Physical trauma is responsible for over six million deaths annually, and of these roughly 40 percent result from acute traumatic coagulopathy (ATC) occurring in the first few hours of incidence. Patients who have developed ATC have significantly improved survivability when treated with tranexamic acid (TXA), a chemical inhibitor of the clot lysing enzyme plasmin. Current methods of detecting ATC are inadequate, lacking in either efficient speed, sensitivity, or cost. Hyperfibrinolysis (HF) is a key component of ATC and can be a result of excess plasmin activity. The following study observes effects of plasmin on hemostasis, and explores the use of silicon-based polydimethylsiloxane (PDMS) microfluidics measuring changes in electrical resistance as a method to detect HF. Coagulation was characterized by measuring turbidity of solutions containing fibrinogen and thrombin, and plasmin was incorporated to observe fibrinolysis and other plasmin-induced effects. It was found that high concentrations of plasmin caused a delay in the turbidity increase during coagulation. This lag phase may be a contributing factor to HF and ultimately ATC. Finally, the use of PDMS microfluidics to measure changes in electrical resistance to detect coagulation and fibrinolysis activity was supported. Resistance change adhered closely to traditional substrate-enzyme kinetics and plasmin-induced effects mimicked those which were observed in turbidity measurements. Further investment and development of this method of measurement could provide a faster, more accurate, and more inexpensive alternative to current techniques for measuring fibrinolysis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that acts as a molecular link between the coagulation and fibrinolytic cascades. TAFI can be activated by thrombin and plasmin but the reaction is enhanced significantly when thrombin is in a complex with the endothelial cofactor thrombomodulin (TM). The in vitro properties of TAFI have been extensively characterized. Activated TAFI (TAFIa) is a thermally unstable enzyme that attenuates fibrinolysis by catalyzing the removal of basic residues from partially degraded fibrin. The in vivo role of the TAFI pathway, however, is poorly defined and very little is known about the role of different activators in regulating the TAFI pathway. In the present study, we have constructed and characterized various TAFI mutants that are resistant to activation by specific activators. Based on peptide sequence studies, these mutants were constructed by altering key amino acid residues surrounding the scissile R92-A93 bond. We measured the thermal stabilities of all our mutants and found them to be similar to wild type TAFI. We have identified that the TAFI mutants P91S, R92K, and S90P are impaired in activation by thrombin or thrombin-TM, thrombin alone, and thrombin alone or plasmin, respectively. The TAFI mutants A93V and S94V were predicted to be resistant to activation by plasmin but this was not observed. The triple mutant, DVV was not activated by any of the aforementioned activators. Finally, we have used in vitro fibrin clot lysis assays to evaluate the antifibrinolytic potential of our variants and were able to correlate their effectiveness with their respective activation kinetics. In summary, we have developed activation resistant TAFI variants that can potentially be used to explore the role of the TAFI pathway in vivo. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-30 11:44:37.191

This study reports the isolation, purification and partial characterisation of the ostrich serpin, a2AP, as well as its target enzyme, ostrich plasmin, in its active and inactive proenzyme, viz. plasminogen, forms. Three different procedures were undertaken to isolate and purify ostrich a2AP. The first one involved L-lysine-Sepharose chromatography, ammonium sulfate fractionation, ion-exchange chromatography on Toyopearl Super-Q 650S, and ostrich plasminogen-Sepharose affinity chromatography. The second procedure replaced the latter chromatographic step with gel filtration on Sephadex G-200 and hydroxylapatite chromatography, while the third one employed instead the theoretically more efficient LBSI-Sepharose chromatographic step. The third procedure yielded purified ostrich a2AP, but the degree of purity and yield were relatively low. Ostrich plasminogen was highly purified after L-lysine-Sepharose chromatography and ostrich plasmin was obtained by the urokinase-activation of the purified ostrich plasminogen Ostrich a2AP revealed an Mr of 77-84 K and two isoelectric forms of pI 3.85 and 6.18. Nterminal sequence analysis showed ostrich a2AP to have only 2 out of 11 residues in common with both those of human and bovine a2AP. Ostrich a2AP showed the largest inhibitory effects on ostrich plasmin, followed by comm. bovine chymotrypsin, trypsin and plasmin, in that order, and it appeared to be a much less potent plasmin inhibitor than bovine aprotinin, but a much more potent one than the synthetic inhibitors, DFP and EACA. Ostrich plasminogen showed an Mr of 92 K and multiple isoelectric forms (~7) in the pI range 6.01-9.18, with a major one of pI 6.01. It showed a total of 775 amino acid residues and its N-terminal sequence showed ~53 percent identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin revealed an Mr of 78 K, two isoelectric forms of pI 4.07 and 6.01, and a total of 638 amino acid residues. N-terminal sequence analysis showed that 2-4 residues are identical to the 5 of human, cat, dog, rabbit, and ox plasmins. The pH and temperature optima of ostrich plasmin were determined as 8.0 and 40 oC, respectively. The thermodynamic and kinetic parameters of ostrich plasmin were computed, and plasmin was shown to prefer Lys to Arg residues in the S1 position. In conclusion, ostrich a2AP, plasminogen and plasmin showed definite similarities to their mammalian counterparts, but there were also significant differences.

At sites of vessel injury, thrombin mediates coagulation by catalyzing fibrin clot formation and platelet activation. Conversely, plasmin facilitates fibrinolysis by catalyzing the degradation of fibrin clots. Thrombin generation is most frequently measured in plasma samples using small molecule substrates, these substrates have low free thrombin efficiency and specificity, cannot measure thrombin’s exosite interactions and have limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells, require anticoagulation and the addition of supraphysiological concentrations of calcium. To overcome these limitations, we have designed and characterized a fluorescence resonance energy quenching (FREQ)-based thrombin sensor (FTS) protein. Compared to small molecule substrates, the FTS demonstrated high specificity for thrombin because it is not cleaved by thrombin inhibited by α2-macroglobulin and interacts with thrombin’s anion binding exosite I. The FTS can effectively measure thrombin generation in plasma and in whole blood. The FTS does not inhibit standard thrombin generation assays. Lastly, FTS-based thrombin generation in non-anticoagulated finger-prick blood is delayed but enhanced compared to citrated plasma. Similarly, plasmin generation is also restricted to plasma samples and measured using the fluorogenic Boc-Glu-Lys-Lys-AMC, which have low free plasmin efficiency and specificity, cannot measure plasmin’s kringle domain interactions and are insensitive to the effects of plasminogen activator inhibitor-1 (PAI-1). Here, we describe the design and characterization of a (FREQ)-based plasmin sensor (FPS) protein that demonstrated high efficiency for plasmin that is not inhibited by α2-macroglobulin when compared to Boc-Glu-Lys-Lys-AMC and interacts with plasmin’s kringle domain 5. The FPS measures plasmin generation in plasma, where it demonstrated greater sensitivity to tranexamic acid compared to Boc-Glu-Lys-Lys-AMC as well as sensitivity to PAI-1 and the effects of fibrin. Therefore, the FTS and FPS will broaden our understanding of thrombin and plasmin generation in ways that are not attainable with current methods. / Thesis / Doctor of Philosophy (PhD) / Increased blood clot formation leads to strokes and heart attacks while the inability to form blood clots when needed leads to bleeding disorders. In the body, thrombin makes blood clots while plasmin breaks them down. Therefore, clotting disorders occur in the absence of proper thrombin and plasmin function and appropriate diagnostics of these processes help to determine appropriate treatment. Currently available tests of thrombin and plasmin do not just measure their active forms and are restricted to plasma assays that do not reflect physiological settings and are not useful for quick diagnosis. Here, we describe the production of novel sensors for thrombin and plasmin that are specific for thrombin and plasmin’s active forms. These sensors have potential to be developed into portable diagnostic tests of thrombin and plasmin activity and to be valuable research tools for the development of better treatment options of blood clotting diseases.

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / This paper describes two methods for utilizing cancer associated proteases for targeting cancer therapy to the tumor. The first method is designing a drug delivery system based on liposomes that are sensitive to cancer associated proteases. Upon contact with the protease, the liposome releases its contents. The second method is designing a prodrug that is based on a porin isolated from Mycobacterium smegmatis. The porin is modified with protease consensus sequences, inhibiting its toxicity. Upon contact with the protease, the drug is activated. Protease sensitive liposomes were synthesized that were sensitive to urokinase plasminogen activator. This was done by synthesizing a cholesterol-anchored, uPA consensus – sequence-containing, acrylic acid block copolymer and using it to form a covalently bound polymer cage around the outside of a hypertonic liposome. Liposomes were synthesized that had a diameter of 136 nm. Upon addition of the polymer the diameter increased by 2.69 nm, indicating it had successfully embedded into the liposome membrane. After crosslinking with either a short peptide containing a lysine (so that it is a diamine) or ethylenediamine, the diameter increased between 5.33 nm and 14.1 nm (depending on the type and amount of the crosslinked). Fluorescence release assays showed that the polymer cage could add in excess of thirty atmospheres of osmotic pressure resistance, and, under isobaric conditions, would prevent release of much of the liposomal contents. Upon treatment with uPA, the polymer caged liposomes released a significantly larger amount of their contents making the liposomes protease sensitive. MspA was shown to be a very stable protein able to be imaged by AFM. AFM imaging demonstrated that MspA is able to form native pore structures in membranes making it a good imitator of the membrane attack complex. MspA was demonstrated to be highly cytotoxic, but poor at distinguishing between cells. Pro-MspA was synthesized by adding a hydrophilic peptide to MspA that prevents insertion. A uPA cleavage sequence embedded causes the MspA to become activated at the cancer site. This was demonstrated in tests against uPA and non-uPA producing cell lines.

Bovine plasmin activity was measured on H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide by following absorbance changes at 405 nm. Steady-state kinetic parameters Vmax, Km, KI, and KI' were estimated. Bovine plasmin is competitively inhibited by casein and has a Kcat of .0158 ΔA405/min/nM, Km of .107 mM substrate, and KI of .86 mg/ml casein. Bovine plasmin can be measured directly in bovine milk without interference from casein. A total of 380 milk samples from nineteen Holstein (one herd) and nineteen Jersey (one herd) cows was collected monthly during one lactation period. Samples from each cow were analyzed for fat, protein, plasmin activity, plasminogen, pH, SCC, clotting time, curd firming rate, and final curd firmness. Three age groups form each breed/herd were chosen; first, third, and fourth and later lactations. Plasmin activity in milk was most affected by lactation number, with milk from fourth- and later-lactation cows having higher activity than milk from first- or third- lactation cows. Plasmin activity in milk increased during lactation but was not affected by breed/herd, pH, protein, or fat. Plasminogen averaged 5.4 times the plasmin activity in milk and increased during the first five months of lactation. Plasmin activity was higher in milk collected ruing summer and fall but plasminogen was higher in milk collected during fall and winter. Percentage of the total (plasmin+plasminogen) enzyme activated to plasmin increased in late-lactation milk and in milk from fourth- and later-lactation cows. Plasmin activity did not affect any milk clotting parameters in this study. Increased protein in milk resulted in shorter clotting times. When statistically adjusted for protein content, clotting time was longer in milk from the Holstein herd compared to the Jersey herd. Curd firming rate was increased in milk with higher protein and fat. Milk samples collected in the fall had faster firming rates than milk from other seasons. Firming rates remained constant during lactation but increased with higher protein and fat content. Jersey herd milk produced firmer curd than Holstein herd milk and milk collected in the fall had firmer curd than during the other seasons.

No description available.

Hematopoietic cells are recruited to and co-opted by the growing tumor making expansive tumor growth possible. Although several cell types become associated with the growing tumor, macrophages play a fundamental role. The movement of macrophages across the basement membrane and through the extracellular matrix to the tumor site requires the activation of proteases, such as plasmin, at their cell surface. The proteolytic aspect of macrophage recruitment may represent an exploitable aspect of tumor growth in terms of therapeutic strategies. Here I show that the S100A10 protein facilitates the infiltration of macrophages into the site of tumor growth by stimulating the generation of the protease plasmin at their surface. Using a mouse model in which wild-type (WT) and S100A10-null mice are inoculated with tumor cells, a decrease in tumor-associated macrophages (TAMs) and greatly diminished tumor growth in tumors grown in S100A10-null mice was observed. Although tumor growth in S100A10-null mice could be restored by intraperitoneal injection of WT macrophages, S100A10-null macrophages only restored tumor growth when directly injected into the tumor. Lastly, selective depletion of macrophages from a WT mouse by liposome encapsulated clodronate treatment resulted in similar tumor growth deficits as in the S100A10-null mouse. These results highlight a new role for the S100A10 protein in the recruitment of TAMs to the tumor site and demonstrate a potential therapeutic strategy in which the tumor associated cells may be targeted.

The role of plasmin and plasminogen activators (PA) in bovine lactation and porcine ovulation has been examined. There is no difference in the activation pattern of plasminogen to plasmin throughout the whole range of somatic cell counts (SCC) and from third to ninth month in lactation. The ratio of (plasminogen + plasmin)/plasmin, which serves as an index of the activation process, was 7.27 during early (first and second month) and 4.23 during late lactation (tenth month) and both values are different (p $<$ 0.01) from all the other ratios throughout the whole range of SCC and from third to ninth month in lactation suggesting limited and increased activation of plasminogen to plasmin during early and late lactation, respectively. Macrophages produce but they do not secrete urokinase-PA, suggesting a minor role in influencing milk plasmin. Somatotropin administration resulted in a suppression of milk plasmin in vivo. Insulin-like growth factor-1 (IGF-1), the most likely mediator of the effects of somatotropin on the bovine mammary gland, inhibited the induction of tissue-PA (t-PA) production which is observed when mammary epithelial cells are cultured in the absence of IGF-1. Plasmin and t-PA increased while PA inhibitor-1 decreased in porcine granulosa, theca interna cells and follicular fluid just prior to the time of expected ovulation suggesting a role for plasmin in follicle rupture.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.