Search

Platelets have an important role in tumor angiogenesis, growth, and metastasis. The reciprocal interaction between cancer and platelets results in changes of several platelet characteristics. It is becoming clear that analysis of these platelet features could offer a new strategy in the search for biomarkers of cancer. Here, we review the human studies in which platelet characteristics (e.g., count, volume, protein, and mRNA content) are investigated in early-stage cancer. The main focus of this paper is to evaluate which platelet features are suitable for the development of a blood test that could detect cancer in its early stages.

Current antiplatelet drugs for the treatment of arterial thrombosis often coincide with increased bleeding risk. Several tyrosine kinase inhibitors (TKIs) for cancer treatment inhibit platelet function, with minor reported bleeding symptoms. The aim of this study was to compare the antiplatelet properties of eight TKIs to explore their possible repurposing as antiplatelet drugs. Samples of whole blood, platelet-rich plasma (PRP), or isolated platelets from healthy donors were treated with TKI or the vehicle. Measurements of platelet aggregation, activation, intracellular calcium mobilization, and whole-blood thrombus formation under flow were performed. Dasatinib and sunitinib dose-dependently reduced collagen-induced aggregation in PRP and washed platelets; pazopanib, cabozantinib, and vatalanib inhibited this response in washed platelets only; and fostamatinib, axitinib, and lapatinib showed no/limited effects. Fostamatinib reduced thrombus formation by approximately 50% on collagen and other substrates. Pazopanib, sunitinib, dasatinib, axitinib, and vatalanib mildly reduced thrombus formation on collagen by 10–50%. Intracellular calcium responses in isolated platelets were inhibited by dasatinib (>90%), fostamatinib (57%), sunitinib (77%), and pazopanib (82%). Upon glycoprotein-VI receptor stimulation, fostamatinib, cabozantinib, and vatalanib decreased highly activated platelet populations by approximately 15%, while increasing resting populations by 39%. In conclusion, the TKIs with the highest affinities for platelet-expressed molecular targets most strongly inhibited platelet functions. Dasatinib, fostamatinib, sunitinib, and pazopanib interfered in early collagen receptor-induced molecular-signaling compared with cabozantinib and vatalanib. Fostamatinib, sunitinib, pazopanib, and vatalanib may be promising for future evaluation as antiplatelet drugs.

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 alpha(IIb)beta(3) activation and secretion. Common sequence variation of GP6 and FCER1G, associated with GPVI-induced alpha(IIb)beta(3) 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.

Background: Low plasma levels of protein C or protein S are associated with venous thromboembolism rather than myocardial infarction. The high coagulant activity in patients with thrombophilia with a (familial) defect in protein C or S is explained by defective protein C activation, involving thrombomodulin and protein S. This causes increased plasmatic thrombin generation.Objective: Assess the role of platelets in the thrombus- and fibrin-forming potential in patients with familial protein C or protein S deficiency under high-shear flow conditions.Patients/Methods: Whole blood from 23 patients and 15 control subjects was perfused over six glycoprotein VI-dependent microspot surfaces. By real-time multicolor microscopic imaging, kinetics of platelet thrombus and fibrin formation were characterized in 49 parameters.Results and Conclusion: Whole-blood flow perfusion over collagen, collagen-like peptide, and fibrin surfaces with low or high GPVI dependency indicated an unexpected impairment of platelet activation, thrombus phenotype, and fibrin formation but unchanged platelet adhesion, observed in patients with protein C deficiency and to a lesser extent protein S deficiency, when compared to controls. The defect extended from diminished phosphatidylserine exposure and thrombus contraction to delayed and suppressed fibrin formation. The mechanism was thrombomodulin independent, and may involve negative platelet priming by plasma components.

Objective: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets’ inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s−1. Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro. Conclusions: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

Background Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation. Methods Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca2+]i, aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed. Results Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation. Conclusion Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding.

All irreversible Bruton tyrosine kinase (Btk) inhibitors including ibrutinib and acalabrutinib induce platelet dysfunction and increased bleeding risk. New reversible Btk inhibitors were developed, like MK-1026. The mechanism underlying increased bleeding tendency with Btk inhibitors remains unclear. We investigated the effects of ibrutinib, acalabrutinib and MK-1026 on platelet function in healthy volunteers, patients and Btk-deficient mice, together with off-target effects on tyrosine kinase phosphorylation. All inhibitors suppressed GPVI- and CLEC-2-mediated platelet aggregation, activation and secretion in a dose-dependent manner. Only ibrutinib inhibited thrombus formation on vWF-co-coated surfaces, while on collagen this was not affected. In blood from Btk-deficient mice, collagen-induced thrombus formation under flow was reduced, but preincubation with either inhibitor was without additional effects. MK-1026 showed less off-target effects upon GPVI-induced TK phosphorylation as compared to ibrutinib and acalabrutinib. In ibrutinib-treated patients, GPVI-stimulated platelet activation, and adhesion on vWF-co-coated surfaces were inhibited, while CLEC-2 stimulation induced variable responses. The dual inhibition of GPVI and CLEC-2 signalling by Btk inhibitors might account for the increased bleeding tendency, with ibrutinib causing more high-grade bleedings due to additional inhibition of platelet-vWF interaction. As MK-1026 showed less off-target effects and only affected activation of isolated platelets, it might be promising for future treatment.© 2021 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.

Thrombosis and haemostasis 726-738 (2021). doi:10.1055/s-0041-1735972, Published by Thieme, Stuttgart

Most agonists stimulate platelet Ca2+ rises via G-protein coupled receptors (GPCRs) or ITAM-linked receptors (ILRs). Well studied are the GPCRs stimulated by the soluble agonists thrombin (PAR1, PAR4), ADP (P2Y(1), P2Y(12)), and thromboxane A(2) (TP), signaling via phospholipase (PLC)beta isoforms. The platelet ILRs glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2), and Fc gamma RIIa are stimulated by adhesive ligands or antibody complexes and signal via tyrosine protein kinases and PLC gamma isoforms. Marked differences exist between the GPCR- and ILR-induced Ca2+ signaling in: (i) dependency of tyrosine phosphorylation; (ii) oscillatory versus continued Ca2+ rises by mobilization from the endoplasmic reticulum; and (iii) smaller or larger role of extracellular Ca2+ entry via STIM1/ORAI1. Co-stimulation of both types of receptors, especially by thrombin (PAR1/4) and collagen (GPVI), leads to a highly enforced Ca2+ rise, involving mitochondrial Ca2+ release, which activates the ion and phospholipid channel, anoctamin-6. This highly Ca2+-dependent process causes swelling, ballooning, and phosphatidylserine expression, establishing a unique platelet population swinging between vital and necrotic (procoagulant 'zombie' platelets). Additionally, the high Ca2+ status of procoagulant platelets induces a set of additional events: (i) Ca2+ dependent cleavage of signaling proteins and receptors via calpain and ADAM isoforms; (ii) microvesiculation; (iii) enhanced coagulation factor binding; and (iv) fibrin-coat formation involving transglutaminases. Given the additive roles of GPCR and ILR in Ca2+ signal generation, high-throughput screening of biomolecules or small molecules based on Ca2+ flux measurements provides a promising way to find new inhibitors interfering with prolonged high Ca2+, phosphatidylserine expression, and hence platelet procoagulant activity.

While in recent trials the dual pathway inhibition with aspirin plus rivaroxaban has shown to be efficacious in patients with atherosclerotic cardiovascular disease, little is known about the effects of this combination treatment on thrombus formation and vascular remodelling upon vascular damage. The aim of this study was to examine the effects of aspirin and/or rivaroxaban on injury-induced murine arterial thrombus formation in vivo and in vitro, vessel-wall remodelling, and platelet-leukocyte aggregates. Temporary ligation of the carotid artery of C57BL/6 mice, fed a western type diet, led to endothelial denudation and sub-occlusive thrombus formation. At the site of ligation, the vessel wall stiffened and the intima-media thickened. Aspirin treatment antagonized vascular stiffening and rivaroxaban treatment led to a positive trend towards reduced stiffening. Local intima-media thickening was antagonized by both aspirin or rivaroxaban treatment. Platelet-leukocyte aggregates and the number of platelets per leukocyte were reduced in aspirin and/or rivaroxaban treatment groups. Furthermore, rivaroxaban restricted thrombus growth and height in vitro. In sum, this study shows vascular protective effects of aspirin and rivaroxaban, upon vascular injury of the mouse artery.

Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr−/− mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions., Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient (Ldlr−/−) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr−/− mice and CONV-R Ldlr−/− mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr−/− mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr−/− mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr−/− mice on HFD were diminished compared to CONV-R Ldlr−/− controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr−/− mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr−/− mice relative to CONV-R Ldlr−/− mice. Ex vivo, this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr−/− mice on type III collagen.

Item does not contain fulltext Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-kappaB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.

Background: Sunitinib is an oral tyrosine kinase inhibitor used for cancer treatment. Patients treated with sunitinib are at higher bleeding risk. As tyrosine kinases are essential for platelet signalling, the effects of sunitinib on platelet function in vitro and in cancer patients on treatment were investigated.Patients and methods: Blood samples were collected from eight healthy volunteers and eight patients diagnosed with metastatic renal cell cancer (RCC) before and 2 weeks on treatment with sunitinib. Platelets from 15 additional healthy individuals were preincubated with sunitinib or vehicle to perform in vitro experiments. Immunofluorescence imaging, western blotting, light transmission aggregometry, whole blood perfusion over collagen, flow cytometry and ELISA were performed.Results: Confocal microscopy indicated that platelets sequester sunitinib in vitro and in patients. In platelets from healthy controls, tyrosine phosphorylation was inhibited by sunitinib. Also, sunitinib dose dependently reduced collagen- and ADP-induced aggregation, collagen-dependent thrombus formation and collagen-induced secretion of platelet-derived growth factor and β-thromboglobulin. In blood from RCC patients before treatment, thrombus formation and procoagulant activity under flow were 47% and 80% higher than in healthy controls. After 14 d of sunitinib treatment, platelet count was moderately, but significantly decreased (from 243 to 144 × 109/l). At the same time, collagen-induced platelet aggregation as well as thrombus formation and phosphatidylserine exposure under flow were significantly reduced (by 45%, 16% and 61%, respectively).Conclusions: Sunitinib uptake by platelets inhibits collagen receptor-induced aggregation and thrombus formation via reduction of protein tyrosine phosphorylation and α-granule secretion. This dysfunction may contribute to the higher bleeding tendency observed in sunitinib-treated patients.

Objective— Beyond their eminent role in hemostasis and thrombosis, platelets are recognized as mediators of inflammation. Platelet cluster of differentiation 40 (CD40) ligand (CD40L and CD154) plays a key role in mediating platelet-induced inflammation in atherosclerosis. CD40, the receptor for CD40L, is present on platelets; however, the role of CD40 on this cell type is until now undefined. Approach and Results— We found that in both mice and humans, platelet CD40 mediates the formation of platelet–leukocyte aggregates and the release of chemokine (C-X-C motif) ligand 4. Leukocytes were also less prone to adhere to CD40-deficient thrombi. However, platelet CD40 was not involved in platelet aggregation. Activated platelets isolated from Cd40 −/− Apoe −/− mice adhered less to the endothelium upon injection into Apoe −/− mice when compared with CD40-sufficient platelets. Furthermore, lack of CD40 on injected platelets led to reduced leukocyte recruitment to the carotid artery as assayed by intravital microscopy. This was accompanied by a decrease in endothelial vascular cell adhesion molecule-1, platelet endothelial cell adhesion molecule, VE-cadherin, and P-selectin expression. To investigate the effect of platelet CD40 in atherosclerosis, Apoe −/− mice received thrombin-activated Apoe −/− or Cd40 −/− Apoe −/− platelets every 5 days for 12 weeks, starting at the age of 17 weeks, when atherosclerotic plaques had already formed. When compared with mice that received Apoe −/− platelets, those receiving Cd40 −/− Apoe −/− platelets exhibited a >2-fold reduction in atherosclerosis. Plaques of mice receiving CD40-deficient platelets were less advanced, contained less macrophages, neutrophils, and collagen, and displayed smaller lipid cores. Conclusions— Platelet CD40 plays a crucial role in inflammation by stimulating leukocyte activation and recruitment and activation of endothelial cells, thereby promoting atherosclerosis.

We would like to promote the fact that platelets are increasingly emerging as a rich source of potential biomarkers for cancer. Blood platelets contain vast amounts of bioactive proteins, such as growth factors, chemokines, and cytokines. These proteins are either synthesized by the megakaryocytes that produce the platelets or are sequestered by the circulating platelets from the blood, in which case these proteins may originate from the tumor. Recent studies in patients have demonstrated that the presence of cancer influences multiple platelet characteristics (e.g., platelet count, volume, activation status, proteins, and RNA content). Interestingly, these changes happened already in early stages of the disease before metastasis had occurred. Additionally, exploiting these platelet alterations enabled discrimination of patients with early-stage cancer from healthy sex- and age-matched individuals. Therefore, we challenge clinicians and researchers to look beyond traditional fluid sources such as plasma or serum, and to take platelets and their content into account as they may become the holy grail in cancer blood biomarker research.

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 &alpha, 2&beta, 1. 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.

Pazopanib is an angiostatic tyrosine kinase inhibitor (TKI) presently used for cancer treatment, particularly in patients with renal cell carcinoma (RCC). This treatment can be accompanied by mild bleeding as an adverse effect. Given the role of protein tyrosine kinases in platelet activation processes, we investigated whether and how pazopanib can affect platelet functions in purified systems and during treatment of advanced RCC patients. In isolated platelets from healthy volunteers, pazopanib dose-dependently reduced collagen-induced integrin activation and secretion, as well as platelet aggregation. Pazopanib addition diminished glycoprotein (GP) VI-dependent tyrosine phosphorylation of multiple platelet proteins, including the tyrosine kinase Syk. Furthermore, pazopanib inhibited GPVI-induced Ca2+ elevation, resulting in reduced exposure of the procoagulant phospholipid phosphatidylserine (PS). Upon perfusion of control blood over a collagen surface, pazopanib inhibited thrombus size as well as PS exposure. Blood samples from 10 RCC patients were also analyzed before and after 14 days of pazopanib treatment as monotherapy. This treatment caused an overall lowering in platelet count, with 3 out of 10 patients experiencing mild bleeding. Platelets isolated from pazopanib-treated patients showed a significant lowering of PS exposure upon activation. In addition, platelet procoagulant activity was inhibited in thrombi formed under flow conditions. Control experiments indicated that higher pazopanib concentrations were required to inhibit GPVI-mediated PS exposure in the presence of plasma. Together, these results indicated that pazopanib suppresses GPVI-induced platelet activation responses in a way partly antagonized by the presence of plasma. In treated cancer patients, pazopanib effects were confined to a reduction in GPVI-dependent PS exposure. Together with the reduced platelet count, this may explain the mild bleeding tendency observed in pazopanib-treated patients.

Src family kinases (SFKs) coordinate the initiating and propagating activation signals in platelets, but it remains unclear how they are regulated. Here, we show that ablation of C-terminal Src kinase (Csk) and receptor-like protein tyrosine-phosphatase CD148 in mice results in a dramatic increase in platelet SFK activity, demonstrating that these proteins are essential regulators of platelet reactivity. Paradoxically, Csk/CD148-deficient mice exhibit reduced in vivo and ex vivo thrombus formation and increased bleeding following injury rather than a prothrombotic phenotype. This is a consequence of multiple negative feedback mechanisms, including downregulation of the immunoreceptor tyrosine-based activation motif (ITAM)- and hemi-ITAM-containing receptors glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain and CLEC-2, respectively and upregulation of the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B and its interaction with the tyrosine phosphatases Shp1 and Shp2. Results from an analog-sensitive Csk mouse model demonstrate the unconventional role of SFKs in activating ITIM signaling. This study establishes Csk and CD148 as critical molecular switches controlling the thrombotic and hemostatic capacity of platelets and reveals cell-intrinsic mechanisms that prevent pathological thrombosis from occurring.

Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano) 6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca2+-dependent K(Ca)3.1Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6min (control 6.4min, P 0.05) with reduced PS exposure (265 to 90%); 2) lowered Ca2+-dependent swelling (280%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.

The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations.

Platelets can contribute to tumor progression and metastasis. Cancer patients are at increased risk of thrombosis, and advanced stages of cancer are associated with thrombocytosis or increased platelet reactivity. Tyrosine kinase inhibitors (TKIs) are widely used as a targeted strategy for cancer treatment, with the aim of prolonging progression-free survival of the patients. Because of their broad kinase target spectrum, most TKIs inevitably have off-target effects. Platelets rely on tyrosine kinase activity for their activation. Frequently observed side effects are lowering of platelet count and inhibition of platelet functions, whether or not accompanied by an increased bleeding risk. In this review, we aim to give insights into: (i) 38 TKIs that are currently used for the treatment of different types of cancer, either on the market or in clinical trials; (ii) how distinct TKIs can inhibit activation mechanisms in platelets; and (iii) the clinical consequences of the antiplatelet effects of TKI treatment. For several TKIs, the knowledge on affinity for their targets does not align with the published effects on platelets and reported bleeding events. This review should raise awareness of the potential antiplatelet effects of several TKIs, which will be enhanced in the presence of antithrombotic drugs.

Platelets play an important role in tumor growth and, at the same time, platelet characteristics are affected by cancer presence. Therefore, we investigated whether the platelet proteome harbors differentially expressed proteins associated with early-stage cancer. For this proof-of-concept study, patients with early-stage lung (n = 8) or head of pancreas cancer (n = 4) were included, as were healthy sex- and age-matched controls for both subgroups. Blood samples were collected from controls and from patients before surgery. Furthermore, from six of the patients, a second sample was collected two months after surgery. NanoLC-MS/MS-based proteomics of gel-fractionated platelet proteins was used for comparative spectral count analyses of patients to controls and before to after surgery samples. The total platelet proteome dataset included 4384 unique proteins of which 85 were significantly (criteria Fc > 1.5 and p < 0.05) changed in early-stage cancer compared to controls. In addition, the levels of 81 platelet proteins normalized after tumor resection. When filtering for the most discriminatory proteins, we identified seven promising platelet proteins associated with early-stage cancer. In conclusion, this pioneering study on the platelet proteome in cancer patients clearly identifies platelets as a new source of candidate protein biomarkers of early-stage cancer. Biological significance: Currently, most blood-based diagnostics/biomarker research is performed in serum or plasma, while the content of blood cells is usually neglected. It is known that especially blood platelets, which are the main circulating pool of many bioactive proteins, such as growth factors, chemokines, and cytokines, are a potentially rich source of biomarkers. The current study is the first to measure the effect of early-stage cancer on the platelet proteome of patients. Our study demonstrates that the platelet proteome of patients with early stage lung or head of pancreas cancer differs considerably compared to that of healthy individuals of matched sex and age. In addition, the platelet proteome of cancer patients normalized after surgical resection of the tumor. Exploiting platelet proteome differences linked to both tumor presence and disease status, we were able to demonstrate that the platelet proteome can be mined for potential biomarkers of cancer.

Objective— To investigate the roles and signaling pathways of CD40L and CD40 in platelet–platelet interactions and thrombus formation under conditions relevant for atherothrombosis. Approach and Results— Platelets from mice prone to atherosclerosis lacking CD40L ( Cd40lg −/− Apoe −/− ) showed diminished α IIb β 3 activation and α-granule secretion in response to glycoprotein VI stimulation, whereas these responses of CD40-deficient platelets ( Cd40 −/− Apoe −/− ) were not decreased. Using blood from Cd40lg −/− Apoe −/− and Cd40 −/− Apoe −/− mice, the glycoprotein VI-dependent formation of dense thrombi was impaired on atherosclerotic plaque material or on collagen, in comparison with Apoe −/− blood. In all genotypes, addition of CD40L to the blood enhanced the growth of dense thrombi on plaques and collagen. Similarly, CD40L enhanced glycoprotein VI–induced platelet aggregation, even with platelets deficient in CD40. This potentiation was antagonized in Pik3cb R/R platelets or by inhibiting phosphatidylinositol 3-kinase β (PI3Kβ). Addition of CD40L also enhanced collagen-induced Akt phosphorylation, which was again antagonized by absence or inhibition of PI3Kβ. Finally, platelets from Chuk1 A/A Apoe −/− mice deficient in IκB kinase α (IKKα), implicated in CD40 signaling to nuclear factor (NF) κB, showed unchanged responses to CD40L in aggregation or thrombus formation. Conclusions— Under atherogenic conditions, CD40L enhances collagen-induced platelet–platelet interactions by supporting integrin α IIb β 3 activation, secretion and thrombus growth via PI3Kβ, but not via CD40 and IKKα/NFκB. This role of CD40L exceeds the no more than modest role of CD40 in thrombus formation.

Background: Detection of early-stage cancer significantly improves patient survival. As platelets play an important role in cancer progression, we aimed to investigate whether platelets can be used for the discovery of early-stage cancer.Methods: Patients with lung (n = 86) or head of pancreas (n = 42) cancer were included, as were healthy sex-and age-matched controls (n = 92). Blood was collected before initiation of treatment. Platelet count, volume and activation status were quantified in whole blood. Next, concentrations of vascular endothelial growth factor, platelet-derived growth factor, platelet factor 4, thrombospondin-1 and connective tissue-activating peptide III were measured in both platelets and plasma. Using the results, two multivariable diagnostic models were developed and internally validated.Findings: Multiple platelet features, including platelet count, volume and protein content, were significantly changed in lung and head of pancreas cancer patients. However, the pattern of changes differed between both groups. The diagnostic model developed for lung cancer discriminated very well between patients and controls (AUC = 88.7%). Addition of smoking as a variable significantly increased the AUC of the model to 94.5%. The diagnostic model for head of pancreas cancer also performed well (AUC = 82.7%). Both models were internally validated, resulting in optimism-corrected AUC's of 86.8% and 80.8%, respectively.Interpretation: In patients with lung or head of pancreas cancer, several platelet characteristics are changed compared to healthy sex-and age-matched controls. A cancer type-specific combination of these platelet features can be used to discriminate between patients with early-stage cancer and healthy individuals. (C) 2017 Elsevier Ltd. All rights reserved.

SummaryThe Calibrated Automated Thrombogram (CAT) assay that measures thrombin generation (TG) in platelet-poor and -rich plasma, is increasingly being recognised as a more sensitive tool to determine the overall function of the haemostatic system. We developed a method enabling the measurement of TG in a small aliquot of blood. The objective was to validate this assay in mouse blood and to examine the rate and extent of TG in a mouse model of premature aging. TG was assayed in blood from 20– to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. Bmal1-KO mice are known to display symptoms of premature aging. TG was initiated by adding calcium, tissue factor and a thrombin specific substrate. After TG, the samples were prepared for scanning electron microscopy (SEM). The intra-assay variations (%) in mouse blood of the endogenous thrombin potential (ETP), peak height, lag time, time-to-peak and velocity index were 10% or less (n=24). We found that Bmal1-KO mice have a significantly (p

Activation of AMP-activated protein kinase (AMPK) in cardiomyocytes induces translocation of glucose transporter GLUT4 and long-chain fatty acid (LCFA) transporter CD36 from endosomal stores to the sarcolemma to enhance glucose and LCFA uptake, respectively. Ca2+/calmodulin-activated kinase kinase-β (CaMKKβ) has been positioned directly upstream of AMPK. However, it is unknown whether acute increases in [Ca2+]istimulate translocation of GLUT4 and CD36 and uptake of glucose and LCFA or whether Ca2+signaling converges with AMPK signaling to exert these actions. Therefore, we studied the interplay between Ca2+and AMPK signaling in regulation of cardiomyocyte substrate uptake. Exposure of primary cardiomyocytes to inhibitors or activators of Ca2+signaling affected neither AMPK-Thr172phosphorylation nor basal and AMPK-mediated glucose and LCFA uptake. Despite their lack of an effect on substrate uptake, Ca2+signaling activators induced GLUT4 and CD36 translocation. In contrast, AMPK activators stimulated GLUT4/CD36 translocation as well as glucose/LCFA uptake. When cardiomyocytes were cotreated with Ca2+signaling and AMPK activators, Ca2+signaling activators further enhanced AMPK-induced glucose/LCFA uptake. In conclusion, Ca2+signaling shows no involvement in AMPK-induced GLUT4/CD36 translocation and substrate uptake but elicits transporter translocation via a separate pathway requiring CaMKKβ/CaMKs. Ca2+-induced transporter translocation by itself appears to be ineffective to increase substrate uptake but requires additional AMPK activation to effectuate transporter translocation into increased substrate uptake. Ca2+-induced transporter translocation might be crucial under excessive cardiac stress conditions that require supraphysiological energy demands. Alternatively, Ca2+signaling might prepare the heart for substrate uptake during physiological contraction by inducing transporter translocation.

Objective— Platelets abundantly express the membrane receptor CD36 and store its ligand thrombospondin-1 (TSP1) in the α-granules. We investigated whether released TSP1 can support platelet adhesion and thrombus formation via interaction with CD36. Approach and Results— Mouse platelets deficient in CD36 showed reduced adhesion to TSP1 and subsequent phosphatidylserine expression. Deficiency in either CD36 or TSP1 resulted in markedly increased dissolution of thrombi formed on collagen, although thrombus buildup was unchanged. In mesenteric vessels in vivo, deficiency in CD36 prolonged the time to occlusion and enhanced embolization, which was in agreement with earlier observations in TSP1-deficient mice. Thrombi formed using wild-type blood stained positively for secreted TSP1. Releasate from wild-type but not from TSP1-deficient platelets enhanced platelet activation, phosphatidylserine expression, and thrombus formation on collagen. The enhancement was dependent on CD36 because it was without effect on thrombus formation by CD36-deficient platelets. Conclusions— These results demonstrate an anchoring role of platelet-released TSP1 via CD36 in platelet adhesion and collagen-dependent thrombus stabilization. Thus, the TSP1–CD36 tandem is another platelet ligand–receptor axis contributing to the maintenance of a stable thrombus.

Background and Objective: Platelets abundantly express glycoprotein CD36 with thrombospondin-1 (TSP1) and oxidized low-density lipoprotein (oxLDL) as proposed ligands. How these agents promote platelet activation is still poorly understood. Methods and Results: Both TSP1 and oxLDL caused limited activation of platelets in suspension. However, immobilized TSP1 and oxLDL, but not LDL, strongly supported platelet adhesion and spreading with a major role of CD36. Platelet spreading was accompanied by potent Ca2+ rises, and resulted in exposure of P-selectin and integrin activation, all in a CD36-dependent manner with additional contributions of alpha(IIb)beta(3) and ADP receptor stimulation. Signaling responses via CD36 involved activation of the protein tyrosine kinase Syk. In whole blood perfusion, co-coating of TSP1 or oxLDL with collagen enhanced thrombus formation at high-shear flow conditions, with increased expression on platelets of activated alpha(IIb)beta(3), P-selectin and phosphatidylserine, again in a CD36-dependent way. Conclusions: Immobilized TSP1 and oxLDL activate platelets partly via CD36 through a Syk kinase-dependent Ca2+ signaling mechanism, which enhances collagen-dependent thrombus formation under flow. These findings provide novel insight into the role of CD36 in hemostasis.

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

Summary. Background: Agonist-induced platelet activation involves different signaling pathways leading to the activation of phospholipase C (PLC) β or PLCγ2. Activated PLC produces inositol 1,4,5-trisphosphate and diacylglycerol, which trigger Ca2+ mobilization and the activation of protein kinase C, respectively. PLCβ is activated downstream of Gq-coupled receptors for soluble agonists with only short interaction times in flowing blood. In contrast, PLCγ2 becomes activated downstream of receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI or activated integrins. Objective and methods: We speculated that PLCγ2 activity might be optimized for sustained but submaximal signaling to control relatively slow platelet responses. To test this hypothesis, we analyzed platelets from mice heterozygous for a gain-of-function mutation in the Plcg2 gene (Plcg2Ali5/+). Results: Plcg2Ali5/+ platelets showed enhanced Ca2+ mobilization, integrin activation, granule secretion and phosphatidylserine exposure upon GPVI or C-type lectin-like receptor-2 stimulation. Furthermore, integrin αIIbβ3 outside-in signaling was markedly enhanced in the mutant platelets, as shown by accelerated spreading on different matrices and faster clot retraction. These defects translated into virtually unlimited thrombus formation on collagen under flow in vitro and a prothrombotic phenotype in vivo. Conclusions: These results demonstrate that the enzymatic activity of PLCγ2 is tightly regulated to ensure efficient but limited platelet activation at sites of vascular injury.

In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cγ2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLCγ2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

Objective— Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. Methods and Results— High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated αIIbβ3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active αIIbβ3. Conclusions— These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication.

Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to αIIbβ3 activation. Strikingly, PKC suppressed Ca2+ signal generation and Ca2+-dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca2+ signaling and procoagulant activity. In murine platelets lacking Gqα, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca2+-dependent phosphatidylserine exposure, and consequently thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.

Platelets stably interact with collagen via glycoprotein (GP)VI and α2β1integrin.With α2-null mice, we investigated the role of α2β1 in thrombus formation and stability in vivo and in vitro. Using a FeCl3-induced thrombosis model, in arteries from α2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects ,while the thromboxaneA2 analogue U46619 was borderline effective in suppressing the embolisation in α2-null mice. In vitro, perfusion of α2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from α2-null mice.We conclude that integrin α2β1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A2 release. The increased embolization in α2-null mice may argue against the use of α2β1 integrin inhibitors for antithrombotic therapy.. See also the following videos: Video 1 Video 2 Video 3 Video 4

Objective— Both collagen and tissue factor can be initiating factors in thrombus formation. We investigated the signaling pathway of collagen-induced platelet activation in interaction with tissue factor–triggered coagulation during the thrombus-forming process. Methods and Results— In murine blood flowing over collagen, platelet exposure of phosphatidylserine and procoagulant activity, but not adhesion, completely relied on each of the following signaling modules: glycoprotein VI (GPVI), FcR γ-chain, Src kinases, adaptor protein LAT, and phospholipase Cγ2 (PLCγ2). On flow in the presence of tissue factor, these signaling components were essential for platelet aggregation and greatly enhanced fibrin clot formation. Collagen-stimulated thrombin generation relied on the presence and activity of GPVI, FcR γ-chain, Src kinase, LAT, and PLCγ2. The physiological importance of this GPVI pathway was shown in a FeCl 3 -induced in vivo murine thrombosis model. In both venules and arterioles, signaling through GPVI, FcR γ-chain, and Src kinases enhanced the formation of phosphatidylserine-exposing and fibrin-rich thrombi. Conclusions— The GPVI-PLCγ2 activation pathway regulates collagen-dependent coagulation in venous and arterial thrombus formation.

Collagen is a unique agonist of platelets, because it acts as an immobilized ligand that only causes platelet activation after stable adhesion. This review addresses the present understanding of how platelet interaction with collagen supports the process of thrombin generation and coagulation. Only some of the collagen-adhered platelets, that is, those showing profound changes in shape and shedding microparticles (resembling apoptotic cells), appear to contribute to the procoagulant activity of platelets. The main signaling receptor for collagen, glycoprotein VI, plays a key role in the platelet procoagulant response during thrombus formation; this is a reason why new anti-glycoprotein-VI antibodies are promising antithrombotic tools.

Objective— High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of α2β1 and αIIbβ3 integrin activation during thrombus formation on collagen. Methods and Results— Blockade of glycoprotein (GP) VI by 9O12 antibody or of P2Y purinergic receptors permitted platelet adhesion but reduced aggregate formation, fibrinogen binding, and activation of α2β1 and αIIbβ3, as detected with antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of these integrins. Combined blockade of GPVI and P2Y receptors and thromboxane formation abolished integrin activation but still allowed adhesion of morphologically unstimulated, nonprocoagulant platelets. Exogenous ADP partly restored the suppressive effect of GPVI blockade on integrin α2β1 and αIIbβ3 activation. Adhesion was fully inhibited only with simultaneous blocking of GPVI and α2β1, indicating that the integrin can support platelet–collagen binding in the absence of its activation. Blockade or absence of GPIbα only moderately influenced integrin activation and adhesion unless GPVI was inhibited. Conclusions— GPVI- and autocrine-released ADP induce affinity changes of α2β1 and αIIbβ3 during thrombus formation on collagen under flow. These integrin changes are dispensable for adhesion but strengthen platelet–collagen interactions and thereby collagen-induced platelet activation.

Vessel wall damage exposes collagen fibres, to which platelets adhere directly via the collagen receptors glycoprotein (GP) VI and integrin α2β1 and indirectly by collagen-bound von Willebrand factor (vWF) via the GPIb-V-IX and integrin αIIbβ3 receptor complexes. Platelet–collagen interaction under shear stimulates thrombus formation in two ways, by integrin-dependent formation of platelet aggregates and by surface exposure of procoagulant phosphatidylserine (PS). GPVI is involved in both processes, complemented by α2β1. In mouse blood flowing over collagen, we investigated the additional role of platelet–vWF binding via GPIb and αIIbβ3. Inhibition of GPIb as well as blocking of vWF binding to collagen reduced stable platelet adhesion at high shear rate. This was accompanied by delayed platelet Ca2+ responses and reduced PS exposure, while microaggregates were still formed. Inhibition of integrin αIIbβ3 with JON/A antibody, which blocks αIIbβ3 binding to both vWF and fibrinogen, reduced PS exposure and aggregate formation. The JON/A effects were not enhanced by combined blocking of GPIb–vWF binding, suggesting a function for αIIbβ3 downstream of GPIb. Typically, with blood from FcR γ-chain +/− mutant mice, expressing 50% of normal platelet GPVI levels, GPIb blockage almost completely abolished platelet adhesion and PS exposure. Together, these data indicate that, under physiological conditions of flow, both adhesive receptors GPIb and αIIbβ3 facilitate GPVI-mediated PS exposure by stabilizing platelet binding to collagen. Hence, these glycoproteins have an assistant procoagulant role in collagen-dependent thrombus formation, which is most prominent at reduced GPVI activity and is independent of the presence of thrombin.

We have generated transgenic mice overexpressing the human P2X1 ion channel in the megakaryocytic cell lineage. Platelets from transgenic mice exhibited a gain of P2X1ionotropic activity as determined by more prominent P2X1-mediated Ca2+ influx and platelet shape change. P2X1 overexpression enhanced platelet secretion and aggregation evoked by low doses of collagen, convulxin, or the thromboxane A2 mimetic U46619. In contrast, transgenic platelet responses to adenosine diphosphate (ADP) or thrombin were normal. Perfusing whole blood from transgenic mice over collagen fibers at a shear rate of 1000 seconds−1 resulted in increased P2X1-dependent aggregate formation and phosphatidylserine exposure. Platelet hyperreactivity to collagen was correlated with up-regulated extracellular signal-regulated kinase 2 (ERK2) phosphorylation. Accordingly, the MEK1/2 inhibitor U0126 potently inhibited the collagen-induced aggregation of transgenic platelets when stirred or when perfused over a collagen surface. In a viscometer, shear stress caused potent aggregation of transgenic platelets under conditions in which wild-type platelets did not aggregate. In an in vivo model of thromboembolism consisting of intravenous injection of a low dose of collagen plus epinephrine, transgenic mice died more readily than wild-type mice. Preinjection of U0126 not only fully protected transgenic mice against thrombosis, it also enhanced the survival of wild-type mice injected with a higher collagen dose. Hence, the platelet P2X1 ion channel plays a role in hemostasis and thrombosis through its participation in collagen-, thromboxane A2-, and shear stress–triggered platelet responses. Activation of the ERK2 pathway is instrumental in these processes.

Objectives: Membrane cholesterol is known to modulate a variety of cell signaling pathways and functions. While cholesterol depletion by High-Density-Lipoproteins (HDL) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain ill defined.Methods: Human and murine macrophages were pre-incubated with human reconstituted (apolipoproteinA-I/phosphatidylcholine) or native HDL.Results: HDL pre-incubation significantly decreased LPS-induced anti-inflammatory IL-10 production, while the opposite was observed for the pro-inflammatory mediators IL-12 and TNF. We show that these effects are mediated by passive cholesterol depletion and lipid raft disruption, without involvement of ABCA1, ABCG1, SR-BI or CD36. These pro-inflammatory effects are confirmed in vivoin peritoneal macrophages from ApoA-I transgenic mice, which have high circulating HDL levels. Native and reconstituted HDL enhances Toll-Like-Receptor-induced signaling by activating protein kinase C (PKC), since inhibition of PKC ablated the observed HDL effects. Using macrophages from NF-κB luciferase mice, we observed that HDL induces NF-κB activation. Western blot analyses showed that in particular the p65 subunit was activated. Using specific knock-out mice, we show that the observed HDL effects are independent of IKK, NIK and CKII. Furthermore, we observed that STAT1 is involved in the pro-inflammatory HDL effects on IL-10 and IL-12. On the other hand, we show that HDL enhances ADAM protease activity, thereby mediating TNF-α release.Conclusions: HDL exerts pro-inflammatory effects on macrophages via passive cholesterol depletion by activation of PKC-NF-kB/STAT1. These pro-inflammatory activities on macrophages could at least partly underlie the disappointing therapeutic potential of HDL raising therapy in current cardiovascular clinical trials.

In this review, we presume that the process of thrombus formation, as assessed in whole blood flow studies and in experimental (murine) thrombosis studies, reflects the platelet responses in human haemostasis and thrombosis. Following this concept, we give an up-to-date overview of the main platelet receptors and signalling pathways that contribute to thrombus formation and are used as targets in (pre)clinical intervention studies to prevent cardiovascular disease. Discussed are receptors for thrombin, thromboxane, ADP, ATP, prostaglandins, von Willebrand factor, collagen, CLEC-2 ligand, fibrinogen and laminin. Sketched are the consequences of receptor deficiency or blockage for haemostasis and thrombosis in mouse and man. Recording of bleeding due to (congenital) platelet dysfunction or (acquired) antiplatelet treatment occurs according to different protocols, while common laboratory methods are used to determine platelet function.