Protein Phosphorylation In Platelets Research Articles

Aging-related alterations in mechanistic target of rapamycin signaling promote platelet hyperreactivity and thrombosis

BackgroundAging is an independent risk factor for the development of cardiovascular, thrombotic, and other chronic diseases. However, mechanisms of platelet hyperactivation in aging remain poorly understood. ObjectivesHere, we examine whether and how aging alters intracellular signaling in platelets to support platelet hyperactivity and thrombosis. MethodsQuantitative mass spectrometry with tandem mass tag labeling systematically measured protein phosphorylation in platelets from healthy aged (>65 years) and young human (<45 years) subjects. The role of platelet mechanistic target of rapamycin (mTOR) in aging-induced platelet hyperreactivity was assessed using pharmacologic mTOR inhibition and a platelet-specific mTOR-deficient mouse model (mTORplt−/−). ResultsQuantitative phosphoproteomics uncovered differential site-specific protein phosphorylation within mTOR, Rho GTPase, and MAPK pathways in platelets from aged donors. Western blot confirmed constitutive activation of the mTOR pathway in platelets from both aged humans and mice, which was associated with increased aggregation compared with that in young controls. Inhibition of mTOR with either Torin 1 in aged humans or genetic deletion in aged mice reversed platelet hyperreactivity. In a collagen–epinephrine pulmonary thrombosis model, aged wild-type (mTORplt+/+) mice succumbed significantly faster than young controls, while time to death of aged mTORplt−/− mice was similar to that of young mTORplt+/+ mice. Mechanistically, we noted increased Rac1 activation and levels of mitochondrial reactive oxygen species in resting platelets from aged mice, as well as increased p38 phosphorylation upstream of thromboxane generation following agonist stimulation. ConclusionAging-related changes in mTOR phosphorylation enhance Rac1 and p38 activation to enhance thromboxane generation, platelet hyperactivity, and thrombosis.

Multi-phased Kinetics and Interaction of Protein Kinase Signaling in Glycoprotein VI-Induced Platelet αIIbβ3 Integrin Activation and Degranulation.

Platelet glycoprotein VI (GPVI) stimulation activates the tyrosine kinases Syk and Btk, and the effector proteins phospholipase Cγ 2 (PLCγ2) and protein kinase C (PKC). Here, the activation sequence, crosstalk, and downstream effects of this Syk-Btk-PKC signalosome in human platelets were analyzed. Using immunoblotting, we quantified 14 regulated phospho-sites in platelets stimulated by convulxin with and without inhibition of Syk, Btk, or PKC. Convulxin induced fast, reversible tyrosine phosphorylation (pY) of Syk, Btk, LAT, and PLCγ2, followed by reversible serine/threonine phosphorylation (pS/T) of Syk, Btk, and downstream kinases MEK1/2, Erk1/2, p38, and Akt. Syk inhibition by PRT-060318 abolished all phosphorylations, except Syk pY352. Btk inhibition by acalabrutinib strongly decreased Btk pY223/pS180, Syk pS297, PLCγ2 pY759/Y1217, MEK1/2 pS217/221, Erk1/2 pT202/Y204, p38 pT180/Y182, and Akt pT308/S473. PKC inhibition by GF109203X abolished most pS/T phosphorylations except p38 pT180/Y182 and Akt pT308, but enhanced most Y-phosphorylations. Acalabrutinib, but not GF109203X, suppressed convulxin-induced intracellular Ca2+ mobilization, whereas all three protein kinase inhibitors abolished degranulation and αIIbβ3 integrin activation assessed by flow cytometry. Inhibition of autocrine ADP effects by AR-C669931 partly diminished convulxin-triggered degranulation. Kinetic analysis of GPVI-initiated multisite protein phosphorylation in human platelets demonstrates multiple phases and interactions of tyrosine and serine/threonine kinases with activation-altering feedforward and feedback loops partly involving PKC. The protein kinase inhibitor effects on multisite protein phosphorylation and functional readouts reveal that the signaling network of Syk, Btk, and PKC controls platelet granule exocytosis and αIIbβ3 integrin activation.

Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2, and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand

BackgroundClustering of the receptors glycoprotein receptor VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), low-affinity immunoglobulin γ Fc region receptor II-a (FcγRIIA), and platelet endothelial aggregation receptor 1 (PEAR1) leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signaling cascades. GPVI, CLEC-2, and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase. Current ligands for these receptors have an undefined valency and show significant batch variation and, for some, uncertain specificity. ObjectivesWe have raised nanobodies against each of these receptors and multimerized them to identify the minimum number of epitopes to achieve robust activation of human platelets. MethodsDivalent and trivalent nanobodies were generated using a flexible glycine-serine linker. Tetravalent nanobodies utilize a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerize the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, adenosine triphosphate secretion, and protein phosphorylation were analyzed using standardized methods. ResultsMultimerization of the nanobodies led to a stepwise increase in affinity with divalent and higher-order nanobody oligomers having sub-nanomolar affinity. The trivalent nanobodies to GPVI, CLEC-2, and PEAR1 stimulated powerful and robust platelet aggregation, secretion, and protein phosphorylation at low nanomolar concentrations. A tetravalent nanobody was required to activate FcγRIIA with the concentration-response relationship showing a greater variability and reduced sensitivity compared with the other nanobody-based ligands, despite a sub-nanomolar binding affinity. ConclusionThe multivalent nanobodies represent a series of standardized, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays.

Heparin and heparin proteoglycan-mimetics activate platelets via PEAR1 and PI3Kβ

BackgroundPlatelet endothelial aggregation receptor 1 (PEAR1) is a single-transmembrane orphan receptor primarily expressed on platelets and endothelial cells. Genetic variants of PEAR1 have repeatedly and independently been identified to be associated with cardiovascular diseases, including coronary artery disease. ObjectivesWe have identified sulfated fucoidans and their mimetics as ligands for PEAR1 and proposed that its endogenous ligand is a sulfated proteoglycan. The aim of this study was to test this hypothesis. MethodsA heparin proteoglycan-mimetic (HPGM) was created by linking unfractionated heparin (UFH) to albumin. The ability of the HPGM, UFH and selectively desulfated heparins to stimulate platelet aggregation and protein phosphorylation was investigated. Nanobodies against the 12th to 13th epidermal growth factor-like repeat of PEAR1 and phosphoinositide 3-kinase (PI3K) isoform-selective inhibitors were tested for the inhibition of platelet activation. ResultsWe show that HPGM, heparin conjugated to an albumin protein core, stimulates aggregation and phosphorylation of PEAR1 in washed platelets. Platelet aggregation was abolished by an anti-PEAR1 nanobody, Nb138. UFH stimulated platelet aggregation in washed platelets, but desulfated UFH did not. Furthermore, HPGM, but not UFH, stimulated maximal aggregation in platelet-rich plasma. However, both HPGM and UFH increased integrin αIIbβ3 activation in whole blood. By using PI3K isoform-selective inhibitors, we show that PEAR1 activates PI3Kβ, leading to Akt phosphorylation. ConclusionOur findings reveal that PEAR1 is a receptor for heparin and HPGM and that PI3Kβ is a key signaling molecule downstream of PEAR1 in platelets. These findings may have important implications for our understanding of the role of PEAR1 in cardiovascular disease.

Rac Inhibition Causes Impaired GPVI Signalling in Human Platelets through GPVI Shedding and Reduction in PLCγ2 Phosphorylation.

Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and reduced PLCγ2 activation, but not phosphorylation. The objective of our study is to investigate the role of Rac1 in GPVI-dependent human platelet activation and downstream signalling. Therefore, we used human platelets stimulated using GPVI agonists (collagen and collagen-related peptide) in the presence of the Rac1-specific inhibitor EHT1864 and analysed platelet activation, aggregation, spreading, protein phosphorylation, and GPVI clustering and shedding. We observed that in human platelets, the inhibition of Rac1 by EHT1864 had no significant effect on GPVI clustering on collagen fibres but decreased the ability of platelets to spread or aggregate in response to GPVI agonists. Additionally, in contrast to what was observed in murine Rac1-deficient platelets, EHT1864 enhanced GPVI shedding in platelets and reduced the phosphorylation levels of PLCγ2 following GPVI activation. In conclusion, Rac1 activity is required for both human and murine platelet activation in response to GPVI-ligands, but Rac1’s mode of action differs between the two species.

Water-Soluble Tomato Extract Fruitflow Alters the Phosphoproteomic Profile of Collagen-Stimulated Platelets.

Platelet hyperactivity is a risk factor for cardiovascular disease and thrombosis. Recent studies reported that the tomato extract Fruitflow inhibited platelet function, but the molecular mechanism is still unclear. The present study used proteomics to quantitatively analyze the effect of fruitflow on the inhibition of collagen-stimulated platelets and validated the involvement of several signaling molecules. Fruitflow significantly inhibited human platelet aggregation and P-selectin expression that were induced by collagen. Proteomics analysis revealed that compared fruitflow-treated collagen-stimulated platelets with only collagen-stimulated platelets, 60 proteins were upregulated and 10 proteins were downregulated. Additionally, 66 phosphorylated peptides were upregulated, whereas 37 phosphorylated peptides were downregulated. Gene Ontology analysis indicated that fruitflow treatment downregulated phosphoinositide 3-kinase (PI3K)/protein kinase B and guanosine triphosphatase-mediated signal transduction in collagen-activated platelets. Biological validation indicated that fruitflow decreased Akt, glycogen synthase kinase 3β, p38 mitogen-activated protein kinase (MAPK), and heat shock protein (Hsp27) phosphorylation in collagen-stimulated platelets. Fruitflow recovered cyclic adenosine monophosphate levels in collagen-activated platelets and reduced protein kinase A substrate phosphorylation that was induced by collagen. These findings suggest that fruitflow is a functional food that can inhibit platelet function, conferring beneficial effects for people who are at risk for platelet hyperactivity-associated thrombosis.

A dichotomy in platelet activation: Evidence of different functional platelet responses to inflammatory versus haemostatic stimuli

IntroductionPlatelets participate in inflammatory disorders through a variety of different functional responses, including chemotaxis, platelet-leukocyte complex formation and facilitation of leukocyte recruitment that are thought to be distinct from platelet aggregation. This may account for why classical anti-platelet drugs have failed to ameliorate inflammatory disorders where platelets are known to participate, suggesting that distinct pathways may control inflammatory and haemostatic functions of platelets. In the present study, we have therefore investigated the effect of different stimuli on several different functions of platelets preferentially involved either in haemostasis or in inflammation. Materials and methodsHuman platelets were stimulated with either inflammatory (fMLP, histamine, IL-1β, LPS, MDC/CCL22, SDF-1α/CXCL12 and 5-HT) or haemostatic (ADP, collagen, convulxin, epinephrine, TRAP-6 and U46619) stimuli. Aggregation, platelet-leukocyte complex formation, platelet migration and platelet protein phosphorylation were assessed. ResultsHaemostatic stimuli induced platelet aggregation, whilst inflammatory agonists induced platelet migration. The haemostatic stimuli, with the exception of epinephrine, and some of the inflammatory stimuli induced platelet-leukocyte complex formation, even if to a different extent. Furthermore, inflammatory stimuli induced a shorter lasting profile of platelet protein phosphorylation compared with haemostatic stimuli. ConclusionsStimulation of platelets with inflammatory stimuli triggers the activation of non haemostatic functions different from those induced by haemostatic stimuli, supporting the existence of alternative platelet responses depending on the stimulus (haemostatic or inflammatory). A deeper understanding of the biochemical pathways behind these functional differences may lead to the development of novel therapeutic options targeting the inflammatory actions of platelets, without affecting their critical role in haemostasis.

Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients.

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.

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

AbstractAdenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein–coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbβ3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.

CLEC‐2‐dependent activation of mouse platelets is weakly inhibited by cAMP but not by cGMP

The activation of platelet CLEC-2 by podoplanin on lymphatic endothelial cells (LECs) has a critical role in prevention of mixing of lymphatic and blood vasculatures during embryonic development. Paradoxically, LECs release cAMP and cGMP-elevating agents, prostacyclin (PGI2 ) and nitric oxide (NO), respectively, which are powerful inhibitors of platelet activation. This raises the question of how podoplanin is able to activate CLEC-2 in the presence of the inhibitory cyclic nucleotides. We investigated the influence of cyclic nucleotides on CLEC-2 signaling in platelets. We used rhodocytin, CLEC-2 monoclonal antibody, LECs and recombinant podoplanin as CLEC-2 agonists on mouse platelets. The effects of the cyclic nucleotide-elevating agents PGI2 , forskolin and the NO-donor GSNO were assessed with light transmission aggregometry, flow cytometry, protein phosphorylation and fluorescent imaging of platelets on LECs. We show that platelet aggregation induced by CLEC-2 agonists is resistant to GSNO but inhibited by PGI2 . The effect of PGI2 is mediated through decreased phosphorylation of CLEC-2, Syk and PLCγ2. In contrast, adhesion and spreading of platelets on recombinant podoplanin, CLEC-2 antibody and LECs is not affected by PGI2 and GSNO. Consistent with this, CLEC-2 activation of Rac, which is required for platelet spreading, is not altered in the presence of PGI2 . The present results demonstrate that platelet adhesion and activation on CLEC-2 ligands or LECs is maintained in the presence of PGI2 and NO.

Phospholipase C-γ2 via p38 and ERK1/2 MAP kinase mediates diperoxovanadate-asparagine induced human platelet aggregation and sCD40L release

ObjectiveRedox imbalance either inside platelets or in their immediate surroundings prove detrimental to their physiologic functions during haemostasis. This study was therefore aimed to assess the effect of peroxide radicals on platelet functions and underlying signalling mechanisms using asparagine-conjugated diperoxovanadate (DPV-Asn).MethodsPlatelet aggregation, ATP secretion, TxB2 release, intra-platelet calcium mobilization, protein tyrosine phosphorylation, GPIIbIIIa activation by PAC1 labelling and sCD40L release (enzyme-linked immunosorbent assay) was monitored using various concentrations of DPV-Asn. Cell viability was assessed by Annexin V labelling, MTT assay, LDH leakage and mitochondrial membrane potential by JC-1.ResultsPlatelet aggregation induced by DPV-Asn was chiefly regulated by dense granule secretion, thromboxane A2 (TxA2) generation, intra-platelet [Ca2+] influx, GPIIbIIIa activation and sCD40L release, which were significantly reduced in presence of U73122 (PLC inhibitor), aspirin (COX), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor). This was further corroborated by enhanced tyrosine phosphorylation of numerous platelet proteins including PLC-γ2, which apparently played a central role in transducing peroxide signals to regulate [Ca2+] influx and phosphorylation of p38 and ERK1/2 MAP kinase.DiscussionPeroxide radicals critically regulate the thrombo-inflammatory functions of platelets via the PLCγ2-p38-ERK1/2-TxA2 pathway, which closely resembles the clinical scenario of various pathologies like hyperglycemia and atherosclerosis during which oxidative stress disrupts platelet functions.

Mechanism of platelet factor 4 (PF4) deficiency with RUNX1 haplodeficiency: RUNX1 is a transcriptional regulator of PF4

Platelet factor 4 (PF4) is an abundant protein stored in platelet α-granules. Several patients have been described with platelet PF4 deficiency, including the gray platelet syndrome, characterized by a deficiency of α-granule proteins. Defective granule formation and protein targeting are considered to be the predominant mechanisms. We have reported on a patient with thrombocytopenia and impaired platelet aggregation, secretion, and protein phosphorylation, associated with a mutation in the transcription factor RUNX1. Platelet expression profiling showed decreased transcript expression of PF4 and its non-allelic variant PF4V1. To understand the mechanism leading to PF4 deficiency associated with RUNX1 haplodeficiency, we addressed the hypothesis that PF4 is a transcriptional target of RUNX1. Chromatin immunoprecipitation and gel-shift assays with phorbol 12-myristate 13-acetate-treated human erythroleukemia (HEL) cells revealed RUNX1 binding to RUNX1 consensus sites at -1774/-1769 and -157/-152 on the PF4 promoter. In luciferase reporter studies in HEL cells, mutation of each site markedly reduced activity. PF4 promoter activity and PF4 protein level were decreased by small interfering RNA RUNX1 knockdown and increased by RUNX1 overexpression. Our results provide the first evidence that PF4 is regulated by RUNX1 and that impaired transcriptional regulation leads to the PF4 deficiency associated with RUNX1 haplodeficiency. Because our patient had decreased platelet albumin and IgG (not synthesized by megakaryocytes) levels, we postulate additional defects in RUNX1-regulated genes involved in vesicular trafficking. These studies advance our understanding of the mechanisms in α-granule deficiency.

Omega-3 fatty acids modulate collagen signaling in human platelets

Dietary intake of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) results in cardioprotective benefits. However, the cellular and physiological bases for these benefits remain unclear. We hypothesized that EPA and DHA treatments would interfere with collagen-mediated platelet signaling. Thirty healthy volunteers received 28 days of 3.4 g/d EPA+DHA with and without a single dose of aspirin. Clinical hematologic parameters were then measured along with assays of collagen-stimulated platelet activation and protein phosphorylation. Omega-3 therapy led to a small but significant reduction in platelets (6.3%) and red blood cells (1.7%), but did not impair clinical time-to-closure assays. However, collagen-mediated platelet signaling events of integrin activation, α-granule secretion, and phosphatidylserine exposure were all reduced by roughly 50% after omega-3 incorporation, and collagen-induced tyrosine phosphorylation was significantly impaired. The diminished platelet response to collagen may account for some of the cardioprotective benefits provided by DHA and EPA.

&amp;lt;italic&amp;gt;L&amp;lt;/italic&amp;gt;-amino acid oxidase from &amp;lt;italic&amp;gt;Naja atra&amp;lt;/italic&amp;gt; venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase Cgamma2. Unlike convulxin, Fc receptor gamma chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

Calpain-mediated regulation of platelet signaling pathways

There is considerable interest in understanding the function and mechanism of calpains in platelet aggregation, spreading, and granular secretion pathways. Recent insights from the calpain-1 knockout platelets suggest a pivotal role of these cysteine proteases in the regulation of outside-in signaling, aggregation, and clot retraction. The calpain-1 knockout mouse provided direct evidence for the role of calpain-1 in platelet aggregation and clot retraction. Reduced tyrosine phosphorylation of platelet proteins correlated with reduced platelet aggregation and clot retraction. Future investigations of the mechanism of platelet defects in calpain-1 null mice may unveil the physiological functions of this important and elusive protease in mammalian cells. This review focuses on the role of calpains in platelets with a particular emphasis on recent findings in calpain-1 null platelets. Previous studies used synthetic inhibitors to study the role of calpains in platelet function yielding useful information about the identification of calpain substrates. The development of calpain-1 null mice demonstrated that calpain-1 plays an important function in the regulation of platelet aggregation and clot retraction. Since the combined deletion of calpain-1 and calpain-2 genes results in embryonic lethality, the calpain-1 null mouse remains the only experimental model available to study the physiological role of calpains in mammalian cells.

Threonine phosphorylation of integrin β 3 in calyculin A-treated platelets is selectively sensitive to 5′-iodotubercidin

Exposure of platelets to toxins (calyculin A or okadaic acid) that inhibit protein serine/threonine phosphatases types 1 and 2A, at concentrations that block aggregatory and secretory responses, results in the phosphorylation of several platelet proteins including integrin β 3. Since protein phosphorylation represents a balance between kinase and phosphatase activities, this increase in phosphorylation reflects either the removal of phosphatases that oppose constitutively active kinases known to reside in the platelet (e.g., casein kinase 2) or the activation of endogenous kinases. In this study, we demonstrate that the addition of calyculin A promotes the activation of several endogenous platelet protein kinases, including p42/44 mapk, p38 mapk, Akt/PKB, and LKB1. Using a pharmacologic approach, we assessed whether inhibition of these and other enzymes block phosphorylation of β 3. Inhibitors of p38 mapk, casein kinase, AMP kinase, protein kinase C, and calcium–calmodulin-dependent kinases did not block phosphorylation of β 3 on thr 753. In contrast, 5′-iodotubercidin, at 50 μM, blocks β 3 phosphorylation without affecting the efficacy of calyculin A to inhibit platelet aggregation and spreading. These data dissociate threonine phosphorylation of β 3 molecules and inhibition of platelet responses by protein phosphatase inhibitors.

Effect of homocysteine on platelet activation induced by collagen

Objective The present study investigated the effects of homocysteine on platelet activation induced by collagen and the downstream signaling pathways potentially involved in these effects. Methods Washed human platelets were incubated with homocysteine and collagen type I. The effects of homocysteine on platelet aggregation and adhesion and the tyrosine phosphorylation of total platelet proteins, Src kinase, and phospholipase-Cγ2 (PLCγ2) were studied. Results Homocysteine (10 to 100 μM) increased collagen-induced aggregation of washed platelets. Upon homocysteine (50 to 100 μM) treatment, platelet deposition to collagen-coated surface was significantly augmented under the low shear rate model (100/s) but not under the high shear rate model (1600/s). Collagen-stimulated total protein tyrosine phosphorylation in platelets was further enhanced by incubation with homocysteine. This effect was almost abrogated by genistein. Homocysteine potentiated collagen-stimulated tyrosine phosphorylation of the Src kinase and PLCγ2, which was partly decreased by integrin β 1 blocking antibody. Conclusion Homocysteine (at 10 to 100 μM) potentiates collagen type I induced-platelet activation through signaling components of glycoprotein VI and integrin α 2β 1 pathway. Our results suggested that upregulation of tyrosine phosphorylation of proteins such as Src and PLCγ2 is involved in the downstream signaling events of homocysteine stimulation in human platelets.

Role of Lipid Rafts in GPVI Agonist-Induced Platelet Signaling.

The collagen receptor glycoprotein VI (GPVI), plays an essential role in platelet activation and the regulation of hemostasis. Microdomains within the plasma membrane, called lipid rafts, have been implicated in GPVI signaling. The GPVI receptor has been shown to associate with the lipid rafts in both resting and activated platelets. It has been reported that there is a reduction in GPVI signaling in raft-disrupted platelets following activation with various GPVI agonists, especially at low to moderate agonist concentrations. Since platelet aggregation is potentiated by secreted adenosine 5′-diphosphate (ADP) at low concentrations of convulxin and at all concentrations of collagen and collagen-related peptide (CRP), we wanted to determine whether the decrease in GPVI signaling found in platelets with disrupted rafts was due to the loss of agonist potentiation by ADP. We compared platelet aggregation, protein phosphorylation, and calcium mobilization in platelets with intact and disrupted lipid rafts following activation with the GPVI agonists, collagen, convulxin and CRP. We show that lipid raft disruption inhibits aggregation induced by collagen and convulxin, but this inhibition is no longer apparent in the presence of ADP feedback inhibitors. Furthermore, raft-disrupted platelets had the same level of phosphorylation of proteins involved in GPVI signaling (i.e. Syk, LAT, and PLCγ2) and the same ability to mobilize calcium following activation with collagen or convulxin. Therefore, the effects of lipid raft disruption on aggregation can be attributed to the loss of ADP feedback. Interestingly, however, raft disruption directly inhibited aggregation and Syk phosphorylation induced by CRP in the presence and absence of ADP feedback. We propose that these differences are due to the fact that CRP is a relatively small, synthesized peptide of 37 amino acids, while collagen and convulxin are large ligands. These agonists are all able to bind the GPVI receptor, but they may not have the same ability to simultaneously cluster multiple receptors due to their size differential. The lipid rafts may be important for CRP stimulation, but not for collagen or convulxin, because they may have a higher density of the GPVI receptor than nonraft membrane regions, allowing CRP to cluster multiple receptors and activate the GPVI signaling cascade. When we disrupt the lipid rafts, we are reducing the effective concentration of GPVI available for activation by CRP but not by collagen or convulxin.

The Development of Small Molecule Inhibitors of Collagen Binding to the Integrin α2β1 as Antithrombotic Drugs.

Platelets tether to collagen in the subendothelial matrix that is exposed by vascular damage. Collagen is a particularly important matrix component in this context, not only because it is a substrate for platelet adhesion, but because it is an agonist for platelet aggregation and secretion as well. There are two platelet collagen receptors, the immunoglobulin gene superfamily member GPVI and the integrin α2Β1. Both are involved in adhesion to exposed collagen and generate downstream activating signals. α2Β1 is widely expressed and has been implicated in hemostasis and thrombosis, as well as cancer metastasis, wound healing, and angiogenesis. In mice, α2Β1 deficiency results in decreased ex vivo platelet aggregation, but normal bleeding times. In mouse tumor models, α2Β1 blockade reduces both metastasis and angiogenesis. Humans lacking α2Β1 have a mild bleeding diathesis. Given this background, α2Β1 appears to be an appropriate target for the development of small-molecule inhibitors to serve as relatively safe anti-platelet and anti-tumor agents, either acting alone or in synergy with other anti-platelet or anti-tumor agents. We have developed two classes of small-molecule α2Β1 inhibitors. The first class is targeted against the collagen binding site located on the α2 I-domain and was designed using molecular modeling to superimpose a dipeptide scaffold onto the published crystal structure of the I-domain bound to a collagen-mimetic peptide (GFOGER). These molecules block recombinant human I-domain binding to immobilized collagen type I with IC50s as low as 10 μM. Although the molecules inhibit platelet adhesion to collagen only at higher concentrations, they readily inhibit melanoma cell adhesion to collagen mimetics. It is also noteworthy that the molecules induce platelet protein phosphorylation and potentiate platelet aggregation induced by other platelet agonists, both of which can be prevented by pre-incubating platelets with monoclonal antibodies directed against the α2 I-domain, but not against GPVI. The second class of molecules was derived from proline-substituted 2,3-diaminopropionic acids and is directed against the Β1 I-like domain, an allosteric site that regulates ligand binding. These molecules are potent inhibitors of platelet adhesion to immobilized soluble collagen type I with IC50s of 10–50 nM and inhibit the adhesion of melanoma cells to collagen mimetics with IC50s of 250–350 nM. These molecules do not inhibit platelet aggregation, nor do they inhibit I-domain binding to immobilized collagen type I, behavior consistent with binding to the Β1 I-like domain. In a murine model of ferric-chloride induced carotid thrombosis, the molecules synergize with aspirin to prevent arterial thrombosis. In summary, we have developed two classes of small molecule inhibitors that impair the interaction of collagen with the integrin α2Β1. Although both classes of inhibitors bind to α2Β1, their effects on its function are substantially different, indicating that there are multiple potential strategies for inhibiting integrin function pharmacologically. Further development of these inhibitors may lead to agents that will be clinically useful in the treatment of thrombosis and cancer.

Increased Tyrosine Phosphorylation of Platelet Proteins Including pp125FAK Suggests Endogenous Activation and Aggregation in Pulmonary Hypertension

Despite of several lines of evidence indicating a pathophysiologic role of platelets in pulmonary hypertension, the occurrence of chronic endogenous platelet activation has been a matter of debate. It was hypothesized that the pattern of tyrosine phosphorylation of platelet proteins examined ex vivo could provide information on the state of platelet activation. This was examined in 10 patients with pulmonary arterial hypertension aged 18 to 53 years. Phosphotyrosine density and the amounts of specific proteins were analyzed in resting platelets after reaction with anti-phosphotyrosine, anti-pp60(s-src), anti-pp125(FAK), and anti-alphaIIbbeta3 antibodies. There was a 79% increase in protein-associated phosphotyrosine in patients in comparison to levels in controls (p<0.05). In particular, phosphorylation on tyrosine residues of pp120 and pp125(FAK) increased 24% and 57%, respectively (p<0.05). Although pp60(s-src)-associated phosphotyrosine was not altered in the patient group as a whole, it was clearly decreased in three subjects. Platelet content of beta3 integrin, pp60(s-src), and pp125(FAK), was not altered. This pattern of phosphorylation suggests an ongoing process of platelet activation. Because phosphorylation of pp125(FAK) is a late, integrin-dependent event, results suggest that platelet activation and aggregation occur in vivo in these patients.