Abdul Rahim Hussein

In vitro capacity of different grades of chitosan derivatives to induce platelet adhesion and aggregation

Chitosan-derived hemostatic agents with various formulations may have distinct potential in hemostasis. This study assessed the ability of different grades and forms of chitosan derivatives as hemostatic agents to enhance platelet adhesion and aggregation in vitro. The chitosan derivatives utilized were 2% NO-CMC, 7% NO-CMC (with 0.45 mL collagen), 8% NO-CMC, O-C 52, 5% O-CMC-47, NO-CMC-35, and O-C 53. Samples of chitosan derivatives weighing 5mg were incubated at 37°C with 50 μL of phosphate buffer saline (PBS) (pH 7.4) for 60 min. The morphological features of the platelets upon adherence to the chitosan were viewed using scanning electron microscope (SEM), and the platelet count was analyzed with an Automated Hematology Analyzer. For platelet aggregation, we added an adenosine diphosphate (ADP) agonist to induce the chitosan-adhered platelets. O-C 52 bound with platelets exhibited platelet aggregates and clumps on the surface of the membrane layer with approximately 70-80% coverage. A statistically significant correlation (p<0.01) for the platelet count was identified between the baseline value and the values at 10 min and 20 min. The results indicate that O-C 53 and O-C 52 were able to promote clotting have the potential to induce the release of platelets engaged in the process of hemostasis.

Glycoprotein IIb/IIIa and P2Y12 Induction by Oligochitosan Accelerates Platelet Aggregation

Platelet membrane receptor glycoprotein IIb/IIIa (gpiibiiia) is a receptor detected on platelets. Adenosine diphosphate (ADP) activates gpiibiiia and P2Y12, causing platelet aggregation and thrombus stabilization during blood loss. Chitosan biomaterials were found to promote surface induced hemostasis and were capable of activating blood coagulation cascades by enhancing platelet aggregation. Our current findings show that the activation of the gpiibiiia complex and the major ADP receptor P2Y12 is required for platelet aggregation to reach hemostasis following the adherence of various concentrations of chitosan biomaterials [7% N,O-carboxymethylchitosan (NO-CMC) with 0.45 mL collagen, 8% NO-CMC, oligochitosan (O-C), and oligochitosan 53 (O-C 53)]. We studied gpiibiiia and P2Y12 through flow cytometric analysis and western blotting techniques. The highest expression of gpiibiiia was observed with Lyostypt (74.3 ± 7.82%), followed by O-C (65.5 ± 7.17%). Lyostypt and O-C resulted in gpiibiiia expression increases of 29.2% and 13.9%, respectively, compared with blood alone. Western blot analysis revealed that only O-C 53 upregulated the expression of P2Y12 (1.12 ± 0.03-fold) compared with blood alone. Our findings suggest that the regulation of gpiibiiia and P2Y12 levels could be clinically useful to activate platelets to reach hemostasis. Further, we show that the novel oligochitosan is able to induce the increased expression of gpiibiiia and P2Y12, thus accelerating platelet aggregation in vitro.

Effect of the Novel Biodegradable N, O-Carboxymethylchitosan and Oligo-Chitosan on the Platelet Thrombogenicity Cascade in von Willebrand Disease

INTRODUCTION Von Willebrand disease (vWD) is the second least common hemostatic disorder in Malaysia, and it has a low prevalence. This study examined the underlying platelet thrombogenicity cascades in the presence of different formulations of chitosan-derivatives in vWD patients. This paper aimed to determine the significant influence of chitosan biomaterial in stimulating the platelet thrombogenicity cascades that involve the von Willebrand factor, Factor 8, Thromboxane A2, P2Y12 and Glycoprotein IIb/IIIa in vWD. MATERIALS AND METHODS Variable chitosan formulations of N,O-Carboxymethylchitosan (NO-CMC) and Oligo-Chitosan (O-C) were tested. Fourteen vWD subjects voluntarily participated in this study after signing informed consent forms. The patients demographic profiles, family history, type of vWD, clinical symptoms and laboratory profiles were recorded and analyzed. Enzyme-linked immunosorbent assay, flow cytometry and Western blot tests were used to determine the level of the chitosan-adhered-platelet-mechanisms. RESULTS The study revealed that most patients were predominantly affected by vWD type I. The O-C group of chitosans scaffold pores is sufficient to allow for nutrients and cells. The O-C-stimulated-mediators are capable of initiating the platelet actions and were detected to expedite the blood coagulation processes. The oligo-group of chitosans was capable of amplifying and triggering more platelet activators pathways via the studied mediators. The present findings suggest that the ability of each type of chitosan to coagulate blood varies depending on its chemical composition. CONCLUSION The oligo group of chitosans is potentially capable of triggering platelet thrombogenicity cascades by activating platelets in vWD patients to form a platelet plug for hemostasis process.

Characterisation and Clinical Significance of FLT3-ITD and non-ITD in Acute Myeloid Leukaemia Patients in Kelantan, Northeast Peninsular Malaysia

BACKGROUND Mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor gene may promote proliferation via activation of multiple signaling pathways. FLT3-internal tandem duplication (FLT3-ITD) is the most common gene alteration found in patients diagnosed with acute myeloid leukaemia (AML) and has been associated with poor prognosis. MATERIALS AND METHODS We performed mutational analysis of exons 14-15 and 20 of the FLT3 gene in 54 AML patients using PCR-CSGE (conformational sensitive gel electrophoresis) followed by sequencing analysis to characterise FLT3 mutations in adult patients diagnosed with AML at Hospital USM, Kelantan, Northeast Peninsular Malaysia. RESULTS FLT3 exon 14-15 mutations were identified in 7 of 54 patients (13%) whereas no mutation was found in FLT3 exon 20. Six ITDs and one non-ITD mutation were found in exon 14 of the juxtamembrane (JM) domain of FLT3. FLT3-ITD mutations were associated with a significantly higher blast percentage (p-value=0.008) and white blood cell count (p-value=0.023) but there was no significant difference in median overall survival time for FLT3-ITD+/FLT3-ITD- within 2 years (p-value=0.374). CONCLUSIONS The incidence of FLT3-ITD in AML patients in this particular region of Malaysia is low compared to the Western world and has a significant association with WBC and blast percentage.

Effects of DPSS green laser irradiation on anaemic red blood cells

This study investigated the effect of DPSS green laser on normal anaemic red blood cells at different exposure times The results show that green laser light has different optimum time in affected normal and anemic blood samples. It requires short exposure time to affect anaemic blood samples compared to normal blood samples.

Conformational Sensitive Gel Electrophoresis (CSGE) as a method for NPM1 mutational screening in patients with Acute Myeloid Leukaemia

Abstract Introduction Nucleophosmin gene ( NPM1 ) exon 12 is the most common mutated gene found in Acute Myeloid Leukaemia (AML), which is present in 25–35%. NPM1 gene encodes for nucleophosmin (NPM) protein, which contains 294 amino acids. The NPM protein is an abundant nucleolar phosphoprotein that shuttles between the nucleus and cytoplasm. It contributes to promotion of ribosome biogenesis, regulation of centrosomal duplication during cell division, interaction with tumor suppressor gene and control of various nuclear proteins. Objective To screen for NPM1 exon 12 mutationsin AML patients diagnosed at Hospital Universiti Sains Malaysia (HUSM) using Conformational Sensitive Gel Electrophoresis (CSGE). Methods Total genomic DNAs were obtained from bone marrow aspirates or peripheral blood samples taken at diagnosis from 67 AML patients diagnosed at HUSM [French-American-British (FAB) subtypes: M0=1, M1=2, M2=10, M3=22, M4=9,M5=12,M6=2,M7=1, Not Otherwise Spesified (NOS)=8]. Patients were screened for NPM1 exon 12 mutations by CSGE. Cases displaying abnormal CSGE profiles were directly sequenced. Results & Discussion Mutation of NPM1 exon 12 was present in 7/67 AML cases (10%) and all cases were females. They consisted of two M1, two M5b, one M2, one M3 and one NOS FAB subtypes. Most of the mutations identified as insertional (ins) mutation (5/7), 4 patients having ins 962–963 CTGG (2), CATG (1) and CTGT (1). One patient having ins at 969 CATG and followed with stop codon (TGA). One patient having single base deletion (del) at 966 and became TGA (stop codon) while another patient having ins at 956 TGGA and followed with del 958–969. Conclusion Our data showed high frequency of NPM1 in AML-M1 and M5b, which is highly associated in female patients. The CSGE was found to be an inexpensive, simple and reliable test to be used in screening of NPM1 mutations.