198 (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (21) (25) (26) (27) (28) (29) (30) (31)
WALTER H . SEEGERS
HEPP,0.: Z. ges. exptl. Med. 99, 709 (1935). HERZOG, R . O., ILLIG,R . , AND KCDAR,H.: Z . physik. Chem. A167, 329 (1933). HOLMBERG, C. G.: Arkiv. Kemi, Mineral. Geol. 17A, No. 26 (1944). LONGSWORTH, L. G.: Chem. Rev. 30, 323 (1942). MCFARLANE, A. S.: Biochem. J. 29, 407, 660, 1175, 1202 (1935). MEHL,J. W . , ORCLEY, J. L . , ASD SrrmA, R . : Science 92, 132 (1940). MUTZENBECHER, R . VON: Biochem. Z . 266, 226, 250, 259 (1933). ONCLEY, J . L.: Ann. N. Y . .4cad. Sci. 41, 136 (1941). OXLEY,J. L.: Chem. Rev. 30, 433 (1942); see also chapter 22 of reference 4. ONCLEY, J. L., MELIN,M., AND GROSS,P. M., JR.: I n preparation. ONCLEY, J. L., MELIN,M., RICHERT, D . A , , CAMERON, J. W., A N D GROSS,P. M.,JR. In preparation. PEDERSEN, K . 0.: Ullracentrifugal Studies o n S e r u m and S e r u m Fraclions. Alrnqvixt and Wiksells Boktryckeri AB, Upsala, Sweden (1915). PERRIN,F . : J. phys. radium [71 7, 1 (1936). PHILPOT,J. ST. L . : Nature 141, 283 (1938). PICKELS,E. G.: Rev. Sci. Instruments 9, 358 (1938); 13, 426 (1942). SCATCHARD, G., BATCHELDER, A. C., AND BROWN,A , : J. Clin. Investigation 23, 458 (1944); J. Am. Chem. Soc. 68, 2320 (1946). SIMHA,R . : J. Phys. Chem. 44, 25 (1940). STRONG, L . E., SURGESOR, D . M., TAYLOR, H . L . , A N L COHN,E. J . : Personal coni. munication. SVECBERG, T., IND PEDERSEN, K. 0 . :T h e Tlltracenlrifuge. Oxford University Press, Kew Pork (1940). TISELIUS, A , : Biochem. J. 31, 1464 (1937).
MCLTIPLE PROTEIN INTERACTIONS AS EXHIBITED BY T H E BLOOD-CLOTTING MECHANIShP WALTER H . SEEGERSl
College of ,Medicine, W a y n e University, Detroit, Michigan Received A u g u s t 8 , 1946
There is now emerging, from the researches of the past century, a tangible concept of a whole system of protein interactions involved in the blood-clotting mechanism. An undertaking of these reactions will lead to new viewpoints in protein chemistry. From the advances and contributions already made, it is apparent that the techniques of physical chemistry in conjunction with quantitative measurements of protein reactivity will be drawn upon heavily for the elucidation of the problems involved. It is the purpose of this paper to organize the existing information as briefly as possible, to show the relationships of the components of the system, and to incorporate new contributions from our labora1 Presented a t the Twentieth Sational Colloid Symposium, which was held a t Madison, Wisconsin, May 26-29, 19iC. 2 This work was aided by a grant from the Research Department, Parke, Davis, and Company, Detroit, Michigan.
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tory. If it is obvious that this field of knowledge is incomplete, it is not so much a reflection on the labors of the past, as an indication that previous efforts have born fruit and that the opportunities for future investigators are promising. I. A SYSTEM O F INTERACTIONS
The reactions under consideration are correlated in figure 1 . Prothrombin, a glycoprotein (35), reacts with thromboplastin, a macromolecular lipoprotein (6, 7,8), to yield thrombin (33) and perhaps other products not identified as yet. The reaction requires the presence of calcium ion (1, 15) in optimum concentration. Only strontium can serve as a substitute, and even it is not as efficient as calcium (23). I t has been shown that the reaction is stoichiometric (26) in
PROFl0RlNOLYSlN
200
WALTER E. SEEOERS
many similarities to equation 4, in which tne solid fibrin clot is also attacked by fibrinolysin. I n the latter case there are also a t least two decomposition products (42), and since it is likely that they are the same derivatives as those obtained from fibrinogen, it is possible to use this information in explaining reaction 2. We have discovered that this same fibrinolysin will also destroy prothrombin (equation 6). This topic will be discussed in succeeding paragraphs. It has also been shown, with the use of purified systems, that thrombin destroys prothrombin as represented by equation 5 (27). The complication extends even farther. Both fibrinolysin and thrombin, which are destructive to prothrombin, can be destroyed by a specific factor, which is in each instance another plasma protein. The specific plasma factor which destroys thrombin (equation 7) is plasma antithrombin, a factor long recognized but still not isolated. The reaction is greatly accelerated by heparin (31,37), but the quantity of thrombin which can be destroyed by a given amount of antithrombin is definitely limited (40). In the case of fibrinolysin the specific inhibitor normally present in plasma (equation 8) alters fibrinolysin in such a manner as to make it incapable of attacking fibrinogen, fibrin (10, 29), or, as will be shown below, prothrombin. Finally, fibrinolysin has its origin in profibrinolysin (9, 11, 24, 28), a plasma protein. The best experiments completed to date indicate that activation of profibrinolysin is probably accomplished by a specific activator (lo), as represented by equation 9. Such an activator can be prepared from streptococci (18, 28). This activation is in a sense the counterpart of prothrombin activation by thromboplastin, as represented by equation 1. Eventually it will be necessary to explain how the animal organism provides for the activator when it is needed. Just as platelets and tissue extracts supply thromboplastin for the activation of prothrombin (33,45),a special sou~ceof fibrinolysin activator must be present in some tissue(s), in which, however, it has not been recognized as yet. In the laboratory, chloroform serves the purpose of an activator (13). 11. COEXISTENCE
In the organization plan of figure 1 calcium ion and certain proteins have been enclosed in rectangles. This designates factors which coexist in plasma. Although each of these protein molecules has reactive groups, their properties are such that they will not react with each other. Now, let us consider what is involved when prothrombin is activated to thrombin. (1) It must be altered, a t some point in the molecule, by thromboplastin. ( 2 ) It can attack fibrinogen. (3) I t can attack prothrombin. (4) It can no longer be destroyed by fibrinolysin. ( 5 ) It can be destroyed by antithrombin. A single protein and its derivative are thus seen to be involved in a variety of reactions, and the whole system is interrelated in such a manner as to suggest common configurational patterns. Similarly, when profibrinolysin is activated the following is involved : ( 1 ) It must be altered, a t some point in the molecule, by profibrinolysin activator. ( 2 ) It can attack fibrinogen. (3) It can dissolve fibrin. ( 4 ) It can destroy prothrombin. ( 5 ) It can itself be destroyed by fibrinolysin inhibitor.
MULTIPLE PROTEIN INTERACTIONS IN BLOOD CLOTTING
201
Until something is known about the kinetics of these rea dons and the size and shape of the molecules involved, both before and after the various reactions, it is not likely that satisfactory explanations will be derived only from studies of --T\”*, -S-S--, -COOH, aromatic hydroxyl, phosphoric ester, guanidonium, or imidazole groups. 111. ADSORPTION OF THROMBIN ON FIBRIN
The protein preparations used and the methods developed for measuring their reactivity have already been described. Bovine prothrombin (35, 36, 41),
UNITS THROMBIN REMAINING
FIG.2. Quantity of thrombin remaining when various concentrations of thrombin are used to clot 1 cc. of a 1 per cent fibrinogen solution. With very high concentrations of thrombin, clotting is too rapid for thorough mixing and consequently two points on the curve are obviously irregular.
thrombin (35, 36, 39, 41), fibrinogen (42), and fibrinolysin (24) were prepared and determined quantitatively (23, 24, 38, 42, 47). To a series of test tubes containing 1 cc. of the fibrinogen (1 per cent in 0.9 per cent sodium chloride) was added 1 cc. of thrombin solution of various known concentrations. Mixing was done as rapidly as possible. After 10 min. the resulting fibrin clot was removed with a glass rod, and the remaining thrombin activity was measured. In all cases the remaining fluid volume was considered to be 2 cc. The amount of thrombin remaining bears a logarithmic relationship to the amount removed by the fibrin (figure 2). This is the relationship expected in adsorption phenomena and consequently constitutes presumptive evidence that the thrombin was removed by adsorption. To give further support to this conclusion the thrombin can be eluted in a
202
WALTER H. SEEGERS
special way: namely, with the aid of fibrinolysin, which has recently been prepared in concentrated form (24). Instead of removing the fibrin clot by mechanical means, it can be removed with fibrinolysin according to equation 5 of figure 1. One cubic centimeter of thrombin (3320 units) was mixed with 1 cc. of fibrinolysin, and then 1cc. of fibrinogen mas also added. h clot formed immediately and within 8 min. was dissolved by the fibrinolysin. Analysis showed 3300 units of thrombin in solution. When 1 cc. of the thrombin from the same thrombin stock solution was mixed with 1 cc. of fibrinogen, only 2000 units of thrombin could be accounted for after removal of the clot by mechanical means. It can, therefore, be concluded that thrombin is adsorbed on fibrin during the clotting process.
WITH FIBRINOLYSIN C 5 UNITS PER CC. h Z . 8 UNITS PER CC.
TIME IN MINUTES
FIG.3. Inactivation of purified prothrombin with fibrinolysin, derived from plasma, a t room temperature, pH 7.2, and in 0.9 per cent sodium chloride solution.
The above experiment also proves the important fact that fibrinolysin does not destroy thrombin. This could not have been predicted, since the circumstances are quite different in the analogous case of prothrombin. IV. REACTION O F PROTHROMBIN WITH FIBRIKOLYSIN
The fact that fibrinolysin will destroy prothrombin was discovered by serendipidy. The rate of prothrombin disappearance is shown in figure 3. With the lower concentration of fibrinolysin used in these experiments some prothrombin remained even after 2 hr., but it can be shown that the remainder was altered. It is far more refractory to the action of thromboplastin (equation 1, figure 1) than ordinary purified prothrombin or native plasma prothrombin. To describe such altered prothrombin we propose to use the term puruprothrombin. It is one of the subtle changes in the protein molecule which can be detected by a study of reaction rates. For unavoidable reasons the analytical methods which must be used are not entirely satisfactory, but are quite adequate to prove the point.
203
IlULTIPLE PROTEIW ISTERACTIOI\'S I N BLOOD CLOTTIKG
V. PARAPROTHROMBIN
To demonstrate the existence of paraprothrombin two solutions are needed: ( 1 ) purified prothrombin, and (2) prothrombin acted upon by fibrinolysin specifically as indicated a t the point of the arrow on figure 2. Each prothrombin preparation is suitably diluted so that reaction 2 can be employed t o follow the rate of reaction 1 (figure 2). I n other words, the two-stage analytical technique (47) is applied to such diluted solutions. The purified prothrombin is completely activated ivithin 15 min. (figure 4). I n contrast, the fibrinolysin-treated prothrombin continues to be converted to thrombin over a period of more than 30 min. The reaction rate nith thromboplastin is thus seen t o be retarded. To appreciate the magnitude af the differ-
45
I C PURIFIED PROTHROMBIN
v)
*
30
PARAPROTHROMBIN
1
- o O
10
20
25
30
35
40
PROTHROMBIN ACTIVATION TIME IN MINUTES
I.'IG. 4. Comparison of activation rate of purified prothromhin and purified prothrombi fibrinolysin, a t room temperature.
II
I rLsntedv i t h
ence between the txvo curves in figure 4 it must be realized that n ciotting timc oi 5 see. involves more than twice as much thrombin as a clotting time of 15 sec. VI. QUAKTITY O F PROTHROIIBIX DESTROYED BY FIBRISOLTSIX
One aspect of the destruction of prothombin by fibrinolysin is atypical when compared with more familiar reaction types. Prothrombin seems tc be resistant to small amounts of fibrinolysin. It is efficiently but incompletely destroyed by intermediate quantities, and rather inefficiently and still incompletely dePtroyed by strong solutions of fibrinolysin. The data are presented in table 1. With 0.2 unit of fibrinolysin no prothrombin activity was lost and no paraprothrombin could be detected. With 0.8 unit of fibrinolysin, 2100 units, or approsimately half the prothrombin \vas destroyed and paraprothrombin was present. Severtheless a tenfold increase in fibrinolysin concentration still did not result in the destruction of all the prothrombin. These relationships are similar to adsorption reactions, in which adsorption is proportionately greater from very dilute
204
WALTER H. SEEGERS
solutions than from more concentrated solutions. The destruction (“adsorption”) of prothrombin is proportionately greater with small amounts of fibrinolysin than with larger amounts of fibrinolysin. Such a relationship between proteins has been encountered previously: namely, in the case of the inactivation of thrombin by antithrombin. The latter reaction (equation 7, figure 1) also follow logarithmic relationships (40). VII. FIBRINOLYSIX INHIBITOR, FIBRINOLYSIK, AND PROTHROMBIN
We have been able to confirm the observation that blood plasma contains a powerful inhibitor (10, 29) which is capable of destroying fibrinolysin so that TABLE 1 Quantity of prothrombin destroyed by various amounts of fibrinolysin, with 15-min. Prothrombin activation time lPOIEPOXBIN DEStPOYEl
PEXAPKJ
unilr per cc
4ooo 4000 4000 4000 4000 4000
0.4 0.6
0,s 2.0 4.0
6.0
Sone None 450 2100
No paraprothrombin Some paraprothrombin
2950 3725 3800 3850 3880
Much paraprothrombin
the latter can no longer attack fibrinogen or fibrin. It remains to be shown, however, whether or not the inhibitor can also protect prothrombin from being inactivated by means of fibrinolysin. A number of experiments have been performed and they all show that the inhibitor does furnish protection. One of these was the following: 1 cc. of plasma was mixed with 1 cc. of fibrinolysin solution (60 units per cubic centimeter) and allowed to stand 1 hr. Then 6000 units of dry prothrombin were dissolved in the mixture. The prothrombin retained its full potency for several hours. Without the plasma inhibitor this amount of fibrinolysin destroys most of the prothrombin within 10 min. (figure 2, and table 1). SUMMARY
Prothrombin can be destroyed by fibrinolysin and during the destruction an intermediate, paraprothrombin, is formed. The inactivation of prothrombin is proportionately greater with small amounts of fibrinolysin than with more concentrated quantities. Fibrinolysin inhibitor protects prothrombin from being destroyed by fibrinolysin. Thrombin is not destroyed by fibrinolysin.
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When thrombin clots fibrinogen, some of the thrombin is adsorbed on fibrin.
It can be recovered by dissolving the clot with fibrinolysin. The physical-chemical characteristics of most of the proteins provided for the blood-clotting mechanism have not been determined. That is also true for the reaction products. Since most of these proteins exhibit activity, further study of such activity in conjunction with studies of physical-chemical characteristics presents a unique opportunity for broadening our understanding of protein interactions and protein structure. REFEREXCES ARTHPS,hf., AND PAGES, C . : Arch. physiol. norm. path. 2, 739 (1890). BAILEY,K., ASTBURY, W.T . , A K D RVDALL, IC.M,: Kature 161, 716 (1943). BOSWORTH, A. W.: J. Biol. Chem. 20, 91 (1915). BRAND, E . , KASSELL, B., . 4 S D SAIDEL, L. J . : J. Clin. Investigation 23, 437 (1944). BRINKHOUS, K. M., S I l I T I f , H. P., W.4RSER, E . D., A N D SEEGERS, H . : Am. J . Physiol. 126, 683 (1939) (6) CHARGAFF, E., ZIFF, hI., A m S f O O R E , D . H . : J . Biol. Chem. 139, 383 (1941). ( i ) CHARGAFF, E., MOORE,D. H . , ASD BEXDICK, A : J. B i d . Chem. 146, 593 (1942). E . , BENDICK, A , , A N D COHEN, S.: J. Biol. Chem. 166, 161 (1944). (8) CHARGAFF, L. R.: J . Gen. Physiol. 28, 363 (1945). (9) CHRISTESSEN, (IO) CHRISTENSES, L. R . , ASD MACLEOD, C. M.:J. Gen. Physiol. 28, 559 (1045). L. R . : J. Bact. 41, 65 (1944). (11) CHRISTESSEN, rv. L.,J R . ,A N D .4RMSTRONG, H . s., (12) COHN, E . J., ONCLEY, J. L., S T R O S G , L. E . , HUGHES, J R . : J. Clin. Investigation 23, 4 1 i (1944). (13) DELEPESSE,C., ASD POZERSKI, E . : Compt. rend. soc. biol. 66, 690 (1903). C . , A N D K N ~ C H ERL. ,: Arch. ges. Physiol. (Pfliiger's) 243,65 (1940). (14) EBBECKE, (15) FERGUSOS,J. H . : Physiol. Rev 16, 640 (1936). (16) FERGUSOS, J . H . , A N D RALPH,P. H . : Am. J. Physiol. 138, 648 (1043). J. F . , SCiiEINBERG,H., AND EDSALL, J. T.:Federation Proc. 3 , 57 (1944). (17) FOSTER, (18) GARSER,R . L . , ASD TILLETT,W. S.: J. Esptl. Bled. 60, 239 (1934). W. H.: Am. J. Physiol. 28, 453 (1910). (19) HOWELL, (20) HOWELL, W. H . : Ani. J. Physiol. 40, 526 (1916). W. H . : Am. J. Physiol. 36, 143 (1914). (21) HOWELL, W. H., AND HOLT,E.: Am. J . Physiol. 41, 328 (191s). (22) HOWELL, (23) LOOMIS,E . C., AND S E E G E R S , w.H . : Arch. Biochem. 6, 265 (19.u). C., JR.,RYDER,A , , A N D XIEFT, M, L.: Arch. Biochem., in (24) LOOUIS,E. C., GEORGE, press. (25) MELLANBY, J.: Proc. Roy. Soc. (London) B I N , 1 (1934). (26) MERTZ,E . T., SEEGERS, W.H., A N D SMITH,H . P . : Proc. SOC.Esptl. Biol. Bled. 42, (1) (2) (3) (4) (5)
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604 (1939). (27) MERTZ,E. T.,SEEGERS, H., AND SMITH, H . P.: Proc. soc. Esptl. Biol. Bled. 41, 657 (1939). (28) MILSTONE,H.: J. Immunol. 42, 109 (1941). (29) OPIE, E . L.: Physiol. Rev. 2, 552 (1922). (30) QUICK,A . J . : Proc. Soo. Esptl. Biol. N e d . 36, 391 (1936). (31) QCICK,A . J.: Am. J. Physiol. 123, 712 (1938). L. A , : Am. J. Physiol. 24, 406 (1909). (32) RKTTGER, A , : Arch. gcs. Physiol. (Pfliiger's) 6, 413 (1872). (33) SCHMIDT, (34) ROBBISS,K. C.: Am. J. Physiol. 142, 581 (1944). W. H . : J . B i d . Chem. 136, 103 (1940). (35) SEEGERS, W. H., A S D SMITH,H. P.: J. Biol. Chem. 140, 6 i i (1941). (36) SEEGERS, W. H . , WARNER, E . D., BRINKHOUS, K. >I ASD .,SMITH,H. P.: Science 96, (37) SEEGERS, 300 (1942).
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H. SEEGERS
(38) SEEGERS, W. H., ASD SMITH,H. P.: Am. J. Physiol. 137,348 (1942). W.H., ASD MCGISTY,D. M.: J. Biol. Chem. 146,511 (1942). (39) SEEGERS, (40) SEEGERS, W.H., A N D SMITH,H. P.: Proc. SOC.Exptl. Biol. Med. 62, 159 (1943). (41) SEEGERS, W . H., LOOUIS,E. C . , A N D VAKDENBELT, J. hl.: Arch. Biochem. 6,85 (1945). W.H., SIEFT, hl. L.,A K D VANDENBELT, J. M . : Arch. Biochem. 7, 15 (1945).. (42) SEEGERS, (43) S T ~ B E X L ,. : Arch. ges. Physiol. (Pfliiger’s) 181, 285 (1920). (44) TOCANTINS, L. hI.: .4m. J. Physiol. 114, 709 (1936). (45) T O C A N T I N S , L. hI.: Medicine 17, 155 (1938). (46) WILSOK,S. J.: Arch. Internal X e d . 69, 647 (1942). (4i) WARNER, E.D., BRISKHOUS, K. M.,ASD SMITH,H. P.: Am. J . Physiol. 114,667 (1936).
HEJIOCYASIKS OF THE GASTROPODS1 SVEN BROHLLT The Institute o.f Physical C h e m i s t i y , Cniuersity of Cpsala, C p s a l a , Sweden Receiued August 8 , 1946
Svedberg and collaborators (9, 13, 16) have shown that the hemocyanins are giant molecules with molecular Tyeights from about half a million up t o about ten million. They have also found that the hemocyanins may dissociate into Jyell-defined submultiples upon a change in the pH, this dissociation often being reversible. Among the hemocyanins the largest molecular weights are observed for the Gastropods (9). The dissociation reactions of these hemocyanins have been investigated more in detail (3, 4). It was then found that for certain species (e.g., Helix pomatia) the dissociation depends not only upon the p H but also upon the nature and the amount of electrolytes or non-electrolytes present in the solution. For other species (e.g., Paludina vivipara) the dissociation is influenced only by the pH. Helix pomatia hemocyanin (abbreviation, H.P.h.) and some other hemocyanins of the Gastropods have been investigated by the author (3) and by Borgman and the author (4,5). Paludina oivipara hemocyanin (abbreviation, P.V.h.) has been studied by Ekvall and the author ( 7 , 6). I. MOLECULAR XYEIGHT AND MOLECULAR SHAPE
The molecular constants have been determined for the two species Helix pomatia and Paludina civipara.
A . Xolecular weight The sedimentation constant, s, the diffusion constant, D, and the partial specific volume, V , are given in table 1 for H.P.h. and P.V.h. and for their 1 Presented a t the Twentieth Sational Colloid Symposium, which was held a t Madison, Wisconsin, May 28-29,1946.
