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

Background: Even when large doses of heparin are administered during cardiopulmonary bypass, thrombin is produced. Thrombin is a powerful protease that is associated with the thrombotic and bleeding complications of open heart surgery and is produced by cleavage of prothrombin by factor Xa. This study assessed the ability of a specific inhibitor of factor Xa, recombinant tick anticoagulant peptide (rTAP), alone or in combination with standard heparin and a low-molecular-weight heparin, enoxaparin, to suppress thrombin formation and activity during in vitro extracorporeal circulation., Methods and Results: Fresh, anticoagulated human blood was recirculated for 2 hours in an extracorporeal membrane oxygenator perfusion circuit at 37 degrees C. Four anticoagulant protocols were evaluated; porcine heparin (3.75 U/mL); enoxaparin (17.5 U/mL); rTAP (4 mumol/L); and porcine heparin plus rTAP (2 mumol/L). Blood samples were obtained for analysis from the donor, after anticoagulation, and after 5, 30, 60, and 120 minutes of recirculation. There were no significant differences between groups in platelet count, response to adenosine diphosphate, or prothrombin fragment (F1.2) production. rTAP plus heparin reduced beta-thromboglobulin release; fibrinopeptide A concentrations were significantly higher with rTAP alone. Enoxaparin strongly and significantly inhibited complement C5b9 production and neutrophil elastase release and was associated with significantly increased concentrations of C1-C1 inhibitor and kallikrein-C1 inhibitor complexes., Conclusions: rTAP does not reduce thrombin formation or activity during in vitro extracorporeal circulation. Enoxaparin markedly inhibits formation of the complement membrane attack complex and neutrophil elastase release, possibly by accelerating C1 inhibitor activity.

Activation of humoral and cellular participants in inflammation enhances the risk of postoperative bleeding and multiple organ damage in cardiopulmonary bypass (CPB). We now compare the effects of heparin alone in combination with nafamostat mesilate (NM), a protease inhibitor with specificity of trypsin-like enzymes, in an extracorporeal circuit which simulates CPB. NM significantly inhibits the release of platelet beta-thromboglobulin (beta TG) at 60 and 120 min. Platelet counts do not differ. ADP-induced aggregation decreases in circuits with NM, which is due to a direct effect of NM on platelet function. NM prevents any significant release of neutrophil elastase; at 120 min, plasma elastase-alpha 1-antitrypsin complex is 0.16 micrograms/ml in the NM group and 1.24 micrograms/ml in the control group. NM completely inhibits formation of complexes of C1 inhibitor with kallikrein and FXIIa. NM does not alter markers of complement activation (C1-C1-inhibitor complex and C5b-9), or indicators of thrombin formation (F1.2). However, at 120 min, thrombin activity as measured by release of fibrinopeptide A is significantly decreased. The data indicate that complement activation during CPB correlates poorly with neutrophil activation and that either kallikrein or FXIIa or both may be more important agonists. The ability of NM to inhibit two important contact system proteins and platelet and neutrophil release raises the possibility of suppressing the inflammatory response during clinical CPB.

Cardiopulmonary bypass prolongs bleeding time, increases postoperative blood loss, and triggers activation of plasma proteolytic enzyme systems and blood cells referred to as the "whole body inflammatory response". Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, contact and complement systems. Contact and complement pathway proteins both induce neutrophil activation. alpha 1-antitrypsin Pittsburgh (Met358-->Arg), a mutant of alpha 1-antitrypsin, is a potent inhibitor of plasma kallikrein and thrombin. We investigated whether this recombinant mutant protein inhibited platelet activation, as well as contact and/or complement-induced neutrophil activation during simulated extracorporeal circulation. Arg358 alpha 1-antitrypsin did not prevent the 34% drop in platelet count at 5 min of recirculation, did not block the 50% decrease in ADP-induced platelet aggregation at 120 min of recirculation, nor inhibit the release of 6.06 +/- 1.07 micrograms/ml beta-thromboglobulin at 120 min of recirculation suggesting that the inhibitor had little effect on platelet activation. However, Arg358 alpha 1-antitrypsin totally blocked kallikrein-C1-inhibitor complex formation but not C1-C1-inhibitor complex formation. Most importantly, Arg358 alpha 1-antitrypsin decreased the release of 1.11 +/- 0.16 micrograms/ml human neutrophil elastase by 43%. The attenuation of neutrophil activation in the absence of an effect on complement activation via the classical pathway, supports the concept that kallikrein is a major mediator of neutrophil degranulation during cardiopulmonary bypass.

Aprotinin reduces blood loss after cardiac operations and decreases the bleeding time. The mechanism of action of aprotinin that produces these effects is not clear. During simulated extracorporeal circulation the contact and complement systems, platelets, and neutrophils are activated. We investigated the effect of aprotinin on kallikrein-C1-inhibitor complex and C1-C1-inhibitor complex formation, neutrophil degranulation, and platelet release and aggregation during simulated extracorporeal circulation. Fresh heparinized human blood was recirculated at 37 degrees C for 2 hours in a spiral coil membrane oxygenator-roller pump perfusion circuit. Changes in platelet count, leukocyte count, platelet response to adenosine diphosphate, and plasma levels of beta-thromboglobulin, kallikrein-C1-inhibitor complexes, C1-C1-inhibitor complexes, and neutrophil elastase were measured before and at 5, 30, 60, and 120 minutes of recirculation at 0, 0.015, 0.03, 0.06, and 0.12 mg/ml doses of aprotinin. Platelet counts decreased to 36% +/- 12% of control values at 5 minutes and increased to 56% +/- 13% at 120 minutes without aprotinin. Aprotinin did not affect platelet counts, but it did prevent the decrease in sensitivity of platelets to adenosine diphosphate and it attenuated beta-thromboglobulin release. In the absence of aprotinin, kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively to 0.53 +/- 0.14 U/ml and 2.38 +/- 0.33 U/ml, respectively, at 120 minutes. Kallikrein-C1-inhibitor complexes were completely inhibited and C1-C1-inhibitor complexes were partially inhibited at aprotinin concentrations of 0.03 mg/ml or greater. Release of neutrophil elastase was partially but not completely inhibited at the highest dose of aprotinin and was 50% inhibited at a dose of 0.03 mg/ml. Because activation of the fibrinolytic system does not occur in this system, the changes were independent of the inhibition of plasmin. We conclude that aprotinin in high doses completely inhibited kallikrein-induced activation of neutrophils and partially inhibited complement-induced activation. Aprotinin did not directly affect platelet adhesion or aggregation, but it indirectly preserved platelet sensitivity to agonists and also attenuated release of alpha-granule contents. The data indicate that in the presence of aprotinin platelet function was partially preserved, kallikrein production was totally inhibited, complement activation was partially inhibited, and neutrophil release was partially inhibited, thus attenuating the "whole body inflammatory response" associated with cardiopulmonary bypass.

Certain forms of extracorporeal circulation exemplified by cardiopulmonary bypass require continuous high-dose anticoagulation to prevent thromboembolic complications. We hypothesized that monocytes may be stimulated to express tissue factor (TF) during prolonged simulated extracorporeal circulation. TF was identified both by flow cytometry by using three TF-specific monoclonal antibodies and functional assay of procoagulant activity (PCA). TF significantly increased between 2 and 6 hours of simulated extracorporeal circulation by both analyses. Relative fluorescence on monocytes increased from a control value of 100 to 313 +/- 79 on cells from the simulated extracorporeal circuit (p < 0.05). PCA increased from 21 +/- 8 to 775 +/- 326 pg TF/10(6) monocytes (p < 0.05) and was blocked 99.6% by preincubation of cells with a mixture of monoclonal antibodies to TF. By 6 hours, the number of leukocytes in the circuit was decreased by 43%. The cells were recovered from the oxygenator membrane by washing with EDTA. Compared with initial values, by 6 hours, both TF antigen at 378 +/- 90 (p < 0.05) and PCA at 1,357 +/- 280 pg TF/10(6) monocytes (p < 0.01) were highest in the recovered cells. Cells incubated for 6 hours and not subjected to simulated extracorporeal circulation did not increase TF. Examination of monocytes for the adhesive receptor CD11b/18 (Mac-1) paralleled TF expression, providing an additional putative receptor for the coagulant proteins, factor X and fibrinogen or fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

The leukocyte integrin Mac-1 (alpha m beta 2, CD11b/18 CR3, MO1), in addition to binding iC3b, has been shown to be the receptor for the coagulation proteins fibrinogen, factor X, and high molecular weight kininogen. Mac-1 is known to be upregulated by agonists that stimulate neutrophils or monocytes. Previous studies from this laboratory have documented neutrophil activation during cardiopulmonary bypass. We therefore used an experimental model for cardiopulmonary bypass, a simulated extracorporeal circulation, to study the surface expression of Mac-1 on peripheral blood leukocytes by immunofluorescence flow cytometry. The number of Mac-1 receptors in polymorphonuclear leukocytes had increased 2.2 times and 2.9 times baseline by 2 hours at 37 degrees C and 28 degrees C, respectively (p < 0.001). Neutrophil elastase-alpha 1-proteinase inhibitor complexes (a measure of neutrophil degranulation) increased more than sixfold from 41 micrograms/L to 256 micrograms/L after 2 hours at 28 degrees C. The number of Mac-1 receptors expressed on polymorphonuclear leukocytes correlated with polymorphonuclear leukocyte elastase complexes (r = 0.93). Mac-1--bearing lymphocytes did not display any change in receptor number in the simulated extracorporeal circulation. By 2 hours, monocytes at 37 degrees C showed only insignificant increase; at 28 degrees C they displayed a 1.6 times increase (p < 0.05). By maintaining a temperature-matched standing control samples, we could prove that, unlike the Mac-1 in polymorphonuclear leukocytes, this increase in monocyte Mac-1 is not from bypass but is rather an effect of temperature. These data suggest that a receptor for procoagulant proteins on circulating polymorphonuclear leukocytes (Mac-1) is upregulated during simulated extracorporeal circulation.

Previous studies indicate that exposure of fibrinogen receptors associated with glycoprotein IIb/IIIa complex contributes to platelet loss during cardiopulmonary bypass. Recently, we isolated a number of RGD (Arg-Gly-Asp)-containing, low molecular weight, cysteine-rich peptides from viper venoms. These peptides, which we propose to call "disintegrins," block platelet-fibrinogen interaction and platelet aggregation. We compared the effect of RGDS (Arg-Gly-Asp-Ser) and four disintegrins (echistatin, flavoridin, albolabrin, and bitistatin) on platelet behavior in a membrane oxygenator. During simulated extracorporeal circulation for 2 hours, platelet count decreased to about 30% of initial values. Addition of echistatin (60-200 nM), albolabrin (60-200 nM), bitistatin (60 nM), and flavoridin (45 nM) significantly inhibited platelet loss in the circuit. RGDS (33 microM) did not show any significant inhibitory effect. ADP-induced platelet aggregation was inhibited in samples of platelet-rich plasma taken from the circuits containing disintegrins. However, echistatin appeared to be a more potent inhibitor of platelet aggregation, whereas albolabrin and flavoridin interfered more selectively with platelet loss from the circuit. Echistatin prevented the accumulation of glycoprotein IIIa on the surface of the circuit. Echistatin (60-200 nM), flavoridin (45 nM), bitistatin (60 nM), and albolabrin (200 nM) significantly inhibited the loss of beta-thromboglobulin from platelets into circulating plasma. Electron microscopy studies demonstrated shape change but not degranulation in platelets circulating in the presence of 200 nM echistatin. On the other hand, this peptide (up to 1,000 nM) did not prevent loss of alpha granules and beta-thromboglobulin from thrombin-stimulated platelets, although it prevented their aggregation. In conclusion, disintegrins protect platelets in the circuit by preventing their adhesion to surfaces and, therefore, preventing fragmentation of adhered platelets under the shear stress of flowing blood. This study indicates that disintegrins may be potential candidates for platelet protection during cardiopulmonary bypass.

The interaction between human platelets and nonbiologic surfaces was studied during in vitro recirculation of 500 ml of fresh, heparinized human blood in four different perfusion circuits. Circuits differed in surface area (0.1 m2 or 0.9 m2) and in surface composition. No important differences were observed between standard silicone-rubber and filler-free, silicone-rubber surfaces. Platelet counts decreased to 85% of control in 0.1- m2 circuits, but retained normal sensitivity to aggregating agents and released only small amounts of platelet factor 4 (PF4). In contrast, platelet counts in 0.9-m2 circuits decreased to 20% of control within 2 min and platelet sensitivity was depressed out of proportion to the fall in platelet count. Plasma PF4 progressively increased and platelet PF4 content progressively decreased during 6 h of recirculation. The results indicate that human platelets may exist in three conditions during extracorporeal circulation. Some platelets are unaltered, some are less sensitive to aggregating agents, and others have undergone extensive release. The ratio of blood volume to surface area appears to be an important determinant of platelet-surface interaction.

Contact between blood and synthetic surfaces of an extracorporeal circuit results in extensive alterations in platelet function. These platelet abnormalities have been reproduced in vitro by recirculating heparinized (5 units/ml) human blood at 37 degrees C in a silicone rubber circuit (0.1 m2) containing a spiral coil membrane oxygenator (0.9 m2). During control recirculation trials, platelet counts fell to 29% +/- 8% (mean +/- standard error of the mean) of initial levels within 30 minutes, and sensitivity to the soluble agonists, adenosine diphosphate and epinephrine, disappeared. Plasma levels of the platelet-specific protein platelet factor 4 rose to 2,600 +/- 200 ng/ml, indicating extensive platelet granule release. Similarly, plasma concentrations of thromboxane B2 rose from less than or equal to 100 pg/ml to 500 +/- 200 pg/ml at 120 minutes, and transmission electron microscopy demonstrated disruption of platelet subcellular architecture. In contrast, when ilioprost, a stable prostacyclin derivative (1 ng/ml), was added to the circuit prior to recirculation, the thrombocyte count remained at 85% +/- 4% of initial values. Platelets incubated or recirculated for 2 hours in the presence of iloprost responded normally to both adenosine diphosphate and epinephrine after separation from the drug by gel-filtration. Furthermore, plasma concentrations of platelet factor 4 remained below 300 ng/ml, plasma levels of thromboxane B2 failed to reach detectable levels, and platelet ultrastructure remained intact. Thus, iloprost effectively preserves the circulating platelet count, prevents platelet granule release, and preserves platelet functional and morphological integrity during simulated extracorporeal circulation.

Contact with surfaces results in activation of formed blood elements. This study demonstrates that during extracorporeal circulation, release of lysosomal enzymes occurs concomitantly with platelet granule secretion. Fresh, heparinized human blood was recirculated for 2 hours at 1,000 ml/min at 37 degrees C in silicone rubber circuits containing a membrane oxygenator (0.85 m2). The plasma levels of a platelet-specific protein, low affinity platelet factor 4 (LA-PF4), rose from less than 0.5 to 16 +/- 3 SEM microgram/ml plasma, indicating extensive release of platelet alpha granule contents. Concurrently, plasma activity of acid phosphatase and N-acetyl-beta-glucosaminidase increased nearly fivefold. Platelet inhibition prevented release of LA-PF4 and reduced acid phosphatase levels, but failed to alter the levels of N-acetyl-beta-glucosaminidase. Lidocaine (10 to 20 microgram/ml), however, prevented the rise in N-acetyl-beta-glucosaminidase activity and diminished acid phosphatase levels without altering secretion of LA-PF4. The failure of platelet inhibitors to block the increase in N-acetyl-beta-glucosaminidase, and the efficacy of lidocaine to do so without altering LA-PF4 secretion, suggest that leukocytes, not platelets, are the primary source of this lysosomal enzyme. Although acid phosphatase activity measured in plasma my be derived from leukocytes, platelet membranes, and not lysosomes, appear to be a more likely source. Release of hydrolytic enzymes from formed blood elements during cardiopulmonary bypass may cause endothelial cell injury and thus contribute to the increased vascular permeability associated with extracorporeal circulation.

Temporary inhibition of platelet function with prostaglandin E1 (PGE1) prevents platelets loss and functional alterations during extracorporeal circulation with a membrane oxygenator. This study evaluated the ability of PGE1 to prevent platelet injury in circuits containing a bubble oxygenator. During in vitro recirculation of human blood, the circulating platelet count, expressed as a percent of initial levels, decreased to 29%; platelets became insensitive to adenosine diphosphate (ADP) and to epinephrine; and plasma levels of low-affinity platelet factor 4 (LA-PF4) progressively rose to 7.4 microgram per milliliter. With PGE1 (1.2 micron), platelet counts remained stable at 92%; platelet reactivity remained normal for 1 hour; and plasma levels of LA-PF4 rose to only 3.3 microgram per milliliter. In rhesus monkeys that underwent cardiopulmonary bypass using a bubble oxygenator without PGE1, platelet counts, expressed as a percent of the prebypass platelet count, declined to 38%; platelets became insensitive to ADP; plasma levels of LA-PF4 progressively rose to 8.4 microgram per milliliter; and the mean postoperative bleeding time was 4.6 minutes. In monkeys that received PGE1, platelet counts declined to only 65%; platelets remained threefold more sensitive to ADP; platelets demonstrated delayed release of LA-PF4 and the mean postoperative bleeding time was 2.7 minutes. This report demonstrates that in a bubble oxygenator system, PGE1 reduces platelet loss, mitigates platelet injury, and shortens postoperative bleeding times following extracorporeal circulation.

The role of platelet fibrinogen receptors and platelet-protein interaction in platelet consumption during simulated cardiopulmonary bypass was investigated. In five recirculation experiments, with whole blood, the platelet count fell to 13% of initial values and in two experiments, with blood from patients with Bernard-Soulier syndrome, to 3% of normal values. However, in three experiments with blood from the patients with Glanzmann's thrombasthenia, the platelet count decreased to 69% of initial values. The extent of platelet consumption in normal blood was diminished to only 72% by addition of 0.5 microM prostaglandin E1 (PGE1) (5 experiments) and to 80% by precoating surfaces of the circuit with 2.5% human albumin (5 experiments). beta-Thromboglobulin antigen (beta TG) loss from platelets was associated with thrombocytopenia. The extent of beta TG loss was significantly reduced by the addition of PGE1 to blood or precoating surfaces with albumin. Proteins adsorbed on the surface of the circuit exposed to normal blood were removed with 0.5% Triton X-100. Some of these proteins were identified to be glycoprotein IIIa (GPIIIa) (3.4-4.3 micrograms/ml), beta TG (1.0-1.6 micrograms/ml), and fibrinogen (1.9-3.7 micrograms/ml). The amount of GPIIIa recovered in the Triton X-100 eluates correlated with the number of platelets lost during recirculation. These studies indicate that exposure of fibrinogen receptors associated with GPIIb-GPIIIa complex contributes to platelet consumption during cardiopulmonary bypass.

The accumulation of extravascular lung water was related to changes in plasma colloid osmotic pressure and pulmonary hydrostatic pressures in 12 normal dogs and 13 dogs that had venovenous perfusion for 2 hours at 45 to 70 ml. per kilogram per minute. The venovenous perfusion system included a membrane oxygenator and a roller pump. Net intravascular filtration pressure was calculated from plasma colloid osmotic pressure and pulmonary hydrostic pressures. Rapid accumulation of extravascular lung water occurred in control and bypass animals when net intravascular filtration pressure exceeded zero. At lower filtration pressures, venovenous perfusion did not affect accumulation of extravascular lung water.

A 5-year-old boy with acute lymphoblastic leukemia in complete continuous remission developed life-threatening varicella pneumonia and acute respiratory insufficiency (ARI). The child recovered after 92 hours of partial venoarterial perfusion with a membrane oxygenator. Functional asplenia developed. Serial pulmonary function tests after perfusion indicate moderately severe restrictive lung disease which has slightly improved during an 18 month period.

Cardiopulmonary bypass prolongs bleeding time and increases postoperative blood loss. During in vitro recirculation in an extracorporeal circuit containing a membrane oxygenator and primed with fresh heparinized human blood, we previously observed thrombocytopenia, impaired platelet aggregation, and depletion of granular contents, all of which were prevented with prostaglandin E1 (PGE1). To investigate these changes further, we studied the number and affinity of platelet alpha 2-adrenergic receptors by measuring the binding of 3H-yohimbine. Before recirculation, we found 235 +/- 28 alpha 2-adrenergic receptors per platelet, a Kd of 3.37 +/- 0.78 nmol/L, complete aggregation with 1.04 mumol/L epinephrine, and a platelet count of 281,000 +/- 33,000 microliters-1. After 2 minutes of recirculation, 9.44 mumol/L epinephrine was required to produce complete aggregation, and the platelet count was 104,000 +/- 22,000 microliters-1 (44% of control). The number of binding sites significantly decreased to 139 +/- 16 per platelet, but the affinity did not change (Kd = 3.78 +/- 0.44 nmol/L). After 2 hours of recirculation, the platelet count had increased to 123,000 +/- 21,000 microliters-1. However, epinephrine did not induce platelet aggregation even at 100 mumol/L. Moreover, alpha 2-adrenergic binding sites were not detectable, and affinity for yohimbine could not be calculated. Two minutes after PGE1 0.3 mumol/L was added to the circuit, platelet numbers, response to epinephrine, alpha 2-adrenergic binding sites per platelet, and affinity for yohimbine were not significantly different from control values. At 2 hours, the number of alpha 2-adrenergic sites was not significantly changed from control, but the affinity of yohimbine for platelets was significantly decreased 2.5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiopulmonary bypass, especially when prolonged, may result in hemostatic failure and pulmonary dysfunction, which has been attributed to changes in platelets and leukocytes, respectively. It has been well documented that contact of blood with synthetic surfaces causes platelet activation. In this report, we explore mechanisms of the activation of neutrophils during simulated in vitro extracorporeal circulation and document the release of neutrophil lactoferrin and elastase during clinical cardiopulmonary bypass (CCB). Inhibition in the simulated circuit by prostaglandin E1 (PGE1) and lidocaine suggests different mechanisms for release of neutrophil-specific proteins. During CCB with a bubble oxygenator it was observed that platelet counts fell to 42% +/- 2% of baseline. In addition, beta-thromboglobulin antigen (beta TG), a platelet-specific, alpha-granule protein marker reflecting the release reaction, increased from 0.15 +/- 0.05 to 0.84 +/- 0.11 microgram/mL. Neutrophil counts decreased to 67% +/- 7% of prebypass levels but then gradually rose as bypass continued. Both lactoferrin, a neutrophil-specific granule marker, and neutrophil elastase, an azurophilic granule marker, increased in plasma threefold to 1.66 +/- 0.33 micrograms/mL and 1.65 +/- 0.68 microgram/mL, respectively, just before bypass was stopped. When fresh heparinized human blood was recirculated within an extracorporeal membrane oxygenator bypass circuit for 120 minutes, plasma beta-TG rose to 5.13 micrograms/mL, lactoferrin increased from 0.13 +/- 0.04 to 1.62 +/- 0.22 micrograms/mL, and neutrophil elastase rose from 0.05 +/- 0.02 to 1.86 +/- 0.41 micrograms/mL. At 120 minutes, lidocaine (100 mumol/L), which inhibits neutrophil activation, delayed release of lactoferrin (1.33 +/- 0.26 micrograms/mL) and markedly inhibited release of elastase (0.24 +/- 0.05 microgram/mL) but did not inhibit release of beta-TG antigen (5.66 micrograms/mL at 120 minutes). PGE1 (0.3 mumol/L) inhibited significantly the release of beta-TG (0.31 microgram/mL) and elastase (0.52 +/- 0.11 microgram/mL) and attenuated the release of lactoferrin (1.57 +/- 0.45 micrograms/mL).

In vitro recirculation of fresh human heparinized blood in an extracorporeal circuit with a membrane oxygenator decreased fibrinogen-induced platelet aggregation and diminished the number of fibrinogen receptors and glycoprotein IIb/IIIa (GPIIb/GPIIIa) antigenic sites on the platelet surface. In seven experiments, the mean +/- SD Km value for fibrinogen (i.e., molar concentration of fibrinogen required to cause 50% of the maximal rate of aggregation) was 1.58 X 10(-7) mol/L +/- 0.68 X 10(-7) mol/L. After recirculation, this value increased to 3.8 X 10(-7) mol/L +/- 1.94 X 10(-7) mol/L (P less than or equal to 0.025). The maximal aggregation rate of chymotrypsin-treated platelets decreased by 40% after 2 hours of recirculation (P less than or equal to 0.025). The number of fibrinogen receptors on platelets, which were treated with chymotrypsin after a recirculation, decreased from 41,370 +/- 24,000 to 13,230 +/- 10,230/platelet under the same conditions (P less than or equal to 0.025). The number of antigenic sites for monoclonal antibody reacting with GPIIb/GPIIIa complex of adenosine diphosphate-stimulated platelets decreased from 34,200 +/- 5,940 to 19,500 +/- 9,680/platelet after recirculation (P less than or equal to 0.025). Prostaglandin E1 (0.3 mumol/L) in the perfusion circuit preserved the ability of platelets to react with fibrinogen. In conclusion, the loss of fibrinogen receptors from the surface of platelet membranes results from the interaction of platelets with the surfaces of perfusion circuits.

Prolonged extracorporeal circulation (ECC) using heparin as anticoagulant may be associated with pronounced thrombocytopenia and excessive bleeding. We, therefore, tested the hypothesis that reversible inhibition of platelet function, in lieu of heparinization, might preserve platelets and prevent coagulation in a perfusion circuit. When 500 ml of fresh heparinized (one U/ml) human blood was recirculated in a perfusion circuit constructed of standard silicone rubber components and a membrane oxygenator (0.95 M2), platelet counts declined to 9 +/- 2 (SEM) % of initial levels within 15 mins; plasma levels of the platelet specific protein LA-PF4 rose to 15 +/- 2 micrograms/ml within one hour indicating extensive release of platelet granule contents, and leukocyte counts declined to 91 +/- 4% within 15 mins. Prostacyclin (PGI2, greater than or equal to 25 eta M) or prostaglandin E1 (20 microM) and theophylline (12 mM) prevented platelet loss and release of granule contents. When heparin was reversed with protamine, however, immediate coagulation ensured. This occurred despite the absence of detectable activation of Hageman factor as evidenced by stability of plasma concentrations of prekallikrein in systems anticoagulated with heparin or citrate and despite our inability to detect thromboplastin-like properties in isolated leukocytes. Thus, coagulation in the presence of platelet inhibition suggests that alternative pathways, independent of platelet activation may exist. Platelet inhibition does preserve platelets preventing contact initiated release, but cannot serve by itself for anticoagulation.

Extracorporeal membrane oxygenation uses peripherally placed cannulas and a streamlined circuit without a venous reservoir. This study tests the flow dynamics of venous catheters connected without a reservoir directly to a centrifugal pump. During in vitro testing, a 30 cm segment of collapsible tubing interposed between the reservoir and pump simulates the vein. In five sheep, flow was measured between catheters placed in the right atrium and inferior vena cava from peripheral sites. Catheter tip design (four types) does not affect flow within a simulated vein in vitro. Maximum pump flow is independent of filling pressures (6 to 21 mm Hg) in vitro and in vivo when the catheter tip is in a tank reservoir or the right atrium. However, when the catheter tip is within a collapsible segment or in the inferior vena cava, maximal flow is significantly influenced by filling pressure (6 to 18 mm Hg) and by the ratio of catheter outer diameter to venous diameter. At all filling pressures, maximal flow in vivo is significantly reduced when this ratio is greater than 0.5. During extracorporeal membrane oxygenation, central venous pressure and catheter/vein ratio, not catheter size alone, control flow through peripheral venous catheters.

Extracorporeal membrane oxygenation (ECMO) reduces the systolic stress integral (SSI) in the normal left ventricle. We tested the hypothesis that the SSI does not decrease in poorly contracting, dilated, ejecting hearts during ECMO. In 14 sheep, four pairs of ultrasonic crystals measured changes in left ventricular (LV) wall thickness and three LV diameters. Volume calculations were validated by balloon distention of the ventricles after death (slope = 0.85; r = 0.85). SSI was measured during ECMO flows of 20 to 100 ml/kg/min in both normal and dilated, poorly contracting hearts produced by 30 minutes of warm ischemia. After warm ischemia, end-systolic elastance, an index of contractility, decreased from 8.3 +/- 0.6 mm Hg/ml to 2.9 +/- 0.4 mm Hg/ml (p = 0.001) and peak systolic pressure decreased from 47.4 +/- 0.7 mm Hg to 37.5 +/- 0.08 mm Hg (p = 0.01). In normal hearts, as ECMO flow increased, SSI decreased from 10.5 +/- 2.2 mm Hg.sec to 7.7 +/- 0.8 mm Hg.sec at 60 ml/kg/min (p = 0.001). However, in postischemic hearts, SSI progressively increased from 6.6 +/- 0.3 mm Hg.sec before ECMO to 12.4 +/- 1.8 mm Hg.sec at ECMO = 100 ml/kg/min. These studies indicate that the initial effect of ECMO on the poorly contracting, dilated heart increases LV wall stress and that the increase in stress is proportional to ECMO flow. The increase in stress is primarily due to an increase in afterload, which more than offsets decreases in systolic and diastolic volumes.