Anwar Alhaq

Heterozygous factor XI deficiency associated with three novel mutations

To determine the utility of single‐stranded conformation polymorphism (SSCP) analysis for screening mutations in the factor XI (fXI) gene, we investigated three patients with heterozygous factor XI deficiency. DNA sequence analysis confirmed three novel mutations; a CGC → TGC (Arg308Cys) mutation in exon 9, a GCT→GTT (Ala412Val) mutation in exon 11 and an AGC → AGA (Ser576Arg) mutation in exon 15. We postulated on the structural implications of these missense mutations. Our results demonstrated that genotypic analysis is a useful tool for conclusive differentiation between heterozygous factor XI deficiency and normal subjects.

Identification of a novel mutation in a non-Jewish factor XI deficient kindred

The role of factor XI (FXI) in blood coagulation has been clarified in recent years by descriptions of FXI‐ deficient patients who are prone to excessive bleeding after haemostatic challenge. We have studied a large kindred of an Italian FXI‐deficient patient with a previously undescribed mutation. The propositus, a 68‐year‐old woman, presented with a cerebral thromboembolic event but had no history of bleeding (FXI activity 1.6 U/dl). A sensitive ELISA failed to detect FXI antigen in the propositus. Sequence analysis of the entire FXI gene revealed a TGG to TGC transversion in codon 228 of exon 7 (FXI‐W228C). This missense mutation results in a Trp to Cys substitution within the third apple domain of FXI. We conclude that this novel mutation occurred in a structurally conserved region and may therefore have interfered with either chain folding and secretion or stability of FXI and was responsible for the inherited abnormality seen in this kindred. It is unclear why this kindred does not exhibit a bleeding tendency but it may correlate with a FXI‐like antigen and factor IX binding activity expressed on platelets.

Eighteen unrelated patients with factor XI deficiency, four novel mutations and a 100% detection rate by denaturing high‐performance liquid chromatography

Summary.  Factor XI (FXI) deficiency is an autosomal bleeding disorder of variable severity. Inheritance is not completely recessive as heterozygotes may display a distinct, if mild, bleeding tendency. Eighteen unrelated FXI‐deficient patients were screened blind by fluorescent single‐stranded conformation polymorphism (F‐SSCP) analysis and denaturing high‐performance liquid chromatography (dHPLC). Mutations were detected in 14 of the 18 patients (∼78%) by F‐SSCP and in all 18 patients by dHPLC. Dideoxy sequencing confirmed the mutations in all 18 patients: eight of the mutations being novel (four of which were in previously reported patients). This showed dHPLC to be a highly sensitive, reliable technique for mutation screening in heterogeneous disorders.

Real-Time quantitative PCR analysis of factor XI mRNA variants in human platelets

Summary.  Coagulation factor XI (FXI) plays an essential role in blood coagulation. A deficiency of FXI is an unusual hemorrhagic diathesis in that the bleeding tendency can be highly variable, ranging from severe deficiencies with no symptoms to mild and moderate deficiencies requiring multiple blood transfusions for hemorrhages. This variability in bleeding has been attributed to a number of factors including the presence of a novel form of FXI associated with platelets, which ameliorates the bleeding in some cases of FXI deficiency. However, the nature of this platelet FXI molecule is controversial. Hsu et al. (J Biol Chem 1998; 273: 13787–93) suggest that it is a product of normal FXI – but lacking exon V whilst Martincic et al. (Blood 1999; 94: 3397–404) were unable to detect this alternatively spliced variant using RT‐PCR. In order to resolve this controversy, we have employed the highly sensitive technique of real‐time quantitative RT‐PCR using RNA isolated from FXI‐deficient patients. Our results indicate that the platelets of both normal and FXI deficient individuals contain FXI mRNA that is identical to the mRNA found in liver. An exon V deleted splice variant was not detected. Thus the FXI message is not alternatively spliced in platelets and therefore would not be able to produce an unusual FXI protein.

The profibrinolytic effect of plasma thrombomodulin in factor XI deficiency and its implications in hemostasis

Summary.  Bleeding tendency in factor (F)XI deficiency may result from premature clot lysis due to insufficient thrombin activatable fibrinolysis inhibitor (TAFI) activation. Thrombomodulin (TM), upon binding to thrombin, is capable of modulating TAFI activation. In this study, we investigated the effects of plasma TM on fibrinolysis in FXI‐deficient patients. A clot lysis assay showed the defective down‐regulation of fibrinolysis in FXI‐deficient patients as compared with normal controls. To evaluate the effects of plasma TM on fibrinolysis, a monoclonal anti‐TM IgG was preincubated with plasma for 30 min. The presence of anti‐TM IgG significantly prolonged the clot lysis times both in the FXI‐deficient and normal plasma, indicating that plasma TM stimulated fibrinolysis. Furthermore, the presence of anti‐TM IgG not only reduced protein C activation, but also increased thrombin generation and TAFI activation. The profibrinolytic effect of plasma TM was inhibited in the assay by including either a monoclonal anti‐TAFI IgG or a specific TAFI inhibitor – carboxypeptidase inhibitor (CPI). Our results indicate that the impaired thrombin generation in FXI‐deficient patients leads to the defective down‐regulation of fibrinolysis, and that plasma TM stimulates fibrinolysis through APC pathway which inhibits TAFI activation. The profibrinolytic effect of plasma TM may contribute to the bleeding tendency observed in some FXI‐deficient patients.

Characterisation of five factor XI mutations.

A large scale factor XI (FXI) mutation screening program identified a number of novel candidate mutations and previously reported mutations and polymorphisms. Five potential missense mutations were selected for further study; these included two novel missense mutations - Met-18Ile (p.Met1Ile) and Met102Thr (p.Met120Thr), two previously reported missense mutations - Tyr133Ser (Tyr151Ser) and Thr575Met (Thr593Met), and one amino acid substitution previously reported as a polymorphism - Arg378Cys (Arg396Cys). The substitutions were recreated by the site-directed mutagenesis of a FXI cDNA and stably expressed in a BHK-570 cell line. Subsequent analysis of both the conditioned media and cell lysates showed that three of the substitutions, Met-18Ile, Met102Thr andTyr133Ser, prevented secretion of the mutated protein from the transfected cell line, resulting in a cross-reactive material negative (CRM-) phenotype. The remaining two mutants, Thr575Met and Arg378Cys, secreted significant levels of FXI into the conditioned media; however, these mutant FXIs were shown to have negligible factor IX activation activity in an APTTbased assay. These results confirmed all five of the missense mutations as being causative of factor XI deficiency, despite one having been previously reported as a polymorphism (Arg378Cys) and one (Tyr133Ser) as a mild mutation - FXI:C 38 U/dl in a homozygous patient.