Blood coagulation is regulated through protein-protein and protein-lipid interactions that occur at the sub-endothelium following vascular damage. Soluble clotting proteins bind to membrane components in a phosphatidylserine (PS) dependent manner to assemble multi-protein complexes that regulate clot formation; however, PS is of limited abundance physiologically. In this manuscript, we investigate synergy between PS and phosphatidylethanolamine (PE)-a lipid of much higher abundance naturally. Using a label-free, silicon photonic technology, we constructed arrays of Nanodiscs having variable lipid composition and probed the binding interactions of seven different clotting factors with GLA domains that have never been studied in tandem experiments before. The factors studied were prothrombin, activated factor VII, factor IX, factor X, activated protein C, protein S, and protein Z. Equilibrium dissociation constants (Kd) for each coagulation factor binding to Nanodiscs with unique compositions of PE and PS were determined. While all factors showed greater binding affinities in the presence of PS and PE, the most dramatic improvements in binding were observed when PS quantities were lowest. This demonstrates that synergy is effective in promoting coagulation factor binding under physiological lipid compositions, as opposed to the artificially high PS content probed in most in vitro activity studies.

Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo, we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi.

The general prognosis of patients with hepatocellular carcinoma (HCC) remains extremely dismal, due to the high frequency of metastasis. Since 2003, our research group has explored the gene expression profiles of metastasized HCC tissue samples and identified a significant upregulation of CCN3. However, the role and precise pathological function of CCN3 remains elusive. We showed that CCN3 is associated with the poor prognosis of patients with HCC, the malignant phenotype of HCC, and vascular thrombosis. We further evaluated the negative roles of CCN3 in vitro and in vivo, and identified osteopontin (OPN), and coagulation factors tissue factor (TF) and thrombin as the leading genes downstream of CCN3, that are positively associated with HCC cell stemness. We demonstrated that overexpressed CCN3 in HCC cells leads to enhanced survival and increased number of pulmonary metastases in vivo. The elevated levels of OPN and TF were associated with signal activation of nuclear factor κB (NFκB) and extracellular signal-regulated kinases (ERK). Our findings suggest CCN3 is a potential therapeutic target that would affect the upregulation of OPN and coagulation factors, which would lead to an enhanced stemness and blood coagulation microenvironment in HCC tissue.

Silver nanoparticles (AgNPs) have tremendous potentials in medical devices due to their excellent antimicrobial properties. Blood compatibility should be investigated for AgNPs due to the potential blood contact. However, so far, most studies are not systematic and have not provided insights into the mechanisms for blood compatibility of AgNPs. In this study, we have investigated the blood biological effects, including hemolysis, lymphocyte proliferation, platelet aggregation, coagulation and complement activation, of 20 nm AgNPs with two different surface coatings (polyvinyl pyrrolidone and citrate). Our results have revealed AgNPs could elicit hemolysis and severely impact the proliferation and viability of lymphocytes at all investigated concentrations (10, 20, 40 μg/mL). Nevertheless, AgNPs didn't show any effect on platelet aggregation, coagulation process, or complement activation at up to ~40 μg/mL. Proteomic analysis on AgNPs plasma proteins corona has revealed that acidic and small molecular weight blood plasma proteins were preferentially adsorbed onto AgNPs, and these include some important proteins relevant to hemostasis, coagulation, platelet, complement activation and immune responses. The predicted biological effects of AgNPs by proteomic analysis are mostly consistent with our experimental data since there were few C3 components on AgNPs and more negative than positive factors involving platelet aggregation and thrombosis.

Myocardial injury after non-cardiac surgery (MINS) is common. We investigated the incidence and outcomes of MINS, and mechanistic underpinnings using pre-operative whole blood gene expression profiling in a prospective cohort study of individuals undergoing lower extremity revascularization (LER) for peripheral artery disease (PAD). Major adverse cardiovascular and limb events (MACLE) were defined as a composite of death, myocardial infarction, stroke, major lower extremity amputation or reoperation. Among 226 participants undergoing LER, MINS occurred in 53 (23.5%). Patients with MINS had a greater incidence of major adverse cardiovascular events (49.1% vs. 22.0%, adjusted HR 1.87, 95% CI 1.07-3.26) and MACLE (67.9% vs. 44.5%; adjusted HR 1.66, 95% CI 1.08-2.55) at median 20-month follow-up. Pre-operative whole blood transcriptome profiling of a nested matched MINS case-control cohort (n = 41) identified upregulation of pathways related to platelet alpha granules and coagulation in patients who subsequently developed MINS. Thrombospondin 1 (THBS1) mRNA expression was 60% higher at baseline in patients who later developed MINS, and was independently associated with long-term cardiovascular events in the Duke Catheterization Genetics biorepository cohort. In conclusion, pre-operative THBS1 mRNA expression is higher in patients who subsequently develop MINS and is associated with incident cardiovascular events. Pathways related to platelet activity and coagulation associated with MINS provide novel insights into mechanisms of myocardial injury.

The indications for laparoscopic liver resection have expanded; however, the safety and benefits of laparoscopic repeat hepatectomy (LRH) remain unclear. We retrospectively reviewed data from 137 patients who underwent partial hepatectomy or left lateral sectionectomy without thoracotomy. We compared patients' clinical factors using a difficulty scoring system for LRH. We defined factors associated with blood loss volumes in the 75th percentile or above as risk factors for bleeding in open repeat hepatectomy, and determined whether these factors were useful for LRH risk assessment. Open repeat hepatectomy and LRH was performed in 96 and 41 patients, respectively. Four of 41 (9.8%) patients undergoing LRH were converted to laparotomy. Blood loss volume was significantly greater in the intermediate-risk group than in the low-risk group (P = 0.046). Multivariate analysis revealed that the presence of tumours located adjacent and caudal or dorsal to the primary tumour site was an independent risk factor for bleeding in LRH (odds ratio 3.21, 95% confidence interval 1.16-8.88, P = 0.024). Our study validated the usefulness of a difficulty scoring system, identified patient factors that predicted the difficulty of LRH, and presented a novel difficulty scoring system for LRH based on an existing difficulty scoring system.

Prothrombin complex concentrates (PCC) are fractionated plasma protein drugs that reverse warfarin anticoagulation. PCC may control more general bleeding. We sought to identify the dominant procoagulant factor in PCC in vivo. We tested PCC or coagulation factor (F) treatment in CD1 mice made coagulopathic by exchange of whole blood for washed red cells. Anesthetized mice were transfused with murine fresh-frozen plasma (mFFP), PCC, mixtures of human vitamin K-dependent proteins (VKDP) (prothrombin, FVII, FIX, or FX), or purified single human VKDP, immediately prior to tail transection (TT), liver laceration (LL), or intravascular laser injury (ILI). Plasma donor mice were treated with vehicle or control antisense oligonucleotide (ASO-CON) or ASO specific for prothrombin (FII) (ASO-FII) to yield mFFP or ASO-CON mFFP or ASO-FII mFFP. Blood losses were determined spectrophotometrically (TT) or gravimetrically (LL). Thrombus formation was quantified by intravital microscopy of laser-injured arterioles. PCC or four factor- (4F-) VKDP or prothrombin significantly reduced bleeding from TT or LL. Omission of prothrombin from 4F-VKDP significantly reduced its ability to limit bleeding. Mice transfused with ASO-FII mFFP demonstrated inferior haemostasis versus those transfused with ASO-FII following TT, LL, or ILI. Prothrombin is the dominant procoagulant component of PCC and could limit bleeding in trauma.

Neisseria meningitidis (meningococcus) causes invasive diseases such as meningitis or septicaemia. Ex vivo infection of human whole blood is a valuable tool to study meningococcal virulence factors and the host innate immune responses. In order to consider effects of cellular mediators, the coagulation cascade must be inhibited to avoid clotting. There is considerable variation in the anticoagulants used among studies of N. meningitidis whole blood infections, featuring citrate, heparin or derivatives of hirudin, a polypeptide from leech saliva. Here, we compare the influence of these three different anticoagulants, and additionally Mg/EGTA, on host innate immune responses as well as on viability of N. meningitidis strains isolated from healthy carriers and disease cases, reflecting different sequence types and capsule phenotypes. We found that the anticoagulants significantly impact on cellular responses and, strain-dependently, also on bacterial survival. Hirudin does not inhibit complement and is therefore superior over the other anticoagulants; indeed hirudin-plasma most closely reflects the characteristics of serum during N. meningitidis infection. We further demonstrate the impact of heparin on complement activation on N. meningitidis and its consequences on meningococcal survival in immune sera, which appears to be independent of the heparin binding antigens Opc and NHBA.

Prothrombin time (PT) and the associated international normalized ratio (INR) are routinely tested to assess the risk of bleeding or thrombosis and to monitor response to anticoagulant therapy in patients. To measure PT/INR, conventional coagulation testing (CCT) is performed, which is time-consuming and requires the separation of cellular components from whole blood. Here, we report on a portable and battery-operated optical sensor that can rapidly quantify PT/INR within seconds by measuring alterations in the viscoelastic properties of a drop of whole blood following activation of coagulation with thromboplastin. In this study, PT/INR values were measured in 60 patients using the optical sensor and compared with the corresponding CCT values. Our results report a close correlation and high concordance between PT/INR measured using the two approaches. These findings confirm the accuracy of our optical sensing approach for rapid PT/INR testing in whole blood and highlight the potential for use at the point-of-care or for patient self-testing.

Psychosocial stressors - life events that challenge social support and relationships - represent powerful risk factors for human disease; included amongst these events are relocation, isolation and displacement. To evaluate the impact of a controlled psychosocial stressor on physiology and underlying molecular pathways, we longitudinally studied the influence of a 28-day period of quarantine on biomarkers of immune signalling, microbial translocation, glycaemic health and blood transcriptome in the wild-born vervet monkey. This event caused a coordinated, mostly transient, reduction of circulating levels of nine immune signalling molecules. These were paralleled by a massive dysregulation of blood transcriptome, including genes implicated in chronic pathologies and immune functions. Immune and inflammatory functions were enriched among the genes downregulated in response to stress. An upregulation of genes involved in blood coagulation, platelet activation was characteristic of the rapid response to stress induction. Stress also decreased neutrophils and increased CD4 + T cell proportions in blood. This model of psychosocial stress, characterised by an immune dysregulation at the transcriptomic, molecular and cellular levels, creates opportunities to uncover the underlying mechanisms of stress-related diseases with an immune component, including cardiovascular diseases and susceptibility to infections.

A single tumor marker is not enough to predict the breast pathologic complete response (bpCR) after neoadjuvant chemotherapy (NAC) in breast cancer patients. We aimed to establish a nomogram based on multiple clinicopathological features and routine serological indicators to predict bpCR after NAC in breast cancer patients. Data on clinical factors and laboratory indices of 130 breast cancer patients who underwent NAC and surgery in First Affiliated Hospital of Xi'an Jiaotong University from July 2017 to July 2019 were collected. Multivariable logistic regression analysis identified 11 independent indicators: body mass index, carbohydrate antigen 125, total protein, blood urea nitrogen, cystatin C, serum potassium, serum phosphorus, platelet distribution width, activated partial thromboplastin time, thrombin time, and hepatitis B surface antibodies. The nomogram was established based on these indicators. The 1000 bootstrap resampling internal verification calibration curve and the GiViTI calibration belt showed that the model was well calibrated. The Brier score of 0.095 indicated that the nomogram had a high accuracy. The area under the curve (AUC) of receiver operating characteristic (ROC) curve was 0.941 (95% confidence interval: 0.900-0.982) showed good discrimination of the model. In conclusion, this nomogram showed high accuracy and specificity and did not increase the economic burden of patients, thereby having a high clinical application value.

Extracellular histones promote platelet aggregation and thrombosis; this is followed by induction of coagulation disorder, which results in exhaustion of coagulation factors. Complement component 5 (C5) is known to be associated with platelet aggregation and coagulation system activation. To date, the pathological mechanism underlying liver injury has remained unclear. Here, we investigated whether C5 promotes liver injury associated with histone-induced lethal thrombosis. C5-sufficient and C5-deficient mice received single tail vein injections of purified, unfractionated histones obtained from calf thymus (45-75 μg/g). Subsequently, the mice were monitored for survival for up to 72 h. Based on the survival data, the 45 μg/g dose was used for analysis of blood cell count, liver function, blood coagulation ability, and promotion of platelet aggregation and platelet/leukocyte aggregate (PLA) production by extracellular histones. C5-deficient mice were protected from lethal thrombosis and had milder thrombocytopenia, consumptive coagulopathy, and liver injury with embolism and lower PLA production than C5-sufficient mice. These results indicate that C5 is associated with coagulation disorders, PLA production, and embolism-induced liver injury. In conclusion, C5 promotes liver injury associated with histone-induced lethal thrombosis.

Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development. The M358R variant of the serpin alpha-1 antitrypsin (AAT) inhibits both FXIa and other proteases. Our aim was to enhance the specificity of AAT M358R for FXIa. We randomized two AAT M358R phage display libraries at reactive centre loop positions P13-P8 and P7-P3 and biopanned them with FXIa. A bacterial expression library randomized at P2'-P3' was also probed. Resulting novel variants were expressed as recombinant proteins in E. coli and their kinetics of FXIa inhibition determined. The most potent FXIa-inhibitory motifs were: P13-P8, HASTGQ; P7-P3, CLEVE; and P2-P3', PRSTE (respectively, novel residues bolded). Selectivity for FXIa over thrombin was increased up to 34-fold versus AAT M358R for these single motif variants. Combining CLEVE and PRSTE motifs in AAT-RC increased FXIa selectivity for thrombin, factors XIIa, Xa, activated protein C, and kallikrein by 279-, 143-, 63-, 58-, and 36-fold, respectively, versus AAT M358R. AAT-RC lengthened human plasma clotting times less than AAT M358R. AAT-RC rapidly and selectively inhibits FXIa and is worthy of testing in vivo. AAT specificity can be focused on one target protease by selection in phage and bacterial systems coupled with combinatorial mutagenesis.

The aim of the present study was to compare different diets used to induce obesity in a head-to-head manner with a focus on insulin resistance and vascular dysfunction. Male C57BL/6J mice were put on standard chow diet (SCD), normal-fat diet (NFD), cafeteria diet (CAF) or high-fat diet (HFD) for 12 weeks starting at the age of 6 weeks. Both CAF and HFD led to obesity (weight gain of 179% and 194%, respectively), glucose intolerance and insulin resistance to a comparable extent. In aortas containing perivascular adipose tissue (PVAT), acetylcholine-induced vasodilation was best in the NFD group and worst in the CAF group. Reduced phosphorylation of endothelial nitric oxide synthase at serine 1177 was observed in both CAF and HFD groups. Plasma coagulation activity was highest in the HFD group and lowest in the SCD group. Even the NFD group had significantly higher coagulation activity than the SCD group. In conclusions, CAF and HFD are both reliable mouse diets in inducing visceral obesity, glucose intolerance and insulin resistance. CAF is more effective than HFD in causing PVAT dysfunction and vascular dysfunction, whereas hypercoagulability was mostly evident in the HFD group. Coagulation activity was higher in NFD than NCD group.

Non-traumatic osteonecrosis of the femoral head (ONFH) relies on multiple pathogenic factors, including intravascular coagulation, osteoporosis and lipid metabolism disorders. Despite extensively explored from various aspects, genetic mechanism underlying non-traumatic ONFH has not been fully elucidated. We randomly collected blood and necrotic tissue samples from 32 patients with non-traumatic ONFH as well as blood samples from 30 healthy individuals for whole exome sequencing (WES). Germline mutation and somatic mutation were analyzed to identify new potential pathogenic genes responsible for non-traumatic ONFH. Three genes might correlate with non-traumatic ONFH: VWF, MPRIP (germline mutations) and FGA (somatic mutations). Germline or somatic mutations in VWF, MPRIP and FGA correlate with intravascular coagulation, thrombosis, and consequently, ischemic necrosis of the femoral head.

Blood coagulation is a vital process for humans and other species. Following an injury to a blood vessel, a cascade of molecular signals is transmitted, inhibiting and activating more than a dozen coagulation factors and resulting in the formation of a fibrin clot that ceases the bleeding. In this process, the Coagulation factor V (FV) is a master regulator, coordinating critical steps of this process. Mutations to this factor result in spontaneous bleeding episodes and prolonged hemorrhage after trauma or surgery. Although the role of FV is well characterized, it is unclear how single-point mutations affect its structure. In this study, to understand the effect of mutations, we created a detailed network map of this protein, where each node is a residue, and two residues are connected if they are in close proximity in the three-dimensional structure. Overall, we analyzed 63 point-mutations from patients and identified common patterns underlying FV deficient phenotypes. We used structural and evolutionary patterns as input to machine learning algorithms to anticipate the effects of mutations and anticipated FV-deficiency with fair accuracy. Together, our results demonstrate how clinical features, genetic data and in silico analysis are converging to enhance treatment and diagnosis of coagulation disorders.

This in vitro study evaluated the potential hemostatic effect of fresh frozen plasma (FFP) ultrafiltration on clotting factors, coagulation parameters, and plasma properties. ABO-specific units of FFP (n = 40) were prepared for the concentrated FFP and cryoprecipitate. Plasma water was removed from FFP by ultrafiltration using a dialyzer with a pump running at a 300 mL/min. The aliquot of each concentrated FFP after 50, 100, 200, and 250 mL of fluid removal were measured the standard coagulation assay, clotting activity, and plasma properties to compare those parameters of cryoprecipitate. Concentrated FFP contained 36.5% of fibrinogen in FFP with a mean concentration of 7.2 g/L, lower than the cryoprecipitate level. The levels of factor VIII (FVIII), von Willebrand factor (VWF):antigen (Ag), and VWF:ristocetin cofactor (RCo) were also lower in concentrated FFP, whereas the levels of factor V, factor IX, factor XIII, antithrombin and albumin was higher in concentrated FFP. Maximum clot firmness (MCF) in thromboelastometry was approximately one-half of that in cryoprecipitate. Although the levels of VWF:Ag, VWF:RCo, and FVIII differed depending on the ABO blood types, fibrinogen levels, and MCF were not significantly different among the ABO blood groups in FFP and concentrated FFP.

Blood coagulation factor VIII is a glycoprotein cofactor that is essential for the intrinsic pathway of the blood coagulation cascade. Inhibitory antibodies arise either spontaneously or in response to therapeutic infusion of functional factor VIII into hemophilia A patients, many of which are specific to the factor VIII C2 domain. The immune response is largely parsed into "classical" and "non-classical" inhibitory antibodies, which bind to opposing faces cooperatively. In this study, the 2.61 Å resolution structure of the C2 domain in complex with the antigen-binding fragment of the 3E6 classical inhibitory antibody is reported. The binding interface is largely conserved when aligned with the previously determined structure of the C2 domain in complex with two antibodies simultaneously. Further inspection of the B factors for the C2 domain in various X-ray crystal structures indicates that 3E6 antibody binding decreases the thermal motion behavior of surface loops in the C2 domain on the opposing face, thereby suggesting that cooperative antibody binding is a dynamic effect. Understanding the structural nature of the immune response to factor VIII following hemophilia A treatment will help lead to the development of better therapeutic reagents.

Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1-/- mice, but not Epac2-/- mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1-/- mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1-/- mouse platelets (thrombocytes) had unbalanced expression of key components of the glycoprotein Ib-IX-V (GPIb-IX-V) complex, with decrease of GP1bβ and no change of GP1bα. This complex is critical for platelet adhesion under arterial shear conditions. Furthermore, Epac1-/- mice have reduced levels of plasma coagulation factors and fibrinogen, increased size of circulating platelets, increased megakaryocytes (the GP1bβ level was decreased also in Epac1-/- bone marrow) and higher abundance of reticulated platelets. Viscoelastic measurement of clotting function revealed Epac1-/- mice with a dysfunction in the clotting process, which corresponds to reduced plasma levels of coagulation factors like factor XIII and fibrinogen. We propose that the observed platelet phenotype is due to deficient Epac1 activity during megakaryopoiesis and thrombopoiesis, and that the defects in blood clotting for Epac1-/- is connected to secondary hemostasis.

Although coagulation abnormalities, including microvascular thrombosis, are thought to contribute to tissue injury and single- or multiple-organ dysfunction in severe influenza, the detailed mechanisms have yet been clarified. This study evaluated influenza-associated abnormal blood coagulation utilizing a severe influenza mouse model. After infecting C57BL/6 male mice with intranasal applications of 500 plaque-forming units of influenza virus A/Puerto Rico/8/34 (H1N1; PR8), an elevated serum level of prothrombin fragment 1 + 2, an indicator for activated thrombin generation, was observed. Also, an increased gene expression of oxidized low-density lipoprotein (LDL) receptor-1 (Olr1), a key molecule in endothelial dysfunction in the progression of atherosclerosis, was detected in the aorta of infected mice. Body weight decrease, serum levels of cytokines and chemokines, viral load, and inflammation in the lungs of infected animals were similar between wild-type and Olr1 knockout (KO) mice. In contrast, the elevation of prothrombin fragment 1 + 2 levels in the sera and intravascular thrombosis in the lungs by PR8 virus infection were not induced in KO mice. Collectively, the results indicated that OLR1 is a critical host factor in intravascular thrombosis as a pathogeny of severe influenza. Thus, OLR1 is a promising novel therapeutic target for thrombosis during severe influenza.