Biomedical subjects

R Porche-Sorbet

Publications and source records attributed to R Porche-Sorbet.

5 recordsLinked to original sources

PubMed2000

Directed glycosylation of human coagulation factor X at residue 333. Insight into factor Va-dependent prothrombin catalysis.

Based on homology, amino acids 326-336 (143-154 in chymotrypsin numbering) of factor X (fX) comprise a flexible surface loop, which is susceptible to self-proteolysis and influences substrate catalysis. To investigate the role of this autolysis loop in fX function, a recombinant variant with a new site for asparagine-linked glycosylation has been produced by changing glutamine 333 to asparagine. Q333N fX is activated normally by factor VIIa and tissue factor, factors IXa and VIIIa, and Russell's viper venom. Proteolysis of the loop is prevented by the mutation. Reactivity of the free enzyme toward substrates and inhibitors is attenuated 4-20-fold; relative to wild type fXa, Spectrozyme Xa(TM) hydrolysis is 25%, inhibition by antithrombin III and the tissue factor pathway inhibitor is approximately 20%, and prothrombin activation in the absence of the cofactor Va is only 5%. Surprisingly, activities of the variant and wild type enzymes are equivalent when part of the prothrombinase complex. N-Glycanase cleaves the new oligosaccharide from Q333N fXa leaving aspartic acid. Q333D fXa is approximately 1.6-fold more reactive with Spectrozyme Xa(TM), antithrombin III and tissue factor pathway inhibitor, and prothrombin than its glycosylated counterpart, Q333N fXa, but still quite abnormal relative to wild type fXa. Like Q333N fXa, Q333D fXa is fully functional as part of the prothrombinase complex. We conclude that Gln-333 is geographically close to a site of proteolytic degradation but not to activator, cofactor, or membrane binding sites. Mutation of Gln-333 impairs catalytic function, but given normal prothrombin activation by the complexed enzyme, the importance of Gln-333 for catalysis is not manifest in the prothrombinase assembly, suggesting a conformational change in complexed fXa.

Amino Acid Substitution↗

PubMed2000

Definition of a factor Va binding site in factor Xa.

We reported previously that residue 347 in activated fX (fXa) contributes to binding of the cofactor, factor Va (fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39, 2861-2867). Four additional residues that participate in fVa binding have now been identified by mutagenesis. All five resulting fX species, fX(R306A), fX(E310N), fX(R347N), fX(K351A), and fX(K414A), are activated and inhibited normally. However, the rate of inhibition by antithrombin III in the presence of submaximal concentrations of heparin is reduced for all the enzymes. In the absence of fVa, all of the enzymes bind and activate prothrombin similarly except fXa(E310N), which has a reduced apparent affinity ( approximately 3-fold) for prothrombin compared with wild type fXa (fXa(WT)). In the absence of phospholipid, fVa enhances the catalytic activity of fXa(WT) significantly, but the response of the variant enzymes was greatly diminished. On addition of 100 nm PC:PS (3:1) vesicles, fVa enhanced fXa(WT), fXa(R306A), and fXa(E310N) similarly, whereas fXa(R347N), fXa(K351A), and fXa(K414A) demonstrated near-normal catalytic activity but reduced apparent affinity for fVa under these conditions. All enzymes function similarly to fXa(WT) on activated platelets, which provide saturating fVa on an ideal surface. Loss of binding affinity for fVa as a result of the substitutions in residues Arg-347, Lys-351, and Lys-414 was verified by a competition binding assay. Thus, Arg-347, Lys-351, and Lys-414 are likely part of a core fVa binding site, whereas Arg-306 and Glu-310 serve a less critical role.

Antithrombin III↗

PubMed2000

Substitution of asparagine for arginine 347 of recombinant factor Xa markedly reduces factor Va binding.

Herein we describe a recombinant factor X (fX) with a single substitution at position 347 (fXR347N). Activated fXR347N had a reduced affinity for factor Va (fVa), although the catalytic impact of fVa binding remained intact. The mutation was selective as demonstrated by normal activation and inhibition, except in the presence of subsaturating heparin where the rate of inhibition by antithrombin III (ATIII) was 15% of normal. The reactivity of fXaR347N toward prothrombin was equivalent to wild-type fXa (fXaWT) in the absence of fVa and phospholipid. Addition (without phospholipid) of fVa dramatically increased the catalytic efficiency of fXaWT toward prothrombin but had a negligible effect on fXaR347N. On addition of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1) vesicles, fXaR347Ndisplayed an increased catalytic activity in response to fVa, but the apparent affinity for fVa on the phospholipid surface was 5-20-fold lower than that of fXaWT. On an activated platelet surface, however, fXaWT and fXaR347N activated prothrombin similarly. In a competitive binding assay that measures the displacement of radiolabeled fXa from fVa on a phospholipid surface, fXaR347N was approximately 10-fold less effective than fXaWT. Substitution of fXa at position 347 selectively attenuates the interaction between fXa and fVa without affecting its catalytic activity.

Amino Acid Substitution↗

PubMed1997

Expression, purification, and characterization of recombinant human factor X.

A system is described for producing recombinant factor X with properties very similar to human plasma factor X. Optimization of the expression system for factor X resulted in the finding that human kidney cells (293 cells) are superior to the widely utilized baby hamster kidney cells (BHK cells) for the expression of functional factor X. It was also determined that production of factor X by 293 cells requires the substitution of the -2 residue (Thr-->Arg) which affords the removal of the factor X propeptide. Purification of recombinant and plasma factor X is accomplished using a calcium-dependent monoclonal antibody directed against the gla domain. The proteins are comparable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rate and extent of activation by the factor X coagulant protein from Russell's viper venom and by factors IXa and VIIIa are similar; activation of the recombinant protein by VIIa and tissue factor is mildly faster. The activated enzymes have the same activity toward a chromogenic substrate and the biologic substrate, prothrombin. Both enzymes have the same apparent affinity for the activated platelet surface as judged by their ability to activate prothrombin. Finally, inhibition by antithrombin, with or without heparin, and inhibition by the tissue factor pathway inhibitor are equivalent. Recombinant factor X produced by this method is therefore well suited for probing structure-function relationships by mutational analysis.

Antithrombin III↗

PubMed1996

Factor XSt. Louis II. Identification of a glycine substitution at residue 7 and characterization of the recombinant protein.

A molecular defect in factor X (fX) results from a point mutation that causes glycine substitution for gamma-carboxylated glutamic acid at position 7. The variant (fXSt. Louis II) and wild type (fXWT) proteins were produced in a mammalian expression system and characterized. fXSt. Louis II has <1% and approximately 3% of normal clotting activity in modified prothrombin time and partial thromboplastin time assays, respectively. The rate of activation of fXSt. Louis II by factor VIIa and tissue factor is undetectable under conditions that result in complete activation of fXWT; activation by factors VIIIa and IXa is approximately 30% of normal activation. The X-activating protein from Russell's viper venom activates fXSt. Louis II completely but at a reduced rate. Thrombin generation on phoshopolipid vesicles or activated platelets is approximately 30% or approximately 5%, respectively. Membrane-dependent autolysis is markedly reduced for fXSt. Louis II. In reactions that are not surface-dependent, fXSt. Louis II is nearly identical to that of fXWT. The rate of inhibition by antithrombin is indistiguishable, as is the rate of thrombin formation in the absence of phospholipid, with or without factor Va.

Antithrombin III↗