Effects of heparin and N-acetyl heparin on ischemia/reperfusion-induced alterations in myocardial function in the rabbit isolated heart.

Evidence is presented that heparin pretreatment produces protective effects on myocardial tissue distinct from its anticoagulant activity. The present study examines the ability of heparin sulfate and N-acetyl heparin (a derivative of heparin devoid of anticoagulant effects) to protect the heart from injury associated with global ischemia and reperfusion. Male New Zealand White rabbits were administered either heparin sulfate (n = 7, 300 U/kg i.v.), N-acetyl heparin (n = 6, 1.73 mg/kg i.v.), or vehicle (n = 6). Two hours after treatment, the hearts were removed, perfused on a Langendorff apparatus, and subjected to 30 minutes of global ischemia, followed by 45 minutes of reperfusion. During reperfusion, creatine kinase concentrations in the coronary sinus effluent were greater in hearts from vehicle-treated rabbits compared with hearts from N-acetyl heparin-treated and heparin-treated rabbits. Left ventricular end-diastolic pressure after 45 minutes of reperfusion in the vehicle-treated group was 64 +/- 15 mm Hg compared with 17 +/- 4 and 10 +/- 3 mm Hg in the heparin-pretreated and N-acetyl heparin-pretreated groups, respectively. Heparin, but not N-acetyl heparin, increased the activated partial thromboplastin time, consistent with its known anticoagulant action. Heparin and N-acetyl heparin inhibited complement-mediated erythrocyte lysis in a concentration-dependent manner. The glycosaminoglycans, in contrast to r-hirudin, reduced complement activation-induced injury in the rabbit isolated heart. The results demonstrate that heparin or N-acetyl heparin, administered to the intact rabbit, protects the isolated heart from subsequent myocardial dysfunction secondary to ischemia/reperfusion. The cardioprotective effects of heparin and N-acetyl heparin are independent of an antithrombin mechanism.

Chimeric 7E3 prevents carotid artery thrombosis in cynomolgus monkeys.

BACKGROUND AND PURPOSE: We compared the current antithrombotic strategy of antiplatelet therapy with aspirin, and anticoagulant therapy with heparin, with a specific genetically engineered chimeric antibody (c7E3 Fab) directed against the human glycoprotein IIb/IIIa receptor in an animal model of arterial thrombosis. METHODS: Anesthetized cynomolgus monkeys (Macaca fascicularis) were instrumented for monitoring of arterial blood pressure, heart rate, and carotid artery flow velocity. Animals were treated with saline (n = 6), aspirin (25 mg PO daily for 3 days; n = 6), heparin (100 U/kg i.v. plus infusion adjusted to maintain activated partial thromboplastin time at 2 to 3 times baseline; n = 6), aspirin plus heparin (as administered separately, n = 6), or c7E3 Fab (0.10 mg/kg i.v., n = 7; 0.15 mg/kg i.v., n = 6; 0.20 mg/kg i.v., n = 6; 0.25 mg/kg i.v., n = 6). Thrombus formation via anodal electrolytic stimulation (100 microA) to the intimal surface of the right carotid artery was initiated 15 minutes after drug administration and continued for 180 minutes. Electrolytic injury to the left carotid artery began 210 minutes after drug administration and continued for 180 minutes. Whole blood cell counts, glycoprotein IIb/IIIa receptor blockade, ex vivo platelet aggregation, template bleeding time, and activated partial thromboplastin time were assessed at various time points throughout the experimental protocol. RESULTS: Hemodynamic and hematologic parameters were comparable among groups at baseline. Treatment with c7E3 Fab inhibited ex vivo platelet aggregation, increased bleeding time, decreased thrombus weight, and increased time to occlusion in a dose-dependent manner in both vessels. Treatment with aspirin, heparin, or the combination of aspirin plus heparin was ineffective for the prevention of carotid artery thrombosis in this model. CONCLUSIONS: Inhibition of the platelet glycoprotein IIb/IIIa receptor with c7E3 Fab was found to be safe and effective for the prevention of primary thrombus formation, whereas treatment with either aspirin or heparin or the combination of the two agents failed to protect against occlusive thrombus formation in cynomolgus monkeys.

Purification and characterization of platelet aggregation inhibitors from snake venoms.

Proteins that inhibit glycoprotein (GP) IIb/IIIa mediated platelet aggregation have been purified from the venom of two snake species. A small platelet aggregation inhibitor (p1.AI), multisquamatin (Mr = 5,700), was purified from Echis multisquamatus venom by hydrophobic interaction HPLC and two steps on C18 reverse phase HPLC. A larger p1.AI, contortrostatin (Mr = 15,000), was purified by a similar HPLC procedure from the venom of Agkistrodon contortrix contortrix. Both p1.AIs inhibit ADP-induced human, canine and rabbit platelet aggregation using platelet rich plasma (PRP). Multisquamatin has an IC50 of 97 nM, 281 nM and 333 nM for human, canine and rabbit PRP, respectively. Contortrostatin has an IC50 of 49 nM, 120 nM and 1,150 nM for human, canine and rabbit PRP, respectively. In a competitive binding assay using 125I-7E3 (a monoclonal antibody to GPIIb/IIIa that inhibits platelet aggregation) both contortrostatin and multisquamatin demonstrated GPIIb/IIIa specific binding to human and canine platelets. The IC50 for contortrostatin displacement of 7E3 binding to human and canine GPIIb-/IIIa is 27 nM and 16 nM, respectively and for multisquamatin it is 3 nM and 63 nM, respectively. Our results indicate that both p1.AIs inhibit platelet aggregation by binding with high affinity to GPIIb/IIIa.

Antiarrhythmic agent, MS-551, protects against pinacidil + hypoxia-induced ventricular fibrillation in Langendorff-perfused rabbit isolated heart.

We studied the electrophysiologic and antifibrillatory effects of the class III agent MS-551 in a rabbit isolated heart model in which ventricular fibrillation (VF) occurs reproducibly under conditions of hypoxia/reoxygenation in the presence of the ATP-dependent potassium channel opener, pinacidil. Ten minutes after MS-551 or vehicle administration, addition of pinacidil (1.25 microM) to the buffer was followed by a 12-min hypoxic period and 40-min reoxygenation. At a low concentration of MS-551 (1.0 microM), VF occurred in 5 of 6 hearts, the same incidence as in the control group (5 of 6). In contrast 0 of 6 hearts treated with 15 microM MS-551 developed VF (p < 0.05 vs. vehicle). Ventricular effective refractory period (VERP) was determined in a separate group of isolated hearts (n = 13). Pinacidil alone, during normoxic perfusion, decreased VERP 48 +/- 11% (p < 0.05) 15 min after exposure. Five minutes of hypoxia alone also decreased VERP (57 +/- 8%, p < 0.05). Under normoxic conditions, MS-551 increased ERP 31 +/- 10% (p < 0.05 vs. baseline). VERP prolongation by MS-551 was reduced in the presence of pinacidil but remained 22 +/- 6% (p < 0.05) above baseline. The results suggest that VERP shortening owing to pinacidil-mediated ATP-dependent K+ channel opening is associated with development of VF in isolated heart. MS-551 attenuates the pinacidil-mediated decrease in VERP and prevents pinacidil+hypoxia-reoxygenation-induced VF. Because pinacidil and hypoxia open myocardial KATP channels, putatively decreasing VERP, MS-551 may exert its antifibrillatory effect through partial blockade of KATP channels.

Antifibrillatory effects of ibutilide in the rabbit isolated heart: mediation via ATP-dependent potassium channels.

This study determined if ibutilide, a drug with class III activity, exhibited antifibrillatory effects in an isolated heart model of ventricular fibrillation (VF). Langendorff-perfused hearts were randomized among six groups. Group I (n = 9) served as the vehicle-treated control group. Groups II (n = 6), III (n = 10) and IV (n = 9) were pretreated with ibutilide 0.1; 1.0 or 3.0 microM, respectively. Ten minutes after perfusion in the presence of vehicle or ibutilide, hearts were perfused with the ATP-dependent potassium channel opener, pinacidil (1.25 microM) and subjected to a 12-min hypoxic period followed by 40 min of reoxygenation, or until the onset of VF. Groups V and VI were used to investigate electrophysiological effects of ibutilide (n = 12), as well as its chemical defibrillatory activity (n = 9), respectively. Additional experiments involved isometric tension recordings from canine atrial pectinate muscle exposed to increasing concentrations of pinacidil (3-300 microM) in the presence of ibutilide (3-30 microM). Ibutilide decreased the incidence of VF in a concentration-dependent manner; eight of nine control hearts developed VF vs. two of nine hearts (P = .018 chi 2) treated with 3.0 microM ibutilide. In atrial pectinate tissue, ibutilide attenuated the negative inotropic effect of pinacidil. An unexpected finding was the ability of ibutilide to achieve chemical defibrillation when added to the perfusion medium after the electrical induction of ventricular fibrillation in the isolated heart. The antifibrillatory effect of ibutilide may result from inhibition of the ATP-dependent potassium channel made susceptible to opening by pinacidil during hypoxia.