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Studies on interfaces between primary and secondary hemostasis
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Our conceptual understanding of hemostasis is still heavily influenced by outdated experimental models wherein the hemostatic activity of platelets and coagulation factors are understood and studied in isolation.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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. , 80 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1544
National Category
Biochemistry and Molecular Biology Pharmacology and Toxicology Medicinal Chemistry Basic Medicine
Identifiers
URN: urn:nbn:se:liu:diva-132413DOI: 10.3384/diss.diva-132413ISBN: 9789176856635 (Print)OAI: oai:DiVA.org:liu-132413DiVA: diva2:1045501
Public defence
2016-11-12, Berzeliussalen, Campus US, Linköping, 11:00 (Swedish)
Opponent
Supervisors
Available from: 2016-11-09 Created: 2016-11-09 Last updated: 2016-11-09Bibliographically approved
List of papers
1. Thrombin-induced platelet activation via PAR4: pivotal role for exosite II
Open this publication in new window or tab >>Thrombin-induced platelet activation via PAR4: pivotal role for exosite II
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2014 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 112, no 3, 558-565 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Schattauer Gmbh, 2014
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-111269 (URN)10.1160/TH13-12-1013 (DOI)000341547000015 ()24990072 (PubMedID)
Note

Funding Agencies|Swedish Research Council [K2010-65X-15060-07-3, K2013-65X-15060-10-3]; Swedish Heart and Lung Foundation [20100219, 20120263]

Available from: 2014-10-15 Created: 2014-10-14 Last updated: 2016-11-09Bibliographically approved
2. Putting polyphosphates to the test: evidence against platelet-induced activation of factor XII
Open this publication in new window or tab >>Putting polyphosphates to the test: evidence against platelet-induced activation of factor XII
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2013 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 122, no 23, 3818-3824 p.Article in journal (Refereed) Published
Abstract [en]

The recent claim that stimulated platelets activate the intrinsic pathway of coagulation by the release of polyphosphates has been considered a breakthrough in hemostasis research. In little more than 3 years, the original publication by Muller et al has been cited greater than100 times. However, none of the citing articles has sought to independently validate this potentially paradigm-shifting concept. To this end, we performed extensive experimentation in vitro and in vivo in an attempt to verify the claim that factor XII (FXII) is primarily activated by stimulated platelets. In contrast to the original assertion, platelet-derived polyphosphates were found to be weak activators of FXII, with a FXIIa-generating activity of less than10% compared with equivalent concentrations of kaolin. Using different coagulation assays, it was shown that platelet contribution to whole blood coagulation was unrelated to the generation of activated FXII in vitro. Additionally, key results used to verify the hypothesis in the original study in vivo were found to be irreproducible. We conclude that platelet-derived polyphosphates are not physiologically relevant activators of FXII.

Place, publisher, year, edition, pages
American Society of Hematology, 2013
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-104301 (URN)10.1182/blood-2013-05-499384 (DOI)000329735000021 ()
Available from: 2014-02-17 Created: 2014-02-14 Last updated: 2016-11-09
3. Response: platelets do not generate activated factor XII--how inappropriate experimental models have led to misleading conclusions
Open this publication in new window or tab >>Response: platelets do not generate activated factor XII--how inappropriate experimental models have led to misleading conclusions
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2014 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 124, no 10, 1692-1694 p.Article in journal, Letter (Other academic) Published
Place, publisher, year, edition, pages
American Society of Hematology, 2014
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-111531 (URN)10.1182/blood-2014-04-566067 (DOI)000342762300027 ()25190755 (PubMedID)
Available from: 2014-10-22 Created: 2014-10-22 Last updated: 2016-11-09Bibliographically approved
4. Contact activation: important to consider when measuring the contribution of tissue factor-bearing microparticles to thrombin generation using phospholipid-containing reagents
Open this publication in new window or tab >>Contact activation: important to consider when measuring the contribution of tissue factor-bearing microparticles to thrombin generation using phospholipid-containing reagents
2014 (English)In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836, Vol. 12, no 4, 515-518 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Wiley, 2014
Keyword
cell-derived microparticles; thromboplastin; blood coagulation; thrombin; factor XII
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-106682 (URN)10.1111/jth.12503 (DOI)000334157000012 ()
Available from: 2014-05-21 Created: 2014-05-19 Last updated: 2016-11-09

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