Ann Thorac Surg 1998;66:1216-1223
© 1998 The Society of Thoracic Surgeons
Original articles: cardiovascular
Recombinant human megakaryocyte growth and development factor attenuates postbypass thrombocytopenia
Masanori Nakamura, MDa,
Christopher F. Toombs, PhDa,
Ignacio G. Duarte, MDa,
Russell S. Ronson, MDa,
L. Susan Schmarkey, BSa,
Sara L. Katzmark, BSa,
Jill Robinson, BSa,
Dirck L. Dillehay, PhDb,
Jakob Vinten-Johansen, PhDa,
Robert A. Guyton, MDa
a Section of Cardiothoracic Surgery, Department of Surgery, Carlyle Fraser Heart Center of Emory University, Cardiothoracic Research Laboratory, Atlanta, Georgia, USA
b Division of Animal Resources, Department of Pathology, Emory University, Atlanta, Georgia, USA
Address reprint requests to Dr Vinten-Johansen, Cardiothoracic Research Laboratory, 550 Peachtree St, NE, Atlanta, GA 30365-2225
Presented at the Poster Session of the Thirty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 26–28, 1998.
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Abstract
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Background. Cardiopulmonary bypass contributes to platelet loss and dysfunction by exposure to shear stresses, foreign surfaces, and hypothermia. This study tested the hypothesis that pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) accelerates recovery of the platelet population after hypothermic extracorporeal circulation (HEC).
Methods. In a blinded study, subcutaneous injections of drug or placebo were given to dogs daily for 3 days preoperatively (day 0, 1, and 2) with no drug on day 3. On day 4, the animal was prepared for arteriovenous HEC. After heparinization, HEC was initiated at 30 to 40 mL · kg-1 · min-1. Hypothermic extracorporeal circulation (25°C) was continued for 90 minutes.
Results. Preoperative platelet count (x103 platelets/µL) did not differ from predrug count in placebo (256 ± 27 versus 255 ± 20) or PEG-rHuMGDF (271 ± 30 versus 291 ± 38). During 60 minutes of HEC, the platelet count decreased to 
10% of baseline in placebo (29 ± 5) and PEG-rHuMGDF (46 ± 8), and recovered to 70% of baseline after rewarming (90 minutes of HEC: placebo, 185 ± 17, versus PEG-rHuMGDF, 169 ± 22). After HEC, platelet count was greater in PEG-rHuMGDF–treated animals (p < 0.05) without altering function (aggregation responses). Within the first 6 hours after HEC, platelet count in PEG-rHuMGDF–treated animals was rising and increased to 260 ± 29 (p < 0.01), but was unchanged in placebo animals (186 ± 21). Thereafter, platelet count in placebo animals declined to a nadir of 124 ± 15 (72 hours after HEC), whereas platelet count in PEG-rHuMGDF animals approximated the preoperative value (>200) at all times.
Conclusions. Appropriately timed presurgical administration of PEG-rHuMGDF counteracts post-HEC relative thrombocytopenia without increasing platelet population and enhancing aggregation preoperatively or during extracorporeal circulation.
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Introduction
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Bleeding complications are frequently encountered after extracorporeal circulation. One important cause of this complication is thrombocytopenia after extracorporeal circulation [1]. Surgical procedures that require extracorporeal circulation can induce thrombocytopenia not only by hemodilution, but also by the exposure of circulating platelets to foreign surfaces and shear stresses. Adherence of platelets to extracorporeal tubing, oxygenators, and filters can cause fragmentation of platelet membranes with a loss of surface receptors, degranulation, and aggregation, thus further reducing the number of functional platelets available to participate in the hemostatic process after extracorporeal circulation [1, 2]. Systemic hypothermia further accelerates platelet dysfunction [1, 3]. Most of the events during extracorporeal circulation that profoundly depress platelet function appear to take place initially in the platelet membrane, and may decrease survival time of the platelets after extracorporeal circulation [2]. To prevent platelet dysfunction and destruction and to reduce blood loss, several studies have reported beneficial effects of the protease inhibitor aprotinin [4] and an antagonist of glycoprotein IIb/IIIa [2], but these effects are still controversial. Extracorporeal circulation is generally used in major cardiac and thoracic aortic operations, as a bridge to transplantation, or as a support mechanism (extracorporeal membrane oxygenator) in patients with potentially lethal hemodynamic and respiratory instability. Transfusion of platelets or other blood products is frequently needed postoperatively to maintain hemostasis. Especially in difficult cases like extensive repair of thoracic aortic aneurysm, postoperative bleeding might be a main cause triggering postoperative multiple systemic organ failure.
Thrombopoietin is the endogenous regulator of platelet population [5] and the endogenous ligand for the cytokine receptor Mpl (the c-mpl receptor). Thrombopoietin was discovered in 1994 and indeed fulfilled all the criteria for the theoretical thrombopoietin that was speculated to exist [6, 7], which stimulates platelet production in vivo. The c-mpl receptor was known to be expressed in megakarocytes, and is present on the platelet membrane [8]. The recombinant derived form of the endogenous c-mpl ligand, human megakaryocyte growth and development factor (rHuMGDF), is produced by Escherichia coli and is able to stimulate production of platelets either in vitro [9, 10] or in vivo [11, 12]. It directly increases platelet function through the c-mpl receptor via signal transduction mechanisms [13]. In the mouse [14], rabbit [11], and baboon [12, 15], rHuMGDF stimulates a dose-dependent production and maturation of megakarocytes, resulting in higher circulating platelet count. The clinical utility of polyethylene glycol derived rHuMGDF (PEG-rHuMGDF) as a treatment for thrombocytopenia secondary to chemotherapy was demonstrated recently by Basser and associates [16] and Fanucchi and colleagues [17], who collectively reported that PEG-rHuMGDF accelerates platelet production in cancer patients. Because PEG-rHuMGDF requires several days for the increased platelet generation to become manifest, pretreatment regimens that affect the success of the outcome may be initiated on a prescribed schedule as a pretreatment to patients undergoing elective operative procedures requiring extracorporeal circulation. The administration of PEG-rHuMGDF at a properly selected dose and treatment interval may increase platelet production so that the appearance of new platelets potentially avoids extracorporeally induced thrombocytopenia. In addition, sustained emergence of new platelets from the marrow should enhance recovery from thrombocytopenia postoperatively.
This study tested the hypotheses that (1) preoperative administration of PEG-rHuMGDF counteracts thrombocytopenia after hypothermic extracorporeal circulation (HEC) and (2) the timing and dose of this preoperative administration of PEG-rHuMGDF can be adjusted to avoid overstimulating platelet population and function preoperatively or during extracorporeal circulation.
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Material and methods
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Twenty adult mongrel dogs weighing 18 to 32 kg (mean, 25.5 ± 0.8 kg) were used for these studies. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Experimental protocols
Drug or placebo was administered three times over a 4-day period preoperatively. The investigators were unaware of the identity of treatment until after the study was complete. In one group (n = 8), vehicle was given for 3 consecutive days preoperatively (placebo group). In a second group of dogs (n = 12), PEG-rHuMGDF was given for 3 consecutive days preoperatively at a dose of 10 µg · kg-1 · day-1. Dogs received daily subcutaneous (at 24-hour intervals) injections of either PEG-rHuMGDF or vehicle for a total of 3 days preoperatively (days 0, 1, and 2). On the day before the surgical procedure (day 3), the drug was not administered to avoid a potential overshoot in platelet count. On day 4, the animal was prepared surgically for HEC.
Operative procedure
Animals were premedicated with 4 mg/kg morphine and 0.02 mg/kg atropine and then anesthetized with 20 mg/kg sodium pentothal intravenously. Each dog was endotracheally intubated and the lungs were ventilated with oxygen-enriched room air. Anesthesia was maintained with isoflurane (1% to 1.5%) throughout the protocol. Arterial and venous cannulas were placed in the femoral artery, the femoral vein, and the right jugular vein for connection to the HEC circuit. A 14F William Harvey arterial perfusion cannula (C.R. Bard, Inc, Billerica, MA) was used in the femoral artery and 18F or 20F thin single venous return cannulas (Research Medical, Inc, Midvale, UT) were used in the femoral and jugular veins. The HEC perfusion circuit consisted of an SMO/INF #16267 Infant Membrane Oxygenator (3M/Sarns, Ann Arbor, MI) and a Sarns 5000 modular perfusion pump (Sarns, Ann Arbor, MI). The perfusion circuit was primed with 600 to 700 mL volume of Hespan (6% Hetastarch; Abbott Laboratories, Chicago, IL). The dog was anticoagulated with 250 U/kg heparin (porcine heparin sodium; Elins-Sinn Inc, NJ) to maintain the activated clotting time greater than 400 seconds. Hypothermic extracorporeal circulation was initiated at a flow rate of 30 to 40 mL · kg-1 · min-1 and was continued for a total of 90 minutes. After stabilization at normothermia for 15 minutes, animals were cooled to 25°C over 15 minutes, maintained at 25°C for 30 minutes, and then rewarmed to 36°C over 30 minutes. The mean blood pressure was kept between 60 and 80 mm Hg and measured every 15 minutes throughout the experiment. Heart rate and mean arterial pressure were averaged from 12-second data files captured on computer and analyzed with a videographics program (SPECTRUM; Wake Forest University, Winston-Salem, NC). At the termination of HEC, the venous and arterial cannulas were withdrawn. The femoral arteriotomy and venotomy sites were repaired with 6-0 Prolene (Ethicon, Somerville, NJ), and a small-bore PE-240 (polyethylene) tube (Becton Dickinson, NJ) was inserted via the right jugular vein to the right atrium to withdraw blood for postoperative evaluation. We did not administer protamine sulfate to reverse systemic anticoagulation. The inguinal and cervical incisions were closed in layers in the usual fashion.
Polyethylene glycol derived recombinant human megakaryocyte growth and development factor (Amgen Inc, Thousand Oaks, CA) is a recombinant-derived truncated c-mpl ligand encompassing the receptor-binding domain covalently bonded with polyethylene glycol at the N-terminus, and supplied as a sterile, clear, aqueous solution. In this study, we used PEG-rHuMGDF because pegylation increases the half-life of this peptide and has been shown to enhance the in vivo potency of rHuMGDF approximately fivefold to tenfold [12, 15].
Postoperative care
Dogs were given 0.5 g of cephazoline subcutaneously each day for 3 postoperative days. At 1 hour, 6 hours, 1 day, 2 days, 3 days, and 4 days (at 24-hour intervals) after the termination of HEC, venous blood was withdrawn for complete blood cell measurements, ex vivo platelet aggregation, and coagulation studies.
Data points and measured variables
Complete blood cell measurements were performed at the following intervals: before drug administration, preoperatively, every 15 minutes during HEC, and 1 hour, 6 hours, 1 day, 2 days, 3 days, and 4 days after the termination of HEC. Complete blood cell measurements were performed on 1 mL of citrated whole blood and analyzed with a Baker 9110+ Hematology Analyzer.
Venous blood samples were withdrawn for coagulation study before drug administration, preoperatively, and 1 hour, 1 day, and 4 days after the termination of HEC. Coagulation studies consisting of prothrombin time, activated partial thromboplastin time, and fibrinogen levels were performed by centrifugation of 6 mL of citrate whole blood at 2,500 g to produce approximately 3 mL of platelet-poor plasma. Platelet-poor plasma was stored at -80°C until analyzed. Coagulation parameters were determined with an Electra 900C automated coagulation timer (MLA, Pleasantville, NY) and optical detection of fibrin clots. The one-stage prothrombin time was determined in duplicate from a 100-µL volume of plasma. Activation of the extrinsic pathway was initiated by the addition of a 200-µL volume of a rabbit brain thromboplastin reagent (Ortho, Raritan, NJ). The activated partial thromboplastin time test was determined in duplicate from a 100-µL volume of plasma. Contact activation of the intrinsic pathway was initiated by the addition of 100 µL of an activating reagent consisting of ellagic acid and phospholipid to substitute for platelet membrane phospholipid (THROMBOFAX Reagent; Ortho). To enable the coagulation pathway to proceed to form a fibrin clot, a 100-µL volume of 0.02 mol/L calcium chloride solution was subsequently added. Plasma fibrinogen was determined with a thrombin clotting time test and the reagents for a quantitative fibrinogen assay (Ortho). Plasma samples were first diluted 1:10 in 3-(N-morpholino)propane sulfonic acid buffered saline solution and duplicate determinations were made on 200 µL of the diluted plasma. The thrombin clotting time was determined after the addition of 100 µL of a 50 U/mL thrombin solution. The resulting clotting time was used to interpolate a fibrinogen content from a standard curve generated with known quantities of fibrinogen in dilute plasma.
Venous blood samples were withdrawn for platelet aggregation at regular intervals. Samples of whole blood were used to prepare platelet-rich plasma for optical aggregometry. Platelet-rich plasma was prepared by low-speed centrifugation (800 rpm for 15 minutes at 20°C). The plasma supernatant was aspirated and counted in a cell counter to determine platelet count. The platelet-rich plasma depleted layer was recentrifuged at 2,200 rpm for 15 minutes at 20°C to obtain platelet-poor plasma. Platelet count in platelet-rich plasma was adjusted to 200,000 platelets per microliter by using autologous platelet-poor plasma as a diluent. Platelet aggregation studies were performed with a Chrono Log optical aggregometer. The platelet-rich plasma (250 µL) was stirred at 1,000 rpm and maintained at 37°C. Increases in light transmission were recorded on an analog chart recorder and converted to percent platelet aggregation by using the optical absorbance of platelet-poor plasma as reference for 100% platelet aggregation. Platelet aggregation was tested on a range of concentrations of adenosine diphosphate (0.1, 0.3, 0.6, 1, 3, 6, 10, and 30 µmol/L) and a single concentration of collagen (10 µg/mL).
Venous blood was withdrawn to measure the plasma concentration of immunoreactive c-mpl ligand at regular intervals. Serum samples were assayed for c-mpl ligand content by an enzyme-linked immunoabsorbance assay as previously described [11].
Histologic examination of femoral arterial and venous cannulation sites
The animals were sacrificed by overdose of nembutal (100 mg/kg). Two-to 3-cm segments of the femoral artery and vein at the cannulation sites were excised and immersed in a solution of 10% formalin. The vessels were stained by hematoxylin and eosin and examined by a veterinary pathologist (Dirck L. Dillehay). The typical histologic features of microthrombi are as follows: (1) a mass of fibrin containing cells neutrophils, lymphocytes, and erythrocytes that was attached to the vascular wall, and (2) endothelial denudation of the vascular wall caused by cannulation [18]. This examination was done at a place distant from the suture placements.
Data analysis and statistics
All data are reported as mean ± standard error of the mean and considered statistically significant if p is less than 0.05. Statistically significant changes were determined with two-way repeated-measures analysis of variance. One-way analysis of variance followed by a Bonferroni posthoc test was used to determine at what point in time the changes in discrete end points of interest were significantly different.
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Results
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A total of 20 dogs were used for this experiment. No animals were excluded from data analysis.
Plasma concentration of c-mpl ligand
Figure 1 depicts serum concentration of immunoreactive c-mpl ligand at each time point. The c-mpl ligand concentration in the PEG-rHuMGDF group was significantly increased preoperatively (2.28 ± 0.6 ng/mL) and at 1 hour postoperative (1.68 ± 0.4 ng/mL). However, the levels at both time points were less than 3 ng/mL, the threshold for enhancement of agonist-induced platelet aggregation by PEG-rHuMGDF [19].
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Fig 1. Plasma concentration of c-mpl ligand (endogenous megakaryocyte growth and development factor plus polyethylene glycol derived recombinant human megakaryocyte growth and development factor [PEG-rHuMGDF]) during the course of the experiment. The plasma level of c-mpl ligand was significantly increased in the PEG-rHuMGDF group before the operation (Pre Op) and 1 hour after the operation (Po 1 h). However, plasma titers were less than 3 ng/mL, a level at which PEG-rHuMGDF does not affect platelet function. (Po = postoperative; *p < 0.05 versus previous time in PEG-rHuMGDF; +p < 0.05 between groups.)
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Intraoperative hemodynamics
Bypass flow, blood pressure, heart rate, and rectal temperature are shown in Table 1. There were no group differences in any variable at any time. Rectal temperature decreased to 24°C in both groups during HEC, and rewarmed to baseline. Heart rate increased significantly after EC but decreased at 45 minutes of HEC and returned to approximately the initial heart rate in both groups. During HEC, phenylephrine was administered in 2 dogs in the placebo group (25%) and in 3 dogs in the PEG-rHuMGDF group (25%) because of hypotension (<50 mm Hg mean arterial pressure).
Complete blood cell data
Figures 2 and 3 summarize hematocrit and white blood cell data during the course of the experiment. Hematocrit and white blood cell count were comparable between groups, suggesting there were no differences in the degree of hemodilution between groups and no effect on erythrocyte or leukocyte population in the presence of absence of rHuMGDF. There was an approximately 50% decrease in white blood cell count during HEC, which rebounded to values modestly greater than predrug values. However, white blood cell count returned to baseline 4 days postoperatively.
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Fig 2. Immediately before (Pre Op) and after (Po 1 hr) extracorporeal circulation, the hematocrit (Hct) was significantly decreased relative to the previous time point. However, Hct was comparable between the groups throughout the experiment, suggesting there was no difference in the degree of hemodilution and no effect of polyethylene glycol derived recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on the erythrocyte population. (EC = extracorporeal circulation; NS = not significant; *p < 0.05 versus previous time in placebo; +p < 0.05 versus previous time in PEG-rHuMGDF.)
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Fig 3. White blood cell (WBC) count was increased 6 hours after extracorporeal circulation (EC) in both groups. However, WBC counts were comparable between the groups, suggesting polyethylene glycol derived recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) did not have any effect on the WBC population. (NS = not significant; *p < 0.05 versus previous time in placebo. +p < 0.05 versus previous time in PEG-rHuMGDF.)
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Platelet count
Figure 4 depicts the platelet count. Preoperative platelet count (x103 platelets/µL) did not differ from predrug counts (0 day) in the placebo group (256 ± 27 versus 255 ± 20) or the PEG-rHuMGDF group (271 ± 30 versus 291 ± 38). During HEC, platelet counts decreased to approximately 10% of the predrug value (60 minutes: placebo, 29 ± 5 versus PEG-rHuMGDF, 46 ± 8), progressively increased during rewarming, and recovered to 70% of baseline after rewarming (90 minutes: placebo, 185 ± 17, versus PEG-rHuMGDF, 169 ± 22). There was no difference between groups during HEC, suggesting PEG-rHuMGDF did not alter the platelet population during HEC. After HEC, platelet counts were greater in PEG-rHuMGDF–treated animals (p < 0.05 by repeated-measures analysis of variance). Within the first 6 hours after HEC, the platelet count in PEG-rHuMGDF–treated animals increased to 260 ± 29 from 169 ± 22 (p < 0.01), but was unchanged in placebo-treated animals (186 ± 21 versus 185 ± 17). On postoperative day 3, platelet count showed a second phase decrease to 124 ± 15 in the placebo group, while increasing progressively to 249 ± 33 in the PEG-rHuMGDF group; the value in the PEG-rHuMGDF group remained greater than that in the placebo group on postoperative day 4 (304 ± 31 versus 158 ± 18). These results suggest that the presurgical administration of PEG-rHuMGDF stabilized postoperative platelet counts to preoperative levels without increasing platelet population preoperatively or during extracorporeal circulation.
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Fig 4. The preoperative platelet count did not differ from predrug count in placebo or polyethylene glycol derived recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) groups. During 60 minutes of hypothermic extracorporeal circulation (EC), platelet count decreased to 10% of baseline and at 90 minutes platelet count during hypothermic EC recovered to 70% of baseline. Platelet count was comparable between the groups before and during EC. However, after EC, platelet counts were greater in the PEG-rHuMGDF group than in the placebo group. Within the first 6 hours after hypothermic EC, platelet count increased in drug-treated animals but was unchanged in the placebo group. In the placebo group at 72 hours after hypothermic EC, platelet count showed a second phase decrease while increasing progressively in the PEG-rHuMGDF group (*p < 0.05 versus placebo.)
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Platelet aggregation
Platelet aggregation to adenosine diphosphate (0.1 to 30 µmol/L) was individually tested in samples of platelet-rich plasma at each time point. Ex vivo platelet aggregatory responsiveness to adenosine diphosphate was significantly decreased immediately after extracorporeal circulation. However, there was no significant difference between groups after administration of PEG-rHuMGDF or after discontinuation of extracorporeal circulation (Fig 5). At 1 hour postoperative, PEG-rHuMGDF tended to modestly enhance platelet aggregation induced by collagen (10 µg/mL), but this was not significant (Fig 6). There were no significant group differences in postoperative platelet aggregation. These data suggested that PEG-rHuMGDF did not significantly increase platelet function in this model.
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Fig 5. Platelet aggregation response to 10 µmol/L of adenosine diphosphate (the peak response) at each point. Extracorporeal circulation tended to impair platelet function at 1 hour postoperatively (Po), which recovered by the first Po day. There were no significant differences between groups after administration of drug or discontinuation of extracorporeal circulation. (PEG-rHuMGDF = polyethylene glycol derived recombinant human megakaryocyte growth and development factor, Pre Op = preoperative; *p < 0.05 between Po 1 h and Po 4 days in placebo.)
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Fig 6. Platelet aggregation response to collagen. Polyethylene glycol derived recombinant human karyocyte growth and development factor (PEG-rHuMGDF) slightly enhanced platelet aggregation induced by collagen, 10 µg/mL, but there were no significant group differences at any time. (Po = postoperative; Pre Op = preoperative; *p < 0.05 between time points.)
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Coagulation times and fibrinogen level
Table 2 depicts the changes in prothrombin time, activated partial thromboplastin time, and fibrinogen level. There was no group difference at any time, suggesting PEG-rHuMGDF did not alter the level of coagulation factors.
Pathologic examination of femoral arterial and venous cannulation sites
These vessels were removed at 7.7 ± 1.4 days in the placebo group and at 7.5 ± 0.7 days in the PEG-rHuMGDF group. Two cases of a total of 7 placebo cases (29%) had histologically detected microthrombi in the arterial lumen, whereas 3 of 10 cases (30%) in the PEG-rHuMGDF group had microthrombi in the arterial lumen. In venous samples, microthrombi were detected in 66% (4/6) of cases in placebo, whereas the incidence was 44% (4/9) in the PEG-rHuMGDF group. This suggests preoperative administration of PEG-rHuMGDF did not increase the incidence of thrombosis in vessels mechanically damaged by cannulations.
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Comment
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Recombinant human megakaryocyte growth and development factor has been found highly effective in stimulating megakaryocyte and platelet production in experimental animals [11, 12, 14, 15], and early clinical data show that PEG-rHuMGDF is effective in stimulating platelet production [16, 17]. Diseases for which rHuMGDF was indicated and tested were related to deterioration of platelet production by chemotherapy. Here we have investigated the use of rHuMGDF to counteract thrombocytopenia after HEC. Hypothermic extracorporeal circulation is associated with an acute consumption of platelets caused by foreign surface interaction and aggregation, shear and mechanical stresses, and hypothermia-induced platelet dysfunction by increased membrane fragility [1]. In the present study, we found that HEC produced a decrease in both platelet population and in vitro function. The relative thrombocytopenia was biphasic, decreasing dramatically during HEC and returning to 70% of baseline immediately after rewarming and discontinuation of HEC, followed by a sustained and slow decrease over the next 3 days. After HEC, platelet counts were greater in PEG-rHuMGDF–treated animals. Administration of PEG-rHuMGDF accelerated recovery of platelet count either because of production of new platelets or by increasing survival time of old platelets by protecting the platelet membrane. We did not measure the survival time of platelets in this study, although Harker and associates [15] reported that administration of PEG-rHuMGDF accelerated the turnover but did not alter the survival time of platelets in normal baboon. Therefore, PEG-rHuMGDF might not increase survival time of old platelets in this HEC-induced thrombocytopenia model. Platelet function was not altered by rHuMGDF. Therefore, proper administration of rHuMGDF (1) avoided preoperative and intraoperative platelet augmentation without the concern for stimulating overactive aggregation and (2) avoided postoperative thrombocytopenia.
Overstimulation of platelet population or function before or during an operation should be prevented. Further, reactive thrombocytosis (two to three times normal) after coronary bypass grafting was identified by Schmuziger and associates [20] as a risk factor for postoperative vein graft occlusion and myocardial infarction. Although additional risk factors (smoking, hyperlipidemia, and previous myocardial infarction) were also present in greater proportion in the reactive thrombocytosis group, the study suggests that a high postoperative platelet count is to be avoided. In the present study, the timing of preoperative administration of drug successfully avoided preoperative and intraoperative thrombocytosis and increased platelet aggregation during the observation period. Postoperatively, platelet count recovered more rapidly in the PEG-rHuMGDF group, but did not exceed the preoperative counts during the observation period and therein mitigate some concern that may be raised based on Schmuziger and associates’ study [20]. The dose and timing of administration of PEG-rHuMGDF was based on a previous in vivo study by Lott and associates [11] and by mathematical modeling of the pharmacokinetic and pharmacodynamic effect of PEG-rHuMGDF. Lott and associates’ study [11] indicated that administration of rHuMGDF in rabbits at the dose of 1.0 µg or 10 µg · kg-1 · day-1 (L. Roskos, personal communication) increased platelet counts significantly on the fourth day of treatment [21]. This suggests that at least 4 days are needed for rHuMGDF to stimulate significant formation of platelets from megakarocytes to increase circulating platelet population. The platelets generated under rHuMGDF stimulation are mature and functionally competent [11]. In pilot studies, administration of PEG-rHuMGDF 4 days preoperatively caused platelet population to increase, which was not observed when treatment was initiated 3 days before the operation. It is not known whether a preoperative increase in platelets would alter the extent of intraoperative thrombocytopenia induced by HEC or would cause an overshoot postoperatively.
Polyethylene glycol derived rHuMGDF augments agonist-induced platelet aggregation in a concentration-dependent manner, but has no agonist activity itself at the concentrations used. Because the c-mpl receptor is localized on the platelet membrane [8], exogenous c-mpl ligand in the form of PEG-rHuMGDF produces tyrosine phosphorylation of platelet proteins with apparent molecular weights of 85 kDa (c-mpl itself) and 130 kDa (Jak2), and these phosphorylated proteins appear to convey an intraplatelet "signal" that may augment functional responses [13]. A plasma rHuMGDF concentration of 10 ng/mL or greater is necessary to enhance agonist-induced platelet aggregation [19]. However, in the present study, the preoperative PEG-rHuMGDF plasma concentration was less than 3 ng/mL, which is below the concentration eliciting increased aggregation responses. Accordingly, agonist-induced platelet aggregation in the canine model was not different in the PEG-rHuMGDF group compared with the placebo group. If PEG-rHuMGDF had augmented platelet responses to in vivo triggers, the exposure of circulating platelets to foreign surfaces might cause platelet aggregation during extracorporeal circulation, resulting in more severe thrombocytopenia; however, this was not observed. In fact, intraoperative platelet counts were 46 ± 8 x 103/µL for the PEG-rHuMGDF group versus 29 ± 5 x 103/µL for placebo. Harker and colleagues [15] reported that the terminal half-life for PEG-rHuMGDF is 21.5 ± 10 hours in baboons. In the present study, plasma levels of c-mpl ligand (including PEG-rHuMGDF) were normalized 24 hours postoperatively. These data showed that our strategic administration of 10 µg · kg-1 · day-1 PEG-rHuMGDF starting 4 days preoperatively with a 24-hour drug-free interval immediately before operation is appropriate.
We examined the cannulation sites in the femoral artery and vein for the presence of histologically evident microthrombi. The endothelium of these vessels was damaged and denuded as a result of cannulation and therefore these sites were thrombogenic, possibly because of loss of endothelium-derived antiplatelet factors. The present study showed that the incidence of microthrombi in arterial and venous lumena at cannulation sites was comparable between groups, indicating that the degree of difference in platelet count observed and the lack of functional alteration did not induce excessive rates of microthrombus. Before PEG-rHuMGDF is considered for clinical trials in the perioperative setting, further data should be obtained in other models in which vascular injury may stimulate in vivo platelet aggregation. These models of vascular endothelial injury include surgical revascularization after coronary artery occlusion [22, 23] or surgical anastomosis of venous or arterial vascular grafts.
Evaluation of the thrombotic consequences associated with the administration of PEG-rHuMGDF is of potential clinical relevance because older atherosclerotic patients undergoing operations may be at risk of the development of thromboembolism, especially when other prothrombotic cofactors are present. These include hyperlipidemia, hypertension, and diabetes mellitus, which are associated with endothelial dysfunction and have been shown to alter thrombocytic responses to extracorporeal circulation and cardiac operations [24, 25].
In summary, we report in the present study that rHuMGDF, administered preoperatively in a strategic time frame to avoid preoperative increased platelet counts, counteracted thrombocytopenia without altering platelet function in the days after HEC. This study, which was designed to test the concept of countering post–cardiopulmonary bypass thrombocytopenia, used a model of partial bypass and moderate hypothermia to achieve transient and moderate (although biphasic) thrombocytopenia. However, the concept of treatment with strategic administration of PEG-rHuMGDF needs to be tested in a more clinically relevant model simulating a cardiac operation in which prolonged total cardiopulmonary bypass, profound cardiac hypothermia, and ischemia-reperfusion may trigger severe thrombocytopenia, which is manifested during the early postoperative period. In clinical cardiac surgery, postoperative blood loss and platelet dysfunction are significant after hypothermic cardiopulmonary bypass [4], and may be counteracted by an appropriate pretreatment with PEG-rHuMGDF. The clinical relevance of effective use of PEG-rHuMGDF is in the avoidance of postbypass thrombocytopenia and the need for platelet transfusions or other blood-based products.
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Acknowledgments
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We are grateful for the technical contributions of Steven T. Shearer and for the assistance of Gail H. Nechtman in preparing the manuscript. The Cardiothoracic Research Laboratory is indebted to the Carlyle Fraser Heart Center of Crawford Long Hospital, Emory University for their continued support.
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Footnotes
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This work was supported by a grant from Amgen, Inc, Thousand Oaks, CA. Doctor Toombs is an employee of Amgen, Inc.
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References
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Abstract
Introduction
