Activation of coagulation during therapeutic whole body hyperthermia

THROMBOSIS RESEARCH 43; 353-360, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

ACTIVATION OF COAGULATION DURING THERAPEUTIC WHOLE BODY HYPERTHERMIA

S. Vance Strother, Joan M. C. Bull, Susan A. Branham Department of Internal Medicine, Section of Hematology/Oncology University of Texas Health Science Center at Houston U.S.A. (Received 3.4.1986; Accepted in revised form 8.5.1986 by Editor M.W. Mosesson)

ABSTRACT

Coagulation parameters were initially monitored in 8 patients receiving whole body hyperthermia (WBH). Patients were heated by the warm water blanket technique to 41.8'C (Tmax), maintained at this temperature for 2 hours, then allowed to cool. A fall in platelets was apparent by the time Tmax was achieved and continued during the 18 hours after WBH. Levels of betathromboglobulin (BTG) and platelet factor 4 rose by 56% and 191% by the end of treatment but returned to baseline 18 hours Fibrinogen, plasminogen and -antiplasmin levels iEEI5ned and FDP and fibrinopeptide Aai!$? levels increased during WBH. Factor XII and Factor VIII:C fell moderately during WBH while Factors VIII R:Ag, VIII:RC and V did not change or showed a late rise. Factor VII levels fell in 7 of 8 patients, reaching levels of 30% of normal in four. To better define the sequence of these coagulations perturbations, earlier and more frequent timepoints were studied in an additional 3 patients. This revealed that decreases in fibrinogen and plasminogen and increases in FPA and BTG occur very early (by the time the patient reaches 39'C). On the other hand, a decrease in Factor VII activity was not apparent until patients had reached Tmax. WBH is therefore associated with a consumption coagulopathy. Possible mechanisms are discussed and extrapolations to the situation seen in heat stroke are suggested.

Reprint requests:

Dr. Strother, Mount Sinai I?edicalCenter, University of Wi consin Medical School, Milwaukee Clinical Campus, 950 N. 12th St., Milwaukee, Wisconsin 5301

Key words:: Hyperthemia,

consumption coagulopathy, platelet activation. 353

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INTRODUCTION Whole body hyperthermia (WBH) is an investigational antineoplastic treatment based on the differential sensitivity of normal tissue and malignant tumors to heat. One of the reported toxicities of WBH is coagulopathy. Ludgate et al (1) observed a decrease in platelet count in the majority of 43 patients treated with WBH, but only an occasional patient had other evidence of disseminated intravascular coagulation as manifested by prothrombin time prolongation, increased levels of fibrin degradation products and hypofibrinogenemia. In contrast, in a phase I study of WBH involving 14 patients (2), monitoring of prothrombin time, partial thromboplastin time, thrombin time, factors VIII, X and XI, fibrinogen, fibrin degradation productions, euglobulin lysis time plasminogen and platelet count revealed no significant alterations in any of these parameters. We have noted unequivocal coagulation changes in many patients receiving WBH. The present study was designed to better define the frequency, nature and sequence of coagulation abnormalities that occur in patients undergoing WBH.

METHODS The study was conducted in two phases. During the first phase a battery of coagulation tests was monitored at four different timepoints in eight patients receiving hyperthermia. In the second phase, selected tests were performed at eight timepoints in three additional patients. The age, sex, and diagnosis of the patients is shown in the table. All were studied during their first WBH treatment. None received chemotherapy in conjunction with WBH and no patient had an objective tumor response to their first course of WBH.

TABLE PATIENT CHARACTERISTICS

AGE 54 44 43 68 32 44 34 41

FIRST SEX M F F M F F F M

PHASE DIAGNOSIS Melanoma Breast Cancer Sarcoma-Unclassified Colon Cancer Melanoma Colon Cancer Melanoma Melanoma

AGE 21 64 36

SECOND PHASE SEX DIAGNOSIS Leiomyosarcoma M Pancreatic Carcinoma M Rhabdomyosarcoma M

Patients were anesthetized during WBH with nitrous oxide, pancuronium, thiopental and either butorphanol or fentanyl. Some received glycopyrralate to prevent sweating. WBH was induced with a warm water heating blanket . Patients were heated over a period of about three hours to a core temperature of 41.8"C (Tmax), maintained at Tmax for two hours and then allowed to cool.

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During the first phase of the study blood was drawn by fresh venipuncture at the follow timepoints: a. pretreatment, b. as soon as core temperature reached 41.8"C, a% the start of cooling (2 hours after timepoint b.) and C. d. 18 hours after timepoint c. Time points for the second phase were: a. pretreatment, b. as soon as core temperature reached 39"C, as soon as core temperature reached 40.8"C, C. d. as soon as core temperature reached 41.8"C, e. after l+ hours at 41.8"C, f. 1% hours after the start of cooling (2 hours after timepoint e.), g* 18 hours after the start of cooling and h. 42 hours after the start of cooling. Beta-thromboglobulin (BTG) and platelet factor 4 (PF4) were measured by radioimmunoassay using commercially available kits (Amersham, Arlington Heights, Illinois and Abbot, North Chicago, Illinois). Factors IT,V,VII, VIII:C and XII were measured by standard functional assays utilizing specific factor-deficient substrates. Factor VIIIR:Ag was measured by Laurel1 immunoelectrophoresis (3). Measurement of factor VIII:RC activity was based on the capacity of patient plasma to support the agglutination of fixed human platelets in the presence of ristocetin. Fibrinopeptide A (FPA) was measured using a radioimmunoassay kit (Mallinckrodt, St. Louis, Missouri). Fibrinogen was quantified with a standard clotting assay. Fibrin/fibrinogen degradation products (FDP) were assayed by latex particle agglutination (American Scientific Products, McGaw Park, Illinois). Plasminogen and alpha2-antiplasmin were measured by functional assays utilizing a fluorometric substrate (American Scientific Products, McGaw Park, Illinois).

RESULTS The results of the first phase of the study are shown in Figure 1. Levels of the platelet alpha granule proteins BTG and PF4 (Figure la.) rose sharply during WBH but had returned almost to baseline 18 hours after the end of treatment. Platelet counts declined progressively throughout the observation period, so that the greatest change, a mean decrease of 35%, was seen 18 hours-post-therapy. Levels of FPA (Figure lb.) were measured as a means of monitoring the generation of thrombin. Increases in FPA were observed by the time patients reached Tmax and levels continued to rise through treatment and the 18 hours afterward. Thrombin generation was accompanied by simultaneous activation of fibrinolysis, manifested by consumption of fibrinogen, plasminogen and alpha2-antiplasmin (Figure lc.) and increases in titers of FDP (Figure Id.). The components of the Factor VIII complex (Figure le.) changed little in response to WBH. Factor VIII:C levels showed a mild decrease to 17% below baseline by the end of treatment. Levels of Factor VIIIR:Ag and Factor VIII:RC showed little change during WBH and were modestly elevated 18 hours afterward.

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FIG. 1 Coagulation changes in eight patients treated with WBH. Changes in FDP are expressed as the mean increase in positive tube dilutions. Other changes are expressed as the mean percent change relative to baseline values. Timepoints are: a. pretreatment (pre), b. arrival at Tmax (Tmax), c. start of cooling (Tmax +2), and d. 18 hours post-WBH (Tmax +20). Bars indicate the standard error of the mean.

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Treatment did not significantly affect levels of Factor V but did result in a decrease in levels of Factor XII and VII (Figure If.) Factor XII levels were reduced by 31X at the end of WBH but had begun to recover 18 hours postWBH. Factor VII levels declined to a lesser degree during treatment but continued to fall afterward. Four of eight patients had Factor VII levels below 0.30 units/ml when tested 18 hours after treatment. These four patients had baseline Factor VII levels that were subnormal or near the lower end of the normal range. The second phase of the study was useful in defining the sequence of changes in coagulation parameters. Levels of BTG were found to increase very early during WBH, before platelet counts began to decline (Figure 2a.). Also evident early in the course of treatment were increases in levels of FPA (Figure 2b.) and decreases in levels of fibrinogen and plasminogen (Figure 2c.) Factor VII levels (Figure 2d.) began to fall relatively later and declinetlmost dramatically after the end of WBH.

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FIG. 2 Coagulation changes in three additional patients treated with WBH. Changes are expressed as the mean percent change relative to baseline values. Timepoints are: a. pretreatment (pre), b. arrival at 39'C (39), c. arrival at 40.8'C (40.8), d. arrival at 41.8"C (Tmax), e. after 1% hours at Tmax (Tmax+ 1.5), f. 1% hours post-WBH (Tmax +3.5), g. 18 hours post-WBH (Tmax +20), and h. 42 hours post-WBH (Tmax +44). Bars indicate the standard error of the mean.

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DISCUSSION All eleven patients monitored during this study exhibited alterations in coagulation parameters. Changes in platelet counts appear to result from several different factors. In some patients there is an initial early rise in count which most likely reflects release of platelets from the spleen in response to stress (4). Very early during treatment there is -in vivo platelet activation, as evidenced by increased levels of platelet-specific proteins. The continued fall in platelet count following WBH, when PF4 and BTG levels are returning toward normal, suggests that platelet production may be decreased during the post-treatment period. Megakaryocyte damage has been reported to be a sequela of therapeutic hyperthermia (5) and heat stroke (6). Activation of the fibrinolytic system is also apparent very early during WBH. This is most likely a secondary response to fibrin formation since a rise in levels of FPA is seen at the same point that levels of fibrinogen and plasminogen begin to decline. It is likely that thrombin is initially generated via the intrinsic clotting pathway because the decline in levels of Factor VII, which participates exclusively in the extrinsic pathway, is delayed compared to the increase in FPA. This delayed activation of the extrinsic pathway may result from tissue factor release from damaged tissues or blood monocytes. Hepatic dysfunction may also play an important role in the genesis of low Factor VII levels. Although patients did not show significant changes in serum levels of hepatic enzymes and bilirubin (data not shown), it is possible that sublethal thermal damage can result in temporary impairment of cellular processes such as protein synthesis and gamma-carboxylation. The coagulopathy observed in our patients is clearly complex and of a consumptive nature. Although it is apparent that platelet activation and thrombin generation are early events, the mechanism by which coagulation is initiated by heat cannot be deduced at this point. One possibility is that heat can directly activate platelets and that fluid phase coagulation is secondarily recruited. Alternatively, the initial event might be thermally-induced endothelial cell damage or dysfunction with consequent exposure of subendothelium or impairment of prostacyclin production. One clinical entity to which information about WBH-induced coagulation changes might be cautiously extrapolated is heat stroke. Although heat stroke involves less controlled hyperpyrexia and additional factors such as dehydration, core body temperatures are similar to those used in WBH, usually in the range of 41 to 43°C. A number of studies have demonstrated perturbations in various coagulation parameters in most victims of heat stroke (6-15). Autopsy findings in heat stroke include multiple thrombi and hemorrhages and widespread endothelial damage (16). It thus is likely that a consumption coagulopathy is an important factor in the pathophysiology of heat stroke and that this coagulopathy is similar in etiology to that seen In individuals treated with WBH. A better understanding of the coagulation changes seen during WBH may ultimately suggest means of early therapeutic intervention in heat stroke.

ACKNOWLEDGEMENTS Supported by a University of Texas Biomedical Research Support Grant.

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