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Biochemical sudies on a low molecular weight antiplasmin of human blood
ARCHIVES
OF BIOCHEMISTRY
Biochemical
AND
179,
166-173 (1977)
Sudies on a Low Molecular Human Blood’ PAUL
Laboratory
BIOPHYSICS
T. K. MUP
of Hematology, St. Jude Department of Biochemistry,
Children’s University Memphis,
AND PANKAJ
Weight
Antiplasmin
of
GANGULY3
Research Hospital, of Tennessee Center Tennessee 38163
Memphis, Tennessee 38101 for the Health Sciences,
and
Received May 17, 1976 We previously reported a low molecular weight antiplasmin present in human platelets and plasma. This material as isolated by ultrafiltration was heterogeneous. The fraction showing maximum inhibition has been further purified by countercurrent distribution. The N-terminal residue of this purified inhibitor is alanine. Amino acid analysis showed glycine to be the predominant residue, and no basic amino acid was detected. Kinetic studies suggested a competitive inhibition of plasmin via enzymeinhibitor complex formation. Carbohydrate and phosphate could not be detected. These data were similar for both platelet antiplasmin and serum antiplasmin. The relationship between the platelet and the serum inhibitor remains unclear. This low molecular weight antiplasmin may contribute to the consolidation of the thrombus.
When bleeding occurs, circulating blood platelets adhere to the exposed fibrillar material and subsequently aggregate to form a hemostatic plug. Fibrin is then generated which becomes a part of the final thrombus. It is well established that a platelet-rich clot is more stable than a platelet-poor clot. Thus, any intrinsic inhibitor of fibrinolysis in platelets may play a significant role at the initial stage in the stabilization of the thrombus. Since the observation of Johnson and Schneider (l), several investigators have confirmed that materials inhibitory to the fibrinolytic enzyme system are closely associated with platelets (2-7). Furthermore, the possible modification of these inhibitors in pathological conditions has been recognized (3, 4, 8, 9). However, there is very little infor1 Supported by Training Grant CA 05176 and Research Grant HL 15090 from NIH and by ALSAC. This work was presented, in part, at the 67th Annual Meeting of the American Society of Biological Chemists, June 1976, San Francisco, California. 2 The work reported in this paper was excerpted from the dissertation of FTKM. Present address: Department of Experimental Medicine, Naval Medical Research Institute, Bethesda, Maryland 20014. 3 To whom reprint requests should be sent. Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
166
mation available in the literature concerning the identity and molecular properties of any such platelet inhibitor. This study was initiated in an attempt to provide a biochemical basis for the observed antiplasmin property of platelets. We have previously demonstrated that antiplasmin activity in platelets is associated with a low molecular weight material (10-12). Plasma contains several high molecular weight inhibitors of fibrinolysis. Their properties and mechanisms of action have been studied in detail (13-18). However, very little work has been done on the low molecular weight materials present in plasma which may have biological signiticame. We have shown in a previous paper that a plasmin inhibitor from platelets or serum could be isolated by ultrafiltration through a UM-10 membrane (12). The antiplasmin activity was found in the ultrafiltrate. Paper chromatography of the ultraflltrate showed six ninhydrin-positive spots. Using the fibrin plate method, three of these components were observed to have an inhibitory property. Spots 4 and 6 from both sources had approximately 30% inhibitory activity, while a maximum activ-
ANTIPLASMIN
OF HUMAN
ity of about 50% could be detected with spot 5. The objective of this study was to focus on the fraction with maximal inhibitory activity. In this report, we present results on the purification, kinetics, and some structural properties of the inhibitor from these two sources. MATERIALS AN’D METHODS Preparation of human platelet extract and serum. Platelet concentrates were obtained from normal blood with citrate as the anticoagulant. The procedure for the preparation of platelet extract has been described (20, 21). Platelets were washed twice with 1% w/v ammonium oxalate-0.1% w/v EDTA, pH 7.5, and once with 0.1 M Tris-HCl-0.1 M NaCl, pH 7.5. The washed platelets were homogenized at 4’C in a Waring Blendor. The homogenate was centrifuged and the supernatant, which formed the platelet extract, was recovered. To prepare serum, blood from volunteer donors was collected by venipuncture and allowed to clot at 37°C. The suspended material was removed by centrifugation, and the supernatant, which formed the serum, was collected. Isolation andpurification ofantiplasmin. The low molecular weight antiplasmin material of platelets or serum was isolated by ultrafiltration through a UM-10 membrane (Amicon Corp., Lexington, Massachusetts) as described previously (12). Further purification of these inhibitors was carried out by countercurrent distribution. An automatic apparatus (H.O. Post Scientific Instrument Co., Inc., Middle Village, New York) with 1000 tubes, each of 4-ml capacity, was used. The solvent system consisted of n-butanol, glacial acetic acid, and water in a volume ratio of 63:25:100. The pH of the upper and lower phases, as measured with a glass electrode, was approximately 2.18 and 1.91, respectively. Two milliliters of the aqueous phase of the solvent system was added to tubes 31 through 500. Three grams of the ultrafiltrate from platelets or serum was dissolved in 60 ml of the lower phase solvent. Two milliliters of this solution was mixed with an equal volume of the upper phase solvent and placed in tubes 1 through 30. The distribution was carried out at 25°C with the machine set for lo-min intervals with 1 tip/min for equilibration. To avoid any phase shift, a concurrent of 0.1 ml of the aqueous phase for each transfer was added. The distribution profile after 500 transfers was analyzed by descending paper chromatography (12). A test sample from every 10th tube was evaporated to dryness in vacua over KOH pellets at 5OC. The residue was reconstituted with water and an appropriate portion was applied to Whatman 3MM paper. The ninhydrin-positive pattern of the material from each tube was compared with the control. Fractions showing a clear ninhydrin-positive spot corresponding to spot 5 of
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the control were pooled. The pooled material was evaporated to dryness as described above. To remove any residual solvent, the dried material was redissolved in distilled water and the drying process was repeated in the same manner. Enzyme assays. (i) Fibrin plate method: The preparation of fibrin plates and the assay of plasmin were carried out as described by Ekert et al. (61. Standard human plasmin (American National Bed Cross, Bethesda, Maryland) prepared in different concentrations was placed in a series of small test tubes. Different volumes of the purified inhibitor or buffer (control) were added. Sufficient buffer was added to bring all solutions to the same final volume. An equal volume (in the microliter range) of each test sample was applied to the wells. The plates were covered and incubated at 37°C. Fibrinolytic activity was determined at different time intervals by measuring the diameter of the circular zones of clearance around the wells to the nearest 0.05 mm, using an immunoplate viewer. (ii) *ZSI-labeled tibrinolytic method: Purified plasma fibrinogen was labeled with radioactive iodine (22, 23). Aliquots of 0.1 ml were placed in wells (0.16 mg of protein) of lml capacity in a plastic tray (Linbro Chemical Co., New Haven, Connecticut). Fibrinogen was converted to fibrin by treating with thrombin. Fibrin was allowed to dry and was then washed gently. The effect of serum or platelet ultrafiltrate on plasmin, trypsin, and urokinase was tested using these lz51labeled fibrin plates. Each test enzyme mixed with an equal volume of the inhibitor or buffer (control) was held for 10 min at 25°C. Three-tenths milliliter of each sample was applied to the wells and the fibrinolytic activity was measured after 18 h of incubation. Fity microliters of each sample was withdrawn from the wells and was counted in an AutoGamma Spectrometer (Packard Instrument Co., Downers Grove, Illinois). (iii) Esterolytic method: Urokinase activity was determined as described by Johnson et al. (24), using acetylglycyl+lysine methyl ester (ALME) as the substrate. (iv) Thrombin-time method: Thrombin activity was determined according to the method of Hardisty and Ingram (25). Amino acid analysis. The purified platelet antiplasmin and serum antiplasmin in 2 ml of redistilled HCl containing 2 ~1 of phenol (saturated with water) were placed in ampules and flushed with nitrogen several times. The ampules were sealed in vacua and maintained at 110°C for 24 h. Phenol was added to prevent varying loss of tyrosine (26). Amino acid analyses of the samples were performed in a Beckman Model 120-C automatic amino acid analyzer. A control for each inhibitor was prepared in the same manner, except for hydrolysis, to assess the presence of any contaminating free amino acid in the sample. Dansylation and thin-layer chromatography. The
168
MUI AND GANGULY
modified method of Airhart et al. (27) was employed for dansylation and the products were separated by thin-layer chromatography (28). The purified antiplasmins in 50 ~1 of 0.33% Na,CO, were mixed thoroughly with 250 ~1 of a dansyl chloride solution (10% w/v in dry acetone). The dansylation was carried out at 37°C and was considered complete when the yellow color of the reaction mixture disappeared. The dansyl product was evaporated to dryness in uacl~o and was then reconstituted in 50 ~1 of acetone:glacial acetic acid (3:2). For amino-terminal studies, the dansyl product was hydrolyzed as described above. For amino acid composition studies, the samples were first hydrolyzed and then dansylated. A control (unhydrolyzed) sample was also processed in the same manner to detect any free amino acid contaminant. Determination of phosphate and carbohydrate. The method of Fisk and SubbaRow (29) was employed for the detection of phosphate, while the method of Hendler (30) was used to test for carbohydrate. RESULTS
In this study, purification of the fraction with maximum antiplasmin activity present in the ultrafiltrate of serum or platelet extract was attempted by countercurrent distribution. This method has the advantage that a large amount of the starting material might be used and interference caused by a high salt concentration may be avoided. After 500 transfers, the component with maximal antiplasmin activity from both platelet and serum ultrafiltrates appeared between transfer numbers 140 and 160. The chromatographic patterns of selected fractions are shown in Fig. 1. The purified material (spot 5) showing a single spot was assayed under different conditions for antiplasmin activity. These materials were quite stable and retained their inhibitory activity during fractionation. With 3 g of ultrafiltrate as the starting material, the average yield of the purified antiplasmin was about 3 mg for serum and 1 mg for platelets. The kinetics of interaction of plasmin with fibrin in the presence and in the absence of the inhibitor were studied by the fibrin plate method. Fibrinolytic activity was determined at 3, 6, 12, and 24 h. The velocity of interaction was estimated as the increase in diameter of the lysed zone in millimeters per hour. The rate of inhibi-
tion was observed to be nearly constant for the first 12 h, after which a slow decline became evident. Typical LineweaverBurk plots are shown in Figs. 2a and 2b for the purified platelet and serum antiplasmins, respectively. The data suggest that, for both antiplasmins, the inhibition is competitive in nature. The difference in slope between the two lines was highly significant, with P < 0.001. Preliminary studies on the amino acid composition of the two inhibitors were carried out. Since free amino acid contaminants might be present, amino acid analyses were performed before and after hydrolysis of the test samples. By comparing the two patterns, the true amino acid composition of the antiplasmins could be determined. The main impurity in the unhydrolyzed material was observed to be valine. A trace of tyrosine could also be detected. 0 i61
A 161
C
0 141
00 IJI
0 I61
171
00 161
191
FIG. 1. Descending paper chromatographic pattern of selected fractions obtained by countercurrent distribution of platelet (P) and serum (S) ultrafiltrates after 500 transfers. Transfer numbers are indicated at the top. Aqueous phase (A), organic phase (01, control (C). The arrow indicates the desired fraction (ninhydrin-positive spot 5) with maximal antiplasmin activity. This fraction occurred between transfer numbers 140 and 160 with both samples.
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OF HUMAN
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FIG. 2. Lineweaver-Burk plots illustrating the competitive nature of the inhibition of plasmin by the purified platelet antiplasmin (a) and serum antiplasmin (b). Stock concentration for both samples was 1.0 mg/ml. Plasmin activity was measured by the fibrin plate method. Different concentrations of plasmin were incubated with the inhibitor or buffer for 10 min at room temperature. Twenty microliters of each test sample was applied to the well and incubated at 37°C for 12 h. The unner line (A) presents the results in the presence of the inhibitor; the lower line (0) is the -control.
There was no appreciable increase in either valine or tyrosine after the samples were hydrolyzed, suggesting that these two amino acids are not a part of the antiplasmin molecule. There were six prominent amino acids, alanine, aspartic acid, glutamic acid, glycine, serine, and threonine, which could be reproducibly detected in both platelet and serum antiplasmins. Glycine was the most predominant, comprising about one-half of the amino acid residues. No basic amino acid could be detected. Further quantitative studies could not be done due to a lack of materials. As an additional support to the amino acid composition data, the dansylation method was employed. The platelet and serum antiplasmins were first hydrolyzed and then dansylated. A control mixture of amino acids, including those indicated by the analyzer to be present in the antiplasmin, was used as a standard. The results of these studies are shown in Fig. 3.4 Excluding the contaminants, valine and tyrosine, six of these spots were identified as the amino acids described above. There were two additional spots, the identity of which remains unknown. Carbohydrate 4 Abbreviation used: DNA, naphthalenesulfonyl.
&dimethylamino-
and phosphate were not detected in the purified antiplasmin material from either platelets or serum. The N-terminal residue studies on the purified platelet and serum antiplasmins were carried out by the dansyl method. The dansylated products were analyzed by two-dimensional thin-layer chromatography. The tmhydrolyzed material was dansylated and chromatographed to determine whether or not any free amino acid contaminant was associated with the samples. In the unhydrolyzed materials, both valine and tyrosine were again found to be the contaminants. Either valine or tyrosine alone, or in combination, was tested for antiplasmin activity. No inhibitory activity was observed. To determine the Nterminal residue, the sample was first dansylated, followed by acid hydrolysis. The results are shown in Fig. 4. The Nterminal residue for both antiplasmins was found to be alanine. A limited number of studies were carried out with the crude ultrafiltrates of serum and platelets to determine whether or not these materials act as general serine protease inhibitors and as antiactivators. Using the 1251-labeled fibrinolytic method, there was no antitrypsin or antiurokinase
FIG. 3. Amino acid composition analysis of platelet antiplasmin (P) and serum antiplasmin (S) by the dansyl microliters of each sample was applied to a 15 x l&cm polyamide thin-layer plate. 1st and 2nd indicate chromatogram was developed by ascending chromatography and photographed under long-wavelength ultraviolet ‘I’hr; E, Gly; F, Ala; G, Phe; H, Tyr; 1, Leu; J, Val; K, Ile; N, DNS-NH,; U, DNS-OH.
P method. C denotes control. Five the direction of migration. The light. A, Asp; B, Glu; C, Ser; D,
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OF HUMAN
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2nd FIG. 4. N-terminal analysis of platelet antiplasmin (P) and serum antiplasmin (Sl by the dansyl method. In pattern P, 2 ~1 of the sample was applied to a 15 x 15-cm polyamide thinlayer plate. In pattern S, 0.5 ~1 of DNS-Ala and of DNS-NH,, in addition, were mixed with 2 ~1 of the sample. 1st and 2nd indicate the direction of migration. The chromatogram was developed by ascending chromatography and photographed under long-wavelength ultraviolet light. A, Ala, V, Val, T, Tyr, N, DNS-NH,, U, DNS-OH.
activity under conditions where antiplasmin activity was observed. In addition, employing the esterolytic method, the activity of urokinase was unaltered by these materials. Further, thrombin-induced clotting time of fibrinogen remained unaffected in the presence of these materials. These results suggest that the materials am not general inhibitors or antiactivators. DISCUSSION
It is generally accepted that platelets contain inhibitors to the fibrinolytic system. The antiplasmin property of bovine blood platelets was first reported by Johnson and Schneider (1). Stefanini and Murphy observed that patients with quantitative or qualitative platelet abnormality had lower antifibrinolytic activity, but the antifibrinolysin could be removed by washing the platelets (2). Using the caseinolytic method, Alkjaersig distin-
guished between platelet and serum antiplasmins (3). Jurgens and Przybilski observed extremely low values of platelet inhibitory activity in 15 patients with von Willebrand’s disease, in whom plasma antiplasmin was normal (4). The above evidence would indicate that platelet antiplasmin is different from that of plasma. Two distinct inhibitors of plasmin in human platelets have been reported by McDonagh et al. (5). Based on gel filtration studies, one of these inhibitors eluted approximately in the same position as factor XIII and the other was found to have a higher molecular weight. Wakabayashi et al. described a protease inhibitor in rabbit platelets which acted against plasmin, trypsin, and chymotry-psin, but not against kallikrein or thrombin. This inhibitor was reported to have a molecular weight of 40,000 (31). There are at least four inhibitors of plasmin present in human plasma, called al-
172
MU1 AND GANGULY
antitrypsin (a-globulin), inter-a-trypsin inhibitor, ar,-antiplasmin (cYz-macroglobulin), and antithrombin III. The cy,-macroglobulin reacts immediately as a competitive inhibitor, while the a-globulin reacts more slowly but firmly with plasmin to form an inactive enzyme-inhibitor complex (13, 15). Antithrombin III, in addition to inhibiting thrombin and factors X, and IX,, inhibits plasmin by forming a nonreversible complex (17). Several antiactivators present in plasma have been reported (19, 32). However, their molecular properties and mechanisms of action have not been completely elucidated. The above plasma proteins with antifibrinolytic properties are well known and have high molecular weights. There has been very little study on any naturally occurring low molecular weight inhibitors of fibrinolysin in man. An inhibitor (M, 20,000) of tissue plasminogen activator and plasmin in human serum was reported (33). However, these investigators later failed to substantiate their previous observation and suggested adsorption of the activator and plasmin to glass as a possible explanation (34). It is evident that, in spite of a considerable amount of work, the origin and biochemical basis of the observed antiplasmin activity of intact platelets remain unexplained. Similarly, there is no definitive report of any low molecular weight antiplasmin present in human serum. We have demonstrated previously that a low molecular weight material in platelet extract possesses an antiplasmin property (11, 12). The inhibitor is dialyzable and a similar substance is present in plasma or serum. In the present study, the fraction with maximal antiplasmin activity has been purified by countercurrent distribution. As judged by this procedure and by other studies, the inhibitor from platelets or serum appears to be stable over a wide range of pH, between 2 and 8, and is stable at 50°C for at least 3 days. Preliminary studies by gel filtration through Sephadex suggest a molecular weight of about 1000 for the inhibitor. The kinetics of inhibition of plasmin by the two inhibitors have been described. Double-reciprocal plots indicate competi-
tive inhibition of the enzyme by these materials. The rate of inhibition is approximately constant for the first 12 h, after which-. a slow decline occurs. This small decline in antiplasmin activity may reflect the high affinity of plasmin for fibrin, resulting in progressive dissociation of the enzyme-inhibitor complex in favor of the enzyme-substrate complex (35). Studies on the amino-terminal residue of platelet and serum antiplasmins revealed alanine in both instances. Amino acid analysis by two independent methods showed that the two materials have a similar amino acid composition. Glycine was observed to be the predominant residue and no basic amino acid could be detected. Since lysyl and arginyl peptide bonds are hydrolyzed by plasmin, the inhibitor could not be, on this basis, a competitive sub strate. The mechanism of inhibition might be enzyme-inhibitor complex formation. Under different conditions, the low molecular weight inhibitors at equivalent concentrations did not have any apparent inhibitory activity toward trypsin, urokinase, or thrombin. There is general agreement that plasma contains the major fibrinolytic inhibitors. Although the absolute amount of platelet antiplasmin is small relative to that in plasma, the local concentration of this inhibitor might be appreciably higher during aggregation of a large number of platelets. There is some evidence suggesting that platelets might release antiplasmin materials during Viscous metamorphosis” (9, 36). This low molecular weight platelet antiplasmin may thus contribute to the stabilization of the thrombus. Chromatographic, electrophoretic, Nterminal amino acid, and amino acid composition studies, as well as inhibitory characteristics of the platelet antiplasmin and the serum antiplasmin, are similar. It is reasonable to suggest that the two materials are identical. The origin and the relationship between the platelet and the serum inhibitor remain unknown. ACKNOWLEDGMENTS Thanks are due to Dr. R. J. Hill for his help in performing the countercurrent distribution experi-
ANTIPLASMIN
OF HUMAN
ments, to Dr. M. Kh. Dabbous and Sister Burcharda for their assistance in performing the amino acid analysis, and to Dr. H. L. James for his valuable suggestions. We also wish to express appreciation to the personnel of St. Jude Children’s Research Hospital Blood Bank and to the blood donors and personnel of the U.S. Naval Air Station and Technical Training Command, Memphis, Tennessee. REFERENCES 1. JOHNSON, S. A., AND SCHNEIDER, C. L. (1953). Science 117, 229-230. 2. STEFANINI, M., AND MURPHY, I. S. (1956) J. Clin.
Invest.
35, 355-361.
3. ALKJAERSIG, N. (1961) in Blood Platelets (Johnson, S. A., Monto, R. W., Rebuck, J. W., and Horn, R. C., eds.), pp. 329-336, Little, Brown, Boston. 4. JIJRGENS, J., AND F’FPXBILSKI, R. (1968) Exp. Biol. Med. 3, 181-188. 5. MCDONAGH, J., KIESSELBACH, T. H., AND WAGNER, R. H. (1969)Amer. J. Physiol. 216, 508 513. 6. EKERT, H., FRIEDLANDER, I., AND HARDISTY, R. M. (1970) Brit. J. Haemutol. 18, 575-584. 7. MURRY, J., CRAWFORD, G. P. M., OGSTON, D., AND DOUGLAS, A. A. (1974) Brit. J. Haematol. 26, 661-668. 8. DEN OTTOLANDER, G. H. H., LEIGNSE, B., AND CREMER-ELFRINK, H. M. J. (1969) Thromb. Diath. Haemorrh. 21, 26-34. 9. ULUTIN, S. B., AND AKTUGLU, G. (1974) New Istanbul Contr. Clin. Sci. 11, 24-30. 10. GANGULY, P. (1972) Clin. Chim. Acta 39, 466468. 11. Mur, P. T. K., JAMES, H. L, AND GANGULY, P. (1973) Fed. Proc. 32, 289 (abstr.). 12. MUI, P. T. K., JAMES, H. L., AND GANGULY, P. (1975) Brit. J. Haematol. 29, 627-637. 13. NORMAN, P. S., AND HILL, B. M. (1958) J. Exp. Med.
14. HEIDE,
108, 639-649.
K., HEIMBIJRGER,
N., AND HAUPT, H. (1965) CZin. Chim. Actu 11, 82-85. 15. RIMON, A., SHAMASH, Y., AND SHAPIRO, B. (1966) J. Biol. Chem. 241, 5102-5107. 16. RINDERKNECHT, H., AND GEOKAS, M. C. (1973)
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17. HIGHSMITH,
Actn
295, 233-244.
R. G., AND ROSENBERG,
R. D. (19’74)
J. Biol. Chem. 249, 4335-4338. 18. GALL~MORE, M. J. (1975) Brit. J. Haematol. 31, 217-231. 19. AOKI, N., AND KAWANO, T. (1972) Amer. J. Physiol. 223, 1334-1337. 20. GANGULY, P., AND MOORE, R. (1967) Clin. Chim. Acta 17, 153-161. 21. GANGULY, P. (1972) J. BioZ. Chem. 247, 1809-
1816. 22. MCFARLANE, A. S. (1963) J. Clin. Znuest. 42, 346-361. 23. JAMES, H. L., AND GANGULY, P. (1973) B&him. Biophys.
Acta
328, 448-455.
24. JOHNSON, A. J., CLINE, V. L., AND ALKJAERSIG, N. (1969) Thromb. D&h. Haemorrh. 21, 259272, 25. HARDISTY, R. M., AND INGRAM, G. I. C. (1965) in Bleeding Disorders, pp. 285-286, Blackwell Scientific Publications, Oxford, England. 26. SANGER, F., AND THOMPSON, E. 0. (1963) B&him. Biophys. Acta 71, 468-471. 27. AIRHART, J., SIBIGA, S., SANDERS, H., AND KHAIRALLAH, E. A. (1973) Anal. Biochem. 53, 132-140. 28. Woons, K. R., AND WANG, K. T. (1967)Biochim. Biophys. Acta 133, 369-370. 29. FISK, C. H., AND SU,BBAROW, Y. C. (1925) J. Biol.
30. HENDLER, 842.
Chem.
66, 375-400.
R. W. (1956) J. Biol.
Chem.
223, 832-
31. WA-BAYASHI, K., FUJIKAWA, K., AND ABE, T. (1970) Thromb. Diath. Haemorrh. 24,76-84. 32. OGSTON, D., BENNETT, B., HERBERT, R. J., AND DOUGLAB, A. S. (1973) Clin Sci. 44, 73-79. 33. SIEGEL-RALSTON, A., AND A~TRUP, T. (1971) Nature New Biol. 234, 180-181. 34. ASTRUP, T., SEELICK, T., KOK, P., AND SEIGELRALSTON, A. (1973) Nature New Biol. 243, 51. 35. AMBRAUS, C. M., AND MARKUS, G. (1960)Amer. J. Physiol. 199, 491-494. 36. JOIST, J. H., NEIWIAROWSKI, S., AND MUSTARD, J. F. (1971) in Thrombolytic Therapy (Mam-
men, E. F., Anderson, G. F., and Barnhart, M. I., eds.), pp. 114-119, Schattauer-Verlag, Stutgart, New York.
OF BIOCHEMISTRY
Biochemical
AND
179,
166-173 (1977)
Sudies on a Low Molecular Human Blood’ PAUL
Laboratory
BIOPHYSICS
T. K. MUP
of Hematology, St. Jude Department of Biochemistry,
Children’s University Memphis,
AND PANKAJ
Weight
Antiplasmin
of
GANGULY3
Research Hospital, of Tennessee Center Tennessee 38163
Memphis, Tennessee 38101 for the Health Sciences,
and
Received May 17, 1976 We previously reported a low molecular weight antiplasmin present in human platelets and plasma. This material as isolated by ultrafiltration was heterogeneous. The fraction showing maximum inhibition has been further purified by countercurrent distribution. The N-terminal residue of this purified inhibitor is alanine. Amino acid analysis showed glycine to be the predominant residue, and no basic amino acid was detected. Kinetic studies suggested a competitive inhibition of plasmin via enzymeinhibitor complex formation. Carbohydrate and phosphate could not be detected. These data were similar for both platelet antiplasmin and serum antiplasmin. The relationship between the platelet and the serum inhibitor remains unclear. This low molecular weight antiplasmin may contribute to the consolidation of the thrombus.
When bleeding occurs, circulating blood platelets adhere to the exposed fibrillar material and subsequently aggregate to form a hemostatic plug. Fibrin is then generated which becomes a part of the final thrombus. It is well established that a platelet-rich clot is more stable than a platelet-poor clot. Thus, any intrinsic inhibitor of fibrinolysis in platelets may play a significant role at the initial stage in the stabilization of the thrombus. Since the observation of Johnson and Schneider (l), several investigators have confirmed that materials inhibitory to the fibrinolytic enzyme system are closely associated with platelets (2-7). Furthermore, the possible modification of these inhibitors in pathological conditions has been recognized (3, 4, 8, 9). However, there is very little infor1 Supported by Training Grant CA 05176 and Research Grant HL 15090 from NIH and by ALSAC. This work was presented, in part, at the 67th Annual Meeting of the American Society of Biological Chemists, June 1976, San Francisco, California. 2 The work reported in this paper was excerpted from the dissertation of FTKM. Present address: Department of Experimental Medicine, Naval Medical Research Institute, Bethesda, Maryland 20014. 3 To whom reprint requests should be sent. Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
166
mation available in the literature concerning the identity and molecular properties of any such platelet inhibitor. This study was initiated in an attempt to provide a biochemical basis for the observed antiplasmin property of platelets. We have previously demonstrated that antiplasmin activity in platelets is associated with a low molecular weight material (10-12). Plasma contains several high molecular weight inhibitors of fibrinolysis. Their properties and mechanisms of action have been studied in detail (13-18). However, very little work has been done on the low molecular weight materials present in plasma which may have biological signiticame. We have shown in a previous paper that a plasmin inhibitor from platelets or serum could be isolated by ultrafiltration through a UM-10 membrane (12). The antiplasmin activity was found in the ultrafiltrate. Paper chromatography of the ultraflltrate showed six ninhydrin-positive spots. Using the fibrin plate method, three of these components were observed to have an inhibitory property. Spots 4 and 6 from both sources had approximately 30% inhibitory activity, while a maximum activ-
ANTIPLASMIN
OF HUMAN
ity of about 50% could be detected with spot 5. The objective of this study was to focus on the fraction with maximal inhibitory activity. In this report, we present results on the purification, kinetics, and some structural properties of the inhibitor from these two sources. MATERIALS AN’D METHODS Preparation of human platelet extract and serum. Platelet concentrates were obtained from normal blood with citrate as the anticoagulant. The procedure for the preparation of platelet extract has been described (20, 21). Platelets were washed twice with 1% w/v ammonium oxalate-0.1% w/v EDTA, pH 7.5, and once with 0.1 M Tris-HCl-0.1 M NaCl, pH 7.5. The washed platelets were homogenized at 4’C in a Waring Blendor. The homogenate was centrifuged and the supernatant, which formed the platelet extract, was recovered. To prepare serum, blood from volunteer donors was collected by venipuncture and allowed to clot at 37°C. The suspended material was removed by centrifugation, and the supernatant, which formed the serum, was collected. Isolation andpurification ofantiplasmin. The low molecular weight antiplasmin material of platelets or serum was isolated by ultrafiltration through a UM-10 membrane (Amicon Corp., Lexington, Massachusetts) as described previously (12). Further purification of these inhibitors was carried out by countercurrent distribution. An automatic apparatus (H.O. Post Scientific Instrument Co., Inc., Middle Village, New York) with 1000 tubes, each of 4-ml capacity, was used. The solvent system consisted of n-butanol, glacial acetic acid, and water in a volume ratio of 63:25:100. The pH of the upper and lower phases, as measured with a glass electrode, was approximately 2.18 and 1.91, respectively. Two milliliters of the aqueous phase of the solvent system was added to tubes 31 through 500. Three grams of the ultrafiltrate from platelets or serum was dissolved in 60 ml of the lower phase solvent. Two milliliters of this solution was mixed with an equal volume of the upper phase solvent and placed in tubes 1 through 30. The distribution was carried out at 25°C with the machine set for lo-min intervals with 1 tip/min for equilibration. To avoid any phase shift, a concurrent of 0.1 ml of the aqueous phase for each transfer was added. The distribution profile after 500 transfers was analyzed by descending paper chromatography (12). A test sample from every 10th tube was evaporated to dryness in vacua over KOH pellets at 5OC. The residue was reconstituted with water and an appropriate portion was applied to Whatman 3MM paper. The ninhydrin-positive pattern of the material from each tube was compared with the control. Fractions showing a clear ninhydrin-positive spot corresponding to spot 5 of
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the control were pooled. The pooled material was evaporated to dryness as described above. To remove any residual solvent, the dried material was redissolved in distilled water and the drying process was repeated in the same manner. Enzyme assays. (i) Fibrin plate method: The preparation of fibrin plates and the assay of plasmin were carried out as described by Ekert et al. (61. Standard human plasmin (American National Bed Cross, Bethesda, Maryland) prepared in different concentrations was placed in a series of small test tubes. Different volumes of the purified inhibitor or buffer (control) were added. Sufficient buffer was added to bring all solutions to the same final volume. An equal volume (in the microliter range) of each test sample was applied to the wells. The plates were covered and incubated at 37°C. Fibrinolytic activity was determined at different time intervals by measuring the diameter of the circular zones of clearance around the wells to the nearest 0.05 mm, using an immunoplate viewer. (ii) *ZSI-labeled tibrinolytic method: Purified plasma fibrinogen was labeled with radioactive iodine (22, 23). Aliquots of 0.1 ml were placed in wells (0.16 mg of protein) of lml capacity in a plastic tray (Linbro Chemical Co., New Haven, Connecticut). Fibrinogen was converted to fibrin by treating with thrombin. Fibrin was allowed to dry and was then washed gently. The effect of serum or platelet ultrafiltrate on plasmin, trypsin, and urokinase was tested using these lz51labeled fibrin plates. Each test enzyme mixed with an equal volume of the inhibitor or buffer (control) was held for 10 min at 25°C. Three-tenths milliliter of each sample was applied to the wells and the fibrinolytic activity was measured after 18 h of incubation. Fity microliters of each sample was withdrawn from the wells and was counted in an AutoGamma Spectrometer (Packard Instrument Co., Downers Grove, Illinois). (iii) Esterolytic method: Urokinase activity was determined as described by Johnson et al. (24), using acetylglycyl+lysine methyl ester (ALME) as the substrate. (iv) Thrombin-time method: Thrombin activity was determined according to the method of Hardisty and Ingram (25). Amino acid analysis. The purified platelet antiplasmin and serum antiplasmin in 2 ml of redistilled HCl containing 2 ~1 of phenol (saturated with water) were placed in ampules and flushed with nitrogen several times. The ampules were sealed in vacua and maintained at 110°C for 24 h. Phenol was added to prevent varying loss of tyrosine (26). Amino acid analyses of the samples were performed in a Beckman Model 120-C automatic amino acid analyzer. A control for each inhibitor was prepared in the same manner, except for hydrolysis, to assess the presence of any contaminating free amino acid in the sample. Dansylation and thin-layer chromatography. The
168
MUI AND GANGULY
modified method of Airhart et al. (27) was employed for dansylation and the products were separated by thin-layer chromatography (28). The purified antiplasmins in 50 ~1 of 0.33% Na,CO, were mixed thoroughly with 250 ~1 of a dansyl chloride solution (10% w/v in dry acetone). The dansylation was carried out at 37°C and was considered complete when the yellow color of the reaction mixture disappeared. The dansyl product was evaporated to dryness in uacl~o and was then reconstituted in 50 ~1 of acetone:glacial acetic acid (3:2). For amino-terminal studies, the dansyl product was hydrolyzed as described above. For amino acid composition studies, the samples were first hydrolyzed and then dansylated. A control (unhydrolyzed) sample was also processed in the same manner to detect any free amino acid contaminant. Determination of phosphate and carbohydrate. The method of Fisk and SubbaRow (29) was employed for the detection of phosphate, while the method of Hendler (30) was used to test for carbohydrate. RESULTS
In this study, purification of the fraction with maximum antiplasmin activity present in the ultrafiltrate of serum or platelet extract was attempted by countercurrent distribution. This method has the advantage that a large amount of the starting material might be used and interference caused by a high salt concentration may be avoided. After 500 transfers, the component with maximal antiplasmin activity from both platelet and serum ultrafiltrates appeared between transfer numbers 140 and 160. The chromatographic patterns of selected fractions are shown in Fig. 1. The purified material (spot 5) showing a single spot was assayed under different conditions for antiplasmin activity. These materials were quite stable and retained their inhibitory activity during fractionation. With 3 g of ultrafiltrate as the starting material, the average yield of the purified antiplasmin was about 3 mg for serum and 1 mg for platelets. The kinetics of interaction of plasmin with fibrin in the presence and in the absence of the inhibitor were studied by the fibrin plate method. Fibrinolytic activity was determined at 3, 6, 12, and 24 h. The velocity of interaction was estimated as the increase in diameter of the lysed zone in millimeters per hour. The rate of inhibi-
tion was observed to be nearly constant for the first 12 h, after which a slow decline became evident. Typical LineweaverBurk plots are shown in Figs. 2a and 2b for the purified platelet and serum antiplasmins, respectively. The data suggest that, for both antiplasmins, the inhibition is competitive in nature. The difference in slope between the two lines was highly significant, with P < 0.001. Preliminary studies on the amino acid composition of the two inhibitors were carried out. Since free amino acid contaminants might be present, amino acid analyses were performed before and after hydrolysis of the test samples. By comparing the two patterns, the true amino acid composition of the antiplasmins could be determined. The main impurity in the unhydrolyzed material was observed to be valine. A trace of tyrosine could also be detected. 0 i61
A 161
C
0 141
00 IJI
0 I61
171
00 161
191
FIG. 1. Descending paper chromatographic pattern of selected fractions obtained by countercurrent distribution of platelet (P) and serum (S) ultrafiltrates after 500 transfers. Transfer numbers are indicated at the top. Aqueous phase (A), organic phase (01, control (C). The arrow indicates the desired fraction (ninhydrin-positive spot 5) with maximal antiplasmin activity. This fraction occurred between transfer numbers 140 and 160 with both samples.
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FIG. 2. Lineweaver-Burk plots illustrating the competitive nature of the inhibition of plasmin by the purified platelet antiplasmin (a) and serum antiplasmin (b). Stock concentration for both samples was 1.0 mg/ml. Plasmin activity was measured by the fibrin plate method. Different concentrations of plasmin were incubated with the inhibitor or buffer for 10 min at room temperature. Twenty microliters of each test sample was applied to the well and incubated at 37°C for 12 h. The unner line (A) presents the results in the presence of the inhibitor; the lower line (0) is the -control.
There was no appreciable increase in either valine or tyrosine after the samples were hydrolyzed, suggesting that these two amino acids are not a part of the antiplasmin molecule. There were six prominent amino acids, alanine, aspartic acid, glutamic acid, glycine, serine, and threonine, which could be reproducibly detected in both platelet and serum antiplasmins. Glycine was the most predominant, comprising about one-half of the amino acid residues. No basic amino acid could be detected. Further quantitative studies could not be done due to a lack of materials. As an additional support to the amino acid composition data, the dansylation method was employed. The platelet and serum antiplasmins were first hydrolyzed and then dansylated. A control mixture of amino acids, including those indicated by the analyzer to be present in the antiplasmin, was used as a standard. The results of these studies are shown in Fig. 3.4 Excluding the contaminants, valine and tyrosine, six of these spots were identified as the amino acids described above. There were two additional spots, the identity of which remains unknown. Carbohydrate 4 Abbreviation used: DNA, naphthalenesulfonyl.
&dimethylamino-
and phosphate were not detected in the purified antiplasmin material from either platelets or serum. The N-terminal residue studies on the purified platelet and serum antiplasmins were carried out by the dansyl method. The dansylated products were analyzed by two-dimensional thin-layer chromatography. The tmhydrolyzed material was dansylated and chromatographed to determine whether or not any free amino acid contaminant was associated with the samples. In the unhydrolyzed materials, both valine and tyrosine were again found to be the contaminants. Either valine or tyrosine alone, or in combination, was tested for antiplasmin activity. No inhibitory activity was observed. To determine the Nterminal residue, the sample was first dansylated, followed by acid hydrolysis. The results are shown in Fig. 4. The Nterminal residue for both antiplasmins was found to be alanine. A limited number of studies were carried out with the crude ultrafiltrates of serum and platelets to determine whether or not these materials act as general serine protease inhibitors and as antiactivators. Using the 1251-labeled fibrinolytic method, there was no antitrypsin or antiurokinase
FIG. 3. Amino acid composition analysis of platelet antiplasmin (P) and serum antiplasmin (S) by the dansyl microliters of each sample was applied to a 15 x l&cm polyamide thin-layer plate. 1st and 2nd indicate chromatogram was developed by ascending chromatography and photographed under long-wavelength ultraviolet ‘I’hr; E, Gly; F, Ala; G, Phe; H, Tyr; 1, Leu; J, Val; K, Ile; N, DNS-NH,; U, DNS-OH.
P method. C denotes control. Five the direction of migration. The light. A, Asp; B, Glu; C, Ser; D,
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2nd FIG. 4. N-terminal analysis of platelet antiplasmin (P) and serum antiplasmin (Sl by the dansyl method. In pattern P, 2 ~1 of the sample was applied to a 15 x 15-cm polyamide thinlayer plate. In pattern S, 0.5 ~1 of DNS-Ala and of DNS-NH,, in addition, were mixed with 2 ~1 of the sample. 1st and 2nd indicate the direction of migration. The chromatogram was developed by ascending chromatography and photographed under long-wavelength ultraviolet light. A, Ala, V, Val, T, Tyr, N, DNS-NH,, U, DNS-OH.
activity under conditions where antiplasmin activity was observed. In addition, employing the esterolytic method, the activity of urokinase was unaltered by these materials. Further, thrombin-induced clotting time of fibrinogen remained unaffected in the presence of these materials. These results suggest that the materials am not general inhibitors or antiactivators. DISCUSSION
It is generally accepted that platelets contain inhibitors to the fibrinolytic system. The antiplasmin property of bovine blood platelets was first reported by Johnson and Schneider (1). Stefanini and Murphy observed that patients with quantitative or qualitative platelet abnormality had lower antifibrinolytic activity, but the antifibrinolysin could be removed by washing the platelets (2). Using the caseinolytic method, Alkjaersig distin-
guished between platelet and serum antiplasmins (3). Jurgens and Przybilski observed extremely low values of platelet inhibitory activity in 15 patients with von Willebrand’s disease, in whom plasma antiplasmin was normal (4). The above evidence would indicate that platelet antiplasmin is different from that of plasma. Two distinct inhibitors of plasmin in human platelets have been reported by McDonagh et al. (5). Based on gel filtration studies, one of these inhibitors eluted approximately in the same position as factor XIII and the other was found to have a higher molecular weight. Wakabayashi et al. described a protease inhibitor in rabbit platelets which acted against plasmin, trypsin, and chymotry-psin, but not against kallikrein or thrombin. This inhibitor was reported to have a molecular weight of 40,000 (31). There are at least four inhibitors of plasmin present in human plasma, called al-
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MU1 AND GANGULY
antitrypsin (a-globulin), inter-a-trypsin inhibitor, ar,-antiplasmin (cYz-macroglobulin), and antithrombin III. The cy,-macroglobulin reacts immediately as a competitive inhibitor, while the a-globulin reacts more slowly but firmly with plasmin to form an inactive enzyme-inhibitor complex (13, 15). Antithrombin III, in addition to inhibiting thrombin and factors X, and IX,, inhibits plasmin by forming a nonreversible complex (17). Several antiactivators present in plasma have been reported (19, 32). However, their molecular properties and mechanisms of action have not been completely elucidated. The above plasma proteins with antifibrinolytic properties are well known and have high molecular weights. There has been very little study on any naturally occurring low molecular weight inhibitors of fibrinolysin in man. An inhibitor (M, 20,000) of tissue plasminogen activator and plasmin in human serum was reported (33). However, these investigators later failed to substantiate their previous observation and suggested adsorption of the activator and plasmin to glass as a possible explanation (34). It is evident that, in spite of a considerable amount of work, the origin and biochemical basis of the observed antiplasmin activity of intact platelets remain unexplained. Similarly, there is no definitive report of any low molecular weight antiplasmin present in human serum. We have demonstrated previously that a low molecular weight material in platelet extract possesses an antiplasmin property (11, 12). The inhibitor is dialyzable and a similar substance is present in plasma or serum. In the present study, the fraction with maximal antiplasmin activity has been purified by countercurrent distribution. As judged by this procedure and by other studies, the inhibitor from platelets or serum appears to be stable over a wide range of pH, between 2 and 8, and is stable at 50°C for at least 3 days. Preliminary studies by gel filtration through Sephadex suggest a molecular weight of about 1000 for the inhibitor. The kinetics of inhibition of plasmin by the two inhibitors have been described. Double-reciprocal plots indicate competi-
tive inhibition of the enzyme by these materials. The rate of inhibition is approximately constant for the first 12 h, after which-. a slow decline occurs. This small decline in antiplasmin activity may reflect the high affinity of plasmin for fibrin, resulting in progressive dissociation of the enzyme-inhibitor complex in favor of the enzyme-substrate complex (35). Studies on the amino-terminal residue of platelet and serum antiplasmins revealed alanine in both instances. Amino acid analysis by two independent methods showed that the two materials have a similar amino acid composition. Glycine was observed to be the predominant residue and no basic amino acid could be detected. Since lysyl and arginyl peptide bonds are hydrolyzed by plasmin, the inhibitor could not be, on this basis, a competitive sub strate. The mechanism of inhibition might be enzyme-inhibitor complex formation. Under different conditions, the low molecular weight inhibitors at equivalent concentrations did not have any apparent inhibitory activity toward trypsin, urokinase, or thrombin. There is general agreement that plasma contains the major fibrinolytic inhibitors. Although the absolute amount of platelet antiplasmin is small relative to that in plasma, the local concentration of this inhibitor might be appreciably higher during aggregation of a large number of platelets. There is some evidence suggesting that platelets might release antiplasmin materials during Viscous metamorphosis” (9, 36). This low molecular weight platelet antiplasmin may thus contribute to the stabilization of the thrombus. Chromatographic, electrophoretic, Nterminal amino acid, and amino acid composition studies, as well as inhibitory characteristics of the platelet antiplasmin and the serum antiplasmin, are similar. It is reasonable to suggest that the two materials are identical. The origin and the relationship between the platelet and the serum inhibitor remain unknown. ACKNOWLEDGMENTS Thanks are due to Dr. R. J. Hill for his help in performing the countercurrent distribution experi-
ANTIPLASMIN
OF HUMAN
ments, to Dr. M. Kh. Dabbous and Sister Burcharda for their assistance in performing the amino acid analysis, and to Dr. H. L. James for his valuable suggestions. We also wish to express appreciation to the personnel of St. Jude Children’s Research Hospital Blood Bank and to the blood donors and personnel of the U.S. Naval Air Station and Technical Training Command, Memphis, Tennessee. REFERENCES 1. JOHNSON, S. A., AND SCHNEIDER, C. L. (1953). Science 117, 229-230. 2. STEFANINI, M., AND MURPHY, I. S. (1956) J. Clin.
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