©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Fluorescent Probe Study of Plasminogen Activator Inhibitor-1
EVIDENCE FOR REACTIVE CENTER LOOP INSERTION AND ITS ROLE IN THE INHIBITORY MECHANISM (*)

(Received for publication, October 21, 1994; and in revised form, December 9, 1994)

Joseph D. Shore (1)(§) Duane E. Day (1) Ann Marie Francis-Chmura (1) Ingrid Verhamme (1) Jan Kvassman (1) Daniel A. Lawrence (2) David Ginsburg (2) (3)(¶)

From the  (1)Division of Biochemical Research, Henry Ford Hospital, Detroit, Michigan 48202-2689 and Departments of (2)Internal Medicine and (3)Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan 48109-0650

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A mutant recombinant plasminogen activator inhibitor 1 (PAI-1) was created (Ser-338 Cys) in which cysteine was placed at the P(9) position of the reactive center loop. Labeling this mutant with N,N`-dimethyl-N(acetyl)-N`-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) provided a molecule with a fluorescent probe at that position. The NBD-labeled mutant was almost as reactive as wild type but was considerably more stable. Complex formation with tissue or urokinase type plasminogen activator (tPA or uPA), and cleavage between P(3) and P(4) with a catalytic concentration of elastase, all resulted in identical 13-nm blue shifts of the peak fluorescence emission wavelength and 6.2-fold fluorescence enhancements. Formation of latent PAI showed the same 13-nm spectral shift with a 6.7-fold fluorescence emission increase, indicating that the NBD probe is in a slightly more hydophobic milieu. These changes can be attributed to insertion of the reactive center loop into the beta sheet A of the inhibitor in a manner that exposes the NBD probe to a more hydrophobic milieu. The rate of loop insertion due to tPA complexation was followed using stopped flow fluorimetry. This rate showed a hyperbolic dependence on tPA concentration, with a half-saturation concentration of 0.96 µM and a maximum rate constant of 3.4 s. These results demonstrate experimentally that complexation with proteases is presumably associated with loop insertion. The identical fluorescence changes obtained with tPAbulletPAI-1 and uPAbulletPAI-1 complexes and elastase-cleaved PAI-1 strongly suggest that in the stable protease-PAI-1 complex the reactive center loop is cleaved and inserted into beta sheet A and that this process is central to the inhibition mechanism.


INTRODUCTION

Plasminogen activator inhibitor-1 (PAI-1) (^1)is a member of the serpin class of protease inhibitors, which includes most of the plasma inhibitors regulating blood coagulation and fibrinolysis. These inhibitors have extensive homology and form very tight 1:1 complexes with their target proteases(1) . They share a number of common characteristics. All of the inhibitory proteins have a reactive bond contained within a loop extending from the protein; upon cleavage of this bond, the residues of the loop are inserted into the beta sheet A at the face of the molecule making it more stable(2) . This form of the molecule can be created by cleavage with catalytic concentrations of elastase (3) or dissociation of the enzyme-inhibitor complex(4) . Cleaved serpins are no longer able to bind proteases and inhibit them(2) . Structural studies of alpha(1)-protease inhibitor indicate that after cleavage at the P(1)-P(1)` bond of the reactive center loop and insertion of the P through P(4) residues into the A-beta sheet, the P(1) and P(1)` residues are separated by approximately 70 Å(5) .

Until recently, the model for an active serpin has been based on the crystallographic structure of ovalbumin, a non-inhibitory member of the serpin family(6) . Recently, the structures of an active form of antithrombin dimerized with an inactive form (7) and of the active form of an antichymotrypsin mutant (8) have been reported and are consistent with previous concepts of the structure of active serpin molecules. It has been proposed that loop insertion is rate-limiting and required for formation of a stable complex(2) . The hypothesis that the reactive center loop of serpins is inserted into the beta sheet A after reaction with proteases is supported by immunologic evidence with antithrombin III in which unique epitopes were exposed due to binding to proteases and cleavage of the reactive bond(9) .

Plasminogen activator inhibitor 1 (PAI-1) is the primary inhibitor of tPA and uPA, reacting at diffusion-limited rate constants(10) . It is also reactive with thrombin, plasmin, and trypsin, with somewhat slower bimolecular rate constants(11, 12) . An unique feature of PAI-1, compared with other serpins, is that it spontaneously converts from an active to a latent form, with a half-life of 3 h at pH 7.4 and 37 °C (17) . This form can be partially reactivated by denaturation. The structure of the latent form, determined by x-ray crystallography, indicates that residues P to P(4) of the reactive center loop are inserted into the beta sheet A(13) . Residues P(2)-P` form an extended loop, which is stretched along the surface of the protein.

Wild type PAI-1 contains no cysteine residues. Thus, the availability of a recombinant mutant with a sufhydryl group provides an unique opportunity for reaction with iodoacetamide derivatives of fluorescent labels and probes. We have reported studies with fluorescein-labeled P(1)` PAI-1(14) , Strandberg and Ny (15) have used this approach to label the P(3) and P(18) positions of PAI-1 with NBD, and Gettins et al.(16) labeled the P(1) position of antithrombin III with NBD to demonstrate the transfer of a heparin-induced conformational change to the region of the reactive bond. Fluorescent probe molecules such as NBD are sensitive to the polarity of the milieu that surrounds them and respond to a more hydrophobic environment by an enhanced and blue-shifted fluorescence emission spectrum.

In the present study, we developed an active PAI-1 mutant with a cysteine residue at the P(9) position in the reactive center loop, which was then available for modification with an NBD probe molecule. This permitted studies of the effects of tPA and uPA binding, formation of latent PAI-1, and cleavage of the reactive center loop at the P(3)-P(4) position by elastase, on the fluorescence properties of the label on the P(9) residue. The changes in fluorescence spectra and yields, due to these interactions of the PAI-1 molecule, demonstrated that binding with target proteases results in changes in the milieu of the reactive center loop. Consistent with our earlier studies(17) , the kinetics of these changes indicate that loop insertion is a critical component of the inhibitory mechanism.


EXPERIMENTAL PROCEDURES

Materials

The two chain form of tPA was prepared from the single chain form (Activase, Genentech) by activation on immobilized plasmin for 30 min; SDS-polyacrylamide gel electrophoresis confirmed complete conversion to the two-chain form. An of 1.92 mlbulletmgbulletcm and molecular weight of 66,000 were used to calculate the enzyme concentration. The S338C (P(9)) mutant PAI-1 was made using the Altered Sites mutagenesis kit (Promega) as described(3) , with the following oligonucleotide: CCTCCTCATGCACAGCTGT. The underlined nucleotide indicates residues deviating from the wild type PAI-1 cDNA. DNA sequence analysis through the mutagenized region confirmed the P(9) substitution. Since the construct was not sequenced throughout the entire PAI-1 coding region, the possibility of additional mutations cannot be completely excluded. However, we have observed no such additional mutations outside of the oligonucleotide-spanning region with this procedure in over 35 kilobase pairs of sequence analysis(3) .

Purification of P(9) Mutant

P(9) PAI-1 bacterial lysate containing between 5 and 20 mg of active PAI-1 was completely reduced by incubation with solid DTT at a final concentration of 10 mM for 30 min at room temperature to ensure complete reduction of cysteine. The sample was then purified on heparin-Sepharose and hydroxylapatite as described previously, (^2)with the exception that a linear gradient was applied to the heparin-Sepharose column from 0.15 M NaCl to 1.0 M NaCl, and all buffers contained 0.5 mM DTT. The active form of PAI-1 was separated from the latent form by hydrophobic chromatography using phenyl-Sepharose and an ammonium sulfate gradient. (^3)Purified samples of 95% active mutant were prepared by concentration on a small column of heparin-Sepharose.

Fluorescent Labeling of S338C PAI-1

Concentrated samples of the purified P(9) PAI-1 (30 µM) in a total volume of 1.0 ml or less were slowly applied to a PD-10 gel filtration column (Bio-Rad) equilibrated in pH 6.6, 0.05 M sodium phosphate containing 0.15 M NaCl, 1 mM EDTA, 0.01% Tween 80, and 150 mM IANBD (Molecular Probes). Because the sample was rapidly exchanged from a buffer containing DTT to the same buffer containing the fluorescent labeling reagent, the amount of final dimerized product was negligible. The sample was allowed to react for 8 h at 25 °C in the dark. Fractions containing the PAI-1 were pooled and applied to a G-25 superfine column (1.5 times 25 cm) (Pharmacia Biotech Inc.) to separate the protein from the free dye.

Quantitation and Characterization of Labeled P(9) PAI-1

The extent of incorporation of NBD fluorophore was determined in 0.1 M Tris buffer, pH 8.5, containing 6 M guanidine and 1 mM EDTA, with absorbance measurements made at 280 and 497 nm. A correction factor of 0.103 (/) was used to correct the 280-nm absorbance of labeled PAI-1 for the contribution of NBD, and extinction coefficients of 26,000 and 43,000 M cm were used to calculate the NBD label and PAI-1 concentrations, respectively. In all cases, stoichiometric incorporation was observed. Covalent incorporation of probe fluorescence into protease/inhibitor complex bands was confirmed by SDS-polyacrylamide gel electrophoresis (data not shown).

Fluorescence Measurements

Fluorescence measurements were performed with an SLM 8000 fluorimeter with an excitation wavelength of 480 nm. Enhancements were calculated from the fluorescence emission at 529 nm, the peak wavelength after the spectral shift. Experiments were performed in 0.1 M HEPES buffer, pH 7.4, containing 0.1 M NaCl, 1 mM EDTA, and 0.1% polyethylene glycol 8000 in acrylic cuvettes (Sarstedt) coated with polyethylene glycol 20,000. NBD-labeled PAI-1 at a concentration of 0.2 µM was reacted with tPA and uPA in excess (0.4 µM) for 10 min to ensure complete complexation of the PAI-1. An experiment with 0.7 µM uPA gave an identical fluorescence enhancement. In a separate experiment, 0.025 µM elastase was added to 0.22 µM NBD PAI-1 and the digestion was monitored for over an hour to determine the maximal fluorescence increase.

Kinetic Measurements

The same solution of P(9) NBD PAI-1 that was used to monitor fluorescence changes was used to follow inhibitory activity as described previously(11) . Stopped flow fluorimetry was performed on an instrument designed and built by Dr. David Ballou at the University of Michigan. Excitation was at 480 nm, and a filter with a cutoff below 495 nm was used to monitor fluorescence emission. Stopped flow experiments were performed under pseudo first order conditions with tPA in at least a 5-fold excess over the PAI-1 concentration. Values for k were obtained by fitting the time constants and amplitude factors of a double exponential growth function to the recorded traces. The calculated k values were plotted against enzyme concentration and were used to obtain values for k(2) and K by non-linear least squares fitting to the equation for a rectangular hyperbola.


RESULTS

Characterization of Fluorescent-labeled PAI-1

Sitedirected mutagenesis was used to incorporate a cysteine residue at the P(9) position (Ser-338 Cys). The results presented in Table 1show that the P(9) cysteine mutant was about 40% as reactive as wild type PAI-1 with tPA, and 70% as reactive with uPA. In both cases, derivatization with NBD showed no significant effect. The tPA and uPA inhibition rate constants of the mutant and its fluorescent derivative were thus in the same range as the values for wild type PAI-1.



Fig. 1shows the fluorescence emission spectra of free iodoacetamido-NBD (IANBD), labeled Ser-338 Cys (P(9)) PAI-1, and the labeled PAI-1 bound to tPA. The free IANBD had an emission peak at 550 nm with excitation at 480 nm, which shifted to 542 nm when linked to the PAI-1 mutant, accompanied by a 58% enhancement of fluorescence. The enhancement and blue shift indicate that the probe is in a more hydrophobic milieu when linked to the protein than when it is free in solution.


Figure 1: Altered NBD probe fluorescence due to local environmental changes. The excitation wavelength was 480 nm. Figure shows emission spectra of: A, free IANBD (0.2 µM); B, NBD P(9) PAI-1 (0.2 µM); C, NBD P(9) PAI-1 (0.2 µM) after addition of r-tc-tPA (0.4 µM).



Spectral Changes of NBD-PAI-1

The emission spectral change observed when the NBD-labeled P(9) mutant PAI-1 is bound to tPA is shown in Fig. 1. Interaction with tPA resulted in a large blue shift of the emission spectrum peak, from 542 nm to 529 nm, and a 6.2-fold fluorescence emission increase. As compiled in Table 2, identical changes occurred when the labeled PAI-1 was reacted with uPA. In order to evaluate interactions of the reactive center loop with the PAI-1 beta sheet A, the loop was cleaved by catalytic elastase concentrations at the P(3)-P(4) (Ser-Val) bond(3) . This also resulted in a 6.2-fold increase in emission intensity and 13 nm shift in peak emission wavelength, to 529 nm. The P(9) NBD derivative of PAI-1 also underwent a transition from active to latent forms. Formation of latent NBD-PAI-1 was accompanied by a 13-nm spectral shift to 529 nm and 6.7-fold enhancement. Fig. 2shows the rate of loss of activity as a tPA inhibitor compared with the rate of increase in NBD fluorescence as the molecule becomes latent. The half-life of labeled PAI-1 is 23.1 ± 2.0 h measured by inhibitory activity and 24.3 ± 0.6 h measured by the fluorescence enhancement, compared with 9.5 h for wild type PAI-1 at pH 7.4 and 25 °C. The half-life of wild type PAI-1 is 3 h at 37 °C(17) , indicating a large temperature effect. The spectral shifts and yield changes for reaction with tPA or uPA, conversion to the latent form, and loop cleavage by elastase are listed in Table 2.




Figure 2: Spontaneous conversion of NBD P(9) PAI-1 (0.9 µM) from an active to latent conformation. Leftaxis (solidcircles), the fluorescence enhancement of NBD probe emission at 529 nm as a function of time. Right axis (invertedtriangles), the amount of active labeled PAI-1 remaining was measured by inhibition of beta-trypsin by the labeled PAI-1 as a function of time.



Using the NBD-labeled P(9) PAI-1 mutant, it was possible to determine the rate of the change in fluorescence emission due to protease binding. The inset to Fig. 3shows a typical reaction with tPA, followed by stopped flow fluorimetry. These reactions were performed with various tPA concentrations under pseudo first order conditions. They fit best to the equation for two exponentials, with the faster one having 87% of the amplitude at the lowest tPA concentration (0.3 µM) and 90% at the highest concentration (5 µM). The line through the data in inset to Fig. 3is from a double exponential fit. The results presented in Fig. 3show the pseudo first order rate constants for the fast phase of the interaction as a function of tPA concentration. The curve fits a hyperbolic dependence on tPA concentration with a limiting rate of 3.4 s and a half-saturation concentration of 0.96 µM.


Figure 3: Stopped flow kinetic analysis of the fluorescent signal generated by reaction of the labeled PAI-1 with increasing concentrations of r-tc-tPA. The pseudo first order rate constants measured for the increase in probe fluorescence are plotted as a function of r-tc-tPA concentration. Inset, a typical stopped flow trace at final concentrations of 0.1 µM NBD PAI-1 and 5.0 µM r-tc-tPA.




DISCUSSION

Replacement of the serine residue at P(9) with cysteine and derivatization with NBD resulted in rate constants for tPA and uPA inhibition that were reduced approximately 2-fold compared to wild type (Table 1), indicating that the substitution has only modest effects on PAI-1 function. Interestingly, the rate constant for transformation of active to latent inhibitor for the derivatized P(9) mutant was less than half that of wild type. This could be due to the bulky NBD molecule causing a slower rate of loop insertion into the beta sheet A of the PAI-1. The spectral shift and enhancement (Fig. 1) due to forming protein-bound NBD, indicating a more hydrophobic milieu, may be due to interactions with adjacent residues on the reactive center loop, or with other parts of the loop. The effect is, however, relatively small compared with the other NBD effects observed in these studies and suggests that even when associated with native PAI-1 the probe is likely to be very solvent-accessible.

The mirror image curves in Fig. 2demonstrate that the process of latent transformation exactly follows the fluorescence change of the labeled PAI-1. In addition, the 13-nm spectral shift and greater than 6-fold fluorescence emission enhancement of the NBD-labeled PAI-1 observed upon this transition as well as with binding to tPA or uPA and catalytic cleavage by elastase, are all very similar. We previously demonstrated that catalytic concentrations of elastase cleave at the P(3)-P(4) bond of PAI-1, resulting in a molecule with physical properties similar to those of latent PAI-1(3) , in which the reactive center loop is fully inserted into the beta sheet A. The fluorescence changes observed upon formation of the latent species must result from the increased hydrophobic milieu surrounding the P(9) position in beta sheet A. These data suggest that the probe on the P(9) position moves into a nearly identical milieu in cleaved PAI-1 as in protease-bound or latent inhibitor, associated with the same conformational change in each case. It should be noted that in the latent structure of PAI-1, alpha helix F sits directly above beta sheet A and would appear to cover beta strands 2, 3, and 4. The similarity of fluorescence changes due to latency, in which the reactive loop is known to be inserted(13) , to those caused by complexation with tPA and uPA or cleavage by elastase, provides evidence that loop insertion is an essential component of the inhibitory mechanism. The emission peak wavelength shift due to formation of latent NBD-PAI-1 is the same as NBD-PAI-1 reacted with tPA or uPA. However, the 6.7-fold enhancement was somewhat higher than that observed with PAI-1 which is cleaved or complexed to proteases. This higher fluorescence yield may be due to a nearly identical position of the P(9) probe in the latent form, with a more hydrophobic milieu. The requirement of insertion of the reactive center loop into beta sheet A for inhibition is also consistent with our previous studies of PAI-1 mutants containing substitutions at the P(14) position of the loop(18) .

The identical 6.2-fold fluorescence increases and blue spectral shift due to cleavage by elastase at the P(3)-P(4) position, and to binding of tPA or uPA, indicate that the NBD probe is in a similar hydrophobic milieu in all three cases. These data suggest that interaction with tPA or uPA results in insertion of the loop into beta sheet A, presumably similar to the structure of latent PAI-1. The identical fluorescence changes resulting from tPA and uPA binding also indicate that these changes derive from alterations in the PAI-1 structure rather than interaction of the probe with each enzyme.

Since the large fluorescence enhancement of the NBD label on the P(9)position is likely to be measuring insertion of the reactive center loop, it was of interest to follow the kinetics of this process. The results in Fig. 3indicate that the rate constant for loop insertion hyperbolically approaches a limiting rate of 3.4 s. The minimum mechanism describing this contains two steps, as shown in .

In this mechanism, E is tPA or uPA, I is PAI-1, and EI* represents the complex after the reactive center loop of PAI-1 has been inserted. The equation for this mechanism would take the following form.

In this equation, K is equal to (k + k(2))/k(1) so that if k k(2), it would be k/k(1), while if k(2) k, K would reduce to k(2)/k(1). Regardless of the rate constants defining K, the rate of inhibition of tPA by NBD-labeled PAI-1 should be equal to k(2)/K. This is indeed true, since the ratio from the data in Fig. 3, 3.4 s/0.96 µM, equals 3.5 times 10^6M s, which is comparable to the value of 3.5 times 10^6 for tPA inhibition determined directly and reported in Table 1. With k(2) equal to 3.4 s, the half-life of the reaction is approximately 0.2 s. Most functional assays and kinetic evaluations of PAI-1 use concentrations in the nanomolar range so that the reaction can be measured over a time scale of minutes. These conditions are very far from the PAI-1 concentration necessary for saturation, which would enable observation of the limiting rate. They are appropriate, however, for determination of the bimolecular inhibition rate constant, which is equal to k(2)/K. The rates of loop insertion at different tPA concentrations were double exponentials, as the excellent fit in Fig. 3(inset) shows. Regardless of the tPA concentration, the rapid phase consisted of approximately 90% of the total reaction amplitude. We used the fast phase for the plot in Fig. 3, which provided a good hyperbolic fit.

The value of k(2), 3.4 s, represents the rate of insertion of the reactive center loop into beta sheet A in the tPA-PAI-1 complex. This is much faster than the rate constant for transformation of NBD-labeled PAI-1 to a latent form, which has a half-life of 23 h at 25 °C. One possible explanation for the rapid insertion rate due to protease binding could be exosite interaction with PAI-1, causing a conformational change in the beta sheet A that facilitates loop insertion. Alternatively, formation of the initial EI complex, involving interaction at the P(1)-P(1)` bond, may alter the mobility or conformation of the loop in a manner that facilitates insertion. Finally, cleavage of the P(1)-P(1)` bond may induce insertion by removing any constraints on the reactive-center loop. This mechanism would be compatible with the stable PAI-1-plasminogen activator complex existing as an acyl enzyme.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL45930 (to J. D. S.) and HL49184 (to D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Henry Ford Hospital, E& Rm. 3126, Division of Biochemical Research, 2799 W. Grand Blvd., Detroit, MI 48202-2689. Tel.: 313-876-3196; Fax: 313-876-2380.

Howard Hughes Medical Institute Investigator.

(^1)
The abbreviations used are: PAI-1, plasminogen activator inhibitor 1; tPA, two-chain tissue plasminogen activator; uPA, two-chain urinary plasminogen activator; NBD, N,N`-dimethyl-N-(acetyl)-N`-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine; IANBD, iodoacetamido-NBD; DTT, dithiothreitol; r-tc-tPA, recombinant two-chain tPA.

(^2)
Eitzman, D. T., Fay, W. P., Lawrence, D. A., Francis-Chmura, A. M., Shore, J. D., Olson, S. T., and Ginsburg, D. (1995) J. Clin. Invest., in press.

(^3)
J. Kvassman and J. D. Shore, submitted for publication.


REFERENCES

  1. Huber, R., and Carrell, R. W. (1989) Biochemistry 28, 8951-8966 [Medline] [Order article via Infotrieve]
  2. Gettins, P., Patston, P. A., and Schapira, M. (1993) Bioessays 15, 461-467 [Medline] [Order article via Infotrieve]
  3. Lawrence, D. A., Olson, S. T., Palaniappan, S., and Ginsburg, D. (1994) J. Biol. Chem. 269, 27657-27662 [Abstract/Free Full Text]
  4. L ö bermann, H., Lottspeich, F., Bode, W., and Huber, R. (1982) Z. Physiol. Chem. 1377-1388
  5. Löbermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R. (1984) J. Mol. Biol. 177, 531-556 [Medline] [Order article via Infotrieve]
  6. Stein, P., Leslie, A. J., Finch, J. T., Turnell, W. G., McLaughlin, P. J., and Carrell, R. W. (1990) Nature 347, 99-102 [CrossRef][Medline] [Order article via Infotrieve]
  7. Schreuder, H. A., deBoer, B., Dijkema, R., Mulders, J., Theunissen, H. J. M., Grootenhuis, P. D. J., and Hol, W. G. J. (1994) Struct. Biol. 1, 48-54
  8. Wei, A., Rubin, H., Cooperman, B. S., and Christianson, D. W. (1994) Struct. Biol. 1, 251-258
  9. Björk, I., Nordling, K., and Olson, S. T. (1993) Biochemistry 32, 6501-6505 [Medline] [Order article via Infotrieve]
  10. Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595 [Abstract/Free Full Text]
  11. Hekman, C. M., and Loskutoff, D. J. (1985) J. Biol. Chem. 260, 11581-11587 [Abstract/Free Full Text]
  12. Keijer, J., Linders, M., Wegman, J. J., Ehrlich, H. J., Mertens, K., and Pannekoek, H. (1991) Blood 78, 1254-1261 [Abstract]
  13. Mottonen, J., Strand, A., Symersky, J., Sweet, R., Danley, D. E., Geoghegan, K., Gerard, R. D., and Goldsmith, E. J. (1992) Nature 355, 270-273 [CrossRef][Medline] [Order article via Infotrieve]
  14. Shore, J. D., Vandenberg, E., Day, D., Olson, S. T., Sherman, R., Ginsburg, D., and Kvassman, J. (1992) Fibrinolysis 6, Suppl. 2, Abstr. 292
  15. Strandberg, L., Johansson, L. B.-A., and Ny, T. (1992) Fibrinolysis 6, Suppl. 2, Abstr. 293
  16. Gettins, P. G. W., Fan, B., Crews, B. C., and Turko, I. V. (1993) Biochemistry 32, 8385-8389 [Medline] [Order article via Infotrieve]
  17. Lawrence, D., Strandberg, L., Grundstrom, T., and Ny, T. (1987) Eur. J. Biochem. 186, 523-533 [Abstract]
  18. Lawrence, D. A., Olson, S. T., Palaniappan, S., and Ginsburg, D. (1994) Biochemistry 33, 3643-3648 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.