Platelet factor XI: intracellular localization and mRNA splicing following platelet activation.

BACKGROUND: The structure and function of platelet factor XI (FXI) protein and the presence of F11 mRNA in platelets are controversial. Although platelets are anucleated cells they contain spliceosome components and pre-mRNAs. Three platelet proteins have been demonstrated to be spliced upon platelet activation. OBJECTIVE: To determine whether FXI is also spliced upon activation and to discern the localization of FXI in platelets. METHODS: Localization of FXI in platelets was assessed by confocal immunofluorescence staining. ELISA, chromogenic assay and western blot analyses were used to measure antigen levels, activity levels and size of FXI in platelets, respectively. Splicing patterns of F11 mRNA were assessed in three states of platelet activation: activated platelets, resting platelets and αIIbß3-integrin activated platelets. RESULTS: Platelet FXI was exhibited in platelet granules. Activated platelets exhibited higher levels of mature F11 mRNA and protein and lower levels of F11 pre-mRNA compared to resting or αIIbß3-integrin activated platelets. CONCLUSIONS: We confirmed the presence of FXI in platelets and showed that it is localized in granules but is not restricted to the same α-granule subtype as von-Willebrand factor and p-selectin. Our study also shows that F11 is present in platelets as pre-mRNA and is spliced upon platelet activation.

An allosteric disulfide bond is involved in enhanced activation of factor XI by protein disulfide isomerase.

Essentials Reduction of three disulfide bonds in factor (F) XI enhances chromogenic substrate cleavage. We measured FXI activity upon reduction and identified a bond involved in the enhanced activity. Reduction of FXI augments FIX cleavage, probably by faster conversion of FXI to FXIa. The Cys362-Cys482 disulfide bond is responsible for FXI enhanced activation upon its reduction. SUMMARY: Background Reduction of factor (F) XI by protein disulfide isomerase (PDI) has been shown to enhance the ability of FXI to cleave its chromogenic substrate. Three disulfide bonds in FXI (Cys118-Cys147, Cys362-Cys482, and Cys321-Cys321) are involved in this augmented activation. Objectives To characterize the mechanisms by which PDI enhances FXI activity. Methods FXI activity was measured following PDI reduction. Thiols that were exposed in FXI after PDI reduction were labeled with 3-(N-maleimidopropionyl)-biocytin (MPB) and detected with avidin. The rate of conversion of FXI to activated FXI (FXIa) following thrombin activation was assessed with western blotting. FXI molecules harboring mutations that disrupt the three disulfide bonds (C147S, C321S, and C482S) were expressed in cells. The antigenicity of secreted FXI was measured with ELISA, and its activity was assessed by the use of a chromogenic substrate. The effect of disulfide bond reduction was analyzed by the use of molecular dynamics. Results Reduction of FXI by PDI enhanced cleavage of both its chromogenic substrate, S2366, and its physiologic substrate, FIX, and resulted in opening of the Cys362-Cys482 bond. The rate of conversion of FXI to FXIa was increased following its reduction by PDI. C482S-FXI showed enhanced activity as compared with both wild-type FXI and C321S-FXI. MD showed that disruption of the Cys362-Cys482 bond leads to a broader thrombin-binding site in FXI. Conclusions Reduction of FXI by PDI enhances its ability to cleave FIX, probably by causing faster conversion of FXI to FXIa. The Cys362-Cys482 disulfide bond is involved in enhancing FXI activation following its reduction, possibly by increasing thrombin accessibility to FXI.

Abnormal plasma clot structure and stability distinguish bleeding risk in patients with severe factor XI deficiency.

BACKGROUND: Factor XI (FXI) deficiency is a rare autosomal recessive disorder. Many patients with even very low FXI levels (< 20 IU dL(-1) ) are asymptomatic or exhibit only mild bleeding, whereas others experience severe bleeding, usually following trauma. Neither FXI antigen nor activity predicts the risk of bleeding in FXI-deficient patients. OBJECTIVES: (i) Characterize the formation, structure and stability of plasma clots from patients with severe FXI deficiency and (ii) determine whether these assays can distinguish asymptomatic patients ('non-bleeders') from those with a history of bleeding ('bleeders'). METHODS: Platelet-poor plasmas were prepared from 16 severe FXI-deficient patients who were divided into bleeders or non-bleeders, based on bleeding associated with at least two tooth extractions without prophylaxis. Clot formation was triggered by recalcification and addition of tissue factor and phospholipids in the absence or presence of tissue plasminogen activator and/or thrombomodulin. Clot formation and fibrinolysis were measured by turbidity and fibrin network structure by laser scanning confocal microscopy. RESULTS: Non-bleeders and bleeders had similarly low FXI levels, normal prothrombin times, normal levels of fibrinogen, factor VIII, von Willebrand factor and factor XIII, and normal platelet number and function. Compared with non-bleeders, bleeders exhibited lower fibrin network density and lower clot stability in the presence of tissue plasminogen activator. In the presence of thrombomodulin, seven of eight bleeders failed to form a clot, whereas only three of eight non-bleeders did not clot. CONCLUSIONS: Plasma clot structure and stability assays distinguished non-bleeders from bleeders. These assays may reveal hemostatic mechanisms in FXI-deficient patients and have clinical utility for assessing the risk of bleeding.

Type I mutation in the F11 gene is a third ancestral mutation which causes factor XI deficiency in Ashkenazi Jews.

BACKGROUND: Factor XI (FXI) deficiency is one of the most frequent inherited disorders in Ashkenazi Jews (AJ). Two predominant founder mutations termed type II (p.Glu117Stop) and type III (p.Phe283Leu) account for most cases. OBJECTIVES: To present clinical aspects of a third FXI mutation, type I (c.1716 + 1G>A), which is also prevalent in AJ and to discern a possible founder effect. METHODS: Bleeding manifestations, FXI levels and origin of members of 13 unrelated families harboring the type I mutation were determined. In addition, eight intragenic and five extragenic polymorphisms were analyzed in patients with a type I mutation, in 16 unrelated type II homozygotes, in 23 unrelated type III homozygotes and in Ashkenazi Jewish controls. Analysis of these polymorphisms enabled haplotype analysis and estimation of the age of the type I mutation. RESULTS: Four of 16 type I heterozygotes (25%) and 6 of 12 (50%) compound heterozygotes for type I mutation (I/II and I/III), or a type I homozygote had bleeding manifestations. Haplotype analysis disclosed that like type II and type III mutations, the type I is also an ancestral mutation. An age estimate revealed that the type I mutation occurred approximately 600 years ago. The geographic distribution of affected families suggested that there was a distinct origin of the type I mutation in Eastern Europe. CONCLUSIONS: The rather rare type I mutation in the FXI gene is a third founder mutation in AJ.

Inherited platelet disorders.

Inherited diseases of the megakaryocyte lineage give rise to bleeding when platelets fail to fulfill their hemostatic function upon vessel injury. Platelet defects extend from the absence or malfunctioning of adhesion (GPIb-IX-V, Bernard-Soulier syndrome) or aggregation receptors (integrin αIIbß3, Glanzmann thrombasthenia) to defects of primary receptors for soluble agonists, secretion from storage organelles, activation pathways and the generation of procoagulant activity. In disorders such as the Chediak-Higashi, Hermansky-Pudlak, Wiskott-Aldrich and Scott syndromes the molecular lesion extends to other cells. In familial thrombocytopenia (FT), platelets are produced in insufficient numbers to assure hemostasis. Some FT affect platelet morphology and give rise to the 'giant platelet' syndromes (e.g. MYH9-related diseases) with changes in megakaryocyte maturation within the bone marrow and premature release of platelets. Diseases of platelet production may also affect other cells and in some cases interfere with development and/or functioning of major organs. Diagnosis of platelet disorders requires platelet function testing, studies often aided by the quantitative analysis of receptors by flow cytometry and fluorescence and electron microscopy. New generation DNA-based procedures including whole exome sequencing offer an exciting new perspective. Transfusion of platelets remains the most common treatment of severe bleeding, management with desmopressin is often used for mild disorders. Substitute therapies are available including rFVIIa and the potential use of thrombopoietin analogues for FT. Stem cell or bone marrow transplantation has been successful for several diseases while gene therapy shows promise in the Wiskott-Aldrich syndrome.

Treatment of inherited platelet disorders.

For patients affected by severe inherited platelet dysfunctions, e.g. Glanzmann thrombasthenia (GT) or Bernard-Soulier syndrome (BSS), platelet transfusion is frequently needed for controlling spontaneous bleeding, and is always needed when trauma occurs or surgery is performed. For the mild-to-moderate bleeding entities, e.g. storage pool disease, thrombaxane A2 receptor defect, platelet transfusion is usually unnecessary. Transfusion of platelets should be used selectively and sparingly because of the substantial risk of alloimmunization against HLA antigens and/or platelet glycoproteins (GP) αIIb, ß(3), or αIIbß(3) in GT, and GPI-IX-V in BSS, which may lead to refractoriness to therapy. To reduce the risk, HLA-matched single donors of platelets should be used. If such donors are unavailable, leucocyte-depleted blood components should be used. Therapy other than platelet transfusion includes: (i) Prevention (vaccination against hepatitis B, avoidance of non-steroidal anti-inflammatory drugs, preservation of dental hygiene, correction of iron deficiency and prenatal diagnosis). (ii) Topical measures (compression with gauze soaked with tranexamic acid, fibrin sealants, splints for dental extractions and packing for nose bleeds). (iii) Antifibrinolytic agents that are useful for minor surgery and as adjuncts for other treatment modalities. (iv) Desmopressin that increases plasma levels of von Willebrand factor and factor VIII giving rise to increased platelet adhesiveness and aggregation associated with shortened bleeding time. (v) Recombinant factor VIIa (rFVIIa). GT patients have been treated for bleeding episodes by rFVIIa with partial success. The mechanism by which rFVIIa arrests bleeding is probably related to increased thrombin generation by a tissue factor-independent process, enhanced platelet adhesion and restoration of platelet aggregation. (vi) Female hormones. Excessive bleeding during menarche in patients with GT or BSS can be controlled by high doses of oestrogen followed by high doses of oral oestrogen-progestin. Menorrhagia later in life can be managed by continuous oral contraceptives. Depo-medroxyprogesterone acetate administered every 3 months is an alternative when combined oral contraceptives are contraindicated.

A mutation in the ß3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIb ß3 to an active state.

BACKGROUND: The cytoplasmic tails of α(IIb) and ß(3) regulate essential α(IIb) ß(3) functions. We previously described a variant Glanzmann thrombasthenia mutation in the ß(3) cytoplasmic tail, IVS14: -3C>G, which causes a frameshift with an extension of ß(3) by 40 residues. OBJECTIVES: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of α(IIb) ß(3) from a resting state to an active state. METHODS: We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild-type (WT) α(IIb) as follows: ß(3) -742stop, a truncated mutant to evaluate the effect of deleted residues; ß(3) -749stop, a truncated mutant that preserves the NPLY conserved sequence; and ß(3) -749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. RESULTS AND CONCLUSIONS: Surface expression of α(IIb) ß(3) of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti-ligand-induced-binding-site 6 [antiLIBS6] or PT25-2). Activation of the α(IIb) ß(3) mutants was only achieved when both PT25-2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the ß(3) tail is important for maintaining α(IIb) ß(3) in a resting conformation.

Point mutations regarded as missense mutations cause splicing defects in the factor XI gene.

BACKGROUND: Point mutations within exons are frequently defined as missense mutations. In the factor (F)XI gene, three point mutations, c.616C>T in exon 7, c.1060G>A in exon 10 and c.1693G>A in exon 14 were reported as missense mutations P188S, G336R and E547K, respectively, according to their exonic positions. Surprisingly, expression of the three mutations in cells yielded substantially higher FXI antigen levels than was expected from the plasma of patients bearing these mutations. OBJECTIVES: To test the possibility that the three mutations, albeit their positions within exons, cause splicing defects. METHODS AND RESULTS: Platelet mRNA analysis of a heterozygous patient revealed that the c.1693A mutation caused aberrant splicing. Platelet mRNA of a second compound heterozygote for c.616T and c.1060A mutations was undetectable suggesting its degradation. Cells transfected with a c.616T minigene favored production of an aberrantly spliced mRNA that skips exon 7. Cells transfected with a mutated minigene spanning exons 8-10 exhibited a significant decrease in the amount of normally spliced mRNA. In silico analysis revealed that the three mutations are located within sequences of exonic splicing enhancers (ESEs) that bind special proteins and are potentially important for correct splicing. Compensatory mutations created near the natural mutations corrected the putative function of ESEs thereby restoring normal splicing of exons 7 and 10. CONCLUSIONS: The present findings define a new mechanism of mutations in F11 and underscore the need to perform expression studies and mRNA analysis of point mutations before stating that they are missense mutations.

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N-terminus of blade 1 of the ß-propeller (Asn2Asp) disrupts a calcium binding site in blade 6.

BACKGROUND: Studies of Glanzmann thrombasthenia (GT)-causing mutations has generated invaluable information on the formation and function of integrin αIIbß(3). OBJECTIVE: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. METHODS AND RESULTS: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the ß-propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbß(3) was found in patients' platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT ß(3). Although the αIIbß(3) was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbß(3) crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbß(3). Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbß(3) surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbß(3), αVß(3) harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVß(3) to calcium chelation compared with αIIbß(3). CONCLUSION: The new GT causing mutation highlights the importance of calcium binding domains in the ß-propeller for intracellular trafficking of αIIbß(3). The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.

Factor XI deficiency in humans.

Factor XI (FXI) deficiency is an autosomal recessive injury-related bleeding tendency, which is common in Jews particularly of Ashkenazi origin. To date, 152 mutations in the FXI gene have been reported with four exhibiting founder effects in specific populations, Glu117stop in Ashkenazi and Iraqi Jews and Arabs, Phe283Leu in Ashkenazi Jews, Cys38Arg in Basques, and Cys128stop in the United Kingdom. Severe FXI deficiency does not confer protection against acute myocardial infarction, but is associated with a reduced incidence of ischemic stroke. Inhibitors to FXI develop in one-third of patients with very severe FXI deficiency following exposure to blood products. Therapy for prevention of bleeding during surgery in patients with severe FXI deficiency consists of plasma, factor XI concentrates, fibrin glue and antifibrinolytic agents. In patients with an inhibitor to FXI, recombinant factor VIIa is useful.

Three residues at the interface of factor XI (FXI) monomers augment covalent dimerization of FXI.

BACKGROUND: Human plasma factor XI is a homodimer, with each monomer comprising a catalytic domain and four homologous 'apple' domains. The monomers bind to each other through non-covalent bonds and through a disulfide bond between Cys321 residues in apple 4 domains. OBJECTIVE: To identify residues essential for dimerization in the FXI monomer interface. METHODS: Specificity-determining residues in apple 4 domains were sought by sequence alignment of FXI and prekallikrein apple domains in different species. Specific residues identified in apple 4 domains were mutagenized and expressed in baby hamster kidney (BHK) cells for evaluation of their effect on FXI dimerization, analyzed by non-reduced sodium dodecylsulfate polyacrylamide gel electrophoresis and size-exclusion chromatography. RESULTS: Among the 19 residues of the FXI monomer interface, Leu284, Ile290 and Tyr329 were defined as specificity-determining residues. Substitutions of these residues or pairs of residues did not affect FXI synthesis and secretion from transfected BHK cells, but did impair dimerization, despite the presence of cysteine at position 321. The double mutant 284A/290A yielded predominantly a monomer, whereas all other single or double mutants yielded monomers as well as disulfide-bonded dimers. CONCLUSIONS: The data suggest that Leu284, Ile290 and Tyr329 in the interface of FXI monomers are essential for forming non-covalently bonded dimers that facilitate formation of a disulfide-bonded stable FXI dimer.

False-positive tests for heparin-induced thrombocytopenia in patients with antiphospholipid syndrome and systemic lupus erythematosus.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy that can be associated with arterial or venous thrombosis and is caused by antibodies against platelet factor 4 (PF4)-heparin complex. Patients with antiphospholipid syndrome (APS) have been reported with positive tests for PF4-heparin complex antibodies by antigen assay. Whether such patients can be treated with heparin is a dilemma. OBJECTIVES: To determine the incidence and nature of the HIT immune reaction in patients with APS and/or systemic lupus erythematosus (SLE). METHODS: Antibodies against PF4-heparin complex were assayed by particle gel immunoassay (PaGIA), or enzyme immunoassay (EIA) with or without an excess of heparin. EIA for PF4 alone was also performed. Functional assays for HIT, that is, heparin-induced platelet activation (HIPA) and heparin-induced platelet aggregation, were also performed. RESULTS: In 32 of 42 patients (76.2%) with APS, APS and SLE, SLE, or SLE with antiphospholipid antibodies, EIA IgG or PaGIA for PF4-heparin complex antibodies were positive. Of these 32 samples, 26 (81.3%) tested positive for anti-PF4 antibodies. All 24 samples that were positive for PF4-heparin complex by EIA IgG were also positive for EIA IgG in the presence of heparin excess, and all were negative by the HIPA and heparin-induced platelet aggregation tests. CONCLUSION: A large proportion of patients with APS and/or SLE give false-positive HIT antigen test results that are presumably related to autoantibodies against PF4, which can be distinguished from true HIT antibodies by EIA for PF4-heparin complexes tested with heparin excess, and by functional assays.

Effects of factor VIII inhibitor bypassing activity (FEIBA), recombinant factor VIIa or both on thrombin generation in normal and haemophilia A plasma.

Factor VIII inhibitor bypass activity (FEIBA) and recombinant factor VIIa (rFVIIa) are the common bypassing agents for treating haemophilia A or haemophilia B patients who developed an inhibitor to factor VIII or IX, respectively. As these preparations differ in their composition and mode of action, combined therapy, either sequential or simultaneous has recently been used for achieving haemostasis during bleeding episodes in patients who became refractory to FEIBA or rFVIIa when each was given alone. In this in vitro study, we show by a sensitive assay of thrombin generation that phospholipids present in FEIBA and other procoagulants contribute to FEIBA's activity and that exogenous phospholipids are essential for the activity of rFVIIa. We also demonstrate that the combination of FEIBA and rFVIIa has a marked synergistic effect on thrombin generation in plasma of a haemophilia A patient with a high titre of an inhibitor. It is conceivable that simultaneous administration of small doses of FEIBA and rFVIIa may be beneficial in treating haemophilia A patients, with an inhibitor to FVIII, who are resistant to conventional therapy.

Trp207Gly in platelet glycoprotein Ibalpha is a novel mutation that disrupts the connection between the leucine-rich repeat domain and the disulfide loop structure and causes Bernard-Soulier syndrome.

BACKGROUND: Bernard-Soulier syndrome (BSS) is a severe inherited bleeding disorder that is caused by a defect in glycoprotein (GP)Ib-IX-V complex, the platelet membrane receptor for von Willebrand factor. PATIENTS: The diagnosis of BSS was made in two members of a Bukharian Jewish family who had life-long thrombocytopenia associated with mucocutaneous bleeding manifestations. METHODS AND RESULTS: Flow cytometry and Western blot analyses showed only trace amounts of GPIb and GPIX on the patients' platelets. Sequence analysis of the GPIbalpha gene revealed a homozygous T > G transversion at nucleotide 709 predicting Trp207Gly substitution in the mature protein. Introduction of the mutation into a mammalian expression construct abolished the surface expression of GPIbalpha in transfected baby hamster kidney cells. The crystal structure of the N-terminus of GPIbalpha (PDB: 1SQ0) indicates that Trp207 is completely buried and located in a disulfide loop structure that interacts with the leucine-rich repeat (LRR) domain. CONCLUSION: A novel mutation, Trp207Gly, causes BSS and predicts disruption of the interaction between a disulfide loop and the LRR domain that is essential for the integrity of GPIbalpha structure.

Diversity of Glanzmann thrombasthenia in southern India: 10 novel mutations identified among 15 unrelated patients.

BACKGROUND: Glanzmann thrombasthenia (GT) is a congenital bleeding disorder caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3. OBJECTIVES: To determine the molecular basis of GT in patients from southern India. PATIENTS: Fifteen unrelated patients whose diagnosis was consistent with GT were evaluated. RESULTS: Platelet surface expression of alphaIIbbeta3 was < 10%, 10%-50%, and > 50% of controls in five, nine, and one patient(s), respectively. Immunoblotting of the platelet lysates showed no alphaIIb in 14 patients, and no beta3 in 10 patients, although severely reduced in four patients. Platelet fibrinogen was undetectable in 13 patients, and severely reduced in one patient. One patient showed normal surface alphaIIbbeta3 expression, and normal alphaIIb, beta3 and fibrinogen levels in the lysate. Ten novel candidate disease-causing mutations were identified in 11 patients. The missense mutations included Gly128Ser, Ser287Leu, Gly357Ser, Arg520Trp, Leu799Arg in alphaIIb, and Cys575Gly in beta3. We have already shown that Gly128Ser, Ser287Leu, and Gly357Ser mutations variably affect alphaIIbbeta3 surface expression. The Cys575Gly mutation may disrupt the disulphide link with Cys586 to cause the GT phenotype. The molecular pathology of the other missense mutations is not clear. Two nonsense mutations, Trp-16Stop and Glu715Stop in alphaIIb, and a 7-bp deletion (330-336TCCCCAG) in beta3 are predicted to result in truncated proteins. An IVS15(-1)G --> A mutation in alphaIIb induced a cryptic splice site as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Thirteen polymorphisms were also identified (five in alphaIIb and eight in beta3), among which five were novel. CONCLUSIONS: While identifying a significant number of novel mutations causing GT, this study confirms the genetic heterogeneity of the disorder in southern India.

Rare bleeding disorders.

Deficiencies of coagulation factors other than factor VIII and factor IX (afibrinogenemia, FII, FV, FV+FVIII, FVII, FX, FXI, FXIII) that cause bleeding disorders (RBDs) are inherited as autosomal recessive traits and are rare, with prevalences in the general population varying between 1 in 500,000 and 1 in 2 million for the homozygous forms. As a consequence of the rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects, and the actual management of bleeding episodes are not as well established as for hemophilia A and B. The study of the genetic basis of these disorders could represent an important tool for prevention through prenatal diagnosis. Treatment of patients with RBDs during bleeding episodes or surgery is a challenge because of the lack of experience and the paucity of data. For some deficiency factor concentrates are still non available and severe complications can occur. These complications can be minimized by assessment of risks of bleeding and thrombosis, use of haemostatic means other than blood components or no therapy at all. The RBDs pose a problem for guideline writers because there are no suitable clinical trials to supply good evidence for how these people are best treated. The lack of adequate information on clinical manifestations, treatment and genetic basis of RBDs could be improved by the collection of data in an International Database (http://www.rbdd.org), linkable to others previously published. This could be a useful tool to fill the gap between clinical data and clinical practice. This article reviews the genetic basis of RBDs, problems and complications of treatment, problems in the preparation of suitable guidelines for treatment and the future perspectives of the International Registry on RBDs.

Prerequisites for recombinant factor VIIa-induced thrombin generation in plasmas deficient in factors VIII, IX or XI.

BACKGROUND: Recombinant factor VIIa (rFVIIa) used for the treatment of hemophilia A or B patients with an inhibitor is hemostatically effective because it induces thrombin generation (TG), despite grossly impaired FVIII- and FIX-dependent amplification of FX activation. Tissue factor (TF) and or activated platelets were shown to be essential for the rFVIIa activity. OBJECTIVE: To evaluate the relative effects of TF and phospholipids on rFVIIa-induced TG in FVIII-, FIX- and FXI-deficient plasmas. METHODS: Phospholipids had an independent effect that was augmented by TF. The contribution of blood-borne TF in FVIII-, FIX- and FXI-deficient plasma to rFVIIa-induced TG was demonstrated by removing microparticles and use of anti-TF antibodies. RESULTS: At increasing concentrations of rFVIIa, the dependence of rFVIIa-induced TG on TF declined, but the presence of phospholipids was essential. rFVIIa was also shown to activate purified FIX and FX in the presence of phospholipids and absence of TF. rFVIIa-induced TG was dramatically augmented in FVIII- or FIX-deficient plasma in which the level of FVIII or FIX was increased to 1 or 2 U dL(-1). CONCLUSIONS: The data indicate that rFVIIa-induced TG is affected by TF, phospholipids, rFVIIa concentration, and the presence of FVIII and FIX.

A 13-bp deletion in alpha(IIb) gene is a founder mutation that predominates in Palestinian-Arab patients with Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by lack or dysfunction of alpha(IIb)beta3 in platelets. GT is relatively frequent in highly inbred populations. We previously identified a 13-bp deletion in the alpha(IIb) gene that causes in-frame deletion of six amino acids in three Palestinian GT patients. In this study, we determined the molecular basis of GT in all known Palestinian patients, examined whether Jordanian patients harbor the same mutations, analyzed whether there is a founder effect for the 13-bp deletion, and determined the mechanism by which the 13-bp deletion abolishes alpha(IIb)beta3 surface expression. Of 11 unrelated Palestinian patients, eight were homozygous for the 13-bp deletion that displayed common ancestry by haplotype analysis, and was estimated to have occurred 300-600 years ago. Expression studies in baby hamster kidney cells showed that substitution of Cys107 or Trp110 located within the deletion caused defective alpha(IIb)beta3 maturation. Substitution of Trp110, but not of Cys107, prevented fibrinogen binding. The other Palestinian patients harbored three novel mutations: G2374 deletion in alpha(IIb) gene, TT1616-7 deletion in beta3 gene, and IVS14: -3C --> G in beta3 gene. The latter mutation caused cryptic splicing predicting an extended cytoplasmic tail of beta3 and was expressed as dysfunctional alpha(IIb)beta(3). None of 15 unrelated Jordanian patients carried any of the described mutations.