<p>This thesis describes the Chandler loop, which makes it possible to conduct studies in vitro of molecular and cellular interactions between whole blood and stents. It was possible to monitor activation and inhibition of the cascades systems, leukocytes and platelets by combining different platelet inhibitors and heparin coating of stents. The clinical study was performed on patients with ACS undergoing PCI and stent implantation. In this study platelet activation markers P-selectin, and αIIb/β3 as well as inflammatory markers were followed from baseline during the first 48 hours post-PCI. The same parameters were evaluated in healthy controls for comparison at baseline. </p><p>In vitro: The activation of blood in the Chandler loops were more pronounced for unmodified stent grafts than for partially heparin coated stent grafts. Heparin coated stent grafts dreated the same activation as the loops alone. This indicated that the Chandler loop system was a feasible tool for evaluation of blood compability of stents. </p><p>Heparin coating of stents significantly reduced TAT, CD11b and platelet activation. The combination of a heparin coated stent and abciximab reduced TAT and contact activation, as compared to abciximab or heparin coating alone. </p><p>Heparin coating of stents in combination with AR-C69931MX resulted in a significant reduction in TAT and preservation of the platelet count but had no effect on contact activation. </p><p>Clinical study: Abciximab resulted in an almost total inhibition of fibrinogen binding to platelets and persisted throughout the observation period. Clopidogrel effectscould be observed at four hours but was more pronounced at 24 hours. </p><p>P-selectin expression did not differ over time between groups, indicating that platelet activation with α-granule secretion was not affected by abciximab treatment. </p><p>The hs-CRP, C3a and sC5b-9 levels increased 24 to 48 hours after PCI in patients with ACS. FXIIa-C1inh was reduced in ACS patients receiving abciximab as compared to controls. The elevated bFGF levels at baseline returned to the levels observed in controls four hours after PCI and stent implantation, whereas an increase in VEGF was observed 24 hours post-PCI.</p>

This thesis describes the Chandler loop, which makes it possible to conduct studies in vitro of molecular and cellular interactions between whole blood and stents. It was possible to monitor activation and inhibition of the cascades systems, leukocytes and platelets by combining different platelet inhibitors and heparin coating of stents. The clinical study was performed on patients with ACS undergoing PCI and stent implantation. In this study platelet activation markers P-selectin, and αIIb/β3 as well as inflammatory markers were followed from baseline during the first 48 hours post-PCI. The same parameters were evaluated in healthy controls for comparison at baseline. In vitro: The activation of blood in the Chandler loops were more pronounced for unmodified stent grafts than for partially heparin coated stent grafts. Heparin coated stent grafts dreated the same activation as the loops alone. This indicated that the Chandler loop system was a feasible tool for evaluation of blood compability of stents. Heparin coating of stents significantly reduced TAT, CD11b and platelet activation. The combination of a heparin coated stent and abciximab reduced TAT and contact activation, as compared to abciximab or heparin coating alone. Heparin coating of stents in combination with AR-C69931MX resulted in a significant reduction in TAT and preservation of the platelet count but had no effect on contact activation. Clinical study: Abciximab resulted in an almost total inhibition of fibrinogen binding to platelets and persisted throughout the observation period. Clopidogrel effectscould be observed at four hours but was more pronounced at 24 hours. P-selectin expression did not differ over time between groups, indicating that platelet activation with α-granule secretion was not affected by abciximab treatment. The hs-CRP, C3a and sC5b-9 levels increased 24 to 48 hours after PCI in patients with ACS. FXIIa-C1inh was reduced in ACS patients receiving abciximab as compared to controls. The elevated bFGF levels at baseline returned to the levels observed in controls four hours after PCI and stent implantation, whereas an increase in VEGF was observed 24 hours post-PCI.

Platelets play a pivotal role in hemostasis and thrombosis. It induces vascular retraction and clot formation through platelets activation and signal transmission, which promote ligands expressing on the surface of platelets, such as glycoproteins and P-selectin. Some surface glycoproteins gatherd to form complexes after activation. It bound to extracellular receptors such as collagen and thrombin to induce aggregation, which could also be induced by releasing agonists, such as arachidonic acid, adenosine diphosphate, epinephrine, serotonin and fibrinogen, to active the nearby platelets. The standard process of plateletapheresis in the Blood Center was to hold the platelets in still for one hour before stored on a vibrator. The process of holding platelets still for one hour before storage was omitted in some hospitals. It was not clear whether to omit the process has any effect on the quality of platelets. The expressions of P-selectin and vWF receptor, CD42b (Gp Ib£\) on platelets were analyzed by flow cytometry in this study. No significant differences (p= 0.77 and p= 0.62, respectively) were found. Similar results were obtained when functions of platelets were evaluated by agonists. It was concluded that leaving the platelets in room temperature for one hour to recover before keeping it on a vibrator would not enhance the functions of platelets aggregation significantly.

Rheumatoid Arthritis (RA) is an autoimmune disease characterized by inflammation and eventual destruction of the synovial joints. The role of platelets in the pathophysiology of arthritis has only recently been established. Because antibodies to CD44 can deplete platelets, we hypothesized that these antibodies might be effective in arthritis through a platelet-depletion mechanism. We examined the K/BxN passive transfer mouse model of arthritis and found that most antibodies against CD44 were capable of depleting platelets. However, anti-CD44 treatment is effective when administered during developing arthritis, while anti-platelet treatment was not. While CD44 antibodies may be therapeutic through platelet-dependant and independent mechanisms, the ability of CD44 antibodies to decrease platelet counts does not seem to be the critical factor in resolving arthritis in the K/BxN model.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 72-104).

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFIa also possesses anti-inflammatory properties. Although liver is the main source of plasma TAFI, platelet-derived TAFI has also been reported. An alternatively spliced TAFI variant resulted from the skipping of exon 6 and a 52-base deletion in exon 10 of CPB2 mRNA (∆6+10) was described to be brain specific. This TAFI variant is reputed to possess a secretase-like activity that cleaves β-amyloid precursor protein to form β-amyloid, a process involved in the onset of Alzheimer's disease. In this thesis, we report the identification of CPB2 mRNA and TAFI protein in various vascular and inflammatory cells. Specifically, we describe the expression of CPB2 mRNA in the megakaryocytic cell lines MEG-01 and Dami, the monocytic cell line THP-1, and peripheral blood mononuclear cells. TAFI protein was detected in differentiated Dami and THP-1 cells. We next describe the effect of external stimuli such as phorbol myristate acetate (PMA) on CPB2 expression in Dami and THP-1 cells. We found that PMA treatment increases both CPB2 mRNA abundance and promoter activity in Dami cells, and decreases both CPB2 mRNA abundance and promoter activity in THP-1 cells. Deletion analysis of the CPB2 promoter indicated cell-type specific regulation of CPB2 gene expression. Finally, we evaluated the expression of alternatively spliced CPB2 mRNA variants in hepatic and non hepatic cells. We found that exon 6 skipping variants are expressed in all cell types of interest. The variant previously reported to be brain specific was also found to be expressed in platelets. We found that the alternatively spliced TAFI variants accumulated inside the cells in a non-secretable, hypoglycosylated form and showed no carboxypeptidase activity. Taken together, this thesis provides further evidence supporting the hypothesis that platelet-derived TAFI is originated from CPB2 gene expression in megakaryocytes. Moreover, our data imply a potential for site-specific anti-inflammatory control provided by macrophage-derived TAFI. Alternative splicing of the CPB2 mRNA may give rise to variants with an intracellular role, perhaps as a peptidase chaperone, and may modulate the synthesis of secretable TAFI. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-09-26 21:22:33.348

Rheumatoid Arthritis (RA) is an autoimmune disease characterized by inflammation and eventual destruction of the synovial joints. The role of platelets in the pathophysiology of arthritis has only recently been established. Because antibodies to CD44 can deplete platelets, we hypothesized that these antibodies might be effective in arthritis through a platelet-depletion mechanism. We examined the K/BxN passive transfer mouse model of arthritis and found that most antibodies against CD44 were capable of depleting platelets. However, anti-CD44 treatment is effective when administered during developing arthritis, while anti-platelet treatment was not. While CD44 antibodies may be therapeutic through platelet-dependant and independent mechanisms, the ability of CD44 antibodies to decrease platelet counts does not seem to be the critical factor in resolving arthritis in the K/BxN model.

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates.

Thesis (Ph.D.) -- McMaster University, 1997 / Includes bibliographical references. Also available via World Wide Web.