Reversibility of fibrinogen fragment D1 binding to human platelets: Comparison with native fibrinogen

THROMBOSIS RESEARCH 68; 259-267,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

REVERSIBILITY OF FIBRINOGEN FRAGMENT D, BINDING TO HUMAN PLATELETS: COMPARISON WITH NATIVE FIBRINOGEN

Ellinor I. B. Peerschke* Department of Pathology, SUNY at Stony Brook Stony Brook, NY 11794-7300 (Received 22.6.1992; accepted in revised form 159.1992 by Editor C.W.Francis) (Received by Executive Editorial Office 30.9.1992) ABSTRACT

To gain further insight into the mechanism responsible for rendering fibrinogen bound to stimulated platelets irreversible to dissociation by EDTA or excess unlabeled fibrinogen, the present study compared the reversibility of platelet interactions with fibrinogen and its plasmic degradation product, fragment D,. Like fibrinogen binding, the binding of fragment D, became progressively less sensitive to dissociation by EDTA, PGE,, or excess unlabeled fibrinogen. Thus in the presence of EDTA, 70 + 19% and 55 + 24% (mean + S.D., n=9) of bound fragment D, failed to dissociate from platelets 60 min after stimulation with 0.15 U/ml thrombin or the combination of 5/uM ADP and 5pM epinephrine, respectively, compared to 75 + 8% and 52 + 17% of platelet-bound, intact with fibrinogen. In contrast, platelet stimulation chymotrypsin or Zn+' failed to support the development of irreversible fragment D, or fibrinogen binding. Only 8 + 6% and 9 + 3% of bound fragment yJ remained associated with Zn --treated platelets, chymotrypsinor respectively, compared to 7 f. 11% and 15 f 6% (mean + S.D., n=3) of platelet-associated fibrinogen. These observations suggest that irreversible fragment D, and fibrinogen binding to platelets occurs by a similar mechanism that requires neither fibrinogen alpha chain 95-97 or 572-574 RGD sequences nor multivalent ligandreceptor interactions.

Key words: Platelets, fibrinogen binding, fragment D, *To whom correspondence should be addressed.

259

260

STABILIZATION OF D, BINDING

Vol.68,No.3

INTRODUCTION The interaction between platelets and fibrinogen is a dynamic process that undergoes qualitative changes over time (1,2). Whereas fibrinogen binding to stimulated platelets is reversible initially, the interaction becomes progressively less reversible in a temperature- and agonist-dependent manner. Although the precise mechanism whereby understood, several this occurs is not (reviewed in 2). contributing factors have been identified Activation of the platelet cytoskeleton appears to be important, and release of alpha granule thrombospondin, although not strictly required, may also contribute. Although fibrinogen binding to several observations GPIIb-IIIa complexes is a prerequisite, suggest that intact GPIIb-IIIa complexes may not be required to mediate "irreversible" platelet fibrinogen interactions (3). Fibrinogen contains known platelet binding domains on both its alpha and gamma chains. The GPIIb-IIIa complex recognizes two RDG sequences in the alpha chain of fibrinogen (residues 95-97 and residues 572-574) to the carboxyterminal (4,5), and binds dodecapeptide sequence (residues 400-411) of the gamma chain (6). Based on these findings, the hypothesis has been advanced that irreversible platelet-fibrinogen interactions involve the multivalent interaction between identified alpha and gamma chain sites in fibrinogen and GPIIb-IIIa receptors. Although previous studies suggested that RGD 572-574 was not required to support irreversible platelet-fibrinogen interactions (3), the relative contribution of RGD 95-97 and the gamma chain dodecapeptide could not be ascertained. The present study addresses these questions by comparing the reversibility of platelet interactions with intact fibrinogen and its plasmic degradation fragment, D,. Fragment D, represents monovalent ligand an apparently containing only the gamma 400-411 platelet-binding sequence (7). It has been shown to retain much of its native conformation (8), and competes with intact fibrinogen for GPIIb-IIIa binding sites (9-11). Fibrinogen fragment D,, however, does not support platelet aggregation (12), presumably due to its monovalent binding capacity. MATERIALS

AND METHODS

Platelet Preoaration Blood from healthy volunteers was collected after obtaining informed consent. Blood was collected into 0.1 vol of 3.2% sodium citrate and 0.05 vol of 1mM aspirin per volume of whole blood. Platelet-rich plasma was obtained by centrifugation (28Og, 15 min). It was incubated 20 min with O.lpM PGE, (Sigma Chemical Co., St. Louis, MO), acidified to pH 6.5 with 1 M citric acid, and centrifuged (lOOOg, 20 min). The platelet pellet was resuspended in 0.01 M HEPES-buffered modified Tyrode's solution (pH 7.5) . . containing 2 mM MgCl,, no added calcium, 2 mg/ml bovine serum albumin, and 0.01 uM PGE,. Platelets were gel-filtered over a column of Sepharose 2B (Pharmacia Fine Chemicals, Piscataway, N.J.) equilibrated with the same buffer but lacking PGE, (HBMT). Fibrinoaen and Fracnnent D, Purification Intact fibrinogen was isolated from fresh frozen plasma as described (13). This material was diluted to a concentration of 1.7 mg/ml, dialyzed against 0.01 M Tris-HCl, 0.15M NaCl pH 8.1., and

Vol.68,No.3

STABILIZATIONOF D,BlNDlNG

261

digested, in the presence of 5 mu CaCl,, with 40 ug/ml plasminogen at 37'C for 48 hours. The digest was dialyzed against 0.025 M Tris/HCl pH 7.8, centrifuged (lOOOg, 30 min) and applied to a column of polybuffer exchanger gel (PBE 94) (Pharmacia LKB, Uppsala, Sweden), equilibrated with the same buffer (14). The column was eluted with polybuffer 74 pH 5.0. Isolated D, fragments were analyzed by SDS-polyacrylamide gel electrophoresis (15). Native fibrinogen and fragment D, (mol wt 100kDa) were radiolabeled with '=iodine using iodobeads (Pierce Chemical Co., Rockford, to manufacturer's instructions. The IL) according following specific activities were attained: fibrinogen, 3600 cpm/pg; fragment D,, 5000 cpm/ g. p Fibrinoaen and Fraoment D, Bindinq Binding was induced using gel-filtered platelets (GFP) and lzI-fibrinogen (10 PM) or lzI-fragment D, (10 PM). Although the development of irreversible platelet-fibrinogen interactions is slightly enhanced at 37°C compared to 22'C (2), studies were performed at 22OC as GFP were more stable at this temperature. Platelets were stimulated with the combination of 5/21M ADP and 5 PM epinephrine, 0.3 mM ZnCl,, or 0.15 U/ml thrombin (human thrombin was the generous gift for Dr. John Fenton, New York State Department of Health, Albany). Thrombin was neutralized with excess hirudin (Sigma) before adding fibrinogen or fragment D,. Additional platelets were stimulated with 0.15 mg/ml chymotrypsin (5 min, 37OC). Chymotrypsin was neutralized with a lo-fold molar excess of phenylmethylsulfonyl were subsequently fluoride. Platelets centrifuged in the presence of 5 mM EDTA and resuspended in HBMT. Ligand binding occurred for 60 min (22'C), as this was previously found to yield optimal stabilization of fibrinogen binding (16). Ligand binding to GPIIb-IIIa was confirmed using a monoclonal antibody, lOE5 (2Opg/ml) (a generous gift from Dr. B. Coller, SUNY at Stony Brook, Stony Brook, NY)(17). Bound ligand was separated from free ligand by centrifugation (12,OOOg, 5 min) of samples through silicon oil (d=1.040). Nonspecific ligand binding was evaluated in the presence of 10 mM EDTA. Specific binding was defined as the amount of total binding less nonspecific binding. Irreversible ligand binding was measured by adding 10 mM EDTA, 2 PM PGE,, or 2 mg/ml intact fibrinogen to platelets 60 min after ligand binding, and quantifying residual binding following a 30 min incubation at 22'C. Nonspecific binding, determined as described above, was subtracted.

plus 1500 U/ml streptokinase,

Qualitative Analvsis of Bound and Unbound Fibrinoaen and Fraoment D, Bound and unbound ligands were analyzed by SDS-polyacrylamide gel electrophoresis (15),25andautoradiography. Platelets bearing I-fragment D, were centrifuged through bound lzI-fibrinogen or silicone oil, the oil and supernatant buffer removed, and the platelet pellet solubilized in a solution containing 1 part 3.3% and 1 part 1% SDS, 12.5 mM SDS and 6 mM N-ethylmaleimide, tris(hydroxymethy1) aminomethane chloride (pH 6.8), 20% glycerol and 0.025% bromophenol blue. Supernatant fractions containing unbound ligands were treated with equal volumes of the above sample Gels were stained with Coomassie buffers and electrophoresed. Brilliant Blue, dried, and exposed to X-ray film (Kodak, Rochester,

262

STABILIZATION

OF D, BINDING

NY).

No qualitative differences in bound and unbound detected by SDS-PAGE (Figure 1).

Vol. 68, No. 3

ligand were

Mol. wt. Markers 18OK 9OK

-

60X 42K 251 -

(1) (2) FIG.

(3)

(4)

1

Autoradiogramludepicting unbouyg (lanes 1,3) and bound (lanes 2,4) I-fragment D, following I-fibrinogen or SDS-PAGE using 7.5% gels. All samples were examined without reduction. RESULTS Platelet stimulation with a variety of agonists supported fragment D, binding. At a final concentration of 10 PM, approximately 8,000 . + 3200 (mean + S.D., n=3) molecules of fragment D, bound to thrombin-stimulated platelets, compared to 57,000 + This difference most likely 11,000 molecules of fibrinogen. reflects previously characterized differences in binding affinities (9-11). Similar to fibrinogen binding, fragment D, binding to thrombin-activated platelets was inhibited (97% + 6%) (mean + S.D., n=4) in the presence of the lOE5 monoclonal antibody which recognizes GPIIb-IIIa. As summarized in Table I, 70% to 75% of bound fragment D, as well as intact associated with thrombinfibrinogen became Similar stimulated manner. platelets in an EDTA-resistant observations were made if the reversibility of fibrinogen and D, binding was assessed in the presence of 2 uM PGE, or 2 mg/ml unlabeled fibrinogen (data not shown). To rule out nonspecific trapping of fragment D, on the surface of thrombin-stimulated platelets by platelet-associated fibrin, similar experiments were conducted in the presence of 0.5 mM gly-pro-arg. No difference in the extent of EDTA-resistant fragment D, binding was observed in the presence or absence of peptide (data not shown).

Vol. 68, No. 3

STABILIZATION OF D, BINDING

TABLE

Comparison

of EDTA-Resistant

AGONIST

ADP + EPI THROMBIN Zn+' CHYMOTRYPSIN

263

I

Fibrinogen

And Fragment D, Binding

EDTA-RESISTANT LIGAND BINDING (% Relative to Total Binding) FRAGMENT D, 55 + 24 70 + 19 9+3 826

FIBRINOGEN 52 + 17 75 + 8 7 + 11 15 + 6

Values represent mean + S.D., n=lO. Platelets were stimulated with 5 &M ADP plus 5 PM epinephrine (EPI), 0.15 U/ml thrombin, 0.3 mM 0.15 U/ml ZnCl, or chymotrypsin. '?-labeled fibrinogen (1OpM) or fragment D, (lO/uM) was added, and ligand binding quantified after 60 min (22'C). EDTA-resistant binding was assessed after incubating samples with 10 mM EDTA for 30 min (22'C). Platelet stimulation with a combination of ADP and epinephrine also supported EDTA-resistant fragment D, and fibrinogen binding. Platelet activation with chymotrypsin or ZnCl,, however, failed to support irreversible fragment D, interactions with platelets. The same agents were previously reported to inhibit formation of EDTAresistant fibrinogen binding (2,16,18). ZnCl, also inhibited the development of EDTA-resistant fragment D, binding to ADP-stimulated platelets in a concentration dependent manner, reaching a maximum near 0.15 mM (Table II). TABLE

II

Effect of Zn+' on ADP-Induced Stabilization of Platelet Interactions with Fibrinogen and Fragment D, ZnCl, (mM) 0 0.0075 0.015 0.0375 0.075 0.15 0.30

EDTA-RESISTANT LIGAND BINDING (% Relative to Total Binding) FIBRINOGEN 55 + 7 51 + 23 45 + 7 39 t 12 29 + 10 14 + 6 15 + 4

Dl

49 50 42 36 20 10 11

+ 12 + 11 + 15 sf:9 + 14 + 5 5 7

were Values represent mean + S.D., n=4. Platelets stimulated with 10,uM ADP in the presence or absence of increasing concentrations of ZnCl,. Ligand binding was assessed after 60 min (22"C), and EDTA resistant binding quantified 30 min (22'C) after adding 10 mM EDTA.

264

STABILIZATION OF D, BINDING

Vol. 68, No. 3

DISCUSSION Human fibrinogen is essential for the GPIIb-IIIa mediated interaction of platelets with each other (13). Fibrinogen binding to platelets has been described as a multiphasic process, culminating in irreversible binding, resistant to dissociation by EDTA or exchange with excess unbound fibrinogen (1,2). This occurs via noncovalent interactions, and may play a role in clot retraction and formation of large platelet aggregates (2,16,19). To better understand the mechanism(s) responsible for the development of irreversible platelet-fibrinogen interactions, the present study compared the binding of fibrinogen and its plasmic degradation product, fragment D,. This comparison addressed the role of fibrinogen RGD sequences in irreversible fibrinogen binding to platelets, and examined the potential requirement for multivalent platelet-fibrinogen interactions. The data suggest a similar agonist specificity for the induction of both EDTA-resistant fibrinogen and fragment D, binding. Agonists including combinations and of ADP and epinephrine, thrombin supported the formation of EDTA-resistant fibrinogen and fragment D, binding, whereas agents including chymotrypsin and ZnCl, failed to do so. In the present study ZnCl, also inhibited the ADPinduced stabilization of fragment D, interaction with platelets. This inhibition may reflect the action of Zn+2 on GPIIb-IIIa calcium binding sites and/or platelet protein kinase C (20,21), which can activate GPIIb-IIIa receptors but appears unable to support the full spectrum of post-fibrinogen binding events (18). Previous studies have shown that the fibrinogen alpha chain RGD 572-574 sequence is not necessary for the formation of irreversible platelet-fibrinogen interactions (3). The present study confirms these observations and demonstrates the lack of requirement for RGD 92-95 as well. The data further suggest that multivalent associations between fibrinogen and the platelet membrane are not a prerequisite for the development of EDTAresistant fibrinogen binding. The presence of multiple, symmetrically distributed platelet binding sites in native fibrinogen (4-6), however, is thought to confer specificity and complexity to fibrinogen interactions with platelets, as well as support post-fibrinogen binding events including platelet aggregation (12). The present study demonstrates that a single gamma 400-411 sequence, present in fragment D,, may be sufficient to support the development of EDTA-resistant interactions with GPIIb-IIIa. This peptide sequence has been shown by chemical crosslinking studies to interact with GP IIb near the second calcium binding domain (22). The potential involvement of GPIIIa in supporting EDTA-resistant plateletfibrinogen interactions cannot be ruled out, however. In addition to the observed stabilization of platelet-fibrinogen interactions, other post-fibrinogen binding events have been described. One of these involves conformational changes in the GPIIb-IIIa receptor. These can be detected by antibodies that bind preferentially to GPIIb or GPIIIa when the receptor is occupied with fibrinogen or RGD containing peptides (23,24). Conformational changes have also been described in bound fibrinogen using monoclonal antibodies (25). Conceivably, conformational changes in both ligand and receptor may directly or indirectly participate in Conferring EDTA-resistance to fibrinogen and fragment D, binding

Vol. 68, No. 3

STABILIZATION

OF D, BINDING

265

to platelets.

ACKNOWLEDGEMENT This work was supported by NIH grant HL28183 from the National Heart, Lung, and Blood Institute. The author is grateful to Jean Ann Wainer for expert technical assistance, and Dr. D.K. Galanakis, SUNY at Stony Brook, Stony Brook, NY for assistance with the preparation of fibrinogen D, fragments. REFERENCES 1.

MARGUERIE, GA, EDGINGTON, TS, and PLOW, EF: Interaction of fibrinogen with its platelet receptors as part of a multistep reaction in ADP-induced platelet aggregation. J Biol Chem 255:154-161, 1980.

2.

PEERSCHKE, EIB: Events occurring after thrombin-induced fibrinogen binding to platelets. Semin Thromb Hemost 18~34-43, 1992.

3.

PEERSCHKE, EIB: Irreversible platelet-fibrinogen of fibrinogen alpha interactions occur independently chain degradation and are not mediated by intact platelet membrane glycoprotein IIb-IIIa complexes. J Lab Clin Med 111:84-92, 1988.

4.

DOOLITTLE, RF, WATT, KWK, COLLTRELL, BA, STRONG, DD, and RILEY, M: The amino acid sequence of the gamma chain of human fibrinogen. Nature 280:460-468, 1979.

5.

KLOCZEWIAK, M, TIMMONS, S, LUKAS, T, and HAWIGER, J: Platelet receptor recognition site in human fibrinogen. Synthesis and structure-function relationshipofpeptides corresponding to the carboxy-terminal segment of the alpha chain. Biochemistry 23:1767-1774, 1984.

6.

HAWIGER, J, TIMMONS, S, KLOCZEWIAK, M, STRONG, DD, and DOOLITTLE, RF: Gamma and alpha chains of human fibrinogen possess sites reactive with human platelet receptors. Proc Nat1 Acad Sci (USA) 79:2068-2071, 1982.

7.

AZ: Conformational CIERNIEWSKI, CS, and BUDZYNSKI, equilibria in the gamma chain COOH terminus of human fibrinogen. J Biol Chem 262:13896-13901, 1987.

8.

AZ: M, and BUDZYNSKI, CIERNIEWSKI, CS, KLOCZEWIAK, sites in the D Expression of primary polymerization intact depends on fibrinogen human domain of 261:9116-9121, 1986. conformation. J Biol Chem

266

STABILIZATION OF D, BINDING

Vol. 68, No. 3

9.

MARGUERIE, GA, ARDAILLOU, N, CHEREL, G, and PLOW, EF: The binding of fibrinogen to its platelet receptor. Involvement of the D domain. JBiol Chem 257:11872-11875, 1982.

10.

NACHMAN, RL, LEUNG, LLK, KLOCZEWIAK, M, and HAWIGER, J: Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain. J Biol Chem 159:8584-8588, 1984.

11.

KIRSCHBAUM, NE, MOSESSON, MW, and AMRANI, DL: Characterization of the gamma chain platelet binding site on fibrinogen fragment D. Blood 79:2643-2648, 1992.

12.

HAWIGER, J: Adhesive interactions of platelets and their blockade. Ann N Y Acad Sci 614:270-278, 1991.

13.

PEERSCHKE, EI, ZUCKER, MB, GRANT, RA, EGAN, JJ, and JOHNSON, MM: Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood 55:841847, 1980.

14.

TERUKINA, S, MATSUDA, M, HIRATA, H, TAKEDA, Y, MIYATA, T, TAKAO, T, and SHIMONISHI, Y: Substitution of gamma Arg-275 by Cys in an abnormal fibrinogen, "Fibrinogen Osaka II." Evidence of a unique solitary cystine structure at the mutation site. J Biol Chem 263:1357913587, 1988.

15.

LAEMMLI, UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680685, 1970.

16.

PEERSCHKE, EIB, and WAINER, JA: Examination of irreversible platelet-fibrinogen interactions. Amer J Physiol 248:C466-C472, 1985.

17.

COLLER BS, PEERSCHKE EIB, SCUDDER LE, and SULLIVAN CA: A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa. J Clin Invest 72:325338, 1983.

18.

PEERSCHKE, EIB: Stabilization of platelet-fibrinogen interactions: Effect of agonist. Thromb Haemost: in press, 1992.

19.

PEERSCHKE, EIB: Stabilization of platelet fibrinogen interactions: Modulation by divalent cations. Circulation: in press, 1992.

20.

FINDIK, D, and PRESEK, P: Zn++ enhances protein kinase activity of human platelet membranes. FEBS LETT 235:5156, 1988.

Vol. 68, No. 3

STABILIZATION OF D, BINDING

267

21.

MARAKAMI, K, WHITELEY, MK, and ROUTTENBERG, A: Regulation ofprotein kinase C activity by cooperative interaction of Zn2+ and Ca2+. J Biol Chem 262:13902-13906, 1987.

22.

D'SOUZA, SE, GINSBERG, MH, BURKE, TA, and PLOW, ED: The ligand binding sites of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit. J Biol Chem 265:3440-3446, 1990.

23.

FRELINGER, AL III, COHEN, I, PLOW, EF, SMITH, MA, ROBERTS, J, LAM, SC-T, and GINSBERG, MH: Selective inhibition of integrin function by antibodies specific for ligand occupied receptor conformers. J Biol Chem 265:6346-6352, 1990.

24.

FRELINGER, AL III, LAM, SC-T, PLOW, EF, SMITH, MA, LOFTUS, JC, and GINSBERG, MH: Occupancy of an adhesive glycoprotein expression of an receptor modulates antigenic site involved in cell adhesion. J Biol Chem 263:12387-12402, 1989.

25.

ZAMARRON, C, GINSBERG, MH, and PLOW, EF: Monoclonal antibodies specific for a conformationally altered state of fibrinogen. Thromb Haemost 64:41-46, 1990.