Background: Heparin-induced thrombocytopenia (HIT) is an iatrogenic immune-mediated prothrombotic disorder that is a direct consequence of heparin therapy. In HIT, antibodies are generated against complexes of platelet factor-4 (PF4) and heparin. Immunoglobulin G (IgG) antibodies bind to PF4/heparin complexes and cause Fc-receptor-mediated activation of platelets and monocytes. PF4 binds endogenous heparin-like polyanions to reveal cross-reactive epitopes that can also bind anti-PF4/heparin antibodies. Based on this observation, researchers have suggested that exposure to PF4/polyanion complexes can sensitize immune cells to become activated to produce HIT antibodies following iatrogenic heparin exposure. Research objective: The objective of this study is to determine whether the preoperative PF4-dependent immune response is associated with postoperative anti-PF4/heparin antibody production in a cohort of patients undergoing cardiopulmonary bypass surgery. Materials and methods: To assess the preoperative immune response to PF4, we utilized two assays: (1) a 3H-thymidine uptake assay to measure peripheral blood mononuclear cell (PBMC) proliferation in response to in vitro stimulation with PF4 and (2) a PBMC ELISPOT assay to measure the preoperative frequency of PF4-specific antibody-secreting cells. Proliferation was quantified as a stimulation index (SI). We then utilized a PF4/heparin-dependent enzyme immunoassay to measure the in vivo levels of anti-PF4/heparin antibodies produced by these patients in the postoperative period. Results: Our findings suggest that preoperative PF4-dependent proliferation is not associated with postoperative polyspecific anti-PF4/heparin antibody production [Spearman’s ρ (95% CI) = –0.02 (–0.32, 0.28), P = 0.91]. PF4-dependent proliferation had a weak negative association with postoperative anti-PF4/heparin IgG antibody production [Spearman’s ρ (95% CI) = –0.31 (–0.56, –0.02), P = 0.04], but was not associated with postoperative IgM or IgA anti-PF4/heparin antibody production [IgM: Spearman’s ρ (95% CI) = –0.04 (–0.33, 0.26), P = 0.78; IgA: Spearman’s ρ (95% CI) = –0.05 (–0.34, 0.25), P = 0.73]. Qualitative analysis demonstrated that two patients who had the strongest preoperative PF4-dependent proliferation responses produced the highest postoperative levels of anti-PF4/heparin IgM antibodies, but this relationship was not observed with postoperative anti-PF4/heparin IgG antibodies. Moreover, the preoperative frequency of PF4-specific antibody-secreting cells (ASCs) was also not associated with postoperative levels of anti-PF4/heparin IgM or IgG antibodies [IgM: Spearman’s ρ (95% CI) = 0.30 (–0.79, 0.93), P = 0.683; IgG: Spearman’s ρ (95% CI) = –0.21 (–0.92, 0.83), P = 0.600]; however, this was only completed on five patients and so the sample size should be increased before any meaningful conclusions can be drawn. We also demonstrated that PF4-dependent proliferation increases 5–6 days following cardiopulmonary bypass surgery [geometric mean (GM) postoperative PF4 alone proliferation (in SI) vs. GM preoperative PF4 alone proliferation (in SI) ± SEM: 23.7 ± 1.3 vs. 6.9 ± 1.5, P = 0.009]. Conclusions: Based on our findings, we conclude that preoperative PF4-dependent proliferation is unable to predict postoperative anti-PF4/heparin antibody production in this cohort of cardiopulmonary bypass patients. Due to the small sample size, we are unable to make conclusive statements regarding the relationship between preoperative PF4-specific ASC frequency and postoperative anti-PF4/heparin antibody production, but our findings would suggest that an association does not exist between these two variables in this patient cohort. Cardiopulmonary bypass surgery, however, may mobilize the postoperative immune cell repertoire to become activated against the self-protein PF4 and may therefore contribute to the postoperative HIT immune response. / Thesis / Master of Science (MSc) / Background: Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that is a direct consequence of heparin therapy. In HIT, antibodies are generated against complexes of platelet factor-4 (PF4) and heparin. Antibodies bind to PF4/heparin complexes and cause activation of platelets and monocytes. Researchers have suggested that exposure to PF4/polyanion complexes can sensitize immune cells to become activated to produce HIT antibodies following iatrogenic heparin exposure. Research objective: The objective of this study is to determine whether the preoperative PF4-dependent immune response is associated with postoperative anti-PF4/heparin antibody production in a cohort of patients undergoing cardiopulmonary bypass surgery. Materials and methods: To assess the preoperative immune response to PF4, we measured cellular proliferation in response to PF4 stimulation and the preoperative frequency of PF4-specific antibody-secreting cells. We also measured the level of anti-PF4/heparin antibodies following surgery. Results: Our findings suggest that preoperative PF4-dependent proliferation is not associated with postoperative anti-PF4/heparin antibody production. Moreover, the preoperative frequency of PF4-specific antibody-secreting cells (ASCs) was also not associated with postoperative levels of anti-PF4/heparin antibodies; however, this was only completed on five patients and so the sample size should be increased before any meaningful conclusions can be drawn. We also demonstrated that proliferation increases 5–6 days following cardiopulmonary bypass surgery. Conclusions: Based on our findings, we conclude that preoperative proliferation is unable to predict postoperative anti-PF4/heparin antibody production in this cohort of patients. Due to the small sample size, we are unable to make conclusive statements regarding the relationship between preoperative ASC frequency and postoperative anti-PF4/heparin antibody production. Cardiopulmonary bypass surgery, however, may mobilize the postoperative immune cell repertoire to become activated against the self-protein PF4 and may therefore contribute to the HIT immune response.

Enoxaparin, a low-molecular-weight heparin, is commonly used as an anticoagulant in dogs, and is currently dosed at 0.8mg/kg every 6 hours. With an increase in individual enoxaparin doses, less frequent dosing may be possible, thereby reducing owner inconvenience and expense. The three phases of this study investigated the appropriate dose (Phase one- 0.8mg/kg, SQ once; Phase two- 2mg/kg, SQ once; Phase three- 1.3 mg/kg, SQ q8h for 7 total doses) and dosing interval needed for maximum effectiveness of enoxaparin. A Sonoclot® analyzer and factor Xa activity were used to assess level of anticoagulation in six healthy dogs. Anticoagulation was inconsistent at the 0.8mg/kg dose, while the 2mg/kg dose showed a high level of anticoagulation, and the 1.3mg/kg dose provided more reliable anticoagulation than the other dosages and dosing intervals. Small sample size and the use of same-breed healthy dogs potentially affected the strength of the results.

No description available.

No description available.

<p>Surface-induced thrombosis is a continuing issue in the development of biomaterials for blood contacting applications. Protein adsorption is a key factor in thrombosis since it occurs rapidly upon contact of a material with blood, initiating coagulation and other adverse reactions including platelet adhesion. The research presented in this thesis explores the use of a unique antithrombin-heparin covalent complex (ATH) for surface modification to provide antithrombogenicity. ATH was tethered to surfaces by various methods. Polyethylene oxide (PEO) was investigated as a linker-spacer molecule for surface attachment of ATH as well as for its antifouling properties.</p> <p>In the first phase of the work gold was used as a model substrate. ATH was attached by three different methods: direct attachment, attachment via a short chain linker, and attachment via PEO. Analogous heparin-modified surfaces were prepared for comparison. Surfaces were characterized using contact angle measurements, x-ray photoelectron spectroscopy (XPS), ellipsometry and quartz-crystal microbalance (QCM). The data suggested that the heparin moiety of ATH was directed away from the surface, in an orientation allowing ready interaction with blood components. The ATH-modified surfaces showed greater antithrombin binding than the heparin-modified surfaces as measured by radioactive labelling and Western blotting analysis. Antithrombin binding was found to occur predominantly through the active pentasaccharide sequence of the heparin moiety of ATH, demonstrating the potential of the ATH for catalytic anticoagulant function. From measurements of the ratio of total heparin to active heparin (anti-factor Xa assay), ATH-modified surfaces were shown to have greater bioactivity than heparin-modified surfaces. The adhesion of platelets to gold and modified gold surfaces was measured from flowing whole blood <em>in vitro</em> using a cone-and-plate device and was lower on all of the modified surfaces compared to bare gold. PEO-ATH surfaces were also shown to prolong plasma clotting times compared to control and heparinized surfaces.</p> <p>In subsequent work, surface modification methods were developed for polyurethane (PU) substrates. Isocyanate groups were introduced into the PU surface for attachment of PEO and ATH was attached to the “distal” end of the PEO. Surfaces using PEO of varying molecular weight and end group were investigated to determine conditions for maximum anticoagulant activity and minimum non-specific protein adsorption. Surfaces were characterized using contact angle measurements and XPS, and protein interactions were studied using radiolabelling. The optimum balance of bioactivity and protein resistance was found to occur with PEO of low to mid range MW (ie. MW 300-600). These PU-PEO-ATH surfaces showed low fibrinogen adsorption and high selectivity for antithrombin. Consistent with results using gold substrates, platelet adhesion remained low when ATH was attached to polyurethane surfaces grafted with PEO. A hetero-bifunctional amino-carboxy-PEO (PEO-COOH surface) was compared with a “conventional” homo-bifunctional dihydroxy-PEO (PEO-OH surface) with respect to their effectiveness as linkers for attachment of ATH. The PEO-COOH-ATH surface was shown to bind slightly greater amounts of antithrombin, indicating higher catalytic anticoagulant activity. Thrombin binding was measured to determine whether the surfaces could provide direct anticoagulant activity. The PEO-OH-ATH surface bound high amounts of thrombin, indicating potential for direct thrombin inhibition. It is hypothesized that the PEO properties (MW and functional end group) may have an effect on the orientation of ATH on the surface thus influencing its "preference" for catalytic vs. direct anticoagulant function.</p> <p>This thesis provides new information regarding the interactions of proteins and platelets with ATH immobilized on biomaterials. ATH-modified surfaces were superior to analogous heparin-modified surfaces with respect to antithrombin binding and catalytic anticoagulant ability. Immobilized ATH was also shown to bind thrombin, suggesting potential for direct anticoagulant activity. It can thus be seen as a unique surface modifier with dual functioning anticoagulant activity. The modification of polyurethane with ATH using PEO as a protein resistant linker-spacer, may provide a material of improved antithrombogenicity for the construction of blood contacting devices.</p> / Doctor of Philosophy (PhD)

Coagulation factor XI plays an important role in the consolidation phase of blood coagulation. Previous studies from our laboratory and others have demonstrated that zymogen factor XI (FXI) binds to heparin with moderate (KD~110 nM) affinity via residues (K252, K253 and K255) located in the apple 3 (A3) domain of the molecule. In contrast, the enzyme, factor XIa (FXIa), was shown to bind to heparin with significantly higher affinity (~1.5 nM by ELISA) via residues (K529, R530 and R532) within the catalytic domain (CD). The interaction between heparin and FXIa potentiates the inhibition of FXIa by protease nexin-2 by 10-fold. In addition, related polyanions heparin and dextran sulfate inhibit the catalytic activity of FXIa. The present study was designed to determine the relative contributions of positively charged residues as well as the dimeric structure of FXI in heparin binding. During this project, wtFXI, FXIR504A, FXIK505A, FXIR507A, FXIR529A, FXIR530A, FXIR532A, and FXIR586A have been expressed and purified. All mutants were homogenous and identical to wtFXI on SDS-PAGE, clotting assays and 1G5 monoclonal antibody binding studied by SPR. In addition, monomeric FXI C321S/K331A was expressed and purified. Utilizing an ELISA assay, no difference in the affinity for heparin between FXIa and FXI was found. Surface plasmon resonance (SPR) data collected for FXI clearly indicate a complex interaction which does not conform to a simple 1:1 Langmuir binding model making it difficult to obtain quantitative information. The complexity of FXI interactions with heparin is likely to arise from the multivalent nature of the binding, in which both protein and heparin have multiple binding sites. Two positively charged residues in the FXI catalytic domain, FXIR507A and FXIR532A, were found to be particularly important for interaction with heparin. The FXIR507A and FXIR532A mutants demonstrated ~ 65% and ~50% decreases respectively in total number of heparin binding sites based on ELISA. Also, the apparent dissociation constants for FXIR507A (KDapp ~13 nM) and FXIR532A (KDapp ~21 nM ) were 6 and 10-fold increased respectively compared with 2.1 nM for the wtFXI. Mutant FXIR586A also demonstrated a defect in affinity (KDapp ~ 13 nM) without an effect on the Bmax. The monomeric FXIC321S/R331A was also characterized for its ability to bind heparin compared with wtFXI. Surprisingly, the monomeric FXI displayed defective binding to heparin according to ELISA (KDapp ~ 30 nM) and SPR methods. Thus, the unique homodimeric structure of FXI in addition to the residues both in its catalytic and A3 domain chains are necessary for high-affinity heparin binding. / Biochemistry

1. Abstract 1.1. Background It is uncertain whether 1) patient’s characteristics (e.g., age, weight, height, and sex) influence anti-Xa heparin levels (hereafter referred to as "heparin levels"), or 2) if heparin levels influence recurrent venous thromboembolism (VTE) or bleeding events, in patients with acute VTE treated with weight-adjusted therapeutic-dose subcutaneous (SC) unfractionated heparin (UFH) or SC low-molecular weight heparin (LMWH). To determine if either association exist, we analyzed data from the Fixed-Dose Heparin (FIDO) study, in which patients were randomized to either SC UFH or SC LMWH, each given in fixed weight-adjusted doses and overlapped with 3 month of warfarin therapy for treatment of acute VTE. 1.2. Methods During the original study, 708 patients were asked to participate in a sub-study that would measure peak heparin levels while they were treated with heparin. 408 patients provided blood samples and met the eligibility criteria for the analyses in this thesis. Linear regression was used to examine the influence of patients’ baseline characteristics (e.g., age, weight, height, body mass index [BMI], sex) on heparin levels. The influence of other factors (e.g., type of heparin [UFH or LMWH]) on heparin levels was also assessed. Logistic regression was used to examine the association of heparin levels with the outcomes of 1) recurrent VTE during 3 months of follow up, and 2) bleeding events in the first 10 days of follow up. 1.3. Results: Mean heparin levels were 0.695 in patients treated with UFH, 0.698 in those treated with dalteparin and 1.034 in those treated with enoxaparin (p<0.001; R2=0.08 for variability accounted for by type of heparin). In a univariable analysis, heparin levels increase by 0.04 IU/ml (95% CI 0.02-0.07; p=0.001; R2=0.03) for every 10-kg increment in weight, by 0.02 IU/ml (95% CI 0.01-0.03; p<0.001; R2=0.04) for each unit of BMI, and by 0.03 IU/ml (95% CI 0.01-0.05; p=0.001; R2=0.03) for every 10 mol/l increment in creatinine. In a multivariable analysis, weight, BMI, and creatinine still influenced heparin levels, after adjusting for type of heparin and timing of blood sample withdrawal. Although heparin levels increased with weight, the magnitude was not large enough to suggest altering the current weight-based dosing method for LMWH. Other baseline factors such as age, height, type of VTE, creatinine clearance and hospitalization status did not influence heparin levels in patients treated with UFH or LMWH. In a univariable analysis, when heparin levels were treated as a continuous variable, higher heparin levels were associated with a lower risk of recurrent VTE at 90-days in patients treated with LMWH (OR 0.04, 95% CI 0.003-0.550, for each 1.0 IU/ml increase in heparin levels), but not in patients treated with UFH (OR 1.46, 95% CI 0.37-5.58, for each 1.0 IU/ml increase in heparin levels). In addition, higher heparin levels were associated with a higher risk of bleeding at 10-days in patients treated with UFH (OR 3.32, 95% CI 1.30-8.46 for each 1 IU/ml increase in heparin levels) but not in patients treated with LMWH (OR 3.77, 95% CI 0.42-33.92, for each 1.0 IU/ml increase in heparin levels). In a multivariable analysis, the association of heparin levels with VTE at 90-days in patients receiving LMWH (lower VTE events) and with bleeding events at 10-days in patients receiving UFH (higher bleeding events) persisted after adjusting for antiplatelet use at baseline and diagnosis of cancer at baseline. When heparin levels were treated as a dichotomous variable (subtherapeutic vs. non-subtherapeutic levels and supratherapeutic vs. non-supratherapeutic levels), the proportion of patient with recurrent VTE was significantly higher in patients with subtherapeutic levels compared with non-subtherapeutic levels in patients receiving LMWH (8.6% vs. 1.3%, p = 0.01). No significant difference was found in the proportion of patients with subtherapeutic levels and non-subtherapeutic levels in patients receiving UFH (0% vs. 3.4%, χ2=0.15, p= 0.70). The test of interaction supported the decision to analyze LMWH and UFH groups separately (p=0.02). Finally, the proportion of patient with bleeding was higher in patients with supratherapeutic compared with non-supratherapeutic heparin levels (6.5% vs. 1.5%, χ2=7.65, p=0.01). The test of interaction did not support the decision to analyze LMWH and UFH groups separately (p=0.13). 1.4. Conclusions Although it was possible to identify factors that were associated with heparin levels in patients who had been treated with weight-adjusted UFH or LMWH, none of these associations were strong enough to suggest that variables other than weight should influence SC heparin dosing. Subtherapeutic heparin levels were associated with a higher risk of recurrent VTE in patients treated with LWMH but not UFH, and supratherapeutic heparin levels were associated with a higher risk of bleeding in patients treated with UFH but not LMWH. Indirectly, these findings suggest that adjusting UFH or LMWH dose in response to heparin levels might improve clinical outcomes. / Thesis / Master of Science (MSc)

Device anchoring of artificial cornea implants, through tissue integration of stromal tissue, is necessary to ensure long-term success. In this work, the delivery of basic fibroblast growth factor (FGF-2), a key modulator in corneal wound healing, via heparin modified materials was investigated as a means of sustained, soluble growth factor delivery for stimulation of device anchorage. Two materials types, commonly used for ophthalmic applications and currently under investigation for use in artificial cornea applications, were utilized. Poly (dimethyl siloxane) (PDMS) is currently under investigation as the base material for keratoprosthetic devices; dendrimer crosslinked collagen has been examined as the basis for use as a tissue engineered corneal equivalent. PDMS surfaces were modified directly or indirectly, through a poly (ethylene oxide) (PEO) spacer, to contain functionalized reactive NSC groups capable of binding heparin and FGF-2 Surface modifications were characterized with attenuated total reflection Fourier transform infrared spectrophotometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and water contact angles. Heparin coverage was assessed with metachromatic and bioactivity assays. Heparinized collagen gels were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and polypropyleneimine octaamine G2 dendrimers. Gel integrity was assessed with water uptake, differential sr::anning calorimetry, and heparin and dendrimer stability. Both materials were exposed to radiolabelled FGF-2 and growth factor immobilization and delivery were quantified. Heparinized PDMS surfaces were capable of binding on average 100 ng/cm2 ofFGF-2, while heparinized collagen gels had higher FGF-2 immobilization, 300 ng, likely attributed to their higher heparin densities and the fact that the bulk gel rather than the surface only was modified. Delivery of FGF-2 from the heparinized materials revealed a first order release profile, with an initial burst of FGF-2, followed by gradual growth factor release. Release rates, over a 2 week period, reached 6.5% and 50%, for 1 day and 3 day FGF-2 exposed heparinized PDMS modified surfaces, while hepruinized dendrimer crosslinked collagen gels released 40%. / Thesis / Master of Applied Science (MASc)

Das Ziel dieser Studie liegt darin, die Vor- und Nachteile der Heparinapplikation nach mikrovaskulär-anastomosierten Transplantaten in der Mund-, Kiefer- und Gesichtschirurgie zu verifizieren und damit einen Nutzen für den klinischen Alltag abzuleiten. In der Zeit von Anfang Februar 2012 bis Ende Februar 2015 erfolgten in der Abteilung für Mund- Kiefer- und Gesichtschirurgie und Plastische Operationen im Klinikum Bremen Mitte 100 mikrovaskulär-anastomosierte Transplan-tationen. Bei 95 % der Fälle wurde die aPTT durch Heparin im Sinne einer Antikoagulation verlängert. Bei 79 % der 100 Fälle konnte ein Verbleiben des Lappens verzeichnet werden. Eine antikoagulative Therapie galt bei mikrovaskulären Transplantaten bisher als ein etabliertes Verfahren zur Vermeidung einer venösen Thrombose des Gefäßstieles. Aufgrund der Kontrollierbarkeit und Steuerbarkeit von hochmolekularem Heparin ist Heparin zur Zeit als das Mittel der Wahl anzusehen in der postoperativen antikoagulativen Therapie bei der mikrovaskulären Lappenchirurgie. Eine antikoagulative Therapie mit Heparin erfordert Aufmerksamkeit und eine intensive Betreuung. Durch die Applikation von Heparin wird das Risiko von Komplikationen erhöht, z.B. eine Heparin-induzierte Thrombozytopenie oder die Gefahr von Blutungen. Inwieweit eine hämostasehemmende / antikoagulative Therapie sinnvoll ist, ist fraglich. Ein signifikanter Unterschied wurde in der eigenen Untersuchung und auch in der neusten Literatur nicht verzeichnet. Die Einflussfaktoren auf das Lappenüberleben scheinen multifaktoriell zu sein. Die Indikationsstellung zur Operation muss gut überlegt sein, um den Erfolg der Operation und das Überleben der Patienten nicht zu gefährden. Patienten, bei denen eine Endothelalteration zu vermuten ist, die zum Beispiel eine vorausgegangene Kombinationsbehandlung mit Strahlentherapie und Chemobehandlung erhalten haben, zeigen ein deutlich höheres Risiko für den Erfolg der Operation im Hinblick auf das Lappenüberleben. Weitere prospektive randomisierte Studien sind erforderlich, um zu beweisen, dass und inwieweit eine hämostasehemmende Therapie einen Einfluss auf das Überleben der Transplantate hat.

Experimentelle Studien zeigten, dass durch die perioperative, intraperitoneale Therapie antiadhärenter und zytotoxischer Substanzen das intra- und extraperitoneale Tumorwachstum nach Operationen vermindert werden kann. Nach intraperitonealen und subkutanen Applikationen von 104 Tumorzellen (DHD/K12/TRb) wurden BD IX Ratten in 14 Gruppen randomisiert: 7 Gruppen wurden mit CO2 laparoskopiert und 7 konventionell operiert. Die Operationszeiten betrugen 30 Minuten. Am Ende der Intervention wurde Ringerlösung, Taurolidin oder Taurolidin/Heparin intraperitoneal oder in die V. femoralis appliziert. Die Veränderungen des Differentialblutbildes auf das Operationstrauma und auf die Applikation der therapeutischen Substanzen wurden ermittelt. Taurolidin und die zusätzliche Therapie mit Heparin reduzierten im Tierexperiment nach intraperitonealer sowie simultaner intraperitonealer und intravenöser Therapie das intraperitoneale Tumorwachstum und die Inzidenz von Trokar- beziehungsweise Inzisionsmetastasen. Die intravenöse Therapie von Taurolidin und der Kombination aus Taurolidin/Heparin hatte keinen tumorsupprimierenden Effekt. Die Verschiebungen der Leukozytenzahlen des Differentialblutbildes wurden hauptsächlich durch das Operationstrauma bewirkt. In diesem Tierversuch wurden nach der Therapie mit Taurolidin und der Kombination mit Heparin keine Nebenwirkungen beobachtet. / Following subcutaneous and intraperitoneal injection of 104 colon adenocarcinoma cells (DHD/K12/TRb) the influences of both taurolidine or taurolidine/heparin on intraperitoneal and subcutaneous tumor growth was investigated in 210 rats undergoing midline laparotomy or insufflation with carbon dioxide. The animals were randomized into 14 groups. To investigate the intraperitoneal (local) influence of either taurolidine or heparin on tumor growth the substances were applied intraperitoneally. Systemic and intraperitoneal effects were evaluated after intravenous injection of the substances. Both application forms were also combined to analyze synergistic effects. Tumor weights, as well as the incidence of abdominal wound metastases were determined four weeks after the intervention. In order to evaluate the effects of the agents blood was taken to determine the peripheral leukocytes counts. Intraperitoneal therapy of either taurolidine or in combination with heparin inhibits local tumor growth and abdominal wound recurrences in rats undergoing midline laparotomy or insufflation with carbon dioxide. Neither the intraperitoneal nor the intravenous application or the combination of the two agents did influence the subcutaneous tumor growth. The substances did not alter the changes of peripheral leukocytes.