Antithrombin Activity of Fucoidan

,

Fucoidan, poly(L-fucopyranose) linked primarily a1 + 2 with either a C3-or a C4-sulfate, is an effective anticoagulant in vitro and in vivo (Springer, G .F., Wurzel, H. A., McNeal, G .M., Jr., Ansell, N. J., and Doughty, M. F. (1957) Proc.SOC.Exp.Biol.Med.94,[404][405][406][407][408][409].We have determined the antithrombin effects of fucoidan on the glycosaminoglycan-binding plasma proteinase inhibitors antithrombin I11 and heparin cofactor 11.Fucoidan enhances the heparin cofactor 11-thrombin reaction more than 3500-fold.The apparent second-order rate constant of thrombin inhibition by heparin cofactor I1 increases from 4 % lo4 (in the absence of fucoidan) to 1.5 % los M" min" as the fucoidan concentration increases from 0.1 to 10 pg/ ml and then decreases as fucoidan is increased above 10 pg/ml.The fucoidan reaction with heparin cofactor 11-thrombin is kinetically equivalent to a "template model."Apparent fucoidan-heparin cofactor I1 and fucoidan-thrombin dissociation constants are 370 and 1 nM, respectively.The enhancement of thrombin inhibition by fucoidan, like heparin and dermatan sulfate, is eliminated by selective chemical modification of lysyl residues either of heparin cofactor I1 or of thrombin.The fucoidan-antithrombin I11 reactions with thrombin and factor Xa are accelerated maximally 285-and 35-fold at fucoidan concentrations of 30 and 500 pg/ml, respectively.Using human plasma and "'Ilabeled thrombin in an ex vivo system, the heparin cofactor 11-thrombin complex is formed preferentially over the antithrombin 111-thrombin complex in the presence of 10 pg/ml fucoidan.Our results indicate that heparin cofactor I1 is activated by fucoidan in vitro and in an ex vivo plasma system and suggest that the major antithrombin activity of fucoidan in vivo is mediated by heparin cofactor I1 and not by antithrombin 111.
Heparin is a glycosaminoglycan that has been widely used as a therapeutic anticoagulant (1).Interaction of heparin with the plasma proteinase inhibitor antithrombin I11 is the primary biochemical basis for the pharmacological effect.It is also thought that heparan sulfate proteoglycans on the surface of endothelial cells serve as the in vivo activator of antithrombin I11 (2).Antithrombin I11 inhibits all of the proteinases involved in the intrinsic coagulation pathway including factors Xa, IXa, XIa, XIIa, kallikrein, and thrombin (3).Heparin cofactor I1 is another plasma protein that inhibits thrombin but no other proteinases involved in intrinsic coagulation (4).
Unlike the antithrombin 111-thrombin reaction, the heparin cofactor 11-thrombin inhibition reaction is enhanced either by heparin or by dermatan sulfate (8,12,13).Structureactivity relationships of polyanion-heparin cofactor I1 interactions have shown that heparin cofactor I1 is activated by various glycosaminoglycans, polysaccharides, and carboxylate-and phosphate-containing polyanions (13).Thus, it appears that the polyanion-binding region in heparin cofactor I1 is less discriminating than the analogous heparin (heparan sulfate)-binding region in antithrombin 111.
In 1957, Springer and associates (15) reported that fucoidan (a polymer of L-fucose linked primarily a1 + 2 with sulfates on the three or four hydroxyls and with M , = 1 X lo6 (14)) from the brown marine alga Fucus uesiculosus, possesses anticoagulant activity in vitro and i n vivo.Recently, Roberts et al. (16) have shown that fucoidan prevents sulfatide binding to the adhesive proteins thrombospondin, laminin, and von Willebrand factor.Fucoidan also reverses bovine aortic endothelial cell spreading, and it has specific effects on other cellcell and cell-matrix adhesion reactions (17,18).In the present paper, we have assessed the interaction of fucoidan with antithrombin I11 and heparin cofactor I1 as a contributing factor in the overall anticoagulant activity of this polysaccharide.We provide evidence that the antithrombin action of fucoidan (ex vivo in a plasma system) is mediated through heparin cofactor I1 and not through antithrombin 111, and this correlates well with the antithrombotic effect of fucoidan observed i n vitro.

Effect of Fucoidan on Heparin Cofactor IZ-and Antithrombin 111-Proteinase Inhibition Reactions-The antithrombin
Portions of this paper (including "Experimental Procedures," Figs.1-7, and

3618
This is an Open Access article under the CC BY license.

Antithrombin Antithrombin Antithrombin Antithrombin Antithrombin
and antifactor Xa activities of heparin cofactor I1 and antithrombin I11 were assessed by measuring the rate of thrombin and factor Xa inhibition in the presence of fucoidan.Addition of fucoidan to heparin-cofactor II-thrombin at pH 8.0 and 25 "C enhanced the rate of thrombin inhibition.As shown in Fig. 1, the rate of thrombin inhibition by heparin cofactor 11 either with fucoidan or with heparin (each at 100 ng/ml) resulted in an acceleration of 30-and 140-fold, respectively, over the heparin cofactor II-thrombin reaction determined in the absence of added polyanion.As found previously with heparin and dermatan sulfate (22), chymotrypsin inhibition by heparin cofactor I1 is not enhanced by fucoidan (data not shown).
The relationship between the rate of heparin cofactor IIthrombin inhibition and fucoidan concentration was determined.The rate constant for thrombin inhibition by heparin cofactor I1 increased in a polyanion-dependent manner from 0.1 to 10 pg/ml, reached a maximum at 10 pg/ml, and then decreased as fucoidan was increased above 10 pg/ml (Fig. 2).Under these reaction conditions, the rate constant ( k ~) determined for heparin cofactor II-thrombin at 10 pg/ml fucoidan was 1.5 X 10' M" min" and represents more than a 3500fold increase over the inhibition rate in the absence of polyanion (4 X IO4 M" min").The shape of the curve suggests formation of a ternary complex ("template model") with binding of both inhibitor and proteinase to fucoidan.This template model has also been suggested for the inhibition of thrombin by heparin cofactor I1 in the presence of heparin and dermatan sulfate (9,13).
Addition of fucoidan (100 ng/ml) to antithrombin IIIthrombin did not accelerate the rate of thrombin inhibition (using similar reaction conditions as in Fig. l), but heparin at the same concentration accelerated thrombin inhibition over 90-fold (data not included).However, at higher concentrations of fucoidan, there was a measurable enhancement of the rate of thrombin inhibition by antithrombin I11 that reached a maximum at about 30 pg/ml fucoidan (kz = 5.7 X lo7 M" min" compared to kz = 2 X 10' M" min" in the absence of polyanion; Fig. 2).The rate of factor Xa inhibition by antithrombin III-fucoidan as a function of fucoidan concentration appeared to follow saturation kinetics, with a rapid rate increase between 1 and 100 pg/ml fucoidan and a maximum (kz = 1 x lo7 M" min-l) as fucoidan approached 1 mg/ml (Fig. 3).An excess of fucoidan (100 pg/ml) decreased the rate of factor Xa inhibition by antithrombin III-heparin (1 pg/ml) (hobs (min") values are shown in parentheses): antithrombin 111 alone (0.31), heparin-antithrombin I11 (0.77), and heparinantithrombin I11 with fucoidan (0.36).The mechanisms for fucoidan acceleration of antithrombin III-thrombin and antithrombin III-factor Xa inhibition appear to be similar to that found for the heparin-catalyzed reactions (9)(10)(11).The results suggest that a ternary complex is formed between fucoidan, antithrombin 111, and thrombin.For factor Xa and antithrombin 111, the data indicate an altered reactivity of either proteinase inhibitor or proteinase in the presence of fucoidan.
Interaction of Fucoidan with Heparin Cofactor 11, Antithrombin III, and Thrombin-The interaction of fucoidan with these glycosaminoglycan-binding proteins was measured by chemical modification of lysyl residues, by altering buffer ionic strength (with mono-and divalent ions), by monitoring changes in intrinsic (antithrombin I11 and heparin cofactor 11) and extrinsic (DEGR-thrombin)' fluorescence, and by evaluating the kinetic mechanism.The fucoidan-catalyzed heparin cofactor II-thrombin inhibition reaction was studied by varying the heparin cofactor I1 (Fig. 4A) and thrombin (Fig. 4 B ) concentration.Saturation kinetics were observed with apparent dissociation constants for heparin cofactor II-fucoidan and thrombin-fucoidan of 370 and 1 nM, respectively (Fig. 4).For comparison, apparent dissociation constants for antithrombin III-heparin, heparin cofactor II-heparin, and thrombin-heparin are 35, 230, and 7 nM, respectively (9,13).As shown for the proposed ternary complex model for the heparin-antithrombin III-thrombin reaction, the overall reaction velocity at optimum heparin concentration depends not only on the concentrations of antithrombin I11 and thrombin but also on the tightness of inhibitor and proteinase binding to heparin (11,28).The strength of the binding constants determined for heparin cofactor I1 and thrombin suggest that fucoidan interaction with both inhibitor and proteinase is important for the catalytic effect.Furthermore, the dissociation constant for heparin cofactor II-fucoidan is below the physiological heparin cofactor I1 concentration and suggests a potential use for this polysaccharide as an antithrombotic compound.
Chemically modified proteins were used to further probe the importance of fucoidan binding to both heparin cofactor I1 and thrombin during the thrombin inhibition reaction.Modification of a limited number of lysyl residues either in heparin cofactor I1 or in thrombin resulted in essentially a complete loss (90 and >95% for heparin cofactor I1 and thrombin, respectively) of the fucoidan-catalyzed activity (Table I).Similar results were obtained when heparin or dermatan sulfate cofactor activity was measured with both modified proteins (Table I).The loss of polyanion cofactor activity following selective lysine modification suggests that these amino acid residues contribute to polyanion binding or that their modification disables these binding sites.3These results also demonstrate the importance of the binding of fucoidan (and heparin and dermatan sulfate) to both heparin cofactor I1 and thrombin for enhancement of the rate of thrombin inhibition.
The inhibition rate of heparin cofactor II-thrombin with fucoidan and heparin was determined as a function of ionic strength.As shown in Fig. 5, there was a marked ionic strength dependence for thrombin inhibition with both the fucoidan and heparin.The loss of accelerated thrombin inhibition by heparin cofactor II-polyanions as a function of ionic strength is likely due to decreased binding of the polyanions to heparin cofactor I1 (heparin cofactor I1 and thrombin elute from immobilized heparin at 0.25 and 0.6 M NaC1, respectively).
It has been shown previously that the heparin-catalyzed antithrombin III-thrombin inhibition rate is greatly decreased by calcium (29).The heparin (100 ng/ml)-catalyzed heparin cofactor II-thrombin reaction was inhibited more than 85%, with kz values of 1.3 x lo7 and 1.7 x IO6 M" min" in the absence and presence of calcium (10 mM), respectively.However, the kz value for the fucoidan (100 ng/ml)-catalyzed heparin cofactor II-thrombin reaction was the same when Further analysis of the phosphopyridoxylation reaction of heparin cofactor I1 in the absence and presence of heparin and fucoidan has been undertaken.Both polysaccharides reduce the extent of protein lysine modification by approximately 1 mol of reagent incorporated per mol of heparin cofactor 11.High performance liquid chromatography peptide maps (tryptic digests of reductively denatured and Scarboxymethylated protein) of phosphopyridoxylated heparin cofactor 11, modified in the presence of heparin or fucoidan, were identical and suggests that these sulfated polysaccharides protected the same of Fucoidan measured in the absence and presence of calcium (10 mM), 3.4 X lo6 M" min".Calcium decreases the apparent binding affinity of heparin for thrombin by binding to the carboxyl groups of heparin (29).Absence of carboxyl groups in fucoidan would explain why calcium does not affect the fucoidancatalyzed heparin cofactor 11-thrombin reaction.
As found previously for heparin and dermatan sulfate, fucoidan did not induce a change in heparin cofactor I1 intrinsic protein fluorescence (data not included).And while the interaction between antithrombin I11 and heparin is readily detected by an intrinsic fluorescence increase, there was no change following the addition of fucoidan to antithrombin I11 (Fig. 6A).However, fucoidan did eliminate the fluorescence enhancement of the antithrombin 111-heparin complex (Fig. 6B).The interaction of fucoidan with DEGR-thrombin resulted in a %fold extrinsic fluorescence enhancement (data not shown).Nesheim et al. (11) showed a similar fluorescence change following the interaction of heparin with DEGRthrombin.The signal change of DEGR-thrombin-heparin most likely represents an event subsequent to heparin-proteinase binding such as intermolecular thrombin interactions (11).
It has been shown recently that the "heparin-induced" conformation in antithrombin I11 is not required to facilitate the inhibition of thrombin (28).Our results show that fucoidan is much less effective than heparin during the antithrombin 111-catalyzed thrombin and factor Xa inhibition reactions and that there is no detectable protein structural change following polysaccharide binding (determined by intrinsic fluorescence).Questions that arise are why does fucoidan accelerate the antithrombin 111-catalyzed thrombin and factor Xa reactions and why does fucoidan eliminate the antithrombin 111-heparin complex enhanced fluorescence signal?One possible explanation is that fucoidan binds to (part of) the heparin binding site yet does not cause the same protein perturbation as does heparin.Another explanation is that steric hindrance follows fucoidan binding which either releases the heparin or modifies the heparin-antithrombin I11 conformation (and quenches the enhanced protein fluorescence).
Heparin cofactor I1 and antithrombin I11 are very distinct thrombin inhibitors yet they share many structural and functional properties.Perhaps their most obvious similarity is the general effect of glycosaminoglycans during thrombin inhibition (although heparin cofactor I1 reacts with a wider variety of polyanions).Much progress has been made on the location of the heparin binding site in antithrombin 111.The putative heparin binding site in antithrombin I11 consists of a region including Pro-41, Arg-47, Trp-49, and the region surrounding lysyl residues 125, 133, and 136 (for review see Ref. 30).Computer-aided three-dimensional modeling studies of antithrombin I11 have revealed that these regions are adjacent to each other (31).4Heparin cofactor I1 is similar to antithrombin I11 both in sequence (32) and in proposed three-dimensional structure (31)4 which may allow identification of homologous polyanion binding sites in heparin cofactor 11.As a further probe of possible similarities in the heparin (polyanion) binding sites of heparin cofactor I1 and antithrombin 111, we have diagrammed specific regions as Edmundson wheel plots (a-helical wheel projections assuming 3.6 amino acid residues/turn of the helix (33)): for antithrombin 111, this region was Ala-124 to Val-141 and for heparin cofactor 11, the regions were Lys-173 to Leu-190 and Phe-183 to Arg-200.As shown in Fig. 7, the putative heparin (polyanion) binding sites for both antithrombin I11 and heparin cofactor I1 yielded amphiphilic a-helices with positively charged and hydrophobic amino acid residues located on opposite sides of the helix.Thus, a paradigm of specific secondary structures (such as an amphiphilic a-helix) and heparin-binding domains might be established for these glycosaminoglycan-dependent proteinase inhibitors.
Fucoidan-catalyzed Thrombin Inhibition by Heparin Cofactor II in Plasma-The anticoagulant activity of fucoidan in plasma was examined using an activated partial thromboplastin time clotting assay.Clotting time was prolonged with increasing amounts of fucoidan (5-100 pg/ml; data not included).Heparin (by weight) was about 12 times more potent as an anticoagulant than fucoidan, essentially in agreement with the results of Springer et al. (15).
We next determined what proteinase inhibitor complexes were formed when thrombin was incubated with plasma in the presence of various amounts of fucoidan.Using this ex vivo system, '251-labeled thrombin was incubated either with plasma or with purified inhibitors and subsequently analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography.Increasing amounts of fucoidan, to 10 pglml, were correlated with preferential incorporation of the radioactive thrombin into a covalent complex with heparin cofactor I1 (Fig. 8).The amount of radiolabeled thrombin incorporated into an antithrombin I11 complex did not increase in the presence of fucoidan (Fig. 8).The physiological role of heparin cofactor I1 remains unclear although McGuire and Tollefsen (34) have recently suggested an in vivo role for thrombin inhibition by heparin cofactor I1 in the presence of extravascular components containing dermatan sulfate proteoglycans.Our data also imply that in vivo thrombin inhibition by heparin cofactor I1 may occur at sites rich in sulfate groups structurally similar to fucoidan.
Concluding Remark-Fucoidan, a sulfated polyfucopyranose, greatly accelerates the in vitro heparin cofactor IIthrombin inhibition reaction.The heparin cofactor II-thrombin complex is rapidly formed in the presence of 10 pg/ml fucoidan when '251-labeled thrombin is incubated with human plasma.These results indicate that the interaction of fucoidan with heparin cofactor 11, not antithrombin 111, is important for the antithrombin activity of this polysaccharide.The FIG. 8. Activation of heparin cofactor I1 in human plasma by fucoidan.Citrated-anticoagulated plasma diluted 1:50 in TNP buffer (50 mM triethanolamine acetate, 100 mM NaCl, and 0.1% polyethylene glycol, pH 8) or purified inhibitors (diluted to plasma concentration levels) were incubated with '251-labeled thrombin (5 nM) for 10 min a t 25 "C with various amounts of fucoidan.The samples were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.An autoradiogram of the gel shows the positions of purified antithrombin 111-thrombin and heparin cofactor 11-thrombin complexes in the presence of 10 pg/ml fucoidan (lanes 1 and 2, respectively); the plasma system with 0, 0.1, 1, and 10 pg/ml fucoidan (lanes 3-6, respectively); purified heparin cofactor I1 and antithrombin 111 mixed with thrombin and 10 pg/ml fucoidan (lane 7); and purified antithrombin 111 and heparin cofactor I1 with thrombin in the absence of fucoidan (lanes 8 and 9, respectively).
Fucoidan Fucoidan characteristics of the fucoidan-catalyzed heparin cofactor IIthrombin reaction are consistent with formation of a ternary complex which implies binding of both proteinase inhibitor and proteinase to fucoidan.Fucoidan and heparin binding sites in heparin cofactor I1 and thrombin are most likely localized to the same region in each protein.Finally, the results illustrate that there are important functional/structural differences and similarities between heparin cofactor I1 and antithrombin 111.factor Xa by antithrombin 111 were performed tn a similar manner Chymotrypsln mhhtion actlvlty of hepar," cofactor I 1 was determlned as detalled previously (22) Control experlmenls Verlfled that fucoldan dld not greatly aflect Substrate hydrolyses by the proteinases used In thls mvestigatlon Apparent pseudo flrst Order (kobs) and second order (k2) rate constants were calculated as descrlbed (19).

of p o l y a n l o n s o n t h r o m b i n i n h i b i t i o n b y heparin Cofactor 11 .
Fig 4 K i n e t i c s of the lucoidan-catalyzed heparin lnhiblllon by heparin cofactor I1 (HC) ~n the presence of 100 nglmL lucoldan was determined.( A ) Initial thrombfn Concentrat~on was 5 nM (B) lnltlal HC concentration was 770 nM Data are plotted as a Hanes-Woolfe plot according to the kmetlc model as descrlbed under 'Exper#mental Procedures'