this period no test observations were made of

Transcription

1 THE COAGULATION DEFECT IN HEMOPHILIA. STUDIES ON THE REFRACTORY PHASE FOLLOWING REPEATED INJEC- TIONS OF GLOBULIN SUBSTANCE DERIVED FROM NORMAL HUMAN PLASMA IN HEMOPHILIA' By FREDERICK J. POHLE AND F. H. L. TAYLOR (From he Thorndike Memorial Laboraory, Second and Fourh Medical Services (Harvard), Boson Ciy Hospial, and he Deparmen of Medicine, Harvard Medical School, Boson) I has been suggesed ha he defec in coagulaion of blood in hemophilia resides in a globulin fracion of he plasma (1, 2). Globulin subsance, prepared by isoelecric precipiaion from ciraed, cellular-free, normal human plasma, posseses marked clo-acceleraing properies for hemophilic blood in viro and in vivo. There is a promp decrease in he blood coagulaion ime when a saline suspension of his maerial is injeced eiher inravenously or inramuscularly ino hemophilic subjecs. The coagulaion ime remains low following such injecions for approximaely 6 hours, when i commences o reurn oward he pre-injecion level. Aemps o mainain a reducion of he coagulaion ime wih repeaed injecions of globulin subsance led o he discovery ha a refracory phase occurred afer several injecions had been given (2). During his phase he coagulaion ime was lile affeced by repeaed injecions alhough i was demonsraed ha he concenraion of his cloacceleraing maerial progressively increased in he circulaing blood of he paien. Recovery from his refracory sae was always complee wihin 24 hours afer he las injecion of globulin subsance. The presen communicaion repors furher sudies concerning he refracory period and offers an explanaion of cerain aspecs of his phenomenon. METHODS. The mehod by which he coagulaion ime of venous blood was deermined has been described elsewhere (2). 1 The expenses of his research were defrayed in par by a gif o Harvard Universiy from Smih, Kline, and French Laboraories of Philadelphia. (Received for publicaion Augus 29, 1938) Conrol period. The invesigaion was carried ou on four hemophilic paiens beween he ages of 18 and 48 years who had been under observaion in his clinic for several years. The shores blood coagulaion ime for hese four paiens was 4 minues and he longes was 18 minues. Prior o each se of observaions a conrol period of 48 hours was esablished. If he coagulaion ime of he blood flucuaed appreciably during his period no es observaions were made of ha subjec. Paiens who had had recen hemorrhages were excluded from his sudy. Preparaion of globulin subsance. The globulin subsance was prepared from ciraed normal human plasma in he same manner as previously described (2). In all experimens he dried maerial was suspended in he same volume of.85 per cen sodium chloride soluion as he volume of plasma from which i was derived, rendered serile and free of pariculae maer by Berkefeld filraion. Each lo of globulin subsance, so prepared, was shown o have maximum clo-acceleraing properies for hemophilic blood in viro (2) before i was used parenerally. Sandard es dose. A sandard es dose of 65 cc. of plasma or an equivalen amoun of saline suspension of globulin subsance was used for in vivo sudies. The sandard es dose of globulin subsance conained approximaely 3 mgm. of he dried maerial. Preparaion of dried maerial by he Flosdorf- Mudd echnique. In cerain insances, plasma or he freshly prepared, mois precipiae of globulin subsance was dried by he lyophilic process as described by Flosdorf and Mudd (3). This porous dry maerial prepared from eiher source was redissolved in an amoun of disilled waer equal in volume o ha of he original plasma. 779

2 78 FREDERICK J. POHLE AND F. H. L. TAYLOR EXPERIMENTAL The effec of repeaed inravenous injecions of plasma. In view of he fac, shown by many observaions, ha a refracory phase followed repeaed injecions of globulin subsance ino hemophilic paiens (Figure 1), i was necessary o observe he effecs of repeaed injecions of he paren plasma.2 Three hemophilic subjecs were given a sandard es dose of ciraed normal human plasma inravenously, repeaed four imes a 6-hour inervals. The resuls obained in one ypical se of observaions are presened in Figure 2. As shown in Figure 2, he blood coagulaion ime was mainained near normal values, and hroughou he enire period of observaion here was no suggesion of he developmen of a refracory period following any injecion of plasma. The effec of dried plasma upon he coagulaion ime of hemophilic blood in viro. Normal ciraed human plasma was dried in open dishes a room emperaure in a curren of air creaed by an elecric fan. The dry maerial, so obained, was suspended in he same volume of disilled waer as he original volume of he plasma, cenrifuged, and hen is clo-acceleraing properies 2 All plasma referred o in his communicaion has been passed hrough a Berkefeld V filer. TABLE I Effec of a soluion of air-dried, ciraed, normal human plasma on he coagulaion ime of hemophilic blood in viro minues 2 cc. hemophilic blood cc. hemophilic blood +.1 cc. suspension of dried plasma cc. hemophilic blood +.3 cc. suspension of dried plasma cc. hemophilic blood +.5 cc. suspension of dried plasma cc. hemophilic blood +.1 cc. suspension of dried plasma cc. hemophilic blood +.2 cc. suspension of dried plasma for hemophilic blood esed in viro. Normal plasma has been shown o reduce he coagulaion ime of hemophilic blood in a quaniaive manner (1, 2). There was a marked loss of hese properies associaed wih he ype of desiccaion described, as shown by Table I. Normal ciraed plasma was hen dried by he lyophilic process. This maerial when redissolved in disilled waer showed maximum clopromoing properies for hemophilic blood in viro (Table II). These observaions indicaed ha he clo-acceleraing properies of plasma I 5O D 4 3 w 2 2 T HOURS 2 INJECrl ON INJECTiON I FIG. 1. EFFECT OF MULTIPLE INTRAMUSCULAR S OF GLOBULIN SUBSTANCE ON THE COAGULATION TIME OF THE BLOOD IN HEMOPHILIA

3 TABLE II Effec of lyophilied, ciraed, normal human plasma on he coagulaion ime of hemophilic blood in viro COAGULATION DEFECT IN HEMOPHILIA minues 2 cc. hemophilic blood cc. hemophilic blood +.1 cc. lyophilic plasma cc. hemophilic blood +.3 cc. lyophilic plasma cc. hemophilic blood +.5 cc. lyophilic plasma cc. hemophilic blood +.1 cc. lyophilic plasma cc. hemophilic blood +.2 cc. lyophilic plasma were no disurbed by he freeing and drying process used in his echnique. The effec of pareneral injecions of lyophilied plasma. A single inravenous or inramuscular injecion of lyophilied plasma redissolved in 65 cc. of disilled waer was given o he same hemophilic paien on four differen occasions. In each insance here was a promp reducion in he coagulaion ime of he blood o normal or near normal values wihin one-half hour. This reducion in he coagulaion ime was mainained for approximaely six hours afer which i gradually reurned o he pre-injecion level. Occa- 781 sionally, he paiens experienced some local pain following he inramuscular injecion bu no hemaomas or general sysemic reacions occurred. A sandard es dose (65 cc.) of a soluion of lyophilied plasma was given inravenously four imes a 6-hour inervals o a hemophilic paien. The resuls were enirely comparable o hose shown in Figure 2. The shorened coagulaion ime was mainained and a refracory period did no develop following any of he injecions. The observaions were repeaed excep ha he plasma was adminisered inramuscularly. The resuls were again enirely similar. The effec of drying globulin subsance by he Flosdorf-Mudd echnique. Globulin subsance was prepared in he usual manner excep ha immediaely afer cenrifugaion he mois precipiae was froen and dried under vacuum by he lyophilic process. This maerial was hen ground, sored in a desiccaor over calcium chloride, and as required suspended in he requisie amoun of saline soluion. The clo-acceleraing properies of his preparaion for hemophilic blood were esed in viro (Table III) and in vio. Globulin subsance dried in his manner reained all of is poency. One hemophilic subjec was given inramuscularly a sandard es dose of globulin subsance m 55 ) F 2o, Ō.-J 1 HOURS O 2 T I I A O IS FIG. 2. EFFECT OF MULTIPLE INTRAVENOUS S OF NORMAL CIRTEID PLASMA ON COAGULATION TIME OF THE BLooD IN HEMOPHILIA

4 782 FREDERICK J. POHLE AND F. H. L. TAYLOR m 5 n 4 W 4 i 3 : - 2 J 1, i i Ts, s s HOURS IS cc.lobuli N GLOBULIN GLOBULIN GLOBULIN PLASMA FIG. 3. EFFECT OF A SINGLE OF LYOPHILE PLASMA ON THE REFRACTORY PHASE FOLLOWING MULTIPLE INJECTONIS OF GLOBULIN SUBSTANCE IN HEMOPHILIA (ALL S INTRAMUSCULAR) dried in his manner every 6 hours for 24 hours. Afer he usual iniial reducion in he coagulaion ime o normal values a refracory phase developed and furher injecions had lile or no effec upon he coagulaion ime. Daa obained were enirely similar o hose expressed graphically in Figure 1. Blood plaele couns aken every 3 hours during his se of observaions showed no flucuaions greaer han normal. The effec of an inramuscular injecion of lyophilied plasma upon he refracory phase. One hemophilic subjec was given a sandard es dose of a saline suspension of globulin subsance TABLE III Effec of a saline suspension of globulin subsance (dried by he lyophilic process) on he coagulaion ime of hemophilic blood in viro minues 2 cc. hemophilic blood cc. hemophilic blood +.1 cc. suspension of globulin subsance cc. hemophilic blood +.3 cc. suspension of globulin subsance cc. hemophilic blood +.5 cc. suspension of globulin subsance cc. hemophilic blood +.1 cc. suspension of globulin subsance cc. hemophilic blood +.2 cc. suspension of globulin subsance repeaed four imes a 6-hour inervals. The usual refracory period developed afer he firs wo injecions and he blood coagulaion ime gradually reurned o he pre-injecion level. The hird and fourh injecions were enirely wihou effec on he coagulaion ime of he blood. Immediaely afer he fourh injecion and a he heigh of he refracory period when furher injecions of globulin subsance have been shown o be ineffecive (2) his subjec received a sandard es dose of lyophilied plasma inramuscularly. There was a sharp reducion in he blood coagulaion ime, and i appeared ha he refracory phase had been abolished by his single injecion of plasma (Figure 3). COMMENT The fac ha many observaions have shown ha repeaed injecions of globulin subsance ino hemophilic paiens produced a refracory phase, as indicaed by failure of furher injecions of globulin subsance o reduce he coagulaion ime of hemophilic blood (2), necessiaed furher invesigaions o deermine he naure of his reacion. Since he proein of he early preparaions could have been denaured his condiion migh be considered a possible cause of he refracory phe-

5 COAGULATION DEFECT IN HEMOPHILIA nomenon. However, repeaed injecions of lyophilied globulin subsance produced a similar refracory phase. Flosdorf and Mudd (3) have shown ha he lyophilic process does no modify he proein complexes, and herefore i is no likely ha denauriaion is responsible for he developmen of he refracory period. Earlier sudies (2) demonsraed ha he concenraion of he clo-promoing facor progressively increased in he blood of he injeced paien during he developmen of he refracory period. I was clear, herefore, ha he concenraion of his maerial in he blood was such ha a reducion of he coagulaion ime should have aken place if globulin subsance was he only maerial deficien or modified in hemophilic blood. From he rapidiy of onse and recovery from he refracory sae shown by he presen and previous sudies he phenomenon could no be a manifesaion of an anigen-anibody reacion. Furhermore, here has been no evidence produced in any of our invesigaions o sugges ha a noncoagulable phase occurs during he refracory phase or a any ime afer he adminisraion of globulin subsance. The presen observaions show clearly ha repeaed injecions of ciraed normal human plasma or lyophilied plasma ino hemophilic subjecs mainains a reduced coagulaion ime of blood so long as he injecions are coninued (Figure 2). These daa sugges ha such plasmas conain cerain subsances acive in blood coagulaion no conained by preparaions of globulin subsance. This supposiion is confirmed by he experience in one case in which lyophilied plasma was injeced a he heigh of he refracory phase wih a reducion of he blood coagulaion ime oward normal limis. The naure of his subsance or hese subsances is a presen under invesigaion. I is possible, however, from an inspecion of he daa presened in his and in a previous repor (2), ha a combinaion beween globulin subsance and his second subsance or subsances may play an essenial role in he reducion of he coagulaion ime of hemophilic blood in vavo. Since globulin subsance, like ciraed normal plasma, exers is clo-promoing effec on hemophilic blood in he es ube in a quaniaive manner (Table III), i would appear 783 ha under hese circumsances he second subsance or subsances was presen in sufficien quaniy o saisfy he requiremens of increasing amouns of globulin subsance. However, during he developmen of he refracory phase in he paien wih hemophilia i is possible ha he increasing concenraion of globulin subsance in he blood finally resuls in he exhausion of his second subsance. The aboliion of he refracory phase wih a single injecion of plasma is probably due o an increase in ier of his second subsance. To wha exen he injecion of such normal plasma will quaniaively affec subsequen injecion of globulin subsance is a presen under invesigaion. CONCLUSIONS 1. In hemophilia, repeaed injecions of lyophilied globulin subsance as well as normal globulin subsance produced a refracory period afer he usual iniial reducion in he coagulaion ime of he blood. 2. Repeaed injecions of eiher normal human plasma or lyophilied plasma ino hemophilics mainained a shorened blood coagulaion ime wihou, however, he developmen of a refracory phase. 3. The refracory phase can be abolished a is heigh by a single injecion of plasma. 4. Boh normal plasma and lyophilied plasma probably conain subsances which play a role in he reducion of he coagulaion ime of blood in vivo and which are no presen in globulin subsance preparaions. The auhors graefully acknowledge he assisance of Miss Nancy Marean. BIBLIOGRAPHY 1. Paek, Arhur J., Jr., and Taylor, F. H. L., Hemophilia. II. Some properies of a subsance obained from normal human plasma effecive in acceleraing he coagulaion of hemophilic blood. J. Clin. Inves., 1937, 16, Pohle, Frederick J., and Taylor, F. H. L., The coagulaion defec in hemophilia. The effec in hemophilia of inramuscular adminisraion of a globulin subsance derived from normal human plasma. J. Clin. Inves., 1937, 16, Flosdorf, Earl W., and Mudd, Suar, Procedure and apparaus for preservaion in " lyophile " form of serum and oher biological subsances. J. Immunol., 1935, 29, 389.