Zinc (Zn2+) is an abundant metal ion that circulates in the body. Within hemostasis, Zn2+ participates in platelet aggregation, coagulation, and fibrinolysis. At the site of injury, Zn2+ released from activated platelets accelerates coagulation and attenuates fibrinolysis. How Zn2+ regulates these processes on a molecular level has not been extensively examined. We hypothesized that Zn2+ released from platelets binds serine proteases involved in coagulation or fibrinolysis and modulates their proteolytic activity, thus controlling the rate of clot formation and lysis. We show that Zn2+ concentrations released from activated platelets are sufficient to modulate clot formation and fibrinolysis. We show in vitro that Zn2+ binds to fibrinogen with high affinity, accelerates fibrin monomer polymerization, and modifies clot structure. Zn2+ promotes clot stability by increasing fiber diameter, reducing fibrin fiber elasticity, and increases clot porosity. Although it might be predicted that these modifications would enhance clot degradation by enabling greater distribution of lytic enzymes through the more porous fibrin network, we showed the opposite. Thus, we demonstrated that Zn2+ binds to plasminogen activators and plasmin with high affinity and down-regulates their protease activity, which delays lysis. This adds to previous studies that showed that both coagulation and fibrinolysis are regulated by Zn2+ ions. These data support the functional role of Zn2+ in hemostasis. / Thesis / Doctor of Philosophy (PhD)

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Platelets permeabilized by high voltage electric discharges have provided a valuable model system in which to analyse the roles of Ca²⁺ ions and guanine nucleotides in the regulation of secretion by exocytosis. In the present study, the effects of fluoride or fluoroaluminate and of vanadate or pervanadate on secretion of platelet dense granule constituents, and the roles of activation of phospholipase D (PLD), phospholipase C (PLC) and protein kinase C (PKC) in secretion, have been investigated. Electropermeabilized human platelets containing [¹⁴C]5-HT in their dense granules were suspended in a glutamate medium containing ATP and incubated for 10 min at 25°C with, variously, Ca²⁺ buffers, KF/AlCl₃, vanadate/H₂O₂, guanine nucleotides and phorbol 12-myristate 13-acetate (PMA). KF/AlCl₃, which activates heterotrimeric G proteins but not low-M, GTP-binding proteins, caused a Ca²⁺ -dependent [¹⁴C]5-HT secretion; maximal effects were obtained with 10 mM KF plus 10 μM AlCl₃ at a pCa of 6, when 53% of [¹⁴C]5-HT was released. Secretion induced by KF/AlCl₃ in the presence of Ca²⁺ correlated with the stimulation of [3H]diacylglycerol accumulation in permeabilized platelets containing [³H]arachidonate-labelled phospholipids. KF/AlCl₃ also stimulated the phosphorylation of pleckstrin (P47) in permeabilized platelets incubated with [γ-³²P]ATP, indicating the activation of PKC. In the absence of Ca²⁺ (pCa > 9), KF/AlCl₃ caused none of the above effects. These actions of KF/AlCl₃ were attnbutable to the activation of PLC, since KF/AlCl₃ also stimulated the formation of [³H]inositol phosphates in [³H]inositol-labelled permeabilized platelets in the presence of Ca²⁺. PLD activity, measured as the formation of[³H]phosphatidylethanol (PEt) from [³H]arachidonate-labelled phospholipids in the presence of ethanol, could not be detected after stimulation of platelets by KF/AlCl₃ in the absence or presence of Ca²⁺. However, KF/AlCl₃ inhibited the [³H]PEt formation (PLD activity) induced by GTPγS. In the absence of Ca²⁺ (pCa >9), the inhibitory effects of KF/AlCl₃ on [¹⁴C]5-HT secretion induced by GTPγS alone or GTPγS plus PMA correlated well with their inhibitory effects on [³H]PEt formation. At pCa 6, KF/AlCl₃ had only a small inhibitory effect on GTPγS-induced secretion and inhibited GTPγS-induced PLD activity more strongly than GTPγS-induced PLC activity. These results suggest that PLD is important for Ca²⁺ -independent secretion, and that, although both PLD and PLC may play roles in Ca²⁺ -dependent secretion, PLC is likely to be the more important. In the presence of Ca²⁺, either vanadate or H₂O₂ caused concentration-dependent stimulations of [¹⁴C]5-HT secretion, [³H]DAG formation and [³H]PEt formation. At pCa 6, low concentrations of vanadate and H₂O₂, which would be expected to form pervanadate, acted synergistically to stimulate [¹⁴C]5-HT secretion, which correlated with [³H]DAG formation. However, vanadate with H₂O₂ had a biphasic effect on PLD activity that did not correlate with secretion. In addition, at pCa 6, GTPγS-induced PLD activity was abolished by vanadate with H₂O₂, whereas GTPγS-induced secretion and PLC activity were only partially inhibited. These results support the idea that both PLC and PLD are involved in the regulation of secretion but have different contributions to Ca²⁺ dependent and Ca²⁺ -independent secretion. The results are consistent with activation of platelet PLC by a heterotrimeric G protein, but suggest that different mechanisms, possibly involving a low-M, GTP-binding protein, may be involved in the regulation of PLD activity. / Thesis / Master of Science (MSc)

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Increased platelet activation and inflammation play a key role in atherothrombosis. Patients with peripheral arterial disease are at increased risk of adverse cardiovascular events, particularly at the time of surgery. We postulated that the increase in peri-operative cardiovascular events is mediated by increased platelet activation and inflammation. We hypothesized that peri-operative dual anti-platelet therapy would improve biomarkers of atherothrombosis without causing unacceptable bleeding in patients undergoing surgery for critical limb ischaemia (CLI). Prior to interventional study, I validated a sensitive flow cytometric technique for the reproducible assessment of in vivo platelet activation in patients with peripheral arterial disease. Thirty patients with stable claudication, attended on two occasions to permit within-day and between-day comparisons. A variety of platelet activation markers were compared to the gold standard of platelet–monocyte aggregation. Platelet-monocyte aggregation demonstrated comparable within-day (mean difference ± co-efficient of reproducibility; 0.9±15.4%) and between-day reproducibility (2.0±12.4%). Plateletmonocyte aggregates correlated well with other platelet activation markers (P selectin r=0.30; Platelet CD40L r=0.41; Platelet microparticles r=0.27; P≤0.026) and monocyte activation markers (monocyte CD40 r=0.27;monocyte CD11b r=0.47; P≤0.026). In a cross sectional study, I demonstrated that resting in vivo platelet activation and inflammation was increased in patients with CLI in comparison to healthy controls, patients with stable claudication and those undergoing treatment for acute coronary syndromes. In addition, platelet activation and inflammation throughout the peri-operative period was markedly increased in CLI patients compared with non-vascular patients undergoing arthroplasty, and exceeded the rise attributable to the stress of surgery itself. In a prospective double-blind randomised controlled trial, 108 patients undergoing infra-inguinal revascularisation or amputation for CLI were maintained on aspirin (75 mg daily) and randomised to clopidogrel (600 mg prior to surgery, and 75 mg daily for 3 days; n=50) or matched placebo (n=58). Peri-operative in vivo platelet activation and inflammation, cardiac-Troponin I (c-TnI) release and bleeding outcomes were recorded. Clopidogrel reduced markers of platelet activation and inflammation before surgery and throughout the post-operative period. Overall, there were 18 troponin-positive events (16.7%), with half of the troponin rises (9) occurring prior to surgery. Patients with postoperative elevations in c-Tn I had significantly greater levels of pre-operative platelet-monocyte aggregation, monocyte CD40, IL-6 and hsCRP. However, despite reducing platelet and inflammatory markers, clopidogrel did not have a direct effect on peri-operative c-Tn I. There was no increase in major life-threatening or minor bleeding, although blood transfusions and wound haematomas were significantly increased. Using sensitive and validated methodologies, I have provided a detailed examination of in vivo platelet activation and inflammation in high-risk vascular surgical patients. This approach has provided the first objective assessment of the risks and benefits of intensive peri-operative anti-platelet therapy in this patient group. Dual anti-platelet therapy reduced biomarkers of atherothrombosis without causing unacceptable bleeding. However, large-scale clinical trials would be required to confirm whether these reductions translate into improvements in clinical outcome.

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Philosophiae Doctor - PhD / Wound healing is a complex process that involves several overlapping and interacting biological pathways. The consequences of delayed or abnormal wound healing may result in tissue formation that has impaired function or structural abnormalities. As a result, clinicians have sought ways to enhance this process. Recently, the use of autologous platelet concentrates have become popular in the management of wound healing sites. However, controversy exists as to how these biomaterials should be prepared and applied. We therefore sought to investigate whether a biologically viable and clinically effective platelet concentrate could be prepared using standard laboratory equipment. The findings are presented in a series of articles that have been published in peer-reviewed journals. The results suggest that the experimental platelet concentrate produced, has a morphological structure that consists of a dense fibrin network intermingled with platelets, has the ability to accelerate cellular growth in-vitro, has no adverse effects on cells in-vitro, can concentrate and release a systemically ingested antibiotic over a period of 24 hours in-vitro, can be stored for at least 60 minutes without showing signs of deterioration, and has shown clinical evidence of accelerating wound healing in sinus augmentation and alveolar ridge preservation procedures. The reduced cost of producing such a biomaterial allows it to be available to resource poor settings and to wider range of healthcare providers as compared to standard platelet concentration techniques. Further studies are required to investigate the clinical potential of this promising biomaterial.

Chronic renal failure patients develop a coagulopathy primarily due to reversible platelet dysfunction. This coagulopathy makes certain anaesthetic techniques and procedures such as neuraxial anaesthesia and invasive line placement possibly contra-indicated or risky. There is no evidence to suggest that the degree of platelet dysfunction is proportional to the degree of renal dysfunction. In this research project the platelet function of 39 end stage renal failure patients, who received regular dialysis and who presented to theatre for vascular access, was assessed using the thromboelastogram. A bleeding time was also performed pre-operatively. A linear regression model was used to determine if the bleeding time, plasma urea, plasma creatinine or creatinine clearance could predict maximum amplitude (and therefore clot strength) on the thromboelastogram. No such regression could be found. The clinical implication of this result is that there exists no "safe" plasma urea or creatinine, below which it is safe to perform procedures which are contra-indicated in coagulopathies. The degree of renal dysfunction did not predict the degree of platelet dysfunction. Since dialysis reverses the platelet dysfunction, the question that should be asked before performing such a procedure is not "how severe is the renal dysfunction?" but rather "has the patient been receiving regular dialysis?"