GPVI Shedding Research Articles

Paclitaxel exerts antiplatelet and antithrombotic activities: Additional benefit from use of paclitaxel-coated balloons and -eluting stents in coronary revascularization and prevention of in-stent restenosis

IntroductionPaclitaxel is a microtubule-stabilizing drug used to treat several types of cancer, including ovarian and breast cancer. Because of its antiproliferative effect on vascular smooth muscle cells, balloons and stents are coated with paclitaxel for use in coronary revascularization and prevention of in-stent restenosis (ISR). However, mechanisms underlying ISR are complicated. Platelet activation is one of the major causes of ISR after percutaneous coronary intervention. Although the antiplatelet activity of paclitaxel was noted in rabbit platelets, the effect of paclitaxel on platelets remains unclear. This study investigated whether paclitaxel exhibits antiplatelet activity in human platelets. Methods and resultsPaclitaxel inhibited platelet aggregation induced by collagen but not that induced by thrombin, arachidonic acid, or U46619, suggesting that paclitaxel is more sensitive to the inhibition of collagen-induced platelet activation. Moreover, paclitaxel blocked collagen receptor glycoprotein (GP) VI downstream signaling molecules, including Lyn, Fyn, PLCγ2, PKC, Akt, and MAPKs. However, paclitaxel did not directly bind to GPVI and cause GPVI shedding, as detected by surface plasmon resonance and flow cytometry, respectively, indicating that paclitaxel may interfere with GPVI downstream signaling molecules, such as Lyn and Fyn. Paclitaxel also prevented granule release and GPIIbIIIa activation induced by collagen and low convulxin doses. Moreover, paclitaxel attenuated pulmonary thrombosis and delayed platelet thrombus formation in mesenteric microvessels without significantly affecting hemostasis. ConclusionPaclitaxel exerts antiplatelet and antithrombotic effects. Thus, paclitaxel may provide additional benefits beyond its antiproliferative effect when used in drug-coated balloons and drug-eluting stents for coronary revascularization and prevention of ISR.

Agitation-dependent biomechanical forces modulate GPVI receptor expression and platelet adhesion capacity during storage

BackgroundContinuous agitation during storage slows down the platelet storage lesions. However, in special circumstances, manual-mixing can be alternatively used to store products for short time periods without compromising platelet quality. Based on this finding, and given the role of shear stress in modulating receptor expression, we were interested in comparing the levels of platelet adhesion receptor, GPVI and platelet adhesion capacity under each storage condition.MethodsPlatelet concentrates (PCs) were divided into three groups: continuously-agitated PCs (CAG-PCs) with or without PP2 (Src kinase inhibitor) and manually-mixed PCs (MM-PCs). Platelet count/MPV, swirling, GPVI and P-selectin expression, GPVI shedding, platelet adhesion/spreading to collagen were examined during 5 days of storage.ResultsWhile MM- and CAG-PCs showed similar levels of P-selectin expression, GPVI expression was significantly elevated in MM-PCs with lower GPVI shedding/expression ratios, enhanced platelet adhesion/spreading and swirling in manually-mixed PCs. Of note, CAG-PCs treated with PP2 also demonstrated lower P-selectin expression and GPVI shedding, higher GPVI expression and attenuated swirling and spreading capability.ConclusionGiven the comparable platelet activation state in MM and CAG-PCs as indicated by P-selectin expression, enhanced platelet adhesion/spreading in MM-PCs, along with relatively higher GPVI expression here, supports previous studies demonstrating a role for biomechanical forces in modulating GPVI-dependent function. Thus, lower GPVI expression in CAG-PCs may be due to shear forces induced by agitation, which keeps this receptor down-regulated while also attenuating platelet adhesion/spreading capacities during storage. Low platelet function in PP2-CAG-PCs also highlights the importance of Src-kinases threshold activity in maintaining platelets quality.

Collagen-dependent platelet dysfunction and its relevance to either mitochondrial ROS or cytosolic superoxide generation: a question about the quality and functional competence of long-stored platelets

BackgroundUpon vascular damage, the exposed subendothelial matrix recruits circulating platelets to site of injury while inducing their firm adhesion mainly via GPVI-collagen interaction. GPVI also supports aggregatory and pro-coagulant functions in arterial shear rate even on the matrix other than collagen. Reactive oxygen species (ROS) modulate these stages of thrombosis; however augmented oxidant stress also disturbs platelet functions. Stored-dependent platelet lesion is associated with the increasing levels of ROS. Whether ROS accumulation is also relevant to collagen-dependent platelet dysfunction is the main interest of this study.MethodsFresh PRP-PCs (platelet concentrates) were either stimulated with potent ROS-inducers PMA and CCCP or stored for 5 days. Intra-platelet superoxide (O2−−) or mitochondrial-ROS and GPVI expression were detected by flowcytometery. GPVI shedding, platelet aggregation and spreading/adhesion to collagen were analyzed by western blot, aggregometry and fluorescence-microscopy, respectively.ResultsMitochondrial-ROS levels in 5 days-stored PCs were comparable to those induced by mitochondrial uncoupler, CCCP while O2−− generations were higher than those achieved by PMA. Shedding levels in 5 days-stored PCs were higher than those induced by these potent stimuli. GPVI expressions were reduced comparably in CCCP treated and 5 days-stored PCs. Platelet adhesion was also diminished during storage while demonstrating significant reverse correlation with GPVI shedding. However, only firm adhesion (indicated by platelets spreading or adhesion surface area) was relevant to GPVI expression. Platelet adhesion and aggregation also showed reverse correlations with both O2−− and mitochondrial-ROS formations; nonetheless mitochondrial-ROS was only relevant to firm adhesion.ConclusionAs a sensitive indicator of platelet activation, GPVI shedding was correlated with either simple adhesion or spreading to collagen, while GPVI expression was only relevant to platelet spreading. Thereby, if the aim of GPVI evaluation is to examine platelet firm adhesion, expression seems to be a more specific choice. Furthermore, the comparable levels of ROS generation in 5 days-stored PCs and CCCP treated platelets, indicated that these products are significantly affected by oxidative stress. Reverse correlation of accumulating ROS with collagen-dependent platelet dysfunction is also a striking sign of an oxidant-induced lesion that may raise serious question about the post-transfusion quality and competence of longer-stored platelet products.

Fibrin exposure triggers αIIbβ3‐independent platelet aggregate formation, ADAM10 activity and glycoprotein VI shedding in a charge‐dependent manner

BackgroundCollagen and fibrin engagement and activation of glycoprotein (GP) VI induces proteolytic cleavage of the GPVI ectodomain generating shed soluble GPVI (sGPVI). Collagen‐mediated GPVI shedding requires intracellular signalling to release the sGPVI, mediated by A Disintegrin And Metalloproteinase 10 (ADAM10); however, the precise mechanism by which fibrin induces GPVI shedding remains elusive. Plasma sGPVI levels are elevated in patients with coagulopathies, sepsis, or inflammation and can predict onset of sepsis and sepsis‐related mortality; therefore, it is clinically important to understand the mechanisms of GPVI shedding under conditions of minimal collagen exposure. ObjectivesOur aim was to characterize mechanisms by which fibrin‐GPVI interactions trigger GPVI shedding. MethodsPlatelet aggregometry, sGPVI ELISA, and an ADAM10 fluorescence resonance energy transfer assay were used to measure fibrin‐mediated platelet responses. ResultsFibrin induced αIIbβ3‐independent washed platelet aggregate formation, GPVI shedding, and increased ADAM10 activity, all of which were insensitive to pre‐treatment with inhibitors of Src family kinases but were divalent cation‐ and metalloproteinase‐dependent. In contrast, treatment of washed platelets with other GPVI ligands, collagen, and collagen‐related peptide caused αIIbβ3‐dependent platelet aggregation and GPVI release but did not increase constitutive ADAM10 activity. ConclusionsFibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin‐induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin‐induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.

Reducing state attenuates ectodomain shedding of GPVI while restoring adhesion capacities of stored platelets: evidence addressing the controversy around the effects of redox condition on thrombosis.

Thrombosis involves different stages including platelet adhesion to the site of injury, aggregatory events governed by integrin activation, pro-inflammatory responses recruiting leukocytes and finally, pro-coagulant activity which results in fibrin generation and clot formation. As important signaling agents, reactive oxygen species (ROS) reduce thrombus volume and growth, however given such a multistage mechanism, it is not well-elucidated how ROS inhibition modulates thrombosis. PRP-platelet concentrates (PCs) were either treated with ROS-reducing agents (1mM NAC or 30μM NOX inhibitor, VAS2870) or kept untreated during storage. Shedding and expression of platelet adhesion receptors in presence of inhibitors, agonists and CCCP (as controls) were analyzed by flow cytometery and western blot respectively. Besides above parameters, platelet adhesion to collagen in stored platelets was examined in presence of ROS inhibitors using fluorescence-microscopy. Highest levels of adhesion receptors shedding were achieved by ionophore and CCCP while collagen induces much more GPVI shedding than that of GPIbα. ROS inhibition reduced receptors shedding from day 3 of storage while enhanced their expressions. ROS inhibition not only did not reduce platelet adhesion capacity but it also enhanced platelets adhesion (in presence of NAC) or spreading (in presence of VAS2870) in 5days-stored PCs. While reducing state significantly inhibits platelet aggregation and thrombus growth, our results indicated that as a first stage of thrombosis, platelet adhesion is resistance to such inhibitory effects. These findings highlight the fact that integrin-dependent platelet activation is much more vulnerable to the inhibition of ROS generation than GPVI-dependent platelet adhesion. Presumably, inhibition of platelet activating signals by ROS inhibitors preserves platelet adhesiveness to collagen due to lessening GPVI shedding.

Continuous-flow mechanical circulatory support induces shedding of platelet adhesion receptors GpIb and GpVI

Hemorrhagic complications, especially mucosal bleedings, are very common for using continuous-flow mechanical circulatory supports (CF-MCS) including Extracorporeal Membrane Oxygenation (ECMO). CF-MCS could impact platelets by exposing this key player in primary hemostasis to high shear stress. A proteolytic cleavage (shedding) of platelet adhesion receptors GpIb and GpVI has been described under high shear stress and could contribute to the hemorrhagic complications in patients with CF-MCS. To assess whether CF-MCS promotes GpIb and GpVI shedding in vitro and in vivo and determine the kinetic evolution during the CF-MCS. First platelet shedding was investigated in vitro using a CF-MCS (Impella-CP®, Abiomed) loop model. Plasma with normal platelet count (plasma-NPC) was obtained by dilution of platelet-rich plasma collected from healthy donors in fresh frozen plasma. Sampling was performed before and after 2, 5, 30, 60 and 180 min perfusion of plasma-NPC in the loop model ( n = 4 runs). Platelet shedding was also investigated for the blood samples collected from 20 ECMO patients (WITECMO-h trial). Platelets of 20 healthy volunteers were evaluated as a control. GpIbα and GpVI shedding was analyzed by flow cytometry. A significant time-dependent decrease of GpIbα ( P < 0.001) and GpVI ( P < 0.001) MFI was observed after 180 min in the Impella®-CP loop model. In vivo baseline GpIbα and GpVI MFIs has been significantly decreased before ECMO implantation compared to the healthy subjects ( P < 0.001). After ECMO implantation, no significant variation of GpIbα and GpVI MFI was observed. CF-MCS induces platelet GpIb and GpVI shedding in vitro. Increased GpIb and GpVI shedding is already present before ECMO implantation and remain significantly elevated after the implantation. Loss of the platelet receptors GPIbα and GPVI in patients with CF-MCS may contribute to hemorrhagic complications observed in these patients.

Reverse correlations of collagen-dependent platelet aggregation and adhesion with GPVI shedding during storage.

Platelet receptor GPVI plays an important role in platelet firm adhesion to site of vascular injury. Receptor ligation with collagen, in company with other agonist/receptor interactions, augments inside out signaling pathways leading to platelet aggregation and thrombus formation. As GPVI expression is significantly modulated by ectodomain shedding, this study aimed to examine whether GPVI shedding functionally affects collagen-mediated platelet activation during storage. 6 PRP-platelet concentrates were subjected to adhesion analysis on collagen matrix under mild stirring condition as well as collagen-induced aggregation on day 1, 3 and 5 post-storage. Concurrently, platelet supernatants of same samples were fractionated by ultra-centrifugation and obtained micro-particle-free samples were subjected to western blot analysis for the evaluation of GPVI shedding. We showed a direct correlation between collagen-dependent platelet aggregation and adhesion (r = 0.8, p = 0.0001). The increasing levels of GPVI shedding during storage were in reverse correlation with collagen-induced platelet aggregation (r = - 0.82, p = 0.0004) which was significantly reducing during storage. Platelet adhesion to collagen matrix significantly decreased post-storage while it was also reversely correlated with the levels of GPVI shedding during 5 days storage of platelets (r = - 0.69, p = 0.002). Data presented here demonstrated that progressive shedding of surface adhesion receptor GPVI can affect its functional activities in stored platelets. Thereby considering the crucial role of GPVI in platelet adhesion to the site of injury, whether the therapeutic efficacy of banked platelet products could be influenced by storage-dependent shedding of this receptor, remains to be answered in future studies.

Association of Oxidative Stress and Platelet Receptor Glycoprotein GPIbα and GPVI Shedding During Nonsurgical Bleeding in Heart Failure Patients With Continuous-Flow Left Ventricular Assist Device Support.

Nonsurgical bleeding (NSB) in heart failure (HF) patients with continuous-flow left ventricular assist device (CF-LVAD) support is the most common clinical complication. The aim of this study was to investigate the association between oxidative stress and platelet glycoproteins GPIbα and GPVI shedding on the incidence of NSB in CF-LVAD patients. Fifty-one HF patients undergoing CF-LVAD implantation and 11 healthy volunteers were recruited. Fourteen patients developed NSB (bleeder group) during 1 month follow-up duration, while others were considered nonbleeder group (n = 37). Several biomarkers of oxidative stress were quantified at baseline and weekly intervals in all patients. Surface expression and plasma elements of platelet receptor glycoproteins GPIbα and GPVI were measured. Oxidative stress biomarkers and platelet GPIbα and GPVI receptor-shedding (decreased surface expression and higher plasma levels) were found to be preexisting conditions in baseline samples of both groups of HF patients when compared with healthy volunteers. Significantly elevated oxidative stress biomarkers and platelet glycoprotein receptor shedding were observed in postimplant bleeder group temporarily when compared with nonbleeder group. Strong significant associations between biomarkers of oxidative stress and platelet glycoprotein receptor shedding were observed, suggesting a possible role of oxidative stress in platelet integrin shedding leading to NSB in CF-LVAD patients. Receiver operating characteristic analyses of GPIbα and GPVI indicated that the likelihood of NSB had a predictive power of bleeding complication in CF-LVAD patients. In conclusion, elevated oxidative stress may play a role in GPIbα and GPVI shedding in the event of NSB. Thus, oxidative stress and GPIbα and GPVI shedding may be used as potential biomarkers for bleeding risk stratification in those patients.

Soluble fibrin causes an acquired platelet glycoprotein VI signaling defect: implications for coagulopathy

Background In coagulopathic blood, circulating thrombin may drive platelet dysfunction. Methods/Results Using calcium dye-loaded platelets, the effect of thrombin exposure and soluble fibrin generation on subsequent platelet GPVI function was investigated. Exposure of apixaban-treated platelet-rich plasma (12% PRP) to thrombin (1-10nm), but not ADP or thromboxane mimetic U46619 exposure, dramatically blocked subsequent GPVI activation by convulxin, collagen-related peptide or fibrillar collagen. Consistent with soluble fibrin multimerizing and binding GPVI, the onset of convulxin insensitivity required 200-500s of thrombin exposure, was not mimicked by exposure to PAR-1/4 activating peptides, was not observed with washed platelets, and was blocked by fibrin polymerization inhibitor (GPRP) or factor XIIIa inhibitor (T101). PAR-1 signaling through Gαq was not required because vorapaxar blocked thrombin-induced calcium mobilization but had no effect on the ability of thrombin to impair GPVI-signaling. Convulxin insensitivity was unaffected by the metalloprotease inhibitor GM6001 or the αIIb β3 antagonist GR144053, indicating negligible roles for GPVI shedding or αIIb β3 binding of fibrin. Thrombin treatment of washed platelets resuspended in purified fibrinogen also produced convulxin insensitivity that was prevented by GPRP. Exposure of apixaban/PPACK-treated whole blood to thrombin-treated fibrinogen resulted in >50% decrease in platelet deposition in a collagen microfluidic assay that required soluble fibrin assembly. Conclusions Conversion of only 1% plasma fibrinogen in coagulopathic blood would generate 90nm soluble fibrin, far exceeding ~1nmGPVI in blood. Soluble fibrin, rather than thrombin-induced platelet activation throuh PAR-1 and PAR-4, downregulated GPVI-signaling in response to stimuli, and may lead to subsequent hypofunction of endogenous or transfused platelets.

GPVI modulation during platelet activation and storage: its expression levels and ectodomain shedding compared to markers of platelet storage lesion

ABSTRACTPlatelet storage is associated with deleterious changes leading to the loss of platelet reactivity and response. During storage, platelets experience increased expression and shedding of P-selectin and CD40L as specific markers of platelet activation, whereas GPIbα decreases due to ectodomain shedding. As an important adhesive receptor, GPVI contributes significantly to thrombus formation while its expression and shedding levels during storage of platelet products have not been well characterized yet. This study investigated the modulation of GPVI during platelet storage.For this study, samples obtained from 10 PRP-platelet concentrates (PCs) were subjected to flow-cytometry analysis to examine the expression of platelet activation markers and GPVI on days 1, 3, and 5 post-storage. To examine the levels of etcodomain shedding of these molecules, microparticle (MP)-free supernatants were also analyzed by either ELISA or Western blot methods.According to results, the expression levels of P-selectin and CD40L as well as the amounts of their soluble forms significantly increased during storage. The expression of GPIbα and GPVI decreased whereas their shedding significantly increased post-storage. The expression and shedding levels of these two receptors were significantly correlated. Negative correlations between the expressions of GPIbα or GPVI and P-selectin have been observed whereas their shedding levels were significantly relevant together. In a control study, the use of biotinylated platelet resuspended in Tyrode’s buffer in the presence of ionophore with/without EDTA, confirmed the role of calcium in receptors shedding. In citrated PRP-PCs, recalcification of platelets also enhanced shedding levels of both GPIbα and GPVI. Intriguingly, the shedding levels of GPVI in stored PRP-PCs were much higher than those of ionophore-treated controls obtained from washed platelets. The ratios of sGPVI in stored platelet to ionophore-treated controls were also at least six times higher than those of GPIbα during storage. In conclusion, here we showed significant decreases of GPVI expression associated with its increasing levels of shedding during storage, suggesting GPVI as a valid marker of platelet storage lesion. Importantly, we found higher levels of GPVI shedding in stored platelets than those of ionophore-treated non-stored control samples. This suggests whereas platelet receptor shedding is mainly modulated by calcium-dependent signals, either platelet–surface interactions with the container walls during storage or induced shear stress under long-term agitation, might be also involved in the excessive shedding of GPVI during the storage of PCs.

In vivo evidence for platelet-induced physiological angiogenesis by a COX driven mechanism.

We sought to determine a role for platelets in in vivo angiogenesis, quantified by changes in the capillary to fibre ratio (C∶F) of mouse skeletal muscle, utilising two distinct forms of capillary growth to identify differential effects. Capillary sprouting was induced by muscle overload, and longitudinal splitting by chronic hyperaemia. Platelet depletion was achieved by anti-GPIbα antibody treatment. Sprouting induced a significant increase in C∶F (1.42±0.02 vs. contralateral 1.29±0.02, P<0.001) that was abolished by platelet depletion, while the significant C∶F increase caused by splitting (1.40±0.03 vs. control 1.28±0.03, P<0.01) was unaffected. Granulocyte/monocyte depletion showed this response was not immune-regulated. VEGF overexpression failed to rescue angiogenesis following platelet depletion, suggesting the mechanism is not simply reliant on growth factor release. Sprouting occurred normally following antibody-induced GPVI shedding, suggesting platelet activation via collagen is not involved. BrdU pulse-labelling showed no change in the proliferative potential of cells associated with capillaries after platelet depletion. Inhibition of platelet activation by acetylsalicylic acid abolished sprouting, but not splitting angiogenesis, paralleling the response to platelet depletion. We conclude that platelets differentially regulate mechanisms of angiogenesis in vivo, likely via COX signalling. Since endothelial proliferation is not impaired, we propose a link between COX1 and induction of endothelial migration.

An Acquired Defect Associated with Abnormal Signaling of the Platelet Collagen Receptor Glycoprotein VI

Introduction: Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. Aim: Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. Methods: Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. Results: Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an ∼40-kDa Syk fragment. Conclusions: These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.

Diesel exhaust particles impair platelet response to collagen and are associated with GPIbα shedding

ObjectiveAir pollution with fine particulates (PM10 and PM2.5) is associated with an increased incidence of cardiovascular events. The proposed mechanisms include indirect proinflammatory and procoagulant reactions involving activation of pulmonary macrophages, endothelial cells and the TNF/TF pathway, or direct procoagulant effects. Our laboratory has observed a reduction of the platelet responsiveness to collagen after exposure to diesel exhaust particles (DEP). HypothesisDEP directly interfere with platelet–collagen interactions by selectively inducing the shedding of platelet signaling receptors via metalloproteinases, which would represent a novel mechanism for DEP action on platelets. MethodsCitrated blood from healthy volunteers was exposed to highly standardized DEP at concentrations of 0.1, 2.5 and 5.0μg/ml or ultrafine carbon black (ufCB, 0.1μg/ml) and the plasmatic and platelet response was analysed. The closure times with the PFA-100 device and the platelet aggregation in response to a variety of agonists were monitored. Interleukins (IL)-1β and IL-8 levels were determined by ELISA and soluble P-selectin by the Luminex bead assay. Thrombin activity was measured as the endogenous thrombin potential (ETP) by fluorescence spectrometry. Soluble GPVI and GPIbα (glycocalicin) ectodomain fragments were measured by ELISA. ADAMTS13 activity was determined by a FRETS based assay and plasmin activity with Spectrozyme PL. ResultsAggregation assays where platelets were treated with low dose DEP or ultrafine carbon black (ufCB) revealed a significantly increased response to low doses of collagen (p<0.05, n=5). At higher doses, however, collagen induced aggregation was suppressed by DEP treatment: at 2.5μg/ml, the inhibition was 34±12% (p<0.01, n=10). Aggregations with cross-linked collagen related peptide (CRPxl), convulxin and with the monoclonal antibody 9O12.2 (all known to specifically bind to and activate GPVI) were also diminished. Ristocetin, arachidonic acid and ADP responses were normal at all DEP concentrations. No cleavage of GPVI ectodomain was detected (soluble GPVI 27.8±3 vs. 28±4μg/ml mean±SEM, n=10); however increased plasma glycocalicin (GPIbα ectodomain) was detected upon diesel exposure (2.58±0.11 vs. 2.28±0.03μg/ml p<0.01, n=10). ADAMTS13 and plasmin activity remained unaffected by DEP under the conditions tested. Platelets were not activated by either DEP or ufCB as soluble P-selectin was insensitive to these. ConclusionsDEP specifically and directly interferes with platelet–collagen interactions. The functional consequences are biphasic and include enhance platelet aggregation at lower DEP concentrations and inhibition at a higher dose. Our data indicate that this interaction does not involve P-selectin or GPVI shedding. It is however associated with an increase in GPIb cleavage.

Pathological Shear Regulates ADAM10 Activity on Circulating Platelets

Abstract 2194Metalloproteinase-mediated ectodomain shedding of platelet-specific receptors for collagen (GPVI) and von Willebrand factor (GPIbα of the GPIb-IX-V complex) is triggered by either ligand-induced platelet activation-dependent pathways, or by activation-independent pathways mediated by Factor Xa or induced by the thiol-modifying agent, N-ethylmaleimide. We recently reported that shed soluble GPVI (sGPVI) was elevated in plasma of 159 ischaemic stroke patients compared with 159 community-based controls (P=0.0168), and in 29 patients with disseminated intravascular coagulation compared with healthy donors (n=25, P=0.002), consistent with a pathophysiological role for GPVI shedding from human platelets. Our new studies now show that transient exposure of human platelets to arterial or pathological shear rates of 3000–10,000 s−1 for 1–5 min ex vivo in a cone-plate viscometer, in the absence of GPVI ligand or platelet activation, activated sheddases producing a 2- to 3-fold increase in plasma sGPVI and a corresponding loss of surface GPVI from sheared platelets. Shear-induced GPVI shedding was blocked by GM6001 or GI254023, a selective inhibitor of ADAM10. In contrast to shear-induced platelet aggregation, shedding was unaffected by inhibitors of aggregation (VWF-blocking anti-GPIbα mAb, AK2, or the αIIbβ3 antagonist, RGD peptide) or by the absence of VWF in a patient with von Willebrand’s disease Type III (VWF antigen levels <1%). Further, shedding of GPVI increased for up to 10 min after cessation of a short exposure of platelets to shear even when signalling, secretion and aggregation was blocked by inhibiting intracellular kinases (PP2, piceatannol), thromboxane generation (aspirin), ADP (apyrase) and calcium flux (BAPTA). Together, the combined results provide the first evidence that receptor sheddase activity can be regulated by hydrodynamic shear stress independent of cellular activation. This may represent a novel protective mechanism for down-regulating platelet reactivity as a response to pathological shear. Disclosures:No relevant conflicts of interest to declare.

Differentially regulated GPVI ectodomain shedding by multiple platelet–expressed proteinases

AbstractGlycoprotein VI (GPVI) mediates platelet activation on exposed subendothelial collagens at sites of vascular injury and thereby contributes to normal hemostasis, but also to the occlusion of diseased vessels in the setting of myocardial infarction or stroke. GPVI is an attractive target for antithrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and/or ectodomain shedding. Metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family have been proposed to mediate this ectodomain shedding, but direct evidence for this is lacking. Here, we studied GPVI shedding in vitro and in vivo in newly generated mice with a megakaryocyte–specific ADAM10 deficiency and in Adam17ex/ex mice, which lack functional ADAM17. We demonstrate that GPVI cleavage in vitro can occur independently through either ADAM10 or ADAM17 in response to distinct stimuli. In contrast, antibody (JAQ1)–induced GPVI shedding in vivo occurred in mice lacking both ADAM10/ADAM17 in their platelets, suggesting the existence of a third GPVI cleaving platelet enzyme. This was supported by in vitro studies on ADAM10/ADAM17 double–deficient platelets. These results reveal that ectodomain shedding of GPVI can be mediated through multiple differentially regulated platelet–expressed proteinases with obvious therapeutic implications.

Proteolysis of platelet receptors in humans and other species

In the past 5 years, metalloproteinase-mediated ectodomain shedding of platelet receptors has emerged as a new mechanism for modulating platelet function. By regulating surface expression of the platelet-specific receptors, glycoprotein (GP)VI that binds collagen, and GPIbalpha (the major ligand-binding subunit of the GPIb-IX-V complex) that binds von Willebrand factor (VWF) and other procoagulant and proinflammatory ligands, shedding not only irreversibly downregulates GPVI/GPIbalpha function, but generates proteolytic fragments that might be unique biomarkers or modulators in plasma. This is potentially significant because GPVI and GPIbalpha are involved in initiating thrombotic diseases such as heart attack and stroke, as well as autoimmune diseases where anti-platelet antibodies result in thrombocytopenia. Altered expression levels of GPIbalpha/GPVI are associated with both thrombotic propensity and platelet aging, suggesting an additional role in platelet clearance. Although emerging data are elucidating molecular mechanisms underlying GPIbalpha/GPVI shedding, evidence for the functional consequences of shedding in vivo, either clinically or in animal models, is far more limited. Here we consider recent published evidence for GPVI or GPIbalpha shedding in humans, nonhuman primates and mice, and whether conservation of sheddase cleavage sites across species points to a functional role for metalloproteolytic shedding in vivo.

Measuring soluble platelet glycoprotein VI in human plasma by ELISA

Recent experimental evidence demonstrates that the platelet-specific collagen receptor, glycoprotein (GP)VI is essentially all uncleaved on normal circulating platelets, but is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligands (including collagen), anti-GPVI antibodies or activation at the platelet Fc receptor, FcγRIIa. This raises the question of whether shed ectodomain fragment in plasma could be a useful biomarker of thrombotic risk and/or autoimmune thrombocytopenia. In this study, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring soluble GPVI in human plasma, using rabbit anti-GPVI polyclonal antibody in the solid-phase, murine anti-GPVI monoclonal antibody (1A12) in the fluid-phase and horseradish peroxidase (HRP)-coupled anti-mouse antibody and enhanced chemiluminescence (ECL) for detection. The ELISA was optimized for sensitivity, reproducibility, inter- and intra-assay precision, addition and recovery and detected GPVI in plasma with a lower detection limit of ∼1 ng/mL. Effects of different anti-coagulants (trisodium citrate, acid-citrate-dextrose or EDTA) were negligible. In ten healthy donors, soluble plasma GPVI levels were 18.9 ± 4.1 ng/mL. Treating normal platelet-rich plasma with a GPVI ligand (collagen-related peptide, CRP), calmodulin inhibitor W7 (that induces GPVI shedding without platelet activation) or N-ethylmaleimide (that directly activates platelet sheddases), under conditions previously shown to induce GPVI shedding, also increased plasma GPVI levels by up to ∼7-fold, compared to previously reported autoimmune (anti-GPVI) patient plasma where soluble GPVI was ∼10-fold higher than normal. Characterization of this sensitive ELISA should facilitate analysis of functional/diagnostic role(s) for soluble GPVI in human plasma associated with thrombotic/immune dysfunction.

The highly specific platelet glycoprotein (GP) VI agonist trowaglerix impaired collagen-induced platelet aggregation ex vivo through matrix metalloproteinase-dependent GPVI shedding.

C-type lectin proteins (CLPs) have diverse targets including platelet GPIb, GPVI and integrin alpha(2)beta(1), and affect platelet function in a various way. In this study, we characterized a huge, heterodimeric venom protein, trowaglerix, which belongs to the CLP family. We purified a potent platelet-aggregation inducer, trowaglerix, from the crude venom of Tropidolaemus wagleri. Biotinylated trowaglerix was used for binding assays, and immunoblotting was used to investigate the signal transduction involved. Two distinct subunits of trowaglerix with similar masses of around 16 kDa were eluted by high-performance liquid chromatography after reduction and alkylation. Trowaglerix induced platelet aggregation of washed human platelets and platelet-rich plasma (PRP) in a concentration-dependent manner. Biotinylated trowaglerix specifically bound to platelet membrane GPVI, but not to GPIb or alpha(2) integrin. Treatment with trowaglerix induced GPVI loss in human platelets in vitro and impaired the platelet aggregation of mouse PRP ex vivo in response to collagen but not in response to adenosine diphosphate (ADP). However, GM6001, a matrix metalloproteinase (MMP) inhibitor, inhibited trowaglerix-induced GPVI cleavage and restored the platelet responsiveness of PRP to collagen. Trowaglerix activates platelets through specific binding to GPVI, leading to kinases-dependent exposure of functional alpha(IIb)beta(3) and platelet aggregation, and also induces MMP-dependent GPVI shedding from platelets.

Transdermal 17-β estradiol replacement therapy reduces megakaryocyte GPVI expression

The platelet-collagen interaction is a critical early event in arterial thrombus formation, and platelet GPVI is the major activating receptor for collagen. We have previously used a mouse model to demonstrate that the estrogen effects on platelets depend upon the agonist, estrogen formulation and route of administration. In the current study we used a model of transdermal estradiol (E2) administration to ovariectomized mice to address the potential inhibitory effects of E2 on platelet GPVI. Platelet GPVI expression was reduced after transdermal E2 replacement therapy (p ≤ 0.001) but no evidence of GPVI shedding was found when platelets were directly incubated with E2. In addition, significantly reduced GPVI-mediated fibrinogen binding and aggregation were observed in platelets from mice subjected to 9 days or longer of in vivo E2 treatment, but not in platelets from mice treated for 3 days or shorter, suggesting an indirect pathway. Studies with mouse bone marrow revealed that E2 replacement in ovariectomized mice reduces megakaryocyte GPVI expression. This data suggest that transdermal E2 is able to affect centrally on megakaryocyte GPVI to regulate platelet GPVI and function.

Molecular regulation of platelet adhesion

Two platelet receptors, the glycoprotein (GP) Ib-IX-V complex and GPVI/FcRγ-chain, are pivotal in initiating signals that propagate both haemostasis and thrombosis. In the arterial circulation when an atherosclerotic plaque ruptures, these receptors initiate platelet adhesion in response to exposed thrombogenic materials by binding vessel wall von Willebrand Factor (VWF) and collagen, respectively. While these adhesive processes and subsequent events in thrombus formation have been the subject of intense investigation, the mechanisms that positively and negatively regulate the function of these and other receptors in activated platelets, and thus act to determine thrombus formation and stability, are only beginning to be understood. Platelet adhesion through the GPIb-IX-V complex is regulated at three distinct levels. First, the binding of VWF to the α-chain of GPIb is regulated by shear. Recent data from our laboratory exploiting analysis of canine-human chimaeras of GPIbα indicate that a negative-charge cluster involving leucine-rich repeats 2-4 plays an important role in the molecular regulation of shear-dependent VWF binding. Second, VWF affinity for the GPIb-IX-V complex is regulated by its association with the scaffolding protein, 14-3-3ζ, and phosphorylation of the α- and β-subunits of the GPIb-IX-V complex. Finally, calmodulin association with GPIbβ and GPV regulates the platelet activation dependent cleavage of GPIbα and GPV by the platelet membrane-associated metalloproteinase, ADAM17. Platelet adhesion through GPVI is primarily regulated by post-ligation-induced shedding of the GPVI ectodomain by ADAM10. Ligand-induced shedding is signaling-dependent and blocked by inhibitors of GPVI-dependent-signaling, Src, phosphoinositide 3-kinase (PI3-kinase) , or the ITAM-related Syk kinase. The calmodulin inhibitor W7 also induces shedding of GPVI, but independently of platelet activation. We have found that, like GPVI, another ITAM-containing receptor, the platelet Fc receptor, FcγRIIa, is cleaved in response to ligands of FcγRIIa (VM58, 14A2 or heparin-induced antibodies) or GPVI (convulxin) , or by W7. Conversely, ligands of FcγRIIa also induce GPVI shedding. FcγRIIa cleavage is blocked not only by Syk inhibition but also by the calpain inhibitor, E64d, and a cytoplasmic calpain-cleavage site was identified upstream of the ITAM domain. Thus, regulation of ligand binding or signaling by these unique platelet receptors through distinct proteolytic mechanisms may play a key role in limiting thrombus size and stability in response to vessel damage or immunological insult.