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Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0
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Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: closed (30 April 2023) | Viewed by 19043

Special Issue Editors

Dr. Kerstin Jurk

E-Mail Website
Guest Editor
Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University of Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany
Interests: coagulation; platelets; haemostasis; omics; signal transduction; thrombo-inflammation; thrombosis; vessel wall
Special Issues, Collections and Topics in MDPI journals
Dr. Marijke J.E. Kuijpers

E-Mail Website
Guest Editor
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands
Interests: coagulation; platelets; haemostasis; omics; signal transduction; thrombo-inflammation; thrombosis; vessel wall
Special Issues, Collections and Topics in MDPI journals
Prof. Dr. Johan W. M. Heemskerk

E-Mail Website
Guest Editor
Synapse Research Institute, Kon. Emmaplein 7, 6214 AC Maastricht, The Netherlands
Interests: coagulation; platelets; haemostasis; omics; signal transduction; thrombo-inflammation; thrombosis; vessel wall
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

In the present decade, we are seeing a rapid increase in available genetics and multiomics information on components of the human and mammalian circulation involved in hemostasis, athero and venous thrombosis, and thrombo inflammation. In this Special Issue, we aim to collect state-of-the-art scientific contributions (reviews and original articles) that provide novel molecular insights into the complex interactions in the blood and the vessel wall in physiological and pathophysiological settings. These include molecular mechanisms contributing to coagulation; platelet activation; leukocyte, erythrocyte, and vascular cell functions; extracellular vesicles; and inter-cellular cross-talk both in vitro and in vivo. Clinical papers without defined molecular mechanisms are not solicited.

The Special Issue 1.0, titled "Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation", has been very successful over the past year, with many submitted and published papers (below). Because of additional submission requests, we have therefore started the second volume on this topic with MDPI.

Dr. Kerstin Jurk
Dr. Marijke J.E. Kuijpers
Prof. Dr. Johan W. M. Heemskerk
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • coagulation
  • inflammation
  • leukocytes
  • platelets
  • proteomics
  • red blood cells
  • vessel wall
  • extracellular vesicles

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Published Papers (21 papers)

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Editorial

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4 pages, 196 KiB  
Editorial
Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation
by Marijke J. E. Kuijpers, Johan W. M. Heemskerk and Kerstin Jurk
Int. J. Mol. Sci. 2022, 23(10), 5825; https://doi.org/10.3390/ijms23105825 - 23 May 2022
Cited by 4 | Viewed by 2807
Abstract
In the present decade, we are seeing a rapid increase in available genetics and multiomics information on blood and vascular components of the human and mammalian circulation, involved in haemostasis, athero- and venous thrombosis, and thrombo-inflammation [...] Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)

Research

Jump to: Editorial, Review, Other

18 pages, 3332 KiB  
Article
Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets
by Pengyu Zhang, Fiorella A. Solari, Johan W. M. Heemskerk, Marijke J. E. Kuijpers, Albert Sickmann, Ulrich Walter and Kerstin Jurk
Int. J. Mol. Sci. 2023, 24(9), 7776; https://doi.org/10.3390/ijms24097776 - 24 Apr 2023
Viewed by 1499
Abstract
Bruton’s tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in [...] Read more.
Bruton’s tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI–Syk–Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0)
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14 pages, 2948 KiB  
Article
Coagulation Factor XIIIa and Activated Protein C Activate Platelets via GPVI and PAR1
by Ilaria De Simone, Constance C. F. M. J. Baaten, Martine Jandrot-Perrus, Jonathan M. Gibbins, Hugo ten Cate, Johan W. M. Heemskerk, Chris I. Jones and Paola E. J. van der Meijden
Int. J. Mol. Sci. 2022, 23(18), 10203; https://doi.org/10.3390/ijms231810203 - 06 Sep 2022
Cited by 2 | Viewed by 1797
Abstract
Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such [...] Read more.
Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the ‘supernatant of (hirudin-treated) coagulated plasma’ (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin αIIbβ3 activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0)
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19 pages, 8777 KiB  
Article
Roles of Focal Adhesion Kinase PTK2 and Integrin αIIbβ3 Signaling in Collagen- and GPVI-Dependent Thrombus Formation under Shear
by Jingnan Huang, Natalie J. Jooss, Delia I. Fernández, Albert Sickmann, Ángel García, Kanin Wichapong, Ingrid Dijkgraaf and Johan W. M. Heemskerk
Int. J. Mol. Sci. 2022, 23(15), 8688; https://doi.org/10.3390/ijms23158688 - 04 Aug 2022
Cited by 3 | Viewed by 1977
Abstract
Glycoprotein (GP)VI and integrin αIIbβ3 are key signaling receptors in collagen-dependent platelet aggregation and in arterial thrombus formation under shear. The multiple downstream signaling pathways are still poorly understood. Here, we focused on disclosing the integrin-dependent roles of focal adhesion kinase (protein tyrosine [...] Read more.
Glycoprotein (GP)VI and integrin αIIbβ3 are key signaling receptors in collagen-dependent platelet aggregation and in arterial thrombus formation under shear. The multiple downstream signaling pathways are still poorly understood. Here, we focused on disclosing the integrin-dependent roles of focal adhesion kinase (protein tyrosine kinase 2, PTK2), the shear-dependent collagen receptor GPR56 (ADGRG1 gene), and calcium and integrin-binding protein 1 (CIB1). We designed and synthetized peptides that interfered with integrin αIIb binding (pCIB and pCIBm) or mimicked the activation of GPR56 (pGRP). The results show that the combination of pGRP with PTK2 inhibition or of pGRP with pCIB > pCIBm in additive ways suppressed collagen- and GPVI-dependent platelet activation, thrombus buildup, and contraction. Microscopic thrombus formation was assessed by eight parameters (with script descriptions enclosed). The suppressive rather than activating effects of pGRP were confined to blood flow at a high shear rate. Blockage of PTK2 or interference of CIB1 no more than slightly affected thrombus formation at a low shear rate. Peptides did not influence GPVI-induced aggregation and Ca2+ signaling in the absence of shear. Together, these data reveal a shear-dependent signaling axis of PTK2, integrin αIIbβ3, and CIB1 in collagen- and GPVI-dependent thrombus formation, which is modulated by GPR56 and exclusively at high shear. This work thereby supports the role of PTK2 in integrin αIIbβ3 activation and signaling. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0)
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17 pages, 3376 KiB  
Article
Antithrombotic Effects of Fostamatinib in Combination with Conventional Antiplatelet Drugs
by Maan H. Harbi, Christopher W. Smith, Fawaz O. Alenazy, Phillip L. R. Nicolson, Alok Tiwari, Steve P. Watson and Mark R. Thomas
Int. J. Mol. Sci. 2022, 23(13), 6982; https://doi.org/10.3390/ijms23136982 - 23 Jun 2022
Cited by 8 | Viewed by 2041
Abstract
New antithrombotic medications with less effect on haemostasis are needed for the long-term treatment of acute coronary syndromes (ACS). The platelet receptor glycoprotein VI (GPVI) is critical in atherothrombosis, mediating platelet activation at atherosclerotic plaque. The inhibition of spleen tyrosine kinase (Syk) has [...] Read more.
New antithrombotic medications with less effect on haemostasis are needed for the long-term treatment of acute coronary syndromes (ACS). The platelet receptor glycoprotein VI (GPVI) is critical in atherothrombosis, mediating platelet activation at atherosclerotic plaque. The inhibition of spleen tyrosine kinase (Syk) has been shown to block GPVI-mediated platelet function. The aim of our study was to investigate if the Syk inhibitor fostamatinib could be repurposed as an antiplatelet drug, either alone or in combination with conventional antiplatelet therapy. The effect of the active metabolite of fostamatinib (R406) was assessed on platelet activation and function induced by atherosclerotic plaque and a range of agonists in the presence and absence of the commonly used antiplatelet agents aspirin and ticagrelor. The effects were determined ex vivo using blood from healthy volunteers and aspirin- and ticagrelor-treated patients with ACS. Fostamatinib was also assessed in murine models of thrombosis. R406 mildly inhibited platelet responses induced by atherosclerotic plaque homogenate, likely due to GPVI inhibition. The anti-GPVI effects of R406 were amplified by the commonly-used antiplatelet medications aspirin and ticagrelor; however, the effects of R406 were concentration-dependent and diminished in the presence of plasma proteins, which may explain why fostamatinib did not significantly inhibit thrombosis in murine models. For the first time, we demonstrate that the Syk inhibitor R406 provides mild inhibition of platelet responses induced by atherosclerotic plaque and that this is mildly amplified by aspirin and ticagrelor. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0)
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18 pages, 4103 KiB  
Article
Formation of Neutrophil Extracellular Traps by Reduction of Cellular Cholesterol Is Independent of Oxygen and HIF-1α
by Timo Henneck, AhmedElmontaser Mergani, Sabrina Clever, Anna E. Seidler, Graham Brogden, Sandra Runft, Wolfgang Baumgärtner, Katja Branitzki-Heinemann and Maren von Köckritz-Blickwede
Int. J. Mol. Sci. 2022, 23(6), 3195; https://doi.org/10.3390/ijms23063195 - 16 Mar 2022
Cited by 6 | Viewed by 2203
Abstract
Formation of neutrophil extracellular traps (NETs) is a two-faced innate host defense mechanism, which, on the one hand, can counteract microbial infections, but on the other hand, can contribute to massive detrimental effects on the host. Cholesterol depletion from the cellular membrane by [...] Read more.
Formation of neutrophil extracellular traps (NETs) is a two-faced innate host defense mechanism, which, on the one hand, can counteract microbial infections, but on the other hand, can contribute to massive detrimental effects on the host. Cholesterol depletion from the cellular membrane by Methyl-β-cyclodextrin (MβCD) is known as one of the processes initiating NET formation. Since neutrophils mainly act in an inflammatory environment with decreased, so-called hypoxic, oxygen conditions, we aimed to study the effect of oxygen and the oxygen stress regulator hypoxia-inducible factor (HIF)-1α on cholesterol-dependent NET formation. Thus, murine bone marrow-derived neutrophils from wild-type and HIF-knockout mice or human neutrophils were stimulated with MβCD under normoxic (21% O2) compared to hypoxic (1% O2) conditions, and the formation of NETs were studied by immunofluorescence microscopy. We found significantly induced NET formation after treatment with MβCD in murine neutrophils derived from wild-type as well as HIF-1α KO mice at both hypoxic (1% O2) as well as normoxic (21% O2) conditions. Similar observations were made in freshly isolated human neutrophils after stimulation with MβCD or statins, which block the HMG-CoA reductase as the key enzyme in the cholesterol metabolism. HPLC was used to confirm the reduction of cholesterol in treated neutrophils. In summary, we were able to show that NET formation via MβCD or statin-treatment is oxygen and HIF-1α independent. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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18 pages, 3043 KiB  
Article
The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors
by Chek Ziu Koo, Alexandra L. Matthews, Neale Harrison, Justyna Szyroka, Bernhard Nieswandt, Elizabeth E. Gardiner, Natalie S. Poulter and Michael G. Tomlinson
Int. J. Mol. Sci. 2022, 23(5), 2440; https://doi.org/10.3390/ijms23052440 - 23 Feb 2022
Cited by 8 | Viewed by 2786
Abstract
The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a ‘molecular scissor’ that cleaves the [...] Read more.
The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a ‘molecular scissor’ that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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20 pages, 5012 KiB  
Article
Structure-Based Cyclic Glycoprotein Ibα-Derived Peptides Interfering with von Willebrand Factor-Binding, Affecting Platelet Aggregation under Shear
by Johana Hrdinova, Delia I. Fernández, Bogac Ercig, Bibian M. E. Tullemans, Dennis P. L. Suylen, Stijn M. Agten, Kerstin Jurk, Tilman M. Hackeng, Karen Vanhoorelbeke, Jan Voorberg, Chris P. M. Reutelingsperger, Kanin Wichapong, Johan W. M. Heemskerk and Gerry A. F. Nicolaes
Int. J. Mol. Sci. 2022, 23(4), 2046; https://doi.org/10.3390/ijms23042046 - 12 Feb 2022
Cited by 10 | Viewed by 2097
Abstract
The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in [...] Read more.
The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26–34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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15 pages, 3307 KiB  
Article
Heparin-Functionalized Adsorbents Eliminate Central Effectors of Immunothrombosis, including Platelet Factor 4, High-Mobility Group Box 1 Protein and Histones
by Marie Ebeyer-Masotta, Tanja Eichhorn, René Weiss, Vladislav Semak, Lucia Lauková, Michael B. Fischer and Viktoria Weber
Int. J. Mol. Sci. 2022, 23(3), 1823; https://doi.org/10.3390/ijms23031823 - 05 Feb 2022
Cited by 13 | Viewed by 4356
Abstract
Inflammation and thrombosis are closely intertwined in numerous disorders, including ischemic events and sepsis, as well as coronavirus disease 2019 (COVID-19). Thrombotic complications are markers of disease severity in both sepsis and COVID-19 and are associated with multiorgan failure and increased mortality. Immunothrombosis [...] Read more.
Inflammation and thrombosis are closely intertwined in numerous disorders, including ischemic events and sepsis, as well as coronavirus disease 2019 (COVID-19). Thrombotic complications are markers of disease severity in both sepsis and COVID-19 and are associated with multiorgan failure and increased mortality. Immunothrombosis is driven by the complement/tissue factor/neutrophil axis, as well as by activated platelets, which can trigger the release of neutrophil extracellular traps (NETs) and release further effectors of immunothrombosis, including platelet factor 4 (PF4/CXCL4) and high-mobility box 1 protein (HMGB1). Many of the central effectors of deregulated immunothrombosis, including activated platelets and platelet-derived extracellular vesicles (pEVs) expressing PF4, soluble PF4, HMGB1, histones, as well as histone-decorated NETs, are positively charged and thus bind to heparin. Here, we provide evidence that adsorbents functionalized with endpoint-attached heparin efficiently deplete activated platelets, pEVs, PF4, HMGB1 and histones/nucleosomes. We propose that this elimination of central effectors of immunothrombosis, rather than direct binding of pathogens, could be of clinical relevance for mitigating thrombotic complications in sepsis or COVID-19 using heparin-functionalized adsorbents. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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15 pages, 3802 KiB  
Article
Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear
by Gina Perrella, Samantha J. Montague, Helena C. Brown, Lourdes Garcia Quintanilla, Alexandre Slater, David Stegner, Mark Thomas, Johan W. M. Heemskerk and Steve P. Watson
Int. J. Mol. Sci. 2022, 23(1), 493; https://doi.org/10.3390/ijms23010493 - 01 Jan 2022
Cited by 7 | Viewed by 2304
Abstract
Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the [...] Read more.
Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s−1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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19 pages, 6651 KiB  
Article
Temporal Roles of Platelet and Coagulation Pathways in Collagen- and Tissue Factor-Induced Thrombus Formation
by Stefano Navarro, David Stegner, Bernhard Nieswandt, Johan W. M. Heemskerk and Marijke J. E. Kuijpers
Int. J. Mol. Sci. 2022, 23(1), 358; https://doi.org/10.3390/ijms23010358 - 29 Dec 2021
Cited by 17 | Viewed by 2421
Abstract
In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to [...] Read more.
In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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15 pages, 20399 KiB  
Article
PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling
by Valentina Scalise, Chiara Sanguinetti, Tommaso Neri, Silvana Cianchetti, Michele Lai, Vittoria Carnicelli, Alessandro Celi and Roberto Pedrinelli
Int. J. Mol. Sci. 2021, 22(23), 12640; https://doi.org/10.3390/ijms222312640 - 23 Nov 2021
Cited by 27 | Viewed by 3437
Abstract
Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of [...] Read more.
Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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9 pages, 2851 KiB  
Article
Early Adventitial Activation and Proliferation in a Mouse Model of Arteriovenous Stenosis: Opportunities for Intervention
by Jenq-Shyong Chan, Yang Wang, Virgilius Cornea, Prabir Roy-Chaudhury and Begoña Campos
Int. J. Mol. Sci. 2021, 22(22), 12285; https://doi.org/10.3390/ijms222212285 - 13 Nov 2021
Cited by 3 | Viewed by 1529
Abstract
Background: Arteriovenous fistula (AVF) stenosis remains an important cause of AVF maturation failure, for which there are currently no effective therapies. We examined the pattern and phenotype of cellular proliferation at different timepoints in a mouse model characterized by a peri-anastomotic AVF stenosis. [...] Read more.
Background: Arteriovenous fistula (AVF) stenosis remains an important cause of AVF maturation failure, for which there are currently no effective therapies. We examined the pattern and phenotype of cellular proliferation at different timepoints in a mouse model characterized by a peri-anastomotic AVF stenosis. Methods: Standard immunohistochemical analyses for cellular proliferation and macrophage infiltration were performed at 2, 7 and 14 d on our validated mouse model of AVF stenosis to study the temporal profile, geographical location and cellular phenotype of proliferating and infiltrating cells in this model. Results: Adventitial proliferation and macrophage infiltration (into the adventitia) began at 2 d, peaked at 7 d and then declined over time. Surprisingly, there was minimal macrophage infiltration or proliferation in the neointimal region at either 7 or 14 d, although endothelial cell proliferation increased rapidly between 2 d and 7 d, and peaked at 14 d. Conclusions: Early and rapid macrophage infiltration and cellular proliferation within the adventitia could play an important role in the downstream pathways of both neointimal hyperplasia and inward or outward remodelling. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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Review

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19 pages, 1107 KiB  
Review
Dysregulated Hemostasis and Immunothrombosis in Cerebral Cavernous Malformations
by Maria Ascencion Globisch, Favour Chinyere Onyeogaziri, Ross Osborne Smith, Maximiliano Arce and Peetra Ulrica Magnusson
Int. J. Mol. Sci. 2022, 23(20), 12575; https://doi.org/10.3390/ijms232012575 - 20 Oct 2022
Cited by 3 | Viewed by 2986
Abstract
Cerebral cavernous malformation (CCM) is a neurovascular disease that affects 0.5% of the general population. For a long time, CCM research focused on genetic mutations, endothelial junctions and proliferation, but recently, transcriptome and proteome studies have revealed that the hemostatic system and neuroinflammation [...] Read more.
Cerebral cavernous malformation (CCM) is a neurovascular disease that affects 0.5% of the general population. For a long time, CCM research focused on genetic mutations, endothelial junctions and proliferation, but recently, transcriptome and proteome studies have revealed that the hemostatic system and neuroinflammation play a crucial role in the development and severity of cavernomas, with some of these publications coming from our group. The aim of this review is to give an overview of the latest molecular insights into the interaction between CCM-deficient endothelial cells with blood components and the neurovascular unit. Specifically, we underscore how endothelial dysfunction can result in dysregulated hemostasis, bleeding, hypoxia and neurological symptoms. We conducted a thorough review of the literature and found a field that is increasingly poised to regard CCM as a hemostatic disease, which may have implications for therapy. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0)
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16 pages, 994 KiB  
Review
Reversible Platelet Integrin αIIbβ3 Activation and Thrombus Instability
by Jinmi Zou, Frauke Swieringa, Bas de Laat, Philip G. de Groot, Mark Roest and Johan W. M. Heemskerk
Int. J. Mol. Sci. 2022, 23(20), 12512; https://doi.org/10.3390/ijms232012512 - 19 Oct 2022
Cited by 5 | Viewed by 2158
Abstract
Integrin αIIbβ3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbβ3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that [...] Read more.
Integrin αIIbβ3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbβ3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbβ3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbβ3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0)
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15 pages, 1269 KiB  
Review
The Controversial Role of LPS in Platelet Activation In Vitro
by Luca Galgano, Gianni Francesco Guidetti, Mauro Torti and Ilaria Canobbio
Int. J. Mol. Sci. 2022, 23(18), 10900; https://doi.org/10.3390/ijms231810900 - 17 Sep 2022
Cited by 16 | Viewed by 3842
Abstract
Circulating platelets are responsible for hemostasis and thrombosis but are also primary sensors of pathogens and are involved in innate immunity, inflammation, and sepsis. Sepsis is commonly caused by an exaggerated immune response to bacterial, viral, and fungal infections, and leads to severe [...] Read more.
Circulating platelets are responsible for hemostasis and thrombosis but are also primary sensors of pathogens and are involved in innate immunity, inflammation, and sepsis. Sepsis is commonly caused by an exaggerated immune response to bacterial, viral, and fungal infections, and leads to severe thrombotic complications. Among others, the endotoxin lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria is the most common trigger of sepsis. Since the discovery of the expression of the LPS receptor TLR4 in platelets, several studies have investigated the ability of LPS to induce platelet activation and to contribute to a prothrombotic phenotype, per se or in combination with plasma proteins and platelet agonists. This issue, however, is still controversial, as different sources, purity, and concentrations of LPS, different platelet-purification protocols, and different methods of analysis have been used in the past two decades, giving contradictory results. This review summarizes and critically analyzes past and recent publications about LPS-induced platelet activation in vitro. A methodological section illustrates the principal platelet preparation protocols and significant differences. The ability of various sources of LPS to elicit platelet activation in terms of aggregation, granule secretion, cytokine release, ROS production, and interaction with leukocytes and NET formation is discussed. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0)
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30 pages, 1602 KiB  
Review
Beyond Hemostasis: Platelet Innate Immune Interactions and Thromboinflammation
by Jonathan Mandel, Martina Casari, Maria Stepanyan, Alexey Martyanov and Carsten Deppermann
Int. J. Mol. Sci. 2022, 23(7), 3868; https://doi.org/10.3390/ijms23073868 - 31 Mar 2022
Cited by 52 | Viewed by 8630
Abstract
There is accumulating evidence that platelets play roles beyond their traditional functions in thrombosis and hemostasis, e.g., in inflammatory processes, infection and cancer, and that they interact, stimulate and regulate cells of the innate immune system such as neutrophils, monocytes and macrophages. In [...] Read more.
There is accumulating evidence that platelets play roles beyond their traditional functions in thrombosis and hemostasis, e.g., in inflammatory processes, infection and cancer, and that they interact, stimulate and regulate cells of the innate immune system such as neutrophils, monocytes and macrophages. In this review, we will focus on platelet activation in hemostatic and inflammatory processes, as well as platelet interactions with neutrophils and monocytes/macrophages. We take a closer look at the contributions of major platelet receptors GPIb, αIIbβ3, TLT-1, CLEC-2 and Toll-like receptors (TLRs) as well as secretions from platelet granules on platelet–neutrophil aggregate and neutrophil extracellular trap (NET) formation in atherosclerosis, transfusion-related acute lung injury (TRALI) and COVID-19. Further, we will address platelet–monocyte and macrophage interactions during cancer metastasis, infection, sepsis and platelet clearance. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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28 pages, 3124 KiB  
Review
Mechanisms Underlying Dichotomous Procoagulant COAT Platelet Generation—A Conceptual Review Summarizing Current Knowledge
by Lucas Veuthey, Alessandro Aliotta, Debora Bertaggia Calderara, Cindy Pereira Portela and Lorenzo Alberio
Int. J. Mol. Sci. 2022, 23(5), 2536; https://doi.org/10.3390/ijms23052536 - 25 Feb 2022
Cited by 11 | Viewed by 2884
Abstract
Procoagulant platelets are a subtype of activated platelets that sustains thrombin generation in order to consolidate the clot and stop bleeding. This aspect of platelet activation is gaining more and more recognition and interest. In fact, next to aggregating platelets, procoagulant platelets are [...] Read more.
Procoagulant platelets are a subtype of activated platelets that sustains thrombin generation in order to consolidate the clot and stop bleeding. This aspect of platelet activation is gaining more and more recognition and interest. In fact, next to aggregating platelets, procoagulant platelets are key regulators of thrombus formation. Imbalance of both subpopulations can lead to undesired thrombotic or bleeding events. COAT platelets derive from a common pro-aggregatory phenotype in cells capable of accumulating enough cytosolic calcium to trigger specific pathways that mediate the loss of their aggregating properties and the development of new adhesive and procoagulant characteristics. Complex cascades of signaling events are involved and this may explain why an inter-individual variability exists in procoagulant potential. Nowadays, we know the key agonists and mediators underlying the generation of a procoagulant platelet response. However, we still lack insight into the actual mechanisms controlling this dichotomous pattern (i.e., procoagulant versus aggregating phenotype). In this review, we describe the phenotypic characteristics of procoagulant COAT platelets, we detail the current knowledge on the mechanisms of the procoagulant response, and discuss possible drivers of this dichotomous diversification, in particular addressing the impact of the platelet environment during in vivo thrombus formation. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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21 pages, 5287 KiB  
Review
Role of Neutrophils and NETs in Animal Models of Thrombosis
by Estelle Carminita, Lydie Crescence, Laurence Panicot-Dubois and Christophe Dubois
Int. J. Mol. Sci. 2022, 23(3), 1411; https://doi.org/10.3390/ijms23031411 - 26 Jan 2022
Cited by 17 | Viewed by 4941
Abstract
Thrombosis is one of the major causes of mortality worldwide. Notably, it is not only implicated in cardiovascular diseases, such as myocardial infarction (MI), stroke, and pulmonary embolism (PE), but also in cancers. Understanding the cellular and molecular mechanisms involved in platelet thrombus [...] Read more.
Thrombosis is one of the major causes of mortality worldwide. Notably, it is not only implicated in cardiovascular diseases, such as myocardial infarction (MI), stroke, and pulmonary embolism (PE), but also in cancers. Understanding the cellular and molecular mechanisms involved in platelet thrombus formation is a major challenge for scientists today. For this purpose, new imaging technologies (such as confocal intravital microscopy, electron microscopy, holotomography, etc.) coupled with animal models of thrombosis (mouse, rat, rabbit, etc.) allow a better overview of this complex physiopathological process. Each of the cellular components is known to participate, including the subendothelial matrix, the endothelium, platelets, circulating cells, and, notably, neutrophils. Initially known as immune cells, neutrophils have been considered to be part of the landscape of thrombosis for more than a decade. They participate in this biological process through their expression of tissue factor (TF) and protein disulfide isomerase (PDI). Moreover, highly activated neutrophils are described as being able to release their DNA and thus form chromatin networks known as “neutrophil extracellular traps” (NETs). Initially, described as “dead sacrifices for a good cause” that prevent the dissemination of bacteria in the body, NETs have also been studied in several human pathologies, such as cardiovascular and respiratory diseases. Many articles suggest that they are involved in platelet thrombus formation and the activation of the coagulation cascade. This review presents the models of thrombosis in which neutrophils and NETs are involved and describes their mechanisms of action. We have even highlighted the medical diagnostic advances related to this research. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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Other

12 pages, 1787 KiB  
Brief Report
Targeting of a Conserved Epitope in Mouse and Human GPVI Differently Affects Receptor Function
by Stefano Navarro, Andreas Starke, Johan W. M. Heemskerk, Marijke J. E. Kuijpers, David Stegner and Bernhard Nieswandt
Int. J. Mol. Sci. 2022, 23(15), 8610; https://doi.org/10.3390/ijms23158610 - 03 Aug 2022
Cited by 5 | Viewed by 1990
Abstract
Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, [...] Read more.
Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6tg/tg). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation 2.0)
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15 pages, 3289 KiB  
Brief Report
Rac Inhibition Causes Impaired GPVI Signalling in Human Platelets through GPVI Shedding and Reduction in PLCγ2 Phosphorylation
by Raluca A. I. Neagoe, Elizabeth E. Gardiner, David Stegner, Bernhard Nieswandt, Steve P. Watson and Natalie S. Poulter
Int. J. Mol. Sci. 2022, 23(7), 3746; https://doi.org/10.3390/ijms23073746 - 29 Mar 2022
Cited by 3 | Viewed by 1800
Abstract
Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and [...] Read more.
Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and reduced PLCγ2 activation, but not phosphorylation. The objective of our study is to investigate the role of Rac1 in GPVI-dependent human platelet activation and downstream signalling. Therefore, we used human platelets stimulated using GPVI agonists (collagen and collagen-related peptide) in the presence of the Rac1-specific inhibitor EHT1864 and analysed platelet activation, aggregation, spreading, protein phosphorylation, and GPVI clustering and shedding. We observed that in human platelets, the inhibition of Rac1 by EHT1864 had no significant effect on GPVI clustering on collagen fibres but decreased the ability of platelets to spread or aggregate in response to GPVI agonists. Additionally, in contrast to what was observed in murine Rac1-deficient platelets, EHT1864 enhanced GPVI shedding in platelets and reduced the phosphorylation levels of PLCγ2 following GPVI activation. In conclusion, Rac1 activity is required for both human and murine platelet activation in response to GPVI-ligands, but Rac1’s mode of action differs between the two species. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation)
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