Effects of protamine and heparin can be detected and easily differentiated by modified thrombelastography (Rotem Ò ): an in vitro study

Transcription

1 British Journal of Anaesthesia 95 (3): 31 1 (5) doi:1.193/bja/aei197 Advance Access publication July 15, 5 Effects of protamine and heparin can be detected and easily differentiated by modified thrombelastography (Rotem Ò ): an in vitro study M. Mittermayr 1, J. Margreiter 1, C. Velik-Salchner 1, A. Klingler, W. Streif 3, D. Fries 1 and P. Innerhofer 1 1 Clinic for Anaesthesia and Intensive Care Medicine, Division of Theoretical Surgery and 3 Department of Pediatrics, Innsbruck Medical University, Innsbruck, Austria Corresponding author: Clinic for Anaesthesia and Intensive Care Medicine, Innsbruck Medical University, Anichstrasse 35, A- Innsbruck, Austria. markus.mittermayr@uibk.ac.at Background. Precise coagulation monitoring might help prevent heparin protamine mismatch and thus decrease postoperative blood loss. We therefore measured coagulation time (CT) by modified thrombelastography (Rotem Ò ) as a possible differential monitor of the effects of heparin and protamine. Methods. Undiluted and diluted blood samples from healthy volunteers were spiked with increasing concentns of heparin (.1,.,.,. and 1 U ml 1 ). In addition, undiluted blood was spiked with protamine hydrochloride (.1,.,.,. and 1. U ml 1 ), and we tested the effect of protamine on the reversal of heparin. U ml 1. Heparin-containing samples were analysed using the heparin-sensitive test and the heparinase-containing test; protamine series were also analysed with the EXTEM test (tissue factor activation). Results. CT by the test [CT-; median (min/max)] increased significantly and dosedependently with increasing concentns of heparin [control, 175 s (/); heparin, 1. U ml 1 13 s (559/1); P<.1] and protamine [control, 17 s (15/55); protamine, 1. U ml 1 57 s (3/135); P<.1]. Up to heparin concentns of. U ml 1, results were similar in undiluted and diluted blood samples. As expected, CT- remained within the normal range for all tested heparin concentns (median 1 13 s), but increased similarly to CT- for increasing protamine concentns. Conclusion. CT measurement using the Rotem Ò technique appears to be a valuable tool for heparin protamine management. For detection of heparin alone, protamine alone and the two combined, the of CT-:CT- can be used to distinguish the effects of heparin excess (CT-:CT->1) from those of protamine excess (CT-:CT- =1). Br J Anaesth 5; 95: 31 1 Keywords: blood, anticoagulants, heparin; measurement techniques, thrombelastography, Rotem Ò ; protamine Accepted for publication: June 1, 5 Open-heart surgery with cardiopulmonary bypass (CPB) is associated with a number of factors influencing haemostasis that alone and in combination with surgical factors may result in significant postoperative blood loss. Because increased blood loss and transfusion requirement are associated with poor outcome 1 and the therapeutic consequences of surgically- or coagulopathically-induced bleeding are quite different, the immediate and precise diagnosis of the underlying cause of increased bleeding is essential. Coagulopathy following CPB originates from haemodilution, coagulation factor depletion, platelet dysfunction, and activation of the fibrinolytic system. In addition, residual heparin and protamine excess impair the coagulation process and platelet function. The high heparin concentns needed during CPB are commonly monitored by measuring activated clotting time (ACT). However, Murray and colleagues showed that ACT is not very sensitive to the low heparin concentns that might be present after protamine reversal. Moreover, a fixed dose of protamine is Ó The Board of Management and Trustees of the British Journal of Anaesthesia 5. All rights reserved. For Permissions, please journal.permissions@oupjournals.org

2 Effects of protamine/heparin on Rotem Ò measurements usually calculated from the total heparin dose. This is followed by ACT measurement to confirm adequacy of protamine administn. Thus, protamine is frequently administered in repeated doses, although elevated ACT after protamine can be caused by residual heparin, protamine excess or other factors influencing coagulation. 3 Thrombelastographic techniques can be used to determine the dynamics and quality of clot formation in whole blood, and to detect coagulation inhibitors such as heparin. Intrinsic activation plus heparinase neutralization ( test) can be used to identify the presence of heparin and its effects on coagulation by comparison with the intrinsically activated measurement containing no heparinase ( test). 5 In patients after CBP, use of the conventional Thrombelastograph Ò (TEG Ò ) and a modification of it (Rotem Ò ) have been shown to decrease transfusion requirement and facilitate the differentiation of coagulopathic from surgical bleeding. 7 However, in vitro studies using non-activated TEG Ò and human blood spiked with heparin showed that concentns as low as. U ml 1 virtually prevented clot formation, indicating that higher concentns of heparin may not be differentiated from lower concentns of heparin. In addition, few data are available on signs of protamine excess in TEG Ò analysis. The goal of our study was to clarify whether in vitro heparin concentns greater than. U ml 1 can be measured and differentiated using the Rotem Ò -derived measurement of coagulation time (CT). In addition, we wanted to determine whether protamine effects are also detectable, and whether they can be differentiated from heparin effects. Since haemodilution, frequently present in surgical patients, might influence the accuracy of heparin detection, measurements were performed in undiluted and diluted blood spiked with heparin. Methods The study was approved by the Ethics Committee of Innsbruck Medical University and written informed consent for blood withdrawal was given by volunteers ( males, females). Mean age was 3 yr (range 5 7 yr). All volunteers denied taking any medication which might influence coagulation within the last weeks. Blood was drawn without stasis from an antecubital vein through a 19 G butterfly needle and collected in four 1 ml citrate-containing tubes (S-Monovette; Sarstedt, Nümbrecht, Germany) for assessment in four thrombelastograph analysis series. In addition, we performed standard coagulation tests and blood cell counts (prothrombin time; partial thromboplastin time, aptt; concentns of antithrombin, fibrinogen; platelet count, and red blood cell count). Test samples were analysed using modified thrombelastography (Rotem Ò ; Pentapharm, Munich, Germany), which is based on the TEG Ò system, after Hartert, reviewed by Mallet and Cox. 9 Technical details of the Rotem Ò analyser have been described elsewhere. 1 Activated tests accelerate the measurement process and enhance reproducibility compared with conventional TEG Ò analysis of native blood. 11 In addition, activation of test samples also decreases the high sensitivity of native TEG Ò against inhibitors of coagulation, such as heparin. Thus, the probability of discriminating low and moderate heparin concentns might be improved with the Rotem Ò technique. A typical Rotem Ò tracing and its interpretation is given in Figure 1. Undiluted and diluted blood samples containing heparin (with and without protamine) were analysed using the heparin-sensitive test ( ml of CaCl. M, ml of thromboplastin-phospholipid, 3 ml blood) and the test (additional 1 ml heparinase). Samples spiked with protamine alone were analysed using the test and the EXTEM test ( ml ofcacl. M, ml of tissue factor, 3 ml blood), because effects of protamine on aptt and prothrombin time have also been reported. Five series were analysed with the test to confirm that heparinase cannot neutralize protamine effects. All reagents were purchased from Pentapharm, and measurements were performed at 37 C. To investigate the effects of heparin, protamine, both combined and the effects of heparin in % diluted blood with modified 3% gelatin solution (Gelofusin Ò ; Braun, Melsungen, Germany) four test series were performed for each volunteer. Gelatin was used since colloids are known to exert a greater effect on coagulation than do crystalloids and gelatin solution is routinely used in our cardiac patients. Spiked and control aliquots were stored at room temperature for min without agitation and subsequently analysed. To avoid the bias of time effects three Rotem Ò devices were used, each allowing assessment of four measurements and thus parallel assessment of all samples from one series. The Rotem Ò devices used were tested daily for proper function using quality control serum (Pentapharm). To avoid channel bias, the sequence of channels was rotated after each complete measurement series. In addition, the time sequence of measurement series (heparin, protamine, heparin plus protamine, heparin in diluted blood samples) was varied. All measurements were run for at least 5 min. According to previously published thrombelastographic results, it is possible to have undetectable clot formation with increasing heparin concentns. Since minimal clot formation after 35 min is of no clinical relevance, we decided to discontinue measurement when initiation of coagulation, CT time (normal range 1 s) was prolonged to about 1-fold the upper normal value (more than 35 min), and the observation was assigned a CT of 1 s, an a angle of and a maximum clot firmness (MCF) of mm. The five heparin and protamine dilutions tested were prepared fresh daily in sterile containers. The heparin solutions were prepared using.9% sodium chloride from unfractionated porcine heparin (1 IU ml 1 ; Ebewe Pharma, 311

3 Mittermayr et al. 1 min 9 mm mm mm Maximum clot firmness (MCF; MA) (mm) ML Iysis Coagulation time (CT; r) (s) Clot formation time (CFT; k) (s) Fig 1 Variables of Rotem Ò analysis are coagulation time (CT, s), corresponding to the reaction time (r time) in conventional TEG Ò ; clot formation time (CFT, s), corresponding to the coagulation time (k time); maximum clot firmness (MCF, mm), which is equivalent to the maximum amplitude; and the a angle, which measures the speed of clot formation. Maximum lysis (ML, %) is defined as the of the lowest clot strength after reaching MCF, to MCF. CT measurement relies mainly on the concentn of coagulation factors and the presence of inhibitors, and reflects initial thrombin genen and the formation of first trace amounts of fibrin. CFT is defined as the time needed to reach a clot strength of mm and also depends on the concentn of fibrinogen and the numbers and function of platelets. MCF reflects the interaction of fibrinogen/fibrin with platelets and coagulation factor FXIII. Unterach, Austria) and the protamine dilution from Protamin ICN Ò (protamine hydrochloride 1 U ml 1 ; ICN Pharmaceuticals, Frankfurt, Germany). According to the manufacturer s instructions, 1 IU protamine hydrochloride antagonizes 1 IU heparin (1 IU protamine hydrochloride is comparable to.1 mg protamine sulphate). Dilutions were calculated to achieve the final concentns in the various 1 ml aliquots of citrated blood by adding ml of heparin or protamine dilution. For the series measuring combined heparin and protamine, one control aliquot of 1 ml citrated blood was left untreated; heparin was then added to the remaining 9 ml citrated blood at a final concentn of. U ml 1 and 1 ml aliquots were subsequently spiked with protamine. In addition to control measurements, the following final concentns of heparin, protamine or both were investigated using the Rotem Ò analyser: (i) heparin series (heparin.1,.,.,. and 1 U ml 1 ); (ii) heparin in % diluted blood (heparin.1,.,.,. and 1 U ml 1 ); and (iii) protamine series (protamine.1,.,.,. and 1. U ml 1 ); (iv) combined heparin. U ml 1 and protamine series (protamine,.,.,. and 1. U ml 1 ). Statistical methods A non-parametric Friedman analysis of variance was used for the analysis of the influences of heparin and protamine on thrombelastographic variables. For comparison of Rotem Ò results with baseline measurements and between the different investigated concentns of heparin and protamine, the Wilcoxon test was used. All statistical tests were twosided, and P<.5 was considered significant. Results Results of standard coagulation tests and Rotem Ò measurements of control samples were within the normal range in all volunteers (data not shown). Since CT values mainly depend on concentns of coagulation factors and the presence of inhibitors, heparin and protamine effects should be detectable mainly from changes in CT values. Heparin series Compared with control samples, addition of heparin (.1 1. U ml 1 ) resulted in a significant dose-dependent increase in CT- above the upper normal range (P<.1), and all CT values differed significantly from each other (Fig. A). As expected, CT- remained within the normal range and was nearly unchanged for all tested heparin concentns and compared with control. Accordingly, with increasing heparin concentns the CT-:CT- increased significantly (P<.1). Up to a heparin concentn of. U ml 1 a clot was detectable in all samples analysed using the test. At heparin. U ml 1 in / samples and at heparin 1. U ml 1 in / samples no clot was detectable (CT>1 s). Similar results for CT- and CT- were observed when heparin concentns were measured in % diluted blood using a modified gelatin solution (Fig. B). Compared with undiluted blood, CT- and CT- results were comparable up to heparin concentns of. U ml 1. At heparin concentns of. and 1. U ml 1, no clot was detected more frequently in 3

4 Effects of protamine/heparin on Rotem Ò measurements A 5 15 C 1 : Protamine (U ml 1 ) : : Heparin (U ml 1 ) B 5 : 1 D H/P H./P H./P. H./P. H./. H./P1. H/P H./P H./P. H./P. H./. H./P1. Heparin/protamine (U ml 1 ) Heparin-diluted (U ml 1 ) Fig Measurement of coagulation time (CT) in undiluted (A) and % diluted (B) blood samples at various concentns of heparin derived from the test (normal range, 1 s) and the test (left panel). CT- divided by CT- (right panel). Increasing concentns of heparin significantly elevate CT- values in a dose-dependent manner, while CT- remains unchanged. Consequently, the CT-:CT- increases to well above 1 with increasing heparin concentns. Results from undiluted blood were comparable to those from diluted blood up to heparin concentns of. U ml 1. Values are 1th percentile, 5th percentile, median, 75th percentile and 9th percentile. P<.5 compared with baseline; P<.5 between various concentns of heparin. (C) Measurement of coagulation time (CT) at various concentns of protamine derived from the test (normal range, 1 s) and the test (left panel). CT- divided by CT- (right panel). Increasing concentns of protamine significantly elevate CT- values in a dose dependent manner, while CT- increases in parallel. Consequently, the CT-:CT- remains at 1 with increasing protamine concentn. Values are 1th percentile, 5th percentile, median, 75th percentile and 9th percentile. P<.5 compared with baseline; P<.5 between various concentns of protamine. (D) Measurement of coagulation time (CT) at control (H/P), at a fixed dose of heparin. U ml 1 (H./P) and after addition of protamine hydrochloride.,.,., 1. U ml 1 (H./P.; H./P.; H./P.; H./P1.) (1 U protamine hydrochloride neutralizes 1 U heparin) using the and tests (left panel). CT- divided by CT- (right panel). Values are 1th percentile, 5th percentile, median, 75th percentile and 9th percentile. 313

5 Mittermayr et al. diluted samples (/ and 1/ measurements respectively) than in undiluted samples. In order to test the ability of Rotem Ò to detect even small concentns of heparin, we compared the CT- and values of control samples with those spiked with.1 U ml 1 heparin. Assuming that CT- is at least 1% higher than CT- when heparin is present, the CT-:CT- was seen to increase in all samples in the heparin series, meaning that of heparincontaining samples (heparin.1 U ml 1 ) were correctly identified as containing heparin, while 5 of control samples were correctly identified as containing no heparin and one control sample showed a false-positive result of containing heparin. Protamine series Compared with control samples, addition of protamine (.1 1. U ml 1 ) produced a significant dose-dependent increase in CT- (P<.5), while all CT values differed significantly from each other (Fig. C). CT-EXTEM increased inconsistently from 5 s (/5) in control samples to s (/1), 7 s (19/9), s (9/), s (11/39) and s (11/5) at protamine.1,.,.,. and 1. U ml 1 respectively. In contrast to the results for the heparin series, CT- values also increased with increasing protamine concentns, because heparinase cannot neutralize protamine effects (Fig. C). Heparin protamine series As described in Methods, our experiments used protamine hydrochloride; 1 U protamine hydrochloride antagonizes 1 U heparin. As long as heparin concentns were greater than protamine concentns (protamine and. U ml 1 ), CT- values were higher than CT- values, while protamine excess caused a similar increase in CT- and CT- (Fig. D). Other Rotem Ò variables Increasing concentns of heparin resulted in a significant dose-dependent decrease in MCF in the test (P<.1), mostly to values outside the normal range, while variable changes in MCF were observed in the HEP- TEM test and in the test and EXTEM test in the protamine series. These changes were not dose-related and remained mainly within the normal range. In diluted blood samples, MCF ( and ) was already below the low normal range at baseline and changed inconsistently with increasing heparin concentns (Table 1). Measurements of a angle, reflecting the speed of clot formation, changed in a manner similar to those of MCF (data not shown). Discussion The main finding of our study is that the Rotem Ò -derived CT from intrinsically activated tests with () Table 1 Maximum clot firmness (MCF) (normal range 5 73 mm) at various concentns of heparin (in undiluted and diluted blood) and protamine, measured in intrinsically activated tests with () and without () heparinase, or tissue factor activation (EXTEM). Values are median (min/max). P<.5 compared with baseline; P<.5 between concentns MCF- (mm) MCF- (mm) Heparin-undiluted blood Uml (51/) 55. (5/).1 U ml (/3) 5. (/5). U ml (5/3) 5.5 (/3). U ml (1/1) 57. (/5). U ml (/57) 57.5 (7/5) 1. U ml 1. (/5) 55. (/) Heparin-diluted blood Uml 1 3. (7/7) 33. (3/1).1 U ml (9/5) 33. (3/). U ml 1. (33/5) 33. (/). U ml 1 7. (3/53) 33. (/1). U ml (/5) 3. (/) 1. U ml 1. (/39) 3. (/) Protamine MCF- (mm) MCF-EXTEM (mm) Uml (3/5) 5. (/1).1 U ml 1 5. (3/) 5. (3/). U ml (5/1) 55. (7/5). U ml 1 5. (1/1) 57. (7/5). U ml 1 5. (5/) 59. (9/) 1. U ml (1/5) 57.5 (/) and without () heparinase enables precise detection of low to moderate heparin concentns, regardless of whether the blood is diluted. In addition, Rotem Ò measurement permits the detection of protamine effects (alone and in the presence of heparin) on the coagulation system, which can be distinguished from those induced by heparin. Even very low protamine concentns elevate CT- values. However, because the CT-:CT- increases with heparin excess but remains at 1 with protamine excess, and because heparinase cannot neutralize protamine, one can discern whether heparin or protamine is responsible for elevated CT- measurements. Moreover, the increase in CT- was dose-dependent for heparin and protamine. These facts are of clinical importance when deciding whether to adjust the heparin or protamine dose in order to guarantee adequate heparin effects or heparin reversal. Rotem Ò measurement also enables the physician to detect or prevent protamine excess resulting from empirical or ACT-based additional protamine administn. Nielsen 13 also demonstrated the ability of native nonactivated TEG Ò in detecting heparin in blood samples from rabbits receiving heparin stepwise from 1 U kg 1 up to a cumulative dose of 3 U kg 1. In this experiment, TEG Ò variables were more sensitive to changes in heparin activity than were aptt and ACT values. However, the data show that the ability of TEG Ò to discern heparin increases of more than U kg 1 is poor, because no discernible clot can be observed. In the study by Zmuda and colleagues, the in vitro addition of heparin. U ml 1 already abolished clot formation, 3

6 Effects of protamine/heparin on Rotem Ò measurements as measured by non-activated TEG Ò. In contrast, in our study at heparin concentns of. U ml 1 no clot formation occurred in only / samples, whereas this was true for / samples at the heparin concentn of 1 U ml 1. However, it has been shown that thrombelastographic results derived from activated tests demonstrate less variability than those obtained from non-activated native blood samples. 9 In addition, in non-activated TEG Ò, thrombin formation is triggered by the relatively weak contact activation occurring between blood and the plastic surfaces of the pin and cup of the TEG Ò /Rotem Ò analyser. In activated Rotem Ò tests, coagulation is triggered by ellagic acid, an activator of the contact pathway (activation by the factors XII, XI, IX, VIII, X and V). This principle also applies to other activators, such as kaolin and Celite. However, these substances have a much greater tendency to sedimentation and were therefore replaced with ellagic acid in Rotem Ò analysis (communication with Pentapharm). Due to the stronger activation, initiation of coagulation is less sensitive to the presence of inhibitors in activated TEG Ò compared with non-activated TEG Ò, a fact already described by Caprini and colleagues in Protamine binds ionically to the numerous negative charges of heparin. In addition, protamine causes anticoagulant effects that prolong ACT, meaning that ACT measurements can be misleading when deciding whether additional protamine should be used for complete heparin reversal. It is not clear whether this anticoagulant effect of protamine is clinically important and what concentns of protamine are critical for inhibiting coagulation. However, several studies have confirmed the impairment of platelet function in the presence of heparin/protamine complexes or protamine alone Griffin and colleagues also showed that the platelet inhibitory effects of protamine are more pronounced under conditions of high shear stress, and they also observed a prolonged prothrombin time and aptt with increasing protamine concentns. The authors showed that the therapeutic window for protamine is narrower than previously documented, since protamine excess at heparin: protamine s greater than 1:1.5 already contributed directly to platelet dysfunction. In our experiments we also observed that protamine excess prolonged CT values and decreased MCF in the test, which shows good correlation with aptt measurements but changed inconsistently in the tissue-factor-activated EXTEM test. We can only speculate that our observation might be the result of an unspecific, charge-dependent impairment of the amplification step of coagulation by protamine, interfering with activation of factors FVIII and FIX on the surface of activated platelets, which is of minor importance in tests using TF for activation. To our best knowledge, only one study using TEG Ò to monitor the effects of heparin also investigated the effects of protamine by analysing recalcified blood samples without activation. In this study, protamine at.3 mgml 1 showed a similar, albeit incomplete, reversal of heparin-induced prolongation (heparin. or.5 U ml 1 ) of reaction time (with regard to CT) as did the threefold concentn of 5 mg ml 1. However, the protamine concentns tested in this study were both well above a heparin:protamine of 1., meaning that in this study it was mainly the effects of protamine excess that were measured. The present study shows that CT- increases with heparin and protamine. By calculating the CT-:CT-, which increases with heparin excess but remains at 1 with protamine excess, we can distinguish impairment of coagulation caused by heparin or protamine. CT measurements depend also on concentns of coagulation factors that might be critically reduced in a surgical patient. Therefore, it seems necessary to measure CT- before and about 15 min after protamine administn. If CT- is already increased before protamine administn, the influence of coagulation factor depletion on CT measurements needs to be considered. If CT- is prolonged only after protamine administn, the result of CT- and the relation to CT- reflects the effect of excess protamine, because it is unlikely that a patient exhibits coagulation factor depletion during this short period, except in the rare cases of excessive blood loss. The limitation on our study is the fact that we investigated the effects of protamine and heparin on Rotem Ò variables only in vitro. However, it is unlikely that the unspecific binding of heparin to proteins, endothelial cells and other cells or the in vivo pharmacokinetics of heparin and protamine alter the accuracy of this measurement technique in detecting heparin and protamine. In conclusion, we demonstrate here that Rotem Ò analysis can detect heparin with high sensitivity, regardless of whether blood is diluted, and can detect it up to a concentn of 1. U ml 1. We show for the first time that the effect of even low concentns of excess protamine on coagulation can be measured with this technique. Further, the effects of heparin and protamine on coagulation can be clearly distinguished from each other. References 1 Karkouti K, Wijeysundera DN, Yau TM, et al. The independent association of massive blood loss with mortality in cardiac surgery. Transfusion ; : 53 Murray DJ, Brosnahan WJ, Pennell B, et al. Heparin detection by the activated coagulation time: a comparison of the sensitivity of coagulation tests and heparin assays. J Cardiothorac Vasc Anesth 1997; 11: 3 Moorehead MT, Westengard JC, Bull BS. Platelet involvement in the activated coagulation time of heparinized blood. Anesth Analg 19; 3: 39 Tuman KJ, McCarthy RJ, Djuric M, et al. Evaluation of coagulation during cardiopulmonary bypass with a heparinase-modified thromboelastographic assay. J Cardiothorac Vasc Anesth 199; : 9 5 Spiess BD, Wall MH, Gillies BS, et al. A comparison of thromboelastography with heparinase or protamine sulfate added 315

7 Mittermayr et al. in vitro during heparinized cardiopulmonary bypass. Thromb Haemost 1997; 7: Cammerer U, Dietrich W, Rampf T, et al. The predictive value of modified computerized thromboelastography and platelet function analysis for postoperative blood loss in routine cardiac surgery. Anesth Analg 3; 9: Shih RL, Cherng YG, Chao A, et al. Prediction of bleeding diathesis in patients undergoing cardiopulmonary bypass during cardiac surgery: viscoelastic measures versus routine coagulation test. Acta Anaesthesiol Sin 1997; 35: Zmuda K, Neofotistos D, Ts ao CH. Effects of unfractionated heparin, low-molecular-weight heparin, and heparinoid on thromboelastographic assay of blood coagulation. Am J Clin Pathol ; 113: Mallett SV, Cox DJ. Thrombelastography. Br J Anaesth 199; 9: Entholzner EK, Mielke LL, Calatzis AN, et al. Coagulation effects of a recently developed hydroxyethyl starch (HES 13/.) compared to hydroxyethyl starches with higher molecular weight. Acta Anaesthesiol Scand ; : Miller BE, Guzzetta NA, Tosone SR, et al. Rapid evaluation of coagulopathies after cardiopulmonary bypass in children using modified thromboelastography. Anesth Analg ; 9: 13 3 Griffin MJ, Rinder HM, Smith BR et al. The effects of heparin, protamine, and heparin/protamine reversal on platelet function under conditions of arterial shear stress. Anesth Analg 1; 93: 7 13 Nielsen VG. The detection of changes in heparin activity in the rabbit: a comparison of anti-xa activity, thrombelastography, activated partial thromboplastin time, and activated coagulation time. Anesth Analg ; 95: 153 Vig S, Chitolie A, Bevan DH, et al. Thromboelastography: a reliable test? Blood Coagul Fibrinolysis 1; : Caprini JA, Zuckerman L, Cohen E, et al. The identification of accelerated coagulability. Thromb Res 197; 9: 17 1 Mammen EF, Koets MH, Washington BC, et al. Hemostasis changes during cardiopulmonary bypass surgery. Semin Thromb Hemost 195; 11: Mochizuki T, Olson PJ, Szlam F, et al. Protamine reversal of heparin affects platelet aggregation and activated clotting time after cardiopulmonary bypass. Anesth Analg 199; 7: Perkins HA, Osborn JJ, Hurt R, et al. Neutralization of heparin in vivo with protamine; a simple method of estimating the required dose. J Lab Clin Med 195; : 3 19 Barstad RM, Stephens RW, Hamers MJ, et al. Protamine sulphate inhibits platelet membrane glycoprotein Ib-von Willebrand factor activity. Thromb Haemost ; 3: 33 7 Salooja N, Perry DJ. Thrombelastography. Blood Coagul Fibrinolysis 1; :