Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin

The maintenance of normal blood flow depends completely on the inhibition of thrombin by antithrombin, a member of the serpin family. Antithrombin circulates at a high concentration, but only becomes capable of efficient thrombin inhibition on interaction with heparin or related glycosaminoglycans. The anticoagulant properties of therapeutic heparin are mediated by its interaction with antithrombin, although the structural basis for this interaction is unclear. Here we present the crystal structure at a resolution of 2.5 A of the ternary complex between antithrombin, thrombin and a heparin mimetic (SR123781). The structure reveals a template mechanism with antithrombin and thrombin bound to the same heparin chain. A notably close contact interface, comprised of extensive active site and exosite interactions, explains, in molecular detail, the basis of the antithrombotic properties of therapeutic heparin.     

Certain high molecular weight heparin chains have high affinity for vitronectin.

Vitronectin is a 70-kDa protein that is found in both the extracellular matrix as well as serum. Vitronectin is one of the few proteins that regulates both the complement and the coagulation systems. Heparin is known to bind to vitronectin. Review of the literature reveals apparently conflicting outcomes of the interaction of heparin, vitronectin, and the complement system. Previous studies demonstrated that heparin diminishes vitronectin inhibition of complement activity. Numerous studies have also demonstrated that heparin exerts a net inhibitory effect on complement. We used two dimensional affinity resolution electrophoresis (2DARE) to examine this apparent paradox. 2DARE allowed simultaneous determination of binding affinity of heparin for vitronectin as well as the M(r) of the heparin species. In the 2DARE experiment, the interaction of heparin with vitronectin caused retardation of the movement of the heparin through the tube gel in the first dimension. The degree of the retardation of movement was used to calculate the approximate K(d) of that interaction. The heparin from the tube gel was then subjected to a second dimension electrophoresis to determine the M(r) of the heparin. 2DARE analysis of the interaction of heparin with vitronectin clearly demonstrated that a sub-population of heparin chains with M(r) > 8000 bound vitronectin with high affinity whereas most high M(r) chains and all lower M(r) chains showed little to no affinity for vitronectin. Our findings are consistent with the hypothesis that a unique binding domain exists in certain heparin chains for vitronectin.     

Interaction of soluble and surface-bound heparin binding growth-associated molecule with heparin.

The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). ITC studies showed that, in solution, heparin bound HB-GAM with a deltaH of -30 kcal/mole corresponding to a dissociation constant (Kd) of 460 nM. The stoichiometry of interaction was 3 moles of HB-GAM per mole of heparin, corresponding to a minimum heparin binding site for HB-GAM of 12-16 saccharide residues. Kinetic measurements of heparin interaction with HB-GAM made by SPR afforded a Kd of 4 nM, suggesting considerably tighter binding when HB-GAM was immobilized on a surface. Affinity chromatography of a sized mixture of heparin oligosaccharides, having a degree of polymerization (dp) of > 14 saccharide units, on HB-GAM-Sepharose demonstrated that oligosaccharides having more than 18 saccharide residues showed the tightest interaction.     

Histidine-rich glycoprotein modulation of the anticoagulant activity of heparin. Evidence for a mechanism involving competition with both antithrombin and thrombin for heparin binding

Heparin binding to rabbit histidine-rich glycoprotein (HRG) was studied in a purified system, allowing for determination of a heparin dissociation constant of approximately 5.5 X 10(-8) M for the interaction with HRG at pH 7.0. The strong interaction between heparin and HRG was demonstrated to be competitive with the binding of both antithrombin and thrombin to the heparin chain. HRG was further tested as a modulator of the anticoagulant activity of heparin by comparing rates of the heparin-catalyzed reaction between antithrombin and thrombin in the presence and absence of added HRG. The heparin-antithrombin-thrombin reaction was modeled using the formalism of a two-substrate enzyme-catalyzed reaction with heparin as the enzyme and HRG analyzed as an enzyme inhibitor. HRG was shown to compete with both antithrombin and thrombin for binding to heparin by this kinetic analysis. Thus, both the kinetic and heparin-binding data indicate that the mechanism by which HRG modulates heparin anticoagulant activity involves competition for heparin with both the inhibitor and the protease. Inhibition by HRG of the heparin-catalyzed reaction was found to be highly dependent on pH, with a sharp increase in inhibition from about 15% to greater than 90% observed as pH was lowered from 7.4 to 7.0. Since little change in the rate of the heparin-catalyzed inhibition of thrombin by antithrombin occurs in this pH region, the dramatic change in HRG inhibition seen upon pH titration may reflect increasing ionic interaction between heparin and HRG due to the protonation of histidine residues which occurs in this pH region.     

Quantitative characterization of the thrombin-heparin interaction. Discrimination between specific and nonspecific binding models

Equilibrium binding of human alpha-thrombin to heparin was investigated at pH 7.4 as a function of thrombin and heparin concentrations, NaCl concentration, temperature, and heparin chain length with the extrinsic fluorescence probe, p-aminobenzamidine, or by quantitative affinity chromatography, in order to distinguish between sequence-specific and nonspecific electrostatic modes of binding. Analysis of binding data by a nonspecific binding model developed for protein-nucleic acid interactions, or by the discrete binding site model previously used to analyze the thrombin-heparin interaction, indicated that both models described the binding interaction equally well over the range of thrombin binding densities accessible to measurement. However, the strong dependence of the thrombin-heparin binding interaction on NaCl concentration, its minimal dependence on temperature, and the increase in apparent binding affinity with increasing heparin oligosaccharide chain length were best accounted for by a nonspecific electrostatic association of thrombin with 5 to 6 anionic residues contained in a 3-disaccharide binding site of heparin. This interaction was characterized by an intrinsic dissociation constant (KD,obs) of 6-10 microM at physiological ionic strength. Although the nonspecific binding model satisfactorily described the binding of thrombin to heparin chains ranging in size from 3 to approximately 13 disaccharides in terms of a single intrinsic KD,obs, deviations from this model were apparent with longer heparin chains (approximately 22 to approximately 35 disaccharides) from a progressive decrease in the intrinsic KD,obs of up to 4-fold. Sedimentation equilibrium analyses of thrombin-heparin complexes suggested a second weaker binding site on thrombin for heparin, which accounted for these deviations as well as the observed insolubility of thrombin-heparin complexes at high thrombin binding densities.     

Interaction of heparin with annexin V

The energetics and kinetics of the interaction of heparin with the Ca2+ and phospholipid binding protein annexin V, was examined and the minimum oligosaccharide sequence within heparin that binds annexin V was identified. Affinity chromatography studies confirmed the Ca2+ dependence of this binding interaction. Analysis of the data obtained from surface plasmon resonance afforded a Kd of approximately 21 nM for the interaction of annexin V with end-chain immobilized heparin and a Kd of approximately 49 nM for the interaction with end-chain immobilized heparan sulfate. Isothermal titration calorimetry showed the minimum annexin V binding oligosaccharide sequence within heparin corresponds to an octasaccharide sequence. The Kd of a heparin octasaccharide binding to annexin V was approximately 1 microM with a binding stoichiometry of 1:1.     

The heparin binding properties of heparin cofactor II suggest an antithrombin-like activation mechanism

The serpin heparin cofactor II (HCII) is a glycosaminoglycan-activated inhibitor of thrombin that circulates at a high concentration in the blood. The antithrombotic effect of heparin, however, is due primarily to the specific interaction of a fraction of heparin chains with the related serpin antithrombin (AT). What currently prevents selective therapeutic activation of HCII is the lack of knowledge of the determinants of glycosaminoglycan binding specificity. In this report we investigate the heparin binding properties of HCII and conclude that binding is nonspecific with a minimal heparin length of 13 monosaccharide units required and affinity critically dependent on ionic strength. Rapid kinetics of heparin binding indicate an induced fit mechanism that involves a conformational change in HCII. Thus, HCII binds to heparin in a manner analogous to the interaction of AT with low affinity heparin. A fully allosteric 2000-fold heparin activation of thrombin inhibition by HCII is demonstrated for heparin chains up to 26 monosaccharide units in length. We conclude that the heparin-binding mechanism of HCII is closely analogous to that of AT and that the induced fit mechanism suggests the potential design or discovery of specific HCII agonists.     

Exosite occupation by heparin enhances the reactivity of blood coagulation factor IXa

Blood coagulation factor IXa (fIXa) is a trypsin-like serine protease with low inherent activity that is greatly enhanced in the factor X activation complex. Molecular details of the conversion of fIXa from an inactive enzyme into a fully functional procoagulant are unclear. Recent studies have identified a heparin-binding exosite in the protease domain of fIXa. Effects of exosite occupation on fIXa activity are unclear. We used the Kunitz-type inhibitor bovine pancreatic trypsin inhibitor (BPTI) to probe fIXa reactivity in the absence and in the presence of heparin. While fIXa alone was poorly reactive with BPTI (K(i) approximately 0.7 mM), this reactivity was increased roughly 20-fold (K(i) = 37 +/- 6 microM) by heparin. This was reproducible with low-molecular-weight heparin (enoxaparin; K(i) = 70 +/- 12 microM). Surface plasmon resonance studies of the interaction between heparin and BPTI indicated an unstable interaction with very low affinity (K(d) = 172 microM). In contrast, kinetic studies revealed a high-affinity interaction between heparin and fIXa (K(d) = 128 +/- 26 nM) and showed that the enhancement of BPTI inhibition of fIXa by heparin was well described by a competitive inhibition model where heparin acts as an affecter of fIXa reactivity with inhibitor. Fluorescence studies with dansyl-EGR-fIXa supported the high-affinity interaction between heparin and fIXa and suggested an altered environment in the fIXa active-site region upon heparin binding. This modulating effect of heparin was supported by the observation of a heparin-induced increase in reactivity of fIXa toward a pentapeptide substrate. When viewed together, the results imply that specific physiological exosite interactions with heparin can induce alterations in the environment of the extended fIXa active site that can result in increased reactivity.     

Zinc ions promote the interaction between heparin and heparin cofactor II

The effects of bivalent cations on heparin binding, structure, and thrombin inhibition rates of heparin cofactor II were examined. Zn(2+) - and to a lesser extent Cu(2+) and Ni(2+) - enhanced the interaction between heparin cofactor II and heparin as demonstrated by heparin affinity chromatography and surface plasmon resonance experiments. Metal chelate chromatography and increased intrinsic protein fluorescence in the presence of Zn(2+) indicated that heparin cofactor II has metal ion-binding properties. The results are compatible with the hypothesis that Zn(2+) induces a conformational change in heparin cofactor II that favors its interaction with heparin.     

Identification of the heparan sulfate binding sites in the cellular prion protein

Data from cell culture and animal models of prion disease support the separate involvement of both heparan sulfate proteoglycans and copper (II) ions in prion (PrP) metabolism. Though direct interactions between prion protein and heparin have been recorded, little is known of the structural features implicit in this interaction or of the involvement of copper (II) ions. Using biosensor and enzyme-linked immunosorbent assay methodology we report direct heparin and heparan sulfate-binding activity in recombinant cellular prion protein (PrP(c)). We also demonstrate that the interaction of recombinant PrP(c) with heparin is weakened in the presence of Cu(II) ions and is particularly sensitive to competition with dextran sulfate. Competitive inhibition experiments with chemically modified heparins also indicate that 2-O-sulfate groups (but not 6-O-sulfate groups) are essential for heparin recognition. We have also identified three regions of the prion protein capable of independent binding to heparin and heparan sulfate: residues 23-52, 53-93, and 110-128. Interestingly, the interaction of an octapeptide-spanning peptide motif amino acids 53-93 with heparin is enhanced by Cu(II) ions. Significantly, a peptide of this sequence is able to inhibit the binding of full-length prion molecule to heparin, suggesting a direct role in heparin recognition within the intact protein. The collective data suggest a complex interaction between prion protein and heparin/heparan sulfate and has implications for the cellular and pathological functions of prion proteins.     

Crystallographic analysis of calcium-dependent heparin binding to annexin A2

Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.     

Characterization of the interaction between Robo1 and heparin and other glycosaminoglycans

Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ∼650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.     

Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects

Heparin has been shown to accelerate the inactivation of alpha-thrombin by antithrombin III (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by comparing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 degrees C, and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT-heparin complex, either with or without the active site of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor.     

The region of antithrombin interacting with full-length heparin chains outside the high-affinity pentasaccharide sequence extends to Lys136 but not to Lys139

The interaction of a well-defined pentasaccharide sequence of heparin with a specific binding site on antithrombin activates the inhibitor through a conformational change. This change increases the rate of antithrombin inhibition of factor Xa, whereas acceleration of thrombin inhibition requires binding of both inhibitor and proteinase to the same heparin chain. An extended heparin binding site of antithrombin outside the specific pentasaccharide site has been proposed to account for the higher affinity of the inhibitor for full-length heparin chains by interacting with saccharides adjacent to the pentasaccharide sequence. To resolve conflicting evidence regarding the roles of Lys136 and Lys139 in this extended site, we have mutated the two residues to Ala or Gln. Mutation of Lys136 decreased the antithrombin affinity for full-length heparin by at least 5-fold but minimally altered the affinity for the pentasaccharide. As a result, the full-length heparin and pentasaccharide affinities were comparable. The reduced affinity for full-length heparin was associated with the loss of one ionic interaction and was caused by both a lower overall association rate constant and a higher overall dissociation rate constant. In contrast, mutation of Lys139 affected neither full-length heparin nor pentasaccharide affinity. The rate constants for inhibition of thrombin and factor Xa by the complexes between antithrombin and full-length heparin or pentasaccharide were unaffected by both mutations, indicating that neither Lys136 nor Lys139 is involved in heparin activation of the inhibitor. Together, these results show that Lys136 forms part of the extended heparin binding site of antithrombin that participates in the binding of full-length heparin chains, whereas Lys139 is located outside this site.     

Investigation of the mechanism of binding between internalin B and heparin using surface plasmon resonance

Listeria monocytogenes, a food-borne pathogen that infects immunocompromised patients, enters and proliferates within mammalian cells by taking advantage of host cell machinery. While entry into macrophages and other phagocytic cells occurs constitutively, intracellular invasion of nonphagocytic cells, such as epithelial and endothelial cells, occurs through induced phagocytosis. Invasion of these nonphagocytic cell types is under the control of the secreted L. monocytogenes protein internalin B (InlB), which directly associates with and activates the receptor tyrosine kinase Met. Activation of Met by InlB has previously been shown to be potentiated by binding of glycosaminoglycans to the GW domains of this protein. We studied the interaction between heparin and full-length InlB as well as a truncated, functional form of InlB to understand the mode of interaction between these two molecules. InlB preferred long-chain (>or=dp14) heparin oligosaccharides, and the interaction with heparin fit a complicated binding model with a dissociation constant in the nanomolar range. While there are various explanations for this complicated binding model, one supported by our data involves binding and rebinding of InlB to multiple binding sites on heparin in a positive and weakly cooperative manner. This mode is consistent with enhancement of interaction of InlB with glycosaminoglycans for activation of Met.     

The cell surface receptor G6b, a member of the immunoglobulin superfamily, binds heparin

The G6b gene, located in the human Major Histocompatibility Complex, encodes a receptor of the immunoglobulin (Ig) superfamily. In this study, we show using a variety of techniques that the extracellular domain of the G6b protein, containing a single Ig-like domain, binds to heparin with high affinity. In an ELISA assay, this binding was displaceable with soluble heparin with an IC50 value of approximately 0.5 microg/ml. Other sulfated glycans showed weaker or no competition. The observed interaction between G6b and heparin is strongly salt dependent suggesting a mainly electrostatic interaction. Heparin might modulate the interaction of G6b with its as yet unidentified protein ligand.     

Interaction of heparin and heparin-derived oligosaccharides with synthetic peptide analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein

Background

Although protamine is effective as an antidote of heparin, there is a need to replace protamine due to its side effects. HIP peptide has been reported to neutralize the anticoagulant activity of heparin. The interaction of HIP analog peptides with heparin and heparin-derived oligosaccharides is investigated in this paper.

Methods

Seven analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein (HIP) were synthesized, and their interaction with heparin was characterized by heparin affinity chromatography, isothermal titration calorimetry, and NMR.

Results

NMR results indicate the imidazolium groups of the His side chains of histidine-containing Hip analog peptide interact site-specifically with heparin at pH 5.5. Heparin has identical affinities for HIP analog peptides of opposite chirality. Analysis by counterion condensation theory indicates the peptide AC-SRPKAKAKAKAKDQTK-NH₂ makes on average ∼ 3 ionic interactions with heparin that result in displacement of ∼ 2 Na⁺ ions, and ionic interactions account for ∼ 46% of the binding free energy at a Na⁺ concentration of 0.15 M.

Conclusions

The affinity of heparin for the peptides is strongly dependent on the nature of the cationic side chains and pH. The thermodynamic parameters measured for the interaction of HIP peptide analogs with heparin are strongly dependent on the peptide sequence and pH.

General significance

The information obtained in this research will be of use in the design of new agents for neutralization of the anticoagulant activity of heparin. The site-specific binding of protonated histidine side chains to heparin provides a molecular-level explanation for the pH-dependent binding of β-amyloid peptides by heparin and heparan sulfate proteoglycan and may have implications for amyloid formation.     

Heparin influence on the complex of serum amyloid P component and complement C4b-binding protein

Serum amyloid P component (SAP) forms a calcium-dependent complex with C4b-binding protein (C4BP) in human serum. this study demonstrated that heparin interacted with SAP in a calcium-dependent manner and prevented formation of the SAP.C4BP complex. Furthermore, the SAP-heparin interaction interfered with SAP binding to membranes. Therefore, all three of these interactions involved similar sites on SAP, or each interaction sterically obstructed the other binding sites. In addition to heparin, SAP bound to heparan sulfate and chondroitin sulfate. In each case, a distinct multimeric species was generated. Gel filtration and sucrose density gradient ultracentrifugation suggested that heparin and heparan sulfate produced a dimer of SAP. The dimer appeared to be the most stable structure since it was not dissociated by excess heparin. While low molecular weight heparin interacted with SAP and inhibited SAP association with membranes, the SAP dimer was not detected in sucrose density gradient ultracentrifugation studies. Polybrene prevented the interaction between SAP and heparin in both a purified system and in human serum that was enriched in SAP and heparin. In contrast, Polybrene did not seem to alter the SAP.C4BP complex. While the function of the SAP.C4BP complex is unknown, it may be important for regulation of complement and/or transport of SAP to sites in the body. Dissociation of the SAP.C4BP complex by sulfated polysaccharides such as heparin may be a physiological response that could be important during tissue damage or complement activation.     

Two-step mechanism of binding of apolipoprotein E to heparin: implications for the kinetics of apolipoprotein E-heparan sulfate proteoglycan complex formation on cell surfaces

The interaction of apolipoprotein E (apoE) with cell-surface heparan sulfate proteoglycans is an important step in the uptake of lipoprotein remnants by the liver. ApoE interacts predominantly with heparin through the N-terminal binding site spanning the residues around 136-150. In this work, surface plasmon resonance analysis was employed to investigate how amphipathic alpha-helix properties and basic residue organization in this region modulate binding of apoE to heparin. The apoE/heparin interaction involves a two-step process; apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics. Circular dichroism and surface plasmon resonance experiments using a disulfide-linked mutant, in which opening of the N-terminal helix bundle was prevented, demonstrated that there is no major secondary or tertiary structural change in apoE upon heparin binding. Mutations of Lys-146, a key residue for the heparin interaction, greatly reduced the favorable free energy of binding of the first step without affecting the second step, suggesting that electrostatic interaction is involved in the first binding step. Although lipid-free apoE2 tended to bind less than apoE3 and apoE4, there were no significant differences in rate and equilibrium constants of binding among the apoE isoforms in the lipidated state. Discoidal apoE3-phospholipid complexes using a substitution mutant (K143R/K146R) showed similar binding affinity to wild type apoE3, indicating that basic residue specificity is not required for the effective binding of apoE to heparin, unlike its binding to the low density lipoprotein receptor. In addition, disruption of the alpha-helix structure in the apoE heparin binding region led to an increased favorable free energy of binding in the second step, suggesting that hydrophobic interactions contribute to the second binding step. Based on these results, it seems that cell-surface heparan sulfate proteoglycan localizes apoE-enriched remnant lipoproteins to the vicinity of receptors by fast association and dissociation.     

The interaction of platelet factor 4 with heparins of different chain length

The interaction of platelet factor 4 with heparin of varying chain lengths has been investigated by labelling the heparin with fluorescein isothiocyanate and monitoring the change in anisotropy of fluorescence when the protein is added to a solution of the polysaccharide. The shape of the titration curve depends on the Mr of the heparin and chains of Mr greater than 10 000 showed a definite break when the concentration of polysaccharide and protein became equimolar. Evidence is presented to show that most of the fluorescein label is linked to residual serines on the heparin. Similar break-points were observed if total fluorescence or light-scattering was used to monitor the interaction. Unlabelled heparin was used for the latter method. These results together with those obtained in buffer of high ionic strength lead us to propose a model where the heparin is wrapped around the tetrameric protein.