CONTROLLING PLATELET SECRETION TO MODULATE HEMOSTASIS AND THROMBOSIS

Upon vascular injury, activated blood platelets fuse their granules to the plasma membrane and release cargo to regulate the vascular microenvironment, a dynamic process central to platelet function in many critical processes including hemostasis, thrombosis, immunity, wound healing, angiogenesis etc. This granule- plasma membrane fusion is mediated by a family of membrane proteins- Soluble N-ethyl maleimide Attachment Receptor Proteins(SNAREs). SNAREs that reside on vesicle (v-SNAREs) /Vesicle-Associated Membrane Proteins(VAMPs) interact with target/t-SNAREs forming a trans-bilayer complex that facilitates granule fusion. Though many components of exocytic machinery are identified, it is still not clear how it could be manipulated to prevent occlusive thrombosis without triggering bleeding. My work addresses this question by showing how the rates and extents of granule secretion could be regulated by various v-SNAREs. We also show that the granule cargo decondensation is an intermediate to secretion that also contributes to rates of cargo release. Platelets contain four major VAMP isoforms (-2, -3, -7, and -8), however, VAMP-8 and -7 play a primary role while VAMP-2 and -3 are ancillary in secretion. To exploit this heterogeneity in VAMP usage, platelet-specific V-2/3-/- and V-2/3/8-/- mouse models were generated and characterized to understand how secretion influences hemostasis. We found that each VAMP isoform differentially contributes by altering the rates and extents of cargo release. The loss of VAMP-2 and -3 had a minimal impact while the loss of VAMP-2, -3 and -8 significantly reduced the granule secretion. Platelet activation and aggregation were not affected though the spreading was reduced in V-2/3/8-/- platelets indicating the importance of secretion in spreading. Though coagulation pathways were unaltered, PS exposure was reduced in both V-2/3-/- and V-2/3/8-/- platelets suggesting diminished procoagulant activity. In vivo experiments showed that V-2/3/8-/- animals bled profusely upon tail transaction and failed to form occlusive thrombus upon arterial injury while V-2/3-/- animals did not display any hemostatic deficiency. These data suggest that about 40-50% reduction in secretion provides protection against thrombosis without compromising hemostasis and beyond 50% secretion deficiency, the animals fail to form functional thrombi and exhibit severe bleeding. Additionally, detailed structural analysis of activated platelets suggests that the post-stimulation cargo dissolution depends on an agonist concentration and stimulation duration. This process is VAMP-dependent and represents intermediate steps leading to a full exodus of cargo. Moreover, we also show that VAMP-8 is important for compound fusion events and regulates fusion pore size. This is a first comprehensive report that shows how manipulation of the exocytic machinery have an impact on secretion and ultimately on hemostasis. These animals will be instrumental in future investigations of platelet secretion in many other vascular processes

Development of Two-Dimensional Gel Electrophoresis Procedures for Analysis of Cupriavidus Necator H16 Subcellular Proteomes

A thesis presented to the faculty of the College of Science and Technology at Morehead State University in partial fulfillment of the requirements for the Degree of Master of Science by Smita Joshi on June 17, 2011

The Nuts and Bolts of the Platelet Release Reaction

Secretion is essential to many of the roles that platelets play in the vasculature, e.g., thrombosis, angiogenesis, and inflammation, enabling platelets to modulate the microenvironment at sites of vascular lesions with a myriad of bioactive molecules stored in their granules. Past studies demonstrate that granule cargo release is mediated by Soluble NSF Attachment Protein Receptor (SNARE) proteins, which are required for granule-plasma membrane fusion. Several SNARE regulators, which control when, where, and how the SNAREs interact, have been identified in platelets. Additionally, platelet SNAREs are controlled by post-translational modifications, e.g., phosphorylation and acylation. Although there have been many recent insights into the mechanisms of platelet secretion, many questions remain: have we identified all the important regulators, does calcium directly control the process, and is platelet secretion polarized. In this review, we focus on the mechanics of platelet secretion and discuss how the secretory machinery functions in the pathway leading to membrane fusion and cargo release

Female Headship and Women’s Work in Nepal

Literature on household headship has, by and large, tended to homogenize female-headed households (FHHs). We present a mixed methods approach to disaggregating the experiences of female-headed households (FHHs) in the post-conflict period in Nepal. One of four households in Nepal is estimated to be female-headed, either as a result of conflict-related male deaths (de jure, or widow heads) or due to male out-migration (de facto, or wives of migrants). We examine similarities and differences in the work responsibilities and labor market participation of de jure and de facto female heads using quantitative data from the Nepal Living Standards Surveys (NLSS) and qualitative data collected through fieldwork in Nepal. While both types of households share some similar concerns, de jure and de facto female heads face different challenges in negotiating their everyday household and labor market roles. Findings inform policy interventions that can be specifically targeted toward each type of household

Addressing Women’s Sanitation-related Safety Concerns in Slums of Maharashtra, India

Through this paper we explore women’s vulnerability during sanitation activities and the impact that household toilets have on women’s safety-related concerns. This study covers 4 cities in the state of Maharashtra– Pune, Pimpri- Chinchwad, Thane, and Kolhapur - where Shelter Associates has provided many slum households with toilets under its One Home One Toilet (OHOT) programme. A good part of the programme’s intention is to offer women an alternative to using their existing, inadequate public sanitation facilities, a problem that was highlighted during discussions with slum women themselves. Shelter Associates is a Maharashtra-based NGO established in 1993 that provides low cost sanitation and housing to slum residents

A prospective study of association of inflammatory markers with BMI in women with polycystic ovarian syndrome

Background: Many studies have demonstrated the association between polycystic ovary syndrome (PCOS) and chronic low-grade inflammation to be of unknown mechanism or an unclear cause; which could either be due to the underlying obesity, insulin resistance, or the polycystic ovary syndrome itself. The aim of our study was to find if there was any correlation between the inflammatory markers and body mass index (BMI) in women with PCOS. Methods: Our study included 100 women aged between 18-45 years with PCOS and were recruited for the study in the obstetrics and gynecology department, Pacific Medical College and Hospital, Rajasthan from October 2020 to April 2022. The enrolled patients then underwent a series of clinical, ultrasonographic and biochemical investigations. Results: WBC was positively correlated with BMI (r=0.453, p<0.000) Along with that, we were also able to establish a moderate degree positive correlation between BMI and serum CRP levels (r=0.396, p<0.000). A high degree significant positive correlation was found between ESR count and BMI (r=0.537, p<0.000) and platelet count and BMI (r=0.386, p<0.000). Conclusions: We discovered that higher WBC concentration, ESR count, platelet count, and serum CRP levels are linked to PCOS. Increase in serum inflammatory cardiovascular risk markers are brought on by PCOS and obesity. In order to determine the relative contributions of various factors, such as insulin resistance, androgen status, and BMI, further studies need to be carried out with a larger sample size as the mechanism for the chronic low-grade inflammation still remains unclear

Fe(III)-Chloroquine Complex: A New Potent Compound in Wellness Industries of High Antimicrobial and Antimalarial Activities

Fe(III)- Chloroquine complex has been synthesized and screened for its physicochemical, microbial as well as pharmacological activity have been done in solid and aqueous phase. On the basis of elemental analysis, polarographic studies, amperometric titration and IR spectral studies the probable formula for the complex has been determined at 25±1OC and ionic strength of µ= 1.0[KCl]. Raper's paper disc method was used for microbial study against various pathogenic bacteria and fungi. In vivo syudy of Swiss mice [25-30gm] were used for antimalarial activity against Chloroquine and its complex on xyline-Alcoholic activity test Kidney, liver and serum of these rats were also studied. On the basis of observed result it could be concluded that Fe (III)-Chloroquine complex were found to be non-toxic and more potent than pure chloroquine drug

Evaluation of single cell oil (SCO) from a tropical marine yeast Yarrowia lipolytica NCIM 3589 as a potential feedstock for biodiesel

Single cell oils (SCOs) accumulated by oleaginous yeasts have emerged as potential alternative feedstocks for biodiesel production. As lipid accumulation is species and substrate specific, selection of an appropriate strain is critical. Five strains of Y. lipolytica, a known model oleaginous yeast, were investigated to explore their potential for biodiesel production when grown on glucose and inexpensive wastes. All the strains were found to accumulate > 20% (w/w) of their dry cell mass as lipids with neutral lipid as the major fraction when grown on glucose and on wastes such as waste cooking oil (WCO), waste motor oil (WMO). However, amongst them, Y. lipolytica NCIM 3589, a tropical marine yeast, exhibited a maximal lipid/biomass coefficient, Y(L/X) on 30 g L(-1) glucose (0.29 g g(-1)) and on 100 g L(-1) WCO (0.43 g g(-1)) with a high content of saturated and monounsaturated fatty acids similar to conventional vegetable oils used for biodiesel production. The experimentally determined and predicted biodiesel properties of strain 3589 when grown on glucose and WCO, such as density (0.81 and 1.04 g cm(-3)), viscosity (4.44 and 3.6 mm(2) s(-1)), SN (190.81 and 256), IV (65.7 and 37.8) and CN (56.6 and 50.8) are reported for the first time for Y. lipolytica and correlate well with specified standards. Thus, the SCO of oleaginous tropical marine yeast Y. lipolytica NCIM 3589 could be used as a potential feedstock for biodiesel production

Psychrotrophic yeast Yarrowia lipolytica NCYC 789 mediates the synthesis of antimicrobial silver nanoparticles via cell-associated melanin

A psychrotrophic marine strain of the ascomycetous yeast Yarrowia lipolytica (NCYC 789) synthesized silver nanoparticles (AgNPs) in a cell-associated manner. These nanostructures were characterized by UV-Visible spectroscopy and scanning electron microscope-energy dispersive spectrometer (SEM-EDS) analysis. The brown pigment (melanin) involved in metal-interactions was obtained from the cells. This extracted pigment also mediated the synthesis of silver nanoparticles that were characterized by a variety of analytical techniques. The melanin-derived nanoparticles displayed antibiofilm activity. This paper thus reports the synthesis of AgNPs by the biotechnologically important yeast Y. lipolytica; proposes a possible mechanism involved in the synthetic process and describes the use of the bio-inspired nanoparticles as antibiofilm agents