Chimeric derivative of fibrolase, a fibrinolytic enzyme from southern copperhead venom, possesses inhibitory activity on platelet aggregation.

Fibrolase, a metalloproteinase isolated from the venom of Agkistrodon contortrix contortrix (southern copperhead snake), is a direct acting fibrinolytic enzyme that has been used to digest occlusive blood clots in animal models. The snake venom enzyme directly degrades fibrin associated with platelet rich blood clots and does not rely on plasminogen activation. Rethrombosis is a serious complication that is experienced in a significant percentage of patients treated with thrombolytic agents to remove occlusive vascular thrombi. The involvement of platelets in the initiation of rethrombosis is well known. Arg-Gly-Asp-(RGD)-containing agents have been shown to inhibit rethrombosis following thrombus dissolution by plasminogen activators. In an effort to create a more effective fibrinolytic enzyme and to target the enzyme to platelet-rich thrombi, thereby decreasing the potential for rethrombosis, a chimeric derivative of fibrolase has been produced. This report describes the construction and biochemical characterization of the chimeric enzyme and an evaluation of its in vitro activities. The chimera was formed by covalently incorporating an RGD-like peptide into fibrolase. The site of peptide attachment was determined to be a single lysine residue remote from the enzymes active site. Covalent modification of fibrolase with the RGD-like peptide did not inhibit either fibrinolytic activity of the enzyme nor platelet aggregation inhibitory activity of the peptide. The chimera not only retained the same level of enzymatic activity as native fibrolase, but also acquired the ability to inhibit platelet aggregation by binding to the fibrinogen receptor (integrin alphaIIbbeta3) on platelets.

Chimeric fibrolase: covalent attachment of an RGD-like peptide to create a potentially more effective thrombolytic agent.

We have prepared an agent possessing both thrombolytic and antiplatelet properties, by conjugating fibrolase, a direct-acting fibrinolytic enzyme isolated from southern copperhead venom, to a peptide which inhibits platelet aggregation. Heterobifunctional coupling reagents, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or sulfosuccinimidyl 6-[alpha-methyl-alpha-(2-pyridyldithio)-toluamido]hexanoate (Sulfo-LC-SMPT), were used in a molar ratio of 10:1 (coupling agent/fibrolase). The N-hydroxy-succinimide of the coupling agent reacts with surface epsilon-amino groups of lysine residues on fibrolase and provides a dithio group that is highly reactive with small thiol compounds. The derivatives obtained in the first reaction contain approximately two moles of 2-pyridyl disulphide per mole of enzyme. These derivatives were then reacted with the free thiol group in an antiplatelet peptide at a molar ratio of 2:1 (peptide/fibrolase). The peptide-fibrolase conjugate was purified by cation exchange HPLC and analyzed by amino acid analysis. The conjugate contains one mole peptide per mole of fibrolase and retains approximately 85% fibrinolytic activity. The IC50 for inhibition of platelet aggregation in human PRP is 300 nM for the conjugate and 67 nM for the antiplatelet peptide. These results demonstrate the successful formation of a novel chimeric protein with bifunctional activity.

Pre-clinical evaluation of technetium-99m platelet receptor-binding peptide.

UNLABELLED: P748 is a dimeric peptide which incorporates two high affinity GPIIb/IIIa receptor-binding domains and a novel 99mTc binding sequence, which provides the platelet imaging agent 99mTc-P748. The aim of this study was to evaluate 99mTc-P748 preclinically for use as a hot spot scintigraphic thrombus imaging agent. METHODS: Technetium-99m-P748 was prepared by either a ligand exchange or a one-vial kit. The oxorhenium congener, [ReO]P748, was prepared by ligand exchange from Bu4NReOBr4. The binding of P748 peptide and [ReO]P748 to GPIIb/IIIa receptors on activated platelets was assessed by their inhibition of ADP stimulated human platelet aggregation in platelet rich plasma (PRP). The localization of 99mTc-P748 in deep vein and pulmonary thrombi was assessed in a canine thrombosis model and the biodistribution of 99mTc-P748 was determined in rats. RESULTS: P748 peptide inhibited the aggregation of human platelets in PRP by 50% at a concentration (IC50) of 28 nM and [ReO]P748 had an IC50 of 36 nM showing the high in vitro receptor binding affinity of both the peptide and its rhenium complex (and by analogy its technetium complex). Technetium-99m-P748 was readily prepared at room temperature in 15 min in > or = 90% radiochemical yield and purity and provided definitive images of femoral vein thrombi within 20 min and pulmonary thrombi, within 1 hr in the canine model. Femoral vein thrombus-to-blood and thrombus-to-muscle ratios at 4 hr averaged 6.7 and 46, respectively. Pulmonary thrombus-to-blood and thrombus-to-normal lung ratios at 4 hr averaged 29 and 27, respectively. Dog and rat studies both showed rapid clearance of the radiotracer from the blood and with no significant hepatobiliary excretion but with notable early kidney retention. CONCLUSION: The combination of high in vitro receptor-binding affinity, high thrombus uptake and definitive in vivo images of both femoral vein and pulmonary thrombi show that 99mTc-P748 has considerable potential as a clinical imaging agent for the detection of venous thromboembolism.

Thrombus imaging with a technetium-99m-labeled activated platelet receptor-binding peptide.

UNLABELLED: The objective of this work was the preclinical evaluation of 99mTc-P280, a 99mTc-labeled peptide having high affinity and specificity for the GPIIb/IIIa receptor expressed on activated platelets, for use as a thrombus imaging agent. METHODS: The affinity and specificity of P280 peptide for the GPIIb/IIIa receptor was assessed by the inhibition of ADP-stimulated human platelet aggregation, the inhibition of the binding of fibrinogen to the GPIIb/IIIa receptor and the inhibition of the binding of vitronectin to the vitronectin receptor. P280 peptide was radiolabeled with 99mTc by ligand exchange using 99mTc-glucoheptonate. The ability of 99mTc-P280to detect thrombi in vivo was assessed using a canine venous thrombosis model and the biodistribution of 99mTc-P280 was determined in rats and rabbits. RESULTS: P280 peptide had an IC50 of 79 nM for the inhibition of aggregation of human platelets in platelet rich plasma, an IC50 of 6.8 nM for the inhibition of fibrinogen binding to the GPIIb/IIIa receptor and an IC50 of 13 microM for the inhibition of vitronectin binding to the vitronectin receptor, showing the high in vitro receptor binding affinity and specificity of the peptide. 99mTc-P280 was readily prepared in > or = 90% radiochemical and yield purity and provided images of femoral vein thrombin in the canine model by 1 hr postinjection (thrombus-to-blood ratio of 4.4 and thrombus-to-muscle ratio of 11 at 4 hr). Dog, rat and rabbit studies all showed rapid clearance of the radiotracer from the blood and rapid renal excretion. CONCLUSION: The combination of high in vitro receptor-binding affinity and specificity, in vivo thrombus imaging and fast clearance support the evaluation of 99mTc-280 as a clinical imaging agent.

Technetium-99m-white blood cell-specific imaging agent developed from platelet factor 4 to detect infection.

UNLABELLED: We have developed a leukocyte-avid, 99mTc-labeled peptide (P483H) as a potential imaging agent for infection. P483H contains the heparin-binding region of platelet factor-4 (PF-4) and a lysine-rich sequence for rapid renal clearance. Technetium-99m-P483H was evaluated for its ability to selectively label white blood cells (WBCs) in vitro and to detect focal E. coli infections in rabbits. METHODS: Technetium-99m-P483H was incubated with citrated whole human blood, layered onto WBC isolation media and subjected to density gradient centrifugation to measure WBC-associated radioactivity. Indium-111-WBCs and 99mTc-gluceptate were used as controls. In the in vivo model, E. coli infected rabbits were imaged and necropsied 4 hr after administration of 99mTc-P483H. Infected and contralateral control muscles were evaluated for %ID, %ID/g, Imax (muscle sample showing the highest uptake, i.e., %ID/g) and Imax-to-blood and Imax-to-control muscle ratios. Indium-111-WBCs, 111In-DTPA, 131I-albumin (HSA), 99mTc-nanocolloid, 67Ga and 99mTc-gluceptate were evaluated as in vivo controls. RESULTS: Technetium-99m-P483H associated predominantly with WBCs in vitro, and 99m-Tc-P483H provided high contrast images of infection in vivo. In vitro, 73% of 99mTc-P483H radioactivity was associated with WBCs. Technetium-99m-P483H outperformed 111In-WBCs, 111In-DTPA, 131I-albumin, 99mTc-nanocolloid, 67Ga-citrate and 99mTc-gluceptate with an infection Imax average of 0.062 %ID/g (+/- 0.029; n = 48). Technetium-99m-P483H also outperformed all controls, including 111In-WBCs, 111In-DTPA, 131I-albumin, 99mTc-nanocolloid, 67Ga-citrate and 99mTc-gluceptate. The Imax-to-blood and Imax-to-control muscle ratios for 99mTc-P483H averaged 3.1 (+/- 2.4) and 26.8 (+/- 16.8), respectively, and again outperformed all controls. CONCLUSION: Technetium-99m-P483H associates predominantly with WBCs in vitro and identified focal infections in vivo within 4 hr versus conventional imaging agents. Additionally, the agent showed rapid blood clearance and exclusive renal excretion, which provides a clear abdominal field for imaging abdominal infections.

Inhibition of long-chain acylcarnitine accumulation during coronary artery occlusion does not alter infarct size in dogs.

We tested whether inhibition of carnitine acyl-transferase-1 (CAT-1) during coronary artery occlusion can limit infarct size (IS) by suppressing accumulation of long-chain acylcarnitines (LCAs), potentially cytotoxic intermediates of fatty acid metabolism. The CAT-1 inhibitor 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA) was administered to dogs before 90-min occlusion and 4-h reperfusion of the left anterior descending or left circumflex coronary artery (LAD, LCX). Dogs in the LAD occlusion series received 7.5 (n = 5) or 15 (n = 2) mg/kg POCA intravenously (i.v.); dogs in the LCX occlusion series received 15 mg/kg i.v. (n = 7); an equal number were treated with drug vehicle. Biopsies were obtained for determination of myocardial LCAs. The region at risk and IS were delineated by dye injection and tetrazolium staining. In vehicle-treated dogs, myocardial LCAs (in picomoles per milligram of wet weight +/- SEM) increased from 11 +/- 3 to a peak of 75 +/- 24 during LAD occlusion and from 32 +/- 10 to 192 +/- 55 during LCX occlusion. In POCA-treated dogs LCAs increased from 12 +/- 2 to only 33 +/- 13 pmol/mg wet weight during LAD occlusion (p < 0.05 vs. vehicle) and did not increase significantly during LCX occlusion; 22 +/- 8 to 27 +/- 5 pmol/mg wet weight (p < 0.005 vs. vehicle). LCX occlusion resulted in larger areas at risk and larger infarcts (as a percentage of left ventricle) than did LAD occlusion. IS as a percentage of the region at risk did not differ significantly among the experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of reocclusion by MCI-9038, a thrombin inhibitor, following t-PA-induced thrombolysis in a canine model of femoral arterial thrombosis.

We compared a selective thrombin inhibitor (MCI-9038; Argatroban), a thromboxane A2 (TXA2) receptor antagonist (L-670,596) and a serotonin-2 receptor antagonist (ketanserin) for their ability to hasten clot lysis and delay reocclusion in a canine model of femoral arterial thrombosis. Occlusive thrombosis was induced by insertion of a thrombogenic copper coil. Femoral arterial blood flow velocity (FABFV) was monitored directly and continuously by Doppler flowmetry. Thrombolysis was induced with tissue plasminogen activator (t-PA; 0.8 mg/kg, i.v.), starting 60 min after thrombotic occlusion and continued for 90 min. Ten minutes after occlusion, dogs received an intravenous infusion of either vehicle, MCI-9038 (10 micrograms kg-1 min-1), ketanserin (0.1 mg/kg bolus plus 5 micrograms kg-1 min-1), L-670,596 (1 mg/kg bolus plus 17 micrograms kg-1 min-1) or a combination of L-670,596 and ketanserin. All infusions were discontinued 1 h after stopping the t-PA, and were followed by a 30 min observation period. The times to thrombolysis were similar for all treatments (mean +/- SEM = 47 +/- 3; all groups). MCI-9038 prevented reocclusion, defined as permanent cessation of FABFV during the hour after stopping the t-PA. All dogs receiving MCI-9038 reoccluded within 30 min after stopping its infusion (71 +/- 3 min). Reocclusion occurred in all other dogs, except one vehicle-treated dog and a second dog that received L-670,596 plus ketanserin. Vehicle-treated dogs reoccluded within 23 +/- 8 min. Reocclusion was not delayed significantly by ketanserin, L-670,596 or the combination of the two.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual antagonism by L-636,499 of serotonin and thromboxane A2 induced aggregation of canine platelets.

We evaluated the ability of L-636,499 (3-carboxyl-dibenzo-[b,f] thiepen-5,5-dioxide), a compound structurally similar to cyproheptadine, to antagonize U46619 (a TXA2/PGH2 mimetic) and serotonin (5-hydroxytryptamine; 5HT)-induced aggregation of canine platelets in vitro. L-636,499 antagonized competitively and dose-dependently aggregation induced by both 5HT and U46619, with pA2 values of 5.8 +/- 0.6 and 4.8 +/- 0.2, respectively. L-670,596, a potent TXA2/PGH2 receptor antagonist, and ketanser in, a potent 5HT2 receptor antagonist, yielded pA2 values of 7.0 +/- 0.3 and 9.0 +/- 0.2 vs. their respective agonists. These results show that despite its low potency vs. TXA2- and 5HT2-induced aggregation, L-636,499 antagonizes both physiologic mediators comparably.

In vivo models of arterial thrombosis and thrombolysis.

This year approximately 1.5 million Americans will undergo a myocardial infarction (MI). Of those who make it to the hospital (approximately 1.2 million), only about 20% will receive thrombolytic therapy. Multiple factors contribute to this dismaying figure, but most of them are risk/benefit-related. Moreover, of those receiving lytic therapy, the coronary arteries of as many as one-third may not reopen, and of those that do undergo coronary thrombolysis, an unacceptable fraction will experience reocclusion acutely. Thus, despite significant progress, major challenges for antithrombotic and thrombolytic therapy remain. Promising results with aspirin provide some hope that the figures above can be altered favorably. Efforts are under way in industry and academia to develop drugs to accomplish one or more of the following: lower the incidence of MI, prevent the development of unstable angina or retard its progression to frank MI, increase the inclusion window for lytic therapy, raise the percentage of patients undergoing successful thrombolysis, and maintain coronary patency. During the period that thrombolytic agents have come into vogue important advances have been made in our understanding of platelet function, coagulation, and the endogenous fibrinolytic system. These have spurred the development of novel drugs, such as platelet fibrinogen receptor antagonists, plasminogen activators, and inhibitors of factor IIa (thrombin) and XIIIa. Evaluation of these agents for their antithrombotic or profibrinolytic activity requires relevant animal models of thrombosis. Despite appropriate concerns about their clinical relevance, these models bridge the wide gap between test tube assays of aggregation or coagulation and humans.

Enhanced thrombolysis by a factor XIIIa inhibitor in a rabbit model of femoral artery thrombosis.

Factor XIIIa (FXIIIa) catalyzes covalent crosslinking reactions of fibrin, affording the clot additional structural stability and resistance to plasmin-mediated degradation. Thus, inhibition of FXIIIa may render thrombi more susceptible to tissue-plasminogen activator (t-PA)-induced thrombolysis in vivo. We therefore examined thrombus weight and time to lysis in anesthetized rabbits undergoing arterial thrombosis produced by insertion of a copper coil into the lumen of the right femoral artery. The effects of t-PA alone (started 30 min after coil insertion) or in combination with a FXIIIa inhibitor (L722151) started 15 min before, 8 min after or 20 min after coil insertion were determined. Although t-PA alone (2 mg/kg over 2 hrs) lowered thrombus weight significantly, there was no evidence of flow restoration. Addition of L722151 to t-PA before, or 8 min after coil insertion, further lowered thrombus weights and produced thrombolysis in 50% of the animals. This beneficial effect was lost when L722151 administration was delayed until 20 min after thrombus formation, suggesting that the type(s) of crosslinking inhibited by L722151 was complete by this time. Infusion of L722151 alone had no significant effect on thrombus weight. These results demonstrate a time-dependent facilitation of t-PA-induced arterial thrombolysis by FXIIIa inhibition in a small animal model.

Effects of 3-methoxycyproheptadine in a canine model of coronary thrombosis.

The enantiomers of 3-methoxycyproheptadine (3-MeO-Cyp) were evaluated for their ability to abolish cyclic flow reductions (CFRs) in stenosed and partially de-endothelialized coronary arteries of open chest dogs. These enantiomers demonstrate differential serotonin antagonism with the levorotatory (-) enantiomer showing a selective antiserotonin profile. (+)- and (-)-3-MeO-Cyp each were evaluated in 5 dogs, at doses ranging from 0.01 to 0.5 mg/kg i.v. 1 hr after establishing CFRs, which were quantified in terms of frequency (CFR/hour) and severity (average nadir of coronary blood flow). The frequency or severity of CFRs was not affected consistently by 10 or 25 micrograms/kg of (-)-3-MeO-Cyp; however, 50 micrograms/kg practically abolished CFRs. (+)-3-MeO-Cyp was significantly less potent; complete abolition of CFRs required 0.5 mg/kg of this enantiomer in three dogs, and partial abolition occurred in the other two. These differences between (+)- and (-)-3-MeO-Cyp correlated well with a 12-fold difference in potency for inhibition of canine platelet aggregation stimulated by serotonin and ADP. The IC50's of (-)- and (+)-3-MeO-Cyp vs. ADP plus serotonin-induced aggregation of canine platelet-rich plasma were 1.03 +/- 0.39 (mean +/- S.E.) and 12.92 +/- 4.28 microM, respectively. Thus, (-)-3-MeO-Cyp, a 5-HT2 antagonist devoid of anticholinergic activity and less antihistamine activity than either its enantiomer or parent drug, cyproheptadine, exerts dose-dependent antithrombotic effects in this canine model, providing further evidence that serotonin plays an important role in platelet aggregation and thrombosis.

Effects of the serotonin antagonists, cyproheptadine, ketanserin and mianserin, on cyclic flow reductions in stenosed canine coronary arteries.

We compared the abilities of three pharmacologically and structurally dissimilar 5-hydroxytryptamine (5-HT2) antagonists, cyproheptadine, ketanserin, and mianserin, to interrupt cyclic flow reductions (CFRs) in stenosed coronary arteries of open-chest dogs. All three drugs decreased the frequency and severity of CFRs, but differed in the doses required for total abolition. Cyproheptadine and ketanserin both reduced by approximately 50% the frequency of CFRs at 10 micrograms/kg i.v. Mianserin appeared to be less potent; an approximately 50% reduction of the frequency of CFRs required 100 micrograms/kg. These in vivo results correlated well with their abilities to inhibit epinephrine plus 5-HT-induced aggregation of canine platelet-rich plasma in vitro. The IC50 values for inhibition of aggregation stimulated by this combination of agonists were 2.21, 2.84 and 7.38 microM, respectively. Ketanserin also inhibited amplification by 5-HT of ADP-induced aggregation of canine platelets much more potently than mianserin (IC50 = 0.07 vs. 3.18 microM). Mianserin and RX 781094, a selective alpha-2 adrenoceptor antagonist, inhibited epinephrine-stimulated aggregation of human platelets more potently than ketanserin (IC50 values = 6.10, 0.19 and greater than 10 microM, respectively). Thus, the abilities of three 5-HT2 antagonists with diverse chemical structures and pharmacologic profiles (5-HT2 antagonism notwithstanding) to abolish CFRs suggests that amplification by 5-HT of other mediators, e.g. epinephrine and/or ADP, influences coronary blood flow importantly in this model.

Comparative effects of verapamil, diltiazem and felodipine during experimental digitalis-induced arrhythmias.

We compared the abilities of three different calcium (Ca2+) entry blockers, verapamil, diltiazem and felodipine to abolish ouabain-induced ventricular ectopy (100 X ectopic/total beats, VE) in anesthetized, closed-chest dogs. Ventricular tachycardia (VT) was produced in anesthetized, bilaterally vagotomized, closed-chest dogs by an average dose of 65 +/- 19 micrograms/kg ouabain. 30 min after establishing VT, either verapamil (25-50 micrograms/kg + 5-10 micrograms/kg/min), diltiazem (50-100 micrograms/kg + 20-50 micrograms/kg/min), felodipine (3 micrograms/kg + 0.3 micrograms/kg/min) or saline was administered for another 30 min. Verapamil, at the higher dose utilized, practically abolished ouabain-induced VT (97 +/- 3 to 8 +/- 19% VE); diltiazem was moderately effective (96 +/- 4 to 50 +/- 8% ectopy) at 100 micrograms/kg, and felodipine exerted no antiarrhythmic effects in this model. All three Ca2+ entry blockers lowered mean aortic pressure, felodipine lowering this parameter most prominently. Thus, these structurally and electrophysiologically dissimilar Ca2+ entry blockers differed in their abilities to abolish the digitalis glycoside-induced arrhythmias in vivo. The superiority of verapamil may be related to its multiple, additional electrophysiologic effects.

Antagonism of U46619-induced aggregation of human and canine platelets by four TXA2 receptor antagonists.

We compared the abilities of four TXA2/PGH2 receptor antagonists, AH 23,848, SQ 29,548, BM 13.177 and BM 13.505, to inhibit aggregation of human and canine platelet rich plasma (PRP) induced by the stable cyclic endoperoxide analog, U46619, alone (human) or in combination with epinephrine (dog). The rank orders of potency of these antagonists, which correlated well between human (h) and canine (c) preparations were [IC50]: SQ 29,548 [28 nM (h) and 92 nM (c)] greater than AH 23,848 [0.5 microM (h) and 0.6 microM (c)] congruent to BM 13.505 [0.4 microM (h) and 0.8 microM (c)] greater than BM 13.177 [3.9 microM (h) and 4.4 microM (c)]. The second wave of aggregation of human PRP induced by epinephrine and platelet activating factor (PAF) was abolished by similar concentrations of the TXA2/PGH2 antagonists, whereas aggregation of canine PRP induced by ADP, serotonin plus epinephrine, or PAF was unaffected by these concentrations of the TXA2/PGH2 antagonists. Epinephrine plus U46619-stimulated aggregation of canine PRP was abolished by RX 781094 (1 microM) but not by prazosin (10(-4) M), selective alpha 2- and alpha 1-adrenoceptor antagonists, respectively. Thus, four selective TXA2/PGH2 receptor antagonists, compared in two species, yield IC50's ranging from 28 nM to 4 microM and nearly identical rank orders of potency.

Effects of propranolol and diltiazem alone and in combination on the recovery of left ventricular segmental function after temporary coronary occlusion and long-term reperfusion in conscious dogs.

We evaluated the ability of propranolol and diltiazem alone and in combination to enhance the recovery of left ventricular (LV) segmental function during 1 month of reperfusion after two temporary occlusions of the left anterior descending coronary artery (LAD) in conscious dogs instrumented with ultrasonic crystals for the measurement of regional net systolic wall thickening (NET). LV segments were classified according to their contractile function after 1 hr of LAD occlusion: class 1, greater than 67% of preocclusion (control) NET; class 2, 0% to 66.9%; class 3, less than 0% (paradoxical systolic wall thinning). Propranolol (1 mg/kg iv) or diltiazem (20 micrograms/kg/min) was given 65 min after LAD occlusion in dogs that had 2 (group I) or 4 hr (group II) of LAD occlusion. Diltiazem plus propranolol (same doses) were given to another group of dogs that underwent 4 hr (but not 2) of LAD occlusion. Untreated control dogs received 25 ml of saline and underwent 2 or 4 hr of LAD occlusion. The NET of class 2 and 3 segments in group I control dogs increased significantly during 1 month of reperfusion, from 32 +/- 5% and -43 +/- 6% to 66 +/- 9% and 26 +/- 9%, respectively (p less than .05). Neither diltiazem nor propranolol enhanced the long-term recovery of these segments in group I dogs. However, diltiazem prevented further deterioration of contractile dysfunction observed in control dogs immediately after reperfusion in both segment classes. The NET of class 2 segments in group II control dogs after 4 weeks of reperfusion remained at levels observed during LAD occlusion: 30 +/- 4% to 37 +/- 12%. Class 3 NET increased from -33 +/- 5% to 12 +/- 12% with 1 month of reperfusion, but these segments were essentially akinetic. Propranolol or diltiazem alone did not produce significant overall increases in NET, but diltiazem again prevented further declines in NET of class 2 and 3 segments during early reperfusion. However, the combination of diltiazem and propranolol significantly enhanced overall recovery of class 2 NET in group II dogs (44 +/- 3% to 88 +/- 7%) and prevented the worsening of NET associated with early reperfusion. Compared with untreated dogs, propranolol plus diltiazem also significantly decreased the extent of histologic necrosis in class 2 and 3 segments as well as the macrohistochemically determined infarct size in group II dogs.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of diltiazem and propranolol on left ventricular segmental relaxation during temporary coronary arterial occlusion and one month reperfusion in conscious dogs.

Using sonar microcrystals implanted in conscious dogs, we have characterized left ventricular segmental relaxation (LVSR) by measuring the mean rate to half end-diastolic thinning (RHEDT) and the late diastolic thinning fraction (TF). In protocol 1 (five nonischemic dogs), RHEDT correlated with changes in left ventricular dP/dt (r = .87) and systemic arterial pressure (r = -.80) but not with alterations in heart rate. Only systemic arterial pressure importantly influenced TF (r = -.65). In protocol 2 (21 dogs), LVSR paralleled net systolic segmental wall thickness (NET) during both 2 and 4 hr of coronary occlusion followed by 1 month reperfusion. Both LVSR and NET remained depressed during 2 and 4 hr of coronary occlusion and through 24 hr of reperfusion, but both also gradually improved afterwards. In protocol 3, 31 dogs underwent 4 hr of coronary occlusion with 1 month of reperfusion. Among these animals, 11 dogs (group S4) received saline after 1 hr of occlusion, nine dogs (group P4) received propranolol, and 11 dogs (group D4) received diltiazem. Drug therapy was stopped at 2 hr of reperfusion. In segments with mildly and moderately depressed NET, LVSR was significantly increased in group D4 vs group S4 animals during the diltiazem infusion. Expressed as mean percentage of control value +/- SEM, RHEDT of moderately dysfunctional segments in group D4 compared with group S4 measured 53 +/- 10% vs 25 +/- 5%, respectively, at 2 hr of occlusion of the left anterior descending coronary artery (p = .03), 76 +/- 17% vs 28 +/- 8%, respectively, at 4 hr of occlusion (p = .01), and 74 +/- 11% vs 33 +/- 10%, respectively, at 1 hr of reperfusion (p less than .05). The differences in TF at these same time points were 106 +/- 10% vs 70 +/- 9% (p less than .03), 105 +/- 7% vs 65 +/- 16% (p less than .02), and 106 +/- 11% vs 74 +/- 13% (p less than .05), respectively. The improvement in LVSR occurred independently of changes in NET. The values of LVSR in the diltiazem-treated dogs fell to the levels of groups S4 and P4 within 24 hr of stopping the intervention. Propranolol did not significantly alter LVSR over the short or long term. The increase in LVSR during administration of diltiazem did not appear to be mediated by changes in contractility or regional myocardial blood flow, but were probably mediated in part by afterload reduction and possibly by a reduction in calcium entry into ischemic myocardium.

Comparative hemodynamic effects of MK-422, a converting enzyme inhibitor, and a renin inhibitor in dogs with acute left ventricular failure.

The angiotensin-converting enzyme (ACE) inhibitor MK-422 (enalaprilat) was compared with the potent renin inhibitor SCRIP for its ability to improve left ventricular function in closed-chest dogs. Acute left ventricular failure (ALVF) was induced by repeated embolization (EMB) of the left coronary arterial vasculature with 50-micron plastic microspheres. Baseline stability data were obtained in 30 dogs in which the evolution of ALVF was monitored over time. Guided by a progressive rise in left ventricular end-diastolic pressure (LVEDP), a stepwise perturbation of the coronary circulation with microspheres over 30 min caused reductions in left ventricular dP/dt and cardiac output, averaging 47 and 40%, respectively. EMB reduced heart rate (20 beats/min) and mean arterial pressure by approximately 20 mm Hg which, along with other hemodynamic variables remained stable after induction of heart failure. MK-422 (100 micrograms/kg i.v.) given 45 min after ALVF was induced, decreased mean arterial pressure by 20 mm Hg (p less than 0.05) and reduced total peripheral resistance (TPR) from 5,453 to 4,150 dyne X s X cm-5 (p less than 0.05). The decline in LVEDP (from 14 +/- 1 to 11 +/- 1 mm Hg) and TPR suggests that MK-422 dilates resistance and, conceivably, capacitance vessels. In dogs with sham EMB (vehicle injections into coronary circulation), MK-422 reduced arterial pressure but had no important effects on the other hemodynamic indices.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of alpha 2-adrenergic and serotonergic receptor antagonists on cyclic blood flow alterations in stenosed canine coronary arteries.

Platelets possess alpha 2-adrenergic and serotonergic (5-hydroxytryptamine) receptors which are thought to mediate the in vitro proaggregatory effects of epinephrine and serotonin, respectively. However, their importance in platelet aggregation in vivo is uncertain. In the present study, we evaluate the ability of yohimbine and ketanserin, relatively selective alpha 2-adrenergic and serotonin antagonists, respectively, to alter cyclic flow reductions in stenosed coronary arteries in open-chest, anesthetized dogs. These cyclic flow reductions, characterized by progressive declines in coronary blood flow interrupted by abrupt and, often spontaneous, restorations of flow, were produced by cylindrical constrictors placed on the left anterior descending coronary artery. A pulsed Doppler flow probe, placed proximal to the constrictor, was used to measure coronary blood flow. Regional myocardial blood flow was measured with 15-micron radiolabeled microspheres before coronary constriction and when coronary blood flow appeared to be at its nadir and zenith during cyclic flow reductions. After the cyclic flow reductions had been observed for 1 hour, yohimbine (1-2 mg/kg), ketanserin (0.25 or 0.5 mg/kg), or saline was given, and coronary blood flow and hemodynamics were monitored for another hour. The frequency of cyclic flow reductions and the mean of the three lowest nadirs of coronary blood flow (mean +/- SE) were compared between the first and second hours. Ketanserin, at doses of 0.25 and 0.50 mg/kg, virtually abolished cyclic flow reductions in all dogs tested. Yohimbine [1 mg/kg ( n = 14)] was partially effective in reducing the frequency (9.6 vs. 5.5 cyclic flow reductions/hr) and severity of cyclic flow reductions (nadirs of coronary blood flow = 6.2 +/- 2.4 vs. 20.9 +/- 6.1% of control). A higher dose of yohimbine [2 mg/kg (n = 7)] was no more effective. The frequency (9.3 +/- 0.9 vs. 9.3 +/- 1.0 CFR/hr) and severity (17.4 +/- 5.4 vs. 12.4 +/- 3.9% of control coronary blood flow) of cyclic flow reductions were not changed by saline. The relatively selective alpha 1-adrenergic antagonist, prazosin (0.01 mg/kg, iv), and the beta-adrenergic antagonist, propranolol (1-2 mg/kg, iv), did not affect the frequency or severity of cyclic flow reductions. Thus, the abilities of yohimbine to inhibit and ketanserin to abolish cyclic flow reductions in stenosed canine coronary arteries suggest that serotonin and, possibly, alpha 2-adrenergic agonists may influence cyclic flow alterations importantly in this model.

Effects of fluorocarbons with and without oxygen supplementation on cardiac hemodynamics and energetics.

Because of uncertainty about the mechanism by which fluorocarbons ameliorate myocardial ischemia, the effects of a fluorocarbon emulsion, perfluorodecalin and perfluorotripropylamine (Fluosol-DA 20% TM) with and without 100% O2 inhalation, on cardiac hemodynamics and energetics were studied in the anesthetized dog. Left ventricular (LV) intramural partial pressure of oxygen (PmO2) was measured by mass spectrometry before and after intravenous infusion of Fluosol-DA 20% (40 ml/kg), and was compared with measurements made in another group of dogs receiving the volume expander dextran (36 ml/kg). Both groups of dogs were then ventilated with 100% O2 and repeat measurements were performed. In the 11 animals receiving fluorocarbons, there were increases in left atrial pressure, LV myocardial blood flow, and LV myocardial O2 consumption (MVO2) compatible with volume expansion. After 100% O2, LV MVO2 decreased to control values, while PmO2 increased to 127 +/- 48 mm Hg (p less than 0.001). There were no significant changes in heart rate, arterial pressure or first derivative of LV pressure (dP/dt) during the study. In 10 dogs treated with dextran there was no change in heart rate or dP/dt, but arterial and left atrial pressures were higher after dextran infusion and remained elevated after 100% O2 inhalation. LV MVO2 increased with volume expansion, and remained increased after 100% O2. PmO2 (66 +/- 18 mm Hg) after 100% O2 was lower (p less than 0.02) than in the fluorocarbon-treated dogs after O2 inhalation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of inotropic and chronotropic stimuli on acute myocardial ischemic injury. III. Influence of basal heart rate.

The influence of basal heart rate (HR) on the effects of inotropic (dobutamine infusion) and chronotropic (atrial pacing) stimuli during acute myocardial ischemia was assessed by measurement of intramural carbon dioxide partial pressure (PCO2) in open-chest dogs undergoing transient 10-minute left anterior descending coronary artery occlusions. In protocol I, in 5 dogs anesthetized with pentobarbital alone, HR increased from 153 +/- 10 to 182 +/- 7 beats/min between experimental coronary occlusions, but the increase in ischemic zone intramural carbon dioxide tension (delta PmCO2) was not altered by this significant increase in HR. In protocols II to V, dogs were anesthetized with combinations of morphine, thiamylal and pentobarbital and had a basal average HR of 81 beats/min. Atrial pacing in protocol II (13 dogs) increased HR from 76 +/- 21 to 134 +/- 19 beats/min (p less than 0.001); left ventricular (LV) myocardial oxygen consumption (MVO2) rose from 3.9 +/- 1.6 to 4.9 +/- 1.4 ml/min/100 g (p less than 0.05), and delta PmCO2 rose from 42 +/- 14 to 50 +/- 15 mm Hg (p less than 0.01), indicating more severe ischemic injury during the second experimental coronary occlusion. In protocol III, 11 dogs received 20 micrograms/kg/min of dobutamine before the second experimental occlusion, which significantly (p less than 0.02) increased HR, LV dP/dt and MVO2; delta PmCO2 increased from 46 +/- 13 to 63 +/- 18 mm Hg (p less than 0.01). The 7 dogs in protocol IV received 3.9 +/- 1.9 micrograms/kg/min of dobutamine, titrated such that HR was unchanged (84 +/- 10 vs 81 +/- 15 beats/min), but LV dP/dt increased by 92% (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)