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Naphthalimide derivatives have multiple biological activities, including antitumour and anti‐inflammatory activities. We previously synthesized several naphthalimide derivatives; of them, compound 5 was found to exert the strongest inhibitory effect on human DNA topoisomerase II activity. However, the effects of naphthalimide derivatives on platelet activation have not yet been investigated. Therefore, the mechanism underlying the antiplatelet activity of compound 5 was determined in this study. The data revealed that compound 5 (5–10 μM) inhibited collagen‐ and convulxin‐ but not thrombin‐ or U46619‐mediated platelet aggregation, suggesting that compound 5 is more sensitive to the inhibition of glycoprotein VI (GPVI) signalling. Indeed, compound 5 could inhibit the phosphorylation of signalling molecules downstream of GPVI, followed by the inhibition of calcium mobilization, granule release and GPIIb/IIIa activation. Moreover, compound 5 prevented pulmonary embolism and prolonged the occlusion time, but tended to prolong the bleeding time, indicating that it can prevent thrombus formation but may increase bleeding risk. This study is the first to demonstrate that the naphthalimide derivative compound 5 exerts antiplatelet and antithrombotic effects. Future studies should modify compound 5 to synthesize more potent and efficient antiplatelet agents while minimizing bleeding risk, which may offer a therapeutic potential for cardiovascular diseases.

Glycoprotein (GP)Ib that binds von Willebrand factor (vWF) and glycoprotein (GP)VI, that binds collagen play a significant role in platelet activation and aggregation, and are potential targets for antithrombotic treatment. They are targeted by snake venom proteinases. The effect of a such proteinase, mutalysin-II, on platelet aggregation was examined using washed human platelets and platelet-rich plasma. Its proteolytic activity on vWF, on its binding partner GPIbα, and on GPVI was analyzed by SDS-PAGE, and immunodetection with the corresponding antibodies after blotting. Dose- and time-dependently, mutalysin-II inhibits aggregation of washed platelets induced by vWF plus ristocetin and by convulxin, but with no significant effect on platelet-rich-plasma. Furthermore, mutalysin-II cleaves vWF into low molecular mass multimers of vWF and a rvWF-A1 domain to realease a ∼27-kDa fragment detectable by SDS-PAGE and blotting with mouse anti-rvWF-A1-domain IgG. Moreover, GPVI was cut by mutalysin-II into a soluble ∼55-kDa ectodomain and a fragment of ∼35-kDa. Thus, mutalysin-II inhibits vWF-induced platelet aggregation via cleavage of bound vWF-A1, and its receptor GPIbα. The additional cleavage of, GPVI, blocks collagen-induced platelets. Our data highlight mutalysin-II as an interesting platelet-directed tool targeting vWF-GPIbα binding and particularly GPVI. Thus, it might be suited for antithrombotic therapy as its combined inactivation of two receptors does not significantly compromise hemostasis, but shows high efficacy and safety. Studies are needed to further develop and demonstrate its potential benefits.

Rationale Hematoma expansion (HE) aggravates brain injury after intracerebral hemorrhage (ICH) and hypertension is a key contributor to HE. Plasma kallikrein (PK) is involved in hemorrhagic transformation in ischemic stroke mice. This study was conducted to explore the role of PK in HE in hypertensive ICH. Methods Hypertension was achieved by continuous infusion of angiotensin II (Ang II) with an osmotic pump in C57BL/6 mice. ICH was achieved by stereotactic intrastriatal injection of blood. PK-specific antibody and platelet glycoprotein VI (GPVI) agonists were administered to intervene in hematoma expansion. The hematoma volume was indicated by the erythrocyte components hemoglobin and carbonic anhydrase-1 in the ipsilateral brain hemisphere. Results Ang II-induced hypertensive mice showed enhanced hematoma expansion and worsened neurologic deficits after ICH modeling. Moreover, intrastriatal injection of blood from Ang II-treated mice into normal mice increased the area of secondary hemorrhage more than blood from untreated mice. Mechanistically, elevated PK was found in Ang II-infused mice whereas, inhibition of PK and administration of the GPVI agonist convulxin decreased hematoma expansion and improved neurologic deficits after ICH. Conclusions These findings suggest that PK inhibition and GPVI agonist treatment might serve as potential methods to intervene in HE after ICH.

Platelet activation is characterized by shape change, granule secretion, activation of fibrinogen receptor (glycoprotein IIb/IIIa) sustaining platelet aggregation, and externalization of negatively charged aminophospholipids contributing to platelet procoagulant activity. Epinephrine (EPI) alone is a weak platelet activator. However, it is able to potentiate platelet activation initiated by other agonists. In this work, we investigated the role of EPI in the generation of procoagulant platelets. Human platelets were activated with convulxin (CVX), thrombin (THR) or protease-activated receptor (PAR) agonists, EPI, and combination thereof. Platelet aggregation was assessed by light transmission aggregometry or with PAC-1 binding by flow cytometry. Procoagulant collagen-and-THR (COAT) platelets, induced by combined activation with CVX-and-THR, were visualized by flow cytometry as Annexin-V-positive and PAC-1-negative platelets. Cytosolic calcium fluxes were monitored by flow cytometry using Fluo-3 indicator. EPI increased platelet aggregation induced by all agonist combinations tested. On the other hand, EPI dose-dependently reduced the formation of procoagulant COAT platelets generated by combined CVX-and-THR activation. We observed a decreased Annexin-V-positivity and increased binding of PAC-1 with the triple activation (CVX + THR + EPI) compared with CVX + THR. Calcium mobilization with triple activation was decreased with the higher EPI dose (1,000 µM) compared with CVX + THR calcium kinetics. In conclusion, when platelets are activated with CVX-and-THR, the addition of increasing concentrations of EPI (triple stimulation) modulates platelet response reducing cytosolic calcium mobilization, decreasing procoagulant activity, and enhancing platelet aggregation.

Background and aims: Platelets can release extracellular vesicles (EVs) upon stimulation with various agonists. Interestingly, platelets from patients with Glanzmann thrombasthenia have reduced EV release. These platelets lack functional alpha(IIb)beta(3) integrins, indicating that alpha(IIb)beta(3) integrin is critical in vesicle release. Integrin activation is central in platelet function and is associated with e.g. adhesion, aggregation and cytoskeletal rearrangement. However, while platelet activation pathways are widely known, the mechanisms underlying EV release remain uncharacterized. We investigated the role of integrin alpha(IIb)beta(3), phosphatidyl serine (PS) exposure, cytoskeletal rearrangement and their associated signalling pathways in EV release.Methods: EVs were isolated from activated platelets. Platelet activation status was measured by multicolour flow cytometry. A panel of pharmacologic inhibitors was used to interfere in specific signalling pathways. EV release was quantified enzymatically based on membrane PS content and nanoparticle tracking analysis. In addition, real-time visualization of EV shedding with confocal microscopy and EVs with Cryo-TEM imaging was performed.Results: Platelet activation with convulxin resulted in higher EV release than with activation by thrombin. Kinetic measurements indicated that EV release followed the pattern of alpha(IIb)beta(3) integrin activation and subsequent closure paralleled by PS exposure. Prevention of alpha(IIb)beta(3) activation with the inhibitor tirofiban dramatically suppressed EV release. Similar results were obtained using a alpha(IIb)beta(3)-deficient platelets from patients with Glanzmann thrombasthenia. Inhibition of actin cytoskeleton rearrangement decreased EV release, whereas inhibition of individual signalling targets upstream of cytoskeletal rearrangement showed no such effects.Conclusion: Platelet EV release requires three main events: integrin activation and closure, PS exposure, and cytoskeletal rearrangement.

Background: Cutaneous and mucocutaneous leishmaniasis are parasitic diseases characterized by skin manifestations. In Brazil, Leishmania (Leishmania) amazonensis is one of the etiological agents of cutaneous leishmaniasis. The therapeutic arsenal routinely employed to treat infected patients is unsatisfactory, especially for pentavalent antimonials, as they are often highly toxic, poorly tolerated and of variable effectiveness. This study aimed to evaluate in vitro the leishmanicidal activity of toxins isolated from Crotalus durissus terrificus venom as a new approach for the treatment of leishmaniasis. Methods: The comparative effects of crotamine, crotoxin, gyrotoxin, convulxin and PLA2 on bone marrow-derived macrophages infected with L. (L.) amazonensis as well as the release of TGF-β from the treated macrophages were studied. Results and Discussion: Crotamine had the strongest inhibitory effect on parasite growth rate (IC50: 25.65±0.52 μg/mL), while convulxin showed the weakest inhibitory effect (IC50: 52.7±2.21 μg/mL). In addition, TGF-β was significantly reduced after the treatment with all toxins evaluated. Conclusion: The Crotalus durissus terrificus toxins used in this study displayed significant activity against L. (L.) amazonensis, indicating that all of them could be a potential alternative for the treatment of cutaneous leishmaniasis.

Background Platelet dysfunction often accompanies trauma-induced coagulopathy. Because soluble fibrin impairs platelet glycoprotein VI (GPVI) signaling and platelets of trauma patients can display impaired calcium mobilization, we explored the role of fibrinolysis on platelet dysfunction during trauma. Methods Convulxin-induced GPVI calcium mobilization was investigated in healthy platelet-rich plasma (PRP) pretreated with thrombin and tissue plasminogen activator (tPA). Blood samples from healthy participants (n = 7) and trauma patients (n = 22) were tested for platelet calcium mobilization, plasma D-dimer, platelet D-dimer binding (via flow cytometry), and platelet lumi-aggregometry. Results For healthy platelets, maximal platelet dysfunction was observed when cross-linked soluble fibrin (no tPA) or cross-linked fibrin degradation products (FDPs) were generated in suspension before convulxin stimulation. Lack of fibrin polymerization (inhibited by Gly-Pro-Arg-Pro [GPRP]) or lack of factor XIIIa cross-linking (T101-inhibited) restored GPVI signaling, whereas non–cross-linked FDPs only partially blocked signaling induced by convulxin. In addition, D-dimer added to healthy PRP impaired platelet aggregation and dense granule release induced by various agonists. Plasma D-dimer level was strongly correlated (R = 0.8236) with platelet dysfunction as measured by platelet calcium mobilization induced with various agonists. By 48 to 120 h after trauma, plasma D-dimer levels declined, and platelet function increased significantly but not to healthy levels. Trauma platelets displayed elevated D-dimer binding that was only partially reduced by αIIbβ3-inhibitor GR144053. After 60-minute incubation, washed healthy platelets resuspended in plasma from trauma patients captured approximately 10 000 D-dimer equivalents per platelet. Conclusions During trauma, D-dimer and FDPs inhibit platelets, potentially via GPVI and integrin αIIbβ3 engagement, contributing to a fibrinolysis-dependent platelet loss-of-function phenotype.

The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow-cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real-time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo-3 (Ca2+ ), ION NaTRIUM Green-2 (Na+ ), and ION Potassium Green-2 (K+ ). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR-1 agonist), AYPGKF (PAR-4 agonist), convulxin (collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. © 2020 International Society for Advancement of Cytometry.

Objective Platelet surface receptors are also present subcellularly in organelle membranes and can be expressed on the surface upon platelet activation. However, some receptors were reported to be decreased after activation. We analyzed the mechanism of activation‐dependent expression for different receptors. Methods Flow cytometry using platelet‐rich plasma or washed platelets was used to analyze receptor‐expression changes after platelet activation by glycoprotein (GP) VI–specific agonists, crosslinked collagen‐related peptide (CRP‐XL) and convulxin (Cvx), and thrombin. Platelets prelabeled with fluorescent antibody specific for a receptor were allowed to adhere on immobilized collagen or fibrinogen and post‐stained with antibody against the same receptor labeled with another fluorophore, allowing us to differentiate preexisting receptors from newly expressed receptors. Results Surface expression of αIIbβ3 increased in CRP‐XL–, Cvx‐, or thrombin‐stimulated platelets, but GPIb decreased due to shedding and internalization. Both total and dimeric GPVI increased in thrombin‐induced platelets, but decreased in platelets stimulated by Cvx, as a result of internalization. The larger platelets showed a greater increase in surface receptor (α2β1, αIIbβ3, GPVI, GPIb) expression upon activation compared to the smaller ones. Pre‐ and postlabeling with antibody specific for the same receptor, but conjugated with different fluorophores, allowed us to differentiate the receptors expressed on the surface of resting platelets from receptors newly exposed to the surface upon platelet activation. Conclusions Increased receptor expressions after activation are mainly manifested in the larger platelets. On platelets adhered on fibrinogen, the newly expressed receptors, especially GPVI, are localized in the lamellipodia of the spread platelets.

Background Platelet-derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood. Aims The aim of this study was to characterize the role of PDIA1 in human platelets. Methods Proteomic analysis of PDI isoforms in platelets was performed using LC-MS/MS, and the expression of PDIs on platelets in response to collagen, TRAP-14, or ADP was measured with flow cytometry. The effects of bepristat, a selective PDIA1 inhibitor, on platelet aggregation, expression of platelet surface activation markers, thromboxane A2 (TxA2 ) and reactive oxygen species (ROS) generation were evaluated by optical aggregometry, flow cytometry, enzyme-linked immunosorbent assay and dihydrodichlorofluorescein diacetate-based fluorescent assay, respectively. Results PDIA1 was less abundant compared to PDIA3 in resting platelets and platelets stimulated with TRAP-14, collagen or ADP. Collagen, but not ADP, induced a significant increase in PDIA1 expression. Bepristat potently inhibited the aggregation of washed platelets induced by collagen or convulxin, but only weakly inhibited platelet aggregation induced by TRAP-14 or thrombin, and had the negligible effect on platelet aggregation induced by arachidonic acid. Inhibition of PDIA1 by bepristat resulted in the reduction of thromboxane A2 and ROS production in collagen- or thrombin-stimulated platelets. Furthermore, bepristat reduced the activation of αIIbβ3 integrin and expression of P-selectin. Conclusions PDIA1 acts as an intraplatelet regulator of the ROS-TxA2 pathway in collagen-GP VI receptor-mediated platelet activation that is a mechanistically distinct pathway from extracellular regulation of αIIbβ3 integrin by PDIA3.

Snake venoms have evolved primarily to immobilize and kill prey, and consequently, they contain some of the most potent natural toxins. Part of that armory is a range of hemotoxic components that affect every area of hemostasis, which we have harnessed to great effect in the study and diagnosis of hemostatic disorders. The most widely used are those that affect coagulation, such as thrombin-like enzymes unaffected by heparin and direct thrombin inhibitors, which can help confirm or dispute their presence in plasma. The liquid gold of coagulation activators is Russell's viper venom, since it contains activators of factor X and factor V. It is used in a range of clotting-based assays, such as assessment of factor X and factor V deficiencies, protein C and protein S deficiencies, activated protein C resistance, and probably the most important test for lupus anticoagulants, the dilute Russell's viper venom time. Activators of prothrombin, such as oscutarin C from Coastal Taipan venom and ecarin from saw-scaled viper venom, are employed in prothrombin activity assays and lupus anticoagulant detection, and ecarin has a valuable role in quantitative assays of direct thrombin inhibitors. Snake venoms affecting primary hemostasis include botrocetin from the jararaca, which can be used to assay von Willebrand factor activity, and convulxin from the cascavel, which can be used to detect deficiency of the platelet collagen receptor, glycoprotein VI. This article takes the reader to every area of the diagnostic hemostasis laboratory to appreciate the myriad applications of snake venoms available in diagnostic practice.

Factor Xa (FXa) has been reported to activate platelet via interaction with glycoprotein (GP) VI but the underlying mechanism has not been fully elucidated. We investigated if Nox2-derived oxidative stress is implicated in FXa-induced platelet aggregation (PA), and the effect of a FXa inhibitor, namely rivaroxaban, with or without aspirin (ASA), on PA. We performed an in vitro study measuring convulxin-induced PA, thromboxane (Tx) B2 and isoprostanes biosynthesis, soluble Nox2-dp (sNox2-dp), a marker of Nox2 activation, soluble GPVI (sGPVI) and PLA2 activation in platelets from healthy subjects (n = 5) added with and without a Nox2 inhibitor. The same variables were also examined in platelets treated with rivaroxaban (15–60 ng/ml), combined or less with ASA (25 µM). Convulxin-stimulated platelets increased sGPVI, sNox2-dp, H2O2, eicosanoid biosynthesis and PLA2 phosphorylation, which were all inhibited by a Nox2 inhibitor. Rivaroxaban alone significantly reduced PA, sGPVI, TxB2 and isoprostanes biosynthesis, concomitantly with Syk, sNox2-dp and PLA2 activation in a dose-dependent fashion; a significant effect was achieved with 30 ng/ml rivaroxaban. Docking simulation analysis showed that rivaroxaban interacts with GPVI. In platelets co-incubated with ASA, rivaroxaban amplified the ASA antiplatelet effect, which was achieved with 30 ng/ml and prevalently attributable to Nox2 inhibition and impaired isoprostane biosynthesis. Here we show that rivaroxaban, at concentrations achievable in human circulation, inhibits PA via GPVI interaction and eventually Nox2-mediated isoprostanes biosynthesis and amplifies the ASA antiplatelet effect.

Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 μg/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1−/−) platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced β-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation.

Patients with type 1 diabetes mellitus (T1DM) have increased thrombosis and platelet activation. The mechanisms for platelet hyperactivation in diabetes are incompletely understood. T1DM is accompanied by hyperglycemia, dyslipidemia, and increased inflammation in addition to an altered hormonal milieu. In vitro analysis of platelets revealed that normal glucose reduces platelet activation whereas hyperglycemic conditions increase platelet activation. We therefore hypothesized that hyperglycemia increases platelet glucose utilization, which increases platelet activation to promote thrombosis. Glucose uptake and glycolysis were increased in platelets isolated from mice given streptozotocin (STZ) to induce T1DM in concert with induction of GLUT3. Platelets from STZ-induced diabetic mice exhibited increased activation after administration of protease-activated receptor 4 peptide and convulxin. In contrast, platelets isolated from GLUT1 and GLUT3 double-knockout (DKO) mice, which lack the ability to use glucose, failed to increase activation in hyperglycemic mice. Diabetic mice displayed decreased survival in a collagen/epinephrine-induced pulmonary embolism model of in vivo platelet activation relative to nondiabetic controls. Survival after pulmonary embolism was increased in diabetic DKO mice relative to nondiabetic controls. These data reveal that increased platelet glucose metabolism in vivo contributes to increased platelet activation and thrombosis in a model of T1DM.

Introduction: Precision mapping of the functional structure of platelet populations holds great promise for the identification of hyper-reactive subtypes that are likely to be disease drivers, having value in prognostics and as therapeutic targets. However, the ability to measure the intrinsic functional capacity of individual platelets is confounded by potent paracrine cross-talk, resulting in phenotypic remodeling of the entire platelet population, and in doing so obscuring the identity of hyper-reactive platelets.Methods: To address this we have developed a droplet microfluidics strategy for single platelet confinement to exclude paracrine signaling. Consideration of the Poisson distribution was used for high throughput single platelet encapsulation and the preparation of minimal platelet collectives serving as digital models for understanding the role of hyper-reactive platelets coordinating system-level behavior by paracrine signaling. Platelets are retrieved from the droplets for phenotyping using standard flow cytometry. In addition, we have incorporated a staggered herringbone micromixing element for accurate agonist and antibody dispensing in droplets.Results: The methodology was used for characterizing sensitivity distributions from healthy blood donors in response to convulxin (agonist of the GPVI receptor, the major platelet receptor for collagen). P-selectin exposure and α IIb β 3 integrin activation were used as analytical end-points to demonstrate the existence of hyper-reactive platelets that direct 20-fold gains in system level sensitivity. Conclusions: The analytical workflow represents an enabling tool for the accurate classification of platelet subtypes and description of their underlying biology.Supplementary information: The online version of this article (10.1007/s12195-020-00665-6) contains supplementary material, which is available to authorized users.

Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) with that of room temperature–stored platelets (RSP) in vitro, in vivo, and posttransfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared with RSP. We found significantly reduced glycoprotein VI (GPVI) levels after cold exposure of 5 to 7 days. After transfusion into humans, CSP were mostly equivalent to RSP; however, their rate of aggregation in response to the GPVI agonist collagen was significantly lower. In a mouse model of platelet transfusion, we found a significantly lower response rate to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of cold storage and highlight the need for thorough investigations of CSP. This trial was registered at www.clinicaltrials.gov as #NCT03787927.

Objectives: Spinal fusion is often complicated by high blood loss and the clinically observed phenomenon of increased bleeding times throughout the case. Although the pathophysiology of this phenomenon involves hyperfibrinolysis and occurs to a lesser extent in the presence of antifibrinolytics (e.g. TXA), we hypothesize that perioperative changes in platelet function also contribute to this continued observation. Methods: Blood samples were acquired from patients undergoing posterior spinal fusion for correcting scoliosis (5 neuromuscular, 3 idiopathic ). Samples were obtained immediately before the procedure, at 2, 4, and 6 hours post-incision, and the subsequent morning (POD1). Platelet activation in response to stimulation with an intravascular agonist, thrombin, and an extravascular agonist, convulxin, was measured by flow cytometry. Activation of the fibrin-binding integrin glycoprotein IIb/IIIa (GpIIb/IIIa) was reported by PAC-1 binding and expression of the alpha-granule component P-selectin was reported by CD62p binding. CBCs and Thromboelastography (TEG) were performed on samples in parallel. Estimated blood loss (EBL) was calculated using a hematocrit-based analysis of red cell mass. Results:All 5 neuromuscular patients exhibited a marked and progressive decline in platelet count and activity throughout surgery and early postoperative period. Reductions were most notable for thrombin-stimulated GpIIb/IIIa activation (Th PAC1) at 4 hrs (mean 41.7%, p=0.0056) and 6 hrs (50.9%, p=0.0096). Similar trends in mean reductions were observed for thrombin stimulated P- selectin expression (Th P-Selectin) at 6 hrs (22.5%, p=0.0112) and on POD1 (39.2%, p=0.0274). Patients with idiopathic scoliosis demonstrated significant reductions in convulxin-stimulated GpIIb/IIIa activation (32.8%, p=0.0087) and P-selectin expression (35.1%, p=0.0147) at 2 hrs, which recovered to or increased beyond baseline thereafter. These trends also manifested on TEG for all neuromuscular patients as the average time to maximum clot strength increased by 48.8% at 6 hrs (p=0.0058). Furthermore, platelet activity correlated with the calculated EBL for all 8 patients (Th PAC1 at 4 hrs R= -0.673, p=0.146 and 6 hrs: R= -0.623, p=0.099; Th P-Selectin at 6 hrs: R= -0.694 p = 0.056 and POD1: R= -0.713, p=0.047). EBL also significantly correlated with reductions in platelet count at 6 hrs (R= -0.919, p=0.010) and absolute platelet count on POD1 (R= -0.858, p=0.006). Conclusions: We have observed a progressive reduction in platelet activation and fibrin-dependent clot formation throughout scoliosis surgery, particularly in patients with neuromuscular disease. High endothelial injury related to achieving surgical exposure may account for the early drop in response to the extravascular collagen receptor agonist, convulxin. Measures associated with secondary hemostasis, such as the thrombin-stimulated platelet activation and time to maximum clot strength, both demonstrated exhaustion towards the post-operative period. These data suggest that surgically-induced disruption in platelet function may be a key component of the coagulopathic phenomenon described and closely associated with blood loss.

The combination of pharmaceutical lipid excipients with aspirin in a novel liquid oral formulation (Vazalore) limits gastrointestinal toxicity of aspirin. This study was performed to determine whether the lipid excipients influence the pharmacodynamic effects of aspirin and whether the excipients directly affect platelet function. The pharmacodynamic effects of aspirin were assessed over a range of concentrations designed to exert limited to maximal inhibition of cyclooxygenase-1 (COX1) necessary for thromboxane A2 production. Platelet aggregation induced by arachidonic acid and assessed with the use of light transmission aggregometry was used as a direct measure of the inhibition of COX1 by aspirin. Flow cytometry was used to assess the direct effect of excipients on platelet function. Twice the ratio of lipid excipient to aspirin used in the formulation of the novel oral agent was used. Blood was taken from 20 healthy subjects and anticoagulated with trisodium citrate (3.2%, 1:10 vol/vol). Aspirin and excipients were added in vitro and incubated for 10 minutes before performance of light transmission aggregometry and flow cytometry. The excipients did not limit the pharmacodynamic effects of aspirin. When the extent of inhibition of platelet aggregation was limited, the excipients tended to enhance pharmacodynamic effects. The excipients did not activate platelets in the absence of agonist and did not alter activation of platelets in response to adenosine diphosphate, arachidonic acid, thrombin, or convulxin (a collagen mimetic). Lipid excipients used in an oral formulation of aspirin do not impair the pharmacodynamic effects of aspirin and do not alter platelet function.

Septins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbβ3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbβ3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

Background Emerging evidence implicates dysfunctional platelet responses in thrombotic complications in COVID‐19 patients. Platelets are important players in inflammation‐induced thrombosis. In particular, procoagulant platelets support thrombin generation and mediate thromboinflammation. Objectives To examine if procoagulant platelet formation is altered in COVID‐19 patients and if procoagulant platelets contribute to pulmonary thrombosis. Patients/Methods Healthy donors and COVID‐19 patients were recruited from the University of Utah Hospital System. Platelets were isolated and procoagulant platelet formation measured by annexin V binding as well as mitochondrial function were examined. We utilized mice lacking the ability to form procoagulant platelets (CypDplt−/−) to examine the role of procoagulant platelets in pulmonary thrombosis. Results and Conclusions We observed that platelets isolated from COVID‐19 patients had a reduced ability to become procoagulant compared to those from matched healthy donors, as evidenced by reduced mitochondrial depolarization and phosphatidylserine exposure following dual stimulation with thrombin and convulxin. To understand what impact reduced procoagulant platelet responses might have in vivo, we subjected mice with a platelet‐specific deletion of cyclophilin D, which are deficient in procoagulant platelet formation, to a model of pulmonary microvascular thrombosis. Mice with platelets lacking cyclophilin D died significantly faster from pulmonary microvascular thrombosis compared to littermate wild‐type controls. These results suggest dysregulated procoagulant platelet responses may contribute to thrombotic complications during SARS‐CoV‐2 infection.

Investigations into the nature of platelet functional variety and consequences for homeostasis require new methods for resolving single platelet phenotypes. Here we combine droplet microfluidics with flow cytometry for high throughput single platelet function analysis. A large-scale sensitivity continuum was shown to be a general feature of human platelets from individual donors, with hypersensitive platelets coordinating significant sensitivity gains in bulk platelet populations and shown to direct aggregation in droplet-confined minimal platelet systems. Sensitivity gains scaled with agonist potency (convulxin > TRAP-14>ADP) and reduced the collagen and thrombin activation threshold required for platelet population polarization into pro-aggregatory and pro-coagulant states. The heterotypic platelet response results from an intrinsic behavioural program. The method and findings invite future discoveries into the nature of hypersensitive platelets and how community effects produce population level responses in health and disease., Maaike S. A. Jongen et al. combine droplet microfluidics with flow cytometry to resolve single platelet responses to agonists. They demonstrate that hyperactive platelets enhance the platelet population response by paracrine signaling as a function of agonist potency and heterotypic responses result from an intrinsic behavioural program.

Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.

Background: Platelet transfusion is indicated for haemorrhage due to severe thrombocytopenia and for trauma associated coagulopathy. Febrile non-haemolytic transfusion reactions are a common complication of platelet transfusions in people and may be due to accumulated inflammatory cytokines. The present study aimed to determine the cytokine profile of a novel canine lyophilised platelet product following reconstitution, to assess the lyophilised platelets' activation response to physiological platelet agonists and to compare the cytokine profiles of basal and stimulated canine lyophilised platelets.Methods: Cell counts and biochemical analyses were conducted following reconstitution. Cytokine concentrations were measured with a canine-specific multiplex immunocapture assay and with an electrochemiluminescent ELISA. Aliquots of reconstituted product from three separate vials were activated for 10 minutes under non-stirred conditions using adenosine diphosphate, thrombin or convulxin and their cytokine concentrations compared with unactivated samples. Flow cytometry and light-transmission aggregometry were used to evaluate the product's ability to express a procoagulant surface, degranulate and aggregate. Fresh platelet-rich plasma was used as a positive control.Results: The product had a mean±SD particle count of 1.23±0.2×109/ml, contained platelets that expressed surface phosphatidylserine before agonist stimulation and was capable of aggregation in response to thrombin stimulation suggesting that the product may have haemostatic potential following in vivo administration. Cytokine concentrations measured by the immunocapture assay were generally low, while twofold to threefold increases relative to published intervals were noted for several cytokines using the ELISA. Concentrations of chemokine (C-X-C) motif ligand 8 and tumour necrosis factor-α were significantly increased as measured by the ELISA, but not by the immunocapture assay, while concentrations of KC-like were significantly increased as measured by the immunocapture assay. Stimulation with platelet agonists did not affect measured cytokine concentrations.Conclusion: Further study of the effects of administration of this lyophilised platelet product is warranted.

Dyslipidemia is a risk factor for clinically significant thrombotic events. In this condition, scavenger receptor CD36 potentiates platelet reactivity through recognition of circulating oxidized lipids. CD36 promotes thrombosis by activating redox-sensitive signaling molecules, such as the MAPK extracellular signal-regulated kinase 5 (ERK5). However, the events downstream of platelet ERK5 are not clear. In this study, we report that oxidized low-density lipoprotein (oxLDL) promotes exposure of procoagulant phosphatidylserine (PSer) on platelet surfaces. Studies using pharmacologic inhibitors indicate that oxLDL-CD36 interaction–induced PSer exposure requires apoptotic caspases in addition to the downstream CD36-signaling molecules Src kinases, hydrogen peroxide, and ERK5. Caspases promote PSer exposure and, subsequently, recruitment of the prothrombinase complex, resulting in the generation of fibrin from the activation of thrombin. Caspase activity was observed when platelets were stimulated with oxLDL. This was prevented by inhibiting CD36 and ERK5. Furthermore, oxLDL potentiates convulxin/glycoprotein VI–mediated fibrin formation by platelets, which was prevented when CD36, ERK5, and caspases were inhibited. Using 2 in vivo arterial thrombosis models in apoE-null hyperlipidemic mice demonstrated enhanced arterial fibrin accumulation upon vessel injury. Importantly, absence of ERK5 in platelets or mice lacking CD36 displayed decreased fibrin accumulation in high-fat diet–fed conditions comparable to that seen in chow diet–fed animals. These findings suggest that platelet signaling through CD36 and ERK5 induces a procoagulant phenotype in the hyperlipidemic environment by enhancing caspase-mediated PSer exposure.

BACKGROUND Collagen- and thrombin-activated (COAT) platelets (PLTs), generated by dual-agonist stimulation with collagen and thrombin (THR), enhance THR generation at the site of vessel wall injury. There is evidence that higher amounts of procoagulant COAT PLTs are associated with stroke, while a decreased ability to generate them is associated with bleeding diathesis. Our aim was to study PLT functions, particularly the ability to generate COAT PLTs, in PLT concentrates (PCs) from buffy coat. Thus, we investigated the effect of processing, pathogen inactivation treatment (amotosalen-UVA), and PC storage. STUDY DESIGN AND METHODS Two PCs from five donors each were pooled and split in two bags; one of them was pathogen inactivated and the other one was left untreated (n = 5). Flow cytometric analyses were performed immediately after PC preparation (Day 1) and thereafter on Days 2, 5, 7, and 9 in treated and untreated PCs to measure the reactivity of PLTs (CD62P and PAC-1), the content and secretion of dense granule after stimulation with different agonists, and the percentage of COAT PLTs after dual stimulation with convulxin (agonist of the collagen receptor GPVI) and THR. RESULTS Preparation of PCs resulted in a significant decrease of COAT PLTs and in an impaired response to adenosine 5'-diphosphate sodium (ADP). Storage further decreased ADP response. Minor differences were observed between untreated or amotosalen-UVA-treated PCs. CONCLUSION Preparation of PCs from buffy coats decreased the ability to generate COAT PLTs and impaired PLT response to ADP.

A 3-year-old, female Greater Swiss Mountain dog developed a hemoperitoneum following an exploratory laparotomy and ovariohysterectomy. Platelet count, PT, APTT, and plasma von Willebrand factor antigen concentration were within RIs. A buccal mucosal bleeding time (BMBT) was prolonged. Given the probability of a hereditary thrombopathia, the dog was administered desmopressin, fresh platelet transfusions, and aminocaproic acid to control hemorrhage. Subsequently, DNA testing for the P2Y12 receptor gene mutation identified the dog as being a heterozygote (carrier). Further platelet function testing was performed following complete recovery. Results of a repeat BMBT and a point-of-care screening test using the Platelet Function Analyzer-100 (collagen/adenosine-diphosphate [ADP] test cartridge) were within RIs. Flow cytometric studies demonstrated a marked reduction in fibrinogen binding to the dog's platelets in response to ADP - adenosine diphosphate activation. Likewise, turbidimetric aggregometry revealed a complete absence of platelet aggregation in response to ADP. However, there were a normal aggregation response to the platelet agonist convulxin and a mild reduction in amplitude in response to γ-thrombin. This is the first report of a dog heterozygous for the P2Y12 receptor gene mutation exhibiting a bleeding tendency and having evidence of impaired platelet function in vitro in response to ADP activation. Given that the mutant allele for the P2Y12 thrombopathia appears to be widespread in the Greater Swiss Mountain dog breed, veterinarians need to be aware that both homozygotes and heterozygotes for this platelet receptor mutation are at risk of developing life-threatening bleeding following trauma or surgery.

Platelets present a number of intracellular and transmembrane targets subject to pharmacological modulation, either for cardiovascular disease reduction or as an unintended drug response. Microfluidic devices allow human blood to clot on a defined surface under controlled hemodynamic and pharmacological conditions. The potencies of a number of antiplatelet and anti-cancer drugs have been tested with respect to platelet deposition on collagen under flow. Inhibitors of cyclooxygenase-1 (COX-1) reduce platelet deposition, either when added ex vivo to blood or ingested orally by patients prior to testing. Some individuals display a functional “aspirin-insensitivity” in microfluidic assay. When certain nonsteroidal anti-inflammatory drugs (NSAIDs) are taken orally, they block COX-1 acetylation by aspirin with concomitant reduction of aspirin efficacy against platelets in microfluidic assay. Both P2Y1 and P2Y12 inhibitors reduce platelet deposition under flow, as do NO donors and iloprost that target the guanylate cyclase and the prostacyclin receptor, respectively. In a microfluidic assay of 37 kinase inhibitors, dasatinib had potent antiplatelet activity, while bosutinib was less potent. Dasatinib and bosutinib have known profiles against numerous kinases, revealing overlapping and non-overlapping activities relevant to their unique actions against platelets. Also, dasatinib caused a marked and specific inhibition of GPVI signaling induced by convulxin, consistent with a dasatinib-associated bleeding risk. Microfluidic devices facilitate drug library screening, dose-response testing, and drug-drug interaction studies. Kinase inhibitors developed as anticancer agents may present anti-platelet activities that are detectable by microfluidic assay and potentially linked to bleeding risks.

The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIb&alpha, agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase &gamma, 2 (PLC &gamma, 2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca2+. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets.

Platelets primarily participate in hemostasis and antimicrobial host defense. The present study aimed to investigate the effects of leucine-rich repeat flightless-interacting protein-1 (LRRFIP1) on platelet agglutination. The bacterial strain of LRRFIP1 was used to synthesize the recombinant protein and a mouse model of LRRFIP1 gene knockout was established. Platelets were isolated from the mice and divided into the different trial groups according to their treatment with collagen, thrombin receptor SFLLRN, anti-wild-type (w)LRRFIP1monoclonal antibodies and the model of LRRFIP1 gene knockout. The platelets were prepared and platelet agglutination was examined using platelet aggregation apparatus. The active αIIbβ3 integrin was examined by flow cytometry. The results revealed that the combined wLRRFIP1 protein was successfully expressed. wLRRFIP1 treatment significantly triggered platelet agglutination of collagen, thrombin and monoclonal antibody treated platelets. wLRRFIP1 knockout significantly decreased αIIbβ3 levels compared with the wild-type. Platelet agglutination was also significantly inhibited in the LRRFIP1−/−mouse model compared with the wild-type. LRRFIP1 knockout significantly decreased the αIIbβ3 levels in platelets undergoing convulxin treatment. In conclusion, LRRFIP1 treatment triggered platelet agglutination and LRRFIP1 gene knockout inhibited platelet agglutination. In addition, LRRFIP1 gene knockout significantly decreased the levels of αIIbβ3. This suggests that LRRFIP1 my be applied to patients in a clinical setting to trigger platelet agglutination in inflammatory diseases and atherothrombotic diseases.

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients’ megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates collagen-mediated platelet activation through its cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). However, the function of CEACAM1’s extracellular cleavage fragments is currently unknown. In the present study, we used mass spectrometry (MS) to identify 9 cleavage fragments shed by matrix metallopeptidase 12 (MMP-12), and then we synthesized peptides with sequences corresponding to the fragments. QLSNGNRTLT (QLSN), a peptide from the A1-domain of CEACAM1, significantly attenuated collagen-induced platelet aggregation. QLSN also attenuated platelet static adhesion to collagen. Additionally, QLSN reduced human platelet secretion and integrin αIIbβ3 activation in response to glycoprotein VI (GPVI)–selective agonist, convulxin. Correspondingly, QLSN treatment significantly decreased convulxin-mediated phosphorylation of Src, protein kinase B (Akt), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) in human platelets. These data indicate that the CEACAM1-derived peptide QLSN inhibits GPVI-mediated human platelet activation. QLSN could potentially be developed as a novel antiplatelet agent. QLSN inhibit collagen-induced human platelet activation through glycoprotein VI.

Made available in DSpace on 2019-10-03T17:32:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2019. Added 1 bitstream(s) on 2019-10-04T16:21:10Z : No. of bitstreams: 1 S0037-86822019000100328.pdf: 1882340 bytes, checksum: 129de41fd9eb894154ad3863a7ce07db (MD5) AbstractINTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation.METHODS:Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated.RESULTS:The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP.CONCLUSIONS:Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms. Fundação Oswaldo Cruz Laboratório de Imunologia Celular Aplicada à Saúde Instituto Federal de Educação, Ciência e Tecnologia de Mato Grosso Campus São Vicente Centro de Referência de Jaciara Universidade Federal de Rondônia Departamento de Medicina Centro de Estudos de Biomoléculas Aplicadas à Saúde Centro Universitário São Lucas Universidade Estadual de São Paulo Instituto de Biociências Departamento de Física e Biofísica Universidade Estadual de São Paulo Instituto de Biociências Departamento de Farmacologia

Platelets are the smallest blood cells, and their activation (platelet cohesion or aggregation) at sites of vascular injury is essential for thrombus formation. Since the use of antiplatelet therapy is an unsolved problem, there are now focused and innovative efforts to develop novel antiplatelet compounds. In this context, we assessed the antiplatelet effect of an acylhydroquinone series, synthesized by Fries rearrangement under microwave irradiation, evaluating the effect of diverse acyl chain lengths, their chlorinated derivatives, and their dimethylated derivatives both in the aromatic ring and also the effect of the introduction of a bromine atom at the terminus of the acyl chain. Findings from a primary screening of cytotoxic activity on platelets by lactate dehydrogenase assay identified 19 non-toxic compounds from the 27 acylhydroquinones evaluated. A large number of them showed IC50 values less than 10 µM acting against specific pathways of platelet aggregation. The highest activity was obtained with compound 38, it exhibited sub-micromolar IC50 of 0.98 ± 0.40, 1.10 ± 0.26, 3.98 ± 0.46, 6.79 ± 3.02 and 42.01 ± 3.48 µM against convulxin-, collagen-, TRAP-6-, PMA- and arachidonic acid-induced platelet aggregation, respectively. It also inhibited P-selectin and granulophysin expression. We demonstrated that the antiplatelet mechanism of compound 38 was through a decrease in a central target in human platelet activation as in mitochondrial function, and this could modulate a lower response of platelets to activating agonists. The results of this study show that the chemical space around ortho-carbonyl hydroquinone moiety is a rich source of biologically active compounds, signaling that the acylhydroquinone scaffold has a promising role in antiplatelet drug research.

The development of small molecule inhibitors targeting GPVI has promising therapeutic role, as they inhibit arterial thrombosis with limited risk of bleeding. Among the compounds showing in vivo antithrombotic activity, the most active compound 6b (ED50 = 28.36 μmol/kg po in mice) showed improved inhibition for collagen (IC50 = 6.7 μM), CRP-XL (IC50 = 53.5 μM), and convulxin (CVX) (IC50 = 5.7 μM) mediated platelet aggregation as compared to losartan (LOS) (collagen, IC50 = 10.4 μM; CRP-XL, IC50 = 158 μM; CVX, IC50 = 11 μM) than any of its enantiomers S (6c) (collagen, IC50 = 25.3 μM; CRP-XL, IC50 = 181.4 μM; CVX, IC50 = 9 μM) and R (6d) (collagen, IC50 = 126.3 μM; CRP-XL, IC50 > 500 μM; CVX, IC50 = 86.8 μM). Compound 6b also inhibited platelet P-selectin expression and thus may diminish atherosclerosis. The molecular interactions of both enantiomers 6c and 6d at the GPVI receptor have been explained through docking studies.

SummaryPyk2 is a Ca2+-regulated kinase predominantly expressed in neuronal and in haematopoietic cells. Previous studies on Pyk2-null mice have demonstrated that Pyk2 plays a crucial role in platelet activation and thrombus formation, thus representing a possible target for antithrombotic therapy. Very limited information is available about the role of Pyk2 in human platelets, mainly because of the lack of specific pharmacological inhibitors. In this work, we have tested two novel Pyk2 inhibitors, PF-4594755 and PF-4520440, to validate their specificity and to investigate their ability to modulate platelet activation. Both molecules were able to efficiently block Pyk2 activity in human and mouse platelets stimulated with thrombin or with the Ca2+-ionophore. In wild-type murine platelets, PF-4594755 and PF-4520440 reduced thrombin-induced aggregation to the level observed in Pyk2 knockout platelets, but did not affect aggregation induced by GPVI stimulation. Importantly, neither compounds affected the residual thrombin-induced aggregation of Pyk2-null platelets, thus excluding possible off-target effects. In human platelets, PF-4594755 and PF-4520440 significantly reduced aggregation stimulated by thrombin, but not by the GPVI agonist convulxin. Both inhibitors reduced platelet adhesion on fibrinogen and prevented Akt phosphorylation in adherent cells, indicating that Pyk2 regulates PI3K and cell spreading downstream of integrins in human platelets. Finally, the Pyk2 inhibitors significantly inhibited thrombus formation upon blood perfusion on immobilized collagen under arterial flow rate. These results demonstrate that PF-4594755 and PF-4520440 are specific inhibitors of Pyk2 in intact platelets and allowed to reliably document that this kinase plays a relevant role in human platelet activation.Supplementary Material to this article is available online at www.thrombosis-online.com.

Objective— Activation of platelets by subendothelial collagen results in an increase of cytosolic Ca 2+ concentration ([Ca 2+ ] i ) and is followed by platelet activation and thrombus formation that may lead to vascular occlusion. The present study determined the role of phosphoinositide-dependent protein kinase 1 (PDK1) in collagen-dependent platelet Ca 2+ signaling and ischemic stroke in vivo. Approach and Results— Platelet activation with collagen receptor glycoprotein VI agonists collagen-related peptide or convulxin resulted in a significant increase in PDK1 activity independent of second-wave signaling. PDK1 deficiency was associated with reduced platelet phospholipase Cγ2–dependent inositol-1,4,5-trisphosphate production and intracellular [Ca 2+ ] i in response to stimulation with collagen-related peptide or convulxin. The defective increase of [Ca 2+ ] i resulted in a substantial defect in activation-dependent platelet secretion and aggregation on collagen-related peptide stimulation. Furthermore, Rac1 activation and spreading, adhesion to collagen, and thrombus formation under high arterial shear rates were significantly diminished in PDK1-deficient platelets. Mice with PDK1-deficient platelets were protected against arterial thrombotic occlusion after FeCl 3 -induced mesenteric arterioles injury and ischemic stroke in vivo. These mice had significantly reduced brain infarct volumes, with a significantly increased survival of 7 days after transient middle cerebral artery occlusion without increase of intracerebral hemorrhage. Tail bleeding time was prolonged in pdk1 −/− mice, reflecting an important role of PDK1 in primary hemostasis. Conclusions— PDK1 is required for Ca 2+ -dependent platelet activation on stimulation of collagen receptor glycoprotein VI, arterial thrombotic occlusion, and ischemic stroke in vivo.

The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure.

BACKGROUND: Platelet (PLT) concentrates are prophylactically given to prevent major bleeding complications. The corrected count increment (CCI) is currently the only tool to monitor PLT transfusion efficacy. PLT function tests cannot be performed in patients with thrombocytopenia. Therefore, an optimized agonist-induced assay was used to determine PLT function, in patients with severe thrombocytopenia before and after transfusion. STUDY DESIGN AND METHODS: PLT reactivity toward adenosine diphosphate (ADP), thrombin receptor-activating peptide SFLLRN (TRAP), and convulxin (CVX) was assessed by flow cytometry. P-selectin expression was measured on PLTs from 11 patients with thrombocytopenia before and 1 hour after transfusion, on stored PLTs, and on stored PLTs incubated for 1 hour in whole blood from patients ex vivo. RESULTS: The mean (±SEM) CCI after 1 hour was 11.4 (±1.5). After transfusion, maximal agonist-induced PLT P-selectin expression was on average 29% higher for ADP (p = 0.02), 25% higher for TRAP (p = 0.007), and 24% higher for CVX (p = 0.0008). ADP-induced reactivity of stored PLTs increased with 46% after ex vivo incubation (p = 0.007). These PLTs also showed an overall higher P-selectin expression compared to PLTs 1 hour after transfusion (p = 0.005). After normalization for this background expression, a similar responsiveness was observed. CONCLUSIONS: Our study shows recovery of PLT function after transfusion in patients with thrombocytopenia. The majority of functional PLTs measured after transfusion most likely represents stored transfused PLTs that regained functionality in vivo. The difference in baseline P-selectin expression in vivo versus ex vivo suggests a rapid clearance from circulation of PLTs with increased P-selectin expression.