Platelet glycoprotein VI cluster size is related to thrombus formation and phosphatidylserine exposure in collagen-adherent platelets under arterial shear

Background: Collagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation. Objectives: To determine the importance of GPVI clusters for thrombus formation in whole blood under shear. Methods: We utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials. Results: Automated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation. Conclusion: Labeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces

Restraining of glycoprotein VI- and integrin α2β1-dependent thrombus formation y platelet PECAM1

The platelet receptors, glycoprotein VI (GPVI) and integrin α2β1 jointly control collagen-dependent thrombus formation via protein tyrosine kinases. It is unresolved to which extent the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptor PECAM1 and its downstream acting protein tyrosine phosphatase PTPN11 interfere in this process. Here, we hypothesized that integrin α2β1 has a co-regulatory role in the PECAM1- and PTPN11-dependent restraint of thrombus formation. We investigated platelet activation under flow on collagens with a different GPVI dependency and using integrin α2β1 blockage. Blood was obtained from healthy subjects and from patients with Noonan syndrome with a gain-of-function mutation of PTPN11 and variable bleeding phenotype. On collagens with decreasing GPVI activity (types I, III, IV), the surface-dependent inhibition of PECAM1 did not alter thrombus parameters using control blood. Blockage of α2β1 generally reduced thrombus parameters, most effectively on collagen IV. Strikingly, simultaneous inhibition of PECAM1 and α2β1 led to a restoration of thrombus formation, indicating that the suppressing signaling effect of PECAM1 is masked by the platelet-adhesive receptor α2β1. Blood from 4 out of 6 Noonan patients showed subnormal thrombus formation on collagen IV. In these patients, effects of α2β1 blockage were counterbalanced by PECAM1 inhibition to a normal phenotype. In summary, we conclude that the suppression of GPVI-dependent thrombus formation by either PECAM1 or a gain-of-function of PTPN11 can be overruled by α2β1 engagement

Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces.

Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin α2β1. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca2+. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)n and Horm-type collagen evoked Syk-dependent Ca2+ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)n sequence

Targeting GPVI: impact of modulating platelet-collagen interactions on receptor signaling and thrombus formation

Platelets are blood cells that prevent the loss of extensive blood volumes, but also contribute to arterial thrombosis. After a vessel is injured, platelets become activated by the exposed collagen and aggregate together, forming a thrombus. Whilst this is a crucial process in hemostasis, platelets also become activated due to rupture of an atherosclerotic plaque in atherothrombosis. Herein, leading to for example, myocardial infarction, stroke or transient ischemic attacks, mediated by collagen that is present in the atherosclerotic plaque. These events are still leading causes of death world-wide, and patients are usually prescribed anti-platelet drugs to prevent a second thrombosis. Currently used drugs are effective in preventing thrombosis, but they are prone to causing unwanted bleeding events in some of the patients. Therefore, other treatment options are currently investigated. A promising approach is to prevent platelet-collagen interactions via inhibition of the collagen receptor, glycoprotein VI (GPVI). In this thesis, we compared the various approaches used to interfere in the GPVI-dependent interaction of platelets with collagens. This was done by a whole blood flow chamber set up with collagen coatings, assessing the effects on thrombus formation

Stability and Degradation Mechanism of Si-based Photocathodes for Water Splitting with Ultrathin TiO2 Protection Layer

Using transmission and scanning electron microscopy, we study mechanisms which determine the stability of Silicon photocathodes for solar driven water splitting. Such tandem or triple devices can show a promising stability as photocathodes if the semiconductor surface is protected by an ultrathin TiO2 protection layer. Using atomic layer deposition (ALD) with Cl-precursors, 4–7 nm thick TiO2 layers can be grown with high structural perfection. The layer can be electrochemically covered by Pt nanoparticels serving as electro-catalysts. However, Cl-remnants which are typically present in such layers due to incomplete oxidation, are the origin of an electrochemical degradation process. After 1 h AM1.5G illumination in alkaline media, circular shaped corrosion craters appear in the topmost Si layer, although the TiO2 layer is intact in most parts of the crater. The crater development is stopped at local inhomogenities with a higher Pt coverage. The observations suggests that reduced Titanium species due to Cl−/O2− substitution are nucleation sites of the initial corrosion steps due to enhanced solubility of reduced Ti in the electrolyte. This process is followed by electrochemical dissolution of Si, after direct contact between the electrolyte and the top Si layer surface. To increase the stability of TiO2 protected photocathodes, formation of reduced Ti species must be avoided

Characterizing the binding of glycoprotein VI with nanobody 35 reveals a novel monomeric structure of glycoprotein VI where the conformation of D1+D2 is independent of dimerization.

The platelet-signaling receptor glycoprotein VI (GPVI) is a promising antithrombotic target. We have previously raised a series of high-affinity nanobodies (Nbs) against GPVI and identified Nb2, Nb21, and Nb35 as potent GPVI inhibitors. The Nb2 binding site has been mapped to the D1 domain, which is directly adjacent to the CRP binding site. Ligand-binding complementary determining region 3 has only 15% conservation between all 3 Nbs. To map the binding sites of Nb21 and Nb35 on GPVI. We determined the X-ray crystal structure of the D1 and D2 extracellular domains of the GPVI-Nb35 complex. We then looked at the effects of various GPVI mutations on the ability of Nbs to inhibit collagen binding and GPVI signaling using surface binding assays and transfected cell lines. The crystal structure of GPVI bound to Nb35 was solved. GPVI was present as a monomer, and the D1+D2 conformation was comparable to that in the dimeric structure. Arg46, Tyr47, and Ala57 are common residues on GPVI targeted by both Nb2 and Nb35. Mutating Arg46 to an Ala abrogated the ability of Nb2, Nb21, and Nb35 to inhibit collagen-induced GPVI signaling and blocked the binding of all 3 Nbs. In addition, Arg60 was found to reduce Nb21 inhibition but not the inhibition Nb2 or Nb35. These findings reveal key residues involved in the high-affinity binding of GPVI inhibitors and negate the idea that GPVI dimerization induces a conformational change required for ligand binding. [Abstract copyright: Copyright © 2022. Published by Elsevier Inc.

Roles of Focal Adhesion Kinase PTK2 and Integrin αIIbβ3 Signaling in Collagen- and GPVI-Dependent Thrombus Formation under Shear

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 pCIB(m)) or mimicked the activation of GPR56 (pGRP). The results show that the combination of pGRP with PTK2 inhibition or of pGRP with pCIB > pCIB(m) 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 Ca(2+) 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

CLEC-2 Supports Platelet Aggregation in Mouse but not Human Blood at Arterial Shear

C-type lectin-like receptor 2 (CLEC-2) is highly expressed on platelets and a subpopulation of myeloid cells, and is critical in lymphatic development. CLEC-2 has been shown to support thrombus formation at sites of inflammation, but to have a minor/negligible role in hemostasis. This identifies CLEC-2 as a promising therapeutic target in thromboinflammatory disorders, without hemostatic detriment. We utilized a GPIbα-Cre recombinase mouse for more restricted deletion of platelet-CLEC-2 than the previously used PF4-Cre mouse. clec1b (fl/fl) GPIbα-Cre (+) mice are born at a Mendelian ratio, with a mild reduction in platelet count, and present with reduced thrombus size post-FeCl (3) -induced thrombosis, compared to littermates. Antibody-mediated depletion of platelet count in C57BL/6 mice, to match clec1b (fl/fl) GPIbα-Cre (+) mice, revealed that the reduced thrombus size post-FeCl (3) -injury was due to the loss of CLEC-2, and not mild thrombocytopenia. Similarly, clec1b (fl/fl) GPIbα-Cre (+) mouse blood replenished with CLEC-2-deficient platelets ex vivo to match littermates had reduced aggregate formation when perfused over collagen at arterial flow rates. In contrast, platelet-rich thrombi formed following perfusion of human blood under flow conditions over collagen types I or III, atherosclerotic plaque, or inflammatory endothelial cells were unaltered in the presence of CLEC-2-blocking antibody, AYP1, or recombinant CLEC-2-Fc. The reduction in platelet aggregation observed in clec1b (fl/fl) GPIbα-Cre (+) mice during arterial thrombosis is mediated by the loss of CLEC-2 on mouse platelets. In contrast, CLEC-2 does not support thrombus generation on collagen, atherosclerotic plaque, or inflamed endothelial cells in human at arterial shear

CLEC-2 Supports Platelet Aggregation in Mouse but not Human Blood at Arterial Shear

C-type lectin-like receptor 2 (CLEC-2) is highly expressed on platelets and a subpopulation of myeloid cells, and is critical in lymphatic development. CLEC-2 has been shown to support thrombus formation at sites of inflammation, but to have a minor/negligible role in hemostasis. This identifies CLEC-2 as a promising therapeutic target in thromboinflammatory disorders, without hemostatic detriment. We utilized a GPIbα-Cre recombinase mouse for more restricted deletion of platelet-CLEC-2 than the previously used PF4-Cre mouse. clec1bfl/flGPIbα-Cre+ mice are born at a Mendelian ratio, with a mild reduction in platelet count, and present with reduced thrombus size post-FeCl3-induced thrombosis, compared to littermates. Antibody-mediated depletion of platelet count in C57BL/6 mice, to match clec1bfl/flGPIbα-Cre+ mice, revealed that the reduced thrombus size post-FeCl3-injury was due to the loss of CLEC-2, and not mild thrombocytopenia. Similarly, clec1bfl/flGPIbα-Cre+ mouse blood replenished with CLEC-2-deficient platelets ex vivo to match littermates had reduced aggregate formation when perfused over collagen at arterial flow rates. In contrast, platelet-rich thrombi formed following perfusion of human blood under flow conditions over collagen types I or III, atherosclerotic plaque, or inflammatory endothelial cells were unaltered in the presence of CLEC-2-blocking antibody, AYP1, or recombinant CLEC-2-Fc. The reduction in platelet aggregation observed in clec1bfl/flGPIbα-Cre+ mice during arterial thrombosis is mediated by the loss of CLEC-2 on mouse platelets. In contrast, CLEC-2 does not support thrombus generation on collagen, atherosclerotic plaque, or inflamed endothelial cells in human at arterial shear