Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates ...atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4
T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c
dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.
Background
In the intact vessel wall, endothelial cells form a barrier between the blood and the remaining vascular structures, serving to maintain blood fluidity and preventing platelet activation ...and fibrin clot formation. The spatiotemporal space of this inhibition is largely unknown.
Objective
To assess the local inhibitory roles of a discontinuous endothelium, we developed a vessel‐on‐a‐chip model, consisting of a microfluidic chamber coated with the thrombogenic collagen and tissue factor (TF), and covered with patches of human endothelial cells. By flow perfusion of human blood and plasma, the heterogeneous formation of platelet aggregates and fibrin clots was monitored by multicolor fluorescence microscopy.
Results
On collagen/TF coatings, a coverage of 40% to 60% of human umbilical vein endothelial cells resulted in a strong overall delay in platelet deposition and fibrin fiber formation under flow. Fibrin formation colocalized with the deposited platelets, and was restricted to regions in between endothelial cells, thus pointing to immediate local suppression of the clotting process. Fibrin kinetics were enhanced by treatment of the cells with heparinase III, partially disrupting the glycocalyx, and to a lesser degree by antagonism of the endothelial thrombomodulin. Co‐coating of purified thrombomodulin and collagen had a similar coagulation‐suppressing effect as endothelial thrombomodulin.
Conclusions
In this vessel‐on‐a‐chip system with patches of endothelial cells on thrombogenic surfaces, the coagulant activity under flow is regulated by: (a) the residual exposure of trigger (collagen/TF), (b) the endothelial glycocalyx, and (c) to a lesser degree the endothelial thrombomodulin.
In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter- and ...intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: i.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced α
β
activation and secretion. Common sequence variation of
and
, associated with GPVI-induced α
β
activation and secretion, affected parameters of GPVI-and CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.
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 α
β
. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca
. 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)
and Horm-type collagen evoked Syk-dependent Ca
rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)
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)
sequence.
Tyrosine kinase inhibitors (TKIs), such as sunitinib, are used for cancer treatment, but may also affect platelet count and function with possible hemostatic consequences. Here, we investigated ...whether patient treatment with the TKI sunitinib affected quantitative and qualitative platelet traits as a function of the sunitinib level and the occurrence of bleeding.
Blood was collected from 20 metastatic renal cell carcinoma (mRCC) patients before treatment, and at 2 weeks, 4 weeks and 3 months after sunitinib administration. We measured blood cell counts, platelet aggregation, and concentrations of sunitinib as well as its N-desethyl metabolite in plasma, serum and isolated platelets. Progression of disease (PD) and bleeding were monitored after 3 months.
In sunitinib-treated mRCC patients, concentrations of (N-desethyl-)sunitinib in plasma and serum were highly correlated. In the patients' platelets the active metabolite levels were relatively increased as compared to sunitinib. On average, a sustained reduction in platelet count was observed on-treatment, which was significantly related to the inhibitor levels in plasma/serum. Principal component and correlational analysis showed that the (N-desethyl-)sunitinib levels in plasma/serum were linked to a reduction in both platelet count and collagen-induced platelet aggregation. The reduced aggregation associated in part with reported bleeding, but did not correlate to PD.
The sunitinib-induced reduction in quantitative and qualitative platelet traits may reflect the effective sunitinib levels in the patient. These novel results may serve as a proof-of-principle for other TKI-related drugs, where both platelet count and functions are affected, which could be used for therapeutic drug monitoring.
In haemostasis and thrombosis, platelet, coagulation and anticoagulation pathways act together to produce fibrin-containing thrombi. We developed a microspot-based technique, in which we assessed ...platelet adhesion, platelet activation, thrombus structure and fibrin clot formation in real time using flowing whole blood. Microspots were made from distinct platelet-adhesive surfaces in the absence or presence of tissue factor, thrombomodulin or activated protein C. Kinetics of platelet activation, thrombus structure and fibrin formation were assessed by fluorescence microscopy. This work revealed: (1) a priming role of platelet adhesion in thrombus contraction and subsequent fibrin formation; (2) a surface-independent role of tissue factor, independent of the shear rate; (3) a mechanism of tissue factor-enhanced activation of the intrinsic coagulation pathway; (4) a local, suppressive role of the anticoagulant thrombomodulin/protein C pathway under flow. Multiparameter analysis using blood samples from patients with (anti)coagulation disorders indicated characteristic defects in thrombus formation, in cases of factor V, XI or XII deficiency; and in contrast, thrombogenic effects in patients with factor V-Leiden. Taken together, this integrative phenotyping approach of platelet-fibrin thrombus formation has revealed interaction mechanisms of platelet-primed key haemostatic pathways with alterations in patients with (anti)coagulation defects. It can help as an important functional add-on whole-blood phenotyping.
In recent years, considerable progress has been made in understanding the mechanisms involved in platelet activation during hemostasis and thrombosis. Parallel-plate flow chambers and other ...microfluidic devices have markedly contributed to this insight. Conversely, such flow devices are now increasingly used to monitor the combined processes of platelet aggregation, thrombus formation, and coagulation in human blood. Currently, by combining microspotting and multi-color fluorescence microscopy, this technology offers the capability of high-throughput measurement of platelet activation processes, even in small blood samples. Here we review the potential of flow chamber devices for complex (multiparameter) platelet and coagulation phenotyping, focusing on patients with (genetic) platelet- or coagulation-based bleeding disorders as well as monitoring of antithrombotic medication. Animal studies are not discussed.
The chemokines CCL5 and CXCL4 are deposited by platelets onto endothelial cells, inducing monocyte arrest. Here, the fate of CCL5 and CXCL4 after endothelial deposition was investigated. Human ...umbilical vein endothelial cells (HUVECs) and EA.hy926 cells were incubated with CCL5 or CXCL4 for up to 120 min, and chemokine uptake was analyzed by microscopy and by ELISA. Intracellular calcium signaling was visualized upon chemokine treatment, and monocyte arrest was evaluated under laminar flow. Whereas CXCL4 remained partly on the cell surface, all of the CCL5 was internalized into endothelial cells. Endocytosis of CCL5 and CXCL4 was shown as a rapid and active process that primarily depended on dynamin, clathrin, and G protein-coupled receptors (GPCRs), but not on surface proteoglycans. Intracellular calcium signals were increased after chemokine treatment. Confocal microscopy and ELISA measurements in cell organelle fractions indicated that both chemokines accumulated in the nucleus. Internalization did not affect leukocyte arrest, as pretreatment of chemokines and subsequent washing did not alter monocyte adhesion to endothelial cells. Endothelial cells rapidly and actively internalize CCL5 and CXCL4 by clathrin and dynamin-dependent endocytosis, where the chemokines appear to be directed to the nucleus. These findings expand our knowledge of how chemokines attract leukocytes to sites of inflammation.
The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet–endothelium interaction. Using ...high‐shear whole‐blood microfluidics, employing coagulant or non‐coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real‐time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen‐related peptide (CRP‐XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label‐free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP‐XL, and/or thrombin. High‐resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable‐isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium‐promoted activity of a multi‐kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.
Novel regulation of a kinase network in platelets by endothelial cells and platelet agonists.
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