SUMMARY: Background Mutation of the growth factor-independent 1B (GFI1B) fifth DNA-binding zinc-finger domain causes macrothrombocytopenia and α-granule deficiency leading to clinical bleeding. The phenotypes associated with GFI1B variants disrupting non-DNA-binding zinc-fingers remain uncharacterized. Objectives To determine the functional and phenotypic consequences of GFI1B variants disrupting non-DNA-binding zinc-finger domains. Methods The GFI1B C168F variant and a novel GFI1B c.2520 + 1_2520 + 8delGTGGGCAC splice variant were identified in four unrelated families. Phenotypic features, DNA-binding properties and transcriptional effects were determined and compared with those in individuals with a GFI1B H294 fs mutation of the fifth DNA-binding zinc-finger. Patient-specific induced pluripotent stem cell (iPSC)-derived megakaryocytes were generated to facilitate disease modeling. Results The DNA-binding GFI1B variant C168F, which is predicted to disrupt the first non-DNA-binding zinc-finger domain, is associated with macrothrombocytopenia without α-granule deficiency or bleeding symptoms. A GFI1B splice variant, c.2520 + 1_2520 + 8delGTGGGCAC, which generates a short GFI1B isoform that lacks non-DNA-binding zinc-fingers 1 and 2, is associated with increased platelet CD34 expression only, without quantitative or morphologic platelet abnormalities. GFI1B represses the CD34 promoter, and this repression is attenuated by different GFI1B zinc-finger mutations, suggesting that deregulation of CD34 expression occurs at a direct transcriptional level. Patient-specific iPSC-derived megakaryocytes phenocopy these observations. Conclusions Disruption of GFI1B non-DNA-binding zinc-finger 1 is associated with mild to moderate thrombocytopenia without α-granule deficiency or bleeding symptomatology, indicating that the site of GFI1B mutation has important phenotypic implications. Platelet CD34 expression appears to be a common feature of perturbed GFI1B function, and may have diagnostic utility.
AIM OF THE STUDY: The study is aimed at identifying the key ingredients of papaya leaf extract and elucidate the mechanism (s) of action of the identified potent component in mitigating thrombocytopenia (Thp).
MATERIALS AND METHODS: C. papaya leaf juice was subjected for sequential fractionation to identify the anti-thrombocytopenic phytochemicals. In vivo, stable thrombocytopenia was induced by subcutaneous injection of 70 mg/kg cyclophosphamide (Cyp). After induction, rats were treated with 200 and 400 mg/kg body weight papaya leaf juice and with identified fractions for 14 days. Serum thrombopoietin level was estimated using ELISA. CD110/cMpl, a receptor for thrombopoietin on platelets was measured by western blotting.
RESULTS: Administration of cyclophosphamide for 6 days induced thrombocytopenia (210.4 ± 14.2 × 103 cells/μL) in rats. Treating thrombocytopenic rats with papaya leaf juice and butanol fraction for 14 days significantly increased the platelet count to 1073.50 ± 29.6 and 1189.80 ± 36.5 × 103 cells/μL, respectively. C.papaya extracts normalized the elevated bleeding and clotting time and decreased oxidative markers by increasing endogenous antioxidants. A marginal increase in the serum thrombopoietin (TPO) level was observed in Cyp treated group compared to normal and treatment groups. Low expression of CD110/cMpl receptor found in Cyp treated group was enhanced by C. papaya extracts (CPJ) and CPJ-BT. Furthermore, examination of the morphology of bone marrow megakaryocytes, histopathology of liver and kidneys revealed the ability of CPJ and fractions in mitigating Cyp-induced thrombocytopenia in rats.
CONCLUSION: C. papaya leaf juice enhances the platelet count in chemotherapy-induced thrombocytopenia by increasing the expression of CD110 receptor on the megakaryocytes. Hence, activating CD110 receptor might be a viable strategy to increase the platelet production in individuals suffering from thrombocytopenia.