Heparin Promotes the Binding of Thrombin to Fibrin Polymer QUANTITATIVE CHARACTERIZATION OF A

The binding of human cr-thrombin (IIa) to fibrin poly- mer (FnIIp) was studied in the presence and absence of a high affinity 20,300 M, heparin (H) at pH 7.4,10.15, and 23 “C. In the absence of heparin, thrombin inter- acts with a high affinity class of binding sites on fibrin polymer with a dissociation constant of 301 f 36 nM in a manner which is independent of the enzyme active site. Studies of thrombin binding as a function of hep- arin and fibrin polymer concentrations imply that a ternary thrombin-fibrin polymer-heparin complex (IIa.FnIIp*H) is formed. Assembly of the ternary com- plex occurs randomly through the interactions of all three possible intermediate binary complexes; IIaaH, IIa*FnIIp, and FnIIp*H. Using an independently deter- mined value of 280 f 35 nM for the FnIIp*H dissocia- tion constant, global fits of the binding data yield a dissociation constant of 15 f: 6 nM for the IIa*H inter- action and 47 + 9 nM for the IIa*H intermediate binary complex interaction with FnIIp. These studies indicate that heparin

The binding of human cr-thrombin (IIa) to fibrin polymer (FnIIp)  This effect of heparin is also independent of whether it has high or low affinity for antithrombin III.
The demonstration of the formation of a ternary IIa.FnIIp*H complex complements kinetic evidence indicating the formation of an analogous ternary complex with fibrin II monomer (Hogg, P. J., and Jackson, C. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,3619-3623).
The possible implications of these findings for the in viva distribution and actions of thrombin and the clinical efficacy of heparin are also discussed.
Thrombin has central regulatory functions in the maintenance of hemostasis. This serine proteinase interacts with several substrates (fibrinogen, factors V, VIII, XIII, and protein C), inhibitors (antithrombin III, a2-macroglobulin, and * This work was supported by an Established Investigatorshin from the American National Red Cross (to C. M. J.) and theSoutheastern Michigan Regional Blood Services, American Red Cross. 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.
$ To whom requests for reprints should be addressed.
heparin cofactor II), and cell surfaces (platelets and endothelial cells) which together contribute to the delicate balance characteristic of the hemostatic process (1). The ultimate action of thrombin in blood-clotting is the conversion of circulating fibrinogen (AaZBP2y2) to the insoluble fibrin matrix of blood clots by the cleavage of Arg-Gly bonds at positions 16-17 of the Aa! chain and 14-15 of the B/3 chain to release fibrinopeptides A and B (2, 3). In addition to this enzymatic function, thrombin also interacts with fibrin and fibrinogen in a manner distinct from the Michaelis complex necessary for fibrinopeptide release. This was demonstrated some time ago by Seegers and co-workers (4) and Liu et al. (5,6) and more recently by Kaminski and McDonagh (7,8) who found that thrombin action on small substrates and its inactivation by low-molecular weight inhibitors is not detectably perturbed by fibrin binding. Recent investigations have further defined the specificity of the interaction, which appears to involve a binding region (9, 10) on the central E domain of fibrin(ogen) (11). Altered binding of thrombin to fibrin may be responsible for physiological disorders. About 20 cases of dysfibrinogenemia associated with arterial or venous thrombosis have been reported (12, 13) and, in some of these cases, the structural defect in the fibrinogen molecule has been identified. In the cases of fibrinogen New York I (14) and fibrinogen Milan0 II (15), the underlying cause of thrombosis was attributed to defective binding of thrombin to the fibrin formed from the abnormal fibrinogen.
The ability of heparin, a complex glycosaminoglycan isolated from a variety of natural sources, to enhance the inactivation of coagulation proteinases has been established (16)(17)(18)(19) and has proven clinical value (20). Of the proteinases which are modulated by heparin action, experiments in plasma (21-25) indicate that the heparin-catalyzed inactivation of thrombin is of most importance. It has been concluded that this is primarily because of the importance of thrombin for activation of Factors VIII and V. However, the inability of heparin to prevent coronary reocclusion in patients treated with tissue plasminogen activator (26-28) suggests physiological or pathophysiological circumstances where the efficacy of heparin is compromised.
It has been demonstrated that fibrin monomer binding to thrombin decreases the second-order rate constant for the inactivation of thrombin by heparinantithrombin III more than 300-fold at physiological fibrin concentrations (29). These studies suggested that thrombin forms a ternary complex with fibrin and heparin and that the thrombin in this complex possesses altered reactivity toward its substrates and inhibitors.
From measurements of thrombin binding as a function of heparin and fibrin polymer concentrations, we demonstrate and quantify the formation of a ternary thrombin-fibrin polymer-heparin complex and discuss 1) the implications of this binding for the clinical efficacy of This arises because the other two components in the system, e.g. thrombin and fibrin polymer, become saturated with this reactant at high concentration resulting in the formation of noninteracting binary complexes, e.g. IIa. H and FnI1p.H.
An example of this phenomenon, as it relates to this system (Scheme l), is seen in Fig. 4 to fibrin polymer at high heparin concentrations ([HI > &,ii,.n) will be shifted to the right and that the extent of this shift will increase with increasing heparin concentration (47). This feature is a result of the formation of FnIIp. H binary complexes at high heparin concentrations which, like the situation described above in relation to Fig. 4, competitively inhibit the binding of thrombin to fibrin polymer. The solid lines in Fig. 5 are the theoretical fits to the data using Equation 2 with the values for the binary and ternary dissociation constants that have been calculated from Figs. 1-3.

Effect of Heparin Molecular Weight and Heparin Affinity for A~tithrombin
Iif on Ternary Complex Formation-The effect of heparin molecular weight on ternary complex formation has been investigated on the basis of its ability to enhance thrombin ([IIa]T = 3.87 nM) binding to fibrin polymer (190 nM). Heparins of molecular weight 11,200-20,300 behave similarly with respect to their influence on ternary complex formation (Fig. 6). This is judged on the basis of the extrapolated extent of thrombin binding at saturating heparin (Fig. 6B). Whether the heparin has high or low affinity for antithrombin III appears to have no influence on its ability to promote thrombin binding to fibrin polymer. The heparin of molecular weight 11,200 has low affinity for antithrombin III (Fig. 6). Heparin species of molecular weight <11,200 are much less effective in promoting thrombin binding to fibrin polymer.

DISCUSSION
The binding of thrombin to fibrin was first demonstrated by Seegers et al. (4). At that time they associated it with a type of antithrombin mechanism (antithrombin I) whereby the capturing of thrombin by the clot reduces thrombin action on circulating fibrinogen. Indeed, defective binding of thrombin to fibrin has been associated with thrombosis in some cases of dysfibrinogenemia (14,15). From these studies we find that thrombin interacts with a high affinity class of sites on fibrin polymer (expressed as fibrin monomer concentration) with a dissociation constant of 301 f 36 nM (Fig. lA). At higher thrombin concentrations heterogeneous binding is observed (Fig. 1B). This agrees well with results of Liu et al. (5), when their data were refitted by nonlinear regression (46) to take explicitly into account the two classes of sites observed by them. The heterogeneous binding of thrombin to fibrin polymer illustrated in Fig. lA indicates approximately 0.34 high affinity site on fibrin polymer for thrombin.
The dissociation constant determined for this interaction, 301 nM, is a stoichiometric constant (I&) and relates to the intrinsic or site-binding constant (kd) by the relationship, kd = nKd (48) where n is the number of binding sites, 0.34. The site binding constant is therefore calculated to be 102 nM. Assuming that thrombin interacts with one, or perhaps two, sites on a fibrin monomer these findings indicate that the polymerization of fibrin results in a loss of thrombin binding sites; or stated alternatively, only one of every three fibrin monomer sites is accessible on polymerized fibrin. This masking of binding sites upon fibrin polymerization may have important implications for the effects of fibrin on the kinetic actions of thrombin because it alters the partition of thrombin between solution and fibrin polymer during the course of fibrin monomer polymerization (see the accompanying article (64)). Thrombin binds to a variety of negatively charged polysaccharides. The nonspecific binding of heparin to thrombin, in addition to its specific interaction with antithrombin III, has been demonstrated to be a prerequisite for the catalytic efficiency of this glycosaminoglycan in enhancing the inactivation of thrombin by antithrombin III (19). The global best-fit estimate for the thrombin-heparin dissociation constant, 15 + 6 nM (1 S.D.), is in agreement with an independently determined estimate of this constant, 32 + 10 nM (1 S.D.), from unrelated quantitative affinity chromatographic studies. Thrombin similarly interacts with the cell-surface glycosaminoglycans (GAG) of vascular endothelial cells (49)(50)(51)(52). It has been proposed that the binding of thrombin to these structures regulates the actions of this proteinase (53). Several studies of the binding of thrombin to cell surface GAG indicate that the Kd for this interaction is also in the low nanomolar range, 20-30 nM (49)(50)(51)(52).
In addition to characterizing the binary complex interactions between fibrin and thrombin and between heparin and thrombin, we have examined the consequences of the combined interactions of these two reactants on the distribution of thrombin in equilibrium mixtures of thrombin, fibrin polymer, and heparin. The findings are summarized in Scheme 2 and are concordant with the predicted behavior for a binding model in which a ternary thrombin-fibrin polymer-heparin complex is formed. In Scheme 2, the assembly of the ternary complex occurs randomly through the interactions of all three A distinguishing feature of ternary complex formation is the ability of one reactant at high concentrations to inhibit competitively the binding of another reactant (47). The results of Figs. 4 and 5 demonstrate that heparin, at high concentrations, will inhibit competitively the binding of thrombin to fibrin polymer. These experiments are important in that they further establish the adequacy of the model proposed here for the interactions of thrombin, fibrin polymer, and heparin. Heparins of molecular weight 11,200-20,300 behave similarly with respect to their influence on ternary complex formation (Fig. 6). As expected for a nonspecific interaction, the ability of heparin to enhance the binding of thrombin to fibrin polymer is independent of whether the heparin has high or low affinity for antithrombin III (Fig. 6). Because the molecular weight distribution of therapeutic heparin spans the region from -5,000 to 30,000 (54, 55) the studies reported here will be relevant to the type of heparin that is used clinically. The large size of the GAG which have been isolated from vascular endothelium similarly suggests that these interactions with thrombin and fibrin will also be supported by these structures.
Heparins of lower molecular weight (~11,200) are less effective in promoting thrombin binding to fibrin polymer. Assuming an equivalent model (Scheme 1) for the interactions of lower molecular weight heparins, the data suggest that the effect of reduction in molecular weight is a result of an increase in the KIIa.H and Kc(lIIs.~j. rniIp dissociation constants. This is reflected as a decrease in the apparent half-maximal saturation value for heparin and an extrapolated lower maximal extent of thrombin binding, respectively (Fig. 6). The heparin molecular weight dependence demonstrated here resembles the molecular weight dependence of heparin with high affinity for antithrombin III in catalyzing the inactivation of thrombin by antithrombin III (56). This may reflect a correspondence between the heparin molecular weight and affinity of heparin for thrombin, as indicated here, and the ability of the heparin to enhance the inactivation of thrombin by antithrombin III. Correlation between heparin molecular weight and its catalysis of thrombin inactivation by antithrombin III has been suggested by Hoylaerts et al. (18). In addition to its interaction with cell-surface GAG, thrombin also interacts with thrombomodulin (TM), an endothelial cell intrinsic membrane glycoprotein that binds thrombin with high affinity and modulates its substrate specificity (see Ref. 57 for a recent review). With a knowledge of the affinities and the approximate number of TM and GAG binding sites on the endothelial cell for thrombin it is possible to calculate the partition of thrombin between these two cell-surface receptors. This enables an assessment of the influence of fibrin on the partition of thrombin and therefore some idea of the relative importance of these ternary complex interactions for the distribution of thrombin between these two endothelial receptors. In the absence of fibrin, assuming microcirculation TM and GAG concentrations of 500 nM and 1.5 pM, respectively (50,571, approximately 88% of the thrombin will be bound to TM and only 12% to GAG. In the presence of 70 nM fibrin, which corresponds to conversion of only 1% of the local fibrinogen concentration (7 pM) to fibrin, 77% of the thrombin will be bound to TM and 23% to GAG. Similarly, at 10% conversion (0.7 pM fibrin) 37% of the thrombin will be associated with TM and 62% with GAG. These results suggest that fibrin, as a result of ternary complex interactions with thrombin and GAG, may alter the distribution of thrombin between these two receptors and thereby influence the binding of thrombin to the endothelial surface. In the accompanying paper (64), we demonstrate that thrombin binding in this ternary complex differentially affects its actions on its many substrates, and thus this partitioning process may have a very important role in regulating thrombin action. The initial observation that more thrombin is bound to fibrin in the presence of heparin than in its absence was made many years ago (59)(60)(61). Although the significance of this phenomenon for the clinical efficacy of heparin is still not known, it may have important implications for the regulation of thrombin action on fibrinogen and protection of thrombin from inactivation by antithrombin III (29). There are multiple mechanisms in blood clotting for localizing and compartmentalizing reactions (62). During hemostasis and perhaps arterial thrombosis the endothelial and platelet surfaces, in conjunction with fibrin and von Willebrand factor, interact to constrain spatially the coagulation reactions to the injury site. The enhanced binding of thrombin to fibrin polymer in the presence of heparin, or GAG on endothelial cells adjacent to the injury site, may be a mechanism whereby diffusion of thrombin away from the growing clot is restricted thus preventing dissemination of this proteinase throughout the circulation. The protection of thrombin from inactivation by heparin-antithrombin III by fibrin (29) will also act to prolong the action of thrombin on fibrinogen in the region of a clot, in contrast to the situation in the circulation where thrombin is subject to rapid inactivation.
In this way heparin and cell surface GAG may facilitate the formation of a fibrin canopy at the site of injury thereby limiting hemostatic plug and thrombus progression. The protection by fibrin monomer may be of clinical importance as it possibly explains the limited efficacy of heparin in preventing coronary reocclusion in patients treated with fibrinolytic agents (26-28). The combined effects of these two reactants on some other kinetic properties of thrombin are explored in the following paper The ~CIU,LS for ,hrombm binding 10 fibrin polymer in #he abreacs or prcm"cc of hepari", whs" (Fnllp] > > ,IIa], arc expressed I" mrms of pcrcsnt thrombm bound 10 afford a mow familiar reprercntatlo" of the binding da,*. The dependent vanable I" Equation 2 I$ simply erprsrsd as follows, Ekeme Factor XI was preparsd by actwatio" of punficd Factor X (31) with the same prorslnarc from Ruucll's viper venom (3,). isolated by Ssphadcr G-,0, ctomatqraphy, and xtw~.slte rnrrated (31.33).