Ginsberg, M. H., Painter, R. G., Forsyth, J., Birdwell, C. &. Plow, E. F. (1980) Proc. NatI. Acad. Sci. USA 77, 1049-1053. 6. Plow, E. F. & Ginsberg, M. H. (1981) J.
Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 3472-3476, May 1985 Medical Sciences
A monoclonal antibody against human thrombospondin inhibits platelet aggregation (domain/epitope/amino acid sequence)
VISHVA M. DIXIT*t, DoRIs M. HAVERSTICKt, KAREN M. O'ROURKE*, SALLY W. HENNESSY* GREGORY A. GRANT*§, SAMUEL A. SANTOROt1§, AND WILLIAM A. FRAZIER* Departments of *Biological Chemistry, *Pathology, and §Medicine, and tDivision of Laboratory Medicine, Washington University School of Medicine, St. Louis, MO 63110
Communicated by Stuart Kornfeld, January 28, 1985
A monoclonal antibody (C6.7) has been genABSTRACT erated against the calcium-replete form of human platelet thrombospondin (TSP). C6.7 is specific for TSP as determined by both competitive radioimmunoassay and immunoprecipitation. This antibody inhibits both thrombin- and A23187induced aggregation of gel-iftered platelets in a concentrationdependent manner without affecting the secretion of serotonin. The epitope on TSP recognized by C6.7 has been localized to an 18-kDa fragment that is present in mild chymotryptic digests of TSP. This fragment is disulfide-linked to a 120- to 140-kDa fragment in unreduced digests, and both reduction and denaturation are required to separate the 18-kDa peptide from the larger fragments. A 25-kDa heparin binding domain is also present in the chymotryptic digest. However, the 18-kDa peptide is distinct from the hiparin binding domain. The amino acid sequence at the NH2 terminus of the 18-kDa fragment is Asp-Thr-Asn-Pro-Thr-Arg-Ala-Gln-Gly-Tyr-.
for retention of its native structure (24, 25), and we have found that both calcium and magnesium are required for its erythrocytes and platelet agglutinating activity (22, 23) and for its binding to fibrinogen (17). Several lines of evidence suggest that TSP may play a role in the aggregation of live platelets. Inhibitors of the lectin-like activity of TSP, such as glucosamine and galactosamine, inhibit platelet aggregation (26). Studies with platelets from afibrinogenemic individuals have suggested that platelet surface-bound fibrinogen serves as a receptor for TSP (27). Potentially altered forms of TSP have been described in patients with essential thrombocythemia and abnormal platelet aggregation (28). While these data are suggestive, they are not conclusive. The large number of protein components implicated in platelet aggregation (4-8) and the fact that inhibitors of TSP's lectin activity (e.g., sugars with free amino groups and arginine) also inhibit the interaction of glycoproteins IIb-III and fibrinogen (29) and the hemagglutination activities of von Willebrand factor (30) and fibronectin (31) do not allow a simple interpretation of the available data. To further investigate the role of TSP in platelet aggregation, we have prepared mAbs against human platelet TSP. We report here that one of these mAbs, C6.7, inhibits the aggregation of gel-filtered platelets activated with either thrombin or A23187. Serotonin release and, hence, platelet activation are not impaired. The epitope for C6.7 has been localized to an 18-kDa peptide distinct from the 25-kDa heparin binding domain (12, 15) of TSP.
In recent years, considerable progress has been made in understanding the nature of the interactions involved in platelet aggregation (1-4). Fibrinogen, bound to the platelet membrane glycoprotein Ilb-III complex, appears to play a major role in bridging adjacent platelets (4). However, recent data suggest that this simple model is inadequate. For example, fibronectin is secreted by activated platelets (5) and binds to their surface (6), and excess fibronectin can partially
block washed platelet aggregation (7). Furthermore, we have described a monoclonal antibody (mAb) that reacts exclusively with fibronectin and has the ability to block the aggregation of gel-filtered platelets activated by either thrombin or A23187 (8). This mAb, A3.3, recognizes a site on fibronectin that is distinct from all previously identified domains of the fibronectin molecule, including the cell binding domain (8). Another protein that is thought to have a role in platelet aggregation is thrombospondin (TSP), previously called thrombin-sensitive protein (9, 10) or glycoprotein G (2). TSP is secreted from platelet a granules upon activation (11) and is found associated with the surface of activated platelets (2, 10) in the presence of divalent ions. The trimeric glycoprotein is composed of apparently identical subunits (12) of 180,000 Da linked by disulfide bonds (13, 14). Each monomer contains protease-resistant domains representing binding sites for heparin (12, 13, 15), fibrinogen (16, 17), fibronectin (18), and collagen (18, 19). TSP has been identified as the endogenous platelet lectin that is responsible for the hemagglutinating activity of activated platelets (20, 21). Purified TSP agglutinates fixed trypsinized human erythrocytes (22) as well as fixed activated platelets (23). TSP requires calcium
MATERIALS AND METHODS Materials. Calcium-replete TSP was purified from the supernatant of thrombin-activated human platelets as described (12). Human von Willebrand factor was purified as described (32) and human fibrinogen was from Kabi (Stockholm, Sweden). Human plasma fibronectin was purified as described (33). Reagents for NaDodSO4/PAGE were from Bio-Rad. Production of mAbs Against Human TSP. mAbs against human TSP were produced essentially as described by Galfre et al. (34), using myeloma line SP2/AG14. Positive hybridomas were selected by solid-phase radioimmunoassay using TSP immobilized on polyvinyl chloride well plates, and they
were subcloned into soft agar and rescreened. Positive subclones were propagated by intraperitoneal injection into pristane-primed BALB/c mice and ascites fluid obtained 1 week to 10 days later. The mAbs were purified from ascites fluid by precipitation with 50% saturated ammonium sulfate, followed by gel filtration on Sephacryl S-200 or affinity chromatography on a goat anti-mouse y-globulin (Cappel Laboratories, Cochranville, PA) affinity column. The solid-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: mAb, monoclonal antibody; TSP, thrombospondin.
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phase radioimmunoassay was performed as described (8), using immobilized mAb C6.7 and TSP iodinated with 1251 with the Iodobead (Pierce) method. mAbs were typed with an ELISA kit from Zymed Laboratories (South San Francisco, CA), and C6.7 was found to be an IgG. Platelet Preparation and Aggregation Assays. Platelets were obtained from healthy volunteers and they were anticoagulated and prepared as described (8). After gel filtration on a Sepharose 2B column the platelets were obtained in 0.05 M Tris HCl, pH 7.4/0.15 M NaCl (Tris-buffered saline)/0.3% bovine serum albumin/5 mM glucose. Platelet aggregation and [14C]serotonin secretion studies were carried out in a Payton (Buffalo, NY) dual channel aggregometer as described (7) at a final platelet count of 2.5 x 108 per ml. Immunoprecipitation. Radioiodinated samples of activated platelet supernatant, intact TSP, or the chymotryptic digest of TSP were incubated with 25 41 of a 1:100 dilution of specific or control ascites fluid on ice for 1 hr. Rabbit anti-mouse y-globulin (DAKO, Santa Barbara, CA; 50 I-l of a 1:100 dilution of the solution supplied) was added and incubated for an additional 1 hr on ice, at which time 50 tkl of a 50% (wt/vol) slurry of protein A-Sepharose (Pharmacia) was added and incubated for 15 min on ice with stirring. The protein A-Sepharose was collected by centrifugation for 10 sec in a Microfuge (Beckman) and washed 8-10 times with Tris-buffered saline containing 2 M urea, 0.1 M glycine, and 1% Triton X-100. Samples were transferred to new Microfuge tubes and washed once with Tris-buffered saline. The specifically adsorbed material was solubilized with NaDodSO4 sample buffer containing 1% 2-mercaptoethanol and 8 M urea by boiling for 5 min. NaDodSO4/PAGE was performed by the procedure of Laemmli (35) on 5-15% gradient slab gels. Autoradiography was performed at -70°C, using Cronex Lightning Plus intensifying screens and Kodak X-Omat XAR-5 film. TSP was iodinated by the Bolton-Hunter and the lodobead procedures. The chymotryptic digest of TSP was performed using 0.5% N-a-tosyllysine chloromethyl ketone chymotrypsin (Worthington) at 25°C for 30 min, and was stopped with 1 mM phenylmethylsulfonyl fluoride. The digest was separated on both a BioGel AO.SM column equilibrated in Tris-buffered saline with 20 mM dithiothreitol and on a BioGel ASM column equilibrated in the same buffer with 6 M guanidine hydrochloride. The digest was also fractionated on heparin-Sepharose as described (12). For analysis in the Applied Biosystems vapor-phase sequencer, A kDa
3473
the chymotryptic digest was fractionated on NaDodSO4/ PAGE (15% acrylamide) and stained briefly with Coomassie blue, and the 18-kDa band was cut out and electroeluted as described (12). The recovered 18-kDa fragment (=4 ,g) was sequenced through 10 residues as described (12). The initial yield of aspartic acid was 160 pmol, and subsequent yields of stable residues were in the range of 98-102 pmol.
RESULTS Specificity of mAb C6.7. Three approaches were taken to determine the specificity of the mAb designated C6.7. First, the supernatant obtained from thrombin-activated platelets was radioiodinated and subjected to immunoprecipitation. This was necessary, because mAb C6.7 does not react with TSP on immunoblots (not shown). As shown in Fig. 1A, only a protein corresponding in molecular mass to purified TSP is immunoprecipitated by C6.7. To circumvent the possibility that C6.7 might react with a platelet protein that did not iodinate well, a competitive radioimmunoassay was performed (Fig. 1B). The mAb was immobilized on polyvinyl chloride wells, and the binding of iodinated TSP was challenged with purified TSP itself, the crude platelet supernatant, and the supernatant after affinity adsorption with heparin-Sepharose to remove TSP, platelet factor 4, and P-thromboglobulin (12). Fig. 1B shows that only unfractionated platelet supernatant and purified TSP compete. Thus, it is very unlikely that any other protein found in platelet supernatants aside from TSP itself is recognized by C6.7. Also tested as competitors of TSP binding to C6.7 were purified fibronectin, fibrinogen, and von Willebrand factor, none of which react with the mAb (Fig. 1B). C6.7 has also been used to immunoprecipitate metabolically labeled TSP from lysates of endothelial cells, which have been shown to synthesize TSP (36, 37). These immunoprecipitates contain no other radiolabeled cellular proteins (not shown). Effects of mAb C6.7 on Platelet Aggregation. A panel of mAbs against human platelet TSP were tested for their effects on the thrombin-induced aggregation of gel-filtered platelets. For this screening, we used a microtiter well plate aggregation assay (8), which uses small volumes of reagents. Only mAb C6.7 inhibited platelet aggregation in this assay, while seven demonstrably different mAbs against TSP did not. To confirm these results, C6.7 was tested for its effect on the aggregation of thrombin-treated (0.5 units/ml) platelets in the
B 2
3
4
200926843-
V
9
0 E
E E
251812-
1000
Competitor, ng/ml FIG. 1. Specificity of mAb C6.7. (A) Iodinated proteins were immunoprecipitated with C6.7. Lanes: 1, immunoprecipitate of iodinated TSP; 2, iodinated supernatant from thrombin-activated platelets; 3, immunoprecipitate of supernatant shown in lane 2; 4, immunoprecipitate of supernatant shown in lane 2 with a control mAb. (B) Radioimmunoassay in which mAb C6.7 was immobilized on polyvinyl chloride wells and the binding of a constant amount of 1251-labeled TSP was challenged with various proteins. *, Purified TSP; o, supernatant from thrombin-activated platelets; o, same platelet supernatant adsorbed with heparin-Sepharose to remove TSP; w, fibronectin; A, von Willebrand factor; v, fibrinogen.
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(1985)
0)~~~~~~~~~~r E
|i
i0.2mg/m
0.2mg/mi 0.4 mg/mi /ml~ 0.4 mg
+ ~ Thrombin
~~~ lmin
_
~
A23187
~
~
04m/m
mi
FIG. 2. Inhibition of platelet aggregation by mAb C6.7. Gel-filtered human platelets (2.5 x 108 per ml) were activated (arrow) while stirring at room temperature in the presence of 2 mM CaC12 and 2 mM MgCl2. Aggregation of the platelets is indicated by the increase in light transmittance as monitored in a Payton aggregometer. (A) Platelets were activated with 0.5 units of thrombin per ml. In the control (upper tracing), no mAb was added. In the lower tracings, which show progressive inhibition, the concentration of mAb C6.7 is indicated in mg/ml. (5) Platekts were activated with the Ca2+ ionophore A23187 (2.5 mM). Again, mAb C6.7 shows concentration-dependent inhibition.
aggregometer. Fig. 2A shows that at 250C, platelets aggregate in an apparently normal fashion with a slightly slower time
course than that observed at 37°C. mAb A2.5, which had no effect on platelet aggregation in the microtiter assay, did not affect the rate or extent of platelet aggregation seen in the
aggregometer (not shown). The concentration-dependent inhibition of platelet aggregation by mAb C6.7 is depicted in Fig. 2A. As shown in Fig. 2B, C6.7 also blocks the aggregation of platelets stimulated with A23187 with a similar concentration dependence. At physiological temperature, platelet aggregation proceeds more rapidly. However, C6.7 can still inhibit aggregation (not shown), although the concentration dependence exhibits a cooperative (threshold or "all or none") effect instead of the more gradual onset of inhibition seen at 25°C. The secretion of serotonin was unaffected by mAb C6.7 under any of the conditions used, indicating that its effect is not due to the inhibition of platelet secretion.
Lcaizatios of the Epitope for C6.7 on TSP. Digestion of calcium-replete TSP with chymotrypsin (0.5% of TSP) at 25°C (but not at 37°C) for 30 min produced a fragment that contained the epitope for C6.7. Fig. 3A shows the autoradiographs of an immunoprecipitate of intact TSP (lane 1) and the chymotryptic digest before (lane 2) and after (lane 3) immunoprecipitation. The NaDodSO4 gel for this experiment was run under reducing conditions. Thus, it appears that mAb C6.7 recognizes either the large 120- to 140-kDa doublet or the small peptide of 18 kDa, and that these fragments are linked by one or more disulfide bonds. Under nonreducing conditions, the fragments that are specifically precipitated run as a doublet with molecular sizes in excess of 400 kDa (Fig. 3B). To determine which of the chymotryptic fragments, the 120- to 140-kDa or the 18-kDa, contained the epitope for C6.7, it was necessary to separate them under reducing and denaturing conditions. The digest was fractionated in 20 mM dithiothreitol/6 M guanidine hydrochloride on BioGel A5M to obtain a clean separation of the 18-kDa peptide from the 120- to 140-kDa doublet, as well as from a 25-kDa peptide also present in the digest (Fig. 4B). The column fractions were dialyzed against Tris-buffered saline containing 1 mM calcium, and the separated fragments were immunoprecipitated with C6.7 as described above. The results (Fig. 4C) clearly
show that only the 18-kDa peptide is specifically bound by C6.7 and, hence, the epitope is localized to this small region of TSP. Since we had previously identified a heparin binding domain in tryptic and thermolytic digests of TSP with a molecular size in the range of 25 kDa (12), we wanted to determine whether the 18-kDa fragment identified here represented another form of this heparin binding domain resulting from digestion with chymotrypsin. To do this, the chymotryptic digest was passed through heparin-Sepharose under conditions that allow retention of the heparin-binding domain on the column (12). The 18-kDa fragment that is recognized by C6.7 flows through the column (not shown) and, hence, does not contain a heparin binding domain by this functional criterion. The 25-kDa peptide present in the same digest binds to the heparin column and elutes with high salt concentration. A
4 kDa 200-p
92--. 43258i2-
2
B 5
3
6 4.
....0 92-
200-
43251812-
14.14
:-,
:. .
A
FIG. 3. Immunoprecipitation of TSP and its chymotryptic digest by mAb C6.7. Intact TSP was iodinated by the Bolton-Hunter reagent and the Iodobead procedure, using Na'251, and digested with chymotrypsin (0.5% TSP) for 30 min at 25°C. Samples were immunoprecipitated with C6.7, and the material bound to the mAb was analyzed by NaDodSO4/PAGE followed by autoradiography. Lanes: 1-3, samples reduced with 2-mercaptoethanol prior to NaDodSO4/PAGE; 4-6, in the absence of reducing agent. Lanes 1 and 4, intact TSP; lanes 2 and 5, chymotryptic digest before immunoprecipitation; lanes 3 and 6, immunoprecipitate of the digest.
Proc. Natl. Acad. Sci. USA 82
Medical Sciences: Dixit et al. A
kDa
C
B
200-
5
16 20 21 22 23 24 25 26
2 3 4 5 -200
200-
92-t
;92
92-
25I 1825-
4
-25
:.
*
18-
1
-
8
FIG. 4. Identification of the chymotryp tic fragment of TSP containing the epitope for C6.7. (A) Chymotrylptic digest of iodinated TSP analyzed by NaDodSO4/PAGE under re(ducing conditions. (B) Fractions of the digest obtained by gel filtrat ion of the digest on a column of BioGel A5M run in the presence orf 20 mM dithiothreitol and 6 M guanidine HCl. Numbers above t he lanes indicate the fraction number. (C) Immunoprecipitation BioGel A5M column with mAb C6.7 analyzed on Lanes: 1, 120- to 140-kDa doublet (fraction 1 5); 2, isolated 18-kDa fragment (fraction 25); 3, immunoprecipita te of fraction 15- 4, immunoprecipitate of fraction 25; 5, immunoprrecipitate of fraction 25 with a control mAb.
NaDodSO4/PAGEm
To further characterize the 18-kDa pI eptide and to determine whether it resulted from a urnique cleavage by chymotrypsin, the chymotryptic digest was separated on NaDodSO4/PAGE, and the 18-kDa banid was identified by brief staining, excised from the gel, an d electroeluted (12). This eluted material was subjected to automated Edman degradation in the Applied Biosystems (Foster City, CA) vapor-phase sequencer. A single sequenc-e was determined in yields expected for this methodology: 10 5 1
Asp-Thr-Asn-Pro-Thr-Arg-Ala-( iln-Gly-Tyr-. Thus, chymotrypsin cleaves TSP at a un ique site to generate the 18-kDa peptide that contains the epiitope for mAb C6.7.
*J~n-Gy-Ty-
DISCUSSION Since its discovery by Baenziger et al. (9) in 1971, TSP has been thought to have some role in plate let aggregation. It is found at high levels in platelet a-granule s and at low levels in plasma (38), and it is secreted in large aimounts after platelet activation by a variety of stimuli (11). The report that TSP could apparently account for the lectiri activity associated with activated platelets (20, 21) lent fuirther support to the idea that TSP is a causal agent in Iplatelet aggregation. Furthermore, free amino sugars and atrginine, compounds that inhibit the agglutination activity of activated platelets and purified TSP, were also found to, at Ileast partially, inhibit the aggregation of thrombin-stimulatedI platelets (26). The interpretation of these results, however, ,has become clouded by the observations that the bindinig of fibrinogen to glycoproteins Ib-III (29) as well as the lectin activity of factor VIII/von Willebrand factor (30) and fibronectin (31) are all inhibited by amino sugars and ar~ Furthermore, TSP, fibronectin, and von Willebrand fE and are all from a-granules at the time of platelet a deficient in Gray platelet syndrome (39P. in pltele aggegaTo help clarify the role, if any, of TS]P in platelet aggregation and to aid in the identification of; large TSP molecule, we produced mAbs agains tTSPithat had been maintained in the presence of Ca2 aurIng lts purrncation and handling. Of several mAbs ag Jainst TSP, only one (C6.7) had any effect on the aggregaLtion of live washed platelets stimulated by thrombin on A233187. Immunoprecip-
ainine.
activation
(1985)
3475
itation of iodinated platelet supernatants, radioimmunoassay of platelet and plasma proteins, and immunoprecipitation of endothelial cell proteins labeled with [35S]cysteine all indicate that mAb C6.7 recognizes no protein aside from TSP (Fig. 1). Thus, the dramatic effect on platelet aggregation (Fig. 3) must be due to the binding of the mAb to TSP secreted by the activated platelets in response to thrombin or A23187 stimulation. These data provide unequivocal evidence for an important role for TSP in platelet aggregation. Since this paper was submitted, two reports have appeared in which polyclonal antisera against a fragment of bovine TSP (40) and intact human TSP (41) were reported to inhibit the secretiondependent phase of platelet aggregation. These observations further strengthen our conclusion that TSP has a direct role in the aggregation process. The region of the TSP molecule that appears to be involved in this reaction is represented in mild chymotryptic digests as an 18-kDa fragment that is derived from a region of the TSP peptide chain distinct from the heparin binding domain (12, 15). The heparin site resides on a 25-kDa domain that we have found to be located at the NH2 terminus of the TSP polypeptide chain, because both the intact molecule and the isolated heparin binding domain have identical amino acid sequences at their NH2 termini (12, 42). The 18-kDa fragment that contains the epitope for C6.7 is clearly distinct from the heparin binding domain. We have previously noted that the binding site on TSP for erythrocytes (22) and fixed activated platelets (23) appears to reside on a 140-kDa thermolytic fragment and is lost upon further digestion of this fragment to 120 kDa. Thus, it is tempting to suggest that the 18-kDa peptide that binds mAb C6.7 also represents the region of TSP that contains the erythrocyte and platelet binding site(s). If it is indeed derived from one end of the 140-kDa thermolytic fragment, it would lie either quite near, perhaps adjacent to, the NH2-terminal heparin binding domain or very near the COOH terminus of the TSP peptide chain. Electron microscopy of TSP-mAb complexes suggests a location for the C6.7 epitope distant from the amino terminal heparin binding domain (unpublished results). In summary, current data based on the use of antibodies exist for the involvement of TSP (this report, refs. 40 and 41), fibronectin (8), glycoproteins IIb-III (43), and fibrinogen (44) in platelet aggregation. It appears that blocking specific sites on any of these four molecules is sufficient to disrupt the aggregation process. It is hoped that by further defining the sites on these large proteins that are important and the components with which these sites interact, it will be possible to construct models for the specific macromolecular interactions between these proteins and the platelet surface that must be formed correctly for platelets to aggregate. At present, it seems that the aggregation process can be viewed as a rapid and precise assembly of a transient local extracellular matrix derived from contents of the platelets' own a-granules and from circulating plasma factors. Since TSP, fibronectin, and fibrinogen are all packaged together in the a-granules, they may exist in a preformed complex and this
preexisting piece of matrix material may be secreted and added to or modified in the extraplatelet environment. Characterization of the mAb C6.7 described here represents
just one step in elucidating the precise role of TSP in this physiologically important and complex process. We thank Dr. Greg Cole for help in performing the fusion that
produced mAbs against native TSP, and for much helpful advice
the Washington staff ofDirector) the Davie, due Joe are (Dr. work. Thanks this Hybridoma during for their Center University assistance with fusions and initial selection of the hybridomas, and the staff of the American Red Cross (St. Louis Chapter) for their help and cooperation in obtaining outdated platelets for TSP purification. The work reported here was supported by funds from the following
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agencies to the indicated persons: National Institutes of Health Training Grant HL-17138 to D.M.H.; a grant-in-aid from the American Heart Association with funds provided in part by the Missouri Affiliate to S.A.S.; and a grant from the Monsanto Co., St. Louis, MO, to W.A.F., who is an Established Investigator of the American Heart Association. 1. Kaplan, K. L. (1981) in Platelets in Biology and Pathology 2, eds. Dingle, J. T. & Gordon, J. L. (Elsevier/North-Holland, New York) pp. 77-90. 2. Phillips, D. R., Jennings, L. K. & Prasanna, H. R. (1980) J. Biol. Chem. 255, 11629-11632. 3. Fujimura, K. & Phillips, D. R. (1983) J. Biol. Chem. 258, 10247-10252. 4. Berndt, M. C. & Phillips, D. R. (1981) in Platelets in Biology and Pathology 2, eds. Dingle, J. T. & Gordon, J. L. (Elsevier/North-Holland, New York), pp. 43-75. 5. Ginsberg, M. H., Painter, R. G., Forsyth, J., Birdwell, C. & Plow, E. F. (1980) Proc. NatI. Acad. Sci. USA 77, 1049-1053. 6. Plow, E. F. & Ginsberg, M. H. (1981) J. Biol. Chem. 256, 9477-9482. 7. Santoro, S. A. (1983) Biochem. Biophys. Res. Commun. 116, 135-140. 8. Dixit, V. M., Haverstick, D. M., O'Rourke, K., Hennessy, S. W., Broekelmann, T. J., McDonald, J. A., Grant, G. A., Santoro, S. A. & Frazier, W. A. (1985) Proc. NatI. Acad. Sci. USA 82, in press. 9. Baenziger, N. L., Brodie, G. N. & Majerus, P. W. (1971) Proc. NatI. Acad. Sci. USA 68, 240-243. 10. Baenziger, N. L., Brodie, G. N. & Majerus, P. W. (1972) J. Biol. Chem. 247, 2723-2731. 11. Gartner, T. K., Gerrard, J. M., White, J. G. & Williams, D. C. (1981) Blood 58, 153-157. 12. Dixit, V. M., Grant, G. A., Santoro, S. A. & Frazier, W. A. (1984) J. Biol. Chem. 259, 10100-10105. 13. Lawler, J. W., Slayter, H. S. & Coligan, J. E. (1978) J. Biol. Chem. 253, 8609-8616. 14. Margossian, S. S., Lawler, J. W. & Slayter, H. S. (1981) J. Biol. Chem. 256, 7495-7500. 15. Lawler, J. W. & Slayter, H. S. (1981) Thromb. Res. 22, 267-279. 16. Leung, L. L. K. & Nachman, R. L. (1982) J. Clin. Invest. 70, 542-549. 17. Dixit, V. M., Grant, G. A., Frazier, W. A. & Santoro, S. A. (1984) Biochem. Biophys. Res. Commun. 119, 1075-1081. 18. Lahav, J., Schwartz, M. A. & Hynes, R. 0. (1982) Cell 31, 253-262. 19. Mumby, S. M., Raugi, G. J. & Bornstein, P. (1984) J. Cell Biol. 98, 646-652.
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20. Gartner, T. K., Williams, D. C. & Phillips, D. R. (1977) Biochem. Biophys. Res. Commun. 79, 592-599. 21. Jaffe, E. A., Leung, L. L. K., Nachman, R. L., Levin, R. I. & Mosher, D. F. (1982) Nature (London) 295, 246-248. 22. Haverstick, D. M., Dixit, V. M., Grant, G. A., Frazier, W. A. & Santoro, S. A. (1984) Biochemistry 23, 5597-5603. 23. Haverstick, D. M., Dixit, V. M., Grant, G. A., Frazier, W. A. & Santoro, S. A. (1984) Biochemistry, in press. 24. Lawler, J. W., Chao, F. C. & Cohen, C. M. (1982) J. Biol. Chem. 257, 12257-12265. 25. Lawler, J. W. & Simons, E. R. (1983) J. Biol. Chem. 258, 12098-12101. 26. Agam, G., Gartner, T. K. & Levine, A. (1984) Thromb. Res. 33, 245-257. 27. Gartner, T. K., Gerrard, J. M., White, J. G. & Williams, D. C. (1981) Nature (London) 289, 688-690. 28. Booth, W. J., Berndt, M. C. & Castaldi, P. A. (1984) J. Clin. Invest. 73, 291-297. 29. Nachman, R. L. & Leung, L. L. K. (1982) J. Clin. Invest. 69, 263-269. 30. Booth, W. J., Furby, F. H., Berndt, M. C. & Castaldi, P. A. (1984) Biochem. Biophys. Res. Commun. 118, 495-501. 31. Yamada, K. M., Yamada, S. S. & Pastan, I. (1975) Proc. Natl. Acad. Sci. USA 72, 3158-3162. 32. Santoro, S. A. & Cowan, J. F. (1982) Collagen Res. 2, 31-43. 33. McDonald, J. A. & Kelley, D. G. (1980) J. Biol. Chem. 255, 8848-8858. 34. Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W. & Howard, J. C. (1977) Nature (London) 291, 421-423. 35. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 36. Mosher, D. F., Doyle, M. J. & Jaffe, E. A. (1982) J. Cell Biol. 93, 343-348. 37. Raugi, G. J., Mumby, S. M., Abbott-Brown, D. & Bornstein, P. (1982) J. Cell Biol. 95, 351-354. 38. Saglio, S. D. & Slayter, H. S. (1982) Blood 59, 162-166. 39. Gerrard, J. M., Phillips, D. R., Rao, G. H. R., Plow, E. F., Walz, D. A., Ross, R., Harker, L. A. & White, J. G. (1980) J. Clin. Invest. 66, 102-109. 40. Gartner, T. K., Walz, D. A., Aiken, M., Starr-Spires, L. & Ogilvie, M. L. (1984) Biochem. Biophys. Res. Commun. 124,
290-295. 41. Leung, L. L. K. (1984) J. Clin. Invest. 74, 1764-1772. 42. Coligan, J. E. & Slayter, H. S. (1984) J. Biol. Chem. 259, 3944-3948. 43. McEver, R. P., Bennett, E. M. & Martin, M. N. (1983) J. Biol. Chem. 258, 5269-5275. 44. Tollefson, D. M. & Majerus, P. W. (1975) J. Clin. Invest. 55, 1259-1268.
