Protamine Induces Endothelium-Dependent Vasodilatation of the Pulmonary Artery

Transcription

1 Protamine Induces Endothelium-Dependent Vasodilatation of the Pulmonary Artery Paulo R. B. Evora, MD, PhD, Paul J. Pearson, MD, PhD, and Hartzell V. Schaff, MD Section of Cardiovascular Research, Mayo Clinic and Mayo Foundation, Rochester, Minnesota Background. Protamine sulfate, which is used for heparin neutralization, has been reported to induce catastrophic pulmonary vasoconstriction after infusion. However, in the systemic circulation, protamine infusion induces hypotension due to peripheral vasodilatation. Methods. To determine whether protamine also could induce vasodilatation in the pulmonary circulation, third-order canine pulmonary artery segments were studied in vitro in organ chambers. Results. In pulmonary artery segments that were caused to contract with phenylephrine (10 -s mol/l), protamine sulfate (40 to 400 /~g/ml, final organ bath concentration) produced concentration-dependent relaxation in canine pulmonary artery segments with endothelium (to 74% ± 7% of the initial contraction to phenylephrine) that was significantly greater (p < 0.05) than in segments without endothelium (30% - 6~ of the initial phenylephrine contraction). Pretreatment of arterial segments with NG-monomethyl-L-arginine (10 -s mol/l), the competitive inhibitor of nitric oxide synthesis from L-arginine, did not change tension of arterial segments, but NG-monomethyl-L-arginine attenuated the relaxation to protamine. The inhibitory effect of N C- monomethyl-l-arginine could be reversed by the addition of L-arginine (10-4 mol/l) but not D-arginlne (10-4 tool/l). Endothelium-dependent vasodilation to protamine (40 to 400 /~g/ml) also could be inhibited by heparin (8 U/mL, final organ bath concentration). However, the inhibitory effect of heparin could be overcome by adding higher concentrations of protamine. Conclusions. Protamine-mediated pulmonary vasodilatation could be an important mechanism to protect against the constrictive effects of autocoids generated during heparin neutralization. Such a mechanism might be dysfunctional in certain persons and put them at risk for pulmonary vasoconstriction after protamine infusion. (Ann Thorac Surg 1995;60:405-10) p rotamine sulfate commonlv is used to reverse the anticoagulant effect of heparin [1]. However, heparin neutralization with protamine infrequently mediates catastrophic pulmonary vasoconstriction in some persons [2, 3]. This vasoconstriction, thought to be induced by heparin-protamine complexes, can be attenuated by thromboxane receptor antagonist [4-6] or by infusing inhibitors of cyclooxygenase [4, 5[. Thus, thromboxane A2, derived from platelets, neutrophils, or pulmonary cells, is implicated in the pulmonary vasoconstriction [7]. Previous studies of protamine effects on the pulmonary circulation focused primarily on its vasoconstrictive action; however, it is possible that protamine also could mediate pulmonary vasodilatation. Indeed, protamine sulfate infusion decreases peripheral resistance in the systemic circulation [8-13]. Protamine sulfate is a polycationic protein rich in the amino acid L-arginine [14]. Certain arginine-containing polypeptides can induce the release of endotheliumderived relaxing factor in systemic and pulmonary blood vessels [15-17]. The active component of this relaxing factor is the nitric oxide radical [18, 19], which not only functions as an endogenous nitrovasodilator [20] but also inhibits platelet aggregation [21, 22] and adhesion [231 in Accepted for publication April 4, Address reprint requests to Dr Schaff, Mayo Clinic, 200 First St SW, Rochester, MN the blood vessel. If protamine could induce the release of endothelium-derived relaxing factor in the pulmonary artery, it could act to inhibit platelet adhesion and aggregation in the pulmonary circulation in addition to counteracting the constrictive effect of thromboxane A 2 on the vascular smooth muscle. Such a protective mechanism might be dysfunctional in certain individuals and put them at greater risk for thromboxane-mediated vasoconstriction. The purpose of our experiment was to determine whether protamine sulfate induces the release of endothelium-derived relaxing factor in the pulmonary artery and to discover whether the action of protamine is modified by the presence of heparin. Material and Methods Animal Preparation Heartworm-free mongrel dogs (25 to 30 kg) of either sex were anesthetized with pentobarbital sodium (30 mg/kg injected intravenously; Fort Dodge Laboratories, Fort Dodge, 1A) and exsanguinated through the carotid arteries. The chest was opened quickly, and the right and left lungs were harvested and immersed in cool, oxygenated physiologic salt solution with the following composition: NaCI, mmol/l; KC1, 4.7 mmol/l; MgSO4, 1.2 mmol/l; KH2PO4, 1.22 retool/l; CaC12, 2.5 mmol/l; NaHCO3, 25.0 mmol/l; Ca-ethylenediaminetetraacetic 1995 by The Society of Thoracic Surgeons /95]$ (95)00400-F

2 406 EVORA ET AL Ann Thorac Surg ENDOTHELIUM-DEPENDENT VASODILATATION 1995;60: Lung ~ "-~I~..~i ~.~_..._.._JL~, 3rd order pulmonary harvested \ ~ ~" ~_.~'~ artery dissected free Endothelium ~ Endothelium left,ntact -~-- ~... d Vessel ring ~ ~' Vessel ring suspended in organ suspended in organ chamber chamber Fig 1. In vitro study of canine pldmonary artery segments. Right and h'ft lower lobes of canine lung were harvested from anesthetized dogs, and third-order pulmonary artery segments were dissected free qf connective tissue. Rings qf pulmonary artery were prepared for organ chamber studies. In select rings in which w~scular smooth muscle function was to be examined independent of the endothelium, the endothelium was removed by gentle rubbing qf the intimal surface of the blood vessel zi,ith a pair qf watchmaker's forceps. acid, mmol/l; and glucose, 11.1 mmol/l This was the control solution. The procedures and handling of the animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Mayo Foundation. In Vitro Experiments Third-order pulmonary arteries were dissected free of connective tissue and placed in control solution (Fig 1). Segments (4 to 5 mm long) of blood vessel were prepared from each artery. Care was taken not to touch the intimal surface of the segments. n the segments in which vascular smooth muscle function was to be tested without the influence of the endothelium, the endothelium was removed by gently rubbing the intimal surface of the blood vessel with a pair of watchmaker's forceps. This procedure removes endothelium but does not affect the ability of vascular smooth muscle to contract or to relax [24, 25]. Pulmonary artery segments with or without endothelium were suspended in organ chambers (25 ml) filled with control solution maintained at 37 C and bubbled with 95% O 2 and 5% CO 2 (ph, 7.4). Each ring was suspended by two stainless steel clips passed through the vessel lumen. One clip was anchored to the bottom of the organ chamber, and the other was connected to a strain gauge for measurement of isometric force (Grass FTO3; Grass Instrument Company, Quincy, MA). The rings were placed at the optimal point of their length-tension relation by progressively stretching them until contraction in response to potassium ions (20 mmol/l) was maximal at each level of distention [26]. In all experiments, the presence or absence of endothelium was confirmed by determining the response to acetylcholine (10 ~ mol/l) by segments that had contracted in re- Y sponse to potassium ions (20 mmol/l) [24, 25, 27]. After optimal tension was achieved, the arterial segments were allowed to equilibrate for 30 to 45 minutes before administration of drugs. Drugs The following drugs were used: acetylcholine chloride, indomethacin, phenylephrine (Sigma Chemical Company, St. Louis, MO), L-arginine, n-arginine, and N G- monomethyl-l-arginine (Calbiochem, San Diego, CA), heparin sodium (bovine, 1,000 U/mL; Upjohn Company, Kalamazoo, MI), and protamine sulfate (10 mg/ml; E1- kins-sinn, Cherry Hill, NJ). All powdered drugs were prepared with distilled water except for indomethacin, which was dissolved in Na2CO 3 (10 _5 mol/l). The concentrations were expressed as final molar concentration in the organ chambers. To examine endothelium-dependent relaxation to protamine, vascular segments were caused to contract with phenylephrine and then exposed to increasing concentrations of protamine sulfate (40 to 400 ~g/ml, final organ-bath concentration). The heparin concentration of 8 U/mL approximates that of patients anticoagulated for cardiopulmonary bypass. A protamine concentration of 40/zg/mL corresponds to serum concentrations achieved in patients who receive a dose of 2.5 mg/kg. Data Analysis Results were expressed as mean _+ standard error of the mean. In all experiments, n is the number of animals from which blood vessels were taken. In segments caused to contract with phenylephrine, responses were expressed as percent changes from the contracted levels. Statistical evaluation of data was performed by Student's t test for either paired or unpaired observations. Values were considered to be significant when p was less than Results Endothelium-Dependent Relaxation In pulmonary artery, segments with endothelium that were caused to contract with phenylephrine (10 6 moll L), addition of increasing concentrations of protamine sulfate induced vasodilatation, which almost completely counteracted the constrictive effect of phenylephrine on the vascular smooth muscle (Fig 2, top trace). However, protamine sulfate only induced a modest decrease in tension in pulmonary artery segments without endothelium. Protamine sulfate (40 to 400/_tg/mL) produced concentration-dependent relaxation in canine pulmonary artery segments with endothelium (to 74% + 7% of the initial contraction in response to phenylephrine), which was significantly greater than in segments without endothelium (p <: 0.05; n - 7) (Fig 3). Protamine sulfate caused a modest but significant decrease in tension in arterial segments without endothelium (to 30% -+ 6% of the initial phenylephrine contraction; p < 0.05).

3 Ann Thorac Surg EVORA ET AL ;60: ENDOTHELIUM-DEPENDENT VASODILATATION Pretreatment of arterial segments with jn~:-monomethyl-l-arginine (10 5 molll), the competitive inhibitor of nitric oxide synthesis from L-arginine, caused no significant change in tension of arterial segments. However, NC;-monometbyl-L-arginine attenuated the relaxation to protamine in pulmonary artery segments with endothelium but did not modify the response in arterial segments without endothelium (Figs 3, 4). Indeed, in the presence of NC;-monomethyl-L-arginine, pulmonary artery segments with and without endothelium responded in a comparable manner to protamine sulfate. The inhibitory effect of NC'-monomethyl-c-arginine on protamineinduced endothelium-dependent dilatation could be reversed by the addition of L-arginine (10 4 mol/l) but not by D-arginine (10 a mol/l) (see Fig 4). Effect of Indornethacin Indomethacin (10 6 tool/l} pretreatment did not significantly attenuate vasorelaxation to protamine sulfate in pulmonary, artery segments with or without endothelium (see Fig 4). Also, the presence or absence of indomethacin did not alter the inhibitory effect of NG-monomethyl-L - arginine on endothelium-dependent vasorelaxation to protamine. Effect of Heparin Heparin (8 U/mL, final organ-bath concentration) caused no significant change in tension in arterial segments with or without endothelium (n - 6, data not shown). In organ chambers containing heparin (8 U/mL), the addition of protamine sulfate (40 to 400 /~glml) caused the initially clear fluid to turn turbid, demonstrating the formation of protamine-heparin complexes. Heparin at a concentration of 8 U/mL completely inhibited the endotheliumdependent vasodilator response to protamine (40 to 400 /~g/ml) (see Figs 2, 4). However, additional doses of protamine did induce vasorelaxation in pulmonary artery segments with endothelium (see Fig 2). Comment The major findings of our study were (1) protamine induces endothelium-dependent vasodilatation of pul- r- ~c O. O j~, ~_ O Endothelium: with without Corltrol 0 n=8 In the presence of: L-NMMA O n=6 L-NMMA+L-Arginine [3 n=7 I 0 I I I I Protamine sulfate, ~Jg/ml Fig 3. Concentration-response cur~es to protamine sulfate in canine pulmonary, arteries. Segments were caused to contract with phenylephrine (10 ~ mol/l). When the contraction induced by phenylephrine was stable, vascular segments were exposed to increasing concentrations of protamine (40 to 400 ~tg/ml, final organ-bath concentration). Results are expressed as means standard error of the mean. (L-NMMA = N~;-monomethyl-L-arginine [10 ~ tool/l]; L- NMMA - L-Arg N~;-monomethyl-L-arginine [10 s tool/l] and L-ar~,inine 110 ~ tool/l]; *siy, nificantly different from control [untreated] pulmonary artery, segments without endothelium [p < 0.05].) monary artery segments, (2) protamine-mediated vasodilatation can be inhibited by NC-monomethyl-L-argi - nine, the competitive antagonist of nitric oxide synthesis from L-arginine, (3) protamine that was complexed with heparin failed to induce endothelium-dependent vasodilatation of pulmonary artery segments, and (4} protamine induced endothelium-dependent vasodilatation in the presence of heparin as long as the concentration of heparin and protamine was sufficient to prevent complete complexing of heparin and protamine. Protamine sulfate has been reported to induce cata- Control E---f Protamine sulfate, }J~/mL Protamine sulfate, )Jg/mL in the p... %~ ~ ~,0P ~b ~ ~jq,~ ~ o ~ of heparin,80u/ml ~'~ ~ *, ~ % % '1' o ## ~ ~ E" ~ - ~ ~. j o Phenylephrine, 10 6 M i 4 5 min 2gI 2gI Fig 2. Isometric tension recording of the effect of protamine sulfate on canine pulmonary arteries (original trace). Segments qf third-order pulmonary artery with or without endothelium were suspended in organ chambers to measure isometric force. Segments were contracted with phenylephrine (10 6 tool/l). When the contraction to phenylephrine was stable, the vessels were exposed to increasing concentrations qf protamine sulfate. In pulmonary artery segments with endothelium, protamine induced concentration-dependent relaxation that completely reversed the constrictive effect of phenylephrine on the vascular smooth muscle (top trace). Heparin (8 U/mL) inhibited endothelium-dependent relaxation to protamine (40 to 400 I~g/mL, final organ-bath concentration) (bottom trace). However, addition of increasing doses of protamine could overcome the inhibitory effect qf heparin (bottom trace). (E+ with endothelium; E = without endothelium.)

4 408 EVORA ET AL Ann Thorac Surg ENDOTHELIUM-DEPEN DENT VASODILATATION 1995;60: C o 20 ) m o 0 Control L-NMMA+ L-NMMA+ Indomethacin L-NMMA Indomethacin L-Arginine D-Arginlne (10"6M) (10"5M) +L-NMMA (10"4M) (10"4M) Heparln (80 U/ml) 20 o 40 1 n =with endothelium [] =without endothelium Fig 4. Inhibitory e_ffect of N~;-monomethyl-L-arginine (L-NMMA) and heparin on endothelium-dependent relaxation to protamine in the canine pulmonary artery. Results are expressed as means + standard error of the mean and represent maximal percent relaxation or constriction to protamine sulfate (200 i~g/ml, final organ-bath concentration) in third-order pulmonary arterial segments with endothelium that were contracted with phenylephrine (10 " tool~l). (Control - vasodilatation in untreated vascular segments; Heparin - maximal response to protamine in presence qf heparin (8 U/mL, final organ-bath concentration); L-NMMA = maximal response to protamine in presence of N a- monomethyl-l-arginine [10 ~ tool~l]; L-NMMA - D-Arg -- maximal response to protamine in presence of L-NMMA [10 5 mol/l] and D-arginine [10 ~ mol/l]; L-NMMA ~ ~.-Arg - maximal response to proh~mine in presence qf L-NSVIMA [10 "~ tool~l] and L-arginine [10 4 tool/l J; * significantly different from vasodilatation in control [untreated] pulmonary artery segments with intact endothelium [p < 0.05].) strophic pulmonary, vasoconstriction in some individuals [2]. Indeed, this idiosyncratic sequela of protamine infusion on the pulmonary circulation led investigators to view protamine as a covert pulmonary vasoconstrictor. However, our study indicated that protamine can act as a pulmonary vasodilator by inducing the release of endothelium-derived relaxing factor. Endothelium-derived relaxing factor first was described by Furchgott and Zawadzki in 1980 [27[. Since then, the active component of this relaxing factor has been identified to be the nitric oxide radical [18, 19], which is also the active component of nitrovasodilators such as sodium nitroprusside and nitroglycerin [28]. In effect, endothelium-derived relaxing factor acts as an endogenous nitrovasodilator to modulate vascular tone [15, 20]. In our experiment, protamine-sulfate-mediated vasodilatation of isolated pulmonary artery segments is attributed to the stimulated release of endotheliumderived relaxing factor. Indeed, protamine-mediated vasodilatation could be inhibited by NC;-monomethyl-L - arginine, which is a competitive inhibitor of nitric oxide production from the basic amino acid L-arginine [29]. The specificity of NC:-monomethyl-L-arginine for L-arginine metabolism was demonstrated by the reversibility of competitive inhibition by the addition of exogenous k- arginine but not D-arginine. Although the endothelium produces other vasodilators such as prostacyclin, this compound does not have a prominent role in protamine- mediated vasodilatation because the cyclooxygenase blocker indomethacin did not significantly inhibit either vasodilatation in response to protamine or the effect of NG-monomethyl-L-arginine. The mechanism by which protamine induces the release of endothelium-derived relaxing factor remains an enigma. Protamine is rich in the amino acid L-arginine [14], the physiologic precursor of nitric oxide [30]. However, exogenously added L-arginine does not induce endothelium-dependent relaxation in vitro [16, 30]. This suggests that endothelial cells have sufficient precursor or an L-arginine salvage pathway so that substrate availability is not the rateqimiting step in nitric oxide production. It is possible that protarnine binds to an endothelial-cell receptor to induce endothelium-dependent relaxation (Fig 5). This hypothesis is supported by the finding that protamine could not induce the release of endothelium-dependent relaxing factor when it was complexed with heparin. Presumably because of steric hindrance or change in electrical charge, heparinprotamine complexes alter the ability of protamine to bind to endothelial cells. It is possible that protamine-stimulated release of endothelium-derived relaxing factor could have a modulating effect on thromboxane-mediated pulmonary vasoconstriction, which is characteristic of catastrophic pulmonary vasoconstriction after protamine infusion. Perhaps certain persons have lost the ability to release endothe-

5 Ann Thorac Surg EVORA ET AL ;60: EN DOTHELIUM-DEPENDENT VASODILATATION Protamine Protamine/heparin complex 10 'nhibjtiort of platejet , O aggregation and adhesion L-~ / Nitric oxide [ Pulmonary vasodilalion~ Smooth moecle generally similar to that in humans and consistent with in vitro data from this study. Second, isolated blood vessels used in this study were denervated, but vasoreactivity in vivo is modulated by both neural pathways and other circulating hormones. However, the direct vascular responses of protamine observed in the organ chamber undoubtedly occur in vivo and may modulate the effects of these other factors. Finally these in vitro experiments were performed in hypoxic conditions, and pulmonary arterial blood is normally desaturated. It is possible that pulmonary vascular responses to protamine vary with normoxia and hypoxia [31]. In conclusion, protamine sulfate stimulates the release of endothelium-derived relaxing factor from the pulmonary arterial endothelium. This vasodilatory response can be inhibited by comparable concentrations of heparin. The stimulated release of the relaxing factor by protamine sulfate would decrease pulmonary resistance and would counteract the constrictive effect of endogenous autocoids on the pulmonary circulation. I~, Vascular resistance ] Fig 5. Proposed mechanism of endothelium-dependent vasodilatation in the pulmonary artery. Protamine sulfate binds to an unident!fied endothelial-cell receptor (R) to induce the production of nitric oxide (the active component of endothelium-derived relaxin X factor) (R-SNO) from the amino acid ;-ar~inine. Nitric oxide then dlio:uses to the underlyiny: vascular smooth muscle to induce relaxation (vasodilatation). Nitric oxide n,leased into the lunlen would promote thn~mbolysis and inhibit platelet adhesion in the blood vessel. The conversion of L-arginine to nitric oxide can be inhibited by N C:- monomethyl-l-ar~inine (t-nmma), a methylated form of L-are, i- nine. In addition, heparin can inhibit the abili~ qf protamine to release endothelium-derived rehlxing j;wtor, presumably by preventiny, the bindin X qf pn)tamine to R. (cgmp :.'yclic xuanosine monophosphate.) lium-dependent relaxing factor in the pulmonary circulation secondary to preexisting vascular injury or because of reperfusion injury, to the pulmonary endothelium. Indeed, after reperfusion injuw in the heart, the coronary endothelium loses the ability to protect against plateletmediated vasoconstriction [24, 25]. If the production of endothelium-derived relaxing factor in the pulmona~ circulation were impaired, the constrictive effects of endogenous autocoids could be expressed unopposed. It is also possible that pulmonary vascular injury could change the effect of protamine on smooth muscle to a constrictive one. Three limitations of this study should be acknowledged. First is the use of a canine model to study protamine reactions. 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