Thesis (Ph. D.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 166-197).

Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on June 11, 2009). Includes bibliographical references.

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are associated with increased risk of atherothrombotic disease. Lp(a) is a unique lipoprotein consisting of a low density lipoprotein-like moiety covalently linked to apolipoprotein(a) (apo(a)), a homologue of the fibrinolytic proenzyme plasminogen. Apo(a) is extremely heterogeneous in size with small isoforms being independently associated with increased cardiovascular risk. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue-type plasminogen activator (tPA)-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin-dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu1-plasminogen to Lys78-plasminogen. Additionally, abnormal fibrin clot structures have been associated with both an increased risk of cardiovascular disease and elevated Lp(a) levels. Similarly, oxidized phospholipids have been implicated in the development of cardiovascular disease, and are not only preferentially carried by Lp(a) in the plasma but have also been shown to covalently-modify both apo(a) and plasminogen. In this thesis, we built upon the understanding of the role of apo(a) in plasminogen activation on the fibrin/degraded fibrin surface by determining that: (i) apo(a) inhibits plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion and identifying the critical domains in apo(a) responsible for this effect, (ii) apo(a) isoform size does not affect either the inhibition of tPA-mediated plasminogen activation or the inhibition of plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion, (iii) apo(a) modifies fibrin clot structure to form more dense clots with thinner fibers and reduced permeability, modifications that enhance the ability of apo(a) to inhibit tPA-mediated plasminogen activation and (iv) the phosphorus content of apo(a) affects its ability to inhibit tPA-mediated plasminogen activation and the phosphorus content of plasminogen affects its ability to be activated by tPA. By understanding these individual reactions, each of which has the potential to affect the broader fibrin clot lysis process, we have expanded our understanding of the overall effect of Lp(a)/apo(a) in the inhibition of plasminogen activation on the fibrin/degraded fibrin surface and thus broadened our understanding of how Lp(a)/apo(a) may mediate the inhibition of thrombolysis in vivo. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-12-14 08:26:54.99

Lic.-avh. (sammanfattning) Umeå : University. / Härtill 3 uppsatser.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

We observed that platelets, which mediate thrombus formation, also enhance fibrinolysis by single-chain urokinase (scuPA).  Preliminary data suggested that this enhancement was due to platelet thrombospondin (TSP), which, depending upon the specifics of the environment, changed conformation, influencing its role in the fibrinolytic system.  Results showed that the activity of scuPA was enhanced by platelets, regardless of platelet treatment or protein release, and that TSP could not explain the platelet effect.  Investigation of the underlying mechanism, using the non-cleavable mutant scuPA (K158E) and protease inhibitors, showed that the platelet-dependent enhancement of scuPA arose from the activation to uPA by a serine protease.  Factor VII activating protease (FSAP) was not the protease responsible, since inhibition of platelet FSAP with function-blocking antibodies did not inhibit the platelet effect.  Comparison of plasminogen and plasma kallikrein, using an array of inhibitors, showed that both candidates mirrored the platelet effect.  Further results, including those from assessment of protease activity on platelets against chromogenic substrates and from the evaluation of uPA formation over time, were consistent with the involvement of plasminogen.  In addition, experiments with platelets from plasminogen-deficient mice showed that platelets lacking plasminogen no longer activated scuPA.  The enhancement of scuPA was found to be platelet-dependent, even in plasma; scuPA activation was more efficient when plasminogen was associated with the platelet membrane, compared to in solution; and the presence of membranes was essential to induce rapid lysis by scuPA.  Our findings indicate that platelets stimulate fibrinolysis by scuPA via local activation to uPA by platelet-associated plasminogen, which is activated by scuPA, consistent with a system of reciprocal activation.

Effects of phorbol myristate acetate (PMA), dibutyryl cyclic AMP (dbcAMP), 6-dimethylaminopurine (6-DMAP), okadaic acid (OA), cycloheximide (CHX), actinomycin D (AcD) and tunicamycin (TuM) on plasminogen activator (PA) activity and maturation rate in bovine and porcine oocyte-cumulus cell complexes (BOCC and POCC, respectively) in vitro were determined. Plasminogen activator activity was measured by SDS-PAGE, casein-agar zymography and densitometry. Three plasmingen-dependent lytic zones (92-95, 71-73 and 49-51 kD) and one PA inhibitor (52 kD) were observed in BOCC. Immunoprecipitation and amiloride sensitivity suggested that the 49-51 kD protease is a urokinase type PA (uPA), the 71-73 and 92-95 kD proteases are a tissue type PA (tPA) and tPA-PAI complex, respectively, and the PAI is PAI-1. In POCC, two plasminogen activators (71-78 and 93-96 KD) were observed. Lack of amiloride sensitivity suggested that the 71 -78 kD band is a tPA and the 93-96 kD band is possibly a tPA-PAI complex. Increasing dbcAMP in the culture medium increased activity in both BOCC and POCC in dose-dependent fashion (P<0.05). In BOCC cultured with PMA, total PA activity increased, however high concentrations of PMA (10 and 100 ng/ml) decreased tPA activity in matured POCC. Plasminogen activator activity decreased in 6-DMAP, actinomycin D and cycloheximide and oocyte maturation was also inhibited in these treatments. When POCC were treated with 25 nM OA, uPA activity was observed. Plasminogen activator activity increased in either BOCC or POCC treated with up to 25 nM OA, however PA activity decreased at concentrations greater than 75 nM (P<0.05). Incubation of BOCC with tunicamycin reduced the molecular mass of tPA and tPA-PAI complex and PAI-1 by 5-10%, however PA activity was not inhibited. These data suggest that BOCC matured in vitro produce uPA, tPA and PAI-1 however POCC produce only tPA and PAI. The production of PA and PAI by either BOCC or POCC is associated with oocyte maturation and influenced by stimulators of the protein kinase A and C, modulators of intracellular phosphorylation and metabolic inhibitors. / Graduation date: 1993

Graduation date: 1990

published_or_final_version / Biochemistry / Master / Master of Philosophy