The minimization of blood loss represents a significant clinical need in the arena of surgery, trauma, and emergency response medicine. Fibrinogen is our body's native polymer system activated in response to tissue and vasculature injury, and forms the foundation of the most widely employed surgical sealant and hemostatic agent. Non-covalent knob:hole interactions are central to the assembly of fibrin that leads to network and clot formation. This project exploits these affinity interactions as a strategy to direct fibrin polymerization dynamics and network structure so as to develop a temperature-triggered polymerizing fibrin mixture for surgical applications. Short peptides modeled after fibrin knob sequences have been shown to alter fibrin matrix structure by competing with native fibrin knobs for binding to the available holes on fibrinogen and fibrin. The fusion of such knob peptides to a non-native component should facilitate binding of the fused component to fibrinogen/fibrin, and may permit the concomitant modification of the fibrin matrix. We examined this hypothesis in a three-step approach involving (a) analyzing the ability of tetrapeptide knob sequences to confer fibrin(ogen) affinity on a non-fibrin protein, (b) investigating the effect of knob display architecture on fibrin(ogen) structure, and (c) designing a temperature-responsive knob-displaying construct to modulate fibrin(ogen) affinity at different temperature regimes, thus altering fibrin(ogen) structure.

Although platelets are anucleate, they do inherit 1500-3000 mRNA transcripts from their megakaryocyte progenitors, in addition to all the machinery essential for protein synthesis; however, there is little understanding why platelets initiate de novo synthesis of these transcripts. Our group demonstrated that fibrinogen (Fg), a ligand of platelet Glycoprotein (GP)IIb-IIIa (αIIbβ3 integrin), is required for platelet P-selectin expression and that engagement of Fg with GPIIb-IIIa is essential for this process. The present study shows that murine platelets incubated with Fg synthesize P-selectin de novo, and this synthesis is blocked by puromycin. A similar effect is also observed when platelets are incubated with fibronectin, another ligand of GPIIb-IIIa. Furthermore, platelets from both ligand- (Fg−/−, von Willebrand factor−/−, apolipoprotein A-IV−/−) and GPIIb-IIIa-deficient mice have altered proteomes. These data suggest an intricate mechanism by which engagement of platelets with their environment triggers signal-dependent translation of the platelet transcriptome, consequently altering the platelet proteome.

Although platelets are anucleate, they do inherit 1500-3000 mRNA transcripts from their megakaryocyte progenitors, in addition to all the machinery essential for protein synthesis; however, there is little understanding why platelets initiate de novo synthesis of these transcripts. Our group demonstrated that fibrinogen (Fg), a ligand of platelet Glycoprotein (GP)IIb-IIIa (αIIbβ3 integrin), is required for platelet P-selectin expression and that engagement of Fg with GPIIb-IIIa is essential for this process. The present study shows that murine platelets incubated with Fg synthesize P-selectin de novo, and this synthesis is blocked by puromycin. A similar effect is also observed when platelets are incubated with fibronectin, another ligand of GPIIb-IIIa. Furthermore, platelets from both ligand- (Fg−/−, von Willebrand factor−/−, apolipoprotein A-IV−/−) and GPIIb-IIIa-deficient mice have altered proteomes. These data suggest an intricate mechanism by which engagement of platelets with their environment triggers signal-dependent translation of the platelet transcriptome, consequently altering the platelet proteome.

published_or_final_version / Community Medicine / Doctoral / Doctor of Philosophy

With the studies performed in this theses the local drug delivery technique FibMat developed by the biotech company AddBIO, was shown to be applicable to other plasma proteins and drugs than the fibrinogen-bisphosphonate combination that is today being commercialized. Hence the potential for a broader field of application was demonstrated. The application targeted today is as a surface modification giving improved strength to bone around screws used in bone implants. The effect of changing protein and manufacturing conditions was studied with null ellipsometry. It was demonstrated that with changes in incubation temperature, pH and salinity the fibrinogen could be successfully exchanged for the plasma proteins human serum albumin and immunoglobulin G. With liquid scintillation counting it was shown that the developed protein multilayers were able to absorb and release the bone strengthening drug alendronic acid in levels comparable to that of the fibrinogen based ditto. Disk susceptibility tests with the bacteria S. Aureus showed a potential for antibacterial functionalization with gentamicin. The release was, in the case of the fibrinogen multilayer, detectable up to 48 hours. Similar test revealed an inability of silver nanoparticle incorporated protein multilayers to achieve inhibitory levels.

Motivation The objective of the South African Avocado Growers Association (SAAGA) is to increase the demand of avocados by advertising, promoting and other means deemed fit by them. In order to promote and advertise a product, consumer research has to be done to determine the consumers' attitudes towards and beliefs concerning the product. These findings then need to be followed up by scientific studies, targeted at specific problems and target groups to yield scientific evidence. Little consumer research has been done on avocados and studies investigating the health effects of avocados are limited, with available literature only focussing on the cholesterol lowering effect of avocados. Objectives Firstly, the objective is to investigate the beliefs and attitudes of the South African consumer towards avocados and health; to determine whether gender, age group, race or living standard influence the consumers beliefs towards avocados. Secondly, the objective is to dispel the myth that avocados are fattening and should therefore be avoided in energy restricted diets; to examine the effects of avocados, a rich source of monounsaturated fatty acids, as part of an energy restricted diet on weight loss, serum lipids, fibrinogen and vascular function in overweight and obese subjects. Methods Consumer study: One thousand nine hundred and ninety-seven South African individuals, randomly selected from metropolitan areas in South Africa, participated in this survey. Data were weighed to reflect the adult metropolitan population based on gender, age and race distribution. The total population (10 695 000) was representative of both genders (5 423 000 men and 5 272 000 women) and major race groups (2 615 000 whites, 6 252 000 blacks, 1 255 000 coloureds and 573 000 Indians) from different age groups and living standards. The questionnaires were designed by a multidisciplinary team and consisted of seventeen foodrelated questionnaires, of which one questioned the beliefs regarding avocados. Trained field workers administrated questionnaires by conducting face-to-face interviews with consumers. The market research company, MARKINOR, was contracted to collect the data. Quantitative data was statistically analysed in order to generate the relevant descriptive statistics, cross tabulations and statistical tests. SUMMARY Dietary intervention study: Sixty one free-living volunteers (13 men; 48 women), with a mean (standard deviation) body mass index (BMI) of 32 (3.9) kg/m2, participated in this randomised, controlled parallel study. The subjects were paired according to gender, BMI and age and randomly assigned to one of two groups. The experimental group consumed 200 g of avocado (1 avocado) per day, substituting 30 g of other mixed dietary fats, and the control group excluded avocado from their energy restricted diet for six weeks. Seven-day isoenergetic menu plans were given according to mean energy requirements of both genders to provide 30% fat, 55% carbohydrates and 15% protein of total energy intake. Anthropometric measurements, physical activity, dietary intakes, blood pressure and arterial compliance were measured with standard methods at the beginning and end of the intervention. Fasting blood samples were drawn at the beginning and end of the intervention period. Results Consumer study: There were no practical significant differences in the consumers responses in terms of gender or age. Practical significant differences were found between different race and LSM (Living Standard Measure) groups for some variables. The overall response of consumers towards the effect of avocados on health, heart health, children's health and the health effects associated with the fat content of avocados were very positive. However, almost half the consumers are still not convinced of or are uncertain as to the cholesterol content of avocados, while 47% of the consumers still believe that avocados are fattening. More than 80% of the consumers agreed that avocados are a good source of vitamins and minerals, and 76% consider avocados to be a good source of fibre. Almost 70% of the consumers agreed that avocados are good for sportsmen and -women. Avocados were seen by 49% of the consumers to be an aphrodisiac. Dietary intervention study: Fifty-five subjects completed the study. Compliance with avocado intake in the experimental group was 94.6%. Anthropometric measurements (weight, body mass index and percentage body fat) decreased significantly in both groups during the study (p<0.001), and the change was similar in both groups. Serum lipid levels (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides), fibrinogen, blood pressure and arterial compliance did not change significantly within or between the two groups. SUMMARY Conclusions Consumer study: There are still a few myths and misconceptions that exist among some consumers regarding avocados, especially with regard to sexual functioning, cholesterol content, and fattening effect of avocados. The agricultural industry can use these results to plan different marketing campaigns focused on certain target groups to change the misperceptions concerning avocados and convey the positive nutritional value of avocados. Dietary intervention study: The consumption of 200 g avocado per day, within an energy restricted diet, does not compromise weight loss when substituted for 30 g of mixed dietary fat. The serum lipid levels, plasma fibrinogen, arterial compliance, as well as systolic and diastolic blood pressure were not affected by weight loss or avocado intake. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2004.

No description available.

Motivation The objective of the South African Avocado Growers Association (SAAGA) is to increase the demand of avocados by advertising, promoting and other means deemed fit by them. In order to promote and advertise a product, consumer research has to be done to determine the consumers' attitudes towards and beliefs concerning the product. These findings then need to be followed up by scientific studies, targeted at specific problems and target groups to yield scientific evidence. Little consumer research has been done on avocados and studies investigating the health effects of avocados are limited, with available literature only focussing on the cholesterol lowering effect of avocados. Objectives Firstly, the objective is to investigate the beliefs and attitudes of the South African consumer towards avocados and health; to determine whether gender, age group, race or living standard influence the consumers beliefs towards avocados. Secondly, the objective is to dispel the myth that avocados are fattening and should therefore be avoided in energy restricted diets; to examine the effects of avocados, a rich source of monounsaturated fatty acids, as part of an energy restricted diet on weight loss, serum lipids, fibrinogen and vascular function in overweight and obese subjects. Methods Consumer study: One thousand nine hundred and ninety-seven South African individuals, randomly selected from metropolitan areas in South Africa, participated in this survey. Data were weighed to reflect the adult metropolitan population based on gender, age and race distribution. The total population (10 695 000) was representative of both genders (5 423 000 men and 5 272 000 women) and major race groups (2 615 000 whites, 6 252 000 blacks, 1 255 000 coloureds and 573 000 Indians) from different age groups and living standards. The questionnaires were designed by a multidisciplinary team and consisted of seventeen foodrelated questionnaires, of which one questioned the beliefs regarding avocados. Trained field workers administrated questionnaires by conducting face-to-face interviews with consumers. The market research company, MARKINOR, was contracted to collect the data. Quantitative data was statistically analysed in order to generate the relevant descriptive statistics, cross tabulations and statistical tests. SUMMARY Dietary intervention study: Sixty one free-living volunteers (13 men; 48 women), with a mean (standard deviation) body mass index (BMI) of 32 (3.9) kg/m2, participated in this randomised, controlled parallel study. The subjects were paired according to gender, BMI and age and randomly assigned to one of two groups. The experimental group consumed 200 g of avocado (1 avocado) per day, substituting 30 g of other mixed dietary fats, and the control group excluded avocado from their energy restricted diet for six weeks. Seven-day isoenergetic menu plans were given according to mean energy requirements of both genders to provide 30% fat, 55% carbohydrates and 15% protein of total energy intake. Anthropometric measurements, physical activity, dietary intakes, blood pressure and arterial compliance were measured with standard methods at the beginning and end of the intervention. Fasting blood samples were drawn at the beginning and end of the intervention period. Results Consumer study: There were no practical significant differences in the consumers responses in terms of gender or age. Practical significant differences were found between different race and LSM (Living Standard Measure) groups for some variables. The overall response of consumers towards the effect of avocados on health, heart health, children's health and the health effects associated with the fat content of avocados were very positive. However, almost half the consumers are still not convinced of or are uncertain as to the cholesterol content of avocados, while 47% of the consumers still believe that avocados are fattening. More than 80% of the consumers agreed that avocados are a good source of vitamins and minerals, and 76% consider avocados to be a good source of fibre. Almost 70% of the consumers agreed that avocados are good for sportsmen and -women. Avocados were seen by 49% of the consumers to be an aphrodisiac. Dietary intervention study: Fifty-five subjects completed the study. Compliance with avocado intake in the experimental group was 94.6%. Anthropometric measurements (weight, body mass index and percentage body fat) decreased significantly in both groups during the study (p<0.001), and the change was similar in both groups. Serum lipid levels (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides), fibrinogen, blood pressure and arterial compliance did not change significantly within or between the two groups. SUMMARY Conclusions Consumer study: There are still a few myths and misconceptions that exist among some consumers regarding avocados, especially with regard to sexual functioning, cholesterol content, and fattening effect of avocados. The agricultural industry can use these results to plan different marketing campaigns focused on certain target groups to change the misperceptions concerning avocados and convey the positive nutritional value of avocados. Dietary intervention study: The consumption of 200 g avocado per day, within an energy restricted diet, does not compromise weight loss when substituted for 30 g of mixed dietary fat. The serum lipid levels, plasma fibrinogen, arterial compliance, as well as systolic and diastolic blood pressure were not affected by weight loss or avocado intake. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2004.

Carboxypeptidase N (CPN) is a plasma carboxypeptidase that was discovered in the 1960s as a regulator of inflammation and vascular tone. Through the removal of carboxy-terminal basic residues, CPN alters the activity or binding specificity of inflammatory mediators and vasoactive peptides. CPN shares significant homology with carboxypeptidases known to mediate antifibrinolysis through the removal of basic residues from fibrin clots, which would otherwise stimulate fibrinolysis. Despite the similarity of these enzymes, CPN is generally regarded as lacking a role in fibrinolysis. This thesis demonstrates that CPN is indeed a capable antifibrinolytic enzyme, and that the antifibrinolytic activity of CPN was previously undisclosed due to the presence of a circulating CPN inhibitor, which is likely the free CPN2 subunit. This inhibitor is described for the first time here. Furthermore, potential mechanisms of inhibition and mechanisms of enhancing activity of CPN are proposed based upon the additional structural characterization of CPN presented here.

Fibrinogen aggregation is the last step in blood coagulation. Inhibition of fibrinogen aggregation could lead to anticoagulation effects. However, there is no good method for the ready evaluation of fibrinogen coagulation. A commonly used path method is slow and requires an expensive instrument. In this project, we have developed a microplate reader and In evaluating inhibitors of fibrinogen coagulations there is no good method. As an important process in hemostasis, fibrinogen coagulation is often detected by micro-plate reader. In our test of fibrinogen coagulation, we improved the observing and analyzing method by using photograph to see concentration-depended effect of thrombin inhibitors on the coagulation. Three known thrombin inhibitors, AEBSF, APMSF and PMSF, were applied to develop the method for detecting the fibrinogen coagulation. The results showed our method is of accuracy in determination of the amount of fibrin when compared with other types of methods.