Interaction of S-protein of complement with thrombin and antithrombin III during coagulation. Protection of thrombin by S-protein from antithrombin III inactivation

S-protein, the inhibitor in plasma of the membrane attack complex of complement, appears to have a second function in coagulation. S-protein during clotting enters into a trimolecular complex with thrombin and antithrombin III (ATIII). Functionally, S-protein in the presence of low concentrations of heparin, protects thrombin from inactivation by ATIII. Complex formation between S-protein and thrombin, and between S-protein, thrombin, and ATIII, was demonstrated by agarose gel electrophoresis and by two-dimensional immunoelectrophoresis of purified proteins and in recalcified, clotted plasma. Formation of the trimolecular S-thrombin-ATIII complex was strictly dependent on the presence of thrombin. No association was detectable between S-protein and ATIII or between S-protein and prothrombin. Heparin was not required for the formation of the bimolecular S-protein-thrombin complex or the trimolecular S-protein-ATIII complex. The protective effect of S-protein on inactivation of thrombin by ATIII was demonstrated in functional assays with purified proteins and in plasma only in the presence of low concentrations of heparin. Thus, S-protein may mediate its effect by scavenging heparin required for ATIII activation. It is suggested that the protection of thrombin by S-protein from inactivation by ATIII may be of physiological importance.     

A method for rapid quantitation and preparation of antithrombin III-high-affinity heparin fractions

An electrophoretic method for the quantitation and preparation of antithrombin III-high-affinity heparin using agarose beds is described. The method allows the determination of high-affinity heparin fractions in several samples in one single step. The incubation mixture containing heparin and antithrombin III is submitted to agarose gel electrophoresis in 0.06 m barbital buffer, pH 8.6. A sharp separation between free antithrombin III, the complex antithrombin III-heparin, and free heparin occurs under these conditions. Around 30% of heparin molecules present in commerical preparations bind to antithrombin. This bound heparin has an anticoagulant activity of 240 IU. Negligible binding of other sulfated mucopolysaccharides to antithrombin III was observed. The whole procedure takes less than 6 h and can also be used as a semipreparative method for high-affinity heparin.     

Circular dichroism of platelet factor 4

The circular dichroism of platelet factor 4 was investigated and it was found to contain 15% alpha-helix, 25% beta-structure, and the rest of the molecule in unordered conformation. In the presence of heparin, no change in the circular dichroism was observed, suggesting no significant changes in the secondary structure of platelet factor 4 when heparin binds. The CD spectrum of platelet factor 4 was also investigated in the presence of increasing concentrations of guanidine hydrochloride. A two-state transition was observed with midpoints at 0.125 and 2.0 M guanidine hydrochloride. Based on gel filtration studies, the first unfolding transition was correlated with the dissociation of the tetrameric structure. This first unfolding domain was not observed in the presence of heparin, suggesting that heparin stabilizes the tetrameric structure. The second unfolding transition corresponds to the disruption of the overall secondary structure which is generally observed with most proteins. It is concluded that a relatively weak force of attraction holds the tetrameric structure of platelet factor 4 and the dissociation of the subunits is accompanied by loss of some helical secondary structure.     

Important role of arginine 129 in heparin-binding site of antithrombin III. Identification of a novel mutation arginine 129 to glutamine

An hereditary abnormal antithrombin III (ATIII Geneva) with defective heparin cofactor activity was characterized by DNA single strand amplification and subsequent direct sequencing. ATIII Geneva was found to have a G to A transition in Exon IIIa leading to an Arg-129 to Gln mutation. This amino acid is part of the ATIII region comprising residues 114-154, which contains the highest proportion of basic residues (Arg or Lys), and is known from chemical modification studies to be involved in heparin binding. The variant protein did not bind heparin-Sepharose and was isolated from the propositus plasma by immunoaffinity chromatography. High affinity (for ATIII) heparin had only a minimal effect on thrombin and activated factor X inhibition by the purified abnormal ATIII. Taken together, these results demonstrate an important role for Arg-129 in the binding and interaction of ATIII with heparin of high affinity. We propose that a cooperation between Lys-125, Arg-129, Lys-136, and Arg-47 exposed at the surface of the inhibitor allows the binding of the essential pentasaccharide domain of heparin which is specific for the ATIII interaction.     

Thermodynamic analysis of heparin binding to human antithrombin.

The binding of heparin to human antithrombin III (ATIII) was investigated by titration calorimetry (TC) and differential scanning calorimetry (DSC). TC measurements of homogeneous high-affinity pentasaccharide and octasaccharide fragments of heparin in 0.02 M phosphate buffer and 0.15 M sodium chloride (pH 7.3) yielded binding constants of (7.1 +/- 1.3) x 10(5) M-1 and (6.7 +/- 1.2) x 10(6) M-1, respectively, and corresponding binding enthalpies of -48.3 +/- 0.7 and -54.4 +/- 5.4 kJ mol-1. The binding enthalpy of heparin in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.3) was estimated from TC measurements to be -55 +/- 10 kJ mol-1, while the enthalpy in Tris buffer (0.02 M, 0.15 M NaCl, pH 7.3) was -18 +/- 2 kJ mol-1. The heparin-binding affinity was shown by fluorescence measurements not to change under these conditions. The 3-fold lower binding enthalpy in Tris can be attributed to the transfer of a proton from the buffer to the heparin-ATIII complex. DSC measurements of the ATIII unfolding transition exhibited a sharp denaturation peak at 329 +/- 1 K with a van 't Hoff enthalpy of 951 +/- 89 kJ mol-1, based on a two-state transition model and a much broader transition from 333 to 366 K. The transition peak at 329 K accounted for 9-18% of the total ATIII. At sub-saturate heparin concentrations, the lower temperature peak became bimodal with the appearance of a second transition peak at 336 K. At saturate heparin concentration only the 336 K peak was observed. This supports a two domain model of ATIII folding in which the lower stability domain (329 K) binds and is stabilized by heparin.     

Characterization of a chondroitin 4-sulfate proteoglycan carrier for heparin neutralizing activity (platelet factor 4) released from human blood platelets

Platelet heparin neutralizing activity (platelet factor 4) is released from human blood platelets by thrombin in the form of a high molecular weight proteoglycan-platelet factor 4 complex. This complex was partially purified by isoelectric precipitation and gel filtration. At high ionic strength (I = 0.75) the complex dissociates into the active component (mol. wt 29000) and the proteoglycan carrier. The components were separated by gel filtration and the proteoglycan further purified by Na₂SO₄ treatment. The molecular weight of the purified carrier was 59000. The carbohydrate moieties of the proteoglycan isolated after papain digestion and ion-echange chromatography were shown to consist of chondroitin 4-sulfate by chemical, physical and electrophoretic analysis. The multichain proteoglycan consists of four chondroitin 4-sulfate chains (mol. wt 12000) in covalent linkage to a single polypeptide. The molecular weight (350000) of the fully saturated proteoglycan carrier suggests that 4 moles of platelet factor 4 are bound per mole of proteoglycan and that the carrier occurs in the form of a dimer consisting of 8 moles of platelet factor 4 and 2 moles of proteoglycan. The isolated chondroitin 4-sulfate moieties combine with platelet factor 4 at a binding ratio of one mole of platelet factor 4 per carbohydrate chain. Heparin completely displaces platelet factor 4 from both the saturated proteoglycan and chondroitin 4-sulfate complexes. Heparitin sulfate, dermatan sulfate and chondroitin 6-sulfate also combine stoichiometrically with platelet factor 4 and are displaced by equimolar amounts of heparin. Hyaluronic acid did not combine with platelet factor 4. The relative binding capacities of glycosaminoglycans for platelet factor 4 were shown to be: heparin (100), heparitin sulfate (75), chondroitin 4-sulfate (50), dermatan sulfate (50), chondroitin 6-sulfate (50), and hyaluronic acid (o). Chondroitin 4-sulfate was identified as the major glycosaminoglycan in all platelet subcellular fractions; in addition, the soluble fraction contains a minor amount of hyaluronic acid. Subcellular distribution studies revealed that 55% of both the proteoglycan carrier and platelet factor 4 activity were localized in the “granule rich” fraction. This data together with the low recovery of both these components in the membrane fraction, suggest that they occur together as a complex within specific granules and are released in this form under physiologic conditions.     

Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site

Porcine mucosal heparin was fragmented into low-molecular-weight (LMW) heparin by treatment of periodate-oxidized heparin with sodium hydroxide, followed by reduction with sodium borohydride and acid hydrolysis. Gradient polyacrylamide gel electrophoresis analysis showed a mixture of heparin fragments with an average size of eight disaccharide units. 1D 1H NMR showed two-thirds of the N-acetyl groups were lost on periodate cleavage, suggesting cleavage had occurred at the glucopyranosyluronic acid (GlcpA) and idopyranosyluronic acid (IdopA) residues located within and adjacent to the antithrombin III (ATIII) binding site. The N-acetyl glucopyranose (GlcpNAc) residue was lost on workup. The GlcpA residue, within the ATIII binding site, is on the non-reducing side of the N-sulfo, 3, 6-O-sulfo glycopyranosylamine (GlcpNS3S6S) residue. Thus, periodate cleaved heparin should be enriched in GlcpNS3S6S residues. Two-dimensional correlation spectroscopy (2D COSY) confirmed that LMW heparin prepared through periodate cleavage contained GlcpNS3S6S at its non-reducing end. As expected, this LMW heparin also showed reduced ATIII mediated anti-factor IIa and anti-factor Xa activities.     

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.     

The pharmacological profile of the low molecular weight heparin 21-23 in man: anticoagulant, lipolytic and protamine reversible effects

The anticoagulant, lipolytic and protamine reversible effects of high doses of low molecular weight (LMW) heparin 21-23 and unfractionated heparin were compared in man. 7,500 units of each heparin were applied, which corresponds to 90 mg LMW heparin and 48 mg unfractionated heparin. The anticoagulant properties of the LMW heparin are characterized by a doubled half life of factor Xa activity, smaller influence on aPTT and thrombin after intravenous (i.v.) and subcutaneous (s.c.) injection, and higher bioavailability of factor Xa activity after s.c. administration (90% versus 15%). Protamine chloride completely neutralizes the effect on aPTT and thrombin and reduces the anti factor Xa activity by 60%. The bleeding time is prolonged by both normal and LMW heparin by 20%. This effect is normalized by protamine chloride, too. Thrombelastography with recalcified whole blood demonstrates that protamine chloride shortens but not completely normalizes the coagulation time in presence of either unfractionated or LMW heparin. The half life of lipoprotein lipase (LPL) activity is 60 min after i.v. administration of unfractionated heparin and 120 min with LMW heparin. Although the release of lipases (LPL and HTGL) is higher after i.v. and s.c. administration of the LMW heparin they do not induce higher releases of free fatty acids. This indicates that the lipolytic activity of this LMW heparin and unfractionated heparin is similar. The results show an improved anticoagulant pharmacological profile of this LMW heparin as compared to unfractionated heparin. Protamine normalizes the anticoagulant effects of LMW heparin with exception of a residual anti factor Xa activity and normalizes the changes of bleeding time and thrombelastography.     

Search for the heparin antithrombin III-binding site precursor.

The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.     

A peptide model for the heparin binding site of antithrombin III.

A peptide model for the heparin binding site of antithrombin III (ATIII) was synthesized to elucidate the structural consequences of heparin binding. This peptide [ATIII(123-139)] and a sequence-permuted analogue (ATIII random) showed similar conformational behavior (as analyzed by circular dichroism spectroscopy) in aqueous and organic media. In the presence of heparin, however, the peptide ATIII(123-139) assumed a stable conformation, whereas peptide ATIII random did not. Complex formation was saturable and sensitive to salt. The ATIII(123-139)-heparin complex contained beta-structure, rather than helical structure. This finding is incompatible with current models of heparin binding and suggests that heparin binding may induce nonnative structures at the binding site which could, in turn, lead to activation of ATIII. The peptide ATIII(123-139) was able to inhibit the binding of ATIII by heparin, consistent with the notion that this peptide may be a model for the heparin binding site.     

Isolation and amino acid sequence of bovine platelet factor 4

Bovine platelet factor 4 was isolated by affinity chromatography using dextran sulfate Sepharose and purified by subsequent gel filtration. The complete amino acid sequence of this 88-residue, 9505-Da protein was determined by isolation and analysis of the overlapping peptides from tryptic and Staphylococcus aureus hydrolysates of reduced, carboxymethylated, and reductive methylated protein. Primary structure comparison was made between bovine platelet factor 4, human platelet factor 4, and human beta-thromboglobulin. The bovine platelet factor 4 amino-terminal region, which contains two unique phenylalanine residues, is extended by 15 residues relative to human platelet factor 4. The bovine carboxy-terminal region is extended by three residues relative to human platelet factor 4 and differed from beta-thromboglobulin in the absence of two additional terminal residues. Bovine platelet factor 4 shares sequence similarities proportionately with both human platelet factor 4 and beta-thromboglobulin. The sequences of the lysine-rich carboxy-terminal putative heparin binding domains are essentially identical for all three proteins. The heparin neutralizing potencies of bovine and human platelet factor 4 are similar: 40 USP units of heparin neutralized per milligram protein, as measured by a modified chromogenic substrate assay. Heparin neutralization was lost by reduction of the disulfide bonds, but only attenuated by tryptic digestion of the intact protein.     

The distinct roles that Gln-192 and Glu-217 of factor IX play in selectivity for macromolecular substrates and inhibitors

In this paper, we report functional characterization of positions 192 and 217 (chymotrypsinogen numbering system) in human factor IX and discuss the distinction and similarity of these two sites among the blood coagulation factors. Recombinant factor IXQ192E (residue glutamine at position 192 replaced by glutamic acid), IXQ192K, IXE217D, and IXE217R proteins exhibited 11%, 46%, 39%, and 2% of the wild-type factor IX's clotting activity, respectively. Binding of these variants to factor VIIIa (FVIIIa) was inefficient compared to that of wild-type factor IX, and the dissociation constants doubled for IXQ192E, 3-fold higher for IXQ192K and 4-fold higher for both IXE217D and IXE217R. In the presence of FVIIIa, all variant factor IX hydrolyzed factor X at the catalytic efficiencies correlating with respective clotting activities. However, FVIIIa greatly enhanced the catalytic efficiency of both IXE217 variants to a greater extent (approximately 7 x 10(4)-fold) as compared to its effect on the wild-type factor IXa and the other two IXQ192 variants [by a factor of (1-2) x 10(4)]. Moreover, while both IXQ192 variants demonstrated small substrate selectivity similar to that of wild-type factor IXa, the selectivity of both IXE217 variants was greatly altered. Mutations at position 192 disturbed the interaction of factor IXa with physiological inhibitors. Although all variants formed an SDS-stable complex with antithrombin III (ATIII) equally well in the presence of heparin and were readily inhibited by ATIII in the absence of heparin, activated IXQ192K exhibited a slower stable complex formation with ATIII without heparin. On the other hand, only IXQ192E showed decreased interaction with TFPI. Our results demonstrate that positions 192 and 217 play different roles unique to factor IX in specifying the interaction of factor IX with substrates and inhibitors.     

Interaction of platelet factor 4 with human platelets

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.     

Serine base exchange enzyme in porcine lyophilised platelets: enzyme properties and modulation by AIF4-and different types of heparin

Phosphatidylserine is one of the PKC modulators and thus it may play an important role in signal transduction. Regulation of the synthesis of this phospholipid is not yet clarified. The contrasting reports are possibly related to the existence of different enzymes which, in mammalian tissues, catalyse the exchange between free serine and the nitrogen base of a membrane phospholipid. This study demonstrates that serine base exchange reactions of commercially available lyophilised porcine platelets exhibit similar pH optima, temperature and Ca²⁺ dependence as observed in fresh tissues. Analysis of fatty acids composition of the three phospholipid classes involved in base exchange reactions also demonstrated a similarity with fresh platelets. Serine and ethanolamine base exchange enzyme activities were assayed in parallel in platelet lysate subjected to preincubation at various temperatures (30-60°C). When dithioerithrol was omitted from the incubation medium, the two base exchange reactions were inhibited with a similar temperature-dependent pattern. Addition of the reducing agent enhanced the sensitivity to preincubation only for the serine base exchange reaction which was inhibited by 80% after preincubation at 45°C. With respect to its regulation, porcine platelet serine base exchange enzyme(s) was inhibited by fluoroalluminate, a widely used G-protein activator, and stimulated by unfractionated heparin. Low mol. wt. heparin did not influence enzyme activity. Unfractionated heparin greatly stimulated SBEE activity assayed at pH 7.4, a pH value far from the optimal pH.     

Role of platelet-activating factor in the ovine heparin-protamine reaction.

Platelet-activating factor (PAF) infusion into sheep, as well as protamine reversal of heparin anticoagulation, causes thromboxane release into plasma, pulmonary hypertension, hypoxemia, and leukopenia. We investigated the possible role of PAF in the heparin-protamine reaction. Intravenous protamine was administered to neutralize heparin anticoagulation in five awake sheep and caused an increase of mean pulmonary arterial pressure from 16.6 +/- 1 (SE) mmHg at base-line to 47 +/- 9 mmHg at 1 min after protamine injection (P < 0.01) because of a 4.5-fold increase of pulmonary vascular resistance. This neutralization reaction induced a 25% reduction of circulating leukocyte count and arterial PO2. Undetectable blood levels of PAF were measured by bioassay and high-performance liquid chromatography during these heparin-protamine reactions. Infusion of BN 52021 (20 mg/kg), a PAF receptor antagonist, before rechallenging the same sheep with heparin and then protamine did not reduce the level of peak pulmonary hypertension or the degree of hypoxemia and leukopenia. We conclude that the leukopenia and thromboxane-mediated pulmonary vasoconstriction occurring after rapid intravascular formation of heparin-protamine complexes in sheep are not due to the release of PAF.     

Affinity chromatography of sulphated polysaccharides separately fractionated on antithrombin III and heparin cofactor II immobilized on concanavalin A—Sepharose

Three sulphated polysaccharides, dermatan sulphate, fucan and heparin, were fractionated according to their affinity towards antithrombin III (ATIII) and heparin cofactor II (HCII), the two main physiological thrombin (IIa) inhibitors. Both inhibitors were immobilized on concanavalin A—Sepharose, which binds to the glycosylated chains of the proteins while the protein-binding site for the polysaccharide remains free. Each polysaccharide was fractionated into bound and unbound fractions either for ATIII or HCII. The eluted fractions were tested for their ability to catalyse and interactions. The possible presence of a unique binding site for ATIII and HCII, on each sulphated polysaccharide, was also studied.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

Characterization of the heparin-binding site of glia-derived nexin/protease nexin-1.

The interaction of heparin with glia-derived nexin (GDN) has been characterized and compared to that observed between heparin and antithrombin III (ATIII). Heparin was fractionated according to its affinity for immobilized GDN, and the ability of various fractions to accelerate the inhibition rate of thrombin by either GDN or ATIII was examined. Fractions with different affinities for GDN accelerated the thrombin-GDN reaction to a similar extent; heparin with a high affinity for immobilized GDN stimulated the reaction only about 30% more than the fraction that did not bind to immobilized GDN. Slightly greater differences were observed for the effect of these fractions on the thrombin-ATIII reaction; heparin that did not bind to the GDN affinity column was about 60% more effective than heparin with a high affinity for GDN in accelerating the inhibition of thrombin by ATIII. The CNBr fragment of GDN between residues 63 and 144 was able to reduce the heparin-accelerated rate of inhibition of thrombin by GDN indicating that this region of GDN was able to bind the heparin molecules responsible for the acceleration. Shorter synthetic peptides within this sequence did not significantly reduce the rate, suggesting that the heparin-binding activity of fragment 63-144 depends on a specific conformation of the polypeptide chain. Fragment 63-144 was less effective in decreasing the heparin-accelerated rate of inhibition of thrombin by ATIII. The results are discussed in terms of the heparin species that are responsible for the acceleration of the GDN- and ATIII-thrombin reactions and the heparin-binding sites of GDN and ATIII.     

Cloning of the Full-Length cDNA of Porcine Antithrombin III and Comparison with its Human Homolog

The characterization of porcine antithrombin III (ATIII)—a highly powerful anticoagulant—is essential for using porcine liver in xenotransplantation applications. The objective of this study was to clarify the functions of porcine ATIII through comparison with human ATIII. We cloned porcine ATIII and compared its important functional sites with those of human ATIII. The full-length cDNA of porcine ATIII was cloned by screening a porcine liver cDNA library, and the ATIII activities of 23 pigs were determined. The full-length cDNA of porcine ATIII spanned 1498 bp and encoded 463 amino acids. Porcine ATIII shared 87.67% nucleotide identity and 89.06% amino acid identity with human ATIII. Complete identity was found at active center Arg393–Ser394, and remarkably high similarities were found at 2 critical heparin-binding sites (residues 41 through 49 and 114 through 156) and in some key residues involved in heparin binding. An ATIII assay found no significant difference between porcine and human plasma. The high level of similarity between porcine ATIII and human ATIII suggests that porcine ATIII will function in a manner similar to human ATIII in xenotransplantation.Abbreviation: ATIII, antithrombin IIIAntithrombin III (ATIII) is a single-chain glycoprotein found in mammalian plasma that inhibits thrombin and other serine proteinases involved in the blood coagulation cascade, such as factor IX, factor X,²⁰ and plasmin. ATIII is considered the most powerful serine proteinase inhibitor (serpin) and the most important contributor to the anticoagulation system.¹¹ Previous studies have provided detailed knowledge of human ATIII¹ and have identified 2 essential activities as prerequisites for its effective function: (1) recognizing and attacking target proteases and (2) interacting with its cofactor, heparin.²The pig has played an important role in biomedical research,^3,6,15,33 especially as a large animal model for surgical experiments and a promising candidate for xenotransplantation.^9,12Although knowledge of the physiologic features of the porcine coagulation system is important for its successful application, differences (if any) between human and porcine coagulation factors have not been studied in detail.¹⁶ Considering the unknown properties of porcine ATIII when it is secreted into human blood and interacts with human thrombin and heparin after liver or hepatocyte xenotransplantation, there is a clear need for elucidating the properties of porcine ATIII.Treatment with high doses of recombinant human ATIII prevents coagulopathy and protects renal xenografts from early injury in the pig-to-baboon model.⁸ Heparin-dependent inhibition of human factor Xa by porcine arterial endothelial cells is blocked completely by neutralizing ATIII but is unaffected by the antitissue factor pathway inhibitor antibody.¹⁸ These results suggest that human ATIII is an effective anticoagulant in the xenotransplantation model, but porcine tissue factor pathway inhibitor and human Xa are incompatible.¹⁸ However, porcine ATIII has not been evaluated with regard to xenotransplantation models. Consequently, in the present study, we cloned and characterized the full-length cDNA of porcine ATIII. We then compared sequence and function of porcine ATIII cDNA with those of human ATIII to gain insight into the molecular compatibility of porcine and human coagulation-related molecules.