Thymol-enriched extract from Thymus vulgaris L leaves: Green extraction processes and antiaggregant effects on human platelets.

This work focuses on the selection and the optimization of an efficient green-extraction method, used to recover a thymol-enriched extract from thyme (Thymus vulgaris L), as well as the evaluation of the inhibitory effect of this latter on the human platelet aggregation. Different innovative extraction techniques, namely bead milling extraction, ultrasound and microwave assisted extraction, were tested for their ability to recover a high added value extract from thyme. Among all tested eco-extraction techniques, microwave extraction (MAE) was the best method in term of its extraction yield (20.84% ± 0.51), thymol concentration (731.71 mg/g) and total phenolic (23.53 ± 1.83 mg (GAE)/g of extract) and flavonoid (6.22 ± 0.35 mg of QE/g of extract) contents. Moreover, thyme extract obtained by microwave assisted extraction (TMAE) showed the most active antioxidant effect comparing to the other tested extracts. Based on these results, TMAE was chosen to be evaluated for its antiplatelet effect. Thereby, arachidonic acid, collagen and ADP were used to induce the platelet aggregation on human platelet rich plasma taken from healthy controls and results revealed that TMAE strongly inhibited the induced platelet aggregation. Indeed, TMAE exhibited potent antiaggregant activity by inhibiting platelet activation, secretion and aggregation. Additionally, cytotoxicity assay on normal HEK-293 cells showed that TMAE has no cytotoxic effect even at high concentration (8 mg/ml) and can further be taken up to various biomedical applications mainly in the prevention of cardiovascular diseases.

Spectrum of the mutations in Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by defects of the GPIb-IX-V complex, a platelet receptor for von Willebrand factor (VWF). Most of the mutations identified in the genes encoding for the GP1BA (GPIbα), GP1BB (GPIbß), and GP9 (GPIX) subunits prevent expression of the complex at the platelet membrane or more rarely its interaction with VWF. As a consequence, platelets are unable to adhere to the vascular subendothelium and agglutinate in response to ristocetin. In order to collect information on BSS patients, we established an International Consortium for the study of BSS, allowing us to enrol and genotype 132 families (56 previously unreported). With 79 additional families for which molecular data were gleaned from the literature, the 211 families characterized so far have mutations in the GP1BA (28%), GP1BB (28%), or GP9 (44%) genes. There is a wide spectrum of mutations with 112 different variants, including 22 novel alterations. Consistent with the rarity of the disease, 85% of the probands carry homozygous mutations with evidence of founder effects in some geographical areas. This overview provides the first global picture of the molecular basis of BSS and will lead to improve patient diagnosis and management.

Antiplatelet activity and toxicity profile of novel phosphonium salts derived from Michael reaction.

In this work, five novel phosphonium salts derived from the Michael reaction were screened for their antiplatelet activity. Our findings revealed that compounds 2a, 2b, 2c, and 2d significantly inhibit platelet aggregation triggered by ADP or collagen (P < 0.001). Notably, compound 2c inhibited the arachidonic acid pathway (P < 0.001). Moreover, the selected compounds reduce CD62-P expression and inhibit GPIIb/IIIa activation. The interactions of the active compounds with their targets, ADP and collagen receptors, P2Y12 and GPVI respectively were investigated in silico using molecular docking studies. The results revealed a strong affinity of the active compounds for P2Y12 and GPVI. Additionally, cytotoxicity assays on platelets, erythrocytes, and human embryonic kidney HEK293 cells showed that compounds 2a, 2c and 2d were non-toxic even at high concentrations. In summary, our study shows that phosphonium salts can have strong antiplatelet power and suggests that compounds 2a, 2c and 2d could be promising antiplatelet agents for the management of cardiovascular diseases.

Prevalence of factor V Leiden in south Tunisian blood donors.

Venous thrombosis (VT) is a common disease with multifactorial pathogenesis. Factor V Leiden mutation (G1691A) (FVL) is the most common risk factor in venous thrombosis. The prevalence of FVL varies according to geography and ethnicity. Hence, in several countries there is a difference in the frequency of this mutation between the southern, central and north. In Tunisia, no data is available about prevalence of FVL mutation by geographical origin. For this reason, we sought the prevalence of FVL mutation in blood donor of south Tunisia population. FVL has been detected by APCR-test and confirmed by PCR-RFLP and sequencing. Two hundred fifty blood donors, different in age and sex were included in this study to determine the prevalence of FVL in blood donors. FVL mutation was found in 13.6% of the studied population. Thirty-one were heterozygous and three persons were homozygous with a rate of 12.4 and 1.2%, respectively. In conclusion, FVL mutation is very common in south Tunisian population.

The same genetic defect in three Tunisian families with Bernard Soulier syndrome: a probable founder Stop mutation in GPIbß.

GPIbα, GPIbß, and GPIX are three candidate genes for a rare genetic bleeding disorder named Bernard Soulier syndrome (BSS). These genes are unique in the genome and encode for glycoprotein subunits of the GPIb-IX complex. Quantitative or qualitative deficiency in this complex is often associated with BSS. Here, we report the novel variant of BSS in which Ser23 of GPIbß is substituted by a Stop codon causing a premature termination of translation, recently described in one family. This genetic defect is revealed in three unrelated BSS patients. The pedigree was determined for two families (F1 and F2) and revealed the homozygosity of the mutation in the two patients and its heterozygosity in parents. In the third family, the patient DNA was heterozygote with the same Ser23 Stop mutation in addition to two missense heterozygote mutations (Asp 51 Gly) and (Ala 55 to Pro). We studied the effect of the Ser23 Stop mutation on the expression of the complex. Our findings confirm that the identified GPIbß mutation is responsible for the BSS phenotype and hampers the GPIb-IX complex to form on the platelets' surface. Regarding the scarcity of the BSS syndrome, the occurrence of the same mutation in three unrelated families would suggest a BSS founder mutation in Tunisia.

Bernard-Soulier syndrome: novel nonsense mutation in GPIbbeta gene affecting GPIb-IX complex expression.

Bernard-Soulier syndrome (BSS) is a rare autosomal recessive genetic disorder characterized by thrombocytopenia, circulating giant platelets, and prolonged bleeding time. BSS is explained by a defect in primary hemostasis owing to quantitative or qualitative defect in the GPIb-IX-V complex, composed of four subunits: GPIbalpha, GPIbbeta, GPIX, and GPV. In this study, we report a novel GPIbbeta defect in a Tunisian family, in which Serine 23 is substituted by a Stop codon causing a premature termination of translation. This defect was homozygous in the BSS patient and heterozygote in both the parents and sisters of the patient. We studied the effect of this mutation on the expression of the GPIb-IX complex by western blot, flow cytometry, and confocal microscopy: GPIbalpha and GPIX were absent on the surface of platelets, whereas they were present in the cytoplasm. These results led to conclude that the novel Ser 23 Stop mutation in GPIbbeta is responsible of BSS in the studied family and hampers the complex to form on the platelets surface.

Purification of glycocalicin from human plasma.

Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product.

Allelic polymorphisms of human platelets-specific alloantigens in South Tunisian population.

OBJECTIVES: Human platelet-specific alloantigens (HPA) are polymorphic epitopes which vary among ethnic groups. BACKGROUND: In Tunisia, HPA frequencies were determined in North and centre; however, the pattern of HPA in South Tunisian population is not been studied yet. The aim of this work was to determine allelic frequencies of HPA-1, -3, and -5 systems in south Tunisian population, in order to estimate the risk of anti-platelet allo-immunization and to create a register of HPA-typed blood donors. METHODS: Our study concerned 212 unrelated healthy, regular blood donors from southern Tunisia. Allelic polymorphisms of each system were determined using a polymerase chain reaction with sequence-specific primers. RESULTS: Genotype frequencies a/a, a/b, and b/b were, respectively, 0.670, 0.288, and 0.042 for HPA-1 system, 0.430, 0.462, and 0.108 for HPA-3 system, and 0.750, 0.241, and 0.009 for HPA-5 system. The allele frequencies were 0.814 and 0.186 for HPA-1a and -1b alleles; 0.660 and 0.340 for HPA-3a and -3b alleles and 0.870, and 0.130 for HPA-5a and -5b alleles. DISCUSSION: The reported frequencies are more similar to those of Caucasians than those of north Tunisian population.