Although cardiac surgery patients are at risk for hemorrhagic complications such as blood loss and transfusion (1,2), they are, paradoxically, also at risk for thrombotic complications, including coronary and cerebral ischemia (3–7). After cardiopulmonary bypass (CPB), a prothrombotic state exists because of activation of the coagulation, complement, and inflammatory pathways (8–12). Yet, little is known about the genetic variants that contribute to hypercoagulable states, or clinically apparent thrombotic events (13).
The protein C system represents an important endogenous anticoagulant pathway (14). Protein C is converted to activated protein C (aPC) by the thrombin-thrombomodulin complex on endothelium. aPC, in the presence of its cofactor protein S, serves many antiinflammatory, anticoagulant, and profibrinolytic functions, one of which is to down-regulate coagulation activity by inactivating factors Va and VIIIa (14). Resistance to aPC is quantified by the relative inability of aPC to prolong a standard clotting time (15). Decreased plasma levels of protein C, protein S, or resistance to aPC are recognized prothrombotic phenotypes (14,16). Many acquired conditions produce aPC resistance (15,17–21); these include pregnancy, acute phase inflammation, critical illness, increased factor VIII:C levels, and antiphospholipid antibodies. Resistance to aPC can also be hereditary, most often caused by a mutation in the factor V gene known as factor V Leiden (FVL). Resistance to aPC in FVL carriers arises from loss of an aPC cleavage site at amino acid 506 of the factor V protein (22,23).
Relationships between abnormalities in the protein C pathway, such as low plasma aPC levels or aPC resistance, and postoperative complications such as impaired myocardial performance (24) and vein graft occlusion (25) have been suggested by previous authors. Furthermore, aprotinin produces aPC resistance, which has been thought to increase the risk for thrombosis in patients with congenital aPC resistance attributed to FVL (20). Recently, FVL has been associated with decreased postoperative blood loss and risk of transfusion after cardiac surgery (26), again suggesting postoperative hypercoagulability in FVL carriers. Despite these findings, the thrombotic risk of FVL in cardiac surgery remains unclear. The purpose of the current report is to test the hypothesis that FVL is associated with a relatively hypercoagulable postoperative state, as indicated by aPC resistance measurements.
The Vanderbilt Cardiac Surgery Registry is a genetic and clinical data repository for patients undergoing cardiac surgery. Patients are enrolled in the registry under informed consent procedures overseen and approved by the IRB. DNA and plasma samples are then drawn during surgery and clinical data are gathered from chart review after discharge. From a 205-patient subset of the registry, for whom perioperative plasma and DNA were available, and CPB was used, we identified 8 subjects heterozygous for FVL, and defined this group as group L (Leiden). For each FVL subject, we identified at least 1, but as many as 3 noncarrier controls from the registry, matched as closely as possible for aprotinin use, age, sex, and surgical procedure; this group was identified as group MC (matched controls, 18 subjects). To account for possible bias caused by incomplete matching, we also formed a group of unmatched controls (group UC, 11 subjects) by identifying consecutive subjects in the registry, without regard to age, sex, surgical procedure, or other variables.
Perioperative management was conducted as per institutional standard practice, with oral anticoagulants held for 3–5 days before the operation. Anesthesia was achieved with commonly used anesthetics (etomidate, thiopental, isoflurane, pancuronium, fentanyl). Porcine heparin was used for anticoagulation at an initial dose of 400 U/kg supplemented to maintain a kaolin activated clotting time of >400 s. Moderate systemic hypothermia (28°–31°C) and cold retrograde and antegrade cardioplegia solution were applied to all patients. Cardiotomy suction was used to remove excessive blood from the surgical field during the period of heparinization. Heparin was neutralized with protamine after separation from CPB, at an initial dose of 250 mg and an additional 50 mg if activated clotting time remained >140 s. Aprotinin was administered in half-Hammersmith doses to all patients undergoing repeat sternotomy procedures. (The only patient undergoing primary sternotomy and receiving aprotinin was a patient in group L with an atrial germ cell tumor and dense adhesions throughout the pericardium.) All care providers were unaware of the subjects’ individual FVL genotypes.
Blood was drawn for DNA isolation and storage through the arterial line placed at the time of anesthesia induction. DNA was isolated using standard approaches, and stored at 4°C until needed. The FVL mutation was detected using the MnlI restriction fragment length polymorphism as described by Ridker et al. (27). Blood was drawn for plasma isolation into 4.5-mL citrated tubes at 3 time points: before surgical incision (baseline), 10 min after protamine (off CPB), and on postoperative day 1 (POD 1, 24 h after intensive care unit admission). Plasma was separated from cells by centrifugation at 2500g for 15 min at 4°C, and stored at −80°C until analysis.
Resistance to aPC was measured using the commercial assay provided by Chromogenix (Coatest®) as follows: test plasma (50 μL) and an equal volume of contact activator (phospholipid with colloidal silica) were heated in duplicate to 37°C for 3 min. Coagulation was then started by addition of 50 μL of 25 mM CaCl2, and the time to clot formation recorded using a Diagnostica Stago STart-4 coagulation analyzer. A second coagulation time was then recorded, in duplicate, in a similar manner but in the presence of aPC. The ratio of the clotting time in the presence of aPC to the clotting time in the absence of aPC was defined as the aPC ratio. A normal aPC ratio for this assay is >2.0, with aPC ratios <2.0 defined as resistance to aPC (28). The assay was conducted in the presence of hexadimethrine bromide to neutralize the effect of residual heparin.
Differences in aPC ratio across the three time points (baseline, postprotamine, and on POD 1) were tested using analysis of variance, with Dunnett’s test for post hoc comparisons. Comparisons were made with the baseline aPC ratio serving as control. Change in aPC ratio from baseline was compared between FVL subjects and controls using Student’s t-test for independent measures. Comparisons in baseline variables were made using analysis of variance or χ2 where appropriate. Comparisons in outcome variables were made using χ2 for discrete data, and Kruskal-Wallis test for continuous data because of the highly non-Gaussian nature of the distributions. SPSS statistical software (version 11.0; SPSS, Inc., Chicago, IL) was used for all statistical analyses.
A summary of the subjects is shown in (Table 1. Trends toward statistical significance were observed for history of venous thrombosis or pulmonary embolism, and oral anticoagulant use for the FVL subjects, reflecting the increased risk for thrombotic complications in this population 29). Some FVL subjects were matched to more control subjects because their age groups and surgical procedures were more common. In group L, two patients underwent atrial tumor excision. Because these cases were not found in controls, these subjects were matched with controls undergoing atrial septal aneurysm repair, partial anomalous pulmonary venous return repairs, and atrial septal defect closures. A variety of other procedures was performed in addition to the more common valve and coronary procedures; these are listed in (Table 1.
Results from the aPC resistance assays performed on group MC are shown in Figure 1A. A significant effect of cardiac surgery on aPC resistance was observed: subjects showed normal response to aPC at baseline and 10 min after protamine, but became increasingly sensitive to aPC on POD 1 (P = 0.007). Group UC subjects (Fig. 1B) showed similar results, where a normal aPC ratio was observed at baseline and 10 min after protamine, but subjects became more sensitive to aPC on POD 1 (P = 0.021). Data from group L are shown in Figure 1C. Group L subjects exhibited aPC resistance at baseline, and this resistance did not change significantly across the perioperative period (P = 0.867). Overall, the noncarrier control subjects showed a mean increase in aPC ratio from baseline of 0.368 on POD 1, whereas the FVL subjects showed an increase in aPC ratio of only 0.066 in the aPC ratio from baseline to POD 1 (P = 0.002).
Outcome data for the 3 groups are listed in Table 2. There were no differences in quantity of blood loss, incidence of reoperation for bleed, number of blood products transfused, or incidence of blood product transfusion before the blood draw on POD 1.
In this study, we observed that patients without the FVL mutation, undergoing cardiac surgery with CPB, demonstrated an increase in the aPC ratio on POD 1. These findings indicate increased sensitivity to aPC that would promote inhibition of coagulation. In contrast, in patients heterozygous for FVL, aPC sensitivity was unchanged on the day after cardiac surgery, indicating persistent aPC resistance. These data describe a potentially important difference in perioperative coagulation profiles between FVL subjects and noncarriers.
The aPC system represents an essential endogenous regulator of coagulation and inflammation. As mentioned, the anticoagulant effect of aPC has been well characterized and involves inactivation of factors Va and VIIIa 14). Antiinflammatory effects of aPC include decreasing cytokine production and inhibition of leukocyte rolling (14,30). Recent evidence indicates that aPC has antiapoptotic effects on ischemic endothelial cells, and neuroprotective activity in vivo (31). In cardiac surgery, authors have reported correlations between the ratio of aPC to total protein C in coronary sinus blood and postoperative myocardial performance (24), suggesting an important role of aPC in suppressing myocardial ischemia-reperfusion injury.
The mechanism by which FVL noncarriers become increasingly sensitive to aPC after CPB is unclear. CPB is associated with an acute inflammatory response (10), which is typically associated with resistance to aPC (15). Significant dilution of coagulation factors to less than half of their baseline levels occurs during CPB, especially for factors V and VIII:C (32). This is potentially significant because increased factor VIII:C levels are associated with aPC resistance and decreased levels are associated with aPC sensitivity (15). After CPB, patients with FVL may have more normal postoperative levels of factor VIII:C and other coagulation factors, because they tend to bleed less and require less transfusion (26), but in the current set of patients there were no significant differences in blood loss or transfusion. It is unclear why the FVL subjects remained resistant to aPC and did not show any significant change. FVL subjects typically have aPC ratios <2.0, but aPC ratio and thrombosis risk can be modified by other genetic or environmental factors (15,33–35).
The presence of FVL or resistance to aPC still carries an uncertain level of risk in cardiac surgery. Authors have suggested that early coronary graft occlusion may be linked to FVL (25). This association is supported by data from animal vein graft models (36), in which impaired protein C activation renders vein grafts thrombogenic. However, data addressing risks of FVL in cardiac surgery are limited and inconclusive. Moor et al. (25) reported early graft occlusion in 45% of FVL carriers and 20% of controls, a finding of borderline significance because of small sample size. Central venous thrombosis may be associated with FVL (37,38) but, similar to the previous example, conclusive data are lacking because of small sample size and lack of studies in adults. Authors have speculated that aprotinin may be thrombogenic in the presence of FVL (20), an assertion currently lacking confirmation. Data presented here provide a plausible mechanism for increased postoperative thrombotic risk in FVL carriers, but specific perioperative risks associated with FVL await conclusive evidence.
In summary, we present data showing that FVL noncarriers demonstrate an increased sensitivity to aPC after cardiac surgery, whereas patients with FVL display a baseline resistance to aPC that is unchanged in the first postoperative 24 hours. These data lend support to previous speculations regarding postoperative hypercoagulability in FVL subjects.
The author gratefully acknowledges contributions of research assistants Rand S. Valery, BS, and Gwen Wissel, BA (Department of Anesthesiology, Vanderbilt University, Nashville, TN), for their fine technical and informatics assistance. The Vanderbilt University Program in Human Genetics DNA Resource Core also provided technical assistance for this study.
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