Platelet Protein Research Articles

Post-Translational Modifications of Platelet-Derived Amyloid Precursor Protein by Coagulation Factor XIII-A.

The physiological function of amyloid β precursor protein (APP) in platelets has remained elusive. Upon platelet activation, APP localizes to the platelet surface and is proteolytically processed by proteases to release various metabolites, including amyloid β (Aβ) and soluble APP. Synthetic Aβ is a substrate of activated coagulation factor XIII (FXIII-A*), a transglutaminase that is active both inside and on the surface of platelets. Here we tested if platelet APP and its fragments are covalently modified by FXIII-A*. Platelet-derived FXIII-A* and fibrin(ogen) bound to APP, and their bound fractions increased 7- and 11-fold upon platelet activation, respectively. The processing of platelet APP was enhanced when FXIII-A* was inhibited. Soluble APPβ was covalently cross-linked by FXIII-A*. This mechanism regulating APP processing is significant, because controlling the processing of APP, such as by inhibiting specific secretases that cleave APP, is a therapeutic target for Alzheimer's disease.

Derivation and Validation of a Predictive Score for Disease Worsening Inpatients with COVID-19

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease. COVID-19 is associated with excessive inflammation, platelet activation, endothelial dysfunction, blood coagulation activation and fibrin formation. Current management of patients hospitalized in an intensive care unit (ICU) is based on supportive care and the mortality rate is high. Blood hypercoagulability -documented by increase in D-Dimers and decrease in antithrombin (AT) -is frequently encountered among COVID-19 patients.Aim: Identification of patients with COVID-19 being at high risk for clinical deterioration is a challenging issue for an earlier adapted treatment and positive clinical outcome. we aimed to identify the most relevant clinical and haematological risk factors for worsening of COVID-19 in hospitalized patients by and constructing an accurate risk assessment tool, COMPASS-COVID-19.Methods: Patients with confirmed COVID-19 (n=430) were divided in derivation (n=310) and validation (n=120) cohorts. They were stratified to those hospitalized at the conventional ward (C-group) and those with worsening disease (W-group) admitted in ICU. The study endpoint was disease worsening. All patients were routinely evaluated with full blood count, prothrombin time, fibrinogen, D-Dimers, antithrombin and protein C activity. Data from the first hospitalization day at the conventional ward or the ICU were analyzed. Cardiovascular risk factors and comorbidities were routinely registered.Results: In the derivation cohort, compensated DIC was diagnosed in 8.2% of patients in the C-group and in 28.2% of patients in the W-group (p=0.001). Compared to the C-group, the patients in the W-group had significantly lower levels of AT, PC, platelets, lymphocyte, monocyte and red blood cell counts as well as, hemoglobin and hematocrit. They had significantly higher levels of fibrinogen, DDimers, white blood cells and neutrophils counts. No difference was noted in eosinophil and basophil counts. Hematological predictors for disease worsening: Increase of fibrinogen (OR= 9.50, 95% CI:2.23-40.54) and D-Dimer levels (OR=7.65, 95% CI:2.67-21.87), a platelet count lower than 100x109/L (OR=7.60, 95% CI:1.55-37.35) and a positive compensated DIC-ISTH score (OR=4.58, CI:2.09-10.07) were major determinants of disease worsening risk. Deficiency of AT activity (OR = 2.13, 95%CI: 1.18-3.85), PT prolongation (OR=2.43, 95% CI:1.15-5.13), leukocytosis (OR=1.86, 95%CI:1.04-3.33), lymphopenia with lymphocyte count lower than 1.5 G/L (OR=3.37, CI:1.58-7.21) were significant risk factors for worsening disease. The multivariate analysis retained obesity- BMI≥30, (OR=6.56, 95%CI: 2.98-14.46; p<0.001), male gender (OR=2.59, 95% CI:1.29-5.21; p=0.007), compensated DICISTH score ≥5 (OR=2.58, 95% CI:1.07-6.21; p=0.034), lymphocyte count <1 G/L (OR=2.21, 95% CI:1.17-4.19; p=0.015) and Hb<11 g/dL (OR=2.25, 95% CI: 1.13-4.48; p=0.021) as significant predictors of worsening disease . The score at 18 points cut-off value identified as high risk for disease worsening 90% of patients at the W-group and 38% of the patients at the C-group. The sensitivity and the specificity of the score were 94% and 58% respectively and the negative and positive predictive values were 96% and 45% respectively.Conclusion: The COMPASS-COVID- 19 score derived from multivariate analysis and includes obesity, gender, hemoglobin, lymphocyte and the cDIC-ISTH score (including platelet count, prothrombin time, D-Dimers antithrombin and protein C levels). The score has a very good discriminating capacity to stratify patients at high and low risk for worsening disease, with AUC value at 0.77, sensitivity 81% and specificity 60%. Application of the COMPASS-COVID-19 score at the validation cohort showed 96% sensitivity. The COMPASS-COVID-19 score is an accurate clinical decision tool for an easy identification of COVID-19 patients being at high risk for diseaseDisclosuresNo relevant conflicts of interest to declare.

Clinical Investigation and Biological Mechanism of Tigecycline on Coagulation Disorders in Cancer Patients

Tigecycline, a first representative of the new class of glycylcycline antibacterial drugs, has been approved by FDA for using in the treatment of complicated skin and skin structure infections and community-acquired pneumonia in adults in 2005. It has been widely used in hospitals due to its broad-spectrum activity against pathogens. Pre-clinical studies showed that tigecycline has a significant anti-bacterial effect with low and mild adverse drug reactions (ADRs), including nausea, vomit or some other ADRs. Accompany with the huge increase of tigecycline usage in China, more and more side effects began to arouse people's attention. Here, we found that tigecycline can cause coagulation abnormalities in cancer patients, especially with hematological malignance, through clinical observation and retrospective analysis. To analyze the effect and risk factors of tigecycline on coagulation index in severely infected patients with hematologic cancer, we screened the cases admitted to department of hematology from three hospitals treated with tigecycline from Nov, 2015 to Oct, 2019. Then we compared the differences among the prothrombin time (PT), prothrombin time activity (PT %), the international standardization ratio (INR), part of the activation of prothrombin time (APTT), fibrinogen (FBG), thrombin time (TT), D-Dimer, fibrin degradation products (FDP), platelet (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and c-reactive protein (CRP) before and after the tigecycline treatment. Meanwhile, we tend to establish the relationship between coagulation function and the time of tigecycline usage. In addition, for exploring the potential cause of abnormal coagulation induced by tigecycline, we evaluated the underlying biological mechanisms via Chinese hamster ovary (CHO) cells treated by tigecycline in vitro and subsequent RNA-sequencing analysis. A total of 129 qualified cases were included for analysis, including 71 males (55%) and 58 females (45%), with average age of 49.5±15.9 years old. After the treatment with tigecycline, PT, APTT and TT were significantly prolonged (P &amp;lt; 0.05). The INR, D-Dimer and FDP levels also increased (P &amp;lt; 0.05), but the FBG concentration was significantly reduced (P &amp;lt; 0.05) which indicated the possibility of hypofibrinogenaemia. Moreover, the coagulation parameters were tremendously changed in the early stage after intravenous infusion of tigecycline for anti-infection. There was no significant change in PLT count, but the levels of ALT, AST, CRE and CRP in the patients were statistically significant declined (P &amp;lt; 0.05). Most of all, the liver and kidney function of the patients was recovered after the infection was controlled by tigecycline treatment. In addition, the adhesion and stretching function of CHO cells were significantly suppressed after tigecycline treatmentin vitro. Lastly, bulk RNA-sequencing assay showed significant abnormalities in gene sets associated with platelet-activation and coagulation, such as PKFP, PLA2g7, PDGFRA, PDGFRB, PAFAH1b3, PDGFA, PECAM1, PDGFC and MPIG6b. Furthermore, adhesion regulating molecules RPN13, CHL1, ICAM5, TLCN were also downregulated by tigecycline treatment. What's more, these key genes which showed above need to be further validated in blood samples of AML patients when treated with tigecycline. Taken together, tigecycline can cause coagulation disorders in blood cancer patients. As we known, blood cancer could disturb the normal hematopoietic reconstitution in bone marrow, which resulted in abnormal numbers and functions of the peripheral blood lineages. Hence, these ADRs can accelerate the disease progression, cause severe bleeding and increase the risk of mortality in these patients. The biological mechanism study of this abnormality showed that tigecycline could inhibit platelet activation and the expression of adhesion regulators, which may increase the risk of bleeding and threaten the safety of patients. Therefore, the monitoring of coagulation index during tigecycline treatment should be adopted. Disclosures No relevant conflicts of interest to declare.

Prospective Study Reveals Increased Platelet Function Associated with Multiple Myeloma and Its Treatment

Background: Multiple Myeloma (MM) is a rare incurable bone marrow cancer characterised by a malignant proliferation of plasma cells. MM is usually preceded by a premalignant and benign Monoclonal Gammopathy of Undetermined Significance (MGUS). The incidence of arterial and venous thrombosis in MM is substantially higher than in the normal population, however the cause of this increased thrombosis risk and the impact of MM on platelet function is unclear. Treatments for both newly diagnosed and relapsed/refractory patients with MM include Immunomodulatory drugs (IMiDs) such as thalidomide/lenalidomide-based combinations. These treatments improve considerably patient outcomes, however iMiD treatment also increases the risk of thrombotic complications in these patients. Aims: In this prospective study we explored the impact of MM and its treatment on platelet function. Methods: High throughput functional analysis was performed using platelets from normal healthy controls (n=31) and patients with MGUS (n=18), smouldering multiple myeloma (SMM, n= 20), and MM (26). The MM group was further divided into 3 treatment cohorts; (1) no treatment, (2) treatment with proteasome inhibitor (PI) and dexamethasone (Dex), and (3) treatment with PI, Dex, immunomodulatory drug (iMiD) and direct oral anticoagulant. Platelet aggregation and activation (fibrinogen binding and P-selectin exposure) were measured in response to a concentration range of agonists including ADP, the thrombin receptor agonist TRAP-6, collagen, collagen-related peptide (CRP), a thromboxane receptor agonist U46619 and epinephrine. Cereblon protein was detected in platelet protein extracts by immunoblot analysis. Results: Consistent with previous reports, modestly increased VWF and factor VIII levels were detected in MM patients, but no additional differences in coagulation parameters were detected in patient groups compared to normal healthy controls (other than expected due to anticoagulant usage). Platelet aggregation in response to each agonist was increased significantly in the MM patient group compared to the normal healthy controls, suggesting that platelet reactivity is elevated in MM patients through a common mechanism that is shared by different activation pathways or the involvement of multiple mechanisms. P-selectin exposure on platelets from MM patients was not significantly different from normal healthy donors, indicating that enhanced platelet reactivity in MM is specifically through modulation of integrin αIIbβ3 activation, fibrinogen binding and therefore enhanced aggregation. The effects of treatment on platelet function in patients on iMiD vs. non iMiD treatment were assessed. In the iMiD treatment group, patient platelets aggregated in response to lower concentrations of ADP, collagen, epinephrine and CRP in samples taken post-treatment compared to those taken before and during treatment. This demonstrates an increased sensitivity to platelet activation in these patients induced by treatment. Immunoblot analysis revealed that platelets contain cereblon, a therapeutic target of lenalidomide. The potential direct effects of iMiDs on platelets in vitro was therefore explored. Lenalidomide treatment (10mM) increased the ability of platelets to aggregate in response to low concentrations of each agonist tested when compared to normal controls. Conclusions: Platelet reactivity is increased in multiple myeloma and increased further upon iMiD treatment. The presence of the key therapeutic target for iMiDs in platelets and the ability of lenalidomide to modulate platelet function directly, reveals new avenues for investigation to determine the underlying mechanism of action. Disclosures Laffan: CSL: Consultancy; Pfizer: Consultancy; Sobi: Consultancy; Roche: Consultancy; LFB: Consultancy; Shire: Consultancy; Octapharma: Consultancy; Bayer: Speakers Bureau; Roche-Chugai: Speakers Bureau; Takeda: Speakers Bureau; Leo-Pharma: Speakers Bureau; Pfizer: Speakers Bureau. Shapiro:Bayer: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Chugai/Roche: Consultancy, Speakers Bureau; Shire/Takeda: Consultancy, Speakers Bureau. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ramasamy:Takeda: Research Funding; Janssen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Amgen: Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sanofi: Honoraria; Oncopeptides: Honoraria; Takeda: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Bristol Myers Squibb: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers squibb: Membership on an entity's Board of Directors or advisory committees. Gibbins:Bristol Myers Squibb: Research Funding; Arena Pharmaceuticals: Research Funding.

RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival

AbstractG protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

Cysteine sulfenylation by CD36 signaling promotes arterial thrombosis in dyslipidemia

AbstractArterial thrombosis in the setting of dyslipidemia promotes clinically significant events, including myocardial infarction and stroke. Oxidized lipids in low-density lipoproteins (oxLDL) are a risk factor for athero-thrombosis and are recognized by platelet scavenger receptor CD36. oxLDL binding to CD36 promotes platelet activation and thrombosis by promoting generation of reactive oxygen species. The downstream signaling events initiated by reactive oxygen species in this setting are poorly understood. In this study, we report that CD36 signaling promotes hydrogen peroxide flux in platelets. Using carbon nucleophiles that selectively and covalently modify cysteine sulfenic acids, we found that hydrogen peroxide generated through CD36 signaling promotes cysteine sulfenylation of platelet proteins. Specifically, cysteines were sulfenylated on Src family kinases, which are signaling transducers that are recruited to CD36 upon recognition of its ligands. Cysteine sulfenylation promoted activation of Src family kinases and was prevented by using a blocking antibody to CD36 or by enzymatic degradation of hydrogen peroxide. CD36-mediated platelet aggregation and procoagulant phosphatidylserine externalization were inhibited in a concentration-dependent manner by a panel of sulfenic acid–selective carbon nucleophiles. At the same concentrations, these probes did not inhibit platelet aggregation induced by the purinergic receptor agonist adenosine diphosphate or the collagen receptor glycoprotein VI agonist collagen-related peptide. Selective modification of cysteine sulfenylation in vivo with a benzothiazine-based nucleophile rescued the enhanced arterial thrombosis seen in dyslipidemic mice back to control levels. These findings suggest that CD36 signaling generates hydrogen peroxide to oxidize cysteines within platelet proteins, including Src family kinases, and lowers the threshold for platelet activation in dyslipidemia.

Derivation and Validation of a Predictive Score for Disease Worsening in Patients with COVID-19.

The prospective observational cohort study COMPASS-COVID-19 aimed to develop a risk assessment model for early identification of hospitalized COVID-19 patients at risk for worsening disease. Patients with confirmed COVID-19 ( n = 430) hospitalized between March 18 and April 21, 2020 were divided in derivation ( n = 310) and validation ( n = 120) cohorts. Two groups became evident: (1) good prognosis group (G-group) with patients hospitalized at the conventional COVID-19 ward and (2) Worsening disease group (W-group) with patients admitted to the intensive care unit (ICU) from the emergency departments. The study end point was disease worsening (acute respiratory failure, shock, myocardial dysfunction, bacterial or viral coinfections, and acute kidney injury) requiring ICU admission. All patients were routinely evaluated for full blood count, prothrombin time, fibrinogen, D-dimers, antithrombin (AT), and protein C activity. Data from the first hospitalization day at the conventional ward or the ICU were analyzed. Cardiovascular risk factors and comorbidities were routinely registered. Obesity, hypertension, diabetes and male gender, increased fibrinogen and D-dimers, thrombocytopenia, AT deficiency, lymphopenia, and an International Society on Thrombosis and Haemostasis (ISTH) score for compensated disseminated intravascular coagulation score (cDIC-ISTH) ≥ 5 were significant risk factors for worsening disease. The COMPASS-COVID-19 score was derived from multivariate analyses and includes obesity, gender, hemoglobin, lymphocyte, and the cDIC-ISTH score (including platelet count, prothrombin time, D-dimers, AT, and protein C levels). The score has a very good discriminating capacity to stratify patients at high and low risk for worsening disease, with an area under the receiver operating characteristic curve value of 0.77, a sensitivity of 81%, and a specificity of 60%. Application of the COMPASS-COVID-19 score at the validation cohort showed 96% sensitivity. The COMPASS-COVID-19 score is an accurate clinical decision-making tool for an easy identification of COVID-19 patients being at high risk for disease worsening.

Platelet gene expression and function in patients with COVID-19

AbstractThere is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis, with the major difference being increased risk of thrombosis rather than bleeding. However, whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene-expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene-expression changes in pathways associated with protein ubiquitination, antigen presentation, and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, angiotensin-converting enzyme 2 (ACE2), was not detected by messenger RNA (mRNA) or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2 of 25 COVID-19 patients, suggesting that platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared with healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity, which may contribute to COVID-19 pathophysiology.

Parkin Coordinates Platelet Stress Response in Diabetes Mellitus: A Big Role in a Small Cell.

Increased platelet activation and apoptosis are characteristic of diabetic (DM) platelets, where a Parkin-dependent mitophagy serves a major endogenous protective role. We now demonstrate that Parkin is highly expressed in both healthy platelets and diabetic platelets, compared to other mitochondria-enriched tissues such as the heart, muscle, brain, and liver. Abundance of Parkin in a small, short-lived anucleate cell suggest significance in various key processes. Through proteomics we identified 127 Parkin-interacting proteins in DM platelets and compared them to healthy controls. We assessed the 11 highest covered proteins by individual IPs and confirmed seven proteins that interacted with Parkin; VCP/p97, LAMP1, HADHA, FREMT3, PDIA, ILK, and 14-3-3. Upon further STRING analysis using GO and KEGG, interactions were divided into two broad groups: targeting platelet activation through (1) actions on mitochondria and (2) actions on integrin signaling. Parkin plays an important role in mitochondrial protection through mitophagy (VCP/p97), recruiting phagophores, and targeting lysosomes (with LAMP1). Mitochondrial β-oxidation may also be regulated by the Parkin/HADHA interaction. Parkin may regulate platelet aggregation and activation through integrin signaling through interactions with proteins like FREMT3, PDIA, ILK, and 14-3-3. Thus, platelet Parkin may regulate the protection (mitophagy) and stress response (platelet activation) in DM platelets. This study identified new potential therapeutic targets for platelet mitochondrial dysfunction and hyperactivation in diabetes mellitus.

Platelets and Defective N-Glycosylation.

N-glycans are covalently linked to an asparagine residue in a simple acceptor sequence of proteins, called a sequon. This modification is important for protein folding, enhancing thermodynamic stability, and decreasing abnormal protein aggregation within the endoplasmic reticulum (ER), for the lifetime and for the subcellular localization of proteins besides other functions. Hypoglycosylation is the hallmark of a group of rare genetic diseases called congenital disorders of glycosylation (CDG). These diseases are due to defects in glycan synthesis, processing, and attachment to proteins and lipids, thereby modifying signaling functions and metabolic pathways. Defects in N-glycosylation and O-glycosylation constitute the largest CDG groups. Clotting and anticlotting factor defects as well as a tendency to thrombosis or bleeding have been described in CDG patients. However, N-glycosylation of platelet proteins has been poorly investigated in CDG. In this review, we highlight normal and deficient N-glycosylation of platelet-derived molecules and discuss the involvement of platelets in the congenital disorders of N-glycosylation.

Siderophores induce mitophagy-dependent apoptosis in platelets.

Siderophores are major virulent factors of K. pneumoniae, and their roles are iron chelators in the host. Several studies have shown that iron chelation could result in mitochondrial dysfunction and increase the production of reactive oxygen species (ROS), which further induces cell mitophagy and apoptosis. However, the impacts of siderophores on platelets are still unknown. We obtained platelets of healthy volunteers to perform in vitro experiments in our study and treated platelets with different siderophores. Mitophagy related proteins (TOMM20, TIMM23, LC3, and p62), signal proteins (PINK1/Parkin and BNIP3), and apoptosis protein (caspase3) in platelets were analyzed by western blot. The co-localization of mitotracker with LC3-II was analyzed by immunofluorescence assays. The flow cytometer was used to evaluate ROS levels. All four kinds of siderophores (10 μM) secreted by K. pneumoniae increased the expression of LC3 II and reduced the expression of mitochondrial membrane protein, TOMM20, and TIMM23. Immunofluorescence assays revealed that the treatment of enterobactin significantly increased the co-localization of mitotracker with LC3-II. All four kinds of siderophores increased the ROS level in platelets. Mitophagy of platelets was activated through several pathways, including PINK1/Parkin- and BNIP3-dependent pathways. We also proved that siderophores increased the expression of caspase3 in platelets, and the expression of caspase3 significantly decreased after the pathways of mitophagy were blocked. K. pneumoniae siderophores lead to mitophagy in platelets, and mitophagy further induces apoptosis, which may be a potential treatment of thrombocytopenia in infections.

Platelets in Healthy and Disease States: From Biomarkers Discovery to Drug Targets Identification by Proteomics.

Platelets are a heterogeneous small anucleate blood cell population with a central role both in physiological haemostasis and in pathological states, spanning from thrombosis to inflammation, and cancer. Recent advances in proteomic studies provided additional important information concerning the platelet biology and the response of platelets to several pathophysiological pathways. Platelets circulate systemically and can be easily isolated from human samples, making proteomic application very interesting for characterizing the complexity of platelet functions in health and disease as well as for identifying and quantifying potential platelet proteins as biomarkers and novel antiplatelet therapeutic targets. To date, the highly dynamic protein content of platelets has been studied in resting and activated platelets, and several subproteomes have been characterized including platelet-derived microparticles, platelet granules, platelet releasates, platelet membrane proteins, and specific platelet post-translational modifications. In this review, a critical overview is provided on principal platelet proteomic studies focused on platelet biology from signaling to granules content, platelet proteome changes in several diseases, and the impact of drugs on platelet functions. Moreover, recent advances in quantitative platelet proteomics are discussed, emphasizing the importance of targeted quantification methods for more precise, robust and accurate quantification of selected proteins, which might be used as biomarkers for disease diagnosis, prognosis and therapy, and their strong clinical impact in the near future.

COVID-19 Clinical Characteristics, Complications and Comorbidity: An Updated Systemic Review and Meta-Analysis

Background: COVID-19, a novel pneumonia associated with the 2019 coronavirus infected pneumonia suddenly broke out in the world. The aim of this study is to summaries and analysis the clinical characteristics of COVID-19. Methods: Literatures have been systematically searched on Scopus, PubMed, Embase, Web of Science, and The Cochrane Library and also special databases prepared for COVID-19 such as LitCovid Hub, WHO, Elsevier, Medrxiv and CDC from inception up to May 1, 2020. Data searching, extracting and quality appraising were done by two researchers, independently. At last, Random-effects size based on Cochrane test and I2 were used. The Newcastle-Ottawa Scale was used to assess quality, and publication bias was analyzed by Egger and Begg’s test. The review protocol has been registered in PROSPERO with ID: CRD42020173639 and with ethical code IR.GUMS.REC.1398.542. Results: Out of 2464 studies, 75(3.04%) were included. A total of 111490 patients with a mean age of 49.43 years [Confidence Interval(CI)95%: 47.44-51.42] were evaluated. Most of patients was male (52.65%). The body mass index (BMI) in the most was in normal rate (23.26). 33.5% of patients were health care workers. 62.71% of patients had direct exposure history. The most common clinical symptoms were fever, cough, myalgia and dyspnea (82.72%, 57.69%; 25.20% and 20.87%, respectively). The most common comorbidities were acute respiratory distress syndrome, chronic medical illness, hypertension, and diabetes (30.4%, 22.25%, 17.47%, and 11.19% respectively). Among the laboratory abnormalities, the most patients had high levels of leucocytes (24.76%), lymphocyte (39.56%), increasing of neutrophil (14.48%), platelet (39.81%), D-dimer (30.89%), AST (22.09%), Creatinine (48.21%), troponin I (4.12%), urea nitrogen (22.94%), Creatine Kinase (48.21%), and C-reactive proteins (56.8%). About 37.35% of patients had a decreasing in the hemoglobin. CT-Scan funding shows that three fourths of patients had bilateral pneumonia involvement (78.25%), and 58.37% had GGO. Crazy-paving, vascular enlargement, air bronchus gram sign, and air trapping were seen in 22.55%, 61.79%, and 11.76%, respectively. The most common regions of the lung involvement were lower lobe 91.70%. Also, 66.34% of patients had peripheral involvement, and had punctate ground glass opacities (64.73%). Conclusion: COVID-19 is still a leading epidemic infection with comorbidities, clinical, laboratory and radiological findings, and its specific demographic characteristics. We found the rate of mortality of patients with COVID-19 is also decreasing, gradually. It could be related to the early recognition, early intervention, and early centered-quarantine of people in the world. Funding Statement: The author(s) received no specific funding for this work. Declaration of Interest: The authors have declared that no competing interests exist.

Enzyme-powered Janus platelet cell robots for active and targeted drug delivery.

Transforming natural cells into functional biocompatible robots capable of active movement is expected to enhance the functions of the cells and revolutionize the development of synthetic micromotors. However, present cell-based micromotor systems commonly require the propulsion capabilities of rigid motors, external fields, or harsh conditions, which may compromise biocompatibility and require complex actuation equipment. Here, we report on an endogenous enzyme-powered Janus platelet micromotor (JPL-motor) system prepared by immobilizing urease asymmetrically onto the surface of natural platelet cells. This Janus distribution of urease on platelet cells enables uneven decomposition of urea in biofluids to generate enhanced chemophoretic motion. The cell surface engineering with urease has negligible impact on the functional surface proteins of platelets, and hence, the resulting JPL-motors preserve the intrinsic biofunctionalities of platelets, including effective targeting of cancer cells and bacteria. The efficient propulsion of JPL-motors in the presence of the urea fuel greatly enhances their binding efficiency with these biological targets and improves their therapeutic efficacy when loaded with model anticancer or antibiotic drugs. Overall, asymmetric enzyme immobilization on the platelet surface leads to a biogenic microrobotic system capable of autonomous movement using biological fuel. The ability to impart self-propulsion onto biological cells, such as platelets, and to load these cellular robots with a variety of functional components holds considerable promise for developing multifunctional cell-based micromotors for a variety of biomedical applications.

Serum Ceruloplasmin Is the Candidate Predictive Biomarker for Acute Aortic Dissection and Is Related to Thrombosed False Lumen: a Propensity Score-Matched Observational Case-Control Study.

Acute aortic dissection (AAD), one of the fatal diseases observed at the department of vascular surgery, is associated with a great mortality rate at the early stage. Ceruloplasmin (CP) is the plasma protein that functions as a copper transporter. The current retrospective research was carried out to assess CP contents and to examine the possible part in diagnosing patients with AAD. In addition, propensity score matching (PSM) was also utilized for reducing the bias in case screening as well as the clinical confounders. Using PSM, this study included 85 pairs of AAD cases (Stanford A and B dissection) and matched controls, and their CP levels were also detected through enzyme-linked immunosorbent assay (ELISA). Additionally, the relative clinical data were extracted from participants included in this study. After PSM adjustment for clinical variables, including gender, age, body mass index (BMI), heart ratio (HR), smoking, hypertension, diabetes mellitus, coronary heart disease (CHD), and stroke, the serum CP contents among AAD cases were remarkably increased compared with those among the normal subjects. Besides, the CP contents showed independent association with the AAD risk. Typically, the CP level was significantly positively correlated with platelet (R = 0.329) or C-reactive protein (R = 0.340) level. Meanwhile, the area under the receiver operating characteristic (ROC) curve (AUC) was 0.929 when CP was used to diagnose AAD, and the best threshold value was 36.82mg/dL. Serum CP content significantly increased in cases with thrombosed false lumen (FL) relative to those in patent FL cases. Results of logistic regression analysis suggested that a greater CP content indicated an increased thrombosed FL risk (OR = 1.11; 95% CI: 1.01-1.23; P = 0.040). Findings in this study suggest that serum ceruloplasmin contents evidently increased among acute aortic dissection cases. CP shows close correlation with the inflammatory factors among AAD cases. Further, CP may serve as the candidate biomarker to diagnose AAD and to identify an increased risk of thrombosed false lumen.

Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus

AbstractAnticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre− mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

Effects of differences in pre-analytical processing on blood protein profiles determined with SWATH-MS

The purpose of the present study was to assess the differences between the human serum and plasma proteomes, and the stability of human plasma proteins under different storage conditions following blood collection, by means of SWATH-MS analysis. When we compared plasma and serum prepared immediately after blood sampling, 95.5% of 176 quantified proteins differed by less than 1.5-fold. When we compared plasma samples prepared by centrifugation after storage of blood at room temperature for 0, 15 or 30 min, or under refrigeration at 0–5 °C for 1, 4 or 8 h, no protein showed a significant change (q < 0.05) that amounted to 1.5-fold or more, except hemoglobins. Those proteins were greatly increased in a single sample at 8 h, probably due to hemolysis. Comparison of data from the same samples indicates that the blood proteome is more stable than the blood metabolome. The present results suggest that most components of the proteome are essentially the same in plasma and serum, and are stable under the storage conditions examined in the present study. However, it may be important to pay attention to the extent of coagulation, and levels of platelet and hemolysis-related proteins. SignificancePre-analytical processing and storage conditions after blood collection are expected to influence the blood proteome. Therefore, we investigated differences in the proteome between human serum and plasma, as well as the stability of human plasma proteins under different storage conditions: at room temperature for 0–30 min, or at 0–5 °C for 1–8 h, which may reflect the clinical situation of blood collection. Proteomics analysis with SWATH-MS identified 342 proteins, and 176 proteins quantified with two or more unique peptides were compared. The levels of most components of the proteome were similar in plasma and serum, and were stable under the storage conditions examined. However, it is necessary to consider the possibility of coagulation, as this affects the levels of platelet and hemolysis-related proteins. Interestingly, the blood proteome appears to be more stable than the blood metabolome, based on previously reported metabolomics data with same samples. These data will be helpful in designing protocols for blood sampling and for blood biomarker discovery and validation.

Opposite Modulatory Effects of Crataegus aronia Aqueous Extract on Platelet Aggregation in Rats.

To reveal the mechanisms behind the dual effects of Crataegus aronia (C. aronia) aqueous extract on platelet aggregation by focusing on function, regulation, expression, and signaling of platelets P2Y12 receptors. Adult male Wistar rats (120 ± 10 g) were classified as control received the vehicle, C. aronia (200 mg/kg), and C. aronia (2,000 mg/kg)-treated rats. After treatments for consecutive 7 days, hematological and molecular experiments were conducted to detect alterations in platelet aggregation, thromboxane B2 (THXB2) and intracellular reactive oxygen species (ROS) content; protein levels of P2Y12, p-Akt, cyclic adenosine monophosphate (cAMP), phosphorylated vasodilator-stimulated-phosphoprotein (p-VASP), nuclear factor κB (NF-κB), P-selectin, and etc. in platelets were determined by Western blot; mRNA expressions of P2Y12 and some inflammatory markers were determined by real-time polymerase chain reaction. At a concentration of 200 mg/kg, C. aronia inhibited platelet aggregation through multiple interconnected mechanisms including downregulation P2Y12 synthesis and expression, stimulating intracellular cAMP levels and protein levels of p-VASP, inhibiting platelets THXB2 release and protein levels of P-selectin. Also, it inhibited platelets level of ROS and of NF-κB, a major signaling pathway that stimulates the expression of P2Y12 and THXA2 synthesis. Opposite findings were seen in platelets of rats received C. aronia at a concentration of 2,000 mg/kg. Interestingly, co-administration of N-acetylcysteine prevented all hematological and molecular alterations exerted by the high dose of the extract and inhibited platelet aggregation. Oral administration of C. aronia at low dose inhibits platelet aggregation by reducing THXB2 release, expression of P-selectin and activating cAMP and Akt signaling through two major mechanisms including downregulation of P2Y12 and inhibition of ROS-induced activation of NF-κB, an effect that is observed to be in the opposite direction with its high dose.

Expression levels of blood platelet and C-reactive protein in patients with severely pneumonic and their predictive values for efficacy

This paper aims to detect the expression levels of blood platelet (PLT) and C-reactive protein (CRP) in severely pneumonic patients and analyze their correlation. For this purpose, eighty-one severely pneumonic patients were retrospectively selected as an observation group and 106 healthy people as a control group. Pretreatment and post-treatment expression levels of PLT and CRP, their predictive values for efficacy, and correlation of PLT, CRP, and PSI scores in observation group after treatment were analyzed. Before treatment, the expression level of PLT in the observation group was higher than the control group (P&lt; 0.05). In the observation group, the expression level of PLT after treatment was significantly lower than that before treatment (P&lt; 0.05). Before treatment, the expression level of CRP in the observation group was higher than the control group (P&lt; 0.05). In the observation group 1) the pretreatment PLT expression level was higher than that in the control group; 2) the post-treatment PLT expression level was significantly lower than that in the pretreatment one; 3) the pretreatment CRP expression level was higher than that in the control group; and 4) the post-treatment CRP expression level was significantly lower than the pretreatment one (All P-values&lt; 0.05). Based upon the efficacy, the observation group was divided into an effective group and an invalid group. The post-treatment expression levels of PLT and CRP in the effective group were lower than those in the invalid group (P&lt; 0.05). Based upon the ROC curve, the area under curves (AUC) of PLT, CRP, and joint detection were 0.843, 0.864, and 0.886, respectively. When the cut-off point was &gt; 0.579, the best specificity and sensitivity were 98.44 and 70.59%, respectively. According to the Pearson test, positive correlations existed between PLT and CRP, between PLT and PSI scores, and between CRP and PSI scores. In conclusion, the expression levels of PLT and CRP in severely pneumonic patients might be used to evaluate the efficacy and conducive to detection of the disease, which have high application values in clinic.

Polyelectrolyte multilayers containing a tannin derivative polyphenol improve blood compatibility through interactions with platelets and serum proteins.

To develop hemocompatible surfaces, a cationic tannin derivate (TN) was used to prepare polyelectrolyte multilayers (PEMs) with the glycosaminoglycans heparin (HEP) and chondroitin sulfate (CS). The surface chemistry of the PEMs was characterized using X-ray photoelectron spectroscopy and water contact angle measurements. PEMs assembled with chitosan (CHI) and HEP or CS were used as controls. We investigate the hemocompatibility of PEMs by analyzing the adsorption of key blood serum proteins, adhesion and activation of platelets, and blood clotting kinetics. TN- and CHI-based PEMs adsorb similar amounts of albumin, whereas fibrinogen adsorption was more pronounced on TN-based PEMs, due to strong association with catechol groups. However, TN-based PEMs significantly reduce both platelet adhesion and platelet activation, while CHI-based PEMs promote platelet adhesion and activation. The whole-blood clotting kinetics assay also shows lower blood coagulation on TN-based PEMs. TN is an amphoteric, cationic, condensed tannin derivative with resonance structures. It also contains catechol groups, which are similar to those in mussel adhesive protein. These chemical features enable strong association with fibrinogen, which promotes the platelet-repelling effect. This study provides a new perspective for understanding platelet adhesion and activation on biomaterial surfaces, toward the development of new blood-compatible surfaces using a tannin derivative-based polymer.