A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Master of Pharmacy Johannesburg 2001. i / The use of a low molecular weight heparin, enoxaparin was evaluated in the prevention and treatment of deep vein thrombosis. Patterns of use were analysed and measured against pre-determined criteria with a view to promoting optimal use and identifying those factors, which may contribute to safer use of the drug. / IT2018

Patients in the intensive care unit are in critical condition which is often accompanied by a coagulation disorder. Sepsis as a leading cause of death in critically ill patients may be associated with both hypercoagulable state with microtrombi formation in microcirculation and with increased production of endogenous heparinoids with inhibitory effects on blood clotting. Central venous catheter and arterial catheter are established in patients for hemodynamic monitoring and these are flushed with heparin to prevent their closure. Both inputs are used for blood sampling for laboratory tests such as blood count and coagulation parameters, including thromboelastography (TEG). In the first step of the work, arterio-venous differences in coagulation parameters were investigated in patients with sepsis. Higher concentration of D-dimers and lower antithrombin activity were found in venous blood. This finding can be explained by increased antithrombin consumption in hypercoagulable state and reactive hyperfibrinolysis. Inconsistency in the site of blood sampling may then lead to misinterpretation of the pathophysiological processes in the body. No significant differences were found in TEG parameters. In the second step of the work we examined how heparin commonly used for catheter flushing affects TEG-assessed...

Indiana University-Purdue University Indianapolis (IUPUI) / Nanoparticles (NPs) are an emerging technology in biomedical engineering with opportunities in diagnostics, imaging, and drug delivery. NPs can be prepared from a wide range of organic and/or inorganic materials. They can be fabricated to exhibit different characteristics for biomedical applications. The goal of this thesis was to develop NPs with tunable surface properties for selective cell targeting. Specifically, polyelectrolyte complexes composed of heparin (Hep, a growth factor binding glycosaminoglycan) and poly-L-lysine (PLL, a homopolymeric lysine) were prepared via a pulse sonication method. The Hep/PLL core NPs were further layered with additional Hep, tetrazine (Tz) modified Hep, or dextran sulfate (DS). The addition of Tz handle on Hep backbone permitted easy modification of NP surface with norbornene (NB) modified motifs/ligands, including inert poly(ethylene glycol) (PEG), cell adhesive peptides (e.g., RGD), and/or fluorescent marker. Both Hep and DS coated NPs could be readily internalized by J774A.1 monocytes/macrophages, whereas PEGylated NPs effectively reduced cellular uptake/recognition. The versatility of this NP system was further demonstrated by laying DS on the Hep/PLL NP surface. DS-coated NPs were recognized by J774A.1 cells more effectively. Furthermore, DS-layered NPs seemed to reduce IL-10 production on a per cell basis, suggesting that these NPs could be used to alter polarization of macrophages.

Der postoperative Blutverlust stellt für Patienten, die sich einem herzchirurgischen Eingriff unterziehen müssen, ein nicht zu unterschätzendes Risiko dar. Blutverlust erfordert Revisionen und Bluttransfusionen. Beides kann zu einem längeren Krankenhausaufenthalt und zu perioperativen Komplikationen führen. In Anbetracht der dadurch erhöhten Mortalität und auch der hohen Kosten, die sich durch einen solchen Verlauf ergeben können, ist die Minimierung des Blutverlustes ein wichtiges Ziel. Bei Operationen mit einer Herz-Lungen-Maschine ist die Applikation von Heparin zur Hemmung der Blutgerinnung erforderlich. Nach Beendigung der EKZ erfolgt die Antagonisierung durch Protamin. In unserer Studie haben wir den Einfluss des Protamin-Heparin-Quotienten auf den postoperativen Blutverlust und den Transfusionsbedarf untersucht. An einem Kollektiv von 182 Patienten konnten wir retrospektiv zeigen, dass Frauen von einer strengen Antagonisierung profitieren. In Abhängigkeit vom Geschlecht zeigte sich zunächst ein Unterschied bezüglich des Verbrauchs an EK gesamt (p = 0,019) und intraoperativ (p < 0,001). Zudem konnten wir zeigen, dass Frauen, die mit einem großen Protamin-Heparin-Quotienten antagonisiert wurden, eine niedrigere Verlustrate von Blut, erfasst als Drainagemenge pro KG [ml/kg] (p = 0,032) beziehungsweise pro KOF nach Mosteller [ml/m²] (p = 0,040), aufwiesen. Auch der Transfusionsbedarfs an EK war bei diesem Kollektiv geringer (p = 0,048). In einer Extremgruppenanalyse mit Vergleich der Patienten mit dem höchsten und dem geringsten Protamin-Heparin-Quotienten konnten diese Ergebnisse bestätigt werden. Auch hier zeigten sich Unterschiede bezüglich Drainagemenge pro KG [ml/kg] (p = 0,025) und Gesamtbedarf an EK (p = 0,011). Entsprechend der erhobenen Ergebnisse ist ein Verhältnis von Protamin zu Heparin von 1:1 im herzchirurgischen Patientenkollektiv anzustreben . Weitere prospektiv randomisierte und kontrollierte Studien sind nötig, um diese ersten Ergebnisse zu bestätigen und eine Standardisierung der Antagonisierung von Heparin zu erarbeiten. Insbesondere die unterschiedlichen Auswirkungen bei Frauen und Männern bieten einen interessanten Anhaltspunkt, der in der Literatur so noch nicht untersucht wurde. / Objectives: Usage of cardiopulmonary bypass during cardiac surgery influences blood coagulation and leads to increased postoperative bleeding and transfusion requirements. The dosage of protamine used for antagonisation of heparin is still controversial. We analyzed the antagonisation’s influence on postoperative bleeding and transfusion requirement. Methods: 182 patients undergoing coronary artery bypass grafting (CABG), cardiac valve surgery or combined procedures in 2005 and 2006 were included in a retrospective matched pair study. They were matched for age, sex and operative approach. Four groups of similar size were built on the basis of the ratio of protamine to heparin (group 1: m=0,788; group 2: m=0,995; group 3: m=1,117; group 4: m=1,384). Results: Analysis of variance (ANOVA) with the four groups of protamine-heparin-ratio and sex (male vs. female) and its interaction was calculated. The postoperative chest tube drainage per kg weight and requirement of erythrocyte concentrates are significantly increased in female members of groups with lower ratio (p=0,032 and p=0,048). Furthermore the comparison of group 1 and 4 shows significant differences regarding intraoperative usage of platelet concentrates (p=0,039). The multivariate analysis of variance (MANOVA) considering the four groups and sex shows significant increases of chest tube drainage per kg weight (p=0,025) and need for erythrocyte concentrates (p=0,011). Conclusion: According to these results women undergoing on-pump cardiac surgery should be antagonized with a stricter ratio of protamine to heparin. Thus it is possible to reduce both postoperative blood loss and transfusion requirements. We recommend a protamine-heparinratio of 1:1 according to the group showing the lowest chest tube drainage.

No / Background: In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2. Experimental Design: Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity. Results: Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing 16 saccharide residues were investigated. Conclusions: Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.

No description available.

To prevent clotting during dialysis, heparin is used to line the tubing which blood flows through. Unfortunately, many side effects arise from taking heparin, especially when it is used for an extended period of time. As such, long-term exposure for individuals undergoing dialysis every day is unavoidable. To prevent the solubilized heparin from entering the bloodstream, a polymer-based natural material is being investigated. This materials properties include reduction of coagulation and elimination of the long-term effects of heparin such as heparin induced thrombocytopenia and osteoporosis. Cellulose nanocrystals (CNCs) contain the same 1,4 linked pyranose backbone structure as heparin along with desirable mechanical properties, like high stiffness and anisotropic shape. By altering the functionalization on the surface of CNCs to closely mirror that of heparin, it should be possible to make a biomimetic material that counteracts blood clotting, while not introducing soluble small molecule anti-coagulants into the body. Through blood assays and platelet fixing analysis, we have been able to show that this change in functionalization does reduce coagulation. Surface chemistry of CNCs were modified from 'plain' CNCs (70 mmol SO3-/kg residual from hydrolysis) to 500 mmol COO-/kg (TEMPO oxidized) and 330 mmol SO3-/kg CNC (sulfonated CNCs). We will show that by utilizing CNCs reactive functional groups and incredible mechanical properties we are able to create a material that reduces clotting while maintaining the tubing's mechanical strength as well as eliminating heparin's side effects associated with it being a soluble anticoagulant. / MS / To prevent clotting during dialysis, heparin is used to line the tubing which blood flows through. Heparin, an anticoagulant, is more commonly known as a ‘blood thinner’ which is a misnomer because it does not actually thin blood. Heparin works by inhibiting clotting factors in the coagulation cascade pathway which in turn limit the formation of blood clots and create the ‘thinning’ effect mentioned earlier. When dialysis is performed the interaction between blood and the dialyzer tubing initiates the formation of a blood clot. This is where heparin use comes in. Unfortunately, many side effects arise from taking heparin, especially when it is used for an extended period of time. As such, long-term exposure for individuals undergoing dialysis every day is unavoidable. To prevent heparin or its mimics from entering the bloodstream, a polymer-based natural material is being investigated. The properties of this material will include reduction of coagulation and elimination of the long-term effects of heparin. The polymer-based natural material being investigated is cellulose nanocrystals (CNCs). CNCs contain the same ring structure and chemical linkage sites as heparin along with desirable mechanical properties. By altering the surface chemistry on the CNCs to closely mirror that of heparin, it should be possible to make a biomimetic material that counteracts blood clotting, while not introducing a solution based small molecule anticoagulant to the body. Through blood assays and platelet fixing analysis, we have been able to show that this change in functionalization does reduce coagulation. The ‘plain’ CNCs used contained an initial charge density of 70 mmol SO₃⁻ /kg. This residual charge density was a result from the acid hydrolysis performed to acquire CNCs from cellulose. Chemically modified CNCs contained many more negatively charged functional groups with TEMPO oxidized and sulfated CNCs having 500 mmol COO⁻/kg and 330 mmol SO₃⁻ /kg, respectively. We will show that by utilizing CNCs reactive functional groups and incredible mechanical properties we are able to create a material that reduces clotting while maintaining the tubing’s mechanical strength as well as eliminating heparin’s side effects associated with it being a soluble anti-coagulant.

Heparin, a glycosaminoglycan (GAG), is a complex biopolymer of varying chain length and consisting of uronic acid and glucosamine residues, which are sulfated at various positions. The interaction of heparin with antithrombin is the basis for anticoagulation therapy. Heparin accelerates the antithrombin mediated inhibition of factor Xa and thrombin by a conformational activation mechansism and bridging mechanism, respectively. The sequence specific pentasaccharide DEFGH in full length heparin is the most important fragment for high affinity and activation of antithrombin, without which the heparin is incapable of binding to antithrombin. Although heparin is a commonly used anticoagulant, it suffers from serious side effects including bleeding complications, heparin-induced thrombocytopenia, and intra- and inter-patient dose response variability. Desai and co-workers have shown that it is possible to replace the GAG skeleton by small, non-saccharide sulfated molecules as antithrombin activators. However, the designed molecules were found to be weak activators of antithrombin due to their binding to the extended heparin-binding site (EHBS), instead of the pentasaccharide-binding site (PBS), of antithrombin. To design better non-saccharide antithrombin activators, a virtual screening-based approach was employed. Combinatorial virtual screening of 24576 molecules based on tetrahydroisoquinoline core scaffold resulted in 92 hits that were predicted to bind preferentially in the PBS of activated antithrombin with good affinity. The work resulted in a predicted pharmacophore consisting of a 5,6-disulfated bicyclic tetrahydroisoquinoline and a 2′,5′-disulfated unicyclic phenyl ring connected by a 4- to 5-carbon linker. The work has led to several hypotheses, which are being tested in the laboratory through synthesis and biochemical evaluation. To understand the mechanism of heparin binding to thrombin in greater detail, structural biology and molecular modeling approaches were used. More specifically, the nature of the heparin binding to thrombin was studied with a special focus on understanding the specificity of recognition. Comparative analysis was performed with heparin–antithrombin interaction to assess similarities and differences between the two heparin binding systems. In antithrombin, three important amino acids are involved in heparin pentasaccharide binding, while in thrombin, at least seven basic amino acids are predicted to be involved. For biological systems, one would expect greater specificity with more interacting points. However, the heparin–thrombin system interestingly displays a lack of specificity. The molecular basis for this lack of specificity is not clear. A study of antithrombin and thrombin crystal structures with regard to surface exposure, flexibility, and geometry of basic amino acids present in the respective heparin binding site provides the basis for the specificity of recognition (or lack thereof) in the two systems. Interestingly, analysis of thrombin exosite-II showed that Arg101, Arg165 and Arg233 are spatially conserved and form a local asymmetric center. Using in-silico docking techniques, selected tetrasaccharide sequences were found to specifically recognize this triad of amino acids indicating the possibility of specific recognition of thrombin. This hypothesis led to the design of a putative lead sequence that is 50% smaller in size and contains 62.5% fewer charges in comparison to the literature reported known exosite II sequence. The design of novel putative ‘specific’ exosite II sequence challenges the idea that the thrombin–heparin interaction is completely non-specific and gives rise to novel opportunities of designing specific thrombin exosite-II ligands.

<p>Collagen crosslinking with a polypropyleneimine octaamine dendrimers, via carbodiimide chemistry, was further exploited to demonstrate the ability of this technology for various tissue engineering strategies, including tissue engineered corneal equivalents (TECE) and blood-contacting biomaterials. In addition, modification with extracellular matrix components and other biomimetic molecules may enhance tissue-host interactions for greater <em>in vivo </em>compatibility.</p> <p>First, the efficacy of the dendrimer crosslinking technology was further validated with commercially available collagen-based materials, from bovine or human sources (Chapter 4: Paper 1), as determined via transmittance, water uptake, differential scanning calorimetry, collagenase stability and <em>in vitro </em>cell compatibility. Despite gel formation, the matrix integrity was compromised with collagen-based materials manufactured under acidic conditions and purified via freeze-drying.</p> <p>To continue the theme of dendrimer crosslinked collagen gels as TECE materials, growth factor incorporation was investigated with epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), as a method for improving device epithelialization and subsequent host integration. However, given the short half lives of these growth factors, an effective growth factor delivery system is necessary to protect growth factor bioactivity. As heparan sulphate proteoglycans sequester and release heparin-binding growth factors <em>in vivo</em>, the use of heparinized dendrimer crosslinked collagen (CHG) gels for HB-EGF delivery would provide prolonged, controlled delivery, while maintaining growth factor effectiveness (Chapter 5: Paper 2). HB-EGF release was prolonged and capable of inducing human cornea epithelial cell (HCEC) proliferation. Thus, HB-EGF delivery from CHG gels could aid in TECE device retention through enhanced device-host integration via epithelialization.</p> <p>Alternatively, tethering EGF or HB-EGF to dendrimer crosslinked collagen (CG) gels could also supply growth factor stimulation in a manner that maintains bioactivity, while stimulating growth factor receptors continually with minute concentrations (Chapter 6: Paper 3). Growth factor uptake and bioactivity was assessed with radiolabeled growth factor and through <em>in vitro </em>epithelial cell culture, respectively. Surface-modification of CG gels with growth factors demonstrated greater bioactivity, compared to growth factor bulk-modification of CG gels.</p> <p>Finally, dendrimer crosslinked collagen gels, with pre-activated heparin (PH gels) were investigated as a tissue engineered blood-contacting biomaterial (Chapter 7: Paper 4), as we hypothesized that biomaterial induced coagulation is not only influenced by an anticoagulant surface, but also by the underlying material and that improved blood-biomaterial interactions may be achieved by utilizing a natural polymer that emulates biomimetic properties. Pre-activation of heparin was utilized to increase heparin gel content, while antithrombotic properties were evaluated via antithrombin and fibrinogen adsorption and plasma recalcification times. PH gels had increased heparinization, but extensive crosslinking compromised antithrombin-heparin interactions, compared to CHG gels. CHG gels demonstrated improved antithrombotic properties and further evaluation of these gels for blood-contacting applications is warranted.</p> / Doctor of Philosophy (PhD)

Embryonic stem cells (ESCs) are a unique cell population that can differentiate into all three embryonic germ layers (endoderm, mesoderm, and ectoderm), rendering them an invaluable cell source for studying the molecular mechanisms of embryogenesis. Signaling molecules that direct tissue patterning during embryonic development are secreted by ESC aggregates, known as embryoid bodies (EBs). As many of these signaling proteins interact with the extracellular matrix (ECM), manipulation of the ESC extracellular environment provides a means to direct differentiation. ECM components, such as glycosaminoglycans (GAGs), play crucial roles in cell signaling and regulation of morphogen gradients during early development through binding and concentration of secreted growth factors. Thus, engineered biomaterials fabricated from highly sulfated GAGs, such as heparin, provide matrices for manipulation and efficient capture of ESC morphogens via reversible electrostatic and affinity interactions. Ultimately, biomaterials designed to efficiently capture and retain morphogenic factors offer an attractive platform to enhance the differentiation of ESCs toward defined cell types. The overall objective of this work was to examine the ability of microparticles synthesized from both synthetic and naturally-derived materials to enhance the local presentation of morphogens to direct ESC differentiation. The overall hypothesis was that microparticles that mimic the ECM can modulate ESC differentiation through sequestration of endogenous morphogens present within the EB microenvironment.