TY  - JOUR
A1  - Navarro, Stefano
A1  - Starke, Andreas
A1  - Heemskerk, Johan W. M.
A1  - Kuijpers, Marijke J. E.
A1  - Stegner, David
A1  - Nieswandt, Bernhard
T1  - Targeting of a conserved epitope in mouse and human GPVI differently affects receptor function
JF  - International Journal of Molecular Sciences
N2  - 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 (hGP6\(^{tg/tg\)). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.
KW  - glycoprotein VI
KW  - JAQ1
KW  - platelet receptors
KW  - platelet activation
KW  - platelet inhibition
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286227
SN  - 1422-0067
VL  - 23
IS  - 15
ER  - 
TY  - JOUR
A1  - Navarro, Stefano
A1  - Stegner, David
A1  - Nieswandt, Bernhard
A1  - Heemskerk, Johan W. M.
A1  - Kuijpers, Marijke J. E.
T1  - Temporal roles of platelet and coagulation pathways in collagen- and tissue factor-induced thrombus formation
JF  - International Journal of Molecular Sciences
N2  - 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.
KW  - coagulation
KW  - fibrin
KW  - glycoprotein VI
KW  - platelet receptors
KW  - spatiotemporal thrombus
KW  - thrombin
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284219
SN  - 1422-0067
VL  - 23
IS  - 1
ER  - 
TY  - JOUR
A1  - Schuhmann, Michael K.
A1  - Kraft, Peter
A1  - Bieber, Michael
A1  - Kollikowski, Alexander M.
A1  - Schulze, Harald
A1  - Nieswandt, Bernhard
A1  - Pham, Mirko
A1  - Stegner, David
A1  - Stoll, Guido
T1  - Targeting platelet GPVI plus rt-PA administration but not α2β1-mediated collagen binding protects against ischemic brain damage in mice
JF  - International Journal of Molecular Science
N2  - Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2\(^{−/−}\) mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.
KW  - ischemic stroke
KW  - integrin α2
KW  - glycoprotein VI
KW  - recombinant tissue-type plasminogen activator
KW  - intracranial bleeding
KW  - transient middle cerebral artery occlusion
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201700
SN  - 1422-0067
VL  - 20
IS  - 8
ER  - 
TY  - JOUR
A1  - Nagy, Magdolna
A1  - van Geffen, Johanna P.
A1  - Stegner, David
A1  - Adams, David J.
A1  - Braun, Attila
A1  - de Witt, Susanne M.
A1  - Elvers, Margitta
A1  - Geer, Mitchell J.
A1  - Kuijpers, Marijke J. E.
A1  - Kunzelmann, Karl
A1  - Mori, Jun
A1  - Oury, Cécile
A1  - Pircher, Joachim
A1  - Pleines, Irina
A1  - Poole, Alastair W.
A1  - Senis, Yotis A.
A1  - Verdoold, Remco
A1  - Weber, Christian
A1  - Nieswandt, Bernhard
A1  - Heemskerk, Johan W. M.
A1  - Baaten, Constance C. F. M. J.
T1  - Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis
JF  - Frontiers in Cardiovascular Medicine
N2  - Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6β1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.
KW  - arterial thrombus formation
KW  - bleeding
KW  - collagen
KW  - glycoprotein VI
KW  - platelets
KW  - microfluidics
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232194
VL  - 6
ER  -