| 1. |
- Björses, Katarina, et al.
(författare)
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In vitro and in vivo evaluation of chemically modified degradable starch microspheres for topical haemostasis
- 2011
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Ingår i: ACTA BIOMATERIALIA. - : Elsevier Science B.V. Amsterdam. - 1742-7061 .- 1878-7568. ; 7:6, s. 2558-2565
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Tidskriftsartikel (refereegranskat)abstract
- Degradable starch microspheres (DSMs) are starch chains cross-linked with epichlorhydrin, forming glycerol-ether links. DSMs have been used for many years for temporary vascular occlusion and drug delivery in treatment of malignancies. They are also approved and used for topical haemostasis by absorbing excess fluid from the blood and concentrating endogenous coagulation factors, thereby facilitating haemostasis. This mechanism of action is not sufficient for larger bleedings in current chemical formulations of DSMs, and modification of DSMs to trigger activation of platelets or coagulation would be required for use in such applications. Chemical modifications of DSMs with N-octenyl succinic anhydride, chloroacetic acid, acetic anhydride, diethylaminoethyl chloride and ellagic acid were performed and evaluated in vitro with thrombin generation and platelet adhesion tests, and in vivo using an experimental renal bleeding model in rat. DSMs modified to activate platelets in vitro were superior in haemostatic capacity in vivo. Further studies with non-toxic substances are warranted to confirm these results and develop the DSM as a more effective topical haemostatic agent.
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| 2. |
- Boknäs, Niklas, et al.
(författare)
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Contact activation : important to consider when measuring the contribution of tissue factor-bearing microparticles to thrombin generation using phospholipid-containing reagents
- 2014
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Ingår i: Journal of Thrombosis and Haemostasis. - : John Wiley & Sons. - 1538-7933 .- 1538-7836. ; 12:4, s. 515-518
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Tidskriftsartikel (refereegranskat)abstract
- Background: A commercial MP reagent containing phospholipids is used for thrombin generation (TG) measurements to estimate the procoagulant activity of microparticles (MPs). Previous reports have shown that contact activation affects TG when TF levels are low, and that addition of phospholipids might augment this effect.Objectives: To quantify the impact of contact activation on TG in the presence of phospholipids and low/no TF, as is the case using a commercially available MP-reagent. Methods Thrombin generation was analyzed using MP- or platelet-rich plasma (PRP)-reagent in the presence and absence of corn trypsin inhibitor and anti-TF antibodies, respectively. To quantify the impact of different experimental parameters on contact activation, microparticle-depleted plasma was analyzed in the presence of different concentrations of phospholipids, TF and/or contact activating agents (kaolin).Results: Even with low contact activating blood collection tubes, substantial thrombin generation was observed with the MP-reagent, but this was completely inhibited by addition of corn trypsin inhibitor. Control experiments illustrate that the phospholipids in the reagent play a major role in enhancing TG initiated by FXIIa. Even with the PRP-reagent, which is recommended for determining the content of phospholipids from MPs, TG was partly dependent on contact activation.Conclusions: Contact activation plays a major role in TG when using reagents/samples containing phospholipids but little or no tissue factor. This needs to be considered and accounted for in future clinical studies using TG to assess the procoagulant activity of MPs.
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| 3. |
- Boknäs, Niklas, et al.
(författare)
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Flow cytometry-based platelet function testing is predictive of symptom burden in a cohort of bleeders
- 2018
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Ingår i: Platelets. - : Taylor & Francis. - 0953-7104 .- 1369-1635. ; 29:5, s. 512-519
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Tidskriftsartikel (refereegranskat)abstract
- Platelet function disorders (PFDs) are common in patients with mild bleeding disorders (MBDs), yet the significance of laboratory findings suggestive of a PFD remain unclear due to the lack of evidence for a clinical correlation between the test results and the patient phenotype. Herein, we present the results from a study evaluating the potential utility of platelet function testing using whole-blood flow cytometry in a cohort of 105 patients undergoing investigation for MBD. Subjects were evaluated with a test panel comprising two different activation markers (fibrinogen binding and P-selectin exposure) and four physiologically relevant platelet agonists (ADP, PAR1-AP, PAR4-AP, and CRP-XL). Abnormal test results were identified by comparison with reference ranges constructed from 24 healthy controls or with the fifth percentile of the entire patient cohort. We found that the abnormal test results are predictive of bleeding symptom severity, and that the greatest predictive strength was achieved using a subset of the panel, comparing measurements of fibrinogen binding after activation with all four agonists with the fifth percentile of the patient cohort (p = 0.00008, hazard ratio 8.7; 95% CI 2.5-40). Our results suggest that whole-blood flow cytometry-based platelet function testing could become a feasible alternative for the investigation of MBDs. We also show that platelet function testing using whole-blood flow cytometry could provide a clinically relevant quantitative assessment of platelet-related hemostasis.
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| 4. |
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| 5. |
- Boknäs, Niklas, 1979-
(författare)
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Studies on interfaces between primary and secondary hemostasis
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Our conceptual understanding of hemostasis is still heavily influenced by outdated experimental models wherein the hemostatic activity of platelets and coagulation factors are understood and studied in isolation. Although perhaps convenient for researchers and clinicians, this reductionist view is negated by an ever increasing body of evidence pointing towards an intimate relationship between the two phases of hemostasis, marked by strong interdependence. In this thesis, I have focused on factual and proposed interfaces between primary and secondary hemostasis, and on how these interfaces can be studied.In my first project, we zoomed in on the mechanisms behind the well-known phenomenon of thrombin-induced platelet activation, an important event linking secondary to primary hemostasis. In our study, we examined how thrombin makes use of certain domains for high-affinity binding to substrates, called exosite I and II, to activate platelets via PAR4. We show that thrombin-induced platelet activation via PAR4 is critically dependent on exosite II, and that blockage of exosite II with different substances virtually eliminates PAR4 activation. Apart from providing new insights into the mechanisms by which thrombin activates PAR4, these results expand our knowledge of the antithrombotic actions of various endogenous proteins such as members of the serpin superfamily, which inhibit interactions with exosite II. Additionally, we show that inhibition of exosite II could be a feasible pharmacological strategy for achieving selective blockade of PAR4.In my second project, we examined the controversial issue of whether platelets can initiate the coagulation cascade by means of contact activation, a hypothesis which, if true, could provide a direct link between primary and secondary hemostasis. In contrast to previous results, our findings falsify this hypothesis, and show that some of the erroneous conclusions drawn from earlier studies can be explained by inappropriate experimental models unsuitable for the study of plateletcoagulation interfaces.My third project comprised an assessment of the methodological difficulties encountered when trying to measure the ability of platelets to initiate secondary hemostasis by the release of microparticles expressing tissue factor. Our study shows that the functional assays available for this purpose are highly susceptible to error caused by artificial contact activation. These results could help to improve the methodology of future research and thus pave the way for new insights into the roles of tissue factor-bearing microparticles in the pathophysiology of various thrombotic disorders.From a personal perspective, my PhD project has been a fascinating scientific odyssey into the largely unexplored interfaces between primary and secondary hemostasis. Looking forward, my ambition is to continue our work exploring platelet-coagulation interactions and to translate these insights into clinically meaningful information, which may someday improve the treatment of patients with bleeding and/or thrombosis.
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| 6. |
- Boknäs, Niklas, et al.
(författare)
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Thrombin-induced platelet activation via PAR4 : pivotal role for exosite II
- 2014
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Ingår i: Thrombosis and Haemostasis. - Stuttgart, Germany : Schattauer Gmbh. - 0340-6245 .- 2567-689X. ; 112:3, s. 558-565
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Tidskriftsartikel (refereegranskat)abstract
- Thrombin-induced platelet activation via PAR1 and PAR4 is an important event in haemostasis. Although the underlying mechanisms responsible for ensuring efficient PAR1 activation by thrombin have been extensively studied, the potential involvement of recognitions sites outside the active site of the protease in thrombin-induced PAR4 activation is largely unknown. In this study, we developed a new assay to assess the importance of exosite I and II for PAR4 activation with alpha- and gamma-thrombin. Surprisingly, we found that exosite II is critical for activation of PAR4. We also show that this dependency on exosite II likely represents a new mechanism, as it is unaffected by blockage of the previously known interaction between thrombin and glycoprotein Ib alpha.
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| 7. |
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| 8. |
- Claesson, Kjersti, et al.
(författare)
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Counting the platelets: a robust and sensitive quantification method for thrombus formation
- 2016
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Ingår i: Thrombosis and Haemostasis. - : SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN. - 0340-6245 .- 2567-689X. ; 115:6, s. 1178-1190
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Tidskriftsartikel (refereegranskat)abstract
- Flow chambers are common tools used for studying thrombus formation in vitro. However, the use of such devices is not standardised and there is a large diversity among the flow chamber systems currently used, and also in the methods used for quantifying the thrombus development. It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups. Whole blood was drawn over a collagen patch in commercial Ibid or in-house constructed PDMS flow chambers. Five percent of the platelets were fluorescently labelled and z-stack time-lapse images were captured during thrombus formation. Images were processed in a Python script in which the number of platelets and their respective x-, y- and z-positions were obtained. For comparison with existing methods the platelets were also labelled and quantified using fluorescence intensity and thrombus volume estimations by confocal microscopy. The presented method was found less sensitive to microscope and image adjustments and provides more details on thrombus development dynamics than the methods for measuring fluorescence intensity and thrombus volume estimation. The platelet count method produced comparable results with commercial and PDMS flow chambers, and could also obtain information regarding the stability of each detected platelet in the thrombus. In conclusion, quantification of thrombus formation by platelet count is a sensitive and robust method that enables measurement of platelet accumulation and platelet stability in an absolute scale that could be used for comparisons between research groups.
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| 9. |
- Ekblad, Tobias, 1979-, et al.
(författare)
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Patterned Hydrogels for Controlled Platelet Adhesion from Whole Blood and Plasma
- 2010
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Ingår i: Advanced Functional Materials. - : John Wiley & Sons. - 1616-301X .- 1616-3028. ; 20:15, s. 2396-2403
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Tidskriftsartikel (refereegranskat)abstract
- This work describes the preparation and properties of hydrogel surface chemistries enabling controlled and well-defined cell adhesion. The hydrogels may be prepared directly on plastic substrates, such as polystyrene slides or dishes, using a quick and experimentally simple photopolymerization process, compatible with photolithographic and microfluidic patterning methods. The intended application for these materials is as substrates for diagnostic cell adhesion assays, particularly for the analysis of human platelet function. The adsorption of fibrinogen and other platelet promoting molecules is shown to be completely inhibited by the hydrogel, provided that the film thickness is sufficient (>5 nm). This allows the hydrogel to be used as a matrix for presenting selected bioactive ligands without risking interference from nonspecifically adsorbed platelet adhesion factors, even in undiluted whole blood and blood plasma. This concept is demonstrated by preparing patterns of proteins on hydrogel surfaces, resulting in highly controlled platelet adhesion. Further insights into the protein immobilization and platelet adhesion processes are provided by studies using imaging surface plasmon resonance. The hydrogel surfaces used in this work appear to provide an ideal platform for cell adhesion studies of platelets, and potentially also for other cell types.
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| 10. |
- Ericsson, Emma, et al.
(författare)
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Glycerol monooleate-blood interactions
- 2009
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Ingår i: Colloids and Surfaces B. - : Elsevier BV. - 0927-7765 .- 1873-4367. ; 68:1, s. 20-26
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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