Despite the recent additions of novel anticoagulant drugs, heparin remains the mainstay anticoagulant therapy for most cardiac and vascular procedures because it is rapidly reversible with protamine sulfate. Protamine, an arginine-rich basic cationic protein derived from salmon sperm, has high affinity for negatively charged sulfated glycosaminoglycans, including heparin.1,2 Paradoxically, since 1937, protamine has been reported to have anticoagulant activity.3 Previous studies have demonstrated that protamine exerts dose-dependent anticoagulant effects in vitro4–6 and in vivo.7–9 These studies showed that excess protamine inhibits the coagulation cascade, possibly involving different serine proteases5,10 and also affects platelet function.4,6,7,11 The anticoagulant activity of protamine has been attributed to inhibition of contact-activated and tissue factor–induced coagulation.5,9,12,13 Taken together, in addition to the well-known hemostatic action of protamine in reversing heparin-induced anticoagulation, excess protamine can potentially enhance bleeding tendency. However, our knowledge of the mechanism and management of protamine-induced impairment of thrombin generation is rather limited. We hypothesized that (i) protamine limits the propagation of thrombin generation by inhibiting prothrombinase (factor Xa/factor Va complex) or tenase (factor IXa/factor VIIIa complex) and (ii) its anticoagulant effect could be reversed by increasing either extrinsic or intrinsic coagulation factors. Thus, we conducted prothrombin time (PT) and diluted Russell's viper venom time (dRVV) measurements, thrombin generation assay, and thromboelastometry to evaluate the anticoagulant mechanism of protamine (Fig. 1, A–C) and to evaluate the effects of different hemostatic drugs to overcome its anticoagulant activity.
After the institutional approval and informed written consent, blood samples (20 mL per subject) were obtained from 6 healthy volunteers (2 women and 4 men, ages 27–62 years) who had not received any drugs in the preceding 2 weeks and had no history of coagulopathy. Blood samples were collected into the 5-mL Vacutainer tubes (Beckton-Dickinson, Franklin Lakes, New Jersey) containing 3.2% sodium citrate and used immediately for thromboelastometric experiments or centrifuged either for 10 minutes at 150g to obtain baseline platelet-rich plasma or for 20 minutes at 2000g to obtain baseline platelet-poor plasma. The platelet count and hemoglobin in collected blood samples ranged from 174 to 256 × 103/μL and from 13.9 g/L to 17.2 g/L, respectively. Platelet count of platelet-rich plasma was adjusted to 50, 100, and 200 × 103 platelets/μL with autologous platelet-poor plasma using the AcT10 Coulter counter (Beckman Coulter, Miami, Florida). All drugs used in the experiments were freshly prepared and, when appropriate, diluted in saline. The volumes of drugs added to platelet-poor or platelet-rich plasma, and whole blood samples were kept to a minimum and resulted in <2% dilution.
Measurements of PT and dRVVT
For PT measurements, the maximum time allowed by the instrument's program was set to 120 seconds and for dRVVT measurements to 400 seconds. RVV is snake venom that directly activates factor X (Fig. 1B). Commercial dRVVT screen/confirm kits (Diagnostica Stago, Parsippany, New Jersey) were used. The dRVVT screen is prolonged when the lupus anticoagulant interferes with the phospholipids that promote clotting. If protamine were to interfere with phospholipids, dRVVT confirm should be normalized similar to lupus anticoagulants in the presence of excess phospholipids.14
Increasing concentrations of protamine (Abraxis, Schaumburg, Illinois; 0, 4, 8, 12, and 24 μg/mL; corresponding to 0 to 5.3 μM final concentration) were added to 360 μL of freshly isolated platelet-poor plasma and to plasma containing the lupus anticoagulants (George King Biomedical Inc., Overland Park, Kansas). Fifty-microliter aliquots were transferred to disposable cuvettes (Diagnostica Stago, Parsippany, New Jersey). After addition of appropriate PT or dRVVT reagents and preincubation at 37°C, samples were run in duplicate using the STart® 4 instrument (Diagnostica Stago, Parsippany, New Jersey).
Thrombin Generation Assay
The calibrated automated thrombin generation system (Thrombinoscope™, Synapse BV, Maastricht, The Netherlands) measures the onset and the amount of thrombin generation according to fluorescent changes produced by the hydrolysis of a fluorogenic peptide that acts as a substrate for thrombin (Fig. 1C). The effects of protamine on thrombin generation were evaluated in platelet-poor and platelet-rich plasma samples. Samples were run in duplicate according to the method of Hemker et al.15 Briefly, to each well of a 96-well microtiter plate (Microfluor black, ThermoLabsystems, Franklin, Massachusetts), we added 80 μL of platelet-poor or platelet-rich plasma adjusted to 50, 100, and 200 × 103 platelets/μL. Platelet-poor plasma samples were spiked with increasing concentrations of protamine sulfate, 0, 4, 8, 12, and 24 μg/mL, and platelet-rich plasma samples with only the highest protamine concentration. To investigate the reversibility of protamine effects, we also added recombinant factor VIIa (NovoSeven®, NovoNordisk A/S, Bagsbaerd, Denmark; final concentration, 3.0 μg/mL corresponding to 60 nM) or purified factor VIII/von Willebrand factor concentrate (Humate P®, CSL Behring, Marburg, Germany; final concentration, 0.5–3.0 U/mL) to some platelet-poor plasma samples containing 24 μg/mL protamine. Because the effects of factor VIII are better delineated under low tissue factor simulation, we used both 2 and 5 pM tissue factor as triggers. On the basis of the proposed inhibitory activity of protamine on factor V activation,16 we also evaluated hemostatic effects of factor VIII/vonWillebrand factor concentrate in factor V–deficient plasma (FV Enzyme Research Laboratories, South Bend, Indiana). Calibrator wells, in which 20 μL thrombin calibrator (Diagnostica Stago, Parsippany, New Jersey) was added to 80 μL plasma samples, were run in parallel for each plasma sample. Thrombin generation was triggered with 20 μL tissue factor–based platelet-poor plasma reagent (2 and 5 pM tissue factor) or dilute actin FS (dilution 1:20 v/v, Dade Behring, Marburg, Germany) in platelet-poor plasma samples or with platelet-rich plasma reagent (1 pM tissue factor without phospholipids; Diagnostica Stago, Parsippany, New Jersey) in platelet-rich plasma samples. The 2 pM tissue factor reagent was prepared by mixing appropriate volumes of low platelet-poor plasma reagent (1 pM tissue factor) with 5 pM tissue factor. The reaction was started by adding 20 μL/well of CaCl2-subtrate buffer. The fluorescence signals were continuously monitored for 70 to 90 minutes with the fluorescence reader (Fluoroscan Ascent, 390/460 nm excitation/emission wavelengths; Thermo Labsystems, Franklin, Massachusetts). A dedicated software program (Thrombinoscope™, Synapse BV, Maastricht, The Netherlands) was used to record the experiments, and for the calculation of the thrombin generation parameters (lag time, time to peak, and peak thrombin level). In addition, we calculated the average rate of thrombin generation (in nanomolars per minute),17,18 assuming a linear thrombin increase over the period during which thrombin generation is increasing using the following simplified formula: thrombin generation rate = peak thrombin level/(time to peak − lag time).
Thromboelastometry (ROTEM™; Pentapharm, Munich, Germany) measures the viscoelastic clot development from thrombin-mediated fibrin polymerization and platelet activation (Fig. 1C). The ROTEM™ measurement is characterized by specific variables reflecting the rate and extent of clot growth, as has been previously described.19,20 We collected the following variables: (i) coagulation time (CT; in seconds), which corresponds to the onset of clot formation; (ii) angle (α; in degrees), which reflects the rate of fibrin polymerization; and (iii) maximal clot firmness (MCF; in millimeters), which refers to the maximal amplitude of the tracing and reflects the tensile strength of clot. Thromboelastometric tracing was allowed to proceed for at least 30 minutes at a temperature of 37°C. Platelet-poor plasma and whole blood samples were spiked with protamine to get final concentrations of 0, 4, 12, and 24 μg/mL. ROTEM™ analyses were conducted with 300 μL of platelet-poor plasma and 20 μL of 0.2 M CaCl2 using tissue factor (2 μL of EXTEM®; Pentapharm, Munich, Germany) or kaolin (Hemoscope Corporation, Niles, Illinois) as a trigger or with 300 μL of whole blood using thrombin 2 nM (Recothrom®, ZymoGenetics, Seattle, Washington) as a trigger. In addition, we tested the effects of factor VIII/von Willebrand factor concentrate (0.5 and 1.5 U/mL) and recombinant factor VIIa (60 nM) in platelet-poor plasma using kaolin activation.
All experiments had n = 6 per condition, because this number of experiments is typically required to obtain a β ≥0.8 and an α ≤0.05 for most variables in thrombin generation and thromboelastometric experiments, as has been demonstrated in previous in vitro investigations.9,19,20 All data including percentage changes in comparison with baseline were expressed as mean ± SD. Paired data were compared by a 2-sided paired t test. Serial data for increasing protamine concentrations or different activators at the same protamine concentration were evaluated by the analysis of variance (ANOVA) for repeated measurements followed by the 2-sided paired t test with the Bonferroni correction (SPSS® version 16.0, SPSS Inc., Chicago, Illinois). A P value < 0.05 was considered significant for all statistical calculations.
With increasing concentrations of protamine, up to 24 μg/mL, added to normal plasma, PT and dRVVT screen increased from 13.0 ± 0.2 to 15.1 ± 0.3 seconds (P = 0.002), and from 35.1 ± 0.1 to 54.2 ± 0.2 seconds (P < 0.001), respectively. Adding exogenous phospholipids (dRVVT confirm) caused no changes of dRVVT in protamine-treated plasma, but it normalized dRVVT in lupus plasma (Table 1). The dRVVT confirm result in lupus plasma did not return to normal when protamine was present at 24 μg/mL.
Thrombin Generation Assay
In platelet-poor plasma, increasing concentrations of protamine prolonged lag time and time to thrombin peak and decreased peak thrombin in a concentration-dependent manner (Figs. 2 and 3A-B). At 24 μg/mL of protamine in comparison with control, peak thrombin generation decreased by 78% ± 8% and 82% ± 7% after 5 pM tissue factor and actin activation, respectively. Peak thrombin generation at 24 μg/mL of protamine decreased by 87% ± 7%, using 2 pM tissue factor as a trigger (Table 2). Lag times became prolonged by 110% ± 16% and 350% ± 24% after 5 pM tissue factor and actin activation, respectively. In parallel, the calculated rate of thrombin generation decreased protamine concentration dependently from 172 ± 54 to 25 ± 11 nM/min after 5 pM tissue factor activation (a decrease of 87% ± 5%, P < 0.001) and from 258 ± 45 nM/min to 32 ± 12 nM/min after actin activation (a decrease of 87% ± 6%, P < 0.001) (Fig. 4). In platelet-rich plasma with 50, 100, and 200 × 103 platelets/μL, protamine at a concentration of 24 μg/mL had no effect on peak thrombin generation, but significantly prolonged the lag time by 56% ± 7%, 48% ± 6%, and 31% ± 5%, respectively (Table 3). The decrease in the rate of thrombin generation in platelet-rich plasma samples supplemented with 24 μg/mL protamine was significantly affected only when the platelet count was decreased from 200 × 103 platelets/μL to 50 × 103 platelets/μL (15.0 ± 2.0 nM/min vs. 4.9 ± 0.7 nM/min; P < 0.001).
In platelet-poor plasma spiked with protamine 24 μg/mL, increasing concentrations of factor VIII/von Willebrand factor significantly increased peak thrombin regardless of the activator (2 and 5 pM tissue factor or actin), but lag time was shortened only after actin activation (Table 2; Fig. 5A). The addition of 60 nM recombinant factor VIIa failed to increase peak thrombin levels in tissue factor–activated plasma spiked with 24 μg/mL of protamine, although recombinant factor VIIa slightly shortened lag time (P > 0.05; Table 2). Similar to factor VIII/von Willebrand factor concentrate, recombinant factor VIIa was partially effective in shortening the lag time of thrombin generation in protamine-supplemented plasma samples using actin as an activator by 27% ± 4% (P = 0.044) (Table 2; Fig. 5B).
In factor V–deficient plasma, no thrombin generation signals could be obtained using tissue factor as a trigger. With actin activation, the addition of increasing concentrations of factor VIII/von Willebrand factor (0.5–3.0 U/mL) increased peak thrombin by 280% ± 36%, decreased lag time by 79% ± 10%, and improved the calculated rate of thrombin generation from 0.3 ± 0.1 nM/min to 21 ± 4 nM/min, a 72-fold increase (Table 2).
In platelet-poor plasma and whole blood, CT was increased by protamine in a concentration-dependent manner but independent of the activator; however, the increase became significant only after kaolin activation (P < 0.001). The highest protamine concentration (24 μg/mL) increased CT by 106% ± 29% and by 92% ± 39% after kaolin activation and tissue factor activation in platelet-poor plasma, respectively, but only by 49% ± 33% after thrombin activation in whole blood (Table 4). Similarly, angle decreased concentration-dependently after kaolin activation in platelet-poor plasma (P < 0.001) but not with tissue factor activation (P = 0.251) or with thrombin activation in whole blood (P = 0.483). MCF was not relevantly affected by increasing protamine concentrations after any activation in platelet-poor plasma and whole blood (Table 4).
The hemostatic effects of factor VIII/von Willebrand factor concentrate or recombinant factor VIIa were tested at 24 μg/mL of protamine in kaolin-activated platelet-poor plasma. CT increased by 106% ± 29% with protamine 24 μg/mL in comparison with control plasma without protamine. In the presence of protamine 24 μg/mL and factor VIII/von Willebrand factor at 0.5 U/mL and 1.5 U/mL, CT increased only by 65% ± 20% and 29% ± 8%, respectively. In the presence of protamine 24 μg/mL and 60 nM of recombinant factor VIIa, CT increased by 33% ± 11%. The angle and MCF values in the presence of factor VIII/von Willebrand factor or recombinant factor VIIa were similar to protamine only–treated samples.
Protamine exerts anticoagulant effects via extrinsic and intrinsic pathways.4,5,7,9,12 We demonstrated that increasing protamine concentrations affected PT/dRVVT, thrombin generation, and thromboelastometric parameters in a protamine concentration–dependent manner after both tissue factor and contact activation (Tables 1, 2, and 4; Figs. 2 –4).
Overall, contact-activated (intrinsic) pathway assays were more strongly affected by protamine than by tissue factor–activated (extrinsic) ones, suggesting inhibition of thrombin-mediated feedback (propagation) reactions. Contact activated tests (i.e., activated partial thromboplastin time, activated clotting time, and INTEM)4,16,21 are often used in clinical situations in which heparin and protamine are administered, because they seem to better reflect the impact of anticoagulants on thrombin-mediated feedback activation of factors V, VIII, and XI.22 Tissue factor–based clotting tests (e.g., PT and EXTEM)21 are less sensitive to protamine because clotting becomes less dependent on thrombin-mediated feedback activation under supraphysiological tissue factor levels. At more physiological levels of tissue factor (≤5 pM), anticoagulant activity of protamine becomes more evident, as is demonstrated in our present thrombin generation experiments (Table 2), and in diluted tissue factor–triggered thromboelastography (1:10,000 dilution).9 Chu et al. demonstrated that protamine inhibits endotoxin-induced tissue factor activity, but not factors VIIa or Xa per se.5 Their cell-based model did not contain prothrombin, factor V, or factor VIII, and thus their results cannot be directly compared with our data. However, in the preliminary experiment using fluorometric assays,23 we also found that protamine did not directly affect factor Xa activity (data not shown). Our findings complement the report by Ni Ainle et al., demonstrating that protamine inhibits factor V activation by factor Xa or thrombin in a concentration-dependent manner.16 The efficiency of thrombin generation decreases because prothrombinase formation is impaired owing to the reduced factor V activation.24
Our results indicate that the presence of platelets affects the anticoagulant activity of protamine using thrombin generation assay in platelet-rich plasma (Table 3) and whole blood thromboelastometry (Table 4). Accordingly, the peak thrombin generation was not affected by protamine, and the delay in lag time of thrombin generation in platelet-rich plasma at 200 × 103 platelets/μL spiked with 24 μg/mL protamine was only prolonged by 30% ± 11% (Table 3) in relation to 110% ± 16% in platelet-poor plasma samples (Table 2). Experiments in platelet-rich plasma were conducted using platelets with recalcification, and therefore it was not possible to determine the influence of protamine on platelet adhesion and aggregation (in anticoagulated samples).4,6,7,11 Platelets presumably overcome protamine anticoagulation by contributing partially activated factor V from α-granule.25 A recent case report suggested that platelet-derived factor Va may compensate for inborn factor V deficiency.26 In agreement, our thrombin generation and thromboelastometry results show that normal platelets partially mitigate protamine anticoagulation in vitro and that the effect is dependent on the platelet count (Tables 3 and 4). It is important to note that most studies investigating the anticoagulant activity of protamine were previously conducted in platelet-poor plasma.4,5,7,12
Although recombinant factor VIIa is a potent activator of factor X, particularly in the presence of tissue factor,27 the extent of factor Xa generation cannot simply be translated into the increase in thrombin generation.23 In the absence of thrombin-activated factor Va, factor Xa is inefficient in converting prothrombin to thrombin and is more susceptible to antithrombin inhibition. Accordingly, adding recombinant factor VIIa only shortened the lag time, but it did not improve peak thrombin generation in protamine-treated plasma after actin activation (Table 2). Interestingly, Parker et al. previously demonstrated that prothrombin and factor X are extensively (>90%) removed from plasma when exposed to protamine-immobilized Sepharose matrix. It was speculated that protamine directly binds to the γ-carboxyglutamic acid domain of vitamin K–dependent factors, which is necessary for Ca2+-mediated platelet membrane binding.13 However, it is unlikely that protamine directly interacts with factor VIIa because recombinant factor VIIa (60 nM) did not result in a relevant recovery of thrombin generation (Table 2; Fig. 5B). In contrast, increasing factor VIII/von Willebrand factor levels partially reversed decreased peak thrombin generation in protamine-treated plasma and factor V–deficient plasma (Table 2; Fig. 5A). Increased factor VIII levels28 or activated platelets29,30 enhance the intrinsic pathway of coagulation. Activated platelets also provide glycoprotein Ib for von Willebrand factor binding.11 We speculate that adding factor VIII/von Willebrand factor concentrate increased activity of intrinsic tenase (factor IXa/factor VIIIa complex), thereby compensating for low factor Va activity associated with excess protamine.16 It seems unlikely that von Willebrand factor influenced thrombin generation experiments in platelet-poor plasma.31
We also tested the possibility of a direct protamine interaction with the negatively charged phospholipids membranes surface using dRVVTs. If coagulation factors could not efficiently access phospholipids surface because of occupation by cationic protamine, thrombin generation would be reduced. However, unlike lupus anticoagulant, protamine activity was not reversed after adding excess phospholipids in the dRVVT confirm assay (Table 1).
Although adverse reactions to protamine are frequently reported, including anaphylaxis, acute pulmonary vasoconstriction, right heart failure, and hypotension,32 protamine sulfate has been widely used for years and is still the drug of choice for reversal of heparin anticoagulation. However, excessive protamine administration has been associated with anticoagulant activity,4,5,7,12 which is considered a possible cause for bleeding after cardiac surgery.33,34 Furthermore, protamine was reported to increase fibrinolytic tendency.9 We speculate that lower peak thrombin levels due to protamine decrease the activation of thrombin-activatable fibrinolysis inhibitor, making fibrin clot susceptible to fibrinolysis.23,35
The findings in our study have important clinical implications. Despite the availability of protamine titration tests,34 they are still infrequently used in clinical practice. Protamine overdose is not uncommon because prolonged activated clotting time values are often attributed to residual heparin effects, and additional protamine is given.4,21 Protamine is unlikely to improve activated clotting time values affected by hemodilution, coagulation factor(s) depletion, thrombocytopenia, or hypothermia. The concentration range of protamine (4–24 μg/mL) used in our study seems reasonable to simulate excess protamine in a clinical setting and is in agreement with previous studies investigating the anticoagulant effect of this drug.9,16 A single dose of 250 mg protamine leads to plasma concentrations between 10 and 60 μg/mL in patients previously anticoagulated with heparin, and 30 to 40 mg protamine in healthy volunteers without preceding heparin achieves plasma levels of up to 5 μg/mL.36,37 Although protamine elimination is rapid,36,37 excess protamine may exacerbate bleeding tendency in patients with reduced factor V, factor VIII, or platelet activity, as is shown by our data. Indeed, Despotis et al.38showed that after cardiac surgery, factor V activity was particularly low in the subgroup of patients with increased bleeding (factor V 26% vs. 42% in nonbleeding control). In contrast, plasma factor VIII levels are relatively well maintained during and after cardiopulmonary bypass.39,40 However, factor VIII may be reduced in certain patients because of hemodilution, blood type O (lower factor VIII activity in relation to non-O types), or colloid usage.41 Differences in factor VIII concentrations and platelet count as well as platelet dysfunction and heterogeneities in prothrombinase complex binding on platelets surface42 may be partly responsible for variable effects of protamine excess in clinical practice.
Our experimental data indicate that excess protamine effects can be partially reduced by measures to increase plasma factor VIII levels. The use of 1-deamino-8-D-arginine vasopressin (DDAVP) or purified factor VIII/von Willebrand factor concentrate can pharmacologically increase factor VIII/von Willebrand factor to enhance thrombin generation in platelet-rich plasma.31 Because the therapeutic response to DDAVP may not be predictable in part owing to simultaneous release of tissue plasminogen activator (thus promoting fibrinolysis),43 and because of endogenous vasopressin release by surgical stress, purified factor VIII/von Willebrand factor concentrate (40–80 U/mL factor VIII activity and 72–224 U/mL von Willebrand factor–Ristocetin cofactor activity for Humate P® according to manufacturer's specifications) may be more titratable for hemostasis.44 Cryoprecipitate—which contains concentrated fibrinogen, von Willebrand factor, and factor VIII (minimum 70 U factor VIII/mL)—should also be useful when a low fibrinogen state coexists.45
Our study has several limitations. First, several studies have shown an impairment of platelet function by heparin/protamine complexes or by protamine alone.4,6,7,11 These experiments require anticoagulation (of blood) or separation of platelets, but our study was primarily focused on protamine effects on thrombin generation in plasma. Second, functional platelets from healthy volunteers were shown to support thrombin generation in the presence of protamine; however, our findings cannot be extended to clinical patients who are being treated with potent platelet inhibitors.46 Third, in vivo hemostatic effects of recombinant factor VIIa or factor VIII/von Willebrand factor concentrate cannot be fully predicted from our in vitro data. Platelets can potentially affect the efficacy of hemostatic interventions, including recombinant factor VIIa and factor VIII/von Willebrand factor, but our experiments were not designed to evaluate such interactions. Finally, an increased sample size may be necessary to determine the hemostatic effects of recombinant factor VIIa and factor VIII/von Willebrand factor distinctly in clinical samples, especially on thromboelastometry.
In conclusion, we demonstrated in plasma and whole blood that protamine mainly affects the propagation phase of thrombin generation, which can be mitigated in the presence of platelets or increased factor VIII/von Willebrand factor concentrations. An enhanced extrinsic pathway using recombinant factor VIIa did not result in the full recovery of thrombin generation. Our present data suggest that protamine overdose can potentially increase bleeding risks in case of severe thrombocytopenia or low factor VIII, but it seems to be a heterogeneous phenomenon influenced by factor V, factor VIII, or platelet activity.
Jerrold H. Levy is Section Editor of Hemostasis and Transfusion Medicine for the Journal. This manuscript was handled by Martin J. London, Section Editor of Perioperative Echocardiography and Cardiovascular Education, and Dr. Levy was not involved in any way with the editorial process or decision.
This study was in part supported by a research grant from CSL Behring (Marburg, Germany). Daniel Bolliger also serves as the principal investigator in an investigator-initiated study (NCT-00805051) supported by a research grant from CSL Behring, Switzerland. CSL Behring had no role in the design and conduct of this investigator-initiated study, or in the collection, management, analysis, and interpretation of the data, or in the preparation, review, and approval of the manuscript. There is no conflict of interest to declare.
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