 values ranging from 0.025 to 0.092 microM (human, guinea pig, dog, swine, hamster) and 0.88-1.0 microM (rabbit and rat) in platelets obtained from these various species. DMP 802 demonstrated a high degree of specificity for platelet GPIIb/IIIa (alphaIIb/beta3) as compared with other integrins including alpha(v)beta3 (IC50, >10 microM), alpha(v)beta5 (IC50, >100 microM), alpha4beta1 (IC50, >100 microM), and alpha5beta1 (IC50, >10 microM). Oral antiplatelet efficacy of DMP 802 was examined after single oral (0.05-0.20 mg/kg) and after repeated oral dosing at 0.05 mg/kg daily for 5 days in mongrel dogs. Dose-dependent antiplatelet efficacy with an extended duration of antiplatelet efficacy was demonstrated based on ex vivo inhibition of platelet aggregation induced by 100 microM ADP. DMP 802 has an oral bioavailability of 14.9% in dogs. In conclusion, the alpha sulfonamide isoxazoline analog, DMP 802, is a novel oral antiplatelet agent with high affinity, relatively slow dissociation rate and specificity for human platelet GPIIb/IIIa receptors.

Similarities in the phenotypic expression of pericytes and bone cells.

Bovine brain microvessel pericytes, bone cells, and fibroblasts were grown in tissue culture in 3%, 21%, or 60% oxygen for 7 weeks. Alkaline phosphatase activity was highest in bone cells and pericytes grown in 3% oxygen, with the activity higher in the former than the latter. Alkaline phosphatase activity was very low in fibroblasts at every oxygen concentration. Osteocalcin concentration was higher in bone cells than in pericytes, was not detected in fibroblasts, and in bone cells and pericytes the concentration was highest in 21% oxygen. Other bovine brain microvessel pericytes were grown in 3% or 21% oxygen for 3 to 24 days in the presence or absence of bone morphogenetic protein 2 and in the presence or absence of parathyroid hormone. At Day 3 of culture, alkaline phosphatase activity was highest in 21% oxygen in the presence of bone morphogenetic protein 2. By Day 17 of culture, alkaline phosphatase activity was highest in 3% oxygen whether bone morphogenetic protein was present or not. Cyclic adenosine monophosphate production in pericytes in response to parathyroid hormone stimulation was very modest when compared with that of bone cells, and this response was not found to be significantly altered by bone morphogenetic protein 2, duration of culture, or the oxygen concentration during incubation. These findings show that the microvessel pericyte is capable of exhibiting several oxygen dependent, phenotypic characteristics ascribed to osteoblasts.

Helping halt hypothermia. An overview of in-line blood/solution warmers.

Preventing hypothermia when delivering fluids to a patient is of critical importance. Several varieties of fluid warmers are available to help prevent hypothermia. Before deciding which model of warmer to use, health care facilities must decide when they will use this technology.

Selective alpha v beta 3 integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury: evidence for the functional importance of integrin alpha v beta 3 and osteopontin expression during neointima formation.

UNLABELLED: Lumen loss from vascular restenosis remains a leading cause of chronic revascularization failure. OBJECTIVE: We hypothesized that cell-matrix adhesion, migration, and differentiation events that underlie restenosis are mediated by alpha v beta 3 integrin-ligand interactions. METHODS: Using immunohistochemistry and in situ hybridization, we examined the spatial and temporal vessel wall expression of alpha v beta 3 and osteopontin following deep coronary arterial injury. Cell migration and adhesion assays were performed to demonstrate the affinity and specificity of XJ 735 for various vessel wall integrins. The effects of XJ 735 (a selective cyclic Arg-Gly-Asp (RGD) peptidomimetic alpha v beta 3 antagonist) on neointimal hyperplasia and lumen stenosis were tested in a porcine coronary injury model. Normolipemic swine underwent oversized stent injury followed by XJ 735 administration (9 animals, 28 lesions; 1 mg/kg bolus + 7 days 4 mg/kg/d infusion + 21 days 2 mg/kg i.v. bolus 12 hourly) or placebo (10 animals, 30 arterial lesions). RESULTS: Maximal alpha v beta 3 immunoreactivity was observed between 7-14 days following injury in the neointima, media, and adventitia. Maximal osteopontin mRNA signal in the neointima, media, and adventitia was observed at 14, 7 and 28 days respectively. IC50 for XJ 735 alpha v beta 3-mediated inhibition of human and porcine endothelial cell adhesion, and vascular smooth muscle cell migration, ranged from 0.6 to 4.4 microM. In contrast, IC50 for porcine or human alpha IIb/beta 3, alpha 4 beta 1, alpha v beta 5, and alpha 5 beta 1 inhibition exceeded 100 microM. Steady state XJ 735 plasma levels exceeded 5 microM. Despite slightly higher injury scores in XJ 735 treated animals, significant reductions in mean neointima area (43% reduction; p = 0.0009), and mean percent lumen stenosis (approximately 2.9 fold reduction; p = 0.04) were observed in XJ 735 treated animals. XJ 735 treatment did not significantly alter the relative size of the arterial injury and reference sites (geometric remodeling). Comparison of neontima area vs. injury score regression lines revealed significant reductions in slope (p = 0.0001) and intercept (p = 0.0001) for XJ 735. CONCLUSIONS: Selective alpha v beta 3 blockade is an effective anti-restenosis strategy that potently limits neointimal growth and lumen stenosis following deep arterial injury. The co-ordinate spatial and temporal upregulation of alpha v beta 3 expression following vessel wall injury, and the high affinity and specificity of XJ 735 for alpha v beta 3, confirms the importance of this integrin in adhesive and migratory cell-matrix events underlying coronary restenosis.

Antagonists of integrin alpha v beta 3 inhibit retinal neovascularization in a murine model.

Integrin alpha v beta 3 is differentially expressed in angiogenic blood vessels in skin granulation tissue, and alpha v beta 3 antagonists inhibit angiogenesis in chick chorioallantoic membranes. In this study, we investigated the role of alpha v beta 3 in retinal neovascularization. There was no detectable signal for alpha v beta 3 by immunohistochemistry in normal human retina, but neovascular tissue removed from the surface of the retina of patients with diabetic retinopathy showed intense staining for alpha v beta 3 within the endothelial cells of new blood vessels. In a murine model of oxygen-induced ischemic retinopathy, there was intense staining for alpha v beta 3 in endothelial cells participating in neovascularization but no detectable staining in normal retinal blood vessels of adult mice. Synthetic peptides that bind alpha v beta 3 and perturb alpha v beta 3-mediated adhesion in vitro inhibited retinal neovascularization in the murine model when given by intraperitoneal or periocular injections. These data suggest that alpha v beta 3 antagonists may provide a useful adjunct for the treatment of retinal neovascularization.

Novel nonpeptide antiplatelet glycoprotein IIb/IIIa receptor antagonist, DMP754: receptor binding affinity and specificity.

OBJECTIVE: To define the antiplatelet efficacy and specificity of the glycoprotein IIb/IIIa complex (GPIIb/IIIa) antagonist prodrug DMP754 and its free acid form, XV459. METHODS AND MATERIALS: DMP754 has an IC50 > 1 mumol/l, and, upon its conversion with esterases to its free acid form, demonstrated high potency (IC50 20-45 nmol/l) in inhibiting human platelet aggregation induced by 10 mumol/l adenosine diphosphate, 20 micrograms/ml collagen, 1 mmol/l epinephrine, 10 mumol/l platelet activating factor or 0.5 IU/ml thrombin. The in-vitro rate of hydrolysis of DMP754 or XV459 is much faster with human or canine liver esterases (t 1/2 = 2.4-23 min) than with plasma esterases (t 1/2 = 5.5-7.6 h). Platelet GpIIb/IIIa integrin binding affinity and specificity for XV459 were determined using cell binding/adhesion assays. RESULTS: The range of IC50 values of XV459 in inhibiting platelet aggregation in platelet-rich plasma obtained from 12 subjects was 0.035-0.069 mumol/l with a mean IC50 of 0.050 +/- 0.003 mumol/l. Additionally, XV459 inhibited platelets obtained from mongrel dogs, baboons, sheep, guinea pigs, and mice with IC50 in the range 0.024-0.06 mumol/l, and IC50 in the range 0.16-5.8 mumol/l in pigs, rabbits, and rats. XV459 inhibited [125I]-fibrinogen binding to activated human platelets with an IC50 of 0.011 +/- 0.003 mumol/l. XV459 demonstrated a high degree of selectivity in specifically inhibiting fibrinogen binding to the platelet integrin, GPIIb/IIIa (IC50 = 0.00025 +/- 0.00005 mumol/l) compared with inhibiting other integrins (alpha v beta 3, IC50 > 10 mumol/l; or alpha v beta 5, alpha 5 beta 1, or alpha 4 beta 1, for which the IC50 exceeded 100 mumol/l). CONCLUSION: DMP754 is a potent antiplatelet agent in inhibiting platelet aggregation, and has a high specificity and affinity for human platelet GPIIb/IIIa receptors.

Oral antiplatelet, antithrombotic efficacy of DMP 728, a novel platelet GPIIb/IIIa antagonist.

BACKGROUND: Currently used antiplatelet drugs, including aspirin and ticlopidine, are effective against certain but not all of the many endogenous platelet activators. Because of their limited efficacy, a significant number of serious thromboembolic complications still occur, highlighting the need for a more effective therapy. DMP 728 has been characterized as a potent and specific platelet glycoprotein IIb/IIIa complex (GPIIb/IIIa) antagonist. The goals of the present study were to determine the oral antiplatelet and antithrombotic efficacies of DMP 728 in various arterial thrombosis models in dogs. METHODS AND RESULTS: In conscious and anesthetized mongrel dogs, DMP 728 at 0.02 to 1.0 mg/kg PO in gelatin capsules produced dose-dependent antiplatelet effects in inhibiting ex vivo platelet aggregation induced by ADP and prolonging template bleeding time. DMP 728 effects on bleeding time prolongation could be reversed more rapidly than those on platelet aggregation inhibition. A maximal antiplatelet effect for DMP 728 was demonstrated at 1.0 mg/kg PO. DMP 728 demonstrated dose-dependent oral antiplatelet effects with an absolute oral bioavailability of 8% to 12% in dogs. Additionally, the antithrombotic efficacy of DMP 728 was examined after intravenous and oral administration at different doses in various models of arterial thrombosis. In the coronary artery Folts' model in dogs, DMP 728 demonstrated maximal antithrombotic efficacy at 0.01 mg/kg IV and < 0.6 mg/kg PO. Additionally, DMP 728 at 0.1 and 1.0 mg/kg IV or PO demonstrated 60% to 100% prevention of primary thrombosis (P < .01) in an electrolytically induced carotid artery thrombosis model in dogs. CONCLUSIONS: These data suggest that DMP 728, a low-molecular-weight GPIIb/IIIa receptor antagonist, may have therapeutic potential as an oral antithrombotic agent in coronary and carotid artery thromboembolic disorders.

A monoclonal antibody that recognizes the GPIIb/IIIa antagonist DMP 728. Reversal of the effects of DMP 728 on platelet aggregation and bleeding time in the dog.

Since hemorrhagic events represent a major safety concern associated with the use of new antithrombotic therapies such as glycoprotein (GP) IIb/IIIa receptor blockade, we evaluated the ability of a monoclonal antibody recognizing DMP 728 (cyclic [D-2-aminobutyryl-N2-methyl-L-argininyl-glycyl-L-aspartyl-3- aminomethyl-benzoic acid] methanesulfonic acid salt), a potent GPIIb/IIIa receptor antagonist, to reverse the pharmacological actions of DMP 728 in the dog. DC11 was chosen for in vivo evaluation based on its ability to inhibit the binding of [3H]DMP 728 to activated platelets and to attenuate the inhibition of ADP-induced aggregation on platelet-rich plasma ex vivo by DMP 728. After anesthesia mongrel dogs were given DMP 728 (20 micrograms/kg body wt IV) infused into the femoral vein, bleeding times were determined using a Simplate device from incisions on the backside of the tongue, and platelet aggregation was determined ex vivo. Nearly complete inhibition of platelet aggregation was observed for the dogs treated with DMP 728 (20 ug/kg IV) for up to 210 minutes, and bleeding times were prolonged > 15 minutes for 2 hours and remained elevated for more than 4 hours. DC11 (0.2 or 1.0 mg/kg body wt IV) given to dogs 10 minutes after DMP 728 resulted in 50% attenuation of the effect of DMP 728 on aggregation at 3 hours. Approximately 34% inhibition of the DMP 728-mediated bleeding time was achieved at 1 hour with the 0.2 mg/kg dose, whereas approximately 50% inhibition of the bleeding time was observed for the 1 mg/kg dose at 1 hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Opposing actions of angiotensin-(1-7) and angiotensin II in the brain of transgenic hypertensive rats.

Lack of specific antagonists to the amino-terminal heptapeptide angiotensin-(1-7) [Ang-(1-7)] prompted us to evaluate the central effects of delivering a specific affinity-purified Ang-(1-7) antibody on the blood pressure and heart rate of 12-week-old conscious homozygous female rats (n = 12) expressing the mouse submandibular Ren-2d gene [(mRen-2d)27] in their genome. Another group of transgenic hypertensive and strain-matched Sprague-Dawley controls were injected with a specific Ang II monoclonal antibody (KAA8). Cerebroventricular administration of the affinity-purified Ang-(1-7) antibody in conscious transgenic hypertensive rats caused significant dose-related elevations in blood pressure associated with tachycardia. The hypertensive response was augmented in transgenic rats studied 7 to 10 days after cessation of lisinopril therapy. Neutralization of Ang II with the Ang II antibody caused a hemodynamic response opposite to that obtained with the Ang-(1-7) antibody. All doses of the Ang II antibody produced hypotension and bradycardia. The magnitude of the depressor response was significantly augmented in transgenic rats weaned off lisinopril therapy. In contrast, central administration of either the Ang-(1-7) or Ang II antibodies had no effect on normotensive rats. Central injections of an affinity-purified IgG fraction were ineffective in both control and transgene-positive rats. These data suggest that in the brain of transgenic hypertensive rats, Ang-(1-7) opposes the action of Ang II on the central mechanism or mechanisms that contribute to the maintenance of this model of hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Real-time calcium response of cultured bone cells to fluid flow.

Using a parallel-plate flow chamber and fura-2 fluorescence microscopy, intracellular calcium was measured cell by cell in preconfluent primary culture rat calvarial bone cells to 18, 35, and 70 dynes/cm2 of fluid-induced shear stress. A heterogeneous response with respect to peak amplitude and latency was observed for the culture, with an overriding dose-dependent relationship between the mean peak amplitude of response and shear-stress magnitude. A dose dependence was observed between the number of responsive cells (responding > 50% over basal levels) and shear-stress magnitude. Not all cells could be restimulated by repeated exposure to flow. The observed cell response appears to be independent of whether cells are clustered together or isolated. Substratum stretch, hydrostatic pressure, and fluid shear stress have been shown in the literature to increase inositol phosphate (IP3) in bone cells, with IP3 causing the release of calcium from intracellular stores such as the endoplasmic reticulum. Therefore, a 6-fold inhibitory effect observed when calcium release from stores was blocked with 8-(n,N-diethylamino)octyl 1-3,4,5-atrimethoxybenzoate hydrochloride implicates an IP3 biochemical pathway mediating the fluid flow response in bone cells.

Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation.

Cardiopulmonary bypass causes hemorrhagic complications and initiates a biochemical and cellular "whole body inflammatory response." This study investigates whether a variety of selective inhibitors of the contact pathway of intrinsic coagulation modulate complement and neutrophil activation during simulated extracorporeal circulation. After 60 min of recirculation in the presence of the slow tight-binding boronic acid inhibitor, Bz-Pro-Phe-boroArg-OH (10.7 microM), complete inhibition of kallikrein-C1-inhibitor complex formation and marked inhibition of C1-C1-inhibitor complex formation and the release of human neutrophil elastase were observed. Arg15-aprotinin (3.1 microM), Ala357,Arg358 alpha 1-antitrypsin (2.6 microM), and soybean trypsin inhibitor (48.0 microM) either completely or partially inhibited the generation of kallikrein-C1-inhibitor complexes but were less effective inhibitors of human neutrophil elastase release. The second-order rate constants for the inhibition of kallikrein in purified systems are consistent with the order of effectiveness of the inhibitors in blocking human neutrophil elastase release in heparinized blood. Our results suggest that low-molecular-weight selective inhibitors of kallikrein may be effective agents in the attenuation of the contact-mediated inflammatory response in cardiopulmonary bypass.

Constrained peptide libraries as a tool for finding mimotopes.

The monoclonal antibody (mAb) KAA8 recognizes the peptide angiotensin II (AII). The KAA8 mAb was biopanned using two phage-displayed peptide libraries, one unconstrained, the other constrained by a disulfide bond. After several cycles of biopanning, both libraries showed enrichment for phage that bind KAA8. Phage isolated from the unconstrained library contain a consensus sequence that matches the sequence of AII. A consensus sequence was also identified from the constrained library that does not resemble the AII sequence, and represents a mimotope of AII. We have also demonstrated that monovalent phage display can be used to discriminate between modest and high-affinity binding peptides.