Protamine sulfate is the agent most frequently used to reverse heparin-mediated anticoagulation, serving as a therapeutic “crouching clot” in vascular and cardiothoracic surgical settings for decades. However, protamine has also been posited to be a source of coagulopathy, or “hidden hemorrhage” for more than 20 years.1 Protamine has been demonstrated in vitro to decrease thrombin activity,2 decrease activation of factor VII by tissue factor,3,4 enhance tissue-type plasminogen activator– mediated fibrinolysis,4 decrease factor V activation by activated factor X or thrombin,5 and attenuate platelet function by inhibition of glycoprotein Ib–von Willebrand factor activity.6 Concordantly, in vivo protamine-mediated anticoagulation without identification of a specific mechanism(s) has been demonstrated in canine7 and murine8 models. Although these investigations support the concept of a protamine-mediated coagulopathy, and editorials urge careful administration of protamine with concurrent monitoring of heparin presence,9 a definitive study implicating protamine as a source of post–cardiopulmonary bypass bleeding has yet to be performed.
In this issue of Anesthesia & Analgesia, Bolliger et al.10 present us with a multimodal approach to further define the relative hierarchy of the hematological mechanisms that underlie protamine-mediated attenuation of hemostasis. This well-established group utilized standard methods (e.g., prothrombin time) combined with thrombinoscopy (a highly sensitive measure of thrombin generation) and thromboelastometry to test blood obtained from normal subjects and various other factor-deficient plasmas exposed to a range of clinically encountered11,12 protamine concentrations. First, the protamine-mediated decrease in thrombin generation was diminished by increasing platelet count from 50,000 to 200,000 platelets/μL. Furthermore, experiments in platelet poor plasma supported the concept that protamine inhibited the intrinsic pathway, specifically factor V activation as previously reported,5 based on the ability of exogenous factor VIII concentrate to restore thrombin generation in protamine-exposed samples of normal and factor V–deficient plasma.10 The authors did not find a similar response when recombinant, activated factor VII was added. Thus, it appeared that protamine inhibited the intrinsic (or contact protein) coagulation pathway of coagulation to a greater extent than the extrinsic (or tissue factor-factor VII–dependent) coagulation pathway.
Although these conclusions seemed quite reasonable, there existed a body of work that demonstrated that inhibition of tissue factor–mediated activation of factor VII by protamine was far more important than inhibition of the intrinsic pathway in platelet-poor plasma.4 In this investigation, one of the present authors (VGN) demonstrated a nearly complete cessation of tissue factor–initiated coagulation by protamine, whereas the same concentration only delayed the onset of nearly normal coagulation in intrinsic pathway samples as determined by thrombelastography.4 Furthermore, increasing tissue factor concentration completely restored both clot growth and resistance to fibrinolysis parameter values in protamine-exposed samples compared with unexposed plasma.4 During review, Bolliger et al. were of the opinion that small concentrations of tissue factor were used by Nielsen,4 perhaps in the range of 0.6 pM. Such small concentrations of tissue factor were thought to be easily inhibited by protamine, whereas the large concentrations of tissue factor (2–5 pM) used by Bolliger et al. would not be inhibited by protamine, thus the observations by Bolliger et al. would primarily be secondary to inhibition of factor V. However, it should be noted that the thrombelastographic response in plasma has already been described with up to 45 pM tissue factor.13 The growth kinetics of these tissue factor–activated plasma clots were actually slower than those published in the work concerning protamine-mediated inhibition of coagulation,4 supporting the notion that protamine inhibited clot formation with a concentration >45 pM, much larger than 0.6 pM. Furthermore, thrombelastographic experiments defining the coagulation kinetic profiles of factor-deficient plasma demonstrated decreased coagulation in factor V–deficient plasma after activation with similar tissue factor concentrations,14 a sharp contrast to the essential cessation of thrombin generation in factor V–deficient plasma in response to 5 pM tissue factor noted by Bolliger et al.10 Taken as a whole, it seems that whereas Bolliger et al. have demonstrated inhibition primarily of the intrinsic pathway, Nielsen's data support the concept that the extrinsic pathway is more inhibited by protamine. Whether the differences between these 2 bodies of work are based on differing techniques (thrombinoscopy, a more sensitive method to detect thrombin generation than thrombelastography), differing tissue factor concentrations, differing efficiencies of factor VII or recombinant factor VII, different plasma samples (e.g., normal subjects versus pooled normal plasma), or other unidentified variables remains to be determined.
So how do any of these investigations help us clinically? Does it really matter which pathway is most affected? What should we do if requested by surgical colleagues to administer “just a little more” protamine, such as 50 mg IV, in response to possible heparin-mediated bleeding? Given the works of Butterworth et al.,11,12 it may be possible to reach circulating protamine concentrations of 5 to 10 μg/mL with such a bolus, depending on the patient's weight and residual circulating heparin concentrations. Such concentrations have been demonstrated in vitro to adversely affect clot growth and enhance fibrinolysis,4 so it is possible that such cavalier administration of protamine may exacerbate both early and delayed (fibrinolytic) bleeding. Although not yet experimentally proven, the following factors may put a patient at risk for protamine-mediated bleeding:
- Prolonged duration of cardiopulmonary bypass (consumption of clotting factors and platelets)
- Important additions (blood, colloid, crystalloid) to perfusate during cardiopulmonary bypass, with consequent hemodilution
- Rapid speed of administration of protamine
- Excessive dose of protamine
- Administration of a second dose of protamine without important circulating heparin concentration
The common thread to all of these potential risk factors is that an operative wound will be presented with circulating blood with decreased thrombin-generating potential secondary to platelet/clotting factor deficiency and/or excessive protamine concentrations. So how do we transform the “hidden hemorrhage” into the “crouching clot”? First, as previously discussed,9 careful administration of protamine with heparin monitoring would be ideal to prevent what is still only a potential contributing factor to post–cardiopulmonary bypass coagulopathy. Second, the maintenance of adequate plasma coagulation function (factor VII, factor V activity) and functional platelets will likely diminish protamine-mediated coagulopathy. The administration of protein concentrates, such as factor VIII, utilized by Bolliger et al.,10 may indeed improve clinical bleeding after cardiac surgery. However, until we are able to clinically detect protamine excess as a source of bleeding in individual patients and better define the mechanisms by which protamine decreases hemostasis in vivo, we will continue to view the clinical effects of protamine administration as a friendly clot more so than a menacing hemorrhage.
Both authors helped to analyze the data and write the manuscript and approved the final manuscript.
1. Gravlee GP, Haddon WS, Rothberger HK, Mills SA, Rogers AT, Bean VE, Buss DH, Prough DS, Cordell AR. Heparin dosing and monitoring for cardiopulmonary bypass: a comparison of techniques with measurement of subclinical plasma coagulation. J Thorac Cardiovasc Surg 1990;99:518–27
2. Cobel-Geard RJ, Hassouna HI. Interaction of protamine sulfate with thrombin. Am J Hematol 1983;14:227–33
3. Chu AJ, Wang Z, Raicu M, Beydoun S, Ramos N. Protamine inhibits tissue factor-initiated extrinsic coagulation. Br J Haematol 2001;115:392–9
4. Nielsen VG. Protamine enhances fibrinolysis by decreasing clot strength: role of tissue factor-initiated thrombin generation. Ann Thorac Surg 2006;81:1720–7
5. Ni Ainle F, Preston RJS, Jenkins PV, Nel HJ, Johnson JA, Smith OP, White B, Fallon PG, O'Donnell JS. Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation. Blood 2009;114:1658–65
6. Barstad RM, Stephens RW, Hamers MJ, Sakariassen KS. Protamine sulfate inhibits platelet membrane glycoprotein Ib-von Willebrand factor activity. Thromb Haemost 2000;83:334–7
7. Kresowik TF, Wakefield TW, Fessler RD II, Stanley JC. Anticoagulant effects of protamine sulfate in a canine model. J Surg Res 1988;45:8–14
8. Eslin DE, Zhang C, Samuels KJ, Rauova L, Zhai L, Niewiarowski S, Cines DB, Poncz M, Kowalska MA. Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin. Blood 2004;104:3173–80
9. Levy JH, Tanaka KA. Anticoagulation and reversal paradigms: is too much of a good thing bad?. Anesth Analg 2009;108:692–4
10. Bolliger D, Szlam F, Azran M, Koyama K, Levy JH, Molinaro RJ, Tanaka KA. The anticoagulant effect of protamine sulfate is attenuated in the presence of platelets or elevated factor VIII concentrations. Anesth Analg 2010;111:601–8
11. Butterworth J, Lin YA, Prielipp R, Bennett J, James R. The pharmacokinetics and cardiovascular effects of a single intravenous dose of protamine in normal volunteers. Anesth Analg 2002;94:514–22
12. Butterworth J, Lin YA, Prielipp RC, Bennett J, Hammon JW, James RL. Rapid disappearance of protamine in adults undergoing cardiac operation with cardiopulmonary bypass. Ann Thorac Surg 2002;74:1589–95
13. Nielsen VG, Audu P, Cankovic L, Lyerly RT III, Steenwyk BL, Armstead V, Powell G. Qualitative thrombelastographic detection of tissue factor in human plasma. Anesth Analg 2007; 104:59–64
14. Nielsen VG, Cohen BM, Cohen E. Effects of coagulation factor deficiency on plasma coagulation kinetics determined via Thrombelastography®: critical roles of fibrinogen and factors II, VII, X and XII. Acta Anaesthesiol Scand 2005;49:222–31