Synthesis of GPIb beta with novel transmembrane and cytoplasmic sequences in a Bernard-Soulier patient resulting in GPIb-defective signaling in CHO cells.

The molecular defect of a new Bernard-Soulier patient, originating from Morocco and presenting thrombocytopenia with large platelets and an absence of ristocetin-induced platelet agglutination, has been identified and reproduced in transfected heterologous cells. Gene sequencing revealed insertion of a guanine in the domain coding for the transmembrane region of the glycoprotein (GP) Ib beta subunit. This mutation causes a translational frame shift, which creates putative novel transmembrane and cytoplasmic 37 and 125 amino acids domains, respectively. A 34 kDa immunoreactive GPIb beta band, instead of the normal 26 kDa subunit, was detected by Western blotting in lysates from the patient's platelets and from transfected cells and in immunoprecipitates of metabolically labeled cells. The abnormal subunit did not associate with GPIb alpha and was mainly intracellular, although a significant fraction could reach the cell surface. Cells expressing the mutant GPIb-IX complex adhered to a von Willebrand factor matrix but were unable to change shape, unlike cells expressing the wild-type receptor. These results strongly suggest a novel role of the GPIb beta subunit and its transmembrane-intracellular region in GPIb-VWF-dependent signaling, in addition to a role in correct assembly and cell surface targeting of the GPIb-V-IX complex.

Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX.

The multisubunit leucine-rich glycoprotein (GP) Ib-IX-V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard-Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse-chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbalpha undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbalpha and surface expression of the complex. Biosynthetic studies were then performed on a Bernard-Soulier variant based on previous description of abnormal GPIbalpha size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbalpha. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments ('ERGIC') and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

A novel monoclonal antibody against the extracellular domain of GPIbbeta modulates vWF mediated platelet adhesion.

GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.

Role of the leucine-rich domain of platelet GPIbalpha in correct post-translational processing--the Nancy I Bernard-Soulier mutation expressed on CHO cells.

The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbalpha found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbalpha were transfected into CHO cells expressing GPlbbeta and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPlbalphadeltaLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient's platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbalphadeltaLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbalpha leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOalphadeltaLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOalphabetaIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOalphadeltaLeubetaIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbalpha is essential for normal processing and function of the GPIb-IX complex. In addition, these results obtained in a cellular system supported the suspected role of the macroglycopeptide region of GPIbalpha in maintaining a suitable conformation of this multisubunit receptor to perform its adhesive function.

A three-base deletion removing a leucine residue in a leucine-rich repeat of platelet glycoprotein Ib alpha associated with a variant of Bernard-Soulier syndrome (Nancy I).

Leucine-rich repeats are conserved structural motifs present in the four components of the human platelet glycoprotein Ib/IX/V complex receptor for the adhesive protein von Willebrand factor. The absence or abnormality of this complex is responsible for Bernard-Soulier disease, an autosomal recessive bleeding disorder. We report a deletion of leucine 179, located in a highly conserved position of the seventh leucine-rich repeat of GPIb alpha, found in a variant form of Bernard-Soulier disease (Bernard-Soulier Nancy I). Three affected siblings of a family were characterized by absence of ristocetin-induced platelet agglutination, although ADP aggregation was normal. Flow cytometry studies showed detectable amounts of all four members of the GPIb/IX/V complex on the surface of the patients' platelets. Western blotting revealed normal levels of GPIX, decreased levels of GPIb beta and GPV, and < 1% of GPIb alpha. RT-PCR studies showed the presence of mRNA coding for GPIb alpha, GPIb beta, GPIX and GPV. Sequencing showed a three-base deletion which results in the absence of a leucine residue, highly conserved across the seven leucine-rich repeats of GPIb alpha and also within the other members of the leucine-rich glycoprotein family. The absence of the leucine 179 in a patient's GPIb alpha is believed to cause a conformational change in the protein which would account for the lack of binding of most of the MoAbs tested and would be responsible for the absence of von Willebrand factor binding. These results point to the leucine-rich region of GPIb alpha as being required for the correct exposure of the von Willebrand binding site as well as for the correct assembly and stability of the GPIb/IX/V complex on the platelet surface.

DNA amplification of HIV genome in hemophiliacs and in newborns from seropositive mothers.

We evaluated the validity of DNA enzymatic amplification (PCR) in a population at risk for HIV-1 infection, consisting of hemophiliacs and children born to seropositive mothers. All but one of the seropositive hemophiliacs and controls were found positive with the three sets of primers. All the seronegative patients and controls were found negative in PCR. No correlation with the anti-nef serology was found, one seropositive being anti-nef negative and three seronegative anti-nef positive. The results obtained with PCR are in good agreement with classical serology, and this would suggest that the possible period of latency may not be as long as suspected. No seroconversion has been described in hemophiliacs since solvent-detergent inactivated blood products have been in use, and seronegative hemophiliacs no longer constitute a population at risk. Studies on seronegative sexual partners of seropositive patients would be of great interest. For newborns from seropositive mothers, PCR is the only possible technique in early age before seronegativation of the healthy children. Further studies will be required to determine the fiability and sensitivity of the test.

Common intragenic and extragenic polymorphisms of blood coagulation factors VIII and IX are different in Chinese and Caucasian populations.

In order to examine the possibilities of carrier detection and prenatal diagnosis in hemophilia A and B in the Chinese region of Suzhou, we analyzed four different RFLPs within the factor IX gene and two intragenic RFLPs and one extragenic RFLP for the factor VIII gene. The results obtained show important differences between the Chinese and Caucasian populations. No polymorphism was found within the factor IX gene in the Chinese population and the informativity obtained for the factor VIII gene was quite different between the two populations for each RFLP studied.

Ethnic polymorphism of DXS52 (St 14) locus linked to coagulation factor VIII gene in Argentina.

Sixty-five individuals belonging to 16 argentinian families of hemophilia A were studied using the St 14 probe (DXS52 locus). This probe is widely used for carrier detection and prenatal diagnosis, despite the risk of recombination between the factor VIII gene and the DXS52 locus, because of its high informativity. The families are divided in two groups: one group constituted only of metis of Indians according to interview and morphotype and a second group of caucasoids (Spanish essentially and Italian). In this study we have shown some ethnic variations of the TaqI RFLPs in the DXS52 locus. In the allelic system I, (which alleles are numbered from 1 to 8) we have noted an over representation of the larger alleles (2 and 3) and of the allele 8 in both Argentinian groups when compared to the caucasian population already studied in our laboratory. The additional polymorphic TaqI site giving the beta band in the system II (alpha and beta bands) is found more frequently in the Argentinian families than in Caucasians. Some other additional polymorphic sites have been found in generally constant bands giving additional allelic systems, in metis families.

Carrier detection in hemophilia using pedigree analysis coagulation tests and DNA probes.

Hemophilia A and B are hereditary X-linked recessive bleeding disorders due to an anomaly or absence of the gene coding for coagulation factors VIII or IX. Until recently, carrier detection was performed on standard pedigree analysis and clotting factor assays. Due to lyonisation, the results obtained by these methods were only probabilistic. Recombinant DNA procedures have now been applied to the identification of molecular defects and carrier detection in inherited diseases. Because of the great heterogeneity of the molecular defects in hemophilia, the diagnosis of carrier status has to be made by the study of restriction fragment length polymorphisms (RFLP) genetically linked to factor VIII or factor IX genes. In a large number of cases, gene probing provides certain diagnosis. We studied some 300 individuals belonging to 70 families with hemophilia A or B. We used two probes to explore hemophilia A: an intragenic probe, p114.12, which detects an RFLP with the enzyme BclI and the extragenic polymorphic probe, St 14, which reveals an RFLP with the enzyme TaqI. For hemophilia B a genomic probe comprising exons b, c, d was used to detect an RFLP linked to a TaqI site. Despite the risk of recombination due to its extragenic location, the St 14 probe proved to be very useful because of the high informativity obtained in the families with hemophilia A. In contrast, the low informativity of the factor IX probe necessitates a search for other RFLPs in or near the factor IX gene. A comparison of the different methods used for carrier detection showed the possibility of misdiagnosis when using only pedigree analysis and biologic data and the improved certainty of diagnosis by gene probing.

Detección de portadores de hemofilia A: complementación de los estudios convencionales con el análisis de ADN / Detection of haemophilia A patients: complementation of the conventional study with DNA analysis

Con el propósito de evaluar la utilidad de complementar los estudos convencionales mediante el análisis del ADN con sondas extragénicas, para la detección de portadoras, estudiamos 60 individuos pertenecientes a 15 familias argentinas con uno (9) o más (6) miembros afectados de hemofilia. A severa. Realizamos el análisis de segregación de los enfermos polimórficos (RFLP), luego del tratamiento del ADN con TaqI/ST14, en 21 varones (15 hemofílicos) y 39 mujeres - 6 portadoras obligadas (PO) y 33 potenciales (PP) -, además de la determinación de la actividad del factor VIII (FVIII) y del antígeno del factor von Willebrand (vWFAg). La probabilidad de ser portadora de cada consultante, fue asignada según los métodos convencionales (antecedentes familiares y niveles de FVIII y vWFAg) y combinada con los resultados del análisis de los RFLP, expresados en términos de probabilidad. ST14 fue informativa en el 67% de las PO y en el 79% de las PP. El análisis de los RFLP permitió definir el estado portador (4,5% de riesgo de recombinación) en el 53% de las familias. En los casos con antecedentes previos, pudimos clasificar 5 de las 10 PP (1 portadora, 4 no-portadoras). El estado portador pudo ser excluido en 5 de 23 mujeres, parientes de hemofílicos esporádicos. En ciertos casos (5/10) observamos una falta de coincidencia entre el genotipo asignado y el fenotipo, probablemente por efecto del fenómeno de Lyon (inactivación al azar de uno de los cromosomas X), aunque no puede excluirse una recombinación entre el locus ST14 y el gen del FVIII. Los resultados del análisis de los RFLP fueron expresados en términos de probabilidad y combinados con los datos fenotípicos; las probabilidades así estimadas fueron bajas (< 0,13) o altas (> 0,95), contrariamente o lo obtenido al considerar exclusivamente los métodos convencionales (0,01 a 0,99). el análisis de segregación de los RFLP disminuye pues, considerablemente la incertidumbre sobre la probabilidad de ser portadora de cada consultante

Detección de portadores de hemofilia A: complementación de los estudios convencionales con el análisis de ADN / Detection of haemophilia A patients: complementation of the conventional study with DNA analysis

Con el propósito de evaluar la utilidad de complementar los estudos convencionales mediante el análisis del ADN con sondas extragénicas, para la detección de portadoras, estudiamos 60 individuos pertenecientes a 15 familias argentinas con uno (9) o más (6) miembros afectados de hemofilia. A severa. Realizamos el análisis de segregación de los enfermos polimórficos (RFLP), luego del tratamiento del ADN con TaqI/ST14, en 21 varones (15 hemofílicos) y 39 mujeres - 6 portadoras obligadas (PO) y 33 potenciales (PP) -, además de la determinación de la actividad del factor VIII (FVIII) y del antígeno del factor von Willebrand (vWFAg). La probabilidad de ser portadora de cada consultante, fue asignada según los métodos convencionales (antecedentes familiares y niveles de FVIII y vWFAg) y combinada con los resultados del análisis de los RFLP, expresados en términos de probabilidad. ST14 fue informativa en el 67% de las PO y en el 79% de las PP. El análisis de los RFLP permitió definir el estado portador (4,5% de riesgo de recombinación) en el 53% de las familias. En los casos con antecedentes previos, pudimos clasificar 5 de las 10 PP (1 portadora, 4 no-portadoras). El estado portador pudo ser excluido en 5 de 23 mujeres, parientes de hemofílicos esporádicos. En ciertos casos (5/10) observamos una falta de coincidencia entre el genotipo asignado y el fenotipo, probablemente por efecto del fenómeno de Lyon (inactivación al azar de uno de los cromosomas X), aunque no puede excluirse una recombinación entre el locus ST14 y el gen del FVIII. Los resultados del análisis de los RFLP fueron expresados en términos de probabilidad y combinados con los datos fenotípicos; las probabilidades así estimadas fueron bajas (< 0,13) o altas (> 0,95), contrariamente o lo obtenido al considerar exclusivamente los métodos convencionales (0,01 a 0,99). el análisis de segregación de los RFLP disminuye pues, considerablemente la incertidumbre sobre la probabilidad de ser portadora de cada consultante (AU)