Coagulation Dynamics Following Thermal Injury: Effect of Heparin and Protamine Sulfate P. WILLIAM CURRERI, M.D.,* MARY E. WILTERDINK, B.S., M.T., (ASCP), CHARLES R. BAXTER, M.D.
A burned rat model was developed to examine post-burn alterations in coagulation. Fibrin split product concentration, as measured by the staphylococcal clumping test, was noted to rise significantly within the first 24 hours following injury. Prophylactic in vivo systemic anticoagulation with heparin was ineffective in modifying this response. However, systemic administration of protamine sulfate prevented post-burn elevation of fibrin split products. In vitro fibrin split product concentration in burn sera following the addition of heparin and protamine sulfate, was also analyzed. The results of these experiments elucidated the biochemical effects of protamine sulfate on circulating fibrin degradation products in the rat burn model.
p IEVIOUS BlEl'ORS by both Baxter2 and McManus9 have noted the presence of an increased concentration of fibrin split produicts in plasma of seriously burned patients duiring the first post-buirn week. Presumably such increases in fibrin split produict concentration result from activation of plasmin in response to significant intravascular or ex-
travascuilar coaguilation. In this stludy, a rat buirn model was utilized to describe alterations in the coagulation mechanism with time. Since the animals all received standardized scald burns, carefully controlled post-buirn therapy with systemically administered anticoagulants couild be evaluated. Methods One-huindred five, 200 gm male Sprague-Dawley rats were suibjected to scalding water at 98 C for 10 seconds to effect either a 30% or 50% full-thickness burn utilizing the method of Walker and his associates.15 All animals were Siubmitted for publication Jtune 11, 1974.
Stupported by the United States Ptublic Health Service Grants NIH 1ROI-GM19571 and NIH 5-PO1-GM14892 from the National Instituites of General Medical Sciences. ° Recipient of Research Career Development Award from the National Instittutes of Health. Reprint requiests: P. William Cturreri, M.D., Department of Suirgery, The University of Washington School of Medicine, Seattle, Washington 981.95.
From the Department of Surgery, The University of Texas Southwestern Medical School, Dallas, Texas
resuscitated with two 10 ml intraperitoneal injections of lactated Ringer's soluition; one dose delivered immediately post-burn and a second given one hour later. Blood samples were obtained by cannulation of the abdominal aorta. After separation of either plasma or serum, samples were frozen for later laboratory analysis. Fibrinogen determinations were performed according to the method of Ratnoff.1" Platelet counts were performed on a Couilter Couinter. Prothrombin time, partial thromboplastin time, and thrombin times were obtained by standard laboratory techniques. The staphylococcal clumping test as modified by Leavelle7 was utilized to assay fibrin split produict concentration. Seruim samples from 105 burned rats and 55 burned patients were suibjected to quantitative analysis for fibrin split product concentration. Additional thrombin (100 tinits/ml serum) was then added to remove any remaining fibrinogen or large molecular weight X fragments and the fibrin split produict assay repeated. The supernatant was then treated with protamine sulfate (10 mg/ml serum) and incubated for 20 minutes at 4 C to disassociate soluble fibrin monomer complexes. The fibrin monomers then undergo gelation or paracoagulation allowing their removal by centrifugation. The supernatant now containing only Y, D, or E fragments was again assayed by the staphylococcal cltumping test. A 30% bturned rat model was utilized to evaluate the effect of systemic heparin or protamine sulfate on the postbturn appearance of elevated fibrin split product levels. Therapetutic doses of heparin (10 units/100 gm body weight) or protamine (0.1 mg/100 gm body weight) were injected suibcuitaneouisly immediately prior to burning and then every fotur houirs until sacrifice at 24 hours. Separate grouips of unbturned rats and burned rats received sub-
161
Ann. Stirg. Febrtiary 1975 CURRERI, WILTERDINK AND BAXTER 162 cutaneous injections of 0.2 ml physiological saline at the Discussion same time intervals and served as controls. In this study we were able to utilize a highly reproducible animal buirn model for evaluation of alterations in the Results coagulability of blood following controlled buirn injury. Alterations in coagulation parameters with time in the rat Alterations in clotting factors with time were strikingly bturn model are suimmarized in Fig. 1. Fibrinogen concen- similar to those observed in the burned patient as previously tration increased from 194 + 22.3 mg% in the uinburned rat reported by Curreri3 and Baxter.2 to a peak of 691 + 13.7 mg% two days post-burn (p < .001). To our knowledge, characterization of the fibrin split Fibrin split products increased from 2.7 + 0.59 ,ug/ml to products, measured by the staphylococcal clumping assay in 13.0 + 2.65 ,ug/ml at day one post-burn (p < .01). No buirned patients, has not previously been attempted. The significant prolongation of thrombin time, partial throm- staphylococcal clumping assay is known to detect boplastin time, or prothrombin time was observed. In fact, a fibrinogen, X and Y fragments, and complexes of X, Y, and state of hypercoagulability was suggested. Platelet concen- D fragments with soluble fibrin monomers.8 The results of tration was normal during the early post-burn period. this stuidy strongly suggest that the major fibrin split In vitro addition of thrombin to burned rat or human produict measured by the staphylococcal clumping test in serum did not result in any further clotting, and did not both burned animals and burned patients during the early reduice the measuired concentration of fibrin split products. post-burn period is a complex between D fragments and However, the addition of protamine sulfate resulted in the soluble fibrin monomer. When excess thrombin is added to loss of all fibrin split products measured by the staphylococ- serum, all remaining fibrinogen and X fragments are recal clumping test, suggesting the presence of complexes moved. Precipitates during this step were not observed and between D fragments and soluble fibrin (Table 1). no decrease in fibrin split product levels was observed in the In vivo protamine sulfate therapy pre- and post-burn was supernatant. Thuis X fragments cannot account for any subinstituited to determine if the subsequient elevation of cir- stantial portion of the measured fibrin split products. cuilating fibrin split products at 24 hours could be Niewiarowski'0 has shown that protamine sulfate disprevented. In the 30% rat model, burned rats receiving associates soluble fibrin monomer complexes, releasing free systemic protamine sulfate had normal fibrin split product fibrinogen degradation fragments and precipitating levels at 24 houirs post-burn in contrast to rats receiving polymerized soluible fibrin monomer. In this experiment the physiologic saline solution or heparin, which exhibited addition of protamine sulfate was associated with the diselevated sertum concentrations of fibrin split products as appearance of fibrin split products as measured by the measuired by the staphylococcal clumping test (Table 2). staphylococcal clumping test. A complex of soluble fibrin -
FIBRINOGEN (mg/100 ml)
FSP (pg/mi) 20 r
PTT (sec) 30 r *p
A burned rat model was developed to examine post-burn alterations in coagulation. Fibrin split product concentration, as measured by the staphylococcal clumping test, was noted to rise significantly within the first 24 hours following injury. Prophylactic in vivo systemic anticoagulation with heparin was ineffective in modifying this response. However, systemic administration of protamine sulfate prevented post-burn elevation of fibrin split products. In vitro fibrin split product concentration in burn sera following the addition of heparin and protamine sulfate, was also analyzed. The results of these experiments elucidated the biochemical effects of protamine sulfate on circulating fibrin degradation products in the rat burn model.
p IEVIOUS BlEl'ORS by both Baxter2 and McManus9 have noted the presence of an increased concentration of fibrin split produicts in plasma of seriously burned patients duiring the first post-buirn week. Presumably such increases in fibrin split produict concentration result from activation of plasmin in response to significant intravascular or ex-
travascuilar coaguilation. In this stludy, a rat buirn model was utilized to describe alterations in the coagulation mechanism with time. Since the animals all received standardized scald burns, carefully controlled post-buirn therapy with systemically administered anticoagulants couild be evaluated. Methods One-huindred five, 200 gm male Sprague-Dawley rats were suibjected to scalding water at 98 C for 10 seconds to effect either a 30% or 50% full-thickness burn utilizing the method of Walker and his associates.15 All animals were Siubmitted for publication Jtune 11, 1974.
Stupported by the United States Ptublic Health Service Grants NIH 1ROI-GM19571 and NIH 5-PO1-GM14892 from the National Instituites of General Medical Sciences. ° Recipient of Research Career Development Award from the National Instittutes of Health. Reprint requiests: P. William Cturreri, M.D., Department of Suirgery, The University of Washington School of Medicine, Seattle, Washington 981.95.
From the Department of Surgery, The University of Texas Southwestern Medical School, Dallas, Texas
resuscitated with two 10 ml intraperitoneal injections of lactated Ringer's soluition; one dose delivered immediately post-burn and a second given one hour later. Blood samples were obtained by cannulation of the abdominal aorta. After separation of either plasma or serum, samples were frozen for later laboratory analysis. Fibrinogen determinations were performed according to the method of Ratnoff.1" Platelet counts were performed on a Couilter Couinter. Prothrombin time, partial thromboplastin time, and thrombin times were obtained by standard laboratory techniques. The staphylococcal clumping test as modified by Leavelle7 was utilized to assay fibrin split produict concentration. Seruim samples from 105 burned rats and 55 burned patients were suibjected to quantitative analysis for fibrin split product concentration. Additional thrombin (100 tinits/ml serum) was then added to remove any remaining fibrinogen or large molecular weight X fragments and the fibrin split produict assay repeated. The supernatant was then treated with protamine sulfate (10 mg/ml serum) and incubated for 20 minutes at 4 C to disassociate soluble fibrin monomer complexes. The fibrin monomers then undergo gelation or paracoagulation allowing their removal by centrifugation. The supernatant now containing only Y, D, or E fragments was again assayed by the staphylococcal cltumping test. A 30% bturned rat model was utilized to evaluate the effect of systemic heparin or protamine sulfate on the postbturn appearance of elevated fibrin split product levels. Therapetutic doses of heparin (10 units/100 gm body weight) or protamine (0.1 mg/100 gm body weight) were injected suibcuitaneouisly immediately prior to burning and then every fotur houirs until sacrifice at 24 hours. Separate grouips of unbturned rats and burned rats received sub-
161
Ann. Stirg. Febrtiary 1975 CURRERI, WILTERDINK AND BAXTER 162 cutaneous injections of 0.2 ml physiological saline at the Discussion same time intervals and served as controls. In this study we were able to utilize a highly reproducible animal buirn model for evaluation of alterations in the Results coagulability of blood following controlled buirn injury. Alterations in coagulation parameters with time in the rat Alterations in clotting factors with time were strikingly bturn model are suimmarized in Fig. 1. Fibrinogen concen- similar to those observed in the burned patient as previously tration increased from 194 + 22.3 mg% in the uinburned rat reported by Curreri3 and Baxter.2 to a peak of 691 + 13.7 mg% two days post-burn (p < .001). To our knowledge, characterization of the fibrin split Fibrin split products increased from 2.7 + 0.59 ,ug/ml to products, measured by the staphylococcal clumping assay in 13.0 + 2.65 ,ug/ml at day one post-burn (p < .01). No buirned patients, has not previously been attempted. The significant prolongation of thrombin time, partial throm- staphylococcal clumping assay is known to detect boplastin time, or prothrombin time was observed. In fact, a fibrinogen, X and Y fragments, and complexes of X, Y, and state of hypercoagulability was suggested. Platelet concen- D fragments with soluble fibrin monomers.8 The results of tration was normal during the early post-burn period. this stuidy strongly suggest that the major fibrin split In vitro addition of thrombin to burned rat or human produict measured by the staphylococcal clumping test in serum did not result in any further clotting, and did not both burned animals and burned patients during the early reduice the measuired concentration of fibrin split products. post-burn period is a complex between D fragments and However, the addition of protamine sulfate resulted in the soluble fibrin monomer. When excess thrombin is added to loss of all fibrin split products measured by the staphylococ- serum, all remaining fibrinogen and X fragments are recal clumping test, suggesting the presence of complexes moved. Precipitates during this step were not observed and between D fragments and soluble fibrin (Table 1). no decrease in fibrin split product levels was observed in the In vivo protamine sulfate therapy pre- and post-burn was supernatant. Thuis X fragments cannot account for any subinstituited to determine if the subsequient elevation of cir- stantial portion of the measured fibrin split products. cuilating fibrin split products at 24 hours could be Niewiarowski'0 has shown that protamine sulfate disprevented. In the 30% rat model, burned rats receiving associates soluble fibrin monomer complexes, releasing free systemic protamine sulfate had normal fibrin split product fibrinogen degradation fragments and precipitating levels at 24 houirs post-burn in contrast to rats receiving polymerized soluible fibrin monomer. In this experiment the physiologic saline solution or heparin, which exhibited addition of protamine sulfate was associated with the diselevated sertum concentrations of fibrin split products as appearance of fibrin split products as measured by the measuired by the staphylococcal clumping test (Table 2). staphylococcal clumping test. A complex of soluble fibrin -
FIBRINOGEN (mg/100 ml)
FSP (pg/mi) 20 r
PTT (sec) 30 r *p
