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SUMMARY Features characteristic of the hereditary syndrome of testicular feminization (tfm) were observed in a 7-year-old Quarter Horse. The horse had female body habitus and male psychosexual behaviour. Gonads located in the abdomen were testes and the uterus and cervix were absent. The vagina was normal in depth but ended as a blind pocket. The sex chromosome composition of testicular fibroblast and leucocyte cultures was XY. Construction of a family pedigree revealed a pattern of hereditary transmission similar to that reported for tfm in other mammalian species. RÉSUMÉ Des caractères d'un syndrome héréditaire de féminisation testiculaire furent constatés chez un cheval de Selle de 7 ans. Ce cheval avait un comportement psychique de mâle et un aspect de jument. Les gonades se trouvaient dans l'abdomen. L'utérus et le cervix n'existaient pas. Le vagin était normal mais se terminait en cul de sac. Les chromosomes sexuel du tissu testiculaire et des leucocytes étaient de type XY. L'étude du pedigree revela des traits génétiques comparables à ceux relevés pour le même syndrome hezc d'autres mammifères. ZUSAMMENFASSUNG Die charakteristischen Merkmale des hereditären Syndroms der testikulären Verweiblichung konnte bei einem 7 jährigen Quarter Horse beobachtet werden. Das Pferd zeigte einen Stutenhabitus und ein männliches Sexualverhalten. Die abdominal gelegenen Gonaden erwiesen sich als Hoden; Uterus und Cervix fehlten. Die Vagina war von normaler Länge, endete aber als Blindsack. Die Geschlechtschromosomen testikulärer Fibroblasten und von Leukocytenkulturen bestanden aus XY. Ein Familienstammbaum Hess auf einen Erbgang schliessen, wie er auch bei anderen Säugetierarten beschrieben worden ist. [ABSTRACT FROM AUTHOR]

Background Glanzmann's Thrombasthenia (GT) and Bernard Soulier Syndrome (BSS) are the two most common inherited forms of thrombasthenia. They are caused by alterations in expression or function of integrin alphaIIb/β3 or glycoprotein (GP) Ib/IX/V respectively. We identified an individual with chronic mild macrothrombocytopenia (range 59–100 × 109 L−1) but no overt bleeding diathesis. The propositus was found to have atypical binding of monoclonal antibodies (mAb) to β3 integrin and increased expression of GPIbVIX. Methods and Results The exons and flanking intronic sequences of ITGB3, ITGA2B GPIBA, GPIBB, GP9 and GP5 genes were sequenced. Heterozygous, non-synonymous single nucleotide polymorphisms (SNP) were identified in the ITGB3 and the GPIBA genes. These result in the substitution of Histidine for Aspartate at residue 723 (D723H) in ITGB3 and Leucine for Proline at residue 69 (P69L) in GPIBA. Asp723 in ITGB3 is known to form a critical salt-bridge with ITGA2B and charge reversal at residue 723 causes constitutive activation of the integrin in vitro. In agreement with this, mutant alphaIIb/β3-His723 expressing CHO cell lines showed an increased expression of activation dependent epitopes relative to wild-type as measured by mAb PAC-1 binding. Increased PAC-1 binding was also observed on resting platelets of affected individuals compared with non-affected family members. Further investigation of three generations of the propositus’ family identified several individuals with both SNPs each of whom had an identical platelet phenotype. However, in the one family member identified who carried the GPIBA mutation alone, normal platelet size and count were observed suggesting that the ITGB3 D723H substitution is the causative mutation. Genotyping a panel of 1127 unrelated controls for the two SNPs identified five individuals with the GPIBA mutation and none with the ITGB3 mutation. Conclusions We report a novel autosomal dominant cause of a mild form of macrothrombocytopenia caused by a D723H substitution in the beta3 integrin. We postulate that the low platelet count is due to increased consumption of platelets due to enhanced levels of surface expressed, activated GPIIbIIIa. [ABSTRACT FROM AUTHOR]

Dithiothreitol (DTT) is known to induce an active conformation of alphaIIbbeta3 integrin and to promote the aggregation of Chinese hamster ovary (CHO)-alphaIIbbeta3 cells in the presence of soluble fibrinogen (Fg). The aim of this study was to compare adhesion and spreading with Fg or von Willebrand factor (VWF) of CHO-alphaIIbbeta3 cells in the presence or absence of DTT. Our results indicate that DTT treatment was required to induce cell spreading on VWF. In contrast, CHO-alphaIIbbeta3 cell spreading on Fg was already optimal in the absence of DTT. We used a small perfusion chamber coupled to videomicroscopy to demonstrate that CHO-alphaIIbbeta3 cells that were adherent and spread on VWF required DTT activation to resist to detachment under increasing shear rates (50-1600/s). In contrast, untreated or DTT-treated cells spread on Fg were able to resist to extremely high flow rates. These data provide novel evidence that activated alphaIIbbeta3 is absolutely required for spread cells to resist detachment and strengthens the importance of the alphaIIbbeta3 activation step for adhesion and spreading to VWF.

Integrins are alpha/beta heterodimeric cell surface receptors devoid of enzymatic activity. Signal transduction therefore requires the association of cytosolic and cytoskeletal proteins with the integrin subunit intracellular regions. This association is initiated upon ligand binding to the integrin receptor and includes clustering of the integrins and recruitment of focal adhesion-associated proteins. Whether integrin clustering is solely dependent on ligand binding to the integrin extracellular parts or involves also interactions between the intracellular tails of integrins is so far unknown. To investigate intracellular events in integrin clustering, we have used peptides corresponding to the integrin beta1 cytoplasmic region. Loading of cells with the peptides results in a decreased cell adhesion and in an inhibition of cell spreading in agreement with the previously reported dominant negative effect of the beta1 integrin cytoplasmic tail on integrin clustering. Direct protein-protein interaction studies by surface plasmon resonance demonstrate that integrin beta1 cytoplasmic peptides self-associate in contrast to integrin beta3 cytoplasmic tails. Size exclusion chromatography and SDS-PAGE analysis of the peptides further show that the integrin beta1 cytoplasmic parts form oligomers and that they assume alpha helical conformation to the extent of about 13% and that this fraction is increased upon aggregation. Thus self-association of the integrin beta1 subunit cytoplasmic regions may be central to beta1 integrin clustering.

We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with recombinant integrin alphaIIbbeta3 expressed in Chinese hamster ovary (CHO) cells either in its native conformation or following partial reduction by dithiothreitol (DTT). We found that DTT-treated cells aggregated in the presence of soluble VWF as well as Fg, whereas non-treated cells did not. Furthermore, we demonstrated that DTT was required to specifically induce alphaIIbbeta3-dependent cell adhesion to immobilized VWF, while Fg-dependent cell adhesion occurred independently of the activation state of alphaIIbbeta3. By comparing the effects of two potent platelet alphaIIbbeta3 inhibitors, monoclonal antibodies (mAbs) AP2 and 10E5, we highlighted the different blocking properties of these mAbs on VWF or Fg binding to activated alphaIIbbeta3. In particular, AP2 prevented VWF-dependent but not Fg-dependent CHO cell aggregation. Furthermore, AP2 inhibited cell adhesion to VWF, but had no effect on adhesion to Fg. In contrast to this distinct effect of AP2 towards these two ligands, mAb 10E5 inhibited activated alphaIIbbeta3-dependent aggregation completely and adhesion partially, whether in the presence of Fg or VWF. These data provide evidence that interaction of VWF and Fg with DTT-activated alphaIIbbeta3 relies on distinct contact sites exposed on the activated receptor that can be selectively blocked by monoclonal antibodies.