Ann Thorac Surg 2005;79:1326-1332
© 2005 The Society of Thoracic Surgeons
Original articles: Cardiovascular
Nafamostat Preserves Neutrophil Deformability and Reduces Microaggregate Formation During Simulated Extracorporeal Circulation
Yuji Hiramatsu, MD, PhDa,*,
Satoshi Homma, MD, PhDb,
Yukio Sato, MD, PhDc,
Shoko Sato, BMTa,
Kanji Matsuzaki, MDa,
Osamu Shigeta, MD, PhDa,
Yuzuru Sakakibara, MD, PhDa
a Department of Cardiovascular Surgery, University of Tsukuba, Tsukuba, Japan
b Department of Cardiology, University of Tsukuba, Tsukuba, Japan
c Department of Thoracic Surgery, Jichi Medical School, Tochigi, Japan
Accepted for publication September 2, 2004.
* Address reprint requests to Dr Hiramatsu, Dept of Cardiovascular Surgery, University of Tsukuba, 1–1–1 Tennodai, Tsukuba 305–8575, Japan (E-mail: yuji3{at}md.tsukuba.ac.jp).
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Abstract
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BACKGROUND: Initial sequestration of activated neutrophils and platelet microaggregates in capillaries are responsible for the inflammatory response associated with cardiopulmonary bypass. The study assesses the inhibitory effects of nafamostat mesilate on neutrophil and platelet activation, and on the neutrophil deformability change and microaggregate formation during simulated extracorporeal circulation.
METHODS: Fresh heparinized human blood was recirculated for 120 minutes in a membrane oxygenator and a roller pump with and without nafamostat (1.0 mg bolus plus 8.0 mg/h infusion; n = 10 for each group). Neutrophil and platelet counts and platelet aggregation were measured. CD11b, L-selectin, and cytoplasmic F-actin of neutrophils were measured by flow cytometry. The microchannel transit time of whole blood was measured as a marker of neutrophil deformability and microaggregate formation. Neutrophil elastase and complement C4d were measured using enzyme immunoassay.
RESULTS: Nafamostat preserved platelet counts and inhibited platelet aggregation. Nafamostat significantly reduced neutrophil elastase release at 120 minutes of recirculation, and F-actin expression at 30 and 60 minutes. The drug did not modulate the changes of CD11b, L-selectin, or C4d. Whole blood filterability was significantly preserved by nafamostat at 30 and 120 minutes.
CONCLUSIONS: Nafamostat preserves blood filterability during recirculation, possibly by suppression of F-actin expression and platelet activation. Nafamostat may reduce neutrophil sequestration and microaggregate formation in the microcirculation during cardiopulmonary bypass.
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Introduction
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The inflammatory response is exaggerated by cardiopulmonary bypass (CPB), and is responsible for a proportion of morbidity associated with open heart surgery [1]. One of the most important initial events in the development of the inflammatory response is neutrophil sequestration in microvessels of organs [2]. Activated neutrophils sequester in microvessels because of the loss of deformability and the increase of adhesive qualities of neutrophils and endothelial cells. Activated platelets also form microaggregates with circulating monocytes and neutrophils [3, 4]. We have previously reported that activated neutrophils and platelet microaggregates impede blood filterability through simulated microcapillaries during simulated CPB, and that the phenomenon mimics cell sequestration in the microcirculation [5]. Hence, pharmacologic inhibition of both neutrophils and platelets could be an attractive scenario to control cell sequestration in microcapillaries in the inflammatory response associated with CPB.
Nafamostat mesilate (NM; Futhan, 6'-amidino-2-naphthyl-p-guanidinobenzoate dimethane sulfonate, Mr = 540; Torii, Tokyo, Japan) is a synthetic, specific, and reversible serine protease inhibitor [6]. Nafamostat mesilate has a potent inhibitory activity on thrombin, XIIa, Xa, kallikrein, plasmin, C1r and C1s subcomponent proteins of complement system, and trypsin, all classified as trypsin-like serine proteases, which are known to have a substrate specificity for arginyl and lysyl residue–containing substrates. Nafamostat mesilate has a high affinity for these proteases with Ki values of approximately 10–7 to 10–8 mol/L, and has been shown to exhibit a weak or practically no inhibition against other proteases studied [6]. Hydrolysis of NM occurs mainly in the blood and liver, followed by glucuronic acid conjugation, with a half-life of 8 minutes in human plasma [7]. Nafamostat mesilate almost completely inhibits either the formation or activity of XIIa and kallikrein, two of the key enzymes of the contact system, and is thought to interact directly with platelets to reduce aggregability [8]. Nafamostat mesilate inhibits neutrophil elastase release during in vitro CPB [8].
This study assesses the inhibitory effects of NM on neutrophil and platelet activation, and on the deformability change of neutrophils and microaggregate formation during simulated extracorporeal circulation. We measured the expression of neutrophil adhesion molecules, cytoplasmic F-actin, platelet counts and aggregation response, neutrophil elastase, and complement C4d levels. Neutrophil deformability and microaggregate formation were assessed by whole blood transit time through simulated microcapillaries. We hypothesized that NM attenuates neutrophil and platelet activation, preserves neutrophil deformability, and reduces microaggregate formation in simulated microcapillaries during simulated extracorporeal circulation.
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Material and Methods
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Simulated extracorporeal circulation [5] involved a membrane oxygenator (model 60EC, 0.6 m2; MERA, Tokyo, Japan), a polyvinyl chloride reservoir (MERA), and a roller pump (model MS-033, MERA). Each circuit was primed with 250 mL of fresh human blood without dilution. Blood was obtained from healthy, fasting volunteers, who abstained from all medications for at least 2 weeks before donation. One donor was used for each individual bypass. Informed consent was obtained from donors, and the protocol was approved by the Institutional Review Board of the University of Tsukuba.
In the control group (n = 10), blood was drawn into a reservoir bag containing heparin (3.75 U/mL) and dextrose (2.25 mg/mL). In the nafamostat mesilate group (NM group, n = 10), the reservoir contained heparin (3.75 U/mL), dextrose (2.25 mg/mL), and 1.0 mg of NM. Nafamostat mesilate was subsequently administered by continuous infusion into the circuit at 8.0 mg/h during simulated extracorporeal circulation. Blood was recirculated for 120 minutes at 400 mL/min with the temperature maintained at 37°C by immersing the reservoir in a water bath. The oxygenator was ventilated with 95% oxygen and 5% carbon dioxide at 1.0 L/min. Preliminary experiments confirmed that the pH of the circulating blood was maintained at 7.3 to 7.5 and that the activated clotting time was more than 500 seconds throughout the experiment. The circuit pressure was not measured.
Blood samples were obtained for analysis from each donor before any anticoagulant was introduced (donor sample), from the reservoir bag before recirculation (0 minutes), and at 30, 60, and 120 minutes of recirculation. Additionally, a 20-mL standing control sample was collected from the reservoir and incubated for 120 minutes at 37°C. Blood samples were obtained with either 3.8% sodium citrate (for platelet aggregation, 9:1 by volume), 3.8% acid-citrate-dextrose (for CD11b, L-selectin, F-actin, and microchannel analysis, 9:1 by volume) or 1.0% ethylenediaminetetraacetic acid disodium (for neutrophil elastase and C4d, 9:1 by volume). Blood collected with 1.0% ethylenediaminetetraacetic acid disodium was centrifuged for 15 minutes at 2,000 g at 4°C, and the plasma was stored at –80°C for subsequent measurements. Samples for cell counts were collected with ethylenediaminetetraacetic acid disodium.
Blood Cell Counts
Blood cell counts were performed using a counter (T-660, Coulter, Hialeah, FL), and differential white cell counts were made on Wright's stained blood smears by an independent observer. Results were expressed as a percentage of the donor values.
Platelet Aggregation Study
Blood used for platelet aggregation was centrifuged at 150 g for 10 minutes to prepare platelet-rich plasma and then at 15,000 g to prepare platelet-poor plasma. Platelet counts of platelet-rich plasma were performed on a model Z1 Coulter counter (Coulter Electronics, Hialeah, FL). Platelet aggregation in response to adenosine diphosphate was assessed on an aggregometer (PAC-4S; Hema Tracer, Tokyo, Japan) with the use of 150,000 platelets/µL. Threshold doses of adenosine diphosphate were determined as the lowest doses of agonist needed to produce biphasic aggregation of at least 60.0% after 5 minutes in donor samples. Threshold doses of adenosine diphosphate were then used to measure the aggregation as a percentage of light transmittance in subsequent samples. Results were expressed as the percentage change from the donor value.
Plasma Neutrophil Elastase and C4d Assay
Plasma neutrophil elastase in a complex with 
-protease inhibitor was determined with an automated homogeneous enzyme immunoassay (Merck, Darmstadt, Germany). Plasma C4d fragment assay measured the C4d-containing activation fragments of C4 (C4b, iC4b, and C4d). C4d fragment was measured by an enzyme-linked immunosorbent assay (Quidel, San Diego, CA).
F-Actin Content Assay
A 50-µL sample was fixed with formaldehyde, and the cells were permeabilized using IntraPrep (Immunotech Coulter, Marseilles, France). Neutrophils were stained with BODIPY FL phallacidin (Molecular Probes, Eugene, OR). Cells were washed with phosphate-buffered saline solution, and the F-actin content was measured using a flow cytometer [9]. The F-actin content was expressed as the percentage change from the donor value.
Adhesion Molecules Assay
Changes in the surface expression of L-selectin and CD11b of neutrophils were measured as markers of neutrophil activation using flow cytometry, as previously described [9]. One hundred microliters of whole blood samples was incubated for 30 minutes with 2 mg/mL fluorescein isothiocyanate (FITC)-conjugated antihuman CD62L antibody (isotype: IgG1, kappa; BD Biosciences Pharmingen, San Diego, CA) and 1 mg/mL R-phycoerythrin (RPE)-conjugated mouse monoclonal anti-human CD11b antibody (isotype: IgG1, kappa; DAKO, Copenhagen, Denmark) at 4°C. Identical samples were incubated with FITC-conjugated monoclonal mouse IgG1, kappa antibody (DAKO), and RPE-conjugated monoclonal mouse IgG1, kappa antibody (DAKO) as negative controls. The erythrocytes were lysed for 60 seconds with Immuno-lyse, and leukocytes were fixed with Immuno-fix (Coulter Clone, Hialeah, FL). Neutrophils were identified by the typical forward-scatter and side-scatter pattern, and the expression of L-selectin and CD11b was measured as a mean fluorescent intensity of 5,000 cells. The changes of L-selectin and CD11b were expressed as the percentage changes compared with the donor value.
Microchannel Transit Time of Whole Blood
The transit time of whole blood through the silicon microchannel array was measured as a surrogate marker of neutrophil deformability and the rheologic impact by formed microaggregates. The detailed procedures and apparatus have been previously described [5, 10]. In short, the microgrooves in the silicon microchannel chip (Bloody-3S, 2600 channels; width, 6 µm; Hitachi Haramachi Electronics, Hitachi, Japan), which are close to the size of capillaries, were prefilled with saline solution. Blood samples were diluted with phosphate-buffered saline solution (1:1 by volume), and the suspension was made to flow through the microchannels under a pressure difference of 10 cm H2O. The transit time for each 100-µL suspension was determined, and results were expressed as a percentage of the transit time of the donor samples. The blood passage through the channels was recorded on a video-microscope system.
Statistical Analysis
All values are expressed as mean ± standard error of the mean. One-way analysis of variance as compared with the donor value was used for within-group comparison. Comparison of two groups as a function of time was performed by analysis of variance with repeated measures. Data were further compared by the use of the post hoc tests with Bonferroni correction.
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Results
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Changes in measured blood and plasma constituents and microchannel transit times during experiments are shown in Table 1. Neutrophil counts and hematocrit levels did not change significantly in either group throughout the recirculation (hematocrit data not shown). Platelet counts decreased to 23.0% ± 2.4% of the donor value by 120 minutes of recirculation in the control group. Nafamostat mesilate preserved platelet counts at more than 46% of the donor value. There were differences between the two groups at 30, 60, and 120 minutes (Fig 1A; p < 0.001 at all three data points). Platelet aggregation response to adenosine diphosphate was attenuated to 39.1% ± 4.0% of the donor value by 120 minutes in the control group. Nafamostat mesilate strongly inhibited platelet aggregation less than 14% of the donor value after 30 minutes. There were differences between the two groups at 0, 30, 60, and 120 minutes of recirculation (Fig 1B; p < 0.001, p < 0.001, p = 0.004, and p = 0.0014, respectively).
Plasma neutrophil elastase levels showed a 12-fold increase by 120 minutes in the control group. Nafamostat mesilate attenuated this increase of neutrophil elastase. Repeated measure analysis of variance showed differences between the two groups at all times (p = 0.042 for the group effect of analysis of variance), and there were differences at 120 minutes (Fig 2; p = 0.049).
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Fig 2. Changes in plasma neutrophil elastase levels before and during recirculation. Values are expressed as the mean ± standard error of the mean. *p < 0.05, **p < 0.01 by one-way analysis of variance as compared with the donor value.  p < 0.05 by analysis of variance with Bonferroni correction between the nafamostat mesilate group and the control group. (SC = standing control.)
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Plasma C4d levels showed a significant increase at 120 minutes of recirculation in the control group but not in the NM group. However, there were no significant differences between the two groups (p = 0.246 for the group effect of analysis of variance).
The cytoplasmic F-actin content of the neutrophils increased after 30 minutes of recirculation and reached 333.2% ± 57.7% of the donor value at 60 minutes in the control group. Nafamostat mesilate inhibited the increase of F-actin, with differences between the two groups at 30 and 60 minutes (Fig 3; p = 0.010, and p = 0.016, respectively).
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Fig 3. The cytoplasmic F-actin content of the neutrophils before and during recirculation. Values are expressed as the mean ± standard error of the mean. *p < 0.05, **p < 0.01 by one-way analysis of variance as compared with the donor value. p < 0.05 by analysis of variance with Bonferroni correction between the nafamostat mesilate group and the control group. (SC = standing control.)
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In both groups, CD11b on the neutrophil surface increased and L-selectin decreased significantly by 120 minutes of recirculation. No statistically significant differences were observed between the two groups either in CD11b or L-selectin.
The microchannel transit time of whole blood was prolonged after starting recirculation and reached 179.6% ± 23.7% of the donor value at 120 minutes in the control group. The NM group showed a small increase of the transit time only at 30 minutes of recirculation. There were significant differences between the two groups at 30 and 120 minutes (Fig 4; p = 0.018, and p = 0.042, respectively), but not at 60 minutes (p = 0.4). The video-microscope image revealed that NM reduced the plugging of the microchannels with neutrophils and platelet microaggregates at 120 minutes (Fig 5).
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Fig 4. The microchannel transit time of the whole blood before and during recirculation. Values are expressed as the mean ± standard error of the mean. *p < 0.05, **p < 0.01 by one-way analysis of variance as compared with the donor value. p < 0.05 by analysis of variance with Bonferroni correction between the nafamostat mesilate group and the control group. (SC = standing control.)
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Fig 5. The video-microscopic image at 120 minutes of recirculation shows the plugging of microchannels with neutrophils and microaggregates in the control group (left). Nafamostat mesilate reduced the plugging and kept the microchannels clearer than the control at 120 minutes (right).
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Comment
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Previous investigations regarding NM carried out in the mid-1990s did not successfully reveal its entire efficacy as a broad-spectrum protease inhibitor for CPB [8, 11–13]. Nafamostat mesilate has not been widely applied in clinical CPB since then. Our recent clinical CPB study [14] barely demonstrated its limited effects on the inhibition of fibrinolysis. However, we still believe that the inhibition of XIIa and kallikrein by NM would attenuate the activation of neutrophils, because kallikrein is a major mediator that directly activates neutrophils and facilitates cleavage of factor XII in a feedback fashion [1, 15, 16]. Thus, a revived interest in the efficacy of NM induced us to elucidate the impact of NM on the inhibition of neutrophils as well as platelets. Given that NM shows profound inhibition on both neutrophils and platelets, the drug may control the initial cell sequestration and microaggregate formation in microcapillaries during the inflammatory response.
In the present study, NM altered the loss of circulating platelets and showed complete inhibition of platelet aggregability. This short-acting efficacy on platelets is similar to the concept of platelet anesthesia using reversible glycoprotein IIb/IIIa inhibitors [17]. As Sundaram and associates explained [8], if the preservation of platelet function is caused by the inhibitory effect on the release of cathepsin G from neutrophils, platelet sensitivity might be better preserved by NM. Therefore, the attenuation of aggregability probably reflects the direct inhibition of platelet aggregation by NM. There could be a dose issue for our more significant results on platelets compared with those of Sundaram and colleagues [8] or our clinical study [14]. We followed previous clinical protocols [11–14] with doses between 40 and 100 mg/h (or 2.0 mg · kg–1 · h–1), and simulated our 250-mL priming as a circuit for a 4-kg infant. It resulted in a five-fold higher dose of NM than that in the study by Sundaram and associates [8]. In addition, plasma concentrations of NM could be kept at a higher level in vitro than in vivo because the elimination of the drug is minimal in vitro [11]. Regarding neutrophil elastase levels, we have demonstrated significant attenuation by NM in the current in vitro study but not in a previous in vivo CPB study [14]. These two contradictory results may also be explained by a possible discrepancy in plasma concentrations of NM between in vivo and in vitro, although we cannot rule out the fact that there may be another important elastase activator involved that may create the discrepancy between in vivo and in vitro CPB.
The fact that NM completely inhibited the change of F-actin assembly is all the more remarkable. The changes in the neutrophil cytoskeleton with F-actin assembly at the cell periphery are responsible for the neutrophil deformability [18]. During neutrophil activation, rapid and significant changes occur in the structure of the microfilament network inside cells. There is a rapid and transient increase in F-actin assembly from the G-actin pool [18]. The loss of neutrophil deformability contributes to their sequestration, allowing close proximity and adhesion to endothelium [19], and it is documented that clumping of neutrophils in microvessels can be induced by complement fragments, cytokines, or lipid mediators such as platelet-activating factor [20]. The effects of NM on cytokines or platelet-activating factor may lead to attenuation of the cytosolic calcium increase inside neutrophils, which is a pivotal event in neutrophil activation [10]. Although a mechanistic hypothesis to explain how NM acts on F-actin is not yet clear, both the modulation of neutrophil deformability by means of attenuation of F-actin assembly and the inhibition of microaggregate formation could be the main reason for preservation of blood filterability through simulated microcapillaries in the NM-treated group.
It is also unclear why NM did not modulate changes of neutrophil adhesion molecules despite considerable attenuation of neutrophil elastase release and F-actin assembly. It may be explained by the lack of endothelial cells, which have many molecules and function against neutrophil adhesion. In the presence of endothelial cells, it has been shown that a neutrophil elastase inhibitor modulates expression of neutrophil adhesion molecules [21].
It is well documented that complement has close interactions with neutrophil elastase activity [22, 23]. C4d fragment, a unique component of the classic pathway, was chosen as an outcome marker for complement inhibition by NM, because NM inhibits the activity of C1 in solution [6]. In addition, the role of the alternative pathway is not thought to be as important in CPB as it is in hemodialysis [1]. Recent studies, however, report that the alternative pathway seems to be the major mechanism of complement activation during CPB, and is mainly triggered by the contact activation cascade [22, 24]. If this is the case, our finding of nonsignificant reduction in C4d may produce an inconclusive result regarding the effect of NM on overall complement activation. Nevertheless, our observations on suppression of elastase release but no alteration of C4d by NM raise doubts about the primary role of the classic complement pathway on neutrophil elastase activity.
Limitations of this experimental approach include the fact that the simulated extracorporeal circulation model lacks endothelial cells, which potentially amplify the neutrophil adhesion processes. The model does not involve ischemia–reperfusion processes. Moreover, the model lacks information regarding the role of the extrinsic pathway because of absence of a wound. Therefore, the results of this study cannot be immediately applied clinically or directly compared with the results of clinical studies. However, a similar simulated extracorporeal circulation model has been used extensively to screen inhibitors [5, 8, 10, 22]. This in vitro system avoids loss of cells or markers from the circuit, and also avoids new cells being recruited. Another potential criticism of this study is that we failed to assess platelet secretion. The secretion data would verify the theory that NM directly inhibits platelet activation, and may support the hypothesis that F-actin assembly is related to release of platelet-activating factor from platelets. Finally, we failed to show the amount of microaggregates by a quantitative analysis, which is not practicable in the present system.
In summary, the most important incremental knowledge in this study is that NM preserves blood filterability, possibly by suppressing F-actin formation and platelet aggregation. These results have important clinical implications. Nafamostat mesilate may reduce neutrophil sequestration and microaggregate formation in the microcirculation during the inflammatory response induced by CPB. Moreover, because loss of neutrophil deformability and neutrophil granule release are important mechanisms in ischemia–reperfusion and endothelial cell injuries [25], NM may attenuate such injuries and improve the physiologic status of critical patients. Further investigations in an in vivo primate CPB model will be necessary to confirm the efficacy of NM on cell sequestration and neutrophil-related inflammatory reactions. The optimal protocol in clinical CPB needs to be determined to derive the best performance of this promising inhibitor.
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Acknowledgments
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The authors wish to thank Shiro Hinotsu, MD, for statistical assistance and Avi Landau for language assistance. Supported by a University of Tsukuba Institutional Research Grant in 2003.
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Abstract
Introduction
p < 0.001 by analysis of variance with Bonferroni correction between the nafamostat mesilate group and the control group. (B) Changes in platelet aggregation response to adenosine diphosphate before and during recirculation. Values are expressed as the mean ± standard error of the mean. **p < 0.01 by one-way analysis of variance as compared with the donor value. 
p < 0.01,