Vitamin D3 and cardiovascular function in rats.

We have previously identified a receptor for 1,25-dihydroxyvitamin D3 in myocardial cells (Simpson, R.U. 1983. Circulation. 68:239.). To establish the relevance of this observation, we evaluated the role of the prohormone vitamin D3 in regulating cardiovascular function. In rats maintained on a vitamin D3-deficient diet for nine weeks, increases in systolic blood pressure (BP) and serum creatine phosphokinase (CPK) were observed. These increases coincided with a reduction of serum calcium from 10.3 to 5.6 mg/dl. However, while serum calcium remained depressed throughout the study, increases in BP and serum CPK were transient. After nine weeks of vitamin D3-depletion, but not after six weeks, ventricular and vascular muscle contractile function were also markedly enhanced. The increase in ventricular contractile function could not be prevented by maintaining serum calcium at 9.0 mg/dl during the period of D3-depletion. These observations suggest a primary role for the vitamin D3-endocrine system in regulating cardiovascular function.

Involvement of 1,25-dihydroxyvitamin D3 in regulating myocardial calcium metabolism: physiological and pathological actions.

The role of the prohormone vitamin D3 in regulating calcium and phosphate metabolism in the intestine, kidney, and bone has been known for several decades. Recent studies have provided evidence that vitamin D3, may also play an important role in regulating metabolism in other organs, including heart. This role has been suggested by the identification of a specific receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, in these tissues, as well as the presence of a 1,25(OH)2D3-dependent calcium binding protein. Although administration of excessive quantities of vitamin D3 has been shown in many studies to produce myocardial calcinosis and heart failure, the importance of vitamin D3 in regulating myocardial metabolism under normal conditions has only recently been demonstrated. The purpose of the present review is to assess the current status of research regarding the pathological and physiological actions of vitamin D3 on the heart. The initial section of this report will focus on the pathological effects of excessive vitamin D3 on cardiovascular function, while the latter sections will describe recent studies related to the involvement of 1,25(OH)2D3 in regulating calcium homeostasis in ventricular cells and the relationship between vitamin D3 and myocardial contractility.

Regulation of myosin isozyme expression by vitamin D3 deficiency and 1,25-dihydroxyvitamin D3 in the rat heart.

In this report, we demonstrate a significant inverse correlation between contractility and serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels and no correlation between contractility and serum levels of calcium, phosphate, or PTH. We also examined myosin isozyme distribution in vitamin D3-deficient rats, because myosin isozyme distribution can alter contractility. There was a significant increase in the levels of the V1 myosin isozyme in animals raised on a vitamin D3-deficient diet that maintained normal serum calcium and phosphate levels. There was no difference in the relative myosin isozyme distribution in animals raised on a hypocalcemia-yielding vitamin D3-deficient diet vs. animals raised on a control diet. As increased contractility has been observed in both groups of vitamin D3-deficient animals, a shift in myosin isozyme distribution cannot solely explain the increase in contractility previously observed in the vitamin D3-deficient rat heart. To determine whether 1,25-(OH)2D3 directly regulates myosin isozyme levels, we analyzed myosin isozyme distribution in primary cultures of ventricular myocytes. We found that 1,25-(OH)2D3 reduces total myosin levels, but does not alter myosin isozyme distribution. Thus, we show that the influence of vitamin D3 status on myosin isozyme expression in the intact rat involves a complex regulatory system of direct and indirect effects.

Subclasses of cyclic GMP-specific phosphodiesterase and their role in regulating the effects of atrial natriuretic factor.

Two subclasses of cyclic guanosine monophosphate (GMP)-specific phosphodiesterases were identified in vascular tissue from several beds. The activity of one subclass (phosphodiesterase IB) was stimulated severalfold by calmodulin and selectively inhibited by the phosphodiesterase inhibitor TCV-3B. The activity of the other subclass (phosphodiesterase IC) was not stimulated by calmodulin and was selectively inhibited by the phosphodiesterase inhibitor M&B 22,948. To assess the involvement of both subclasses in regulating cyclic GMP-dependent responses, the ability of TCV-3B and M&B 22,948 to potentiate the in vitro and in vivo responses to the endogenous guanylate cyclase stimulator atrial natriuretic factor (ANF) was evaluated. Both TCV-3B and M&B 22,948 relaxed isolated rabbit aortic and pulmonary artery rings and also potentiated the relaxant effect of ANF. In addition, both inhibitors produced small increases in urine flow and sodium excretion in anesthetized rats and potentiated the diuretic and natriuretic responses to exogenous ANF. M&B 22,948 (30 micrograms/kg/min) produced a threefold increase in the natriuretic response to simultaneously administered ANF, and TCV-3B (10 micrograms/kg/min) produced a twofold increase in the response to ANF. The results of the present experiments suggest that both the calmodulin-sensitive and calmodulin-insensitive subclasses of cyclic GMP-specific phosphodiesterase play a role in regulating the in vitro and in vivo response to ANF.

A new generation of phosphodiesterase inhibitors: multiple molecular forms of phosphodiesterase and the potential for drug selectivity.

With several notable exceptions, interest in the area of multiple molecular forms of phosphodiesterase remained relatively dormant during the decade following Thompson's discovery of more than one phosphodiesterase in brain in 1971. Within the last several years, however, over 20 novel agents have been identified that exert selective inhibitory effects on the various molecular forms of phosphodiesterase present within different cells. In addition, several studies have documented that such agents can produce discrete changes in cyclic AMP and cyclic GMP, an action that is not shared by "first generation" phosphodiesterase inhibitors such as theophylline. The purpose of this Perspective is to provide some clarity to this rapidly evolving area of selective phosphodiesterase inhibitors. Thus, we have attempted to characterize the different forms of phosphodiesterase present in various tissues and cells according to their kinetic properties, substrate specificity, etc. and also to characterize those major classes of agents that have been shown to inhibit phosphodiesterase activity, whether selectively or nonselectively. In addition, we have described several therapeutic areas wherein selective phosphodiesterase inhibitors might prove efficacious, paying particular attention to those areas in which selective phosphodiesterase inhibitors have already been shown to exert beneficial effects, namely, stimulation of myocardial contractility, inhibition of mediator release, and inhibition of platelet aggregation. Although focusing on these three areas, it is obvious that the potential therapeutic utility of selective phosphodiesterase inhibitors could conceivably extend to several other areas in which modulation of cyclic nucleotides can have desirable effects, including cancer chemotherapy, analgesia, the treatment of depression, Parkinson's disease, and learning and memory disorders. For example, the selective type III phosphodiesterase inhibitor rolipram has been shown to antagonize reserpine-induced hypothermia and also to potentiate yohimbine lethality, two tests that are indicative of antidepressant activity. In addition, microinjection of the selective PDE III inhibitor Ro 20-1724 into the rat brain stem has been shown to produce analgesia.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiotonic agents. 6. Synthesis and inotropic activity of 2,4-dihydro-5-[4-(1H-imidazol-1-yl)phenyl]-3H-pyrazol-3-ones: ring-contracted analogues of imazodan (CI-914).

A series of 2,4-dihydro-5-[4-(1H-imidazol-1-yl)phenyl]-3H-pyrazol-3-ones was synthesized and evaluated for positive inotropic activity. Only compounds with two small alkyl groups at C-4 showed significant activity. The structure-activity relationships for optimal inotropic activity are presented and compared with those of the 4,5-dihydro-3(2H)-pyridazinone series. The phosphodiesterase inhibitory activity is also reported and correlated with the substitution pattern at C-4 in the pyrazolone ring.

Cardiotonic agents. 7. Inhibition of separated forms of cyclic nucleotide phosphodiesterase from guinea pig cardiac muscle by 4,5-dihydro-6-[4-(1H-imidazol-1-yl)phenyl]-3(2H)-pyridazinones and related compounds. Structure-activity relationships and correlation with in vivo positive inotropic activity.

The structure-activity relationships of a series of 4,5-dihydro-6-[4-(1H-imidazol-1-yl)phenyl]-3(2H)-pyridazinones and related compounds were investigated for the in vivo inhibition of different forms of cyclic nucleotide phosphodiesterase (PDE) isolated from guinea pig ventricular muscle. With few exceptions, these 4,5-dihydropyridazinones were potent inhibitors of cardiac type III phosphodiesterase, which is a low Km, cyclic AMP specific form of the enzyme. The inhibitory effects on cardiac type I and type II phosphodiesterase, both of which hydrolyze cyclic AMP as well as cyclic GMP, were minimal. The most selective PDE III inhibitor was CI-930 (10), the 5-methyl analogue of imazodan (CI-914, 1), with an IC50 of 0.6 microM. The most potent inhibitor of PDE III was the 4,5,6,7-tetrahydrobenzimidazole analogue of 10 (31), with an IC50 of 0.15 microM. This paper describes the structural features that impart both selectivity for inhibiting type III phosphodiesterase and potency of inhibition. In addition, correlations between in vitro PDE inhibitory potency, in vivo positive inotropic potency, and physicochemical properties are discussed.

Cardiotonic agents. 3. Synthesis and biological activity of novel 6-(substituted 1H-imidazol-4(5)-yl)-3(2H)-pyridazinones.

Several 6-(substituted 1H-imidazol-4(5)-yl)-3(2H)-pyridazinones were synthesized and evaluated for positive inotropic activity. The 1H-imidazol-4-yl regioisomers 4,5-dihydro-6-(1-methyl-2-phenyl-1H-imidazol-4-yl)-3(2H)-pyridazinone (25a) and 6-(1-methyl-2-phenyl-1H-imidazol-4-yl)-3(2H)-pyridazinone (28a) were potent positive inotropic agents. By contrast, the corresponding 1H-imidazol-5-yl regioisomers 25b and 28b were only weak positive inotropic agents. Compounds 25a and 28a were also potent inhibitors of cardiac phosphodiesterase fraction III.

Cardiotonic agents. 5. 1,2-Dihydro-5-[4-(1H-imidazol-1-yl)phenyl]-6-methyl-2-oxo-3- pyridinecarbonitriles and related compounds. Synthesis and inotropic activity.

Several 1,2-dihydro-5-(substituted phenyl)-2(1H)-pyridinones were synthesized and evaluated for inotropic activity. 1,2-Dihydro-5-[4-(1H-imidazol-1-yl)phenyl]-6-methyl-2-oxo-3- pyridinecarbonitrile (5a) and the corresponding unsubstituted analogue 14a were the most potent positive inotropic agents in this series. Although the 4,6-dimethyl analogue 6a retained most of the activity of 5a, the 4-methyl analogue 8a was substantially less potent. The synthesis and structure-activity relationships are discussed.

2-(2-Aryl-2-oxoethylidene)-1,2,3,4-tetrahydropyridines. Novel isomers of 1,4-dihydropyridine calcium channel blockers.

The title compounds are novel double bond isomers of 1,4-dihydropyridine-type calcium channel blockers (CCB). These derivatives were prepared by using the Hantzsch dihydropyridine synthesis. The assignment of structure was based on spectroscopic data and a regiochemically unambiguous synthesis. Several of the analogues inhibited [3H]nitrendipine binding with IC50 values as low as 25 nM. By comparison, nifedipine, a clinically useful 1,4-dihydropyridine CCB, inhibits [3H]nitrendipine binding with an IC50 of 1.6 nM. In the Langendorff rat heart preparation, treatment with the more potent derivatives produced marked dose-related increases in coronary flow with little or no effect on heart rate or contractility, except at the highest concentrations tested. The selectivity for vascular versus cardiac effects was similar to that of nifedipine, i.e., the concentration producing vasodilation was approximately 2 orders of magnitude lower than the concentration eliciting cardiodepression. These novel isomers extend the structure-activity relationships for calcium channel blockers into a series closely related to the 1,4-dihydropyridines.

Cardiotonic agents. 9. Synthesis and biological evaluation of a series of (E)-4,5-dihydro-6-[2-[4-(1H-imidazol-1-yl)phenyl]ethenyl]-3 (2H)-pyridazinones: a novel class of compounds with positive inotropic, antithrombotic, and vasodilatory activities for the treatment of congestive heart failure.

A novel series of analogues of (E)-4,5-dihydro-6-[2-[4-(1H-imidazol-1-yl) phenyl]ethenyl]-3(2H)-pyridazinone was synthesized as a variation on the imazodan series. The compounds were evaluated for (i) hemodynamic activity, (ii) cyclic AMP-phosphodiesterase inhibitory activity (human platelets and guinea pig heart tissue), and (iii) platelet aggregation inhibitory activity. The insertion of the ethenyl moiety between the phenyl and dihydropyridazinone rings produced novel compounds that retained the potent inotropic/vasodilator activity of the parent imazodan series and enhanced the platelet aggregation inhibitory potency. Compound 3d, the most potent in this series, demonstrated in vivo antithrombotic activity. The synthesis and the biological activity of these new pyridazinone analogues are reported.

Cardiotonic agents. 8. Selective inhibitors of adenosine 3',5'-cyclic phosphate phosphodiesterase III. Elaboration of a five-point model for positive inotropic activity.

Inhibitors of adenosine 3',5'-cyclic phosphate phosphodiesterase III (cAMP PDE III) were studied by using solid-state, solution, and theoretical methods in order to refine a five-point model for positive inotropic activity. Cyclic AMP PDE III inhibitors bear a striking resemblance to cAMP itself. This investigation supports the importance of an overall planar topography for selective and potent cAMP PDE III inhibition. (Possible reasons for the potency of certain nonplanar compounds are discussed.) Cardiotonics like imazodan (1; CI-914) and 2 (CI-930) can readily achieve essentially planar geometries, as shown with X-ray crystallographic, IR, UV, NMR, and theoretical data. Small alkyl substituents that occupy space corresponding to certain portions of the cAMP sugar region increase potency (see, e.g., 2, 4). Selective inhibition of cAMP PDE III can be achieved by mimicking the attractive electrostatic potential associated with the phosphate group (e.g., with an amide) and by providing an additional attractive potential spatially opposite to the previous one, in the vicinity of the adenine N1 and extending to N3 (e.g., with an imidazole), together with a partial dipole moment comparable to the adenine dipole moment. This extends and better defines our five-point model in terms of cAMP, a natural substrate for PDE.

Calcium channel blocking and positive inotropic activities of ethyl 5-cyano-1,4-dihydro-6-methyl-2-[(phenylsulfonyl)methyl]-4-aryl-3- pyridine-carboxylate and analogues. Synthesis and structure-activity relationships.

The synthesis and pharmacological evaluation of a series of 2-[(arylsulfonyl)methyl]-4-aryl-5-cyano-1,4-dihydropyridine-3-carboxylic acid esters and analogues are described. These compounds possess a unique profile namely, calcium channel blocking and positive inotropic activities in vitro. Compound 54 was selected as the best compound in the series and was studied in detail. The synthesis and biological profiles of enantiomers of 54 are also reported. The data indicate that although the calcium channel blocking property of 54 is stereospecific the positive inotropic activity is not. Examples of 3- and 6-cyano and other closely related 1,4-dihydropyridine derivatives are described and evaluated for comparison and were found to be devoid of dual activities mentioned above.

Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets. Isolation, characterization, and effects of various reference phosphodiesterase inhibitors and cardiotonic agents.

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been identified previously in several tissues and cell types using a variety of different isolation methods. In the present study, the different molecular forms of phosphodiesterase (PDE) were isolated from cardiac muscle (guinea pig left ventricle), vascular smooth muscle (bovine coronary arteries) and human platelets using the same isolation procedure in each instance. These enzymes were then characterized kinetically, and the effects of various reference PDE inhibitors and cardiotonic agents on each form were examined. A low Km, low Vmax form of phosphodiesterase (PDE I) was found in all three tissue/cell types. PDE I activity was stimulated by calmodulin in cardiac and smooth muscle, but not in platelets. In smooth muscle and platelets, PDE I preferentially hydrolyzed cyclic GMP, whereas cardiac muscle PDE I hydrolyzed cyclic AMP and cyclic GMP equally. A high Km, high Vmax form of phosphodiesterase (PDE II) was found in cardiac muscle and platelets, but not in smooth muscle. PDE II activity was not stimulated by calmodulin and there was no substrate specificity. A low Km, low Vmax cyclic AMP-specific form of phosphodiesterase (PDE III) was found in all three tissue/cell types. The activity of PDE III was not stimulated by calmodulin. The reference inhibitors theophylline and papaverine exerted non-specific inhibitory effects on all forms of phosphodiesterase. Other reference inhibitors (M & B 22,948 and dipyridamole) and several cardiotonic agents (AR-L 57, CI-914, CI-930, amrinone, and MDL 17,043) exerted selective inhibitory effects on only one molecular form of phosphodiesterase. The degree of selectivity was often dependent upon the tissue or cell from which the molecular form of phosphodiesterase was isolated. These studies demonstrate that there is heterogeneity regarding the number of phosphodiesterases present in various tissue/cell types, as well as their substrate specificity and their ability to be stimulated by calmodulin, and these different molecular forms of phosphodiesterase can be selectively inhibited by different pharmacological agents. The possibility exists that such selective inhibitors may produce discrete changes in cyclic AMP or cyclic GMP levels, and that these changes may be produced in specific tissues and/or cells.

Differential inhibition of human neutrophil functions. Role of cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase.

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been characterized in various tissues and cells according to their substrate specificity, intracellular location, and calmodulin dependence. The purpose of this study was to evaluate the possible involvement of different molecular forms of phosphodiesterase in regulating the respiratory burst and lysosomal enzyme release responses of human neutrophils. Treatment with the selective cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase inhibitors Ro 20-1724 or rolipram, or the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), resulted in inhibition of respiratory burst stimulated by the chemoattractants formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (IC50 values: 0.71-17 microM) and complement fragment C5a (IC50 values: 61-93 microM), but did not inhibit phagocytosis-stimulated respiratory burst (less than 10% inhibition at 100 microM). Selective inhibitors of calmodulin-dependent phosphodiesterase (ICI 74,917), calmodulin-insensitive, cyclic GMP-specific phosphodiesterase (M & B 22,948), cyclic GMP-stimulated phosphodiesterase (AR-L 57), or cyclic AMP-specific, cyclic GMP-inhibited phosphodiesterase (amrinone and cilostamide) exhibited little or no inhibitory effect on FMLP- or phagocytosis-stimulated respiratory burst (0-42% inhibition at 100 microM). Regulation of neutrophil activation by phosphodiesterase was also response specific, as Ro 20-1724, rolipram and IBMX were less potent inhibitors of FMLP-induced lysosomal enzyme release (0-14% inhibition at 100 microM). Analysis of human neutrophil preparations confirmed the existence of a cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase, which was associated with the particulate fraction of the cell. These results demonstrate a role for the cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase in the regulation of human neutrophil functions, which appears to be both stimulus specific and response specific.

Relaxant effect of human brain natriuretic peptide on human artery and vein tissue.

Brain natriuretic peptide (BNP) is a cardiac-derived peptide hormone with cardiovascular and renal actions that is structurally and functionally related to atrial natriuretic peptide (ANP). Previous studies using rat vascular tissue have demonstrated a direct vasorelaxant effect of BNP. However, species-specific potency issues have precluded an accurate measurement of the effect of human BNP. This report demonstrates the vasorelaxant effects of human BNP on human vascular tissue prepared from internal mammary artery and saphenous vein samples. The vasorelaxant effect of human BNP is compared to the other members of the natriuretic peptide family, human ANP and C-type natriuretic peptide (CNP). With regard to potency and magnitude of effect, human BNP and human ANP were similar in relaxing arterial tissue preconstricted with endothelin-1 (BNP ED50 = 1.9 nmol/L and ANP ED50 = 1.8 nmol/L) or phenylephrine (BNP ED50 = 10 nmol/L and ANP ED50 = 19 nmol/L), while CNP was significantly less effective. All three natriuretic peptides exhibited weak venodilating action. These data demonstrate that human BNP is a potent inhibitor of the vasoconstrictive actions of endothelin-1 and the alpha-adrenergic agonist phenylephrine on isolated human artery tissue preparations.

Two phases of contractile response in rat isolated vas deferens and their regulation by adenosine and alpha-receptors.

Contractions to transmural electrical stimulation and exogenous norepinephrine were recorded in isolated longitudinal segments of rat vas deferens. Electrical stimulation for 30 s produced a biphasic contraction in the vas deferens consisting of a rapid, transient response (Phase I), followed by a slowly developing, sustained contraction (Phase II). N6-Cyclohexyladenosine (CHA), a selective adenosine1 (A1)-receptor agonist, attenuated in a concentration-dependent manner the Phase I contractile response, while having little effect on the Phase II response. In contrast, 2-(phenylamino)adenosine (CV-1808), a selective adenosine2 (A2)-receptor agonist had little effect on either contractile phase. CHA did not inhibit the contraction to exogenous norepinephrine, suggesting that A1-receptors were located at a presynaptic site. The relatively selective alpha 2-receptor agonist clonidine produced the same pattern of contractile inhibition as CHA. The inhibitory effect of CHA on the Phase I contractile response in the vas deferens could be antagonized by the selective A1-receptor antagonist 8-cyclopentyltheophylline, while the selective alpha 2-receptor antagonist idazoxan preferentially antagonized the inhibitory effect of clonidine on the Phase I response. Both the Phase I and Phase II contractile responses were reduced by the selective alpha 1-adrenoceptor antagonist prazosin and the ATP analog alpha, beta-methylene adenosine triphosphate (alpha, beta-methylene ATP), suggesting that norepinephrine and ATP are coreleased as neurotransmitters for both responses. The results of the present study demonstrate that in the rat vas deferens the presynaptic inhibitory effects of adenosine is mediated by the A1-receptor subtype, and that both A1- and alpha 2-receptor agonists exert a selective inhibitory effect on the Phase I contractile response to electrical stimulation.

Studies aimed at elucidating the mechanism of action of CI-914, a new cardiotonic agent.

CI-914 is a novel positive inotropic agent whose cardiotonic activity is not due to inhibition of Na+, K+-ATPase or to stimulation of cardiac beta-receptors. CI-914 also has no direct effect on sarcoplasmic reticulum, mitochondria or adenylate cyclase activity. CI-914 does, however, exert a potent inhibitory effect on cardiac phosphodiesterase activity. In evaluating the effect of this agent on the different molecular forms of phosphodiesterase present in cardiac muscle, CI-914 was found to selectively inhibit PDE III, which is a low Km, cAMP-specific form of the enzyme (IC50 = 6.1 microM). This inhibitory effect was found to be competitive with respect to the substrate. Papaverine and theophylline on the other hand were found to inhibit all three forms of phosphodiesterase present in cardiac muscle. The role of phosphodiesterase inhibition in mediating the positive inotropic response to CI-914 is supported by the finding that this agent: (i) significantly elevates cyclic AMP levels in ventricular tissue; (ii) shifts the normal concentration-response to the beta-receptor stimulant isoproterenol to the left: and (iii) restores contractility to K+-depolarized papillary muscles.

Relationship between inotropic activity and phosphodiesterase inhibition for flosequinan and milrinone.

The goal of this study was to assess the relationship between the positive inotropic response to high concentrations of the vasodilators flosequinan and BTS 53 554 (the sulfone metabolite of flosequinan) and the effect of both compounds on different forms of cyclic nucleotide phosphodiesterase. In addition, the relationship between inotropic activity and phosphodiesterase inhibition for the cardiotonic milrinone was also evaluated. All three agents exerted a positive inotropic effect on human cardiac muscle fibers. The concentration of milrinone required to increase cardiac contractility was comparable to the concentration required to inhibit the milrinone-sensitive subclass of cyclic AMP-specific phosphodiesterase (type III phosphodiesterase). However, no such relationship was observed for flosequinan and BTS 53 554. These results suggest that the cardiac response to high concentrations of flosequinan and BTS 53 554 is not mediated by inhibition of type III phosphodiesterase.

Neutralase reverses the anti-coagulant but not the anti-thrombotic activity of heparin in a rabbit model of venous thrombosis.

Neutralase (heparinase I; E.C. 4.2.2.7) is a heparin-degrading enzyme undergoing clinical evaluation as an alternative to protamine for reversing the anticoagulant effects of heparin in coronary bypass surgery. The objective of this study was to assess the relative effects of Neutralase and protamine on reversal of heparin-dependent elevations in coagulation parameters and inhibition of clot formation in a rabbit vena caval stasis model. Rabbits were treated with saline or heparin (300 U/kg) for 10 minutes, followed by saline, protamine (2.6 mg/kg), or Neutralase (10 or 30 microg/kg, representing 1.23 IU/kg and 3.69 IU/kg, respectively). Twenty minutes later, venous stasis was induced, and vena caval clots were excised, weighed, and characterized. Coagulation parameters [activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT)] and antiFactor IIa and Xa levels were measured throughout the protocol. Both protamine and Neutralase reversed heparin-mediated increases in aPTT (>300 seconds to 26-35 seconds) and TCT (>300 seconds to 29-56 seconds) to values that were not different from saline-treated, nonheparinized animals. Thrombus weight in the nonheparinized saline group was 62+/-7 mg; heparin-treated animals had no detectable clots. Protamine reversal of heparin was associated with clot formation (89+/-20 mg) while Neutralase reversal was not (no clots). Heparin-induced increases in antiFactor IIa activity were reversed similarly by protamine and Neutralase (from 4.3-8.8 U/ml to 0.2-0.3 U/ml) while antiFactor Xa activity was differentially reversed (from 3.9-5.9 U/ml to 0.7-1.3 U/ml Neutralase; 5.5 U/ml to 0.02 U/ml protamine). These results are consistent with a hypothesis that Neutralase cleaves heparin into fragments, which are devoid of antiFactor IIa activity that retain modest antiFactor Xa activity, resulting in reversal of anticoagulant, but not antithrombotic, heparin activity. This property of Neutralase may be beneficial in reducing post-surgical thrombotic events after reversal of heparin.