THE .JOURNAL OF BIOLOGICAL CHEMISTRY ii, 19& by The American Society of Biological Chemists, Inc.

Vol. 259, No. 23, Issue of 'December 10, PP. 14721-14727,1984 Printed in U.S A.

Specific Binding of Blood Coagulation Factor XIII, to Thrombin- stimulated Platelets*

(Received for publication, May 4, 1984)

Charles S. GreenbergSj and Marc A. Shumannll From the $Departments of Medicine, Duke University Medical Center, Durham, North Carolina 27710 and the TUniuersity of California Medical Center, Sun Francisco, California 94143

We studied the binding of lz6I-p1atelet and plasma Factor XI11 (lzSI-Factor XIII) to human platelets. When 1261-Factor XIII was incubated with gel-filtered plate- lets, calcium chloride (5 mM) and thrombin (1 unit/ml) at 37 "C, saturable binding was observed. Half-maxi- mal binding occurred at 1 min. Binding was inhibited 93% by a 100-fold molar excess of unlabeled ligand but not by other purified proteins. Greater than 87% of platelet-bound radioactivity migrated as thrombin- cleaved a-chains (a'-chains) in sodium dodecyl sulfate- polyacrylamide gels indicating that Factor XIII, but not Factor XIII binds to platelets.

'261-Factor XII1,does not bind to unstimulated plate- lets. When platelet secretion was blocked, binding was markedly inhibited. '261-Fa~tor XIII, bound minimally to platelets stimulated with agonists other than throm- bin. Thus, binding is dependent on platelet activation, as well as modification of platelets by thrombin. '"I- Factor XIII, bound to y-thrombin-stimulated platelets, at concentrations which did not clot fibrinogen. There- fore, Factor XII1,is not bound to fibrin associated with platelets.

Binding was only partially reversible. Approxi- mately 12,000 molecules of Factor XIII, were bound per platelet.

'261-Factor XIII, bound normally to platelets from patients with severe Glanzmann's thrombasthenia in- dicating that lZ6I-Factor XIII, does not bind to platelet glycoproteins IIb or IIIa, or platelet-bound fibrinogen. Chymotrypsin treatment of platelets inhibited '"1- Factor XIII, binding by 78% without inhibiting secre- tion.

Methylamine and putrescine, Factor XIII,substrates, and N-ethylmaleimide, an active site inhibitor, did not inhibit binding. Factor XIII, bound to platelets was enzymatically active and catalyzed [3Hjputrescine in- corporation into platelet proteins. The specific binding of Factor XIII, to platelets suggests it may play a role in physiologic reactions involving platelets.

* This work was supported in part by Grant HL 21403 from the National Institutes of Health. A preliminary report of this work was presented a t the Annual Meeting of the American Society of Hema- tology, Washington, D. C., December 1982, and was published in abstract form (Greenberg, C. S., and Shuman, M. A. (1982) Blood 60, 199a). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ American Heart Association Research Fellow, Northern Califor- nia Affiliate. To whom all reprint requests should be addressed at, P. 0. BOX 3934, Duke University Medical Center, Durham, NC 27710.

11 Recipient of Research Career Development Award K04 HL- 00802 from the National Institutes of Health.

Formation of a platelet plug at the site of vascular injury is required for normal hemostasis. Thrombin formed on the platelet surface stimulates platelet secretion and aggregation, as well as fibrin formation (1). Thrombin formation, platelet secretion, and fibrin formation have been shown to be closely coordinated events during in vitro clotting (2). Thrombin facilitates platelet aggregation by exposing receptors on the platelet surface for fibrinogen which is required for platelet aggregation (3). Thrombin also exposes the receptor for von Willebrand's factor (4), a molecule which plays an important role in platelet adhesion (4, 5). Fibronectin, another plasma protein postulated to facilitate platelet adhesion, is also bound to thrombin-stimulated platelets (6).

Because thrombin-activated blood coagulation Factor XI11 (Factor XIII,) cross-links fibrinogen and fibrin molecules to each other (7) and cross-links fibronectin to fibrin (8), we were interested in determining whether Factor XIII. interacts with thrombin-stimulated platelets. In this study, we dem- onstrate that thrombin induces the specific binding of Factor XIII, to human platelets. In addition, Factor XIII, bound to the platelet surface is catalytically active.

MATERIALS AND METHODS AND RESULTS'

DISCUSSION

We have shown that thrombin-stimulated platelets selec- tively bind the a'-chains of lZ5I-Factor XIII,. Several results indicate that '251-Factor XIII, binding is specific and not an artifact of the radiolabeling method. The affinity of labeled and unlabeled Factor XI11 preparation was similar. Moreover, activation of lZ5I-Factor XI11 by thrombin resulted in specific binding of the a'-chains but not the b-chains. In addition, binding of '251-plasma Factor XIII. was specifically inhibited by unlabeled plasma Factor XIII, or afibrinogenemic plasma containing Factor XIII, but not inhibited by serum factors or purified plasma proteins. These studies indicate that there are specific binding sites for Factor XIII, on thrombin-acti- vated platelets. Additional support for this conclusion is our observation that binding is modified by chymotrypsin treat- ment of platelets and that there are a limited number of

Portions of this paper (including "Materials and Methods," "Re- sults,'' Figs. 1-8, and Tables 1-8) are presented in Miniprint at the end of this paper. The abbreviations used are: PGI,, prostaglandin Iz; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, MD 20814. Request Document No. 84M-1345, cite the au- thors, and include a check or money order for $12.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

14721

14722 Factor XII I , Binds to Thrombin-stimulated Platelets

binding sites on platelets. Furthermore, Factor XIII, does not bind to unstimulated platelets or to platelets stimulated by ADP. Therefore, there must be specific binding sites which are exposed after thrombin treatment.

Inhibitors of platelet activation inhibited binding of Factor XIII,. Thus, the effect of thrombin on Factor XIII, binding required exposure of platelets to thrombin as well as subse- quent activation. It remains to be determined whether secre- tion and/or a change in the platelet plasma membrane is required for binding of Factor XIII.. Exposure of platelets to other agonists, ADP or ionophore A23187, resulted in little or no binding of Factor XIII,. Thus the effect of thrombin on platelets is specific and not due to activation alone. Binding of Factor XIII, is not due to conversion of secreted platelet fibrinogen to fibrin by a-thrombin. When platelets were in- cubated with concentrations of y-thrombin a t which fibrino- gen was not converted to fibrin, Factor XIII, binding to platelets was normal.

Binding was only partially reversible. Irreversible binding of several ligands to membrane receptors has been previously reported (22, 23). In preliminary experiments, we have found that binding of lZ5I-Factor XIII. to fibrin is resistant to dissociation unless an anionic detergent (Le. sodium dodecyl sulfate) is present. The nature of the chemical bond between Factor XIII, and fibrin or the platelet receptor has not been characterized.

We have demonstrated that the binding sites for Factor XIII, on thrombin-activated platelets are not surface-bound fibrinogen or fibrin, since platelets from patients with Glanz- mann’s thrombasthenia and Gly-Pro-Arg-Pro-treated normal platelets failed to bind lZ5I-fibrinogen or fibrin but did bind 1251-Factor XIII.. In previous studies, we showed that the zymogen form of Factor XI11 binds to fibrinogen in solution or covalently attached to solid beads (13). On the other hand, preliminary experiments have demonstrated that plasma Fac- tor XI11 does not bind to fibrinogen adsorbed to bentonite particles. These results suggest that the binding domain on fibrinogen for Factor XI11 may be altered when fibrinogen is bound to certain surfaces including platelets and bentonite. It is unlikely that the binding site on platelets for Factor XIII. is fibronectin since binding was not inhibited by anti- fibronectin antisera.

The divalent cation calcium is necessary for optimal bind- ing of Factor XIII,. However, even in the absence of exogenous calcium chloride, the platelets bound Factor XIIL, suggesting that secretion of endogenous platelet cations facilitated bind- ing.

The time course and thrombin concentrations required for binding suggest that plasma Factor XIII, may bind to the platelet surface during blood clotting. Since the platelet sur-

face is the site of thrombin formation, the high concentrations of thrombin on platelets could stimulate Factor XIII, binding. Once Factor XIII. was bound, it would remain, in part, localized to the surface and could function to catalyze the cross-linking of platelet membrane proteins to each other, to plasma proteins or to subendothelial connective tissue. Our observation that Factor XIII, bound to platelets cross-links [3H]putrescine to platelets supports this hypothesis.

Further studies are in progress to characterize the role of Factor XIII. binding to thrombin-stimulated platelets in sta- bilizing the platelet plug during blood clotting.

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Factor XIII , Binds to Thrombin-stimulated Platelets

Factor XIII, Binds to Thrombin-stimulated Platelets

1251-Factor Xlli Binding to Platelets RBqulles Aetivation of Both Platelets

and 1251-F~Ef~r Xnl by Thrombin.

Platelets + mrombm

Factor Xm + mrombin

Platelets + Factor X 1 + Thrombin

8.820

8,480

89.450

Platelets + Fmctor X I In0 thrombin1 9,010

Substance Added (final emeentrstmn) Inhibition of Binding (a, ? S.D.)

Plg. 1

Addilim

Factor XII I , Binds to Thrombin-stimulated Platelets

ADP

A B C D

0.1 z 0.2

18.5 ! 5.2

108.3 12.6

11.3 f 13.1

6.1 z 0.8

14725

1.6 * 0 .8

2.0 1 . 2

N u m d 100 100

G l m z m m Patiml I 97 f o 3 ! 3

G l u u m m n Palien1 2 91 f 7 2 f l

N u m d * GPAP 89 f 10 S f 6

14726 Factor XIII, Binds to Thrombin-stimulated Platelets

PGI2 15 nM) 0.1

1.0

0.1

1.0

Q d o l i n + lndOmethsein 0.1

1.0

lndomethaeln I10 mM) 0.1

1.0

c I I I I I 0 100 200 300

Chymotrypsin l p g / m l )

15 t

Factor XIII, Binds to Thrombin-stimulated Platelets

Inhibitor Inhibition (%I

72s

810

1.280

OB0

1.150

36,850

14727