• View in gallery
    Figure 1—

    Box-and-whisker plots indicating thromboelastography variables (R [reaction time; A], K [clot formation time; B], α angle [C], and MA [D]) for various concentrations of protamine (0, 22, 44, and 66 μg/mL) in blood samples obtained from 8 dogs that were not activated with kaolin. Boxes represent interquartile ranges, horizontal lines inside boxes represent median values, and whiskers represent the range. Open boxes and whiskers (ie, whiskers without horizontal lines indicating maximum or minimum values) represent values for blood samples in which clot formation was completely inhibited. *Value is significantly (P < 0.01) different from the value for blood samples with 0 μg of protamine/mL.

  • View in gallery
    Figure 2—

    Box-and-whisker plots indicating thromboelastography variables (R [A], K [B], α angle [C], and MA [D]) for various concentrations of protamine (0, 22, 44, and 66 μg/mL) in blood samples obtained from 8 dogs that were activated with kaolin. See Figure 1 for remainder of key.

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The effects of protamine sulfate on clot formation time and clot strength thromboelastography variables for canine blood samples

Christopher J. BaileyDepartment of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.

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Amy M. KoenigshofDepartment of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.

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Abstract

Objective—To determine the effects of protamine sulfate on clot formation time and clot strength thromboelastography variables for canine whole blood samples.

Animals—Blood samples obtained from 11 healthy dogs.

Procedures—Blood samples were collected from jugular veins of dogs into syringes with 3.2% sodium citrate (blood to citrate ratio, 9:1). Blood samples were divided into aliquots, and protamine sulfate was added to various concentrations (0 [control], 22, 44, and 66 μg/mL). Prepared samples were activated with kaolin (n = 8) or not activated (8), CaCl2 was added, and thromboelastography was performed. Reaction time (R), clot formation time (K), rate of clot formation (α angle), and maximum amplitude (MA) were measured.

Results—For kaolin-activated and nonactivated blood samples, protamine (66 μg/mL) significantly increased R and K and decreased α angle and MA, compared with values for control samples. Also, protamine (44 μg/mL) decreased MA in nonactivated blood samples and increased K and decreased α angle in kaolin-activated samples, compared with values for control samples.

Conclusions and Clinical Relevance—Results indicated protamine prolonged clot formation time and decreased overall clot strength in a dose-dependent manner; such effects may contribute to a hypocoagulable state in dogs. Kaolin-activated and nonactivated blood samples were appropriate for measurement of the effects of protamine on coagulation. Administration of protamine to reverse the effects of heparin should be performed with caution.

Abstract

Objective—To determine the effects of protamine sulfate on clot formation time and clot strength thromboelastography variables for canine whole blood samples.

Animals—Blood samples obtained from 11 healthy dogs.

Procedures—Blood samples were collected from jugular veins of dogs into syringes with 3.2% sodium citrate (blood to citrate ratio, 9:1). Blood samples were divided into aliquots, and protamine sulfate was added to various concentrations (0 [control], 22, 44, and 66 μg/mL). Prepared samples were activated with kaolin (n = 8) or not activated (8), CaCl2 was added, and thromboelastography was performed. Reaction time (R), clot formation time (K), rate of clot formation (α angle), and maximum amplitude (MA) were measured.

Results—For kaolin-activated and nonactivated blood samples, protamine (66 μg/mL) significantly increased R and K and decreased α angle and MA, compared with values for control samples. Also, protamine (44 μg/mL) decreased MA in nonactivated blood samples and increased K and decreased α angle in kaolin-activated samples, compared with values for control samples.

Conclusions and Clinical Relevance—Results indicated protamine prolonged clot formation time and decreased overall clot strength in a dose-dependent manner; such effects may contribute to a hypocoagulable state in dogs. Kaolin-activated and nonactivated blood samples were appropriate for measurement of the effects of protamine on coagulation. Administration of protamine to reverse the effects of heparin should be performed with caution.

Coagulopathies associated with cardiac bypass procedures in dogs are complex and poorly understood. Known causes include hemodilution, platelet activation, thrombin activation and enhanced fibrinolysis, and enhanced antithrombin III activity by means of interactions with heparin.1–4 Protamine may further contribute to such coagulopathies.5–13 Protamine is a cationic peptide that is administered to reverse the anticoagulant effects of heparin. That drug can be used for patients that have received an overdose of heparin or those in which there is a need to quickly reverse the effects of heparin, but it is most commonly administered to reverse the effects of that drug following cardiac surgery. Administration of high doses of protamine is associated with postoperative hemorrhage and an increased need for blood and platelet transfusions in humans.9–13 Protamine decreases clot strength and increases fibrinolysis in plasma of humans and in vivo.9,13 In healthy dogs, protamine can cause marked thrombocytopenia, neutropenia, systemic hypotension, pulmonary hypertension, and prolongation of activated clotting time and prothrombin time5–8; similar effects have been reported14,15 for humans.

The dose of protamine administered to cardiac bypass patients varies. For humans, either an institution-specific standardized dose of protamine is administered for each adult or child, or patients receive 1 mg of protamine for each 100 U of heparin that is administered.16,17 For dogs, protamine doses range from 2.5 to 6 mg/kg.18,19

Few studies have been conducted to evaluate the effects of protamine on global measures of clot formation and fibrinolysis in dogs. The circulating half-life of protamine in dogs has not been evaluated, to the authors’ knowledge; however, the half-life in humans is short (7.4 minutes in healthy humans and 4.5 minutes in patients undergoing cardiac bypass).17,20 This has made definitive determination of the cause of protamine-induced coagulopathies difficult, although results of other studies9,20 indicate protamine inhibits activation of coagulation factor V and may decrease thrombin generation.9,21

Thromboelastography is an effective tool for global evaluation of hemostasis, clot formation, and fibrinolysis.22–25 Thromboelastography allows for the detection of hypercoagulable or hypocoagulable states, clotting factor deficiencies, and thrombocytopenias and has been used to guide selection of blood products.22,26–29 The use of thromboelastography reduces the total number of blood products administered to human patients.30 Both native (nonactivated) and kaolin-activated blood samples obtained from healthy dogs can be analyzed with that method,30,31 but some authors30–32 have suggested that the use of coagulation activators may mask changes in coagulation variables for patients with coagulopathies. The effects of protamine on thromboelastography variables for dogs have not been determined, to the authors’ knowledge.

The purpose of the study reported here was to determine the effects of protamine on thromboelastography variables for clot formation time and clot strength in whole blood samples obtained from dogs. We hypothesized that protamine would increase the time to clot formation and decrease clot strength. We also hypothesized that the use of kaolin for activation of coagulation during thromboelastography would decrease the effects of protamine.

Materials and Methods

Samples—This study was approved by the Institutional Animal Care and Use Committee of Michigan State University, and written informed consent was obtained from each dog owner. For each of the 2 thromboelastography experiments in this study (assay performed with and without kaolin activation), blood samples were collected from 8 privately owned dogs that were determined to be healthy on the basis of histories and results of physical examinations. Blood samples obtained from 5 dogs were used in both experiments, and blood samples from 6 dogs were used in 1 experiment; therefore, blood samples were collected from a total of 11 dogs for this study. The dogs included 8 castrated males and 3 spayed females. Breeds included 8 mixed-breed dogs, 2 German Shepherd Dogs, and 1 Border Collie. Median age of the dogs was 4 years (range, 3 to 8 years). For the 5 dogs with blood samples that were used in both experiments, at least 24 hours was allowed between collections of blood samples. All animals were fed a maintenance food intended for dogs. Dogs were excluded from the study if they were receiving any medications other than prophylactic parasiticides.

Prior to performance of experiments, thromboelastography variables were determined for each dog; values were within reference intervals for the laboratory. In addition, serum total solids concentration, PCV, and platelet counts were determined for each blood sample. Platelet counts were determined with an established protocol33 by a single investigator (CJB). Briefly, blood smears were prepared with citrated whole blood samples after samples were kept at room temperature for 30 minutes. Slides were examined by means of light microscopy to detect large platelet clumps, and the number of platelets in 10 hpfs (magnification, 1,000×) was counted and multiplied by 15,000; the mean value for the 10 hpfs was determined. Blood samples were not used if substantial platelet clumping was detected or platelet counts were outside the laboratory reference range for dogs.33 Serum total solids concentrations were measured by use of a refractometer. Packed cell volume was measured after centrifugation of heparinized capillary tubesa at 13,000 × g for 3 minutes. Blood samples were determined to have clinically normal platelet counts (median, 146,000 platelets/μL; range, 96,000 to 220,000 platelets/μL), serum total solids concentrations (median, 5.9 g/dL; range, 5.7 to 5.9 g/dL), and PCVs (median, 44%; range, 38% to 50%).

Study protocol—Food was withheld from dogs overnight before collection of blood samples. A blood sample (5.4 mL) was collected from a jugular vein of each dog with a 20-gauge needle into a 6-mL syringe containing 0.6 mL of 3.2% sodium citrate (total sample volume, 6 mL). Each blood sample was collected by means of direct needle entry into the vein, and all samples used in the study were collected on the first attempt (if blood samples were not collected on the first attempt, no further attempt was made to collect blood samples from the dog on that day). Blood samples were kept at room temperature (22.5°C) for 30 minutes prior to preparation for thromboelastography. Blood samples were divided into aliquots for 4 experimental groups of protamine sulfateb concentrations: 0 μg of protamine/mL (control), 22 μg of protamine/mL (intended to simulate the circulating concentration in a dog that received a dose of 2 mg/kg), 44 μg of protamine/mL (intended to simulate a dose of 4 mg/kg), and 66 μg of protamine/mL (intended to simulate a dose of 6 mg/kg). A theoretical total blood volume of 90 mL/kg in each animal was used for calculation of the approximate circulating concentration of protamine that would be achieved in a dog after administration of a dose. For preparation of blood samples with protamine, protamine (10 mg/mL) was diluted with saline (0.9% NaCl) solutionc so that the total volume of each sample was equal, regardless of the protamine concentration. For the 22 μg/mL preparation, 7.5 μL of protamine was mixed with 92.5 μL saline solution. For the 44 μg/mL preparation, 15 μL of protamine was mixed with 85 μL of saline solution. For the 66 μg/mL preparation, 22.5 μL of protamine was mixed with 77.5 μL of saline solution. The final ratio of blood to protamine solution for each aliquot was 33:1.

For performance of thromboelastography without kaolin activation, blood samples were mixed by means of repeated inversion and 990 μL of blood was mixed with 30 μL of the protamine and saline solution mixture prepared for each protamine concentration group. Each mixture was inverted 5 times to ensure proper mixing, and thromboelastographyd was performed.

For performance of thromboelastography with kaolin activation, blood samples were mixed by means of repeated inversion and 1,188 μL of blood was mixed with 36 μL of the protamine and saline solution mixture prepared for each protamine concentration group. Each mixture was inverted 5 times to ensure proper mixing, a 1,000-μL aliquot was pipetted into a tube containing kaolin,e samples were mixed by means of repeated inversion, and thromboelastography was performed.

Thromboelastography—A 340-μL aliquot of each prepared blood sample was added to a warm (39°C) thromboelastography cup, and 20 μL of 0.2M CaCl2f was added. Thromboelastography was performed at 39°C (intended to simulate the approximate rectal temperature of dogs). The R (reaction time), K (clot formation time), α angle (rate of clot formation), and MA (a measure of overall clot strength) were measured for each sample. Each blood sample was assayed in 1 thromboelastography well; duplicate samples were not assayed because only 4 thromboelastography wells were available. The well that was used for each assay was determined by means of a randomization procedure (random number generatorg). Assay-specific quality control samples were assayed in all 4 thromboelastography wells on each day prior to data collection for experimental samples.

Figure 1—
Figure 1—

Box-and-whisker plots indicating thromboelastography variables (R [reaction time; A], K [clot formation time; B], α angle [C], and MA [D]) for various concentrations of protamine (0, 22, 44, and 66 μg/mL) in blood samples obtained from 8 dogs that were not activated with kaolin. Boxes represent interquartile ranges, horizontal lines inside boxes represent median values, and whiskers represent the range. Open boxes and whiskers (ie, whiskers without horizontal lines indicating maximum or minimum values) represent values for blood samples in which clot formation was completely inhibited. *Value is significantly (P < 0.01) different from the value for blood samples with 0 μg of protamine/mL.

Citation: American Journal of Veterinary Research 75, 4; 10.2460/ajvr.75.4.338

Statistical analysis—Statistical analysis was performed with a commercially available statistical software program.h For each assay condition (nonactivated and kaolin-activated thromboelastography), R, K, α angle, and MA values were compared separately. Data were analyzed with a Friedman's test. For very high values that were considered essentially infinite (ie, clots did not form), data were assigned the highest rank. Values of P < 0.05 were considered significant.

Results

Thromboelastography without kaolin activation—The median R and K values were significantly longer for blood samples with 66 μg of protamine/mL (R, 25.6 minutes; K, 28.4 minutes) than they were for samples with 0 μg of protamine/mL (R, 10.9 minutes; K, 8.4 minutes). The median α angle was significantly smaller for blood samples with 66 μg of protamine/mL than they were for samples with 0 μg of protamine/mL (8.1° and 26.6°, respectively). The median MA was significantly smaller for blood samples with 44 or 66 μg of protamine/mL (32.2 and 26.6 mm, respectively) versus samples with 0 μg of protamine/mL (50.9 mm). No significant differences in values were detected between blood samples with 22 μg or protamine/mL and those with 0 μg of protamine/mL or among samples with 22, 44, or 66 μg of protamine/mL.

Figure 2—
Figure 2—

Box-and-whisker plots indicating thromboelastography variables (R [A], K [B], α angle [C], and MA [D]) for various concentrations of protamine (0, 22, 44, and 66 μg/mL) in blood samples obtained from 8 dogs that were activated with kaolin. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 75, 4; 10.2460/ajvr.75.4.338

Thromboelastography with kaolin activation—The median R value was significantly longer for blood samples with 66 μg of protamine/mL (17.1 minutes) than it was for samples with 0 μg of protamine/mL (3.9 minutes). The median K value was significantly longer for blood samples with 44 or 66 μg of protamine/mL (12.6 and 12.2 minutes, respectively) than it was for samples with 0 μg of protamine/mL (3.9 minutes). The median α angle was significantly smaller for blood samples with 44 or 66 μg of protamine/mL (16.2° and 17.9°, respectively) than it was for samples with 0 μg of protamine/mL (44.3°). Blood samples with 66 μg of protamine/mL had a significantly smaller median MA versus those with 0 μg or protamine/mL (37.7 and 45.9 mm, respectively). No significant differences in values of variables were detected between blood samples with 22 μg of protamine/mL and those with 0 μg of protamine/mL or among samples with 22, 44, or 66 μg of protamine/mL.

Discussion

Performance of thromboelastography with whole blood samples allows global assessment of coagulation. Four variables are typically evaluated for thromboelastography results. The R and K times represent the time required for a clot to start to develop, the α angle represents the rapidity of clot formation, and the MA represents overall clot strength. Results of the present study indicated protamine increased R and K times and decreased α angle and MA of blood samples. Therefore, as assessed by means of thromboelastography, protamine delayed clot formation and decreased clot strength and these effects seemed to be dose dependent. Although protamine is known to have anticoagulant properties, the present study is the first in which the effects of protamine on thromboelastography variables for canine whole blood samples were determined, to the authors’ knowledge. A similar study was conducted with human plasma samples; results indicate protamine significantly delays clot formation and decreases clot strength.9 However, because that study was conducted with plasma samples, the effects of protamine on platelets and other blood cells during clot formation were not determined. Results of that other study and those of the present study suggested that protamine is a strong anticoagulant in humans and dogs.

The pharmacokinetics of protamine is poorly understood. However, protamine likely induces a hypocoagulable state by means of inhibition of coagulation factor V and modulation of platelet activity.21,34 Activation of coagulation factor V to factor Va enhances the ability of factor Xa to convert prothrombin to thrombin by a factor of 278,000 and makes factor Xa refractory to inhibition by the anticoagulant, tissue factor pathway inhibitor.35,36 Protamine causes a dose-dependent decrease in factor V activation by α-thrombin, which accounts for most of the anticoagulant effect of protamine on the tissue factor pathway in human plasma; however, protamine has a small effect on plasma containing preformed factor Va.21 That mechanism for inhibition of thrombin generation was likely important in the delay in clot formation and decrease in clot strength caused by protamine in the present study.

The effects of protamine on platelets are poorly understood. Protamine can induce transient thrombocytopenia in dogs and humans that have or have not received heparin.5–7,13,15,34 Administration of protamine causes increased aggregation of platelets and thrombocytopenia in dogs,6 and results of an in vitro study7 indicate protamine decreases adenosine diphosphate–induced platelet aggregation. In platelet-rich plasma, the effects of protamine on thrombin may be attenuated by platelets.37 Such attenuation of the effects of protamine by platelets may be a reason for the less substantial thromboelastography coagulation effects detected in blood samples in the present study, compared with those detected in human plasma samples in another study.9 Although further studies are warranted, the interaction of protamine with platelets likely decreases platelet function and the effectiveness of platelets during clot formation. This likely contributed to the delay in clot formation and decrease in MA detected in blood samples with protamine in the present study.

Results of this study suggested that blood samples that are not activated with kaolin can be used to assess changes in coagulation caused by protamine. Small differences in results were detected between the blood samples activated with kaolin and those that were not activated. Further studies are warranted to determine which type of blood sample preparation is best for testing of clinical patients.

Results of the present study and those of other studies6,9 suggested that doses of protamine should be chosen carefully. When choosing a dose of protamine, the goal should be to achieve a physiologically normal state of coagulation in a patient. However, hypocoagulable states may be worsened after administration of an inappropriate dose of protamine. Protamine is typically administered to dogs at doses up to 6 mg/kg. However, use of activated clotting times and heparin dose-response curves may be appropriate for titration of protamine doses to circulating heparin concentrations.38,39 Results of the present study and those of similar studies for humans suggested such methods could reduce the risk of administration of an excessive dose of protamine and development of associated coagulopathies.

A major limitation of the present study was that it was conducted in vitro and the effects of endothelial cells on coagulation were not determined. Additionally, although clinically normal dogs were included in the study, complete blood analyses (including coagulation tests) were not performed for each dog. In future studies, the effects of heparin-protamine interactions on protamine-induced coagulopathies should be evaluated. In addition, the in vivo effects of protamine on coagulation as assessed with thromboelastography should be determined; in vivo effects of protamine on coagulation may differ from the in vitro effects determined in the present study.

Results of this study indicated protamine significantly reduced clot strength in canine whole blood samples as assessed by means of thromboelastography, and that effect seemed to be dose dependent. Results also suggested that doses of protamine for dogs should be chosen with caution.

ABBREVIATION

MA

Maximum amplitude

a.

Fisher Scientific, Pittsburgh, Pa.

b.

APP Pharmaceuticals LLC, Schaumburg, Ill.

c.

Hospira Inc, Lake Forest, Ill.

d.

TEG 5000, Haemonetics Corp, Niles, Ill.

e.

Kaolin, Haemonetics Corp, Niles, Ill.

f.

0.2M CaCl2, Haemonetics Corp, Niles, Ill.

g.

Microsoft Corp, Redmond, Wash.

h.

GraphPad Software Inc, La Jolla, Calif.

References

  • 1. Sniecinski RM, Chandler WL. Activation of the hemostatic system during cardiopulmonary bypass. Anesth Analg 2011; 113:13191333.

  • 2. Reges RV, Vicente WV & Rodrigues AJ, et al. Retrograde autologous priming in cardiopulmonary bypass in adult patients: effects on blood transfusion and hemodilution. Rev Bras Cir Cardiovasc 2011; 26:609616.

    • Search Google Scholar
    • Export Citation
  • 3. Hirsh J, Raschke R & Warkentin TE, et al. Heparin: mechanism of action, pharmacokinetics, dosing considerations, monitoring, efficacy, and safety. Chest 1995; 108:258S275S.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Cormack JE, Forest RJ & Groom RC, et al. Size makes a difference: use of a low-prime cardiopulmonary bypass circuit and autologous priming in small adults. Perfusion 2000; 15:129135.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Jaques LB. A study of the toxicity of the protamine, salmine. Br J Pharmacol Chemother 1949; 4:135144.

  • 6. Kresowik TF, Wakefield TW & Fessler RD II, et al. Anticoagulant effects of protamine sulfate in a canine model. J Surg Res 1988; 45:814.

  • 7. Lindblad B, Wakefield TW & Whitehouse WM Jr, et al. The effect of protamine sulfate on platelet function. Scand J Thorac Cardiovasc Surg 1988; 22:5559.

    • Crossref
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Contributor Notes

Supported by the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University.

Address correspondence to Dr. Koenigshof (koenig27@msu.edu).