Apolipoprotein A-IV polymorphisms Q360H and T347S attenuate its endogenous inhibition of thrombosis.

Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbß3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s-1) and low (300s-1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hindrance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.

The roles of P-selectin in cancer cachexia.

P-selectin, a cell adhesion molecule of the selectin family, is expressed on the surface of activated endothelial cells (ECs) and platelets. Binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) supports the leukocytes capture and rolling on stimulated ECs and increases the aggregation of leukocytes and activated platelets. Cancer cachexia is a systemic inflammation disorder characterized by metabolic disturbances, reduced body weight, loss of appetite, fat depletion, and progressive muscle atrophy. Cachexia status is associated with increased pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), which activates ECs to release P-selectin. Single-nucleotide polymorphisms (SNPs) loci of P-selectin encoding gene SELP are associated with higher level of plasma P-selectin and increase the susceptibility to cachexia in cancer patients. Elevated P-selectin expression has been observed in the hypothalamus, liver, and gastrocnemius muscle in animal models with cancer cachexia. Increased P-selectin may cause excessive inflammatory processes, muscle atrophy, and blood hypercoagulation, thus facilitating the development of cancer cachexia. In this review, physiological functions of P-selectin and its potential roles in cancer cachexia have been summarized. We also discuss the therapeutic potential of P-selectin inhibitors for the treatment of cancer cachexia.

Platelet-derived circulating soluble P-selectin is sufficient to induce hematopoietic stem cell mobilization.

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF)-mediated mobilization of hematopoietic stem cells (HSCs) is a well-established method to prepare HSCs for transplantation nowadays. A sufficient number of HSCs is critical for successful HSC transplantation. However, approximately 2-6% of healthy stem cell donors are G-CSF-poor mobilizers for unknown reasons; thus increasing the uncertainties of HSC transplantation. The mechanism underlining G-CSF-mediated HSC mobilization remains elusive, so detailed mechanisms and an enhanced HSC mobilization strategy are urgently needed. Evidence suggests that P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) are one of the cell-cell adhesion ligand-receptor pairs for HSCs to keep contacting bone marrow (BM) stromal cells before being mobilized into circulation. This study hypothesized that blockage of PSGL-1 and P-selectin may disrupt HSC-stromal cell interaction and facilitate HSC mobilization. METHODS: The plasma levels of soluble P-selectin (sP-sel) before and after G-CSF administration in humans and male C57BL/6J mice were analyzed using enzyme-linked immunosorbent assay. Male mice with P-selectin deficiency (Selp-/-) were further employed to investigate whether P-selectin is essential for G-CSF-induced HSC mobilization and determine which cell lineage is sP-sel derived from. Finally, wild-type mice were injected with either G-CSF or recombinant sP-sel to investigate whether sP-sel alone is sufficient for inducing HSC mobilization and whether it accomplishes this by binding to HSCs and disrupting their interaction with stromal cells in the BM. RESULTS: A significant increase in plasma sP-sel levels was observed in humans and mice following G-CSF administration. Treatments of G-CSF induced a decrease in the level of HSC mobilization in Selp-/- mice compared with the wild-type (Selp+/+) controls. Additionally, the transfer of platelets derived from wild-type mice can ameliorate the defected HSC mobilization in the Selp-/- recipients. G-CSF induces the release of sP-sel from platelets, which is sufficient to mobilize BM HSCs into the circulation of mice by disrupting the PSGL-1 and P-selectin interaction between HSCs and stromal cells. These results collectively suggested that P-selectin is a critical factor for G-CSF-induced HSC mobilization. CONCLUSIONS: sP-sel was identified as a novel endogenous HSC-mobilizing agent. sP-sel injections achieved a relatively faster and more convenient regimen to mobilize HSCs in mice than G-CSF. These findings may serve as a reference for developing and optimizing human HSC mobilization in the future.

The C-type lectin domain of CD62P (P-selectin) functions as an integrin ligand.

Recognition of integrins by CD62P has not been reported and this motivated a docking simulation using integrin αvß3 as a target. We predicted that the C-type lectin domain of CD62P functions as a potential integrin ligand and observed that it specifically bound to soluble ß3 and ß1 integrins. Known inhibitors of the interaction between CD62P-PSGL-1 did not suppress the binding, whereas the disintegrin domain of ADAM-15, a known integrin ligand, suppressed recognition by the lectin domain. Furthermore, an R16E/K17E mutation in the predicted integrin-binding interface located outside of the glycan-binding site within the lectin domain, strongly inhibited CD62P binding to integrins. In contrast, the E88D mutation that strongly disrupts glycan binding only slightly affected CD62P-integrin recognition, indicating that the glycan and integrin-binding sites are distinct. Notably, the lectin domain allosterically activated integrins by binding to the allosteric site 2. We conclude that CD62P-integrin binding may function to promote a diverse set of cell-cell adhesive interactions given that ß3 and ß1 integrins are more widely expressed than PSGL-1 that is limited to leukocytes.

Impact of P-selectin-PSGL-1 Axis on Platelet-Endothelium-Leukocyte Interactions in Fatal COVID-19.

In critically ill patients infected with SARS-CoV-2, early leukocyte recruitment to the respiratory system was found to be orchestrated by leukocyte trafficking molecules accompanied by massive secretion of proinflammatory cytokines and hypercoagulability. Our study aimed to explore the interplay between leukocyte activation and pulmonary endothelium in different disease stages of fatal COVID-19. Our study comprised 10 COVID-19 postmortem lung specimens and 20 control lung samples (5 acute respiratory distress syndrome, 2 viral pneumonia, 3 bacterial pneumonia, and 10 normal), which were stained for antigens representing the different steps of leukocyte migration: E-selectin, P-selectin, PSGL-1, ICAM1, VCAM1, and CD11b. Image analysis software QuPath was used for quantification of positive leukocytes (PSGL-1 and CD11b) and endothelium (E-selectin, P-selectin, ICAM1, VCAM1). Expression of IL-6 and IL-1ß was quantified by RT-qPCR. Expression of P-selectin and PSGL-1 was strongly increased in the COVID-19 cohort compared with all control groups (COVID-19:Controls, 17:23, P < .0001; COVID-19:Controls, 2:75, P < .0001, respectively). Importantly, P-selectin was found in endothelial cells and associated with aggregates of activated platelets adherent to the endothelial surface in COVID-19 cases. In addition, PSGL-1 staining disclosed positive perivascular leukocyte cuffs, reflecting capillaritis. Moreover, CD11b showed a strongly increased positivity in COVID-19 compared with all controls (COVID-19:Controls, 2:89; P = .0002), indicating a proinflammatory immune microenvironment. Of note, CD11b exhibited distinct staining patterns at different stages of COVID-19 disease. Only in cases with very short disease course, high levels of IL-1ß and IL-6 mRNA were observed in lung tissue. The striking upregulation of PSGL-1 and P-selectin reflects the activation of this receptor-ligand pair in COVID-19, increasing the efficiency of initial leukocyte recruitment, thus promoting tissue damage and immunothrombosis. Our results show that endothelial activation and unbalanced leukocyte migration play a central role in COVID-19 involving the P-selectin-PSGL-1 axis.

A familial case of MYH9 gene mutation associated with multiple functional and structural platelet abnormalities.

Mutations in the MYH9 gene result in macrothrombocytopenia often associated with hemorrhages. Here, we studied the function and structure of platelets in three family members with a heterozygous mutation R1933X in the MYH9 gene, characteristic of closely related disorders known as the May-Hegglin anomaly and Sebastian syndrome. The examination included complete blood count, blood smear microscopy, platelet flow cytometry (expression of P-selectin and active integrin αIIbß3 before and after activation), the kinetics of platelet-driven contraction (retraction) of blood clots, as well as scanning/transmission electron microscopy of platelets. Despite severe thrombocytopenia ranging (36-86) × 109/l, none of the patients had hemorrhages at the time of examination, although they had a history of heavy menstruation, spontaneous ecchymosis, and postpartum hemorrhage. Flow cytometry showed background platelet activation, revealed by overexpression of P-selectin and active αIIbß3 integrin above normal levels. After TRAP-induced stimulation, the fractions of platelets expressing P-selectin in the proband and her sister were below normal response, indicating partial platelet refractoriness. The initiation of clot contraction was delayed. Electron microscopy revealed giant platelets with multiple filopodia and fusion of α-granules with dilated open canalicular system, containing filamentous and vesicular inclusions. The novel concept implies that the R1933X mutation in the MYH9 gene is associated not only with thrombocytopenia, but also with qualitative structural and functional defects in platelets. Platelet dysfunction includes impaired contractility, which can disrupt the compaction of hemostatic clots, making the clots weak and permeable, therefore predisposing patients with MYH9 gene mutations to the hemorrhagic phenotype.

Massive expansion of P-selectin genes in two Venerida species, Sinonovacula constricta and Mercenaria mercenaria: evidence from comparative genomics of Bivalvia.

BACKGROUND: P-selectin is a molecule participating in the inflammatory response through mediating cellular adhesion and essential for wound repair. However, studies regarding P-selectin in Bivalvia are rare. This study identified 90 P-selectin genes among nine bivalve genomes and classified them into 4 subfamilies according to phylogenetic analysis. RESULTS: Notable P-selectin gene expansion was observed in two Venerida species, Sinonovacula constricta and Mercenaria mercenaria. The synteny analysis revealed that P-selectin gene expansion was mostly caused by tandem duplication. In addition, the expression profiles of P-selectin genes in S. constricta showed that many P-selectins were specifically highly expressed in the gills, and the P-selectin expression patterns changed dramatically under low salt stress and ammonia nitrogen stress. CONCLUSIONS: The massive expansion of P-selectins may facilitate the tolerance to environmental stresses. This study sheds light on the characterizations and expression profiles of P-selectin genes in Bivalvia and provides an integrated framework for further investigation of the role of P-selectins in the environmental tolerance of bivalves.

Mechanism by which Huoxue Tongluo Qiwei Decoction improves the erectile function of rats with diabetic erectile dysfunction.

ETHNOPHARMACOLOGICAL RELEVANCE: Huoxue Tongluo Qiwei Decoction is a classical herbal formula, which can improve the symptoms of erectile dysfunction (ED) patients and has a good therapeutic effect on patients with diabetic erectile dysfunction (DIED). The main function of Huoxue Tongluo Qiwei Decoction is to stimulate the blood circulation and dredge collaterals, remove blood stasis, and calm wind. RATIONALE: To further explore the mechanism of Huoxue Tongluo Qiwei Decoction in the treatment of DIED, related animal experiments were designed. MATERIALS AND METHODS: The chemical constituents of Huoxue Tongluo Qiwei Decoction were identified with the help of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A rat model was induced by streptozotocin (STZ) and screened by apomorphine (APO). Serum sE-selectin, lysyl oxidase-1 (LOX-1), malondialdehyde (MDA) and other markers of vascular endothelial injury and related indicators of oxidative stress were studied through enzyme-linked immunosorbent assay (ELISA). The endothelial cells and ultrastructure of the corpus cavernosum were examined by electron microscopy and HE staining. The expression of protein and mRNA was detected by western blotting (WB) and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: The results of the study revealed that the sE-selectin, LOX-1, intercellular adhesion molecule-1 (sICAM-1), endothelial microparticles (EMPs), P-selectin (CD62P), and MDA levels in the serum of group M rats were considerably higher than rats of group K, while the superoxide dismutase (SOD) level showed a significant decrease. In addition, the PKC pathway was activated, and the expression of related proteins and mRNA was increased. After 8 weeks of intervention with Huoxue Tongluo Qiwei Decoction and LY333531, serum level of sE-selectin, LOX-1, sICAM-1, EMPs, CD62P and MDA in L, D and G groups were remarkably lower than group M while SOD level increased significantly, protein kinase C (PKC) pathway was inhibited with the improved erectile function of rats. CONCLUSION: Huoxue Tongluo Qiwei Decoction can inhibit the expression of protein and mRNA of the PKCß signaling pathway related molecules in DIED rats to cure the injury of vascular endothelial, enhance antioxidant capacity, and prevent the activation of platelet, thus improving erectile function in rats with DIED.

Emerging Anti-Atherosclerotic Therapies.

Cardiovascular disease (CAD) is the main cause of morbidity and deaths in the western world. The development of atherosclerosis underlying CAD development begins early in human life. There are numerous genetic and environmental risk factors accelerating its progression which then leads to the occurrence of acute events. Despite considerable progress in determining risk factors, there is still a lot of work ahead since identified determinants are responsible only for a part of overall CAD risk. Current therapies are insufficient to successfully reduce the risk of atherosclerosis development. Therefore, there is a need for effective preventive measures of clinical manifestations of atherosclerosis since the currently available drugs cannot prevent the occurrence of even 70% of clinical events. The shift of the target from lipid metabolism has opened the door to many new therapeutic targets. Currently, the majority of known targets for anti-atherosclerotic drugs focus also on inflammation (a common mediator of many risk factors), mechanisms of innate and adaptive immunity in atherosclerosis, molecule scavengers, etc. The therapeutic potential of cyclodextrins, protein kinase inhibitors, colchicine, inhibitors of p38 mitogen-activated protein kinase (MAPK), lipid dicarbonyl scavengers, a monoclonal antibody targeting interleukin-1ß, and P-selectin inhibitors is still not fully confirmed and requires confirmation in large clinical trials. The preliminary results look promising.

SELP Asp603Asn and severe thrombosis in COVID-19 males.

Thromboembolism is a frequent cause of severity and mortality in COVID-19. However, the etiology of this phenomenon is not well understood. A cohort of 1186 subjects, from the GEN-COVID consortium, infected by SARS-CoV-2 with different severity was stratified by sex and adjusted by age. Then, common coding variants from whole exome sequencing were mined by LASSO logistic regression. The homozygosity of the cell adhesion molecule P-selectin gene (SELP) rs6127 (c.1807G > A; p.Asp603Asn) which has been already associated with thrombotic risk is found to be associated with severity in the male subcohort of 513 subjects (odds ratio = 2.27, 95% Confidence Interval 1.54-3.36). As the SELP gene is downregulated by testosterone, the odd ratio is increased in males older than 50 (OR 2.42, 95% CI 1.53-3.82). Asn/Asn homozygotes have increased D-dimers values especially when associated with poly Q ≥ 23 in the androgen receptor (OR 3.26, 95% CI 1.41-7.52). These results provide a rationale for the repurposing of antibodies against P-selectin as adjuvant therapy in rs6127 male homozygotes especially if older than 50 or with an impaired androgen receptor.

The Proteasome Inhibitor Bortezomib Induces Apoptosis and Activation in Gel-Filtered Human Platelets.

Bortezomib (BTZ) has demonstrated its efficacy in several hematological disorders and has been associated with thrombocytopenia. There is controversy about the effect of BTZ on human platelets, so we set out to determine its effect on various types of platelet samples. Human platelets were investigated in platelet-rich plasma (PRP) and as gel-filtered platelets (GFPs). Mitochondrial inner membrane potential depolarization and phosphatidylserine (PS) and P-selectin expression levels were studied by flow cytometry, while thrombin generation was measured by a fluorescent method. In PRP, BTZ caused negligible PS expression after 60 min of treatment. However, in GFPs, PS expression was dose- and time-dependently increased in the BTZ-treated groups, as was P-selectin. The percentage of depolarized cells was also higher after BTZ pretreatment at both time points. Peak thrombin and velocity index increased significantly even with the lowest BTZ concentration (p = 0.0019; p = 0.0032) whereas time to peak and start tail parameters decreased (p = 0.0007; p = 0.0034). The difference between PRP and GFP results can be attributed to the presence of plasma proteins in PRP, as the PS-stimulating effect of BTZ could be attenuated by supplementing GFPs with purified human albumin. Overall, BTZ induces a procoagulant platelet phenotype in an experimental setting devoid of plasma proteins.

P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression.

Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, its microenvironment plays a crucial role in its progression. Among the brain stromal cells, the microglia were shown to facilitate GB invasion and immunosuppression. However, the reciprocal mechanisms by which GB cells alter microglia/macrophages behavior are not fully understood. We propose that these mechanisms involve adhesion molecules such as the Selectins family. These proteins are involved in immune modulation and cancer immunity. We show that P-selectin mediates microglia-enhanced GB proliferation and invasion by altering microglia/macrophages activation state. We demonstrate these findings by pharmacological and molecular inhibition of P-selectin which leads to reduced tumor growth and increased survival in GB mouse models. Our work sheds light on tumor-associated microglia/macrophage function and the mechanisms by which GB cells suppress the immune system and invade the brain, paving the way to exploit P-selectin as a target for GB therapy.

X­irradiation induces acute and early term inflammatory responses in atherosclerosis­prone ApoE­/­ mice and in endothelial cells.

Thoracic radiotherapy is an effective treatment for many types of cancer; however it is also associated with an increased risk of developing cardiovascular disease (CVD), appearing mainly ≥10 years after radiation exposure. The present study investigated acute and early term physiological and molecular changes in the cardiovascular system after ionizing radiation exposure. Female and male ApoE­/­ mice received a single exposure of low or high dose X­ray thoracic irradiation (0.1 and 10 Gy). The level of cholesterol and triglycerides, as well as a large panel of inflammatory markers, were analyzed in serum samples obtained at 24 h and 1 month after irradiation. The secretion of inflammatory markers was further verified in vitro in coronary artery and microvascular endothelial cell lines after exposure to low and high dose of ionizing radiation (0.1 and 5 Gy). Local thoracic irradiation of ApoE­/­ mice increased serum growth differentiation factor­15 (GDF­15) and C­X­C motif chemokine ligand 10 (CXCL10) levels in both female and male mice 24 h after high dose irradiation, which were also secreted from coronary artery and microvascular endothelial cells in vitro. Sex­specific responses were observed for triglyceride and cholesterol levels, and some of the assessed inflammatory markers as detailed below. Male ApoE­/­ mice demonstrated elevated intercellular adhesion molecule­1 and P­selectin at 24 h, and adiponectin and plasminogen activator inhibitor­1 at 1 month after irradiation, while female ApoE­/­ mice exhibited decreased monocyte chemoattractant protein­1 and urokinase­type plasminogen activator receptor at 24 h, and basic fibroblast growth factor 1 month after irradiation. The inflammatory responses were mainly significant following high dose irradiation, but certain markers showed significant changes after low dose exposure. The present study revealed that acute/early inflammatory responses occurred after low and high dose thoracic irradiation. However, further research is required to elucidate early asymptomatic changes in the cardiovascular system post thoracic X­irradiation and to investigate whether GDF­15 and CXCL10 could be considered as potential biomarkers for the early detection of CVD risk in thoracic radiotherapy­treated patients.

P-selectin deficiency promotes liver senescence in sickle cell disease mice.

Sickle cell disease (SCD) is caused by a homozygous mutation in the ß-globin gene, which leads to erythrocyte sickling, vasoocclusion, and intense hemolysis. P-selectin inhibition has been shown to prevent vasoocclusive events in patients with SCD; however, the chronic effect of P-selectin inhibition in SCD remains to be determined. Here, we used quantitative liver intravital microscopy in our recently generated P-selectin-deficient SCD mice to show that chronic P-selectin deficiency attenuates liver ischemia but fails to prevent hepatobiliary injury. Remarkably, we find that this failure in resolution of hepatobiliary injury in P-selectin-deficient SCD mice is associated with the increase in cellular senescence and reduced epithelial cell proliferation in the liver. These findings highlight the importance of investigating the long-term effects of chronic P-selectin inhibition therapy on liver pathophysiology in patients with SCD.

Association of Thr715Pro P-Selectin Gene Polymorphism in Diabetic Retinopathy in North Indian Population: A Prospective Clinical Study.

INTRODUCTION: The aim of this study was to evaluate the role of Thr715Pro P-Selectin gene polymorphism in patients with Diabetic Retinopathy in North Indian population and establish its role in the pathophysiology as an independent factor. MATERIALS AND METHODS: This is a prospective clinical study conducted on 60 patients at a tertiary care centre in North India over a period of eighteen months. Sixty patients satisfying the inclusion criteria were selected from the Vitreoretina clinic in the department. They were categorised equally in three groups namely Diabetics with diabetic retinopathy (DwDR), Diabetics without diabetic retinopathy (DwoDR), and non diabetics. The non-diabetics group was further divided into healthy controls, Hypertensive Retinopathy (HR) and Non-exudative Age Related Macular Degeneration (NEAMD). All the patients underwent complete ophthalmic evaluation and blood samples were drawn for the genetic study with their informed consent. Data was analysed using SPSS software version 16. RESULTS: The genotypic analysis between DwDR, DwoDR and the three subgroup of controls comprising of healthy controls, HR and NEAMD showed that Thr715Pro (A/C) polymorphism prevalence was significantly high in DwDR (p = 0.003) and DwoDR (p = 0.003) compared to healthy controls. No significant difference was noted between DwDR, DwoDR and the HR and NEAMD groups. CONCLUSION: Thr715Pro P-Selectin gene Polymorphism could not be established as an independent factor in the pathogenesis of diabetic retinopathy, as its association is found with other systemic diseases which create a prothrombotic state.

Fucoidan-coated nanoparticles target radiation-induced P-selectin to enhance chemoradiotherapy in murine colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related death for both men and women, highlighting the need for new treatment strategies. Advanced disease is often treated with a combination of radiation and cytotoxic agents, such as DNA damage repair inhibitors and DNA damaging agents. To optimize the therapeutic window of these multimodal therapies, advanced nanomaterials have been investigated to deliver sensitizing agents or enhance local radiation dose deposition. In this study, we demonstrate the feasibility of employing an inflammation targeting nanoscale metal-organic framework (nMOF) platform to enhance CRC treatment. This novel formulation incorporates a fucoidan surface coating to preferentially target P-selectin, which is over-expressed or translocated in irradiated tumors. Using this radiation stimulated delivery strategy, a combination PARP inhibitor (talazoparib) and chemotherapeutic (temozolomide) drug-loaded hafnium and 1,4-dicarboxybenzene (Hf-BDC) nMOF was evaluated both in vitro and in vivo. Significantly, these drug-loaded P-selectin targeted nMOFs (TT@Hf-BDC-Fuco) show improved tumoral accumulation over multiple controls and subsequently enhanced therapeutic effects. The integrated radiation and nanoformulation treatment demonstrated improved tumor control (reduced volume, density, and growth rate) and increased survival in a syngeneic CRC mouse model. Overall, the data from this study support the continued investigation of radiation-priming for targeted drug delivery and further consideration of nanomedicine strategies in the clinical management of advanced CRC.

Plasmin-Induced Activation of Human Platelets Is Modulated by Thrombospondin-1, Bona Fide Misfolded Proteins and Thiol Isomerases.

Inflammatory processes are triggered by the fibrinolytic enzyme plasmin. Tissue-type plasminogen activator, which cleaves plasminogen to plasmin, can be activated by the cross-ß-structure of misfolded proteins. Misfolded protein aggregates also represent substrates for plasmin, promoting their degradation, and are potent platelet agonists. However, the regulation of plasmin-mediated platelet activation by misfolded proteins and vice versa is incompletely understood. In this study, we hypothesize that plasmin acts as potent agonist of human platelets in vitro after short-term incubation at room temperature, and that the response to thrombospondin-1 and the bona fide misfolded proteins Eap and SCN--denatured IgG interfere with plasmin, thereby modulating platelet activation. Plasmin dose-dependently induced CD62P surface expression on, and binding of fibrinogen to, human platelets in the absence/presence of plasma and in citrated whole blood, as analyzed by flow cytometry. Thrombospondin-1 pre-incubated with plasmin enhanced these plasmin-induced platelet responses at low concentration and diminished them at higher dose. Platelet fibrinogen binding was dose-dependently induced by the C-terminal thrombospondin-1 peptide RFYVVMWK, Eap or NaSCN-treated IgG, but diminished in the presence of plasmin. Blocking enzymatically catalyzed thiol-isomerization decreased plasmin-induced platelet responses, suggesting that plasmin activates platelets in a thiol-dependent manner. Thrombospondin-1, depending on the concentration, may act as cofactor or inhibitor of plasmin-induced platelet activation, and plasmin blocks platelet activation induced by misfolded proteins and vice versa, which might be of clinical relevance.

Supplemental Fibrinogen Restores Platelet Inhibitor-Induced Reduction in Thrombus Formation without Altering Platelet Function: An In Vitro Study.

BACKGROUND: For patients treated with dual antiplatelet therapy, standardized drug-specific 3-to-7 day cessation is recommended prior to major surgery to reach sufficient platelet function recovery. Here we investigated the hypothesis that supplemental fibrinogen might mitigate the inhibitory effects of antiplatelet therapy. METHODS AND RESULTS: To this end blood from healthy donors was treated in vitro with platelet inhibitors, and in vitro thrombus formation and platelet activation were assessed. Ticagrelor, acetylsalicylic acid, the combination of both, and tirofiban all markedly attenuated the formation of adherent thrombi, when whole blood was perfused through collagen-coated microchannels at physiological shear rates. Addition of fibrinogen restored in vitro thrombus formation in the presence of antiplatelet drugs and heparin. However, platelet activation, as investigated in assays of P-selectin expression and calcium flux, was not altered by fibrinogen supplementation. Most importantly, fibrinogen was able to restore in vitro thrombogenesis in patients on maintenance dual antiplatelet therapy after percutaneous coronary intervention. CONCLUSION: Thus, our in vitro data support the notion that supplementation of fibrinogen influences the perioperative hemostasis in patients undergoing surgery during antiplatelet therapy by promoting thrombogenesis without significantly interfering with platelet activation.

Platelet disturbances correlate with endothelial cell activation in uncomplicated Plasmodium vivax malaria.

Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson's correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406-651] vs. 1029 [732-1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.