California CATHOLIC MEDICAL CENjTS* ENTRAL MEDICAL LIBRARY Medicine 88-2I5153rd STREET N~WYORK .11I432q

Blood and the Coumarin Anticoagulant Drugs

PAUL M. AGGELER, M.D., San Francisco

DR. LESLIE L. BENNETT*: Ladies and gentlemen, ing with clotting mechanisms, which appeared in it gives me unusual pleasure to introduce Dr. Paul 1938. Early recognizing the importance of the M. Aggeler as Faculty Research Lecturer from the work of Dam and Almquist on the bleeding dia- San Francisco Division of the Academic Senate thesis of vitamin K-deficient chicks and the prob- for 1966. There is no higher honor that the fac- able significance for human disease of the observa- ulty can bestow upon one of its colleagues than to tion of Greaves and Schmidt of the effectiveness select him to represent publicly the faculty's com- of vitamin K in controlling the bleeding of rats with mon urge toward creativity which primarily mani- biliary fistulas, he began in the late 1930's his in- fests itself in our scientific setting as research. vestigation of the role of vitamin K in controlling Dr. Aggeler is uniquely a Californian. He is one prothrombin levels in patients with jaundice and of those rare individuals, a native son. His birth hepatic disease. This is the first of his classic works occurred in Ferndale among the redwoods of Hum- referred to in the citation of the Research Com- boldt County. After attending St. Ignatius High mittee. School in San Francisco, he enrolled as a pre- In this year, 1966, when there is increased medical student at the University of California awareness of the ethical responsibilities of physi- in Berkeley in 1929 and received the M.D. cians engaged in clinical research, it is important degree from the University of California in to recall that Dr. Aggeler was unusually adept at 1937. Dr. Aggeler interned at the University of doing venipunctures on himself to obtain blood California Hospital and was successively Assistant to set up the controls necessary for his investiga- Resident, Research Fellow in Medicine and Resi- tion of the clotting mechanism in patients with dent in Medicine. Except for one year of training jaundice. His critical observation and studies of spent at the San Joaquin General Hospital, his pro- patients with hemophilia led to his discovery of fessional and scientific training was entirely at the plasma thromboplastin component deficiency and University of California; thus I say he is uniquely resulted in an explosion of knowledge about hemo- a California product. philia. Most recently, the same critical clinical acu- As a medical student, Dr. Aggeler came under men and inquiring mind resulted in his discovery the influence of Dr. Salvatore Lucia and in Dr. of a genetic control of warfarin metabolism. His Lucia's laboratory began a systematic study of national and international reputation is thoroughly factors affecting the prothrombin time as then de- established and I shall not repeat the list of editor- termined by the method devised by Armand Quick. ships he holds, advisory roles he has filled and the These studies resulted in his first publication deal- honors that have come his way. Let me simply say The Ninth Annual Faculty Research Lecture, University of Cali- that I am proud to present my colleague for over fornia San Francisco Medical Center, 23 March 1966. a quarter of a century, the man who completely Submitted 20 January 1967. requests to: Editorial Office, Room 4320, S.F. General meets the criteria for scholar and gentleman, Dr. Hospital,Reprint 1001 Potrero Avenue, San Francisco 94110. *Morris Herzstein Professor of Physiology. Paul Aggeler.

CALIFORNIA MEDICINE 255 DR. PAUL M. AGGELER*1: Professor Bennett, cepts of the blood clotting mechanism as I have members of the Academic Senate, ladies and gen- seen it unfold during the past 30 years. Blood clot- tlemen: I am sensible of the very great honor ting is somewhat unusual among physiologic proc- which the Academic Senate has bestowed on me, esses in that a great deal of what we know about and I am most grateful. I am overwhelmed to be it has come from careful study of its derangement included in the illustrious company of those who in sick patients. I will stress particularly the dis- have preceded me in this distinction. What I have coveries made through investigation of coagulation done seems inconsequential in comparison with defects in such individuals. Because of the limita- their accomplishments. I am pleased, however, that tions of time this must, of necessity, be oversimpli- what I have done and what I have been has seemed fied and must ignore the enormous contributions M ritorious to my colleagues. of many investigators. During the second part of At the outset I must acknowledge, with thanks, the hour I will outline some recent work we have the great debt I owe to many associates, fellows done with a class of drugs which interferes with and technical assistants for their help in my re- blood clotting. search endeavors. I am afraid that much of what I first became interested in blood clotting during I am given credit for is really their work. I am also my internship in this hospital in the year 1936-37. grateful to the Board of Directors and Administra- During that year I was involved in the care of sev- tion of the Children's Hospital of San Francisco eral patients suffering from hemophilia. At that for providing sponsorship and space for much of time the nature of the disease was poorly under- my activities during the past 10 years. Finally, I stood, and its treatment highly unsatisfactory. I would like to express my thanks to my first men- had no special training for research and certainly tor, Dr. Salvatore Lucia, for his stimulation, en- no intention of becoming seriously involved in it, couragement and support when I worked under but I did have an urge to find out more about the him first as a research fellow and later as an In- curious inability of the hemophiliac's blood to clot. structor in the Division of Preventive Medicine. In partial explanation of why, almost 30 years I have approached this lecture with more than later, I am still involved in blood clotting problems, the ordinary amount of apprehension. It would be I would like to quote a portion of the introduction appropriate perhaps to spend some time in philoso- to their book on blood coagulation by Drs. Rose- phizing about medical research, but I do not feel mary Biggs and Roger Macfarlane: "To the un- qualified for that task. In fact, I cringe whenever initiated, the fascination that this work has for its I am referred to as a researcher. For the better devotees must seem difficult to understand. But a part of my career I have held a title in the clinical number of explicable factors operate to develop professorial series. I have been a practicing intern- this attraction, so that once involved in it the new ist specializing in hematology with special interest recruit is almost powerless to draw back. To begin in the hemorrhagic disorders. I have been a mem- with, the spontaneous transformation of fluid ber of that branch of the faculty whose principal blood to solid clot has a stimulating effect on the concern has been with the care of patients. Such curiosity like that produced by a well executed research as I have been able to accomplish has conjuring trick. One feels that so simple and strik- sprung directly from the bedside and has usually ing a phenomenon must have a simple and striking had immediate clinical relevance. With the increas- explanation and the urge to discover it is imme- ing complexity of medical science it has become diate. Next there is the deceptive ease with which more difficult to pursue this kind of career, but I the work can be started. All that is needed is a hope the opportunity will not be completely lost. few glass tubes and a watch, a supply of blood and For me it has been an exciting and rewarding way ingenuity. Later the more mechanically-minded in- of life. vestigator will discover that almost equally good results can be obtained by apparatus of limitless In attempting to shape my discussion this morn- complexity. . . . Thereafter experiments are easy ing into a form which might have some interest for to plan and carry out, and provide unlimited op- the many disciplines represented on this campus, portunity for being most satisfyingly clever." I have decided to discuss two related topics. In the Man's fascination with the phenomenon of blood first part of the hour I will attempt to trace briefly clotting is very old. Hippocrates and Aristotle the history of the development of our present con- thought it was simply a matter of solidification on *lProfessor of Medicine. cooling. Later, a popular theory held that the blood

256 APRIL 1967 * 106 * 4 contained an accumulation of minute particles of Morawitz also recognized that the production of fibrin-like material which were kept widely dis- thrombin from its plasma precursors was in some persed by the circulation, but which would quickly way initiated or at least enhanced by contact with aggregate with the cessation of blood flow. Andrew a foreign surface. Buchanan, whose work was published in Glasgow During the early part of the Twentieth century in 1845, probably deserves credit as the founder a number of investigators including C. H. Mills, of the modern theory of blood coagulation. He was P. Nolf, A. Fischer, J. J. Mellanby, W. H. Howell the first to show that it took two substances to and others proposed conflicting theories. The cause coagulation: "Liquid fibrin" or fibrinogen "classical" theory of Morawitz was by no means as we would now call it, which by itself was stable, universally accepted. and a substance capable of affecting the "liquid Dr. Armand Quick, now Professor Emeritus of fibrin" and transforming it into an insoluble form. Physiology at Marquette University, was one of During the last half of the Nineteenth century a the pioneers in the present era of blood coagula- large number of investigations on blood clotting tion research. The paper shown in Figure 2, pub- were undertaken. The conclusions of different in- lished in 1935, did much to change the course of vestigators were divergent and often contradictory. my own life. In it Dr. Quick reaffirmed the "clas- However, a number of important discoveries were sical" theory of blood clotting as proposed by made: Fibrogen, the soluble precursor of fibrin, Morawitz and introduced his now famous one- was isolated from the plasma by salt precipitation; stage method for the measurement of prothrombin. thrombin, the agent which converted fibrogen to The success of Quick's test depended in large part fibrin, was extracted from fresh serum by alcohol on the use of a stable, standardized tissue throm- precipitation; tissue extracts were shown to hasten boplastin prepared from rabbit brain. In the test, blood clotting but not to initiate it; and the need plasma is recalcified in the presence of an optimum for ionized calcium in the clotting mechanism was amount of this tissue extract. When normal plasma demonstrated. is used in the test it clots in 11 to 12 seconds. He- In a magnificent monograph on blood clotting mophilic plasma clots in a normal time, but plasma published in 1905, containing some 490 refer- from many patients with jaundice requires as long ences, Paul Morawitz summarizes the work done as 30 to 40 seconds or longer. Quick concluded during the previous half century by Andrew Bu- that the prothrombin concentration is reduced in chanan, Alexander Schmidt, E. Bruecke, P. Denis, the plasma of many patients with jaundice but is L. Fredericq, J. Bordet, L. Wooleridge, 0. Ham- normal in the plasma of patients with hemophilia. marsten, M. A. Dastre, C. A. Pekelharing, M. Arthus and many others, and proposed what we Am. J. Med. Sci. 190: 501, 1935 now call the "classical" theory of blood clotting, A STUDY OF THE COAGULATION DEFECT IN HEMOPHILIA shown in Figure 1. AND IN JAUNDICE.* This theory proposes that in the presence of Bi- An.'%Ni5,.v.-1. (QUIC(K. I'll.D., N1.D).. I.NNISTANT PROESSONR (IF I'iiiltMl'llANY, MAROEQTTD I'NIl'FrlIl'rY SCHao. OF ionized calcium and thromboplastin, prothrombin VEDICI(INF S11.'MVIWAU\ EF'.. is converted to thrombin, which then converts ri r r .4011131 ~tr raIIFNItI; ot'lt:N. ii'11-"1'.1t:IFs 1 i^'114. roI,N INl't("O lX ItRI.IC.NI. fibrinogen to fibrin. (The Roman numerals, which I'AITH01.414;V, O'tII F:; s' V'1V\W91N'1 \N D SURI.'14;FN',J ( I1X\1'11|1\ I'NIVEH:SITY. A^ND I have inserted after the names of these substances, NN.FlFl1X\. BANo'10 )FT1, NI .1) refer to the system of nomenclature of blood clot- aI)lhisrIII'tli * o.'FRu, FIF"I'II iF:Nt': 1 11N1S.rI.. 'II (1I'F: 1 \:N.I I' 'I.INI'I. ting factors recently adopted by the International NEF:\' \ t1K CITV'W. Committee on Hemostasis and Thrombosis). (Irinn thr IX-ijartmllit if Smiigryii*i, l FifthAvrtriltll 114isi,ital-)t IPiw; B:Vs ill t he ,Ia1lagellient f henmrrhage ill hemohilia andi( in certain types of obstructive jaundice has been Iniuch impeded by the lack o. a )ractical concept of 11i i1 ( lotting. A distilct nieed Tissue Thromboplastin (111) exists for at simple theory which u1lay slerve as a guide ill the lillical study sof disturbances inl the clotting function (f the lilool. Recently Ca++ (IV) ANohifisch' ill ak thoruigh oll critical review of the coalgillaltiol (if Prothrombin (11) * Thrombin h)lo(xl conistructeil a tIlt4vwhich iln(crl)orates the esselitial i(leals of Bordet.1 \oraWitz3 1'11(Iothers. According to tihis oemicePit tiot- tillg p)roCeds ill tW0o phaVSelms: 1. Prothronunin + thromuulxopilastini + calcium = thromnbin II. Fibrinogen -4+ thronmbin = fibrin Thrombin - Fibrin Fibrinogen (I) Figure 2.-Title page of article by Dr. Armand J. Figure 1.-Morawitz' "classical" theory of blood clot. Quick which introduced the one-stage method for the ting, 1905. measurement of plasma prothrombin.

CALIFORNIA MEDICINE 257 Shortly after this test was introduced both vitamin In -1947,- Dr. Paul Owren published his- mag- K and dicumarol were synthesized and made nificent treatise concerning the investigations on available for general use. Quick's test proved to a new blood clotting factor which he had con- be ideal for measuring their effects. I learned to ducted in seclusion in Nazi-occupied Norway dur- do this simple but very useful little test and there- ing World War II (Figure 3). The subject of his upon became the local blood clotting expert. studies was a 29-year-old woman who had suf- fered all her life from a bleeding tendency charac- ACTA MEDICA terized by severe epistaxis, hematomata, bleeding from lacerations and menorrhagia. She had a pro- longed "prothrombin" time by Quick's test but SCANDINAVICA was shown by other methods to have a normal amount of prothrombin in her blood. Owren SUPPLEMENTUM CXCIV (194) showed that the defect in this woman's blood clot- ting was not a deficiency of prothrombin but rather a delayed conversion of prothrombin to thrombin. Her prothrombin could be converted to thrombin THE COAGULATION if a small amount of normal plasma, from which OF BLOOD all of the prothrombin had been removed by ad- sorption, was added to her plasma. This new clot- INVESTIGATIONS ON ting factor, which Owren called proaccelerin, was A NEW CLOTTING FACTOR not removed from normal plasma by adsorption, and it disappeared rapidly from normal serum. By PAUL A. OWREN At about the same time Quick noted that the OSLO. NORrAX prothrombin time (as measured by his test) grad- Figure 3.-Owren's article in which he described pro- as normal blood was he accelerin (now called Factor V). Source: Acta Medica ually lengthened stored; Scandinavica, Suppl. 194:1-327, 1947. attributed this to the disappearance of what he

J. Clin. Invest. 30: 596, 1951

CONGENITAL SPCA DEFICIENCY: A HITHERTO UNRECOGNIZED COAGULATION DEFECT WITH HEMORRHAGE RECTIFIED BY SERUM AND SERUM FRACTIONS I By B. ALEXANDER, R. GOLDSTEIN, G. LANDWEHR, AND C. D. COOK wITH THE TEHNICAL ASSISTANCE ou EUNICE ADDELSON AND CLAIRE WILSON (From the Yamins Research Laboratory, Betl Israel Hospital; from the Children's Medkca Center, and from the Departments of Mediine and Pediatrics, , Boston, Mass.) (Submitted for publication January 17, 1951; accepted, March 26, 1951)

INTRODUCTION accelerator (spca) described in earlier reports T}he importance of non-prothrombin plasma and (58). serum constituents in the physiological conversion CASE ItEPORT 2 of prothrombin to thrombin has been recognized R (No. 309157), a four year old white girl of Scotch, only recently. A hemorrhagic disorder, called Irish, French and Dutch extraction, was admitted to the "parahenophilia" (Owren's disease), is associated Children's Medical Center, Boston, on June 13, 1950, with bloody stools and hanaeis of three days' and six with an elevated prothrombin time attributable to hours' duration, respectively. The family history was deficiency of a plasma component termed "Fac- cotrbutory only in the fact that the mother stated tor V" (1, 2). Inadequacy of plasma Ac-globu- that she and her brother bruised easily and that the ma- lin, required for rapid evolution of thrombin, has ternal grandfather died of intestinal bleeding following trauma. Figure 4.-Title page of paper by Goldstein et al. which first described SPCA (now called Factor VII).

258 APRIL 1967 * 106 * 4 called the "labile factor" of prothrombin. Walter testing purposes. During the next few years a num- Seegers and his coworkers also found that their ber of cases were diagnosed as instances of con- preparation of purified prothrombin could be con- genital deficiency of Factor VII on the basis of the verted to thrombin much more rapidly in the results of tests employing such plasma. After a presence of a factor they found in beef serum time it became apparent, however, that one group which they called accelerator globulin, or AcG. of these patients differed from the remainder in Within a short time it became apparent that the certain essential details. activities attributed to proaccelerin, labile factor In 1957, Cecil Hougie, E. M. Barrow and John and accelerator globulin all arose from the same B. Graham in North Carolina segregated these factor. In the present system of nomenclature this two types of cases and clearly demonstrated that is known as Factor V. one type was identical to that observed in Alexan- In 1951 Dr. Benjamin Alexander and his asso- der's original patient with Factor VII deficiency, ciates at the Beth Israel Hospital in Boston de- while the other was caused by the absence of a scribed the congenital deficiency of a new clotting previously unknown factor which they called the factor which they called serum prothrombin con- Stuart Factor, after the family name of their first version accelerator or SPCA (now known as pro- patient in whom its absence was demonstrated convertin or Factor VII). This factor was found (This is now known as Factor X) (Figure 5). to be deficient in a four-year-old girl who suffered Their first patient was a 36-year-old farmer who from easy bruising, spontaneous epistaxis and re- had had frequent epistaxis and hematomata, and current bouts of gastrointestinal bleeding (Figure occasional mild hemarthrosis. They showed that 4). They showed that this factor, like Factor V Factor X was required for coagulation of blood was required for the conversion of prothrombin to whether tissue thromboplastin was present or not. thrombin. Unlike Factor V, it was quite stable on Factor VII, on the other hand, was found to be storage, was present in human serum and was required only as a co-factor of tissue thrombo- readily adsorbed onto barium sulfate. Advantage plastin. It was not required for the intrinsic pro- was taken of its adsorbability to prepare an arti- duction of blood thromboplastin. It was apparent, ficially depleted normal plasma as a substrate for then, that at least three factors (Factors V, VII J. Clin. Invest. 36: 485, 1957

STUART CLOTTING DEFECT. I. SEGREGATION OF AN HEREDITARY HEMORRHAGIC STATE FROM THE HETEROGENEOUS GROUP HERETOFORE CALLED "STABLE FACTOR" (SPCA, PROCONVERTIN, FACTOR VII) DEFICIENCY By CECIL HOUGIE,1 EMILY M. BARROW, AND JOHN B. GRAHAM (From the Department of Pathology, University of North Carolina, Chapel Hill, N. C.) (Submitted for publication August 24, 1956; accepted November 29, 1956)

In 1951, Alexander, Goldstein, Landwehr, and ciency and hypoproconvertinemia may be a hetero- Cook (1) described a patient with a congenital geneous group. hemorrhagic diathesis of an unusual type. There The following paper describes a follow-up was a normal prothrombin concentration, but a study of one of the cases (R. S.) previously stud- prolonged prothrombin time which could be cor- ied by Lewis, Fresh, and Ferguson (9). It will rected by serum and serum fractions but not by be shown that the factor deficient in this patient BaSO4-adsorbed plasma or serum. The clotting is similar to but not identical with the one lacking factor deficient in the plasma of this patient dif- in the patient of Alexander (1), and is identical fered, therefore, from prothrombin, Factor V, ac- with the factor Crockett's patient lacks (25). celerator globulin (Ac-G), and antihemophilic fac- These findings establish the heterogeneity of the tor (AHF). The normal analogue of the factor group of patients listed in Table I. The factor Figure 5.-Title page of paper by Hougie et al. in 1957 which described the Stuart Factor (now called Factor X).

CALIFORNIA MEDICINE 259 and X) were required for conversion of prothrom- Factor X bin to thrombin in the presence of an optimal Factor VII amount of tissue thromboplastin and calcium, as Factor V shown in Figure 6. Tissue Thromboplastin (111) It was clear also that extracts of tissues were Ca ++ (IV) capable of supplying optimum amounts of throm- Prothrombin (11) f Thrombin boplastin, but it remained a mystery how this thromboplastic activity developed in blood kept Tlhrombin free of contact with the tissues and allowed to clot Fibrinogen (I) > Fibrin in the test tube. How the defect in hemophilia Figure 6.-Three factors, V, VII and X were found to be required to convert prothrombin to thrombin in the fitted into the classical scheme of clotting remained presence of an optimal amount of tissue thromboplastin an enigma. In the early days it was thought that and calcium. thromboplastin was released from the breakdown of platelets and that in hemophilia the platelets In 1946, Professor E. J. Cohn and his cowork- failed to disrupt in a normal manner. ers at Harvard University published a method for the separation of plasma proteins by alcohol frac- However, in a series of papers from the Thorn- tionation. It was soon found that AHG was present dike Memorial Laboratory in Boston between in Cohn's Fraction I. 1936 and 1939, A. J. Patek, R. P. Stetson, F. H. In 1948, we were engaged in testing the AHG L. Taylor, F. J. Pohle and E. L. Lozner clearly potency of Cohn Fraction I, which was supplied demonstrated that an euglobulin fraction could be to us by the Cutter Laboratories in Berkeley. One prepared from normal platelet-free plasma which of our hemophiliac patients was an 11-year-old would correct the defect when added to hemophilic boy named Kent Kincaid, an only child with no blood (Figure 7). This substance was called anti- family history of bleeding, who had a decidedly hemophilic globulin (AHG) (now known as Factor prolonged clotting time and had been in hospital VIII), and it was thought that the platelets and over a hundred times for major hemorrhagic epi- AHG united in some way to produce blood throm- sodes. He had impressed his pediatrician, Dr. boplastin. George Bates, however, not only by the frequency

J. Clin. Invest. 15: 531, 1936

HEMOPHILIA. I. THE ABNORMAL COAGULATION OF THE BLOOD AND ITS RELATION TO THE BLOOD PLATELETS By ARTHUR J. PATEK, JR., AND RICHARD P. STETSON (Fronm the Thortndike Memorial Laboratory, Second and Fourth Medical Services (Harvard), Boston City Hospital, anid the Department of Medicine, Harvard Medical School, Boston) (Received for publication June 23, 1936) The one abnormality constatntly found in hemo- citrate in distilled water that the final concentration was philia is an inability of the blood to coagulate in 0.25 per cent. Conditions for the observation of clotting a normal manner. This defect is demonstrable times were kept rigidly uniform. Acid-cleaned test tubes usually by a prolonged clotting time, which in 100 X 12 mm. were used. The test blood or blood con- stituent was pipetted to the bottom of the tube, which turn determines the various manifestations of the had been freshly rinsed with 0.85 per cent solution of disease. Further understanding of this disease sodium chloride. Hemophilic blood was then obtained by depends, therefore, upon knowledge of the cause venipuncture, the needle of the syringe removed, and of the prolonged clotting time. Most investiga- exactly 2 cc. added to the tube containing the test mate- tions of the blood clotting nmechanism in hemo- rial-discarding, however, the last three or four cubic philia have been made with methods involving the centimeters of blood in the syringe. Control clotting Figure 7.-Title page from a paper by Patek which first described antihemophilic globulin (now called Factor VIII).

260 APRIL 1967 * 106 * 4 of his hemorrhages but also by the unusually good Blood 3: 101, 1953 response he invariably showed to the transfusion of normal whole blood or plasma. Most of our Plasmtia Thromhoplastin Comilponent (PTC) Fraction I preparation showed good antihemo- A Hitherto Unrecognizetl Blood Coagulation Factor philic potency when tested on one or another of Case RIepsort of ITRC Deficiency /WY SIDNEV G. WHIITE, MAJOi, MA.., t.SA1, PI>.\L M. AGGLAEIt, MI). our hemophilic patients. However, an occasional AND MAIIY BETH (.LEN:DI:NINo, I'ii.D). one was impotent. We did not attach any particu- I 1NlIt.I.11INARNl REPORT)l'S3'we Iesvrilxl a nw hlotnar-liaigic dliause lar significance to the observation when the first 1 -aii$iI Ii, Iii tfi.i iIlilt-o.r a iioxviousii tinre,igiiz(elusli^,lainthnllo- p>lastinl vozmponen^t (lMl() widl ollilled : lItillill or.. the partiail plirifivatio} and; preparation we tried on Kent was incapable of 4ioll itllt liioll of tile I" ( 'fcttr>t. It is lepiltlips .ol lietseit ptltort al. - plify the{ dem.rip)tioti of ilit- livillonl-hal;gir flialtheSis ill Zlil- patlient li tolRd prs4.)1^st. correcting his defect. Some months later we tested the dIetails of ixlr(iexlitiittal p diaiitire ililit' inivestigati sl of hlixs iimgkila- a new lot of Fraction I on Kent; again we found tioll defect. that the preparation had no power to correct his Figure 9.-Further studies on PTC deficiency. coagulation defect, but this time we found that the same lot was potent when tested with the blood of the clotting feature missing in Kent's case (Fig- other hemophiliac patients. It was then but a sim- ure 8). You will note that this was a most co- ple matter to show, by direct plasma-mixing tests, operative venture. Dr. Bates, of the Department that Kent's blood and that of other hemophiliacs of Pediatrics, I have mentioned already. Drs. Er- were mutually corrective. We knew then that Kent nest Page and Mary-Beth Glendening of the De- did not have hemophilia and subsequently we were partment of Obstetrics and Gynecology were in- able to show that he had no other defect in clotting volved at that time in their pioneering studies on time at that time. the phenomenon of disseminated intravascular co- In 1951, Dr. Sidney White joined us as a re- agulation in patients with placentae previa, and we search fellow, and in May of 1952 we published were able to enlist their interest in our strange case. the first report showing the essential features of I would also like to express my particular appre-

Proc. Soc. 1p. *Bl. & Ned. 79:692, 1952 Plasma Thromboplastin Component (PTC) Deficiency: A New Disease 'Resembling Hemophilia.* (19488) I'AUL M. AGGELER, SIDNEY G. WHITE,t 'MARY BETH GLENDENING, ERNEST W. PAGE, TILLIE B. LEAKE, AND GEORGE BATES. From thte Divisions of Medicine, Obstetrics and Gynecology, and Pediatrics, tniversity of Californid School of Medicine, San Francisco. In current concepts of blood coagulation tissues, muscles, joints, gastrointestinal tract, mechanism(l), thromboplastin is said to be pharynx, and retroperitoneal region. In all liberated from fixed tissue sources through in- instances prolonged coagulation time was jury. or from blood itself by interaction of markedly shortened and the hemorrhage a platelet factor and a single inactive plasma rapidly controlled by the transfusion of whole factor (2.3). The coagulation defect in hemo- blood, fresh or frozen plasma, or freshly philia is thought to be due to absence of the lyophilized plasma. The first suspicion that latter plasma factor. We obtained evidence the patient did not have true hemophilia from the study of the coagulation defect of arose when the coagulation time was not sig- a patient. who had previously been thought nificantly shortened by potent preparations to suffer from hemophilia, of the presence of of anti-hemophilic globulin (Cohn's fraction a second plasma factor which appears to be I). The prolonged coagulation time is ac- as important biologically as the anti-hemo- companied by a markedly defective prothrom- philic factor for the production of thrombo- bin utilization(4,5), which can be corrected plastin in the blood. by addition of small quantities of normal Figure 8.-First article by the author and his coworkers on PTC, a new clotting factor (now called Factor IX).

CALIFORNIA MEDICINE 261 ciation of Mrs. Tillie Leake,* who was my very Dec. 27, 1952 BRUTISH capable blood clotting research technician at that MEDICAL JOURNAL time. In February of the following year we published CHRISTMAS DISEASE a more detailed account of these investigations A CONDITION PREVIOUSLY MISTAKEN FOR (Figure 9). We named the factor plasma throm- boplastin component, or PTC. It is now known as BY Factor IX. In contrast to AHF it could be readily ROSEMARY BIGGS, M.D. adsorbed onto barium sulfate, was present in the A. S. DOUGLAS, M.R.C.P. serum and was found in Cohn's Fraction IV rather R. G. MACFARLANE, M.D. than Fraction I, and in the fraction of plasma pre- Radclifle Infirmary. Oxford cipitated between 40 per cent and 50 per cent sat- L. V. DACIE, M.D., M.R.C.P. uration with ammonium sulfate rather than the 0 W. R. PITNEY, M.D., M.R.A.C.P. to 25 per cent saturated fraction. Unlike AHG, this Postgraduate Medical School. LondSont, W. 12 factor was quite stable on storage. Plasma from C. MERSKEY, M.D., M.R.C.P. outdated blood bank blood could be used for treat- UniversitY of Cipetowl'n1 ment, rather than the very fresh or freshly frozen AND in plasma required hemophilia. The beneficial J. R. O'BRIEN, D.M. effect of transfusion on the clotting time persisted South Devois and E;ast Cornw-all Hospital, Plymouthl for a much longer time than in AHG deficiency. In the 27 December 1952 issue of the British lHacmophilia is a severe bleedilng disease of males %xith a sex-linked recessive inheritance. Laboratory tests show Medical Journal, there appeared a paper appropri- a prolonged whole-blood clotting-time and deficient ately entitled "Christmas Disease: A Condition conversion of prothrombin to thrombin durinig clottinig Previously Mistaken for Hemophilia," by Dr. When clinical, genetic, and the usual laboratory features Rosemary Biggs and her associates are all present the diagnosis of haemophilia is generally (Figure 10). believed to be simple. This paper described seven patients with the same condition we had observed in Kent, and in addi- Figure 10.-Tide page of article by Dr. Biggs and her coworkers describing "Christmas" factor (later shown to *Deceased. be same as PTC).

Pr. S c. p. Blol. & Md. 82:171, 1953

New Hemophilia-like Disease Caused by Deficiency of a Third Plasma Thromboplastin Factor.* (23957)

ROBERT L. ROSENTHAL, 0. HERMAN DRESKIN, AND XNATHAN ROSENTHAL.' (Introduced by W. Antopol) From the Laboratories of the Joseph and Ilekn V'eamians Let v Fosndation. Betli Israel Hospital. New Y'ork City and Dept. of Internal iMslioine a'ad Pathology, Jewisk Hospital, Cincinnati, 0.

Hemophilia has been generally recognized with platelets to forn thromboplastin(1-3). as a hereditary disease characterized by the This factor. designated as anti-hemophilic deficiency of a plasma factor which reacts irlobulin (AHG). is essential for the normal conversion of prothrombin to thrombin(4,5). * This work was aided in part by a grant from Recently. evidence for a second plasma throm- the National Heart Institute. National Institutes of boplastin factor has been presented on the Health. Public Health Service. basis of 2 reports of male patients with hemor- + Consulting hematologizt, Mt. Sinai Hospital. rhagic disease and coagulation findings similar . to hemophilia except that the blood of these Figure 11.-Title page of article by Rosenthal et al. describing plasma thromboplastin antecedent (now called Factor XI).

262 APRIL 1967 * 106 * 4 tion showed that it was transmitted as an X-linked recessive trait, exactly as in classical hemophilia. Platelet Phospholipid Their first patient was a five-year-old boy whose Ca++ (IV) family name was AHF (Vil) Christmas. PTC (IX) Bloo A short time later, Dr. Robert Rosenthal of New PTA (XI) Thromboplastin York and his associates described another defect Stuart (X) in clotting in three patients in one family, a 50- Hageman (XII) year-old man and his two nieces, who had shown Proaccelerin (V) since childhood moderate evidences of a bleeding Blood Thromboplastin tendency, marked chiefly by bleeding after tooth Ca (IV) extractions (Figure 11). Like AHF and PTC, this Prothrombin (11) a Thrombin factor seemed to be required for the production of blood thromboplastin but was not necessary for Fibrinogen (I) Thrombin w Fibrin clotting with tissue extracts. They called it plasma Figure 13.-By 1957 a revision of the classical theory thromboplastin antecedent, or PTA. It is now of clotting included all the above. known as Factor XI. Like Factor IX this factor was present in serum but unlike Factor IX it was in the blood clotting field. This factor is now known only partially adsorbed on barium sulfate. It also as Factor XII. Its properties were very similar to seemed to be involved in the activation of the clot- those of Factor XI, and it could be distinguished ting mechanism on exposure to glass and similar clearly from Factor XI only by direct plasma-mix- surfaces. ing tests. Finally in 1955 Oscar Ratnoff and Joan Colopy By 1957 a revision of the classical theory of at Western Reserve University described a new clotting would have to include all of the factors defect in clotting in a 37-year-old railroad brake- shown in Figure 13. However, no one ever sug- man named Hageman and in two housewives aged gested that all of these new factors united together 46 and 50 years (Figure 12). Curiously, although at once to form the prothrombin converting prin- the clotting time was decidedly prolonged, none ciple. From the beginning, attempts had been made of these patients or the many that have been de- to study their manner of interaction. At first it was scribed since then with the same condition have proposed that a series of products might be formed, suffered from abnormal bleeding. Why these peo- as shown in Figure 14. ple do not bleed is one of the remaining enigmas However, the concept of even three or four fac-

J. Clin. Invest. 34: 602, 1955

A FAMILIAL HEMORRHAGIC TRAIT ASSOCIATED WITH A DEFICIENCY OF A CLOT-PROMOTING FRACTION OF PLASMA I By OSCAR D. RATNOFF AJND JOAN E. COLOPY (From the Dertmet of Medkice, t Wester Reserve Uxniersity School of Medice, and Uxiversity HosPitals-Cflewlcnd, Ohio) (Submitted for publication October 11, 1954; accepted Dmber 29, 1954) Recently, three patients have been studied in bled after a tonsillectomy when he was 6 yeanrs old, after whmdthe clotting time of venous blood was greatly an injury to the hand at 10, nor after a dental extrac- None of the patients had significant tion, at 36. He had had symptoms and laboratory evidene PTOlOPmd of a duodenal ulcer since 1943, and was to be opert hemrhagic symptoms. The trait was apparently upon for the relief of pyloric obstruction, but this pro- familial and appeared in both sexes. The pa- cedure was not carried out because of the abnor tients' disorder could not be identified with any clotting time. A few months before he was studied, be known bleeding disease. However, in each case, had had black stools for 2 days, but he was taking bis- Figure 12.-Title page of article by Ratnoff and Colopy describing a new factor in blood clotting (now called Factor XII).

CALIFORNIA MEDICINE 263 STEP tors uniting simultaneously in such a manner as No SYNONYM INTRINSIC SYSTEM this was repugnant to most physiologists, and work during the past several years has centered on at- Surface Contact EXTRINSIC SYSTEM tempts to reduce the blood clotting process into a 1 Hageman Xll-Xlla Ill (Tissue Extrad) series of two-factor reactions. Almost all of the experimental data now available are consistent 2 PTA XI-*Xla VIl (Proconvertin) with the theory expressed by Roger Macfarlane, 3 PTC and by E. W. Davie and Oscar Ratnoff in 1964, as 4 AHF Vlill-aVlla/ shown in Figure 15. 5 Stuart t These investigators have proposed a simple 6 Proacceblrin V_~Va waterfall or cascade sequence to explain the func- tion of the various factors in blood clotting. Ac- 7 Prothrombin 11-*la (Thrombin) cording to this theory, clotting is initiated by sur- 8 Fibrinogen I - la (Fibrin) face contact activation of Factor XII, possibly by Co++ Foctor IV) required for stops 3-6. unfolding the molecule. Then each factor in turn Platelet phospholipid required for step 6 and possibly step 4. is converted to a form that has enzymatic activity. Thrombin accelerotes ond perhaps is required for steps 4 and 6. Figure 15.-The "cascade" or "false waterfall" theory The activation occurs in a step-wise sequence with of the mechanism of blood coagulation as described by each newly formed enzyme reacting with its spe- Macfarlane and by Davie and Ratnoff in 1964. cific substrate, converting it to an active enzyme until the final substrate, fibrinogen, is reached. Recent work also strongly suggests that a small There is still some uncertainty regarding the steps amount of thrombin is required to activate Factors involving the interaction of Factors X, V and phos- V and VIII. How this first tiny bit of thrombin is pholipid. Ratnoff believes that the final prothrom- generated is not known. bin converting factor is activated Factor V, as in- The role of the natural inhibitors of blood clot- dicated in the diagram. Other investigators feel ting is not so clear. It is probable that there are that activated Factor X, Factor V and phospho- two lines of defense against intravascular clotting. lipid combine together to form the final prothrom- One consists of the reticuloendothelial system bin converting factor. which appears capable of removing activated in- The key role of Factor X in the clotting mecha- termediates in the clotting mechanism. The other nism has only recently become apparent. This fac- is a series of natural circulating inhibitors of the tor can be activated either by activated Factor activated factors such as the well known anti- VIII in the intrinsic clotting system or by Factor thrombin III. VII and tissue thromboplastin in the extrinsic sys- tem. It can also be activated by Russell's viper Use of Drugs to Inhibit Clotting venom, trypsin, papain and other enzymes. There is much more one could say about other recent developments in the blood clotting field. Surface Contact However, I have chosen to use the remaining time Hageman (XII) o Contact Factor to discuss some aspects of the use of drugs to in- PTA (IX) J hibit blood clotting. Contact Fador 1 AHF (Vill) A list of the oral anticoagulant drugs most com- PTC (IX) > Internediate Product I monly used here and abroad is shown in Figure Stuart (X) I 16. Since the isolation of dicumarol from spoiled Ca++ (IV) J sweet clover by Karl Paul Link and his students Intermediate product I Platelet phospholipid nte*mediate Prodlud 11 in 1939, a large number of coumarin and indane- Ca++ J dione anticoagulant compounds have been synthe- Intermediate Product (11 sized. Since 1942, many thousands of patients Proaccelerin (V) 3 Blood Thromboplastin have received treatment with these compounds for Ca++ Ca++ (IV) a variety of thromboembolic disorders. The two Prothrombin (11) Blood Thromboplastin Thrombin drugs most commonly used in the United States Fibrinogen (1) Thrombin are warfarin and dicumarol. Figure 14.-One of the theories of the mechanism of The pharmacodynamics of this group of drugs coagulation popular in the early 1960's. varies widely, but they all influence the clotting

264 APRIL 1967 * 106 * 4 mechanism in the same manner; they all interfere Previous investigators showed that the disap- with the production of clotting Factors II, VII, IX pearance rate of dicumarol from the plasma is in- and X in the liver. Their effects can all be neutral- creased by the concomitant administration of bar- ized with large doses of vitamin K. The physio- bital compounds and suggested that this resulted logic response to these drugs can be measured from "enzyme stimulation" in the liver. The abil- conveniently by Quick's original "prothrombin" ity of one drug to reduce the pharmacologic activ- test which reflects the combined activity of Factors ity of another by stimulating its metabolic inactiva- II, V, VII and X. These latter factors are some- tion is well known. Drugs capable of causing this times referred to as the "prothrombin complex." effect act by increasing the amount of drug metab- There are many difficulties associated with the olizing enzymes in liver microsomes. use of these drugs. Several years ago Dr. Robert The administration of large doses of barbitals O'Reilly and I embarked on a study of some of to rats increases the amount of coumarin drug these problems. So far, we have limited our investi- metabolizing enzymes in their liver homogenates. gations to dicumarol and warfarin. We have at- There is no direct proof, however, that a similar tempted to differentiate between conditions which mechanism can be activated by barbitals in man. alter the clotting factor response by influencing We have performed in human volunteers a num- the absorption, distribution or elimination of the ber of experiments which suggest to us that absorp- drugs and those which alter the response by di- tion of dicumarol is impaired by barbital ad- rectly affecting the clotting synthesizing mecha- ministration. We first determined the pattern of nism in the liver. response of an oral dose of 600 mg of dicumarol. Theoretically, incomplete absorption of these The subjects were then given 400 mg of hepta- drugs might occur in patients with vomiting, diar- barbital on each of three evenings; on the morning rhea or malabsorption syndromes. We have ob- of the fourth day the same dose of heptabarbital tained some evidence that barbital compounds may was given, followed by 600 mg of dicumarol an also cause decreased absorption. hour later. The initial rate of absorption of dicu-

GENERIC NAME CHEMICAL NAME TRADE NAME COUMARIN ANTICOAGULANT DRUGS

Sodium Warforin Sodium salt of 3-(a -acetonylbenzyl)-4-hydroxycoumarin Coumadin (Endo) Panwarfin (Abbott) Dicoumarol 3,3-methylenebis [4-hydroxycoumarin]) Dicumarol (Abbott) Bishydroxycoumarin Dicumarol (Lilly)

Phenprocoumon Liquamor (Organon) Marcoumor (Roche)

Nicoumalone 3449 -acetonyl-4-nitrobenzyl)-4-hydroxycoumarin Sintrom (Geigy) Acenocoumarin Cycicoumarol 3,4-(2' methyl-2-methoxy-4'-phenyl)- Cumopyran (Abbott) dihvdroovranocoumarin

Ethyl Biscoumacetate 3,3'-carboxymethylene bis -(4-hydroxycoumarin) Tromexan (Geigy) ethyl ester

INDANEDIONE DERIVATIVES Phenindione 2-ph.nylindan*-1:3-dione Dindevan (Evans) Hedulin (Walker) Danilone (Schieffelin) Diphenadione 2-(diph*nylacetyl)-indote-1:3 dione Dipaxin (Upjohn) Anisindione 2-p-anisyl-indane-1:3 dione Miradon (Schering) Figure 16.-List of the most commonly used oral anticoagulant drugs.

CALIFORNIA MEDICINE 265 marol appeared identical in the two experiments, but when heptabarbital was given the duration of absorption was much shorter and an increased amount of dicumarol was found in the stool. A typical experiment is shown in Figure 17. The results without heptabarbital are shown by the open circles and cross-hatched bars; the peak drug level was 52 mg per liter of plasma and the 50 biologic half-life of the drug was 62 hours; 24 per 40 cent of the dose was excreted in the stool, 9 per cent within the first 24 hours. The results obtained when the subject was pretreated with heptabarbital are shown by the solid circles and bars; the peak blood level was only 40 mg per liter of plasma and the biologic half-life 30 hours; 50 per cent of the dose was excreted in the stool, 48 per cent of it 000 within the first 24 hours. The prothrombin time response was significantly less when the subject was pretreated with heptabarbital. aO 0o P. Now it so happens that dicumarol is one of the 50 1 , lis aoln few drugs known whose disappearance rate is di- rectly proportional to the dose administered. The increased rate of metabolism of dicumarol when 30-o 1 0 2 0 3 the barbital was administered could have been 25- ~ ~ AY caused, therefore, by the smaller amount of di- cumarol that was actually absorbed. It is not neces- sary to invoke the concept of enzyme stimulation to account for it.

"It) i.I LA. Thgue courseA evprients whndc tn the effcsubject qE

now stabilized on a dally maintenance dose of 60

mg of dicumarol, was given heptabarbital is shown

0 in Figure 18. Four hundred milligrams of hepta-

Ux barbital was taken each evening, one hour before the dally dose of dicumarol, on eight successive days. The dicumarol concentration of blood and

stool specimens collected each morning was meas- * WITH HEPTABARBITAL toE 40 a WITHOUT HEPTABARBITAL ured, and the prothrombin complex activity of the T0Q 30 plasma was determined. Allowing a period of 24 So 20 I10 to 72 hours for passage of dicumarol through the m -i gut, the sequence of events appears to be: ( 1) A WITH HEPTABARBITAL decrease in absorption ° WITHOUT HEPTABARBITAL of dicumarol, (2) an in- c U) 35 009 20 crease in its excretion in the stool, (3) a lowered cc0 A L 6- ! 4. M A L RAN.G. 6;-, P.Ioo; concentration of drug in the blood, and (4) a de- I-Liw bE 24 48 72 96 120 t creased prothrombinopenic response in the plasma. HOURS " We do not yet have sufficient data to determine Figure 17.-An experiment indicating the effect of barbital on the metabolism of a single large dose of whether failure of absorption accounts for the dicumarol. Source: Aggeler, P. M., and O'Reilly, R. A.: entire effect of barbital compounds on dicumarol Pharmacologic basis of oral anticoagulant therapy, Thromb. Diath. Haemorrh., 21:227-256, 1966. metabolism or whether the same effect can be

266 APRI L 1967 * 106 * 4 demonstrated with other oral anticoagulant drugs. The disposition of these drugs has been studied directly in a variety of animal species; in general, 10 no unusual pattern has been found, and no organ has shown higher concentration than plasma. In 47 most studies the concentration of anticoagulant tC t 3 drugs has been higher in liver and kidneys than in W 20 other organs. Anticoagulant drug levels in liver tissue and in plasma normally decline at approxi- Il mately the same rates. We have found that the It volume of distribution of warfarin and of dicu- 12 marol when given in small doses is 12.6 per 411Id 40 0 cent of body weight-about the same as for serum 30 18t0).0 Id 20 35 to. albumin. -- 1 LI RANGE skAAi 4.N. : 0 14 ,:.%Onm 09 These drugs are extensively bound to serum 12' Z * V A.M. albumin. They are not present in appreciable 24 48 72 96 120 144 168 amounts in red blood cells or cerebrospinal fluid HOURS Figure 20.-Potentiation of the prothrombinopenic ef- in man. Transplacental transfer and the passage fect of warfarin by phenylbutazone. of unchanged drug into breast milk occurs. Nei- ther dicumarol nor warfarin is excreted unchanged The frequency distribution of the biologic half- in the urine. Normally up to 30 per cent of the life of warfarin in 24 normal subjects is shown in dose of dicumarol may be found in the stool. We Figure 19. It is skewed somewhat toward the more have never found unchanged warfarin in the stool. rapid disappearance rates. We have recently in- We know of no condition which can alter dis- vestigated a patient who required 40 mg of war- tribution of these drugs in the body. However, we farin per day compared with the average daily dose are investigating the possibility that the amount of of 7.5 mg. The biologic half-life of the drug in his plasma protein-binding of these drugs may be case was only five and a half hours. It seems likely changed by the administration of other com- that some instances of extraordinary sensitivity or pounds, and that this may lead to an altered pro- resistance to the prothrombinopenic effects of thrombinopenic response. these drugs may be caused by extreme variations There is a good correlation between the dis- in their rate of catabolism. Such variations could appearance rate of warfarin and the maximum be on either a genetic or an environmental basis. prothrombin response in normal subjects. In a Few diseases are known to affect the elimina- group of 24 normal subjects the mean half-life of tion rate of these drugs. Although impaired liver warfarin was 44 hours and the standard deviation function may profoundly increase the physiologic 9.75 hours. response, the disappearance rate of the drugs themselves is normal in patients with liver disease. 8 The disappearance rate is also normal in patients (f) 7 with severe azotemia. We have been unable to 6 detect unchanged warfarin in the urine even when 1)0 5 massive proteinuria was present. Lf) A pronounced increase in the response to cou- 4 marin anticoagulant drugs occurs when pyrazolone 0~ 3 L& compounds (phenylbutazone and oxyphenylbuta- 2 zone) are administered concomitantly. Murray Weiner and his associates recently stated that oxy- phenylbutazone slowed the disappearance rate of dicumarol in some subjects and that this was re- T2 OF WARFARIN IN HOURS sponsible for the enhanced prothrombinopenic re- Figure 19.-Frequency distribution of biologic half- sponse. They suggested that this might result from life of warfarin in normal subjects. Source: Aggeler, P. inhibition by the pyrazolone drugs of enzymes in M., and O'Reilly, R. A.: Pharmacologic basis of oral anti- coagulant therapy, Thromb. Diath. Haemorrh., 21:227-256, the liver which metabolize the coumarin anti- 1966. coagulants.

CALIFORNIA MEDICINE 267 We have regularly found decided potentiation of the prothrombinopenic effect of warfarin and O N-2 P.Q dicumarol by phenylbutazone and oxyphenylbuta- A N-2 L V. 0 0 P-3 P.O. zone, but, in contrast to Weiner, we have found )h. AP-3 LV. an increase rather than a decrease in the rate of 10 disappearance of the coumarin drugs from the 1L~ plasma. The results of one such experiment are 0iI-u 20 shown in Figure 20. A normal subject was given -K=-0.07 30 200 mg of phenylbutazone thrice daily for 11 days hi. K - 0.06 a a A before and during the entire course of observation 4..' 40 after the oral administration of a standard dose of 1.5 mg of warfarin per kilogram of body weight. s0 A :)0 Although decided potentiation of the prothrom- 2=a binopenic effect occurred in the presence ofphenyl- i0. butazone, the rate of elimination of warfarin from 70 _/ These t the plasma was speeded rather than slowed. I I I I I I I results are shown in Figure 20 by the line drawn qw%f 25 50 100 200 400 700 1500. DOSE OF WARFARIN through the open circles. The control experiment IN mg without phenylbutazone is shown by the line Figure 22.-A comparison of prothrombinopenic re- drawn through the closed circles. We are investi- sponses to increased doses of warfarin in a normal sub- gating the possibility that phenylbutazone poten- ject and a patient with hereditary resistance to the drug. P.O. = Oral. I.V. = Intravenous. Source: O'Reilly, R. A., tiates the action of warfarin by displacing warfarin Aggeler, P. M. Hoag, M. S., Leong, L. S., and Kropatkin, from its binding to plasma albumin in vivo, mak- M. L.: Hereditary transmission of exceptional resistance to coumarin anticoagulant drugs: first reported Kindred, ing increased quantities of free drug available for New Engl. J. Med., 271:809-815, 1964. excretion, biotransformation and biologic action at specific drug receptor sites in the liver. include phenylramidol HCI, clofibrate, chlorpro- Other drugs have been reported to cause an in- mazine and the C-17 alkylated anabolic steroids. creased prothrombinopenic response to the oral In most instances the mechanism of this poten- anticoagulant drugs and doubtless new ones will tiation has not been studied, but in the case of be discovered. Those mentioned in the literature the anabolic steroids Kalevi Pyoriilii reported that it was not associated with any alteration in the ,00 4 metabolism of the coumarin drugs. 70 ;< WARFAtIN, ORALLY i Alterations in the prothrombin response which q L. _ a2.5mg=1.SM9/h9 40 - x. A -A660mg/=I0my/lg "v.* are not dependent upon variations in the metabo- '4- dF;z._ lism of the drugs are well known. These include rIQ e~~~~~ the increased sensitivity of patients with impaired I'. liver function or diseases causing vitamin K defi- c1z ciency. In these conditions the increased response "'4- is caused by the already impaired production of clotting factors in the liver. Extreme variations in the dietary content of vitamin K can also alter the response to the oral anticoagulant drugs. The re- 2 70'r 8 sponse to these drugs is also increased in febrile j" 5 o 9 states and in hyperthyroidism and is decreased in I8 :1z;t 30 hypothyroidism, presumably because of corre- i 20 35 - ., i 0 4 L. L- I ALaat . sponding changes in the turnover rates of the rele- it z ...... ::i, : . ::: . . .i ...... I.o W. iZ c vant clotting factor proteins in these conditions. 0 24 48 72 96 120 144 HOURS The ability of the clotting factor synthesizing Figure 21.-An experiment demonstrating lack of re- mechanism in the liver to respond to oral anti- sponse to a standard dose of warfarin, but the satisfactory response to a very large dose in a patient with geneti- coagulant drugs is also under genetic control. Sev- cally-determined resistance to the action of the drug. eral years ago we discovered a family in whom Source: O'Reilly, R. A., and Aggeler, P. M.: Coumarin anticoagulant drugs: hereditary resistance in man, Fed. extraordinary resistance to the coumarin drugs Proc. 24:1266-1273, 1965. was on this basis. The propositus was a 73-year-

268 APRIL 1967 * 106 * 4 old oil prospector named Harry Martin, who had were given only 1.5 of the drug per kilogram of sustained an acute myocardial infarction and was body weight. When this was done, the concentra- found to be resistant to ordinary doses of warfarin. tion of warfarin in the patient's plasma rose pro- He was given a single standard oral dose of 1.5 portionately (shown by the dotted line in the up- mg of warfarin per kg of body weight (Figure 21 ). per figure). The concentration of warfarin in plasma and the The patient was tested with a series of orally prothrombin activity were determined serially and and intravenously administered doses of warfarin compared with the results obtained with the same ranging from 40 to 1,000 mg (Figure 22). The dose of warfarin in a group of 23 normal subjects results in the patient are shown as open circles and (shown in the cross-hatched area). The concen- triangles and those in a normal subject as solid cir- trations ofdrug in the plasma ofthe patient (shown cles and triangles. In both, the prothrombin re- by the line connecting the solid circles in the upper sponses were proportional to the size of the dose graph) were well within the normal range at all administered, but it required a much larger dose times tested. However, this amount of warfarin had in the patient than in the normal subject to produce only a slight effect on the prothrombin activity in the same effect. the patient (shown in the lower figure). It was During long-term treatment with warfarin over necessary to give the patient a dose of 12 mg of a period of several years it was found that the pa- warfarin per kilogram of body weight before the tient required 145 mg daily to produce a satisfac- prothrombin response (shown by the dotted line tory prolongation of the prothrombin time (Fig- in the lower figure) could be brought within the ure 23). The dose is shown in the lower graph. range of values observed when normal subjects The plasma drug levels are in the middle graph

I00

J 90

.1 so

0

z 60

40 IbI11ilulI*L. JITn I h ~u 30 E =i ITC RAN=OL It10 20 I I I I I I I I I I I

so

1 70

2 ttSO It0)

40

\ 30

iE 20

10

i 200 8 Iso 2 E loo 2 50 I 50 1oo ISO 200 250 300 350 400 450 500 TIME IN DAYS Figure 23.-Plasma concentration of drug and prothrombin response in a patient with hereditary resistance to cou- marin drugs during long-term treatment with warfarin. Source: O'Reilly, R. A., and Aggeler, P. M.: Coumarin anti- coagulant drugs: Hereditary resistance in man, Fed. Proc., 24:1266-1273, 1965.

CALIFORNIA MEDICINE 269 and the prothrombin response in the upper graph. >,. Iut. Both the dose of warfarin and the average plasma S 70 drug concentration (55 mg per liter of plasma) were about 20 times those of the average patient I 40 receiving long-term treatment with this drug. ) 30 The responsiveness of other members of the timoz 20 family was determined and seven of the eight z 15 members tested were found to be resistant. The 0* 1 pedigree of the family showing the incidence of 8. resistance is given in Figure 24. x 60 We have postulated that a pair of allelic genes §Om 40 control the structure or amount of the substances « 20 involved in the synthesis of the clotting factors de- 0 pendent on vitamin K and that resistance to these Z 150 is a drugs transmitted by dominant allele. Whether !tl 100 the gene is located on an X or autosomal chromo- n75 some cannot be discerned from the available data. a00. In terms of the operon theory of F. Jacob and t0 50 the most is that in this J. Monod, likely hypothesis - 30 family an abnormal regulator gene produced an t 20 I I 2tI. I . II6 I II 10I12II I II IA altered repressor substance which has a decreased C 2 4 6 8 10 12 14 affinity for the coumarin anticoagulant drugs, or an DAYS increased affinity for vitamin K or both. Figure 25.-Levels of anticoagulant and prothrombin in this are of more than activity in plasma and the response to vitamin K ther- The findings family apy in a patient suffering from surreptitious ingestion of passing interest, as they document the first instance bishydroxycoumarin. Source: O'Reilly, R. A., and Agge. reported in the literature of determined ler, P. M.: Surreptitious ingestion of coumarin anticoagu- genetically lant drugs, Ann. Intern. Med., 64:1034-1041, 1966. resistance not only to a coumarin but to any drug in man. stopped and prothrombin complex activity re- Finally, I would like to speak for a moment turned to normal. At the time of admission the about a curious hemorrhagic disorder resulting patient's plasma contained a very large amount of from the surreptitious ingestion of these drugs. dicumarol (110 mg per liter). This and subse- The data shown in Figure 25 is from the case of quent dicumarol levels are also shown in Figure a 23-year-old nurse admitted to this hospital in 25. On a number of occasions during her hospital 1962 because of recent onset of generalized pur- stay, the patient denied ingestion of the drug, but pura, epistaxis and gross hematuria. Prothrombin six months later she wrote that she was now under complex activity was reduced to 8 per cent. A de- psychiatric care, that she had been in a severe de- tailed analysis of plasma clotting factors showed a pression and that she had taken 5 gm of dicu- pattern suggesting vitamin K deficiency or the in- marol over a two-week period before entering the gestion of a coumarin drug. Repeated large doses hospital. of vitamin K1 were required before the bleeding The production of factitious disease by a wide variety of means is well known. Individuals have 1 2 3 4 5 6 been found to induce thyrotoxicosis with the self- p * A-t t it administration of large doses of thyroid, diarrhea PROPOSITUS with phenolphthalein and hypokalemia with 2 3 chlorothiazide; some have caused persistent skin II 7 | MALE FEMALE ulcerations by excoriation with acids and others Q 0 AFFECTED BB <3 NOT AFFECTED have produced factitious purpura by self-inflicted o Q NOT TESTED. blunt injury. Within the past four years we have I 2 3 T DECEASED found warfarin or dicumarol in the plasma of 12 patients who had ingested the drug surreptitiously Figure 24.-Pedigree of a family showing incidence of (Figures 26 and 27). All but one were women; resistance to coumarin drugs. Source: O'Reilly, R. A., and Aggeler, P. M.: Coumarin anticoagulant drugs: Heredi- almost all were young adults. One was a physician, tary resistance in man, Fed. Proc., 24:1266-1273, 1965. seven were nurses and one was a pharmacy clerk.

270 APRIL 1967 * 106 * 4 PROTHROMBIN PLASMA Only three of the patients had no medical aflilia- COMPLEX ANTI- CONCENTRATION ADMISSION CASE ACTIVITY COAGULANT OF DRUG OF tion. Two of these, however, had been treated pre- NO. IN % INGESTED IN mg/L AAPPARENT MOTIVE INGESTION viously with anticoagulant drugs. Five of the pa- 1 < 5 WARFARIN 5 TO INDUCE PUPURA NO 2 < 5 of 5 UNKNOWN NO tients had a previous history of bleeding episodes, 3 < 5 8 SUICIDE NO 4 < 5 45 TO INDUCE PUPURA YES which in all likelihood were also caused by sur- 5 <10 1DICUMAROL 22 NO reptitious ingestion of anticoagulant drugs. Wide- 6 <10 ,, 54 UNKNOWN NO 7 <10 80 TO INDUCE PUPURA YES spread ecchymosis was the most common hemor- 8 < 5 *82 UNKNOWN NO rhagic finding, although hematuria, metrorrhagia 9 <10 82 SUICIDE NO 10 <10 ,,1 95 TO INDUCE PUPURA NO and hematochezia also occurred. 11 <10 135 SUICIDE YES The prothrombin complex activity was less than 12 < 5 192 YES Figure 27.-Findings in surreptitious ingestors of cou- 10 per cent in all cases; four of the patients had marin anticoagulant drugs. ingested warfarin and eight dicumarol. An admis- sion of surreptitious ingestion could be obtained situation. In several patients we have been able to from only four patients. People frequently ask us trace purported resistance to coumarin drugs to why anyone would do such a stupid thing as this. simple, willful, secret rejection of the prescribed We really don't know the motive, but in our cases medication. We call this "surreptitious non-inges- it appeared to be either suicide or the production tion" of the drugs. of a factitious hemorrhagic disease in order to at- In summary: I have attempted to show the im- tract attention, to gain sympathy or to obtain nar- portance of the careful study of individual cases cotic drugs. It has been suggested that patients of in the development of knowledge regarding the this type differ from most malingerers and many blood clotting mechanism. I have also reviewed a hysterics in that their disability is not directed to few of the pharmacologic features of the oral anti- immediate material gain or advantage. It has been coagulant drugs which may influence the response theorized that these patients, unlike hysterics, are to therapy, and I have indicated the nature of the capable only of limited repression and dissocia- disorder which results from the surreptitious inges- tion, and that when a certain threshold of mental tion of these drugs. I hope that the intensive inves- conflict is exceeded, they are driven to a consci- tigation of selected patients with blood coagulation ously determined symptomatic solution, whether anomalies will continue to yield information of the original motivating situation is consciously ap- some fundamental significance. preciated or not. Whatever the psychological basis may be, the proper diagnosis of surreptitious in- In closing, I would like to thank my two favor- gestion is important in order that attention may be ite patients, Kent Kincaid and Harry Martin, for focused on the psychiatric rather than the somatic their superb cooperation in our investigations disorder. I would also like to mention that with the which now have extended over a number of years. aid of chemical assays we have found the opposite Mr. Martin is still in good health at the age of 78 years and is probably at this moment out tramping

PREVIC KS the hills in search of that ephemeral oil deposit he ANTl- PREVIOUS CASE COAGULAIkNT HEMORRHAGIC HEMORRHAGIC NO. SEX AGE OCCUPATION THERWA EPIODE MANWSTAIONS swears will some day make us all rich. Kent is 1 M 30 PHYSICIAN NO NO ECCHYMOSES, HNEMA IA now 30 years old and a successful school 2 F 23 NURSE NO NO primary 3 F 38 NURSE's YES NO METRORRHAGIA.KEAH TOCHEIA teacher. He also does part-time work in our labo- 4 F 26 NURSE NO YES ECCHYMOSES, HETOCHEZIA S F 30 PHARMCY CLERK NO NO #0 HEMATURIA ratory. He still has plenty of bleeding problems but 6 F 63 HOUSEWIE YES YES 7 F 28 NURSE NO NO he is very active. Last year he left a picture of S F 39 HOUSEWIFE NO NO 9 F 44 SANK CLERK YES YES himself on skis on my desk, with the cryptic title, 10 F 38 NURSE NO YES 11 F 30 NURSE NO NO ,, HEMATURIA "K.K.-29-yr.-old Caucasian male with no sib- 12 F 30 NURSE NO YES MEIRORRHAGIA lings." However, last night he left me a note on "ALSO PNYSICIAN'S WIFE our laboratory blackboard, which reads "Dear Dr. Figure 26.-Findings in surreptitious ingestors of cou. marin anticoagulant drugs. -w---Www Aggeler, Remember-I don't race cars anymore!"

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