Enhanced Platelet Adhesion Research Articles

Influence of Low Temperature Plasma Oxidizing on the Bioactivity of NiTi Shape Memory Alloy for Medical Applications.

The present study elucidates the impact of glow discharge oxidation within a low-temperature plasma environment on the bioactivity characteristics of an NiTi shape memory alloy. The properties of the produced surface layers, such as structure (TEM observations), surface morphology (SEM observations), chemical and phase composition (EDS and XRD measurements), wettability (optical gonimeter), and the biological response of osteoblasts and platelets to the oxidized surface compared with the NiTi alloy without a surface layer are presented. The presented surface modification of the NiTi shape memory alloy, achieved through oxidizing in a low-temperature plasma environment, led to the creation of a continuous surface layer composed of nanocrystalline titanium oxide TiO2 (rutile). The findings obtained from this study provide evidence that the oxidized layer augments the bioactivity of the shape memory alloy. This augmentation was substantiated through the spontaneous biomimetic deposition of apatite from a simulated body fluid (SBF) solution. Furthermore, the modified surface exhibited improved osteoblast proliferation, and enhanced platelet adhesion and activation. This proposed surface modification strategy holds promise as a prospective solution to enhance the biocompatibility and bioactivity of NiTi shape memory alloy intended for prolonged use in bone implant applications.

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

Proteoglycans form a heterogeneous family of proteins with covalently bound sulfated glycosaminoglycans. The extracellular matrix proteoglycan perlecan has been proposed to bind to the platelet- and megakaryocyte-specific receptor G6bB, co-regulating platelet glycoprotein VI (GPVI) signaling. The derived non-sulfate proteoglycan endorepellin was previously shown to enhance platelet adhesion via the collagen receptor, integrin α2β1. Here, we compared the roles of perlecan and other matrix proteoglycans in platelet responses and thrombus formation. We used multi-color flow cytometry to measure the degranulation and integrin αIIbβ3 activation of washed platelets in response to various proteoglycans and collagen-related peptide (CRP), the GPVI agonist. Perlecan, but not endorepellin, enhanced the CRP-induced activation of platelets in a time- and concentration-dependent manner. Similar to collagen, immobilized perlecan, but not other proteoglycans, supported static platelet adhesion and spreading. In-flowed whole-blood perlecan diminished shear-dependent platelet adhesion, while it enforced GPVI-dependent thrombus formation-to a larger extent than endorepellin-to induce more contracted aggregates of activated platelets. We concluded that the sulfated proteoglycan perlecan enhances GPVI-dependent platelet responses extending to thrombus formation, but it does so at the expense of reduced adhesion of platelets under flow.

Desmopressin as a hemostatic and blood sparing agent in bleeding disorders.

Intranasal, subcutaneous, or intravenous desmopressin can be utilized to release von Willebrand Factor and Factor VIII into circulation, enhance platelet adhesion and shorten bleeding time. Due to these properties, desmopressin can be effective in controlling bleeding in mild hemophilia A, certain subtypes of von Willebrand disease and in acute bleeding from uremia, end stage renal disease, and liver disease. Its use, however, can be complicated by hyponatremia and rarely arterial thrombotic events. While desmopressin has also been used as a prophylactic blood sparing agent in orthopedic, renal, and hepatic procedures, clinical studies have shown limited benefit in these settings. The purpose of this article is to review the evidence for desmopressin in primary hematologic disorders, discuss its mechanism of action and evaluate its utility as a hemostatic and blood sparing product in various bleeding conditions.

Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction.

Endothelial dysfunction is a critical component of both atherosclerotic plaque formation and saphenous vein graft failure. Crosstalk between the pro-inflammatory TNF-α-NFκB signaling axis and the canonical Wnt/β-catenin signaling pathway potentially plays an important role in regulating endothelial dysfunction, though the exact nature of this is not defined. In this study, cultured endothelial cells were challenged with TNF-α and the potential of a Wnt/β-catenin signaling inhibitor, iCRT-14, in reversing the adverse effects of TNF-α on endothelial physiology was evaluated. Treatment with iCRT-14 lowered nuclear and total NFκB protein levels, as well as expression of NFκB target genes, IL-8 and MCP-1. Inhibition of β-catenin activity with iCRT-14 suppressed TNF-α-induced monocyte adhesion and decreased VCAM-1 protein levels. Treatment with iCRT-14 also restored endothelial barrier function and increased levels of ZO-1 and focal adhesion-associated phospho-paxillin (Tyr118). Interestingly, inhibition of β-catenin with iCRT-14 enhanced platelet adhesion in cultured TNF-α-stimulated endothelial cells and in an ex vivo human saphenous vein model, most likely via elevating levels of membrane-tethered vWF. Wound healing was moderately retarded by iCRT-14; hence, inhibition of Wnt/β-catenin signaling may interfere with re-endothelialisation in grafted saphenous vein conduits. Inhibition of the Wnt/β-catenin signaling pathway with iCRT-14 significantly recovered normal endothelial function by decreasing inflammatory cytokine production, monocyte adhesion and endothelial permeability. However, treatment of cultured endothelial cells with iCRT-14 also exerted a pro-coagulatory and moderate anti-wound healing effect: these factors may affect the suitability of Wnt/β-catenin inhibition as a therapy for atherosclerosis and vein graft failure.

Effect of Epitope Specific Antibodies on Single Platelet Physiology with Implications for Immune Thrombocytopenia Purpura

Background: Platelets play a vital role in both hemostasis and thrombosis and dysfunction thereof may lead to uncontrolled bleeding and clotting. Consequently, patients with Immune Thrombocytopenia Purpura (ITP) exhibit extremely low platelet counts caused by enhanced platelet clearance and destruction due to platelet reactive antibodies. However, even though all patients have thrombocytopenia, only 20 percent of ITP patients develop major bleeding episodes, which cannot be reliably predicted by platelet count alone. While platelet auto-antibodies have been investigated previously, whether epitope-specific antibodies directly affect platelet function remains poorly understood. To that end, we explored the possible physiological impacts of antibodies on single platelet adhesion, spreading, morphology and activation. Because 70% of platelet reactive antibodies in ITP are directed toward the integrin GPIIb/IIIa, we leveraged well-characterized monoclonal antibodies toward GPIIb/IIIa to better understand their physiological effects of platelet-fibrinogen interactions via the assays described above. Overall, we found that antibodies toward GPIIb/IIIa alter the functionality of individual platelets on fibrinogen surfaces in an epitope dependent manner. Most notably, antibodies that bound to either the head or tail region of αIIb (MBC 290.5 and MBC 314.5) increased the percent of platelets expressing phosphatidylserine, when compared to the control and antibodies binding to the head or tail region of βIIIa (AP3, AP5, and Libs 2). However, the mean intensity of this expression was on average much weaker than the βIIIa antibodies. This suggesting differing functional consequences of platelets to various epitopes and in turn could help explain the differing effects antibodies could have in ITP. Methods: Healthy donor platelets were diluted to 10 million/mL in Tyrode's modified HEPES buffer to ensure that single platelets were being measured and to reduce the number of platelet aggregates. These platelets were then incubated and adhered on 100 µg/mL human fibrinogen-coated coverslips for 2 hours in the presence of an antibody toward a selected epitope (Figure 1A). Adhered platelets were then stained with a cell membrane stain and Annexin V (PS exposure), fixed and then imaged with fluorescence microscopy. After imaging, thousands of platelets were then counted and analyzed. The antibody treated platelets were then normalized to the non-treated control. Results: Our preliminary data indicates an epitope-specific effect of antibodies on platelet physiology at the single cell level. Using well-characterized antibodies to various epitopes of GPIIb/IIIa (Figure 1A), we found that when compared to the non-antibody treated control, MBC 290.5 and Libs 2 decreased platelet spreading area by 29% and 31% respectively. However, while MBC 290.5 did not alter platelet density (n/mm²) Libs 2 enhanced platelet adhesion by increasing platelet density by 85%. This indicates the possible decrease in functionality of platelets treated with MBC 290.5. Additionally, both MBC 290.5 and MBC 314.5 increased the percentage of platelets that exposed PS by 97% and 87%, respectively, while AP3 and Libs 2 decreased the percentage of PS-exposed platelets by 64% and 49% respectively. Interestingly, although there was a decrease in the percent PS-exposed platelets, the mean intensity of the platelets that were PS exposed was much greater than the control with an increase of 360% and 228% respectively (Figure 1B). Conclusion: Although auto-antibodies cause platelet clearance, leading to low platelet counts, little is understood about the possible ramifications of antibodies on platelet behavior. Importantly, we show here that antibodies have a physiological consequence on platelets and different epitope-specific antibodies exhibit unique signatures for altering single platelet physiology, which could help explain how patients with ITP have varying clinical presentations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Platelet-Activating Factor Promotes the Development of Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a multifaceted clinicopathological syndrome characterised by excessive hepatic lipid accumulation that causes steatosis, excluding alcoholic factors. Platelet-activating factor (PAF), a biologically active lipid transmitter, induces platelet activation upon binding to the PAF receptor. Recent studies have found that PAF is associated with gamma-glutamyl transferase, which is an indicator of liver disease. Moreover, PAF can stimulate hepatic lipid synthesis and cause hypertriglyceridaemia. Furthermore, the knockdown of the PAF receptor gene in the animal models of NAFLD helped reduce the inflammatory response, improve glucose homeostasis and delay the development of NAFLD. These findings suggest that PAF is associated with NAFLD development. According to reports, patients with NAFLD or animal models have marked platelet activation abnormalities, mainly manifested as enhanced platelet adhesion and aggregation and altered blood rheology. Pharmacological interventions were accompanied by remission of abnormal platelet activation and significant improvement in liver function and lipids in the animal model of NAFLD. These confirm that platelet activation may accompany a critical importance in NAFLD development and progression. However, how PAFs are involved in the NAFLD signalling pathway needs further investigation. In this paper, we review the relevant literature in recent years and discuss the role played by PAF in NAFLD development. It is important to elucidate the pathogenesis of NAFLD and to find effective interventions for treatment.

Therapeutic implementation in arterial thrombosis with pulmonary administration of fucoidan microparticles containing acetylsalicylic acid

Several antithrombotic drugs are available to treat cardiovascular diseases due to its high mortality and morbidity worldwide. Despite these, severe adverse effects that can lead to treatment withdrawal have been described, highlighting the importance of new therapies. Thus, this work describes the development of fucoidan microparticles containing acetylsalicylic acid (MP/F4M) for pulmonary delivery and in vitro, ex vivo, and in vivo evaluation. Microparticles were prepared via spray-drying and characterized in vitro (mucoadhesive properties, coagulation time, platelet aggregation, adhesion, and hemolysis) followed by ex vivo platelet aggregation, in vivo arterial thrombosis, and hemorrhagic profile. The formulation physicochemical characterization showed suitable characteristics along with delayed drug release, increased breathable particle fraction, and high washability resistance as well as antiplatelet activity and enhanced platelet adhesion in vitro. In in vivo assays, MP/F4M protected against arterial thrombosis, without changes in the hemorrhagic profile. Finally, no lung changes were observed after prolonged pulmonary administration, whereas isolated ASA led to an inflammatory response. In conclusion, pulmonary administration of fucoidan microparticles with an antiplatelet drug may be an alternative therapy to treat cardiovascular diseases, opening the field for different formulations.

The role of thrombin in haemostasis.

Thrombin is a multifunctional serine protease generated in injured cells. The generation of thrombin in coagulation plays a central role in the functioning of haemostasis. The last enzyme in the coagulation cascade is thrombin, with the function of cleaving fibrinogen to fibrin, which forms the fibrin clot of a haemostatic plug. Although thrombin primarily converts fibrinogen to fibrin, it also has many other positive regulatory effects on coagulation. Thrombin has procoagulant, inflammatory, cellular proliferation and anticoagulant effects. In coagulation system, thrombin has two very distinct roles. Firstly, it acts as a procoagulant when it converts fibrinogen into an insoluble fibrin clot, activates factor (F) XIII, activates thrombin activatable fibrinolysis inhibitor (TAFI) and activates FV, FVIII and FXI. Thrombin also enhances platelet adhesion by inactivating a disintegrin and metalloprotease with thrombospondin type1 motif (ADAMTS13). However, when thrombin activates protein C, it acts as an anticoagulant. A natural anticoagulant pathway that supplies regulation of the blood coagulation system contains protein C, which is the key component. This is accomplished by the specific proteolytic inactivation of FV and FVIII. In this review, the multiple roles of thrombin in the haemostatic response to injury are studied in addition to the cofactors that determine thrombin activity and how thrombin activity is thought to be coordinated.

Multimerin-1 Binds to Specific Motifs in Vessel Wall Collagens and Contributes to Thrombosis: Novel Insights Regarding the Mechanisms That Support Human and Mouse Platelet Adhesion

Multimerin-1 (MMRN1) is a homopolymeric glycoprotein stored in platelets and vascular endothelial cells for regulated release that enhances platelet adhesion to collagen. We tested Mmrn1 contributions to platelet adhesion ex vivo and in vivo using selective deficient mice and triple-helical collagen mimetic peptides to evaluate a putative Mmrn1-selective binding motif. Selective Mmrn1-deficient (Mmrn1-/-) mice were generated by crossing Mmrn1+/- “knockout-first allele” (Mmrn1flxneo/+ from European Mutant Mouse Archive) with flp deleter mice, and then with transgenic global cre deleter mice. The resultant heterozygous Mmrn1+/- mice were crossed with wild type (Mmrn1+/+) mice to remove the cre and flp transgenes. Inbred, gender-mixed, age-matched Mmrn1+/+ and Mmrn1-/- mice were then used for in vitro and in vivo assessments of platelet function. The following highly conserved GPAGPOGPX sequences in fibrillar collagens (that are distinct from collagen motifs that bind von Willebrand factor [VWF], platelet integrin alpha2beta1 or platelet GPVI) were identified to bind MMRN1: GPAGPOGPI (type I), GPAGPOGPV (type II), and GPAGPOGPQ (type III). In static adhesion assays with washed human platelets, triple-helical GPAGPOGPX peptides enhanced platelet adhesion when co-presented with the alpha2beta1-binding peptide GFOGER, like the VWF-binding peptide derived from type III collagen which was tested as a positive control. Static platelet adhesion to GPAGPOGPX peptides was also enhanced by agonists that induce MMRN1 release (p<0.02 for each comparison, where family-wise α=0.05, k=7), and co-presentation of GPAGPOGPX peptides with both GFOGER and III-23 maximized platelet adhesion (p<0.02 for each comparison, where family-wise α=0.05, k=5), providing insights on how MMRN1 and VWF synergistically enhance platelet adhesion. In high shear (1500 s-1) flow assays of platelet adhesion, platelet adhesion to triple-helical GPAGPOGPX peptides that were co-presented with GFOGER was greater than adhesion to GFOGER alone (p=0.015). Mmrn1-/- mice were viable and fertile, with normal blood cell counts and bleeding times (respective medians [ranges] for Mmrn1+/+ versus Mmrn1-/- mice: 300 [60-900] seconds vs. 330 [90-900] seconds, p=0.07), and confirmed Mmrn1 deficiency by Western blot analyses of platelets. In assays of platelet adhesion at high shear (1500 s-1), platelets from Mmrn1-/- mice showed a significant impairment in adhesion to full-length fibrillar collagen (% surface area covered by platelets ± SEM for Mmrn1+/+ versus Mmrn1-/- mice: 37 ± 3% vs. 18 ± 2%, p<0.001) and in collagen-induced aggregation in assays with washed platelets (20-30% reduction in maximal aggregation for Mmrn1-/- platelets compared to Mmrn1+/+ platelets, p<0.05). In assays of washed platelet adhesion, Mmrn1-/- platelets showed minimal static adhesionto the GPAGPOGPX peptide, unlike Mmrn1+/+ platelets(p<0.0001). Assays of platelet adhesion under conditions of flow indicated that co-presentation of GPAGPOGPX with GFOGER enhanced the adhesion of activated Mmrn1+/+ but not Mmrn1-/- platelets. Intravital microscopyexperiments indicated that Mmrn1-/- mice also had impaired platelet adhesion in arterioles treated with ferric chloride (platelets deposited per minute ± SEM for Mmrn1+/+ versus Mmrn1-/- mice: Mmrn1+/+ : 82 ± 4 vs. Mmrn1-/-: 42 ± 6, p<0.001), and the thrombi that formed in Mmrn1-deficient mice were smaller and less stable, with delayed vessel occlusion times (respective medians [ranges] for Mmrn1+/+ versus Mmrn1-/- mice: 17 [10-35] min vs. 35 [22-40] min, p<0.001). Taken together, our studies illustrate that multimerin-1 has functions in supporting and enhancing platelet adhesive function, in vivo and in vitro, and that fibrillar collagens contain highly conserved GPAGPOGPX motifs that bind Mmrn1 and contribute to platelet adhesion. DisclosuresNo relevant conflicts of interest to declare.

Estrogen activates endothelial exocytosis

Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17β-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERβ agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.

Multiparameter microfluidics assay of thrombus formation reveals increased sensitivity to contraction and antiplatelet agents at physiological temperature

IntroductionCurrent developments to assess qualitative and quantitative platelet traits in flowed whole-blood are based on microfluidic devices that mostly operate at room temperature. However, operation at physiological temperature (37 °C) may increase the assay's sensitivity, and facilitates the comparison to other platelet function tests of the diagnostic laboratory. Materials and methodsWe adapted the conventional microspot-based microfluidic device with a simple thermo-coupled pre-heating module. Automated analysis of microscopic images assisted in obtaining five time-dependent parameters of thrombus formation over collagen microspots (shear rate 1000 s−1). These modifications allowed rapid testing of control and patient blood samples at physiological temperature. Results and conclusionThe higher temperature enhanced platelet adhesion and aggregation as well as late thrombus characteristics such as size and contraction, when compared to room temperature. Moreover, assessment at 37 °C indicated a time-dependent impairment of the thrombus parameters in blood from patients taking common antiplatelet medication, i.e. aspirin and/or clopidogrel. This pointed to increased contribution of the autocrine platelet agonists thromboxane A2 and ADP in the buildup of contracted thrombi under flow. Overall, this study underlined the advantage of multiparameter assessment of microfluidic thrombus formation in detecting an acquired platelet dysfunction, when operating at physiological temperature. This work may bring microfluidics tests closer to the diagnostic laboratory.

Multimerin 1 supports platelet function in vivo and binds to specific GPAGPOGPX motifs in fibrillar collagens that enhance platelet adhesion

BackgroundMultimerin 1 (human: MMRN1, mouse: Mmrn1) is a homopolymeric, adhesive, platelet and endothelial protein that binds to von Willebrand factor and enhances platelet adhesion to fibrillar collagen ex vivo. ObjectivesTo examine the impact of Mmrn1 deficiency on platelet adhesive function, and the molecular motifs in fibrillar collagen that bind MMRN1 to enhance platelet adhesion. MethodsMmrn1‐deficient mice were generated and assessed for altered platelet adhesive function. Collagen Toolkit peptides, and other triple‐helical collagen peptides, were used to identify multimerin 1 binding motifs and their contribution to platelet adhesion. ResultsMMRN1 bound to conserved GPAGPOGPX sequences in collagens I, II, and III (including GPAGPOGPI, GPAGPOGPV, and GPAGPOGPQ) that enhanced activated human platelet adhesion to collagen synergistically with other triple‐helical collagen peptides (P < .05). Mmrn1−/− and Mmrn1+/− mice were viable and fertile, with complete and partial platelet Mmrn1 deficiency, respectively. Relative to wild‐type mice, Mmrn1−/− and Mmrn1+/− mice did not have overt bleeding, increased median bleeding times, or increased wound blood loss (P ≥ .07); however, they both showed significantly impaired platelet adhesion and thrombus formation in the ferric chloride injury model (P ≤ .0003). Mmrn1−/− platelets had impaired adhesion to GPAGPOGPX peptides and fibrillar collagen (P ≤ .03) and formed smaller aggregates than wild‐type platelets when captured onto collagen, triple‐helical collagen mimetic peptides, von Willebrand factor, or fibrinogen (P ≤ .008), despite preserved, low shear, and high shear aggregation responses. ConclusionsMultimerin 1 supports platelet adhesion and thrombus formation and binds to highly conserved, GPAGPOGPX motifs in fibrillar collagens that synergistically enhance platelet adhesion.

Desialylation of platelet surface glycans enhances platelet adhesion to adsorbent polymers for lipoprotein apheresis.

Background:Lipoprotein apheresis is an important therapeutic option in homozygous familial hypercholesterolemia, progressive atherosclerosis, or when depletion of lipoprotein(a) is indicated. It is generally regarded as safe, but drops in platelet counts as well as sporadic episodes of thrombocytopenia have been reported. We assessed the influence of platelet desialylation, which may be induced by endogenous or pathogen-derived neuraminidases, on platelet adhesion to polyacrylate-based adsorbents for whole blood lipoprotein apheresis.Methods:Medical grade platelet concentrates were incubated with neuraminidase in vitro and were circulated over adsorbent columns downscaled from clinical application.Results:Cleavage of terminal sialic residues resulted in platelet activation with significantly elevated expression of platelet factor 4 (PF4) and in enhanced platelet adhesion to the adsorbent, accompanied by a pronounced drop in platelet counts in the column flow-through.Conclusion:Alterations in endogenous neuraminidase activity or exogenous (pathogen-derived) neuraminidase may trigger enhanced platelet adhesion in whole blood lipoprotein apheresis.

The involvement of toll-like receptors 2 and 4 in human platelet signalling pathways

In addition to haemostasis, platelets play an essential role in mechanisms of inflammation and in immunological reactions. Platelets express various toll-like receptors (TLR) on their surface, among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. This study compared TLR2- and TLR4-dependent platelet signalling and their effect on platelet function.Platelet-rich-plasma and washed platelets were prepared from peripheral blood samples of healthy donors. Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli) were used for stimulation of TLR2 and TLR4. Intracellular signalling pathways were investigated by Western blot. TLR2- and TLR4-mediated specific transcription factor DNA binding activity was measured by the nuclear factor kappa B (NFκB) transcription factor assay kit. Platelet adhesion and glycoprotein Ib function were assessed by immunofluorescence staining and analysis of ristocetin-induced agglutination.Both, Pam3CSK4 and LPS were able to induce NFκB-mediated and classical activating platelet signalling with a higher stimulatory capacity of TLR2. In addition, TLR2 and TLR4 activation led to a similar activation of inhibitory pathways. In contrast to TLR2, stimulation of TLR4 resulted in decreased Akt/protein kinase B phosphorylation conditioned by enhanced protein phosphatase 2A activity. TLR4-mediated signalling induced platelet adhesion and facilitated ristocetin-induced platelet agglutination.In conclusion, Pam3CSK4 directly induces aggregation via classical activation cascades, whereas LPS enhances platelet adhesion and glycoprotein receptor Ib-dependent platelet agglutination.

Recapitulating atherogenic flow disturbances and vascular inflammation in a perfusable 3D stenosis model

Blood vessel narrowing and arterial occlusion are pathological hallmarks of atherosclerosis, which involves a complex interplay of perturbed hemodynamics, endothelial dysfunction and inflammatory cascade. Herein, we report a novel circular microfluidic stenosis model that recapitulates atherogenic flow-mediated endothelial dysfunction and blood-endothelial cell (EC) interactions in vitro. 2D and 3D stenosis microchannels with different constriction geometries were fabricated using 3D printing to study flow disturbances under varying severity of occlusion and wall shear stresses (100 to 2000 dynecm−2). Experimental and fluid simulation results confirmed the presence of pathological shear stresses in the stenosis region, and recirculation flow post stenosis. The resultant pathological flow profile induced pro-inflammatory and pro-thrombotic EC state as demonstrated by orthogonal EC alignment, enhanced platelet adhesion at the stenosis, and aberrant leukocyte-EC interactions post stenosis. Clinical utility of the vascular model was further investigated by testing anti-thrombotic and immunomodulatory efficacy of aspirin and metformin, respectively. Overall, the platform enables multi-factorial analysis of critical atherogenic events including endothelial dysfunction, platelets and leukocyte adhesion, and can be further developed into a liquid biopsy tool for cardiovascular risk stratification.

In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion

Essentials Platelet adhesion to von Willebrand factor (VWF) is critical for hemostasis and thrombosis. Whether VWF can undergo phosphorylation is unknown. Family with sequence similarity 20 kinase phosphorylates VWF A2 domain at S1517 and S1613. Phosphorylation of VWF and VWF A1A2A3 domain at S1613 enhances platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) mediates platelet adhesion and contributes to hemostasis at sites of vascular injury as well as to arterial thrombosis. The A1A2A3 domains of VWF contain important sites that differentially participate in supporting platelet adhesion. FAM20c (family with sequence similarity 20, member C) has emerged as a serine/threonine kinase, which phosphorylates extracellular proteins containing the S-X-E/pS motifs that are also found within the VWF A domains. This is of interest because we and others have shown that structural modifications within these A domains influence the ability of VWF to support platelet adhesion. Objective We assessed if VWF A domains can be phosphorylated and the functional consequence of phosphorylated VWF. Results Here, we show that FAM20c phosphorylated purified plasma VWF, VWF A1A2A3 protein, isolated A2 domain, but not A1 and A3 domain proteins, invitro. FAM20c phosphorylated the isolated A2 domain at S1517 and S1613 within the S-X-E recognition motif, with S1613 being the major phosphorylation site. Mass spectrometry analysis of purified plasma VWF from healthy donors revealed several phosphorylation sites, including the S1613 in the A2 domain. VWF A1A2A3 domain protein phosphorylated at S1613 promoted stable platelet adhesion and microthrombi at high shear stress. Lastly, under high shear stress VWF treated with FAM20c and ATP robustly supported platelet adhesion, compared to VWF treated with FAM20c in the absence of ATP. Conclusion These outcomes indicate that VWF can be phosphorylated by FAM20c invitro, and this novel post-translational modification enhances the adhesiveness of VWF to platelets.

Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage.

Dengue virus (DENV) infection, the most common mosquito-transmitted viral infection, can cause a range of diseases from self-limiting dengue fever to life-threatening dengue hemorrhagic fever and shock syndrome. Thrombocytopenia is a major characteristic observed in both mild and severe dengue disease and is significantly correlated with the progression of dengue severity. Previous studies have shown that DENV nonstructural protein 1 (NS1), which can be secreted into patients’ blood, can stimulate immune cells via Toll-like receptor 4 (TLR4) and can cause endothelial leakage. However, it is unclear whether DENV NS1 can directly induce platelet activation or cause thrombocytopenia during DENV infection. In this study, we first demonstrated that DENV but not Zika virus cell culture supernatant could induce P-selectin expression and phosphatidylserine (PS) exposure in human platelets, both of which were abolished when NS1 was depleted from the DENV supernatant. Similar results were found using recombinant NS1 from all four serotypes of DENV, and those effects were blocked in the presence of anti-NS1 F(ab’)2, anti-TLR4 antibody, a TLR4 antagonist (Rhodobacter sphaeroides lipopolysaccharide, LPS-Rs) and a TLR4 signaling inhibitor (TAK242), but not polymyxin B (an LPS inhibitor). Moreover, the activation of platelets by DENV NS1 promoted subthreshold concentrations of adenosine diphosphate (ADP)-induced platelet aggregation and enhanced platelet adhesion to endothelial cells and phagocytosis by macrophages. Finally, we demonstrated that DENV-induced thrombocytopenia and hemorrhage were attenuated in TLR4 knockout and wild-type mice when NS1 was depleted from DENV supernatant. Taken together, these results suggest that the binding of DENV NS1 to TLR4 on platelets can trigger its activation, which may contribute to thrombocytopenia and hemorrhage during dengue infection.

Dyslipidemias and Microcirculation.

Dyslipidemia is widely accepted as one of the major risk factors in cardiovascular disease mainly due to its contribution in the pathogenesis of atherosclerosis in medium-sized and large arteries. However, it has become increasingly accepted that high-cholesterol levels can also adversely affect the microvasculature prior to the development of overt atherosclerosis. Moreover, hypercholesterolemia has shown, in preclinical animal models, to exert detrimental effects beyond the vascular tree leading to larger infarcts and adverse cardiac remodeling post-myocardial infarction. At a functional level, hypercholesterolemia has shown to impair endotheliumdependent vasodilation because on defects on nitric oxide bioavailability. The pathogenic mechanisms underlying microvascular dysfunction involve an enhanced arginase activity, enhanced production of free radicals and the activation, recruitment and accumulation of leukocytes, primarily neutrophils, via their diffusion through postcapillary venules. In turn, recruited inflammatory cells and certain inflammatory mediators enhance platelet adhesion, overall inducing a proinflammatory and prothrombotic phenotype. Within the present review, we aim to discuss the existing evidence regarding the presence of dyslipidemia - particularly high low density lipoprotein-cholesterol levels - and the occurrence of microvascular dysfunction, the mechanism by which high cholesterol levels induce functional alterations in the microvascular bed and, finally comment on the impact of dislipidemia-induced microvascular dysfunction at the myocardial level.

Aspirin in primary prevention for patients with diabetes: Still a matter of debate.

Patients with diabetes are at high cardiovascular (CV) risk due to an exaggerated platelet activation and aggregation. In the first 2000s low-dose aspirin was first recommended for primary prevention, but then re-discussed. This short narrative review, based on the material searched for and obtained via PubMed up to February 2018, aims at clarifying this controversial topic. The JPAD2 study has been designed to evaluate the occurrence of any CV event in a cohort of patients with diabetes and concluded that low-dose aspirin did not influence the risk for CV events while increasing the risk for gastrointestinal (GI) bleeding. Reasons for this result can be found in the role of diabetic platelets, which are known to be hyperreactive, thus producing intensified adhesion, activation, and aggregation. In this setting, other associated metabolic conditions can concur to enhance platelet adhesion and activation. Aspirin resistance has been often considered a guilty actor, although many mechanisms have been mistaken for true aspirin resistance, such as patient poor compliance, inadequate dosing, drug interactions, and high-platelet turnover. However, the mere presence of diabetes is not likely to give a net benefit for CV protection with respect to GI bleeding. It appears advisable to follow current guidelines addressing first of all classical risk factors and evaluate aspirin therapy in primary prevention only for patients with type 1 or 2 diabetes at increased CV risk and no risk for GI bleeding. Anyway, additional clinical trials are needed to address the current topic.

The chaperone protein HSP47: a platelet collagen binding protein that contributes to thrombosis and hemostasis

Essentials Heat shock protein 47 (HSP47), a collagen specific chaperone is present on the platelet surface.Collagen mediated platelet function was reduced following blockade or deletion of HSP47.GPVI receptor regulated signalling was reduced in HSP47 deficient platelets.Platelet HSP47 tethers to exposed collagen thus modulating thrombosis and hemostasis. SummaryObjectiveHeat shock protein 47 (HSP47) is an intracellular chaperone protein that is vital for collagen biosynthesis in collagen secreting cells. This protein has also been shown to be present on the surface of platelets. Given the importance of collagen and its interactions with platelets in triggering hemostasis and thrombosis, in this study we sought to characterize the role of HSP47 in these cells.Methods and ResultsThe deletion of HSP47 in mouse platelets or its inhibition in human platelets reduced their function in response to collagen and the GPVI agonist (CRP‐XL), but responses to thrombin were unaltered. In the absence of functional HSP47, the interaction of collagen with platelets was reduced, and this was associated with reduced GPVI‐collagen binding, signalling and platelet activation. Thrombus formation on collagen, under arterial flow conditions, was also decreased following the inhibition or deletion of HSP47, in the presence or absence of eptifibatide, consistent with a role for HSP47 in enhancing platelet adhesion to collagen. Platelet adhesion under flow to von Willebrand factor was unaltered following HSP47 inhibition. Laser‐induced thrombosis in cremaster muscle arterioles was reduced and bleeding time was prolonged in HSP47‐deficient mice or following inhibition of HSP47.ConclusionsOur study demonstrates the presence of HSP47 on the platelet surface, where it interacts with collagen, stabilizes platelet adhesion and increases collagen‐mediated signalling and therefore thrombus formation and hemostasis.