Matrix metalloproteinase 9 is involved in Crohn's disease-associated platelet hyperactivation through the release of soluble CD40 ligand.

BACKGROUND: Patients with Crohn's disease have an increased risk for systemic thromboembolism. Their platelets are hyperactive and possess an elevated endogenous content of CD40 ligand (CD40L), a tumour necrosis factor alpha family protein member. Under basal conditions and after stimulation, these platelets express more CD40L on their surface and release higher amounts of soluble (s)CD40L than control platelets, through a mechanism that might be mediated by matrix metalloproteinases (MMPs). OBJECTIVE: The aim of this work is to study whether enhanced sCD40L release secondary to changes in the platelet content of MMPs contributes to the higher state of activation of platelets from patients with Crohn's disease. METHODS: State of activation, CD40L and metalloproteinases content of platelets isolated from patients with Crohn's disease and age- and sex-matched control individuals were analysed, respectively, by flow cytometry, western blot and gelatin zymography. RESULTS: The hyperactive state of platelets from patients with Crohn's disease might rely on their enhanced release of sCD40L, since its inhibition by a broad-range inhibitor of MMPs (GM6001) reduced fibrinogen binding induced by platelet stimulation. Analysis of the content of MMPs in platelets from patients with Crohn's disease showed an exclusive increase in MMP-9 activity. Moreover, MMP-9 inhibition diminished sCD40L release and fibrinogen binding to activated platelets. CONCLUSIONS: The results suggest that platelets from patients with Crohn's disease release more sCD40L than controls as a consequence of their higher endogenous content of CD40L and of MMP-9, which is involved in CD40L shedding. The increased levels of released sCD40L might be responsible, at least in part, for the high state of activation of platelets from patients with Crohn's disease.

A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia.

This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (

Compound heterozygosity of the GPIbalpha gene associated with Bernard-Soulier syndrome.

We report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibalpha and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbalpha in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbalpha gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution chan-ino Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbbeta and GPIX failed to detect surface expression of the GPIbalpha mutants. Cells transfected with [1418insT]GPIbalpha-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast. transfection of [T715A]GPIbalpha-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbalpha more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.

Effects of thrombopoietin receptor agonists on procoagulant state in patients with immune thrombocytopenia.

Thrombopoietin receptor agonists (TPO-RA) have recently been introduced for the treatment of immune thrombocytopenia (ITP), an anti-platelet-antibodies autoimmune disease. The observation of a low frequency of bleeding episodes despite their thrombocytopenia suggests the existence of a compensatory mechanism. This study aimed to evaluate the effect of TPO-RA treatment on platelet function and on the procoagulant state in ITP patients before (ITP-bR) and after responding (ITP-aR) to treatment. Plasma- and microparticle (MP)-associated procoagulant capacity from ITP patients was similar before and after responding to the TPO-RA regimen but higher than the healthy control values. High MP-associated procoagulant activity did not seem to be due to increased platelet activation, since platelet stimulation by agonists was reduced in ITP-bR and ITP-aR patients. It could be related to increased platelet apoptosis, evaluated in terms of surface phosphatidylserine (PS), observed in both ITP groups. In summary, TPO-RA treatment increased platelet count but did not ameliorate their function and did not change plasma- and MP-associated procoagulant state of ITP patient responders to this therapy.

Podocalyxin enhances the adherence of cells to platelets.

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Delta) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin alphaVbeta3/alphaVbeta5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid.

Cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme gene. Regulatory role of Sp1 and AP-2.

This work reports the molecular cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme (mME) gene. The proximal promoter region has features of housekeeping genes like high G + C-content and absence of TATA or CCAAT boxes. Deletion analysis of the 5' region of the mME showed that maximal transcriptional activity is located within the -205/+86 region. Footprinting analysis showed two protected regions, one comprising potential overlapped AP-1, CREB, and AP-4 sites and a second one encompassing AP-2 and several Sp1 ci-acting elements. Mutation of putative AP-1/AP-4/CREB sites reduced basal promoter activity to less than 50%. Supershift assays demonstrated the specific binding of Sp1 and AP-2 proteins. Moreover, experiments in Drosophila SL2 cells lacking endogenous Sp1 demonstrated that the Sp1 site(s) is essential to maintain a normal basal rate of transcription of this gene. A low-level expression of AP-2 enhanced the activity of a mME promoter construct in HepG2 cells and this effect was prevented by disruption of the putative AP-2 element. In contrast, higher levels of expression of AP-2 induced a DNA-independent inhibitory response. A biphasic regulation of endogenous mME gene is also shown in HepG2 cells transfected with an AP-2 expression plasmid, suggesting that availability of AP-2 protein may control this gene under physiological conditions. A recombinant lambda genomic clone containing a mME pseudogene was also isolated. The high degree of sequence conservation seems to indicate a recent emergency of this human pseudogene.

A 1063G-->A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb.

We report the molecular, genetic and functional analysis of a case of thrombasthenic phenotype. The proband showed absence of platelet glycoprotein (GP)IIb and very low content of GPIIIa, and both his parents showed a marked reduction in the levels of platelet GPIIb-IIIa. Single-stranded conformational polymorphism-polymerase chain reaction (SSCP-PCR) analysis and direct sequencing of PCR-amplified GPIIb exon-12 revealed the presence of a G-->A transition at position 1063 with the expected substitution of glutamate 324 with lysine (K). This mutation did not alter the level of GPIIb mRNA. Co-expression of normal or mutant [324K] GPIIb with normal human GPIIIa in Chinese hamster ovary (CHO) cells failed to show surface exposure of [324K]GPIIb-IIIa complexes. Pulse-chase and immunoprecipitation analysis demonstrated that [324K]GPIIb cDNA was translated into proGPIIb, but neither mutant GPIIb heavy chain (GPIIbH) nor [324K]GPIIb-GPIIIa complexes were detected, suggesting that this mutation is the underlying molecular basis for the thrombasthenic phenotype. Mutation analysis demonstrated that 324E of GPIIb could be replaced by other negatively charged or polar amino acids (AAs) without impairing the surface expression of GPIIb-IIIa. However, substitution of 324E of GPIIb for a positively charged AA other than K prevented the expression of GPIIb-IIIa complexes. These observations suggest that a domain encompassing 324E of GPIIb is essential for heterodimerization with GPIIIa and its substitution for a positively charged residue precludes normal subunit association.

A novel (288delC) mutation in exon 2 of GPIIb associated with type I Glanzmann's thrombasthenia.

This work reports the molecular genetic analysis of two patients who suffer mucocutaneous haemorrhages, prolonged bleeding time and failure of platelets to aggregate, either spontaneously or in response to agonists. The absence of platelet surface glycoprotein (GP)IIb-IIIa complexes confirmed the clinical diagnosis of Glanzmann's thrombasthenia (GT). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of exon 2 of GPIIb showed polymorphic bands caused by the homozygous deletion of a cytosine at position 288 relative to the translation start site. causing a shifting of the reading frame and appearance of a premature termination codon. The heterozygous relatives showed a reduced platelet content of GPIIb-IIIa, and a correlation was found between the levels of GPIIb mRNA and surface expression of GPIIb-IIIa complexes. Unlike other mRNAs carrying a nonsense mutation, (288Cdel)GPIIb does not force alternative splicing of GPIIb mRNA. As expected, co-transfection of Chinese hamster ovary (CHO) cells with cDNAs encoding GPIIIa and (288delC)GPIIb failed to enhance the surface exposure of GPIIIa. It is concluded that the (288delC)GPIIb mutation is responsible for the thrombasthenic phenotype of the patients. In addition, it has also been determined that heterodimerization of GPIIb-IIIa requires the integrity of exons 2 and 3 of GPIIb.

Competition between normal [674C] and mutant [674R] subunits: role of the molecular chaperone BiP in the processing of GPIIb-IIIa complexes.

This work aimed at investigating the function of the [C674R] mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide bridge. Individuals heterozygous for this mutation show a platelet GPIIb-IIIa content approximately 30% of normal controls, which is less than expected from one normal functioning allele. Coexpression of normal [674C]GPIIb and mutant [674R]GPIIb with normal GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary (CHO) cells, suggesting that [674R]GPIIb interferes with the association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the surface exposure of GPIIb-IIIa. However, substitution of 674C for A produced a much lesser inhibition than R, suggesting that a positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in transiently transfected CHO cells. BiP was also found associated with [674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or normal heterodimers. Overexpression of BiP did not increase the surface exposure of [674R]GPIIb-IIIa complexes, indicating that its availability was not a limiting step. Platelets from the thrombasthenic patient expressing [674R]GPIIb-IIIa were found to bind soluble fibrinogen in response to physiologic agonists or dithiothreitol treatment. Thus, the [674R]GPIIb mutation leads to a retardation of the secretory pathway, most likely related to its binding to the molecular chaperone BiP, with the result of a defective number of functional GPIIb-IIIa receptors in the cell surface.

Molecular genetic analysis of a compound heterozygote for the glycoprotein (GP) IIb gene associated with Glanzmann's thrombasthenia: disruption of the 674-687 disulfide bridge in GPIIb prevents surface exposure of GPIIb-IIIa complexes.

This work was aimed at elucidating the molecular genetic lesion(s) responsible for the thrombasthenic phenotype of a patient whose low platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a case of type II Glanzmann's thrombasthenia (GT). The parents did not admit consanguinity and showed a reduced platelet content of GPIIb-IIIa. Polymerase chain reaction (PCR)-single-stranded conformational polymorphism analysis of genomic DNA showed no mutations in the patient's GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a C-->A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C-->A] inherited from the father. The predicted effect of this mutation, insertion of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transcription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband's GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T2113-->C transition that changes Cys674-->Arg674 disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa. Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg674]GPIIb is capable of forming a heterodimer complex with GPIIIa, but the rate of subunit maturation and the surface exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GPIIb is essential for the normal processing of GPIIb-IIIa complexes. The additive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.