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Imaging methods for haemostasis research
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood is a vital part of the human physiology; a transport system that brings nutrients and oxygen to sustain living cells and simultaneously facilitates the removal of carbon dioxide and other waste products from the body. To assure the continuity of these functions, it is of uttermost importance to keep the flowing blood inside the vascular system at any cost. The principal components of the haemostatic system are the blood platelets and the plasma coagulation system, both working in concert to create a blood stopping haemostatic plug when a vessel is ruptured. In modern health care, methods for treatment and diagnostics often implicate the contact between blood and artificial materials (biomaterials). Biomaterial surfaces may activate platelets and the coagulation cascade by exposing a surface that during blood contact shares certain characteristics with surfaces found at the site of vascular injury. Therefore it is of great importance that the mechanisms behind the interactions between foreign surfaces and blood are studied in order to minimize, and if possible, prevent unnecessary reactions that may lead to thrombosis.

This thesis describes two important methods to study blood – surface interactions in terms of surface induced plasma coagulation and platelet adhesion/aggregation. The method ‘Imaging of coagulation’, a coagulation assay based on time-lapse image capture of the coagulation process was developed during the course of this work. The use of images enables the method to answer questions regarding where coagulation was initiated and how fast coagulation propagates. Such questions are highly relevant in the study of blood-biomaterial interactions but also in general haemostasis research. In vivo, platelet adhesion and aggregation are events that always proceed under flow conditions. Therefore we also developed a cone-and-plate flow model to study these mechanisms under similar conditions in vitro. The cone-and-plate setup was found to be a flexible platform and was used for both blood compatibility testing of potential biomaterials as well as for general haemostasis research.

With the above mentioned methods we tested the haemocompatibility of glycerol monooleate (GMO), a proposed substance for use in biomaterial applications. It was found that GMO did not activate coagulation to any great extent either in plasma or in whole blood.

Surface induced coagulation and platelet adhesion was also studied on PEG-containing hydrogels and compared with hydrogels constructed from three different non-PEG-containing monomers. It was concluded that all the grafted hydrogels, in particular those produced from the monomers 2-hydroxyethyl methacrylate (HEMA) and/or PEG- methacrylate (PEGMA), demonstrated good haemocompatibility.

Supported phospholipid bilayers were used to investigate the relationship between surface charge and procoagulant activity. The coagulation process was studied in a straightforward manner using the imaging of coagulation setup. We concluded that the content of negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) in the bilayer must exceed ~ 6% for the bilayer to exert procoagulant activity.

The physiological role of factor XII in human haemostasis and thrombosis was investigated in the imaging of coagulation setup and the cone and plate setup by the use of surfaces with thrombogenic coatings. We found that tissue factor initiated coagulation could be greatly accelerated by the presence of contact activating agents in a platelet dependent manner.

In conclusion, the method ‘Imaging of coagulation’ and platelet adhesion/aggregation in the cone-and-plate flow model are both versatile methods with many possible applications. The combination of the two methods provides a solid foundation for biomaterial and haemostasis research.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 61 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1131
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-19178ISBN: 978-91-7393-621-7 (print)OAI: oai:DiVA.org:liu-19178DiVA: diva2:223531
Public defence
2009-06-01, Aulan, Hälsans hus, Campus US, Linköpings Universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2009-06-12 Created: 2009-06-12 Last updated: 2009-08-21Bibliographically approved
List of papers
1. Imaging of blood plasma coagulation and its propagation at surfaces
Open this publication in new window or tab >>Imaging of blood plasma coagulation and its propagation at surfaces
2008 (English)In: Journal of biomedical materials research. Part A, ISSN 1552-4965, Vol. 85, no 4, 1129-1134 p.Article in journal (Refereed) Published
Abstract [en]

A new method utilizing image capture and processing was developed for the analysis of blood plasma coagulation at surfaces. The coagulation was detected in a cuvette by time-lapse image capture of light scattering from the developing fibrin network. By image processing and computer analysis of the captured image data, both early detection of coagulation at the surface and the propagation phase of coagulation could be measured in the same experiment. It is possible to use both platelet-rich plasma (PRP) and platelet-free plasma (PFP) with the method, and thereby study the platelet contribution to both surface coagulation and propagation of coagulation. Two well-known model surfaces, hydrophilic and hydrophobic glass, were used in combination with PRP and PFP to illustrate the method. Hydrophilic glass activated coagulation significantly faster (PRP: 7.0 +/- 1.7 min, PFP: 5.9 +/- 1.2 min, n= 16) than hydrophobic glass (PRP: 50 +/- 14 min, PFP: 65 +/- 32 min, n = 16) in both PRP and PFP. Hydrophilic surfaces showed a faster initial propagation of coagulation adjacent to the surface (mean velocity: 0.14 +/- 0.05 mm/ minute) compared with the propagation observed further out from the surface (mean velocity: 0.05 +/- 0.01 mm/min). The method is very flexible and can be suitable for screening hemocompatibility of biomaterials.

Keyword
Glycerol monooleate, Blood compatibility, Shear, Ellipsometry
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-19173 (URN)10.1002/jbm.a.31529 (DOI)17907239 (PubMedID)
Available from: 2009-06-12 Created: 2009-06-12 Last updated: 2009-08-18Bibliographically approved
2. Glycerol monooleate-blood interactions
Open this publication in new window or tab >>Glycerol monooleate-blood interactions
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2009 (English)In: Colloids and Surfaces B: Biointerfaces, ISSN 0927-7765, Vol. 68, no 1, 20-26 p.Article in journal (Refereed) Published
Abstract [en]

In the present study the initial blood compatibility of glycerol monooleate (GMO)-coated surfaces was evaluated after deposition to surfaces and in bulk. The model surface was silica onto which multiple layers of fibrinogen or human serum albumin (HSA) was immobilized. The protein-coated surfaces were subsequently dip-coated in GMO in ethanol and used for blood plasma and whole blood experiments. The characterization methods included null ellipsometry, scanning electron microscopy, imaging of coagulation, hemolysis test and whole blood coagulation time by free oscillation rheometry.

The results showed a GMO film thickness of approximately 350 angstrom (similar to 4 mu g/cm(2)) upon dip-coating in ethanolic solution. A major part of the deposited layer detached in aqueous solutions, especially during shear conditions. The coagulation time on GMO was significantly prolonged compared to that on HSA coated silica. Whole blood tests showed that GMO is a very weak hemolytic agent. Deposited GMO detached easily from surfaces upon rinsing or shearing, although a stable layer with undefined phase structure and a thickness of 50-70 angstrom remained on HSA and fibrinogen precoated surfaces. This indicates that GMO has stronger adhesive forces to its substrate compared to the cohesive forces acting within the bulk GMO. The ability of GMO to detach from itself and tentatively form micelles or lipid bilayers when subjected to flowing blood may be of use in extravascular applications. It is concluded that GMO results in weak blood activation, and the material may in spite of this be suitable in selected biomaterial applications, especially as a biosealant and in colloidal dispersions.

Keyword
Glycerol monooleate, Blood compatibility, Shear, Ellipsometry
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16356 (URN)10.1016/j.colsurfb.2008.09.016 (DOI)
Available from: 2009-01-16 Created: 2009-01-16 Last updated: 2009-08-18Bibliographically approved
3. Blood compatibility of photografted hydrogel coatings
Open this publication in new window or tab >>Blood compatibility of photografted hydrogel coatings
2010 (English)In: ACTA BIOMATERIALIA, ISSN 1742-7061, Vol. 6, no 7, 2599-2608 p.Article in journal (Other academic) Published
Abstract [en]

In this work we have evaluated the haemocompatibility of different surface modifications, intended for biomaterials and biosensor applications. Polystyrene slides were coated with thin hydrogel films by self-initiated photografting of four different monomers. The hydrogel surface modifications were thoroughly characterized and tested for their protein resistance and ability to facilitate platelet adhesion and activation of the coagulation system. There was very little protein adsorption from human plasma on the hydrogels formed from poly(ethylene glycol) methacrylate (PEGMA) and 2-hydroxyethyl methacrylate (HEMA). Platelet adhesion tests performed under both static and flow conditions showed that these coatings also demonstrated very high resistance towards platelet adhesion. A small amount of platelets were found to adhere to hydrogels formed from ethylene glycol methyl ether methacrylate (EGMEMA) and 2-carboxyethyl methacrylate (CEA). The polystyrene substrates themselves facilitated large amounts of platelet adhesion under both static and flow conditions. Utilizing a novel setup for imaging of coagulation, it was shown that none of the hydrogel surfaces activated the coagulation system to any great extent. We suggest that this simple fabrication method can be used to produce hydrogel coatings with unusually high blood compatibility, suitable for demanding biomaterials applications.

Place, publisher, year, edition, pages
Elsevier Science B.V. Amsterdam, 2010
Keyword
Hydrogel; Biomaterial; Protein adsorption; Coagulation; Platelet
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-19175 (URN)10.1016/j.actbio.2009.12.046 (DOI)000278868000027 ()
Available from: 2009-06-12 Created: 2009-06-12 Last updated: 2011-03-23Bibliographically approved
4. Activation of blood coagulation at charged supported lipid membranes
Open this publication in new window or tab >>Activation of blood coagulation at charged supported lipid membranes
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The purpose of this work was to investigate the relationship between surface charge of phospholipid membranes and coagulation. Also, we wanted to demonstrate that coagulation at phospholipid membranes could successfully be studied in the method for imaging of coagulation.

Analytical procedure: Supported phospholipid membranes were formed from palmitoyl-oleoyl-glycero-3-ethylphosphocholine (POEPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1- palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) on silicon substrates. The surface charge of the phospholipid membranes was controlled by using different compositions of POPS (negative net charge), POPC (weak negative net charge) and POEPC (positive net charge). Imaging of coagulation experiments were performed on all phospholipid membrane coated surfaces as well as the clean silicon substrate. The experiments were performed in platelet-free plasma (PFP) diluted 50:50 with phosphate-buffered saline (PBS).

Results: Comparing the negatively charged SiO2 surface with the negatively charged POPS (30%)/POPC(70%) we found an interesting difference. Although both surfaces activated coagulation rapidly, the POPS surface facilitated a faster propagation of coagulation from the surface than the SiO2 surface. It was also found that in order for the phospholipid membranes to exert procoagulant properties, the POPS content in the membrane had to exceed ~6 %. It was also found that positively charged phospholipid membranes did not induce activation of coagulation.

Conclusions: The work in this paper demonstrated that the coagulation process at phospholipid membranes can be studied in a straightforward manner using the imaging of coagulation setup. Furthermore, we speculate that the negatively charged phospholipid membranes but not the SiO2 surface can support the binding of coagulation factor complexes, thus facilitating a faster propagation of coagulation. The fact that the POPS content must exceed ~ 6% to fully exert procoagulant properties was also a very interesting result, especially since platelets, when activated, become procoagulant by increasing their negatively charged phosphatidylserine exposure from ~0 % to maximally ~10 %.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-19176 (URN)
Available from: 2009-06-12 Created: 2009-06-12 Last updated: 2015-09-18Bibliographically approved
5. The role of coagulation factor XII in propagation of coagulation
Open this publication in new window or tab >>The role of coagulation factor XII in propagation of coagulation
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The physiological relevance and function of coagulation factor XII (FXII), the first zymogen in the intrinsic pathway, has for a long time been a matter of debate. The aim of this study was to shed some light on the role of factor XII in thrombus formation with a focus on its effect during the propagation phase of coagulation. In order to study propagation of coagulation we utilized a new imaging method to measure propagation rates from an activating surface in both platelet-free plasma and platelet-rich plasma. The most essential results revealed that both FXII and its substrate FXI are located on the surface of activated platelets. The surface of preexisting clots does not support coagulation in a FXII dependent manner. However, we found strong evidence for an accelerated propagation of tissue factor initiated coagulation when contact activation of FXII simultaneously occurred in the proximity. In vivo sources for contact activation may be exposed subendothelial collagen as well as soluble and cell derived poly-anions. If such in vivo contact activation of FXII occurs, even though moderate, it could contribute to in vivo thrombus growth rate and thus be of pathophysiological importance.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-19177 (URN)
Available from: 2009-06-12 Created: 2009-06-12 Last updated: 2015-03-13Bibliographically approved

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