Study of clotting factors in liver homotransplantation

JOHN

Study

of Clotting

Liver

Homotransplantation

OORE

in

F. STREMPLE,M.D.,CLARA V. HU~SEY, M.D.,AND EDWIN H. ELLISON,M.D., Milwaukee, Wisconsin

changes in coagulation were also observed after removal of the liver of the recipient. It was suggested that the increase in fibrinolytic activity might be explained by a decreased inhibition of plasminogen activator. In all but one case, measures were taken to prevent fibrinolysis and hypofibrinogenemia by the administration of fresh blood, fibrinogen, or epsilon-aminocaproic acid. These measures were reported to be helpful in the control of hemorrhage. One patient was heparinized prophylactically at operation to prevent a subsequent period of hypercoagulability. However, diffuse hemorrhage developed and the patient required 18 pints of blood. After canine auxiliary liver homotransplantation, Goodrich and associates [5] reported only one case of unexplained fatal hemorrhage. Other investigators reported no clotting defects or a bleeding diathesis after homotransplantation of the liver in dogs [3,6,7]. Experience with auxiliary homotransplantation in human subjects, however, has been associated with uncontrollable hemorrhage in one of two cases

From the Departments of Surgery andPathology, Marquette University School of Medicine, and the AllenBradley Medical Science Laboratory, Milwaukee, Wisconsin. This study was supported in part by U.S.P.H.S. Grant AM-04150-06.

M

Factors

AND

CO-WORKERS

[I]

&mOnSttYited

no significant coagulation abnormalities after canine orthotopic liver homotransplantation until the time of death. The bleeding tendency which developed as a terminal event and was characterized by an increased prothrombin time was attributed to liver failure. Marchioro and associates [2] reported the presence of clotting defects in all dogs subjected to orthotopic liver allografts. Fatal hemorrhage developed in 80 per cent of these animals. The clotting abnormalities recorded by Marchioro and his co-workers were an increased fibrinolytic activity, as measured by decreased euglobulin lysis time, decreased plasma fibrinogen, and an increased thrombin time. The coagulation abnormalities and subsequent fatal hemorrhage were thought due to the length of extracorporeal perfusion of the donor liver. Similar experience was reported by Starzl and associates [3]. This same group of investigators demonstrated coagulation defects in experience with liver homotransplantation in human subjects [a]. Three of five patients with hepatic allografts died of hemorrhage or had a bleeding diathesis during or shortly after the operative procedure. A study of the clotting mechanisms in human subjects showed an increased fibrinolytic activity, again measured by the decreased euglobulin lysis time, and a rapid dissolution of clot as shown by the thromboelastogram. These changes occurred within minutes after manipulation of the liver of the recipient patient. The

PI.

Coagulation disorders have been recognized as a serious deterrent to successful homotransplantation of the liver in dog and man [3]. It has been suggested [Y] that changes in the coagulation mechanism after liver homotransplantation may be divided into two phases: (1) an acute phase occurring during the operative procedure and immediately afterwards, characterized by defective coagulation; (2) a second phase occurring later in the postoperative period, that has been described as a hypercoagulable state. The study reported herein deals with the 862

American Journal of Sur@~y

Clotting Factors in Liver Homotransplantation

_

Baseline

CoaQulotion

Coagulation Study

Coagulation Study

SKi

ogulation Study

Stud;

FIG. 1. A summary of the experimental method which was carried out in three stages, that is, end to side portacaval shunt, implantation of heterotopic liver allograft, and removal of host liver. mechanism of the bleeding diathesis in the acute or early phase after liver homotransplantation. The study was designed to define

more fully the existence and types of coagulation abnormalities which might occur. It seemed important also to attempt to determine whether early coagulation abnormalities and a bleeding diathesis occur after removal of the host liver or because of the presence of an auxiliary donor liver. METHOD

Twelve adult mongrel dogs were used as recipients in this study. The mean weight of the recipients was 23 kg. and that of the donors 6 kg. The donor and recipient animals were anesthetized with Pentothal@ sodium and were maintained under anesthesia with ether. X group of clotting factors were selected which were thought to test adequately the various aspects of the clotting mechanism. Base line clotting factors were measured before and after the recipient animal had an end to side portacaval shunt. The superior mesenteric vein of the donor animal was cannulated, and slow perfusion of the donor liver was begun with cold Ringer’s lactate solution, containing one million units of aqueous penicillin, 1 cc. of procaine, and 6.0 cc. of epinephrine [IO]. No heparin was used in the perfusate or in the syringes used to draw blood samples. The donor liver was then removed and placed in cold Ringer’s lactate solution and flushed until the effluent was free of blood. Transplantation was performed as described by Gliedman and coworkers [1 I] (Fig. 1) except that in most cases

splanchnic outflow was through the portal vein of the donor liver. Measurement of the various clotting factors was carried out after implantation of the donor liver. Clotting studies were repeated after removal of the recipient animal’s liver. The time interval between the completion of each stage and measurement of clotting factors was one and a quarter to two and three-quarter hours and averaged two hours. If the animal survived the entire procedure, clotting studies were repeated every forty-eight hours. The recipient animals were supported as necessary with normal saline solution and sodium bicarbonate. They received no blood, plasma, or dextran. In some animals, the procedures were staged so that the recipient underwent a portacaval shunt and liver allograft during one operative period and hepatectomy three days later. Other animals underwent all three stages during one operative period. In this group some had hepatectomy before and some after allograft. Sections of the liver from both donor and host were taken for microscopic inspection at autopsy. A control group of eight animals was subjected to a period of ether anesthesia without an operative procedure, and measurements of the same clotting factors were carried out after Pentothal induction and six hours of ether anesthesia. Difficulties involving the interpretation of changes in coagulation factors and fibrinolytic activity are well known. The dynamic and complex interaction of these systems as well as species variation accounts for many of the difficulties. The problem is corn pounded when there is measurement of only a limited number of coagulant and anticoagulant factors as

Stremple, Hussey,

864

well as limited methods of determinations within each system. In evaluation of the clotting factors in this study a variety of assays were utilized with different substrates to measure changes in both fibrinolytic and coagulation systems. It should be pointed out that there is considerable controversy as to the relative accuracy of the methods available for the assay of fibrinolytic activity [12,13]. The following panel of coagulation factor determinations have been used in this study: (1) prothrombin time [14] ; (2) prothrombin time corrected with serum [14]; (3) prothrombin time corrected with serum and factor v [14]; (4) factor v assay [14]; (5) clotting time of recalcified plasma [14]; (6) thrombin time and heparin assay [14] ; (7) euglobulin lysis time [15]; (8) partial thromboplastin time [16]; (9) thromboplastin generation test [17]; (10) differentiation of antithrombin VI and heparin [18]; (11) plasma fibrinogen [19]; (12) platelet count [20]; (13) presence of active fibrinolysin as tested by the method of Hussey [21] (1 ml. of dog serum is added to bovine fibrinogen solution in normal buffer [pH 7.4 and 0.01 M] so that the final concentration was 1 mg. [lo0 mg. per cent] ; the fibrin clot formed after addition of 1 unit of thrombin was observed for lysis for twenty-four hours) ; (14) presence of active fibrinolysin also was tested by observation of the whole blood clot for lysis for eight hours. RESULTS

Of the eight animals given ether anesthesia without an operative procedure, six showed a significant decrease in euglobulin lysis time. In four the decrease in euglobulin lysis time occurred after induction of anesthesia with Pentothal and remained significantly decreased throughout the period of ether anesthesia. There was no evidence of an increase in thrombin time in these animals and, therefore, there was no production of antithrombin or endogenous heparin. Since the euglobulin lysis time was significantly decreased in control animals, it was abandoned in experimental animals. A modified fibrin clot lysis method for measurement of active fibrinolysin was substituted [21]. Twelve animals had clotting factor determinations and auxiliary liver allografts. Four survived long enough to have removal of the host liver. In three of these four animals, the host liver was removed after the allograft and in one, prior to implantation of the auxiliary liver. Six of the twelve animals with an auxiliary liver manifested a hemorrhagic diathesis as evidenced by incoagulability and uncontrollable hemorrhage from vascular anastomoses which had been previously sealed. Five of these six

and Ellison animals had a portacaval shunt and auxiliary liver allograft. One had removal of the host liver prior to liver allograft. The observation of hemorrhage and incoagulability in these animals correlated with an increase in measured clotting times and production of endogenous heparin. Clotting times were significantly increased in five of six animals which showed a hemorrhagic diathesis. These same five animals which demonstrated an increase in clotting time and a hemorrhagic diathesis also were found to have produced endogenous heparin. One animal with an auxiliary liver allograft that showed a bleeding diathesis did not have an elevated clotting time and did not produce endogenous heparin. One animal with an auxiliary liver allograft that did not show a bleeding diathesis but produced endogenous heparin after three days had an elevated clotting time. The mean duration of hepatic anoxia (time between removal of the donor liver and completion of the inflow-outflow anastomosis in the recipient) was recorded in all animals. There was no correlation between the duration of donor hepatic anoxia and the production of endogenous heparin. An attempt was made to relate the donor esophageal temperature to bleeding diathesis. In those animals demonstrating a bleeding diathesis the mean esophageal temperature in the donor immediately prior to removal of the liver was 33.3”~. In those that did not have bleeding diathesis it was 32.1”~. In animals producing endogenous heparin the esophageal temperature of the donor at the time of liver removal was 31”~. In those animals which did not produce endogenous heparin the esophageal temperature as 32’~. at the time of removal of the donor liver. These temperature differences are not statistically significant but perhaps may be interpreted as emphasizing the need for adequate hypothermia of the donor in liver homotransplantation. Plasma fibrinogen and platelets showed an over-all decrease in those animals with a hemorrhagic diathesis. (Table I.) Only those animals manifesting a bleeding diathesis had a change in factor v and prothrombin time, and this change was marked. No active fibrinolysin was present in either group of animals, and no significant change in the partial thromboplastin time was noted. Five of the six animals with bleeding from previously sealed anastomoses American Journal

of Surgery

Clotting TABLE STMMARY

Sturly

Fibrinogen Platelets Factor v I’rothrornbin time Heparin Fibrinolysin

:Inirnals with Bleeding of of of of

83$; 43tA 80f ;, 2 to 6

in 5

in Liver

I

OF COAGULATION

Decrease Decrease Decrease Increase timts Appeared Sane

Factors

STUDIES

.knimals with No Bleeding Decrease of 44y0 Decrease of 205& No change No change Appeared in 1 IiOlW

SOTE: Endogenous hcparin appeared in five of six animals with bleeding. The figures represent the mean per cent change from base line measurement.

produced measurable amounts of endogenous heparin. The increased thrombin time could be corrected with protamine sulfate, and the antithrombin activity was heat stable. These observations ruled out antithrombin VI as a consideration since it is heat labile. Table I shows a significant over-all decrease in plasma fibrinogen of 83 per cent and in platelets of 43 per cent. However, in those animals which exhibited a hemorrhagic diathesis and also produced endogenous heparin, these changes did not occur until the endogenous heparin appeared in the circulation. Only after the appearance of endogenous heparin was there a significant decline in the plasma fibrinogen of 43 per cent in those animals which exhibited a bleeding diathesis as compared with those animals that did not bleed or produce endogenous heparin. In fact, platelets increased Yi per cent until the appearance of endogenous heparin. The entire decrease in platelets of 43 per cent (Table I) occurred after the appearance of endogenous heparin. Even with the over-all decrease the platelet level remained above that usually associated with bleeding. Decrease in factor v usually began prior to the appearance of endogenous heparin. The decrease accelerated, however, after the appearance of endogenous heparin. In only one animal was there a bleeding diathesis after liver allowithout production of endogenous graft heparin. This animal showed a marked over-all decrease in factor v, plasma fibrinogen, and platelets; however, as with the other animals the platelet decrease did not occur until hemorrhage was apparent. Table II shows that in six of twelve animals measurable amounts of endogenous heparin

S&i

Homotransplantation

were found. This observation was noted only after implantation of the donor liver even though one animal underwent the removal of the host liver six hours prior to liver allograft. The quantity of endogenous heparin recorded is an approximation calculated in terms of normal plasma volume. Although the quantity of endogenous heparin is small, it should be recalled that all animals in which it appeared had abnormal clotting times. In one interesting case, abnormal totting time and appearance of endogenous heparin occurred three days after liver allograft. In this animal, no fall in iibrinogen, factor v, or platelets was apparent and n(J bleeding difficulties arose. Endogenous heparin liberated from the liver of the dog in other experiments has been shown to have two and a half times greater activity than commercial heparin [22]. The quantity of endogenous heparin produced by the animals reported in this study is probably an underestimation because its calculation was based on normal plasma volume. However, it should be recalled that five of the six animals were actively bleeding without blood replacement when samples were being taken. Removal of the host liver had no influence on the appearance of a bleeding diathesis, active fibrinolysin, or endogenous heparin. Congestion of the donor liver was grossly apparent after all allografts. There was no correlation between the gross and microscopic congestion of the donor liver and the production of endogenous heparin or a bleeding diathesis. The microscopic examination of the donor livers showed no evidence of central vein congestion or thrombi.

TABLE APPEARANCE

TIME

OF

II

HEPARIN

Animal

Appearance Time (hr.) after Implantation of Donor Liver

1 2 3 4 5 6

1.25 2.5 2.5 2.0 3 days 1 .i

IN

EACH

Quantity (1ng.1

2.i -4.2 >l”.O 3 ” 6 .2

1.4

ANIMAL*

Bleeding

If If 8+

2+ 0 2+

* Removal of the host liver had no influence on appearance of heparin (four of twelve animals).

Stremple, Hussey, and Ellison COMMENTS

Several factors of importance have been found in this study of the hemorrhagic diathesis that occurs after liver homotransplantation. The majority of studies of fibrinolysis have involved the euglobulin lysis test. Ratnoff [23] has questioned the validity of these observations. He has pointed out that the antihemophilic factor, factor v, and fibrinogen are all three sensitive substrates for plasmin. It has been observed that these substances may all be normal in samples which displayed rapid fibrinolysis in vitro. Sherry, Fletcher, and Alkjaersig [13] have shown that plasmin may produce fibrinolysis in the absence of fibrinogenolytic activity but have found this to be very uncommon. Significant decreases in euglobulin lysis time in dogs with anesthesia alone have been demonstrated in this study. Heinrich, Noe, and Vonderheide [24] have confirmed these findings. An abnormality in the euglobulin lysis time does not necessarily mean that the animal will exhibit a bleeding diathesis or that increase in fibrinolytic activity is necessarily the cause of bleeding which is already occurring. Coon and Hodgson [25,26] showed that patients also exhibited increased fibrinolytic activity without the stress of surgery and that this observation was more frequent in patients with extensive malignancy. Some degree of fibrinolytic activity was found in 85 per cent of all postoperative patients in their study. Some patients showed a marked fibrinolytic activity and decreased fibrinogen with a bleeding tendency. Sherry, Fletcher, and Alkjaersig [13] have stated that it has been reported that total activation of plasma plasminogen can be produced and maintained in human subjects without the development of hemorrhagic difficulties. It can be seen that the demonstration of fibrinolytic activity is not necessarily related to bleeding. Care must be used in the interpretation of tests for fibrinolytic activity, particularly when exogenous agents have been used which may affect the system being measured. Starzl and associates [4] stated that in their experience heparin and epsilon-aminocaproic acid would not affect the euglobulin lysis time. However, it is possible that circulating heparin could have established the abnormal euglobulin lysis time reported by these investigators. Others have shown that small amounts of heparin shorten

the euglobulin lysis time and large amounts of heparin and epsilon-aminocaproic acid inhibit euglobulin lysis time [13,27,28]. Since the liver has been shown to contain almost no fibrinolytic activator substances [29], it is unlikely that manipulation of the host liver could cause increased fibrinolytic activity by release of activator. Active fibrinolysin should be readily detected by the methods employed in this study. If active fibrinolysis was present in the animals studied, it would have had to be present in large amounts because incoagulability of the blood was apparent, and there was a marked increase in clotting times. However, no active fibrinolysis could be measured under the conditions of our study in those animals who had a heterotopic liver homotransplant as well as removal of the host liver. It should be mentioned that Grossi, Rousselot, and Panke [30] noted increased fibrinolytic activity and decreased fibrinogen after portacaval shunt in cirrhotic patients. In our study of the normal dog, no evidence of fibrinolytic activity followed portacaval shunt. Bleeding diathesis and hemorrhage were observed in this study with auxiliary liver allograft without concomitant hepatectomy. Drapanas, Shim, and Stewart [31] studied dogs after hepatectomy and found that death occurred in almost every case because of hemorrhage without increased fibrinolytic activity. Recently, Von Kaulla and associates [47] found increased fibrinolytic activity after hepatectomy in the dog prior to vascularization of the orthotopic liver allograft. However, significant increase in thrombin times during this period was also demonstrated but not further investigated for a causative factor such as endogenous heparin. They thought that important factors in the demonstration of fibrinolytic activity were the sensitivity of the euglobulin test employed and the time of sampling. Gans [32] showed that fibrinogen declined rapidly after hepatectomy and noticed an increased plasminogen activator and plasmin activity. Epsilonaminocaproic acid did not correct the drop in fibrinogen and, therefore, defibrination and fibrinolysis activation were attributed to systemic intravascular clotting. Decrease in the clotting factors produced by the liver was observed. These clotting factors are fibrinogen and factor v. Decreased production by the liver of these clotting factors in this American

Journal of Suvgery

Clotting

Factors

in Liver Homotransplantation

study is unlikely since such marked changes occurred very quickly after liver transplantation [33]. These are also the clotting factors along with platelets that are characteristically affected by intravascular thrombosis. Of the animals studied, however, no intravascular thrombosis was observed and platelets increased prior to endogenous heparin production and appearance of incoagulability. .inother possibility for the alterations in the coagulation system could be blood loss. The blood loss might be the result of the endogenous heparin observed after implantation of the donor liver. The possible relation of this increased endogenous heparin and bleeding diathesis and of an early immune reaction is an intriguing one. Howell [34] and Quick [35] produced anaphylactic shock in dogs and noted a complete inhibition of blood clotting. The blood from these animals contained a heparinlike substance. Quick [35] suggested that when the heparinlike substance exceeded the quantity of thrombin, coagulation was completedly inhibited. Quick, Ota, and Baronofsky [36] showed that the release of endogenous histamine and heparin and a decrease in platelets were characteristic of anaphylactic shock in the dog. They further suggested that thrombocytopenia, heparinemia, and histamine release were independent of each other and not related to the severity of the reaction. Riley [37] noted that it is only in the dog that heparin and histamine are bound together in mast cells and released simultaneously. Dragstedt, Wells, and Rocha e Silva [38] showed that the release of trypsin, proteose, or other antigenic substances in anaphylactic reactions increased the amount of circulating heparin. The increase was in excess of that required to prevent coagulation. Smith and associates [39] proposed that the activation of the pituitary-adrenocortical axis induced hyperheparinemia in the stress of anaphylactic shock. Rebound to a hypercoagulable state and increased incidence of thrombosis were secondary observations. The work of Jaques and Waters [ZZ] supported the contention that the production of endogenous heparin was responsible for the incoagulability of the blood in anaphylactic shock. They also showed that the liver was the only source of endogenous heparin. These workers demonstrated appreciable amounts of endogenous heparin occurring within three minutes of the onset of shock.

sAi

Interestingly, Monkhouse, Fidlar, and Barlolr [40] were able to increase the amount (of endogenous heparin produced during an immune reaction by depression of antibody formation through total body irradiation. In another type of immune reaction, Friesen and co-workers [41,42] noted incoagulability and production of a heparinlike substance after incompatible blood transfusions in dogs and in human subjects. McKay and associates 1431 noted disseminated intravascular coagulation after incompatible blood transfusion in the dog. McKay [44] observed that the dog responded to antigen-antibody reactions by the induction of intravascular coagulation. Subsequently, a circulating anticoagulant similar to heparin developed. Hardaway and co-workers [FZ] found that after hemorrhagic shock in dogs, periods of intravascular hypercoagulability causing clotting were followed by periods of incoagulability due to liberation of endogenous heparin. These findings were not confirmed by the study of Smith, Grace, and Hussey [46]. Endogenous heparin produced in our experiments was not likely the response to disseminated intravascular clotting because platelets increased prior to its appearance and no wideIt is appealspread thrombosis was apparent. ing to assume that the hemorrhage and endogenous heparin produced were the result of an immune reaction. The appearance of heparin three days after a liver allograft without manifestation of excessive bleeding or decrease in fibrinogen or platelets suggests that endogenous heparin may be released primarily in an immune response. This response occurs with auxiliary liver allografts in dogs. This immune reaction alters clotting factors and may contribute to a hemorrhagic diathesis. SUMMARY

1. Bleeding diathesis occurred in six of twelve animals after addition of a donor liver and its cause is probably related to implantation of the donor liver. 2. Measurable levels of endogenous heparin were found in six of twelve animals after liver allografts. Five of the six animals had a hemorrhagic diathesis. 3. Removal of the host liver had no influence on the appearance of a bleeding diathesis, active fibrinolysin, or endogenous heparin. 4. Endogenous heparin may contribute to the hemorrhagic diathesis.

868

Stremple,

Hussey, and Ellison

5. Heparin was found three days after a liver allograft in one animal. That animal did not have bleeding diathesis. 6. These observations may be another manifestation of the immune reaction in liver homotransplantation. REFERENCES 1. MOORE, F., WHEELER, H., DEMISSIANOS, H., SMITH, L., BALANKURA, O., ABEL, K., GREENBERG, J., and DAMMIN, G. Experimental wholeorgan transplantation of the liver and of spleen. Ann. ‘surg., 152: 374, 1960. 2. MARCHIORO, T., HUNTLEY, R., WADELL, W., and STARZL, T. Use of extracorporeal perfusion for obtaining postmortem homografts. Surgery, 54: 900, 1963. 3. STARZL, T., MARCHIORO, T., ROWLANDS, D., KIRKPATRICK, C., WILSON, W., RIFKIND, D., and WADDELL, W. Immunosuppression after experimental and clinical homotransplantation of the liver. Ann. Surg., 160: 411, 1964. 4. STARZL, T., MARCHIORO, T., VON KAULLA, K., HERMANN, G., BRITTAIN, R., and WADDELL, W. Homotransplantation of liver in humans. Surg. Gynec. & Obst., 117: 659, 1963. 5. GOODRICH, E., WELCH, H., NELSON, J., BEECHER, T., and WELCH, C. Homotransplantation of the canine liver. Surgery, 39: 244, 1956. 6. MARCHIORO, T., PORTER, K., DICKINSON, T., FARIS, T., and STARZL, T. Physiologic requirements for auxiliary liver homotransplantation. Surg. Gynec. 6 Obst., 121: 17, 1965. 7. MEHREZ, T., NABSETH, D., KEKIS, B., APOSTOLOU, K., GOTTLIEB, L., and DETERLING, R. Homotransplantation of the canine liver: a new technic. Ann. Surg., 159: 416, 1964. 8. GLIEDMAN, M. Personal communication. 9. STARZL, T., MARCHIORO, R., HUNTLEY, D., RIFKIND, D., ROWLANDS, D., DICKINSON, T., and WADDELL, W. Experimental and clinical homotransplantation of the liver. Ann. Neu York Acad. Sci., 120: 739, 1964. 10. MORENO, A., ROUSSELOT,L., BURCHELL, A., BONO, R., and BURKE, J. Studies on the outflow tracts of the liver. II. On the outflow tracts of the canine liver with particular reference to its regulation by the hepatic vein sphincter mechanism. Ann. Surg., 155: 427, 1962. 11. GLIEDMAN, M., PANGAN, J., MINKOWITZ, S., POPWITZ, L., and KARLSON, L. Heterotopic liver transplantation after liver damage. Tr. Am. Sot. Artif:lnt. Organs, 11: 205, 1965. 12. BAUMGARTEN. W.. A~BRUS. C.. MCCALL, K.. and PENNELL, k. Panel disc&ion: assay t&h&sproblems of correlation with results of treatment. Am. J. Curdiol., 6: 447, 1960. 13. SHERRY, S., FLETCHER, A., and ALKJAERSIG, N. Fibrinolysis and fibrinolytic activity in man. Physiol. Rev., 39: 343, 1959. 14. QUICK, A. J. Hemorrhagic Diseases. Philadelphia, 1957. Lea & Febiger. 15. SHERRY, S., LINDEMEYER, R., FLETCHER, A., and ALKJAERSIG, N. Studies on enhanced fibrinolytic activity in man. _7. Cl&. Invest., 38: 810, 1959.

16. LANDGDELL, R., WAGNER, R., and BRINKHOLIS,I;. Effect of antihemophilic factor on one-stage clotting tests: a presumptive test for hemophilia and a simple one-stage antihemophilic factor assay procedure. J. Lab. & C&z. Med., 41: 637, 1953. 17. BIGGS, R. and DOUGLAS, A. The thromboplastin generation test. J. Clin. Path., 6: 23, 1953. 18. ROSZKO~SKI, I., NIEWIAROWSKA, M., and BARPRATKOWSKA, J. Fibrinogen derived coagulation inhibitors in obstetric cases of acute fibrinolysis. Thrombos. Diathes. haemorrh., 13: 25, 1965. 19. FEISSLY, R. Blood platelets. International Symposium, p. 99. Boston, 1961. Little, Brown & Co. 20. HAWK, P., OSER, B., and SUMMERSON,W. Practical Physiological Chemistry, p. 495. New York, 1947. McGraw-Hill Book Co., Inc. 21. HUSSEY, C. Unpublished data. 22. JAQUES, L. and WATERS, E. The identity and origin of the anticoagulant of anaphylactic shock in the dog. J. Physiol., 99: 454, 1941. 23. RATNOFF, 0. Hemostatic mechanisms in liver disease. M. Cl&. North America, 47: 721, 1963. 24. HEINRICH, R., NOE, F., and VONDERHEIDE, E. A rapid method for the estimation of fibrinolytic changes in the dog. Am. J. Physiol., 193: 283, 1958. 25. COON, W. and HODGSON, P. Fibrinolysis in surgery patients. Surg. Gynec. b Obst., 95: 717, 1952. 26. HODGSON, P. and COON, W. Fibrinolysis in surgery patients: fibrinogen-fibrinolysis relationships. S. Forum, 4: 152, 1953. 27. VON KAULLA, K. and MCDONALD, T. The effect of heparin on components of the human fibrinolytic system. Blood, 13: 811, 1958. 28. BUCKELL, M. The effect of citrate on euglobulin methods of estimating fibrinolytic activity. J.

Clin. Path., 11: 403, 1958. 29. ALBRECHTSEN, 0. The fibrinolytic activity of human tissues. Brit. J. Haemat., 3: 284, 1957. 30. GROSSI, C., ROUSSELOT, L., and PANKE, W. Control of fibrinolysis during portocaval shunts: study of patients with cirrhosis of liver. J.A.M.A., 187: 1005, 1964. 31. DRAPANAS, T., SHIM, W., and STEWART, J. Studies of fibrinolytic activity after hepatectomy. Arch. Surg., 87: 64, 1963. 32. GANS, H. Study of fibrinogen turnover rates after total hepatectomy in dogs. Surgery, 55: 544, 1964. 33. RUTHERFORD, R. and HARDAWAY, R. Significance of the rate of decrease in fibrinogen level after total hepatectomy in dogs. Ann. Surg., 163: 51, 1966. 34. HOWELL, W. The purification of heparin and its presence in blood. Am. J. Physiol., 71: 553, 1925. 35. QUICK, A. J. On the coagulation defect in peptone shock. A consideration of antithrombosis. Am. J. PhysioE., 116: 535, 1936. 36. QUICK, A., OTA, R., and BARONOFSKY, I. On the thrombopenia of anaphylactic and peptone shock. Am. J. Physiol., 145: 273, 1946. 37. RILEY, J. Heparin, histamine and mast cells. Blood, 9: 1123, 1954. 38. DRAGSTEDT, L., WELLS, J., and ROCHA E SILVA, M. Inhibitory

effect of heparin upon histamine

re-

American Journal of Surgery

Clotting Factors in Liver Homotransplantation

39.

40.

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lease by trypsin, antigen, and proteose. Proc. Ser. Erper. Biol. & Med., 51: 191, 1942. SMITH, R., MARGULIS, R., BRENNA, M., and MANTO, R. The influence of ACTH and cortisone on certain factors of blood coagulation. Scienre, 112 : 295, 1950. MONKHOVSE, F., FIULAR, E., and BARLOW, J. Release of heparin in anaphylactic shock in irradiated and non-irradiated animals. dm. J. I’irysiol., 169: 712, 1952. FRIBSEN, S., HARSHA, W., and MCCRUSKEY, C. Massive generalized wound bleeding during operation with clinical and experimental evidence of blood transfusion reactions. Surgery, 32: 620, 1952. FRIESES, S., MCCRUSKEY, C., and ROODY, M. Wound bleeding during experimental transfusion reaction in isoimmunized dogs. S. Forum, 4: 209, 1953

S(i!i

43. MCKAY, D., HARDAWAY, R., WAHLE, G., HALL. R.. and BARNS, K. Pathologic study of intrava.scular coagulation following incompatible blood tranbfusion in dogs. II. Intra-aortic injection of illcompatible blood. ilm. /. SlLrg., 91: 32, 19.56. 44. MCKAY, D. Disseminated Intravnscuiar Coqgulation. An Intermediary Mechanism of Disease New York, 1965. Harper & Row. 45. HARDAI~AY, R.. BRUNE, W., GF,EVER, I?.. BUKNS, J., anti MOCK, H. Studies on the role of intravascular coagulation in irreversible hetnorrhagic shock. Aizn. Surg., 155: 241, 1962. 46. SMITH, J., GRACE, R., and HUSSEY, C. Coagulation mechanism and effect of heparin in hemorrhagic shock. rim. J. Physiol., 193: 593. 1958. 47. VON KAULLA, K., KAYE. H., v0N KAULLA, E.. MARCHIORO, T., and STARZL, T. Changes in blood coagulation in hepatectomy and liver transplantation. Arch. Sung., 92: 71, l966.