Proteolytic cleavage by plasmin of the HA polypeptide of influenza virus: Host cell activation of serum plasminogen

VIROLOGY

66, 172-180 (1973)

Proteolytic Influenza

Cleavage Virus: SONDRA

by

Plasmin

of the

Host Cell Activation G. LAZAROWITZ; AND PURNELL

Th.e Rockefeller

HA

of Serum

Polypeptide

of

Plasminogen’

ALLAN R. GOLDBERG: W. CHOPPIN

CJnitTersity, IVew York, ,Vew York IOOQl Accepted July

30, 1973

Cleavage of the HA polypept.ide, the largest glycoprotein of the WSN strain of influenza virus, to polypeptides HA1 plus HA2 occurs when influenza virus is grown in MDBK cells in medium containing 2y0 calf serum, but does not occur in serumfree medium. Addition of highly purified plasminogen in concentrations as low as 1.5 pg/ml to serum-free medium results in complet,e cleavage. The release of plasminogen activat.ors by the cell is essential for the activation of the zymogen and cleavage of the HA polypeptide. Either chick or calf plasminogen can be converted to the active enzyme, plasmin, by activators produced by either infected or uninfected MDBK cells. Cleavage of the HA polypeptide by plasmin can be prevent.ed by trypsin inhibitors from bovine pancreas (Kunitz) or soybean. Cleavage does not occur in the presence of 2yc calf serum from which plasminogen has been removed specifically by affinity chromatography, indicating that plasminogen is the only serum component involved in the conversion of t.he HA polypeptide to HA1 plus HA2. The results obtained indicate that in the MDBK cell-WSN strain system, cleavage of the H-4 polypeptide can be explained by t,he act,ivat.ion of serum plasminogen b) cellular activators, and that cleavage of the influenza virus hemagglut.inin polypeptide is a sensit,ive indicator of the product.ion of plasminogen activators by cells. INTRODUCTION

The hemagglutinin of influenza virus consist,s of a 75400430,000 dalton glycoprotein which can exist as a single polypeptide chain (HA) (Lazarowitz et ab., 1971, 1973; Stanley et al., 1973) or as a disulfide-bonded complex of two polypeptides of molecular weights of 50,00&60,000 daltons (HAl) and 23,000-30,000 daltons (HA2) (Laver, 1971). 1 Supported by research grants CA-13362 from the National Cancer Inst.itute and AI-05600 from the National Inst,itute of Allergy and Infectious Diseases, and a Brown-Hazen grant from The Research Corporation. p National Institutes of Health Predoctoral Fellow 5 FOl GM-48628. 3 Richard King Mellon Foundation Fellow in The Rockefeller University. This research was done in part while A. R. G. was a Special Fellow of the Leukemia Society of America.

Studies of t,he synthesis of influenza virus specific proteins demonstrat,ed that the HA polypeptide is a primary viral gene product, whereas HA1 and HA2 are derived from the proteolytic cleavage of HA (Lazarowitz et ad., 1971; Klenk et al., 197213;Skehel, 1972). Available evidence suggeshs that in cells infected with the WSN strain of influenza Ao virus, the cleavage can occur while the HA polypeptide is associated with the cell plasma membrane (Lazarowitz et al., 1971; Compans, 1973). The proteolytic nature of this cleavage is suggested by the observations that: (1) in vitro incubation of virions with trypsin results in the cleavage of the HA polypeptide to HA1 plus HA2 (Lazarowitz et al., 1973); and, (2) diisopropylphosphofluoridate (DFP), an inhibitor of cellular prot,rases. inhibits the appearance of HA1 and HA2 in fowl plague virus-infected chick 172

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&T 19i3 by hcademic Press, of reproducti~m in any form

PLSSMIN

CLEAVAGE

embryo fibroblasts (Klenk and Rott, 1973). Cleavage of the HA polypeptide is not required for the assembly of infectious, hemagglutinating viriohs (Lazarowitz et al., 1973; Stanley et al., 1973); and both the uncleaved HA polypeptide and the cleavage products HA1 plus HA2 are equally active in hemagglutination (La.zarowitz et al., 1973). The hemagglutinin of WSN virions grown in MDBK cells in the absence of serum has been shown to consist of 285 % uncleaved HA polypept,ide (Rifkin et al., 1972; Lazarowitz et al., 1973). In contrast, the hemagglutinin of WSN virions grown in the presence of 2 % calf serum was demonstrated to be 290 % HA1 plus HA2. However, in vitro incubation in the presence of 2% calf serum of virions containing uncleaved HA polypeptide did not result in t,he cleavage of H-4 to HA1 plus HA2 (Lazarowitz et aZ., 1973). Thus, a direct action of some serum component alone could not explain the above observations. It therefore appeared that an interaction with cellular components was necessary for the proteolysis of the HA polypeptide, and it was suggested that cellular proteases were involved. This was furt,her support,ed by the observations that the extent of cleavage of the HA polypept#ide was both st,rain-dependent and host cell-dependent, and appeared to correlate with the cytopathic effect (CPE) produced in the particular system studied (Lazarowiitz et al., 1973). A serum component that might interact with cellular components and be involved in proteolysis is plasminogen. This zymogen seemed a likely candidate for &is role since it, as well as plasmin, the activat,ed form, is quite stable; it has a low level of self-act8ivat,ion; the act#ivated form has a trypsin-like specificity; and the production by cells of proteases that can activate plasminogen to plasmin is well-known (Robbins and Summaria, 1970). In this report we present, evidence t,hat’ plasminogen and cellproduced plasminogen activators are involved in t’he cleavage of t,he HA polypeptide observed when WSN virions are grown in MDBK cells. The use of the cleavage of the HA polypeptide of influenza virus as a sensitive indicat,or to study protease levels in general, and levels of plasminogcn ac-

173

OF HA POLYPEPTIDE

tivators associated wit#h normal and transformed chick embryo fibroblasts in particular is described elsewhere (Goldberg and Lazarowitz, 1973). MATERIALS

AND

METHODS

Growth of cells and virus. Madin-Darby bovine kidney (MDBK) cells were grown in reinforced Eagle’s medium (REM) (Bablanian et al., 1965) containing 10 % fetal calf serum as described previously (Choppin, 1969). The WSN strain of influenza A,, virus was grown in RIDBK cells under single cycle condit,ions as described by Choppin (1969), except that the viral growth medium was as indicat,ed in t,he text. Virus was harvested 20-22 hr after infection. Radioactively labeled virus was grown and purified as described by Compans and co-workers (1970), except that the virus was centrifuged to equilibrium in an 11 ml 15 % (w/w) to 40% (w/m) potassium tartrate linear gradient at 200,OOOg (38,000 rpm) for 1.25 hr at 4” in the Spinco SW 41 rotsor. In all experiments described in which direct comparison of virion proteins under different growth conditions was made, the protocol was as follows. Confluent MDBK monolayers were washed twice with phosphatehuffered saline (PBS) (Dulbecco and Vogt, 1954) to assure removal of growth medium and were inoculated at, a mult,iplicity of 10 PFU/cell with WSN virions diluted in Eagle’s medium (Eagle, 1959) cont.aining 1%’ bovine serum albumin. After adsorption for 2 hr, the inoculum was removed, and the cells washed once with PBS. The monolayers were then incubated in the various growth media indicated in t,he text. Virions were labeled by growth in [3H]glucosamine and [14C]amino acid containing medium which was composed of 1 part REM, 3 parts RERI wit,hout amino acids and without glucose, 3 &X/ml [3H]glucosamine, and 1 &i/ml [l4C]amino acids with the appropriate additions of sera, plasminogen, or protease inhibitors as indicated in the text. Polyacrylamkle gel electrophoresis. Samples were made 1% in sodium dodecyl sulfate (SDS) and 1% in p-mercaptoethanol, and incubated for 1 min at 100” (Maize1 et al., 1968). The preparation of polyacrylamide gels (Caliguiri et al., 1969) and the

174

LAZAR.OWITZ,

GOLDBElt(:,

!r”pA

CXIOPPIN

the met,hod of Lowry and co-workers (1951) using bovine serum albumin as a sta.ndard. Chm leak arid isotopes. Purified bovine pancreatic inhibit.or (Kunitz) was a kind gift of Dr. Lewis J. Greene of the Brookhaven National Laboratory. The inhibitor preparation was prepared as described by Kassell et al. (1963) and was found to be homogeneous by several different criteria including amino acid analysis and acrylamide gel electrophoresis. S-p-tosyl-L-lgsinechloromethylkct~one-HCl (TLCK) and lima bean trypsin inhibitor were obtained from Sigma Chemical Co., St. Louis, Missouri; soybean trypsin inhibitor (fraction VI) from Miles-Seravac. Iiankakee, Illinois; components for polyacrylamide gels from Canal Industries Corp., Rockville, Maryland. l’C-labeled amino acid mixtures and lysin’e-monohydrochloride from Schwara BioResearch, Inc., Orangeburg, Yew York; [3H]glucosanline from New England Nuclear Corp., Boston, Massachusetts; and Sepha-

of gels for determination of radioactivity (Scheid et al., 1972) have been described. Puri$kation of plasm i,rogen. Plasminogen was purified from calf serum or from chicken serum by affinity chromatography on lysine coupled to Sepharose 4B as first employed by Deutsch and Mertz (1970). Plasminogen purified from chicken serum is referred to as chicken plasminogen and that purified from calf serum as calf plasminogen. Plasminogenfree serum is that, fraction of the serum which constitutes the void volume of a lysine-Sepharose column. ILL vitro incubation oj tfirions. Purified virions (~20 pg/ml) containing ullcleaved HA polypeptide in 0.01 X phosphate buffer, pH 7.2, were incubated for 1 hr at 37” wit.h preincubated plasminogen as indicated below, at a final concentration of -10 pg/ml. Samples were then immediately prepared for analysis on SDS-polyacrylamidr gels. Protein conce~ltraticms mere determined by processing

(A)

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(‘3)

i

NA

8

20

40

60

60

E

‘I

I00

Frachon

M

20

40

60

80

number

FIG. 1. Effect. of plasminogen in the growth medium on the cleavage of the HA polypept,ide. Polyacrylamide gel electropherograms of [3H]glucosamineand [“Clamino acid-labeled WSN virions grown in MDBK cells in the presence and in the absence of plasminogen. (A) Virions grown in unsupplemented REM; (B) virions grown in REM containing 3.5 pg/ml chicken plasminogen. Migration in this and all subsequent figures is from left to right,. The 3H :W ratio is the same in both panels. 3H, O-----O; l*C, o--o.

PLASMIN

CLEAVAGE

rose 4B from Pharmacia, Piscataway, Jersey.

pg/ml. Furthermore, plasminogen appears to be the only serum component involved in this conversion since the presence in the growth medium of 2 % plasminogen-free calf serum does not result in the cleavage of the HA polypeptide, as illustrated in Fig. 2B. The virions grown under these latter conditions contain only the uncleaved form of the HA polypeptide without any detectable HA1 and H-42.

New

RESULTS

Role of Plasnlinogen HA Polypeptide

in th.e Cleavage of the

To determine whether plasminogen might be a factor in the cleavage of the HA polypeptide when WSN is grown in RIDBK cells in the presence of calf serum, the structural proteins of WSN virions grown with either REM or RERI containing 3.5 pg/ml purified chicken plasminogen as growth medium were compared. This concentration of plasminogen is equivalent to the amount present in 2%’ serum. As shown in Fig. lA, t,he hemagglutinin of virions grown in unsupplemented REM is exclusively uncleaved HA polypeptide. Neither HA1 nor HA2 are detectable by either amino acid or glucosamine label. However, growth in the presence of 3.5 pg/ml of chicken plasminogen results in complete cleavage of the HA polypeptide to HA1 plus HA2, as shown in Fig. 1B. As can be seen in Fig. 2A, the results are the same when plasminogen purified from calf serum is present in the viral growth medium (REM) at a conce&ration of 2

h’j’ect of Protease Inhibitors of the HA Polypeptide

on the Cleavage

The involvement of plasmin, the activated form of plasminogen, in the proteolysis of the HA polypeptide was also demonstrated further by the effect of adding protease inhibitors to the viral growth medium. As shown in Fig. 3 and listed in Table 1, the presence of bovine pancreatic trypsin inhibitor, an inhibitor of the action of plasmin, at a concentration of 10 pg/ml in the viral growth medium of REM cont,aining 2 rg/ml of chicken plasminogen resulted in almost complete inhibition of the cleavage of the HA polypeptide to HA1 plus HA2. Only minor amounts of H-41 and HA2 are found

(A) 3H

175

OF H-4 POLYPEPTIDE

(B) P

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NP HA,

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HA,

60

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5

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100

n

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20

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Fraction number FIG. 2. Polyacrylamide gel electropherograms of [3H]glucosamineand [‘4C]amino acid-labeled WSN virions grown in MDBK cells in the presence of calf plasminogen or plasminogen-free calf serum. (A) Virions grown in REM containing 2 rg/ml calf plasminogen; (B) virions grown in REM containing 2% 14C, a---@.’ plasminogen-free calf serum. The 3H.*I% ratio is the same in bot.h panels. 3H, O-----O;

176

LAZAROWITZ, HA 1 5

NP FA, lFA

HA, 1

GOLDBERG,

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ANI> CHOPPIN

partial inhibkion of the plasminogen effect the cleavage of the HA polypeptide. Approximately 23 % of the HA polypeptide remained uncleaved. This concentration of lima bean trypsin inhibkor was found to be somewhat toxic for JIDBK cells in 5 hr. 011

1 - 20

h

Vitro Cleavage of the HA Polypeptide

Plasminogen activators have been shown to be secreted by various cell types including kidney (Williams, 1951; Sugiyama and Okamot,o, 1964; Ali and Lack, 1965; RIaki I - 4 et al., 1969), brain (RIenon et al., 1970), leukocyt,es (Wiinschmann-Henderson et al., r---v 1972), erythrocytes (Semar et al., 1969), 0 20 40 60 SO 100 and stomach and duodenum (Eras et ab., Fracf~on no 1970). Activat,ors of plasminogen also have FIG. 3. Polyacrylamide gel electrophoresis of been demonstrated in cell cultures (Barnet’t [3H]glucosamineand [Y]amino acid-labeled and Baron, 1959; Painter and Charles, WSN virions grown in MDBK cells in REM con1962; Bernik and Kwaan, 1969). We theretaining 2 &ml chicken plasminogen and 10 pgg!ml fore investigated the role of cell-produced bovine pancreabic trypsin inhibitor. 3H, O-----O; plasminogen act,ivators in the cleavage of ‘C, .--0. the HA polypeptide in a series of in vitro TBBLE 1 experiments. Infect#ed and uninfect,ed EFFECT OF VARIOUS PROTE.I~E INHIBITORS ON THE JIDBK cell monolayers were incubated in CLEAVAGE OF INFLUENZA VIRUS HEYAGREJI containing 20 pg/ml chicken plasminoGLUTININ BE- ACTIVATED PLISMINOGEN gen. After 24 hr, the medium was removed and an aliquot added to purified WSN virions. As shown in Fig. 4A, incubation of virions with the plasminogen-containing medium in which infectsed RIDBK cells None >93 had been grown resulted in partial cleavage OC Plasminogen, 2 pg/ml ~0 >95 of the HA polvpeptide to HA1 plus HA2. Plasminogen, 2 fig, ‘ml When the medium from uninfect#ed RIDBIi plus Kunitz in10 91 9 cells was used, the resuks were identical. hibitor Dialysis of the medium had no effect on the plus soybean t,ryp100 90 10 cleavage of t.he HA polypeptide. sin inhibitor The activat,ion of plasminogen by actiplus lima bean 100 23 ii vators released by either uninfected or trypsin inhibitor influenza virus-infected hIDBK cells was a Virus was harvested after a 22.hr growt.h also demonstrated by incubating plasminoperiod in reinforced Eagle’s medium (REM) with gen with serum-free REM obtained from the additions indicated. cells after 24 hr, and t’hen incubating these b Calculated from areas under peaks of radiopreparations with purified virions. Preincuactive profiles of polyacrylamide gel elect.ropherobation of plasminogen under these conditions grams. for 24 hr produced results indistinguishc None detected. able from those shown in Fig. 4A. In a control experiment, plasminogen mixed n-it,h in virions grown under these conditions. fresh REM did not, cause the cleavage of Similar results were obtained when the viral the H.4 polypeptide as shown in Fig. 4B. growth medium contained soybean trypsin of plasminogen with fresh inhibitor (fraction VI) at a concentration of Preincubation 100 pg,/ml. Lima bean trvpsin inhibitor at a REM for as long as 24 hr did not aker this concentration of 100 pg/‘ml also resulted in result,. Thus, it is clear t,hat plasminogen

PLASMIN HA I

(A)

0

20

NPHA, tt

40

60

CLEAVAGE

OF HA POLYPEPTIDE

HA, M I I

(El

SO

100

Fraction

HA ,

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M I

p;“P

40

177

60

SO

100

number

FIG. 4. Polyacrylamide gel electropherograms of [aH]glucosamine- and [“Clamino acid-labeled WSN virions grown in MDBK cells in unsupplemented REM after in vitro incubation at 37” with (A) REM containing 20 pg/ml chicken plasminogen in which WSN-infected MDBK cells had been grown for 24 hr; and, (B) fresh REM containing 20 rg/ml chicken plasminogen. 3H, O-----O ; IJC, @---a.

self-activation in a 24-hr period does not play a major role in the cleavage of the HA polypeptide, and activators released by the cell are essential. We therefore conclude that both WSNinfected and uninfected MDBK cells produce plasminogen activators. It appears that the action of these enzymes on serum plasminogen can result in t,he cleavage of the HA polypeptide to HA1 and HA2 that is observed when WSN is grown in RSDBK cells in the presence of 2 70 calf serum. DISCUSSION

The resuks presented in this report establish that calf serum plasminogen can be converted to plasmin by plasminogen activators produced by either uninfect,ed or influenza virus-infected MDBK cells. Plasmin appears to be the major factor involved in the cleavage of the HA polypeptide of influenza virus when the virus is grown in RIDBK cells in the presence of serum. These conclusions are based on the observations that: (1) the hemagglutinin of WSN virions grown in MDBK cells in REM containing 2 pg/ml plasminogen appears to consist only of HA1 plus HA2; (2) when the viral growth medium is RERI containing plasminogenfree calf serum, the WSN hemagglutinin

appears to consist exclusively of the uncleaved polypeptide HA; (3) the addition of inhibitors of plasmin action, specifically bovine pancreatic trypsin inhibitor and soybean trypsin inhibitor, to the plasminogencontaining viral growth medium results in inhibition of the cleavage of the HA polypeptide to HA1 plus HA2; and (4) plasminogen can be activated in vitro by the cellproduced activators resulting in cleavage of the HA polypeptide. It has been found that plasminogen plays a role in cell growt#h under tissue culture conditions (Goldberg, unpublished observations). Therefore, it seems reasonable that cell produced activators might have a strict specificity for plasminogen as a substrate. Indirect support for such an hypothesis comes from the observation t#hat the addition of tqypsinogen (100 rg/ml) to the viral growth medium does not lead to the cleavage of the HA polypeptide (Lazarowitz and Goldberg, unpublished observations). In contrast, plasrninogen levels as low as 0.15 pg/ml result in almost total cleavage of the HA polypept.ide (Goldberg and Lazarowitz, 1973). The difference in magnitude of these effects indicates that t,he cell-produced activators are likely t,o have a stringent specificity

for

plasminogen

as

a

substrate.

178

LAZAROWITZ.

GOLDBERG.

Furthermore, many different cell types have been reported t’o produce plasminogen activators, and all cell t,ypes that, we have tested also secrete some plasminogen activators, although the levels produced vary (Goldberg, in preparation; Goldberg and Lazarowitz, 1973). The sit,e of action of plasmin cannot be directly deduced from the above data, although the results are suggestive of a plasma membrane location. In WSN-infected cells, the HA polypeptide appears to be cleaved while it is in association with the plasma membrane (Lazarowitz et al., 1971; Compans, 1973). The concentrat,ion of plasminogen capable of resulting in almost total cleavage of the HA polypept,ide under tissue culture conditions is quke low, e.g., 0.15 pg/ml for 20-50 pg of virus produced Goldbefg and (Lazarowitz, 1973). This is in contrast to the much higher concentration of plasminogen (10 pg/ml) required to cause partial cleavage of the HA polypeptide under in vitro conditions. These results suggest that in cell culture the plasmin is concentrated at the site of virus maturation, and are consistent with the sit,e of action of the enzyme being at t,he cell plasma membrane. Further studies are required to clearly answer the question of plasmin binding to and acting at the cell surface. Earlier results demonstrated that the cleavage of the HA polypeptide varies with strain and host cell, appearing t.o correlate with t,he extent of CPE in the system (Lazarowkz et al., 1973). The experiments presented here, while clearly demonstrating that the activation of plasminogen can participate in this cleavage, do not rule out the possible involvement of lysosomal enzymes in this proteolysis, as was suggest’ed previously. It has been reported that plasminogen activators are present in lysosomes, and may be released from cells under conditions of stress (Sugiyama, 1965; Beard et al., 1969). The release of lysosomal enzymes during influenza virus replication could thus result in plasminogen activation. Direct comparison of the levels of plasminogen activators produced by infected and uninfected cells is required to answer this question. Furthermore, the results presented here do not rule out the possibilit

AND

CHOPPIN

that under cert#ain conditions lysosomal enzymes can directly act to cleave the HA polypeptide. Findings consistent with this latter possibility are that: (1) WSN virions grown in RIDBK cells in the absence of serum can cont,ain 10-15s of their hemagglutinin in the form of HA1 plus HA2; (2) trypsin inhibitors from bovine pancreas and soybean were not found to completely ininhibit the prot,eolysis of HA; and, (3) the hemagglutinin of fowl plague virions grown in CEF cells in the absence of serum appears to be complet8ely cleaved (Klenk et al.. 1972a, b; Slcehel, 1972). Furthermore, when WSN virions are grown in embryonated chicken eggs, their hemagglutinin appears to be exclusively in the form of the cleavage products HA1 plus HA2. At present, it is not known which enzymes in the eggs are responsible for this proteolysis. Studies of t,he proteolysis in the absence of plasminogen of the HA polypeptide of different strains of influenza virus and ident,ification of proteases found in the egg are required t,o elucidate fully the role of t,he various agents involved in t#he cleavage of the HA polypeptide. ACKNOWLEDGMENT The authors are indebted to Dr. Andreas Scheid for his many encouraging and insightful discussions. REFERENCES -4~1, 6. P., and L.~K, C. H. (1965). Studies on the tissue activator of plasminogen. Distribution of activator and proteolytic activity in the subcellular fractions of rabbit kidney. Biochem. J. 96, 63-74. B.IBL.~NI.IN, R., EGGERS, H. J., and T.~MM, I. (1965). Studies on the mechanism of poliovirusinduced cell damage. I. The relation bet,ween poliovirus-induced metabolic and morphological alterat,ions in cultured cells. T’irology 26, lOC113. BARNETT, E. V., and BARON, S. (1959). An act,ivator of plaminogen produced by cell culture. Proc. Sot. Exp. Biol. Med. 102, 308-311. BEARD, E. L., BUSUTTIL, R. W., and GOTTSHALK, S. K. (1969j. Stress induced release of plasminogen activator from lysosomes. Thromb. Diath. Haemorrh. 21. 20-25. BERNIK, M. B., and KWAAN, H. C. (1969). Plasminogen activat.or activity in cultures from human tissues. An immunological and histochemical study. J. Cl&. Znaest. 48, 1740-1753.

PLASMIN

CLEAVAGE

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OF HA POLYPEPTIDE

179

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AND

CHOPPIN

R. B. (1951j. The fibrinolytic activit) of urine. &it. J. Exp. Path.ol. 32.530-537. WiiNSCHM.4NN-HENDERSON, B., HORWITZ, LJ. L., and ASTRUP, T. (1972). Release of plasminopen activator from viable leukocytes of mau, baboon, dog, and rabbit. Proc. Sot. Exp. Biol. Med. l-&1,634-638. wILLIlMS,J.