Ultra-high throughput-based screening for the discovery of antiplatelet drugs affecting receptor dependent calcium signaling dynamics.

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules.

Design, manufacturing and testing of a green non-isocyanate polyurethane prosthetic heart valve.

The sole effective treatment for most patients with heart valve disease is valve replacement by implantation of mechanical or biological prostheses. However, mechanical valves represent high risk of thromboembolism, and biological prostheses are prone to early degeneration. In this work, we aim to determine the potential of novel environmentally-friendly non-isocyanate polyurethanes (NIPUs) for manufacturing synthetic prosthetic heart valves. Polyhydroxyurethane (PHU) NIPUs are synthesized via an isocyanate-free route, tested in vitro, and used to produce aortic valves. PHU elastomers reinforced with a polyester mesh show mechanical properties similar to native valve leaflets. These NIPUs do not cause hemolysis. Interestingly, both platelet adhesion and contact activation-induced coagulation are strongly reduced on NIPU surfaces, indicating low thrombogenicity. Fibroblasts and endothelial cells maintain normal growth and shape after indirect contact with NIPUs. Fluid-structure interaction (FSI) allows modeling of the ideal valve design, with minimal shear stress on the leaflets. Injection-molded valves are tested in a pulse duplicator and show ISO-compliant hydrodynamic performance, comparable to clinically-used bioprostheses. Poly(tetrahydrofuran) (PTHF)-NIPU patches do not show any evidence of calcification over a period of 8 weeks. NIPUs are promising sustainable biomaterials for the manufacturing of improved prosthetic valves with low thrombogenicity.

Endothelium-mediated regulation of platelet activation: Involvement of multiple protein kinases.

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.

Restraining of glycoprotein VI- and integrin α2ß1-dependent thrombus formation by platelet PECAM1.

The platelet receptors, glycoprotein VI (GPVI) and integrin α2ß1 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 α2ß1 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 α2ß1 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 α2ß1 generally reduced thrombus parameters, most effectively on collagen IV. Strikingly, simultaneous inhibition of PECAM1 and α2ß1 led to a restoration of thrombus formation, indicating that the suppressing signaling effect of PECAM1 is masked by the platelet-adhesive receptor α2ß1. Blood from 4 out of 6 Noonan patients showed subnormal thrombus formation on collagen IV. In these patients, effects of α2ß1 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 α2ß1 engagement.

Regulation of Glycoprotein VI-Dependent Platelet Activation and Thrombus Formation by Heparan Sulfate Proteoglycan Perlecan.

Proteoglycans form a heterogeneous family of proteins with covalently bound sulfated glycosaminoglycans. The extracellular matrix proteoglycan perlecan has been proposed to bind to the platelet- and megakaryocyte-specific receptor G6bB, co-regulating platelet glycoprotein VI (GPVI) signaling. The derived non-sulfate proteoglycan endorepellin was previously shown to enhance platelet adhesion via the collagen receptor, integrin α2ß1. Here, we compared the roles of perlecan and other matrix proteoglycans in platelet responses and thrombus formation. We used multi-color flow cytometry to measure the degranulation and integrin αIIbß3 activation of washed platelets in response to various proteoglycans and collagen-related peptide (CRP), the GPVI agonist. Perlecan, but not endorepellin, enhanced the CRP-induced activation of platelets in a time- and concentration-dependent manner. Similar to collagen, immobilized perlecan, but not other proteoglycans, supported static platelet adhesion and spreading. In-flowed whole-blood perlecan diminished shear-dependent platelet adhesion, while it enforced GPVI-dependent thrombus formation-to a larger extent than endorepellin-to induce more contracted aggregates of activated platelets. We concluded that the sulfated proteoglycan perlecan enhances GPVI-dependent platelet responses extending to thrombus formation, but it does so at the expense of reduced adhesion of platelets under flow.

Crucial roles of red blood cells and platelets in whole blood thrombin generation.

Red blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to reflect the interactions between plasma coagulation and the various blood cells. Using a new high-throughput method capturing the complete TG curve, we were able to compare TG in whole blood and autologous platelet-rich and platelet-poor plasma to redefine the blood cell contributions to the clotting process. We report a faster and initially higher generation of thrombin and shorter coagulation time in whole blood than in platelet-rich plasma upon low concentrations of coagulant triggers, including tissue factor, Russell viper venom factor X, factor Xa, factor XIa, and thrombin. The TG was accelerated with increased hematocrit and delayed after prior treatment of RBC with phosphatidylserine-blocking annexin A5. RBC treatment with ionomycin increased phosphatidylserine exposure, confirmed by flow cytometry, and increased the TG process. In reconstituted blood samples, the prior selective blockage of phosphatidylserine on RBC with annexin A5 enhanced glycoprotein VI-induced platelet procoagulant activity. For patients with anemia or erythrocytosis, cluster analysis revealed high or low whole-blood TG profiles in specific cases of anemia. The TG profiles lowered upon annexin A5 addition in the presence of RBCs and thus were determined by the extent of phosphatidylserine exposure of blood cells. Profiles for patients with polycythemia vera undergoing treatment were similar to that of control subjects. We concluded that RBC and platelets, in a phosphatidylserine-dependent way, contribute to the TG process. Determination of the whole-blood hypo- or hyper-coagulant activity may help to characterize a bleeding or thrombosis risk.

A signature of platelet reactivity in CBC scattergrams reveals genetic predictors of thrombotic disease risk.

Genetic studies of platelet reactivity (PR) phenotypes may identify novel antiplatelet drug targets. However, such studies have been limited by small sample sizes (n < 5000) because of the complexity of measuring PR. We trained a model to predict PR from complete blood count (CBC) scattergrams. A genome-wide association study of this phenotype in 29 806 blood donors identified 21 distinct associations implicating 20 genes, of which 6 have been identified previously. The effect size estimates were significantly correlated with estimates from a study of flow cytometry-measured PR and a study of a phenotype of in vitro thrombus formation. A genetic score of PR built from the 21 variants was associated with the incidence rates of myocardial infarction and pulmonary embolism. Mendelian randomization analyses showed that PR was causally associated with the risks of coronary artery disease, stroke, and venous thromboembolism. Our approach provides a blueprint for using phenotype imputation to study the determinants of hard-to-measure but biologically important hematological traits.

Antagonistic Roles of Human Platelet Integrin αIIbß3 and Chemokines in Regulating Neutrophil Activation and Fate on Arterial Thrombi Under Flow.

BACKGROUND: Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches. METHODS: Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbß3. RESULTS: We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [N-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca2+]i rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide. CONCLUSIONS: Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.

MAGT1 Deficiency Dysregulates Platelet Cation Homeostasis and Accelerates Arterial Thrombosis and Ischemic Stroke in Mice.

BACKGROUND: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. METHODS: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. RESULTS: MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channel, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GPVI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GPVI signaling, platelet aggregation, and thrombus formation in vivo. CONCLUSIONS: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.

High-throughput microfluidic blood testing to phenotype genetically linked platelet disorders: an aid to diagnosis.

Linking the genetic background of patients with bleeding diathesis and altered platelet function remains challenging. We aimed to assess how a multiparameter microspot-based measurement of thrombus formation under flow can help identify patients with a platelet bleeding disorder. For this purpose, we studied 16 patients presenting with bleeding and/or albinism and suspected platelet dysfunction and 15 relatives. Genotyping of patients revealed a novel biallelic pathogenic variant in RASGRP2 (splice site c.240-1G>A), abrogating CalDAG-GEFI expression, compound heterozygosity (c.537del, c.571A>T) in P2RY12, affecting P2Y12 signaling, and heterozygous variants of unknown significance in the P2RY12 and HPS3 genes. Other patients were confirmed to have Hermansky-Pudlak syndrome type 1 or 3. In 5 patients, no genetic variant was found. Platelet functions were assessed via routine laboratory measurements. Blood samples from all subjects and day controls were screened for blood cell counts and microfluidic outcomes on 6 surfaces (48 parameters) in comparison with those of a reference cohort of healthy subjects. Differential analysis of the microfluidic data showed that the key parameters of thrombus formation were compromised in the 16 index patients. Principal component analysis revealed separate clusters of patients vs heterozygous family members and control subjects. Clusters were further segregated based on inclusion of hematologic values and laboratory measurements. Subject ranking indicated an overall impairment in thrombus formation in patients carrying a (likely) pathogenic variant of the genes but not in asymptomatic relatives. Taken together, our results indicate the advantages of testing for multiparametric thrombus formation in this patient population.

Combining human platelet proteomes and transcriptomes: possibilities and challenges.

The anucleate human platelets contain a broad pattern of mRNAs and other RNA transcripts. The high quantitative similarity of mRNAs in megakaryocytes and platelets from different sources points to a common origin, and suggests a random redistribution of mRNA species upon proplatelet formation. A comparison of the classified platelet transcriptome (17.6k transcripts) with the identified platelet proteome (5.2k proteins) indicates an under-representation of: (i) nuclear but not of other organellar proteins; (ii) membrane receptors and channels with low transcript levels; (iii) transcription/translation proteins; and (iv) so far uncharacterized proteins. In this review, we discuss the technical, normalization and database-dependent possibilities and challenges to come to a complete, genome-wide platelet transcriptome and proteome. Such a reference transcriptome and proteome can serve to further elucidate intra-subject and inter-subject differences in platelets in health and disease. Applications may also lay in the aid of genetic diagnostics.

Blood platelets contain thousands types of mRNAs and proteins. The mRNA composition is similar to the mRNAs of megakaryocytes, from which platelets are derived, suggesting a random redistribution upon platelet formation. First attempts to identify all classified platelet proteins from mass spectral analysis used the genome-wide information of all mRNA types. This analysis revealed that the so far absent proteins in platelets are especially located in the megakaryocyte nucleus, or have low mRNA levels or low copy numbers. In this review, we discuss the possibilities to come to subject-dependent identification of the platelet protein and mRNA composition. Future applications may aid the genetic diagnostics.

Role of SHP2 (PTPN11) in glycoprotein VI-dependent thrombus formation: Improved platelet responsiveness by the allosteric drug SHP099 in Noonan syndrome patients.

INTRODUCTION: The protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. MATERIALS AND METHODS: Washed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. RESULTS: Pharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbß3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11-mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. CONCLUSION: Pharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients.

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.

Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets.

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.

Platelet glycoprotein V spatio-temporally controls fibrin formation.

The activation of platelets and coagulation at vascular injury sites is crucial for haemostasis but can promote thrombosis and inflammation in vascular pathologies. Here, we delineate an unexpected spatio-temporal control mechanism of thrombin activity that is platelet orchestrated and locally limits excessive fibrin formation after initial haemostatic platelet deposition. During platelet activation, the abundant platelet glycoprotein (GP) V is cleaved by thrombin. We demonstrate with genetic and pharmacological approaches that thrombin-mediated shedding of GPV does not primarily regulate platelet activation in thrombus formation, but rather has a distinct function after platelet deposition and specifically limits thrombin-dependent generation of fibrin, a crucial mediator of vascular thrombo-inflammation. Genetic or pharmacologic defects in haemostatic platelet function are unexpectedly attenuated by specific blockade of GPV shedding, indicating that the spatio-temporal control of thrombin-dependent fibrin generation also represents a potential therapeutic target to improve haemostasis.

High-throughput assessment identifying major platelet Ca2+ entry pathways via tyrosine kinase-linked and G protein-coupled receptors.

In platelets, elevated cytosolic Ca2+ is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca2+ response consists of Ca2+ mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca2+ entry pathways. Several channels can contribute to the Ca2+ entry, but their relative contribution is unclear upon stimulation of ITAM-linked receptors such as glycoprotein VI (GPVI) and G-protein coupled receptors such as the protease-activated receptors (PAR) for thrombin. We employed a 96-well plate high-throughput assay with Fura-2-loaded human platelets to perform parallel [Ca2+]i measurements in the presence of EGTA or CaCl2. Per agonist condition, this resulted in sets of EGTA, CaCl2 and Ca2+ entry ratio curves, defined by six parameters, reflecting different Ca2+ ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca2+ entry ratio of 3-7. Strikingly, in combination with Ca2+-ATPase inhibition by thapsigargin, the maximal Ca2+ entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca2+ entry. By pharmacological blockage of specific Ca2+ channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca2+ entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na+/Ca2+ exchange (NCE) > P2×1 (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca2+ carriers regulating GPVI- and PAR-induced Ca2+ entry in human platelets.

An agent-based approach for modelling and simulation of glycoprotein VI receptor diffusion, localisation and dimerisation in platelet lipid rafts.

Receptor diffusion plays an essential role in cellular signalling via the plasma membrane microenvironment and receptor interactions, but the regulation is not well understood. To aid in understanding of the key determinants of receptor diffusion and signalling, we developed agent-based models (ABMs) to explore the extent of dimerisation of the platelet- and megakaryocyte-specific receptor for collagen glycoprotein VI (GPVI). This approach assessed the importance of glycolipid enriched raft-like domains within the plasma membrane that lower receptor diffusivity. Our model simulations demonstrated that GPVI dimers preferentially concentrate in confined domains and, if diffusivity within domains is decreased relative to outside of domains, dimerisation rates are increased. While an increased amount of confined domains resulted in further dimerisation, merging of domains, which may occur upon membrane rearrangements, was without effect. Modelling of the proportion of the cell membrane which constitutes lipid rafts indicated that dimerisation levels could not be explained by these alone. Crowding of receptors by other membrane proteins was also an important determinant of GPVI dimerisation. Together, these results demonstrate the value of ABM approaches in exploring the interactions on a cell surface, guiding the experimentation for new therapeutic avenues.

Pharmacological Inhibition of Glycoprotein VI- and Integrin α2ß1-Induced Thrombus Formation Modulated by the Collagen Type.

BACKGROUND: In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2ß1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2ß1 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs. METHODS: Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and α2ß1. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28. RESULTS AND CONCLUSION: In this first comparison of four inhibitors of platelet-collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2ß1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2ß1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs.

Repeated platelet activation and the potential of previously activated platelets to contribute to thrombus formation.

BACKGROUND: Especially in disease conditions, platelets can encounter activating agents in circulation. OBJECTIVES: To investigate the extent to which previously activated platelets can be reactivated and whether in-and reactivation applies to different aspects of platelet activation and thrombus formation. METHODS: Short-and long-term effects of glycoprotein VI (GPVI) and G protein-coupled receptor (GPCR) stimulation on platelet activation and aggregation potential were compared via flow cytometry and plate-based aggregation. Using fluorescence and electron microscopy, we assessed platelet morphology and content, as well as thrombus formation. RESULTS: After 30 minutes of stimulation with thrombin receptor activator peptide 6 (TRAP6) or adenosine diphosphate (ADP), platelets secondarily decreased in PAC-1 binding and were less able to aggregate. The reversibility of platelets after thrombin stimulation was concentration dependent. Reactivation was possible via another receptor. In contrast, cross-linked collagen-related peptide (CRP-XL) or high thrombin stimulation evoked persistent effects in αIIbß3 activation and platelet aggregation. However, after 60 minutes of CRP-XL or high thrombin stimulation, when αIIbß3 activation slightly decreased, restimulation with ADP or CRP-XL, respectively, increased integrin activation again. Compatible with decreased integrin activation, platelet morphology was reversed. Interestingly, reactivation of reversed platelets again resulted in shape change and if not fully degranulated, additional secretion. Moreover, platelets that were previously activated with TRAP6 or ADP regained their potential to contribute to thrombus formation under flow. On the contrary, prior platelet triggering with CRP-XL was accompanied by prolonged platelet activity, leading to a decreased secondary platelet adhesion under flow. CONCLUSION: This work emphasizes that prior platelet activation can be reversed, whereafter platelets can be reactivated through a different receptor. Reversed, previously activated platelets can contribute to thrombus formation.

Multi-omics approaches to study platelet mechanisms.

Platelets are small anucleate cell fragments (2-4 µm in diameter) in the blood, which play an essential role in thrombosis and hemostasis. Genetic or acquired platelet dysfunctions are linked to bleeding, increased risk of thromboembolic events and cardiovascular diseases. Advanced proteomic approaches may pave the way to a better understanding of the roles of platelets in hemostasis, and pathophysiological processes such as inflammation, metastatic spread and thrombosis. Further insights into the molecular biology of platelets are crucial to aid drug development and identify diagnostic markers of platelet activation. Platelet activation is known to be an extremely rapid process and involves multiple post-translational mechanisms at sub second time scale, including proteolysis and phosphorylation. Multi-omics technologies and biochemical approaches can be exploited to precisely probe and define these posttranslational pathways. Notably, the absence of a nucleus in platelets significantly reduces the number of present proteins, simplifying mass spectrometry-based proteomics and metabolomics approaches.