Ann Thorac Surg 2006;81:1720-1727
© 2006 The Society of Thoracic Surgeons
Original article: Cardiovascular
Protamine Enhances Fibrinolysis by Decreasing Clot Strength: Role of Tissue Factor-Initiated Thrombin Generation
Vance G. Nielsen, MD
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Department of Anesthesiology, The University of Alabama at Birmingham, Birmingham, Alabama
Accepted for publication December 7, 2005.
* Address correspondence to Dr Nielsen, Department of Anesthesiology, The University of Alabama at Birmingham, 619 South 19th Street, Birmingham, AL 35249-6810 (Email: vnielsen{at}uab.edu).
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Abstract
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BACKGROUND: Excessive protamine administration to neutralize heparin after cardiopulmonary bypass has been implicated as a cause of postoperative hemorrhage. Protamine directly inhibits thrombin and tissue factor (TF)-mediated activation of factor VII. However, the half-life of protamine is only 4.5 minutes; thus the purpose of this study was to determine if protamine could enhance fibrinolysis, explaining the delayed, protamine-associated hemorrhage observed in the postoperative period.
METHODS: Human plasma containing 0, 6.25, 12.5, or 25 µg/mL of protamine (n = 6 per condition) was exposed to 0.01% tissue factor and tissue-type plasminogen activator (tPA, 100 U/mL) for 30 minutes, with clot growth and disintegration measured by Thromboelastograph (Haemoscope Corp, Skokie, IL). The TF was increased to 0.1% in additional experiments with plasma containing protamine (25 µg/mL) and tPA.
RESULTS: Protamine significantly (p < 0.05) delayed the time to clot initiation, decreased the speed of clot propagation, and diminished clot strength in a concentration-dependent fashion. The onset of fibrinolysis was significantly (p < 0.05) increased only in samples with 25 µg/mL of protamine, and the rate of clot lysis was not different among the conditions. The clot duration time (from initiation to disintegration) was significantly (p < 0.05) decreased in a concentration-dependent manner by protamine. Increased TF concentration (0.1%) significantly improved clot growth kinetics and prolonged clot duration in samples with 25 µg/mL of protamine compared with samples activated with 0.01% TF.
CONCLUSIONS: Protamine enhanced fibrinolysis by decreasing clot strength by diminishing TF-initiated thrombin generation. Additional, clinical investigation is warranted to mechanistically implicate protamine-mediated enhancement of fibrinolysis to delayed bleeding after cardiopulmonary bypass.
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Introduction
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Protamine administration to neutralize heparin-mediated enhancement of antithrombin activity in the setting of cardiac surgery has long been implicated as a potential factor contributing to perioperative hemorrhage [1–4]. The tacit assumption underlying this contention has been that excessive protamine administration may lead to significant, free circulating protamine concentrations not bound to heparin. In turn, free protamine may directly inhibit thrombin [5], inhibit tissue factor (TF)-mediated activation of factor VII [6] and diminish platelet function [7–9]. However, the circulating half-life of protamine in healthy volunteers [10] and in cardiac patients undergoing cardiopulmonary bypass [11] has been found to be 7.4 minutes and 4.5 minutes, respectively. Given the brief period of protamine administration and small circulating half-life, it was difficult to mechanistically link protamine-mediated inhibition of clot formation with well-documented postoperative bleeding attributed to excessive protamine administration.
Another, up to this time unexplored, hypothesis concerning protamine-associated hemorrhage would be that protamine could enhance fibrinolysis. If free protamine was incorporated into clot during heparin neutralization and in some way accelerated fibrinolysis (eg, increase plasmin activation, inhibit thrombin generation, decrease factor XIII [FXIII]-mediated fibrin cross-linking), then the brief circulating half-life of protamine would no longer teleologically confound the observation that excess protamine administration resulted in delayed bleeding after cardiac surgery. Thus, the purpose of the present study was to determine if protamine could enhance fibrinolysis with a recently described plasma-based in vitro model of fixed fibrinolytic stress wherein clot growth and disintegration kinetics are determined by Thromboelastograph (TEG; Haemoscope Corp, Skokie, IL) [12].
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Material and Methods
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Plasma, unlike whole blood from volunteers, is devoid of individual hemostatic variation mediated by platelets. As the precise activities of individual procoagulants and anticoagulants are known, normal, pooled plasma is used as a standard for most hematologic analyses performed in clinical laboratories. Finally, as the products are commercially available, are noncellular, and cannot be linked to individual donors, institutional ethical approval is not required as per the guidelines of the National Institutes of Health.
Protamine-Mediated Effects on Fibrinolysis After Tissue Factor Activation
Experimentation utilized one lot of pooled, control plasma (George King Bio-Medical, Overland Park, KS) anticoagulated with sodium citrate. The plasma had normal procoagulant enzyme activities (98–118% normal values), 302 mg/dL fibrinogen, 106% normal plasminogen activity, and less than 0.5 µg/mL D-dimer. Experiments with plasma had all conditions represented with n = 6, as this number of experiments is required to obtain a ß greater than or equal to 0.8 with an 
less than 0.05 for most thromboelastographic variables, as demonstrated in previous in vitro investigation [12, 13]. The final volume for all subsequently described plasma sample mixtures was 360 µL. Sample composition consisted of the following: 310 µL of plasma; 10 µL of tissue-type plasminogen activator (tPA, 580 U/µg; Genentech, Inc, San Francisco, CA) diluted with 50 mM potassium phosphate buffer (pH 7.4) for a final activity of 100 U/mL; 10 µL tissue factor (TF) (rabbit brain TF, international sensitivity index 1.1, Trinity Biotech, Ventura, CA) for a final concentration of 0.01%; 10 µL 0.9% NaCl containing protamine (0, 6.25, 12.5, or 25 µg/mL final concentration; corresponding to a 0, 1.35, 2.7, and 5.4 µM concentration, respectively); and 20 µL 200 mM CaCl2. Protamine concentrations of 6.25, 12.5, and 25 µg/mL correspond to the expected unbound protamine concentrations associated with protamine to heparin reversal ratios of 1.04:1, 1.08:1, and 1.15:1, respectively [7]. Additional samples containing 12.5 µg/mL of protamine had 1 U activated factor XIIIa (FXIIIa) added within the 10 µL 0.9% NaCl component of the sample mixture. The concentration of TF and activity of tissue type plasminogen activator (tPA) was chosen as they provided consistent clot growth [12, 13] and disintegration [12] within a 30 minute observation period.
Protamine-Mediated Effects on Coagulation After Celite Activation in FXIII-Deficient and Control Plasma
In order to quantify the TF-independent, direct antithrombin effects of protamine on coagulation without FXIII-mediated effects, FXIII-deficient plasma (George King Bio-Medical) anticoagulated with sodium citrate was exposed to 0, 25, or 50 µg/mL protamine. This plasma was obtained from one congenitally FXIII-deficient donor. These samples consisted of 320 µL FXIII-deficient plasma, 10 µL 1% celite (0.28 mg/mL final concentration), 10 µL 0.9% NaCl containing protamine, and 20 µL 200 mM CaCl2. To quantify the TF-independent, direct antithrombin effects of protamine on coagulation with normal FXIII activity, another lot of pooled, control plasma (George King Bio-Medical) anticoagulated with sodium citrate was utilized. The plasma had normal procoagulant enzyme activities (95–111% normal values), 280 mg/dL fibrinogen, 100% normal plasminogen activity, and less than 0.5 µg/mL D-dimer. Control plasma samples consisted of 320 µL plasma, 10 µL 1% celite (0.28 mg/mL final concentration), 10 µL 0.9% NaCl containing 0, 25, or 50 µg/mL protamine, and 20 µL 200 mM CaCl2.
Protamine-Mediated Effects on Coagulation After Activation With 0.01% and 0.1% TF in Control Plasma
To quantify the summation of protamine-mediated inhibition of TF-dependent FVII activation and inhibition of thrombin on coagulation, pooled, control plasma (George King Bio-Medical) anticoagulated with sodium citrate was utilized. The plasma lot utilized was the same one mentioned in the experiments involving celite activation. The samples consisted of 320 µL plasma, 10 µL TF (0.01 or 0.1% final concentration), 10 µL 0.9% NaCl containing 0 or 50 µg/mL protamine, and 20 µL 200 mM CaCl2.
Protamine-Mediated Effects on Fibrinolysis After Activation With 0.01% and 0.1% TF in Control Plasma
To quantify the summation of protamine-mediated inhibition of TF-dependent FVII activation and inhibition of thrombin on fibrinolysis, pooled, control plasma (George King Bio-Medical) anticoagulated with sodium citrate was utilized. The plasma lot utilized was the same one mentioned in the experiments involving celite activation. The samples consisted of 310 µL plasma, 10 µL of tPA (Genentech, Inc., San Francisco, CA) diluted with 50 mM potassium phosphate buffer (pH 7.4) for a final activity of 100 U/mL, 10 µL TF (0.01 or 0.1% final concentration), 10 µL 0.9% NaCl containing 0 or 25 µg/mL protamine, and 20 µL 200 mM CaCl2.
Thromboelastographic Analyses
Plasma sample mixtures were placed in a disposable cup in a computer-controlled Thromboelastograph (TEG) hemostasis system (Model 5000, Haemoscope Corp, Skokie, IL), with addition of CaCl2 as the last step to initiate clotting. A detailed description of thromboelastography has been previously described [12, 13]. The following standard variables were determined at 37°C: reaction time (R, time to 2 mm of amplitude, the time to clot initiation, seconds); angle (, a measure of the speed of clot strength, degrees); maximum amplitude (MA, a measure of clot strength, mm), clot growth time (CGT, time from R to MA in seconds); clot lysis time (CLT, time from MA to 2 mm amplitude or the end of observation in seconds); and clot duration time (CDT, the sum of CGT and CLT). Additional elastic modulus-based parameters previously described [12] were determined. These parameters used to quantify the velocity of clot growth and disintegration are based on change in elastic modulus (G, dynes/cm2) determined by changes in amplitude (A), expressed by the following equation:
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The various time periods and phases of clot growth and disintegration are subsequently grounded in how G changes over time. The nomenclature used to describe these phenomena is as follows:
Time to Maximum Rate of Thrombus Generation (TMG). This is the time interval (seconds) observed prior to maximum speed of clot growth.
Maximum Rate of Thrombus Generation (MTG). This is the maximum velocity of clot growth observed (dynes/cm2/second).
Total Thrombus Generation (TTG). This is the total area under the velocity curve during clot growth (dynes/cm2), representing the amount of clot strength generated during clot growth.
Time to Maximum Rate of Lysis (TML). This is the time interval (seconds) measured from the beginning of the observation period to the time when the velocity of clot disintegration is maximal. In anticipation of delayed clot initiation secondary to protamine-mediated thrombin inhibition, an R-adjusted TML (TMLR) was calculated as the time from maximum clot strength to maximum velocity of clot disintegration (TMLR = TML – (R+CGT)).
Maximum Rate of Lysis (MRL). This is the maximum velocity of clot disintegration observed (-dynes/cm2/second).
Area of Clot Lysis (ACL). This is the total area under the velocity curve during clot disintegration (-dynes/cm2), representing the amount of clot strength lost during clot disintegration.
These parameters are depicted in Figure 1, with a corresponding thromboelastogram also presented within the graphic. All data were collected for 30 minutes and analyzed with Version 4.2 TEG software (Haemoscope). Plasma samples exposed to fibrinolytic stress were observed for 30 minutes, whereas samples without exposure to tPA were observed until a stable MA was observed for 3 minutes (determined by TEG software) or for a maximum of 30 minutes.
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Fig 1. Elastic modulus-based and time-based thromboelastograph variables of clot growth and disintegration. A clot growth-disintegration velocity curve and corresponding thromboelastogram are depicted. (ACL = area of clot lysis [-dynes/cm2]; CDT = clot duration time [CGT+CLT, seconds]; CGT = clot growth time [seconds]; CLT = clot lysis time [seconds]; MRL = maximum rate of lysis [-dynes/cm2/second]; MTG = maximum rate of thrombus generation [dynes/cm2/second]; TMG = time to maximum rate of thrombus generation [seconds]; TML = time to maximum rate of lysis [seconds]; TTG = total thrombus generation [dynes/cm2]).
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Statistical Analyses
Analyses of the effects of protamine on thromboelastographic variables in the presence of constant fibrinolytic stress were conducted with one-way analysis of variance with the Holm-Sidak post hoc test for multiple comparisons (SigmaStat 3.0, SPSS Inc, Chicago, IL). Analyses of the effects of FXIIIa on clot growth and disintegration were performed with a 2-tailed Student t test. Analyses of the effects of protamine on thromboelastographic variables in plasma activated with celite without fibrinolytic stress were conducted with one-way analysis of variance with the Holm-Sidak post hoc test for multiple comparisons. Analyses of the effects of protamine on thromboelastographic variables in plasma activated with TF without fibrinolytic stress were conducted with Kruskal-Wallis one-way analysis of variance with the Student-Newman-Keuls post hoc test for multiple comparisons. Nonparametric statistics were used for this data set as protamine exposure resulted in a cessation of coagulation and consequent marked differences in variance between the conditions tested (eg, R values of 1,800 seconds [30 minutes of recording]). Graphic representation of the data was generated with commercially available software (Origin 7.5, OriginLab Corp, Northampton, MA). Values were expressed as mean ± SD or median (first–third quartiles) as appropriate. A p value of less than 0.05 was considered significant.
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Results
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Protamine-Mediated Effects on Fibrinolysis After Tissue Factor Activation on Standard and Time-Based Variables During Fibrinolysis
As anticipated, protamine increased the time to clot initiation, decreased the speed of clot formation, and decreased clot strength as indicated by changes in R, , and MA, respectively (Table 1). Clot growth time was significantly decreased by 25 µg/mL of protamine compared with all other concentrations, whereas CLT was significantly decreased between 12.5 and 25 µg/mL compared with 0 to 6.25 µg/mL concentrations. Clot duration time was significantly decreased in a concentration-dependent fashion throughout the range of protamine concentrations tested. Compared with samples exposed to 12.5 µg/mL of protamine, samples exposed to 12.5 µg/mL of protamine and FXIIIa had significantly greater MA, CGT, and CDT values. Representative thromboelastograms with corresponding clot growth-disintegration velocity curves of the thromboelastographic concentration-response to protamine are depicted in Figure 2.
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Fig 2. Representative thromboelastograms and clot growth-disintegration velocity curves of the effects of protamine on fibrinolysis. All plasma samples were exposed to 100 U/mL tissue type plasminogen activator. Data collection occurred over a 30 minute period. (A) and (B) are the thromboelastogram and velocity curve, respectively, of a plasma sample with no protamine added. (C) and (D) are the thromboelastogram and velocity curve of a plasma sample with 25 µg/mL of protamine.
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Protamine-Mediated Effects on Fibrinolysis After Tissue Factor Activation on Elastic Modulus-Based Thromboelastographic Variables During Fibrinolysis
Protamine significantly increased TMG, decreased MTG, and decreased TTG in a concentration-dependent manner (Table 2). As anticipated, protamine considerably, albeit paradoxically, significantly increased the time to maximum rate of lysis. However, TMLR, accounting for changes in clot initiation, demonstrated that samples exposed to 25 µg/mL of protamine had significantly smaller values than all other concentrations tested. With regard to the rate of fibrinolysis, there were no significant differences in MRL between the concentrations of protamine tested. However, there was a significant, concentration-dependent decrease in ACL that mirrored the changes observed in TTG. Last, compared with samples exposed to 12.5 µg/mL of protamine, samples exposed to 12.5 µg/mL of protamine and FXIIIa had significantly greater TTG and ACL values.
Protamine-Mediated Effects on Coagulation After Celite Activation in FXIII-Deficient and Normal Plasma
FXIII-deficient, celite activated plasma exposed to protamine (25–50 µg/mL) had significantly prolonged clot initiation, decreased clot propagation, and diminished clot strength compared with samples not exposed to protamine (Table 3). However, there were no significant differences in clot kinetics between the two protamine concentrations. Similarly, normal plasma exposed to protamine (25–50 µg/mL) had had significantly prolonged clot initiation and decreased clot propagation. However, in contrast to FXIII-deficient plasma, normal plasma exposed to protamine had no significant decreases in clot strength compared with normal plasma not exposed to protamine. Representative thromboelastograms with corresponding clot growth velocity curves of the thromboelastographic concentration-response to protamine in FXIII-deficient and normal plasma activated by celite are depicted in Figure 3, panels (A)–(D).
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Table 3. Effects of Protamine on Standard and Elastic Modulus-Based Thromboelastographic Variables on Normal and Factor XIII (FXIII)-Deficient Plasma Without Fibrinolysis
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Fig 3. Effects of protamine on factor XIII (FXIII)-deficient and normal plasma after celite or tissue factor activation. Representative thromboelastograms with corresponding clot growth velocity curves are displayed for each condition. (A) Celite activated FXIII-deficient plasma, no protamine. (B) Celite-activated FXIII-deficient plasma, 50 µg/mL of protamine. (C) Celite-activated normal plasma, no protamine. (D) Celite-activated normal plasma, 50 µg/mL of protamine. (E) Tissue factor (0.01%) activated normal plasma, no protamine. (F) Tissue factor (0.01%) activated normal plasma, 50 µg/mL of protamine.
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Protamine-Mediated Effects on Coagulation After TF Activation in Normal Plasma
Normal plasma activated with 0.01% TF exposed to protamine 50 µg/mL had clot initiation, propagation, and strength significantly reduced compared with normal plasma not exposed to protamine (Table 4). Notably, there was essentially no coagulation in TF-activated plasma exposed to protamine 50 µg/mL. However, when the concentration of TF was increased to 0.1%, plasma samples exposed to 50 µg/mL of protamine had significantly improved clot initiation, propagation, and strength compared with samples activated with 0.01% TF. Lastly, samples activated with 0.1% TF exposed to protamine had a significantly faster clot initiation time but still diminished propagation and strength compared with plasma not exposed to protamine. Representative thromboelastograms with corresponding clot growth velocity curves of the thromboelastographic concentration-response to protamine in normal plasma activated by 0.01% TF are depicted in Figure 3, panels (E) and (F).
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Table 4. Effects of Tissue Factor (TF) on Protamine-Mediated Alterations of Standard and Elastic Modulus-Based Thromboelastographic Variables in Normal Plasma Without Fibrinolysis
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Protamine-Mediated Effects on Fibrinolysis After Activation with 0.01% and 0.1% TF
Normal plasma exposed to fibrinolytic stress and activated with 0.01% TF demonstrated significant prolongation of clot initiation, decreased propagation, and decreased strength after exposure to protamine 25 µg/mL, just as in the first series of experiments (Table 5). Further, clot lysis time and clot duration time were significantly decreased by protamine, as demonstrated previously. However, when TF concentrations were increased to 0.1% in samples with protamine 25 µg/mL, clot initiation, propagation, and strength were significantly improved compared with plasma activated with 0.01% TF, with or without protamine exposure. Protamine significantly decreased the maximum rate of clot lysis, and increasing TF activation from 0.01 to 0.1% significantly increased MRL in samples exposed to protamine compared with both conditions with activation with TF 0.01%. Nevertheless, samples activated with 0.1% TF with protamine 25 µg/mL had significantly prolonged CLT and CDT compared with samples activated with 0.01% TF with protamine 25 µg/mL. Lastly, samples activated with 0.1% TF with protamine 25 µg/mL had CLT and CDT values that were not different from samples activated with 0.01% TF without protamine. Representative thromboelastograms with corresponding clot growth-disintegration velocity curves of the thromboelastographic concentration-response to protamine and variable TF activation are depicted in Figure 4.
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Fig 4. Effects of increasing tissue factor concentration on protamine-mediated enhancement of fibrinolysis. Representative thromboelastograms with corresponding clot growth velocity curves are displayed for each condition. All plasma samples were exposed to 100 U/mL tissue type plasminogen activator. (A) Tissue factor (0.01%) activated normal plasma. (B) Tissue factor (0.01%) activated normal plasma, 25 µg/mL of protamine. (C) Tissue factor (0.1%) activated normal plasma, 25 µg/mL of protamine.
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Comment
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The principle finding of this investigation was that clinically encountered protamine concentrations significantly delayed clot initiation, decreased the speed of clot growth, decreased final clot strength, and decreased the time required for disintegration in a fibrinolytic environment. The most likely mechanism for protamine-mediated enhancement of fibrinolysis is the combination of decreased thrombin generation by inhibition of TF-mediated FVII activation and subsequently decreased thrombin generation [6] coupled with direct inhibition of thrombin with protamine [5]. Further, protamine may diminish FXIIIa-mediated 2-antiplasmin-fibrin bonding by diminishing FXIII activation, as FXIIIa addition did not delay the onset of fibrinolysis as has been demonstrated previously [12]. The consequent decrease in thrombin-mediated fibrin polymer formation and FXIII activation results in a weak, easily disintegrated clot. The relationships of protamine-mediated attenuation of thrombin generation and thrombin inhibition are depicted in Figure 5. While direct inhibition of thrombin by protamine plays a role in decreasing clot formation kinetics [5], a 10 µM concentration of protamine (50 µg/mL = 10.8 µM) results in a less than 5% reduction in thrombin activity based on fibrin clot formation or amidolytic assay [5]. In contrast, 2 µM protamine significantly prolongs prothrombin time, and 5 µM and 10 µM protamine diminishes TF-initiated clot formation by approximately 50% and 90%, respectively [6]. These findings [5, 6] are consistent with the data of the present study; specifically, celite-activated FXIII-deficient plasma (reflective of thrombin-fibrinogen interactions) had a modest decrease in clot formation kinetics in response to protamine exposure, and celite-activated normal plasma (reflective of thrombin-fibrinogen-FXIII interactions) demonstrated no significant decrease in clot strength after protamine exposure. However, TF-initiated coagulation was essentially extinguished by 50 µg/mL protamine, while increasing TF concentrations tenfold partially (50 µg/mL protamine) or completely (20 µg/mL protamine) restored clot formation kinetics in the absence or presence of fibrinolytic stress, respectively. In sum, while protamine-mediated direct inhibition of thrombin may play a role, the inhibition of TF-initiated thrombin formation and subsequently weakened clot formation is the major mechanism by which protamine exposure results in a clot that is more vulnerable to fibrinolytic attack.
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Fig 5. Mechanism by which protamine decreases clot strength and enhances fibrinolysis. Protamine inhibits tissue factor (TF) mediated activation of factor (F) VII, which in turn decreases activation of factor X and prothrombin, and ultimately decreases thrombin generation. Further, protamine inhibits thrombin-mediated fibrinogen polymerization and factor XIII activation, decreasing fibrin polymer formation and cross-linking that subsequently decreases clot strength.
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The concern that excessive protamine administration is a significant source of postoperative bleeding after cardiopulmonary bypass is long standing [1–4], but the precise mechanisms by which protamine administration increases hemorrhage remains to be determined. Given the small circulating half-life of 4.5 minutes in cardiac patients [11], one is pressed to implicate this mechanism as a significant source of postoperative bleeding during the ensuing hours in the intensive care unit. Nevertheless, Despotis and colleagues [14] demonstrated that a reduction in protamine to heparin ratios resulted in a decrease in bleeding and a reduction in the administration of platelets, fresh-frozen plasma, and cryoprecipitate. Paradoxically, Teoh and colleagues [15] demonstrated that postoperative protamine infusions (25 mg/hour) resulted in a significant decrease in heparin rebound and decreased postoperative bleeding by 13%, 12 hours postoperatively. One way to resolve these potentially contradictory investigations is to consider the various time courses of both protamine administration and the onset of coagulation (and fibrinolysis) in the immediate and postoperative periods after cardiopulmonary bypass. First, protamine administration should not be considered as just a "protamine to heparin ratio"; rather, the rate of administration and total dose should be contemplated. If administered at an ideal rate, very little unbound protamine will exist in the circulation beyond that required to neutralize heparin. However, if administered too rapidly or if the amount of protamine administered is excessive, free protamine could be incorporated into forming a mediastinal clot, perhaps enhancing immediate bleeding or resulting in enhanced fibrinolysis, explaining accelerated clot disintegration in the immediate postoperative period. Further, if adequate hemostasis is obtained with an appropriate rate of administration, then an infusion of protamine at a very slow rate in the postoperative period would likely not affect the established clot, and would be expected to diminish unwanted anticoagulation by heparin rebound [15]. In sum, perhaps the difficulty in establishing a consistent hemostatic outcome with various protamine to heparin ratios may be secondary to our incomplete knowledge of the complex interactions of cardiopulmonary bypass and surgical trespass with the coagulation system, resulting in an inability to optimize protamine administration.
This investigation does have several limitations and its results should not be immediately extrapolated to clinical situations. First, it is a "proof of concept" study, designed with a precise in vitro approach to identify potential protamine-mediated effects on fibrinolysis in normal, pooled plasma, without the confounding effects of individual patient variability or platelet-mediated effects on hemostasis. Further, it is likely that blood samples obtained during protamine administration may provide different, site-specific responses to a fixed fibrinolytic stimulus (eg, blood in the mediastinum may have greater plasminogen activator activity than systemic blood). Indeed, markers of fibrinolysis such as plasmin-2-antiplasmin complexes and D-dimers were found to be fourfold to 14-fold greater, respectively, in the mediastinal cavity compared with the systemic circulation [16]. Finally, the activity of tPA utilized (100 U/mL) was chosen to obtain consistent clot lysis in a period of time that would allow one to obtain data potentially useful at the bedside and was not intended to simulate a clinically encountered scenario. Nevertheless, the data of the present study serve as the rational basis to link excessive protamine administration to fibrinolysis-associated hemorrhage in future, prospective investigations.
In conclusion, the present investigation demonstrated that protamine concentrations clinically encountered (1.35–5.4 µM) significantly enhanced fibrinolysis primarily by the inhibition of TF-initiated thrombin generation. Additional clinical investigation is warranted to mechanistically implicate protamine-mediated enhancement of fibrinolysis to delayed bleeding after cardiopulmonary bypass.
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Acknowledgments
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This investigation was supported by a grant from the Department of Anesthesiology, University of Alabama at Birmingham.
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References
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Abstract
Introduction
