Inhibition of human factor IXa by human antithrombin.

A procedure is presented for the purification of Factor IX from human plasma. The final product is homogeneous as judged by disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Furthermore, it is completely free of other coagulation component activities. Factor IX is converted to its enzymatically active form by the addition of small quantities of Factor IXa in the presence of calcium ions. This activated species is added to purified antithrombin-heparin cofactor and the interaction is studied in the presence and absence of heparin. Antithrombin-heparin cofactor is found to be a progressive, time-dependent inhibitor of Factor IXa and neutralizes approximately 57% of this enzyme's proteolytic activity within 30 min. The addition of heparin dramatically accelerates the rate of this interaction with virtually complete inhibition of Factor IXa occurring within 15 s. Sodium dodecyl sulfate gel electrophoresis of reduced and nonreduced proteins indicates that antithrombin-heparin cofactor functions as a potent inhibitor of Factor IXa by forming an undissociable complex with the enzyme which is stable in the presence of denaturing or reducing agents (or both). This complex represents a 1:1 stoichiometric combination of enzyme and inhibitor. Heparin increases the rate of formation of this complex without affecting its dissociability or stoichiometry.     

Inhibition of bovine factor IXa and factor Xabeta by antithrombin III.

Factor IXa and factor Xabeta are serine proteases which participate in the middle phase of blood coagulation. These two enzymes are inhibited by antithrombin III by the formation of an enzyme-inhibitor complex containing 1 mol of enzyme and 1 mol of antithrombin III. The complex was readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and loss of coagulant or esterase activity at increasing concentrations of inhibitor. The inactivation of factor IXa by antithrombin III was relatively slow, but the reaction was greatly accelerated by the addition of heparin.     

Heparin modulates the 99-loop of factor IXa: effects on reactivity with isolated Kunitz-type inhibitor domains

Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys(98). Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys(98) to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys(98) and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite.     

Inhibition of factor IXa and factor Xa by antithrombin III/heparin during factor X activation

We investigated the kinetics of the inhibitory action of antithrombin III and antithrombin III plus heparin during the activation of factor X by factor IXa. Generation and inactivation curves were fitted to a three-parameter two-exponentional model to determine the pseudo first-order rate constants of inhibition of factor IXa and factor Xa by antithrombin III/heparin. In the absence of heparin, the second-order rate constant of inhibition of factor Xa generated by factor IXa was 2.5-fold lower than the rate constant of inhibition of exogenous factor Xa. It appeared that phospholipid-bound factor X protected factor Xa from inactivation by antithrombin III. It is, as yet, unclear whether an active site or a nonactive site interaction between factor Xa and factor X at the phospholipid surface is involved. The inactivation of factor IXa by antithrombin III was found to be very slow and was not affected by phospholipid, calcium, and/or factor X. With unfractionated heparin above 40 ng/ml and antithrombin III at 200 nM, the apparent second-order rate constant of inhibition of exogenous and generated factor Xa were the same. Thus, in this case phospholipid-bound factor X did not protect factor Xa from inhibition. In the presence of synthetic pentasaccharide heparin, however, phospholipid-bound factor X reduced the rate constant about 5-fold. Pentasaccharide had no effect on the factor IXa/antithrombin III reaction. Unfractionated heparin (1 micrograms/ml) stimulated the antithrombin III-dependent inhibition of factor IXa during factor X activation 400-fold. In the absence of reaction components this stimulated was 65-fold. We established that calcium stimulated the heparin-dependent inhibition of factor IXa.     

Heparin inhibits the intrinsic tenase complex by interacting with an exosite on factor IXa

The specific molecular target for direct heparin inhibition of factor X activation by intrinsic tenase (factor IXa-factor VIIIa) was investigated. Comparison of size-fractionated oligosaccharides demonstrated that an octasaccharide was sufficient to inhibit intrinsic tenase. Substitution of soluble dihexanoic phosphatidylserine (C6PS) for phospholipid (PL) vesicles demonstrated that inhibition by low-molecular weight heparin (LMWH) was independent of factor IXa-factor VIIIa membrane assembly. LMWH also inhibited factor X activation by the factor IXa-PL complex via a distinct mechanism that required longer oligosaccharides and was independent of substrate concentrations. The apparent affinity of LMWH for the factor IXa-PL complex was higher in the absence of factor VIIIa, suggesting that the cofactor adversely affected the interaction of heparin with factor IXa-phospholipid. LMWH did not interact directly with the active site, as it failed to inhibit chromogenic substrate cleavage by the factor IXa-PL complex. LMWH induced a modest decrease in factor IXa-factor VIIIa affinity [K(D(app))] on PL vesicles that did not account for the inhibition. In contrast, LMWH caused a substantial reduction in factor IXa-factor VIIIa affinity in the presence of C6PS that fully accounted for the inhibition. Factor IXa bound LMWH with significantly higher affinity than factor X by competition solution affinity analysis, and the K(D(app)) for the factor IXa-LMWH complex agreed with the K(I) for inhibition of the factor IXa-PL complex by LMWH. Thus, LMWH binds to an exosite on factor IXa that antagonizes cofactor activity without disrupting factor IXa-factor VIIIa assembly on the PL surface. This exosite may contribute to the clinical efficacy of heparin and represents a novel target for antithrombotic therapy.     

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.     

Potentiation by calcium ions of the antithrombin III inhibition of thrombin

Calcium ions potentiated heparin-modulated antithrombin III inhibition of amidolysis catalysed by thrombin. Potentiation by calcium ions of heparin-independent antithrombin III inhibition of thrombin activity appeared to contribute to this effect. These results suggest a complex modulatory role for calcium ions in proteinase-catalysed reactions influenced by anti-proteinases and glycosaminoglycans.     

Mechanism of catalysis of inhibition of factor IXa by antithrombin in the presence of heparin or pentasaccharide

Because of the homology between factor IXa and factor Xa (f.IXa and f.Xa, respectively), and the critical upstream position of f.IXa in the coagulation cascade, the contribution of the heparin-derived pentasaccharide to antithrombin-mediated inhibition of f.IXa was investigated. Pentasaccharide promotes inhibition of both f.IXa and f.Xa generated in recalcified plasma. This result demonstrates that antithrombin is the predominant inhibitor of f.IXa in plasma, and that the activity of antithrombin is promoted by pentasaccharide. Kinetic experiments reveal that pentasaccharide increases the rates of antithrombin-mediated inhibition of both f.IXa and f.Xa by 2 orders of magnitude. These findings indicate that pentasaccharide-induced conformational changes in antithrombin enhance its capacity to inhibit both f.IXa and f.Xa. In the presence of Ca2+, full-length heparin produces an additional approximately 10-fold increase in the rates of inhibition of both enzymes, consistent with a template role of heparin. Heparin binding to f.Xa was previously shown to be promoted in the presence of Ca2+. Binding studies with f.IXa reveal a 10-fold higher affinity for heparin in the presence of Ca2+ compared with its absence. Thus, Ca2+ promotes heparin-catalyzed inhibition of f.IXa and f.Xa by antithrombin by augmenting the template mechanism. These results indicate that heparin-mediated catalysis of f.IXa inhibition by antithrombin reflects both pentasaccharide-induced conformational changes and heparin-mediated bridging of antithrombin to f.IXa. Furthermore, our data suggest that the efficacy of pentasaccharide for prevention and treatment of thrombotic disorders may reflect its action at two sites in the coagulation system.     

Magnesium and calcium ions enhance barotolerance of streptococci

Summary Magnesium and calcium ions were found to enhance barotolerance of Streptococcus faecalis ATCC strain 9790 growing in a complex, glucose-containing medium. Enhancement was indicated both by higher growth rates and yields at 408 atm, and also by an increase in the maximum pressure permitting growth from 550 to 700 atm. The optimum concentration of either ion was ca. 50 mM, and both ions appeared to be equipotent in affecting the same processes by chemically specific interactions. Sodium, potassium, strontium, manganous, chloride, bromide or sulfate ions were all ineffective or only marginally effective in enhancing barotolerance. Mg⁺⁺ and Ca⁺⁺ also enhanced growth of compressed, ribose-degrading cultures. Pressure increased the sensitivity of streptococcal growth to low pH, and there appeared to be two distinct effects of Mg⁺⁺ and Ca⁺⁺ on barotolerance. First, the rate of exponential growth was enhanced prior to the time at which culture acidity began to limit growth. Second, growth was possible in more acid conditions under pressure when the ions were present, and enhanced yields from compressed cultures were related to this partial reversal of the potentiating effect of high pressure on acid inhibition of growth.Barotolerance of Escherichia coli or Saccharomyces cerevisiae was not enhanced by these ions; while tolerance of two types of chain-forming cocci freshly isolated from a rotting mussel was enhanced.     

Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III

The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. IX activation by VIIa/TF is considered to contribute catalyst necessary for further Xa production in the ensuing amplification phase. We have investigated Xa and IXabeta production by VIIa-TF in a system reconstituted with both X and IX and the principal physiologic inhibitors of this pathway TFPI and antithrombin III (AT). Kinetic studies without inhibitors established that IX and X functioned as competitive alternate substrates for VIIa/TF with similar kinetic constants. When both IX and X were present, TFPI significantly inhibited the extent of formation of either IXabeta or Xa. In contrast, AT rapidly depleted active Xa with a small effect on IXabeta formation. When both AT and TFPI were present, active IXabeta formation significantly exceeded the formation of active Xa regardless of the VIIa/TF concentration. These findings could be quantitatively accounted for by a model encompassing the kinetics of the individual activation and inhibition steps. Active Xa formation by this pathway is regulated in a principal way by its rapid inactivation by AT. In contrast, the Xa-dependent inhibitory reactions of TFPI play a primary role in limiting zymogen consumption and the formation of active IXabeta. These regulatory phenomena yield active IXabeta as a major rather than secondary product of VIIa/TF. Our findings raise the possibility that IXabeta produced by the extrinsic pathway, and its ability to function within the intrinsic Xase complex to activate X may play a significant role in producing Xa necessary for both the initiation and sustained phases of the procoagulant response following vascular damage.     

Activation of antithrombin as a factor IXa and Xa inhibitor involves mitigation of repression rather than positive enhancement

Allosteric activation of antithrombin as a rapid inhibitor of factors IXa and Xa requires binding of a high-affinity heparin pentasaccharide. The currently-accepted mechanism involves removal of a constraint on the antithrombin reactive center loop (RCL) so that the proteinase can simultaneously engage both the P1 arginine and an exosite at Y253. Recent results suggest that this mechanism is incorrect in that activation can be achieved without loop expulsion, while the exosite can be engaged in both low and high activity states. We propose a quite different mechanism in which heparin activates antithrombin by mitigating an unfavorable surface interaction, by altering its nature, and by moving the attached proteinase away from the site of the unfavorable interaction through RCL expulsion.     

The interaction of calcium and magnesium ions with deoxyribonucleic acid.

Conductance and equilibrium dialysis studies are reported for the aqueous systems (native calf thymus) DNA-CaCl₂ and DNA-MgCl₂ at various pH values and ionic strengths at 25 °C. Discontinuities occur in the conductance curves at mole ratios of Ca²⁺ and Mg²⁺ to nucleate phosphorus of 0.125, 0.30, and 0.50. The dialysis results show the formation of complexes of stoichiometry 0.50 and 1.00 mol Ca²⁺ or Mg²⁺/mol nucleate phosphorus (2:1 and 1:1 complexes), the latter only in neutral or alkaline solutions, in agreement with the conductance discontinuity at 0.50. The other discontinuities may be due to preferential binding in the formation of the 2:1 complex. Binding constants for the 2:1 complexes are evaluated. Absorption-temperature profiles have been determined for “native” and dialysed DNA in the presence of NaCl, CaCl₂, and MgCl₂. For dialysed DNA at 26 ° C and 260 nm the decrease of absorbance with increased salt concentration was halted for MgCl₂ and CaCl₂ at a concentration corresponding to the formation of the 2:1 complex. The absorbance of “native” DNA did not decrease. T_m and the reciprocal of the hypochromic rise ($\text{1}\text{h}$) increased linearly with log (salt concn). Values of T_m were the same at 230, 260, and 280 nm, but h was greater at 230 and 280 than at 260 nm, which may be due to the existence of alternating blocks of (A + T) and (G ? C) pairs. The entropy of transition was in the order Ca > Mg ? Na.     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.     

Formation constants for interaction of citrate with calcium and magnesium ions

Formation constants for the interaction of citrate ion with calcium and magnesium ions in solution at 37°C and a constant ionic strength of 0.15 were determined by potentiometric titration. Values for the formation of CaL^? and CaHL⁰ complexes were 1.88 × 10³ and 67, respectively. Corresponding constants for MgL^? and MgHL⁰ were 2.19 × 10³ and 42, respectively. The existence of other complexes was not confirmed. Protonation constants were also determined under the same conditions.     

The interaction of bovine factor IX, its activation intermediate, factor IX alpha, and its activation products, factor IXa alpha and factor IXa beta, with acidic phospholipid vesicles of various compositions.

The interactions of bovine factor IX, its activation intermediate, Factor IX alpha, and its activation products, Factor IXa alpha and Factor IXa beta, with phospholipid vesicles, of mean radius of approx. 30 nm, containing various amounts of phosphatidylserine (PS) and phosphatidylcholine (PC), have been examined. For Factor IX, Factor IX alpha, Factor IXa alpha and Factor IXa beta, the dissociation constants, at saturating levels of Ca2+, are independent of the PS concentration in the vesicle after levels of 20-30% (w/w) have been reached, and attain minimum values of approx. 1.7, 1.7, 0.7 and 1.0 microM, respectively, with vesicles containing 50% PS. The amount of protein bound per vesicle particle is independent of the PS content, above 20% PS, for Factor IX and Factor IXa beta, with values of approx. 995-1197 and 1128-1566 molecules/vesicle, respectively. With Factor IX alpha, a dependence on the amount of protein bound with the content of PS is seen, which ranges from 338 to 619 molecules/vesicle with membranes containing 30-50% PS. For Factor IXa alpha, no regularity is noted and a range of 583-1083 molecules of protein/vesicle is observed with the systems employed. Examination of the radii of the proteins on the vesicle demonstrates that Factors IX alpha and IXa alpha occupy considerably more of the surface than do Factors IX and IXa beta, suggesting that a reason for the decreased number of binding sites for the former two proteins on the vesicle may be related to their greater surface spatial requirements.     

The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin.

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.     

The active site of antithrombin. Release of the same proteolytically cleaved form of the inhibitor from complexes with factor IXa, factor Xa, and thrombin

Reactions between near equimolar amounts of antithrombin and Factors IXa or Xa resulted in the formation of a free proteolytically modified, two-chain form of the inhibitor, in addition to the inactive antithrombin-protease complexes. The modified inhibitor produced by either enzyme was electrophoretically identical with that formed in the reaction with thrombin. As in the latter reaction, the formation of the modified antithrombin by Factor Xa was increased in the presence of heparin, while only small amounts were produced by Factor IXa both in the absence and presence of the polysaccharide. NH2-terminal sequence analyses of the isolated modified inhibitor formed by Factor Xa showed that a single Arg-Ser bond in the COOH-terminal end of the inhibitor had been cleaved. This cleavage site is identical with that identified in free thrombin-modified antithrombin. The purified antithrombin-Factor IXa and antithrombin-Factor Xa complexes were dissociated by ammonia or hydroxylamine into free enzyme and a modified two-chain form of the inhibitor. Electrophoresis studies and NH2-terminal sequence analyses showed that the modified antithrombin obtained from either complex was identical with that produced in free form by the two enzymes and also with the modified inhibitor that is released from the antithrombin-thrombin complex. The fact that identical results were obtained for the reactions between antithrombin and three enzymes with different specificities strongly suggests that the observed Arg-Ser cleavage site is the active site of antithrombin.     

Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide

Platelet factor 4 (PF4) is a heparin-binding protein which exhibits anti-heparin activities through the inhibition of antithrombin (AT)-dependent reactions with the serine proteases thrombin and factor Xa. PF4 also neutralizes heparan sulfate (HS), a glycosaminoglycan (GAG) present on the surface of endothelial cells, thereby possibly modulating an anticoagulant response. Previous models of PF4 mechanism did not distinguish whether PF4 causes steric hindrance of AT binding to fXa or of AT binding to the surface of the GAG chain. To shed light on the mechanism of PF4, studies of HS/heparin-catalyzed fXa inactivation by AT were undertaken. The results were consistent with PF4 directly interfering with AT binding to fXa rather than AT binding to the GAG chain, since PF4 did not prevent the heparin-dependent increase in AT intrinsic fluorescence. In fact, PF4 mechanism was competitive with respect to AT and non-competitive with respect to fXa, suggesting inhibition of important regulatory/catalytic interactions of fXa with the polysaccharide. Altogether, the results suggested a model by which PF4 bound to proximal (but distinct) sites to AT, resulting in steric interference of fXa binding to both polysaccharide and AT. It is proposed that PF4 inhibited the sequence of events recapitulated in the template mechanism describing heparin-dependent inhibition of fXa.