Prothrombin protects factor Xa in the prothrombinase complex from inhibition by the heparin-antithrombin complex

Abstract

Heparin is a commonly used anticoagulant drug. It functions primarily by accelerating the antithrombin inhibition of coagulation proteinases, among which factor Xa and thrombin are believed to be the most important targets. There are conflicting results as to whether anticoagulant heparins can catalyze the antithrombin inhibition of factor Xa in the prothrombinase complex (factor Va, negatively charged membrane surfaces, and calcium ion), which is the physiologically relevant form of the proteinase responsible for the activation of prothrombin to thrombin during the blood coagulation process. In this study, a novel assay system was developed to compare the catalytic effect of different molecularweight heparins in the antithrombin inhibition of factor Xa, either in free form or assembled into the prothrombinase complex during the process of prothrombin activation. This assay takes advantage of the unique property of a recombinant mutant antithrombin, which, similar to the wild-type antithrombin, rapidly inhibits factor Xa, but not thrombin, in the presence of heparin. A direct prothrombinase inhibition assay, monitoring thrombin generation under near physiological concentrations of prothrombin and antithrombin in the presence of therapeutic doses of low-and high-molecular-weight heparins, indicates that factor Xa in the prothrombinase complex is protected from inhibition by antithrombin more than 1000 times, independent of the molecular size of heparin. IntroductionThe anticoagulant function of heparin is primarily derived from its ability to accelerate the inhibition of the blood coagulation proteinases by antithrombin. [1][2][3] The mechanism and the extent of this acceleration have been thoroughly investigated for antithrombin inhibition of factor Xa and thrombin. [4][5][6][7][8][9][10][11][12][13][14][15] It is not, however, known whether heparin is effective in catalyzing the inhibition of factor Xa when the proteinase binds to factor Va on negatively charged membrane surfaces in the presence of calcium (the prothrombinase complex) to activate prothrombin to thrombin. [16][17][18][19][20][21][22][23][24] Results of several previous studies suggest that the assembly of factor Xa into the prothrombinase complex is accompanied by the protection of factor Xa from inhibition by the antithrombin-heparin complex. [18][19][20][21] Conflicting reports exist about the extent to which factor Xa in the prothrombinase complex is protected from inhibition by antithrombin in the presence of different molecular weight heparins. These reports range from support for nearly complete protection 19,21 to less than 5-to 10-fold protection of factor Xa in prothrombinase from inhibition by antithrombin in the presence of high-or low-molecular-weight high-affinity heparins. [22][23][24] As expected, these results have generated conflicting hypotheses within the scientific community as to whether the therapeutic heparins are effective in catalyzing prothrombinase inhibition by antithrombin, and if so, whether the effect is dependent...

Partial Activation of Antithrombin without Heparin through Deletion of a Unique Sequence on the Reactive Site Loop of the Serpin

“…Expression and Purification of the Antithrombin Derivatives-Wild type and mutant antithrombin derivatives were expressed in H293 cells and purified to homogeneity by a combination of HPC4 immunoaffinity and HiTrap-Heparin column chromatography as described previously (18,19). Except for des-RVT, which eluted at ϳ0.3 M NaCl, recombinant wild type (rAT) and all other mutants of AT were eluted at ϳ0.7-0.8 M NaCl from the HiTrap-Heparin column (data not shown).…”

“…, were individually (des-R, des-V, and des-T), or in combination of two (des-VT) or three (des-RVT) deleted by standard PCR mutagenesis methods, expressed in H293 cells using RSV-PL4 expression/purification vector system as described previously (18,19). Accuracy of the mutations was confirmed by sequencing prior to expression.…”

“…Accuracy of the mutations was confirmed by sequencing prior to expression. Wild type and mutant serpins were purified from cell culture supernatants by immunoaffinity chromatography using the HPC4 antibody linked to Affi-Gel 10 (Bio-Rad) followed by a HiTrap-Heparin (Amersham Biosciences, Inc.) ion exchange chromatography with a gradient elution from 0.1 to 2.0 M NaCl in 20 mM Tris-HCl, pH 7.4 as described previously (19). Concentrations of the AT derivatives were determined from the absorbance at 280 nm using a molar absorption coefficient of 37,700 M Ϫ1 cm Ϫ1 and by stoichiometric titration of the serpins with calibrated heparin as monitored from changes in intrinsic protein fluorescence (20).…”

The transcriptome of the salivary glands of the female western black-legged tick Ixodes pacificus (Acari: Ixodidae)

“…As a result, the effective concentration of the inhibitor is increased at sites that are predominantly pro-coagulant. Also, we speculate that FXa-which is usually protected from physiologic inhibitors (e.g., TFPI, heparin/ATIII) when the prothrombinase is fully assembled (Mast and Broze, 1996;Rezaie, 2001)-would be more susceptible to these bifunctional molecules. Demonstration that proteins rich in positively charged residues effectively block the coagulation cascade comes from studies performed with a recombinant Rhodnius prolixus salivary lipocalin (nitrophorin-7, NP-7).…”

The status of new anticoagulants

“…Direct factor Xa inhibitors bind directly to the active site of factor Xa and block its interaction with its substrates. Unlike the heparin/antithrombin complex, direct factor Xa inhibitors not only inhibit free factor Xa, but also inactivate factor Xa bound to platelets within the prothrombinase complex (Eisenberg et al, 1993;Brufatto & Nesheim, 2001;Rezaie, 2001). This property may endow these agents with an advantage over indirect factor Xa inhibitors.…”