Defective binding of factor XI–N248 to activated human platelets

Abstract

Variants of factor XI containing Gln226 to Arg (Q226 to R) and Ser248 to Asn (S248 to N) substitutions were first identified in an African American family with a history of excessive bleeding. The substitutions have recently been identified in unrelated individuals, suggesting they are relatively common. Both amino acids are located in the third apple domain of factor XI, an area implicated in binding interactions with factor IX and activated platelets. Recombinant factor XI-R226 and factor XI-N248 were compared with wildtype factor XI in assays for factor IX activation or platelet binding. Factor XI-R226 activates factor IX with a MichaelisMenten constant (K m ) about 5-fold greater than wild-type protein. The catalytic efficiency of factor IX activation is similar to wild-type protein, however, due to an increase in the turnover number (k cat ) for the reaction. Iodinated factor XI-N248 binds to activated platelets with a dissociation constant (K d ) more than 5-fold higher than wild-type protein (55 nM and 10 nM, respectively). Activation of factor XI-N248 by thrombin in the presence of activated platelets is slower and does not progress to the same extent as activation of the wild-type protein under similar conditions. Factor XI-N248 activates factor IX normally in a purified protein system and has relatively normal activity in activated partial thromboplastin time (aPTT) assays. Factor XI-N248 is the first factor XI variant described with a clear functional difference compared with wild-type protein. Importantly, the defect in platelet binding IntroductionCoagulation factor XI is the zymogen of a plasma protease that contributes to hemostasis by activating factor IX. 1 In congenital factor XI deficiency in humans, low plasma activity is almost always associated with a comparable loss of antigen (cross-reactive material-negative [CRM Ϫ ] deficiency). 2,3 Few factor XI-deficient patients with low activity and disproportionately high circulating levels of factor XI antigen (CRM ϩ deficiency) have been reported, 4-6 and the genetic abnormalities in these individuals have not been determined. This distinguishes factor XI deficiency from hemophilias A and B (deficiency of factor VIII or IX, respectively) and von Willebrand disease, where many circulating dysfunctional variants have been described. 7 Another peculiarity of factor XI deficiency that distinguishes it from hemophilia is the poor correlation between plasma factor XI activity and bleeding symptoms. 6,8,9 It is likely that several factors are responsible for this phenomenon. One possibility is that commonly used in vitro tests for factor XI activity do not accurately measure properties of the protein most important for in vivo hemostasis.Previously, we reported 2 amino acid substitutions in factor XI (Q226R and S248N), identified in an African American with a history of excessive bleeding and a modest deficiency in plasma factor XI activity. 10 This patient is a compound heterozygote, with the amino acid substitutions residing on separate alleles. The ...

Spectrum of factor XI (F11) mutations in the UK population – 116 index cases and 140 mutations

“…We were able to study eleven of the repeat mutations, with haplotype analysis showing that at least three, G460R (p.G478R), C398Y (p.C416Y) and the 2 bp deletion at codon 303 (p.321), had arisen independently and were not the result of a founder mutation. Two patients were initially assigned to have a Q226R (p.Q244R) mutation, however, on revision it was ascertained that this had been previously identified as a polymorphism (Sun, et al, 2001). Further investigation identified an alternative causative mutation in one patient, whilst it was determined that the other patient did not have inherited FXI deficiency.…”

Identification of a Binding Site for Glycoprotein Ibα in the Apple 3 Domain of Factor XI

“…To confirm and extend our understanding of this interaction, we developed a solid phase binding assay for examining FXI binding to glycocalicin. cific, reversible, high affinity (K d app ϭ ϳ10 nM) FXI-binding sites (n ϭ 1,500 sites/platelet) that require the presence of HK and ZnCl 2 , whereas in the presence of ZnCl 2 without added HK, FXI binds to about half this number of sites with similar affinity (9,19,21 (19,21,24) and because the FXI-binding site on platelets is in the GPIb␣ subunit of the GPIb-IX-V complex, we initially determined whether glycocalicin (the soluble form of GPIb␣) interacts with the Apple domains by examining the capacity of each of the recombinant, expressed Apple domains of FXI to compete with FXI for binding to glycocalicin in our solid phase assay. Fig.…”

“…Specific ligand-binding functions have been attributed to each of the four FXI Apple domains (1,(15)(16)(17)(18)(19)(20) including the A3 domain, which has been shown to bind the platelet (19,21), heparin (22), and FIX (23). We have previously shown that specific amino acids in the A3 domain (Ser ) mediate the binding of FXI to platelets (21,24), whereas amino acids juxtaposed to the plateletbinding site (Lys 252 and Lys…”

Factor 11 single-nucleotide variants in women with heavy menstrual bleeding

“…However, rs5969 was associated with FXI activity levels comparable to wildtype when tested in an APTT-based assay (Martincic et al 1998). In line with this, the catalytic efficiency (kcat) for FIX activation was shown to be comparable to that of wild-type FXI, thus normalising the APTT result (Sun et al 2001). Various clinical studies as well as studies that examined the structural features of rs5969 have confirmed the minimal effect (Mitchell et al 1999;Mitchell et al 2003;O'Connell et al 2005;Mitchell et al 2006;Saunders et al 2009).…”

“…Besides being compound heterozygous for type II (rs121965063, p.Glu135Ter) and type III (rs121965064, p.Phe301Leu) variants, the abnormal control did not contain the other two non-synonymous variants. The binding affinity between FXI and FIX (Km) differed considerably from wild-type FXI (Sun et al 2001). However, rs5969 was associated with FXI activity levels comparable to wildtype when tested in an APTT-based assay (Martincic et al 1998).…”