Enhanced Coagulation Activation Research Articles

Thrombocytopenia is associated with a dysregulated host response in severe COVID-19

BackgroundThrombocytopenia is associated with increased mortality in COVID-19 patients. ObjectiveTo determine the association between thrombocytopenia and alterations in host response pathways implicated in disease pathogenesis in patients with severe COVID-19. Patients/methodsWe studied COVID-19 patients admitted to a general hospital ward included in a national (CovidPredict) cohort derived from 13 hospitals in the Netherlands. In a subgroup, 43 host response biomarkers providing insight in aberrations in distinct pathophysiological domains (coagulation and endothelial cell function; inflammation and damage; cytokines and chemokines) were determined in plasma obtained at a single time point within 48 h after admission. Patients were stratified in those with normal platelet counts (150-400 × 109/L) and those with thrombocytopenia (<150 × 109/L). Results6.864 patients were enrolled in the national cohort, of whom 1.348 had thrombocytopenia and 5.516 had normal platelets counts; the biomarker cohort consisted of 429 patients, of whom 85 with thrombocytopenia and 344 with normal platelet counts. Plasma D-dimer levels were not different in thrombocytopenia, although patients with moderate-severe thrombocytopenia (<100 × 109/L) showed higher D-dimer levels, indicating enhanced coagulation activation. Patients with thrombocytopenia had lower plasma levels of many proinflammatory cytokines and chemokines, and antiviral mediators, suggesting involvement of platelets in inflammation and antiviral immunity. Thrombocytopenia was associated with alterations in endothelial cell biomarkers indicative of enhanced activation and a relatively preserved glycocalyx integrity. ConclusionThrombocytopenia in hospitalized patients with severe COVID-19 is associated with broad host response changes across several pathophysiological domains. These results suggest a role of platelets in the immune response during severe COVID-19.

Systemic inflammation and portal vein thrombosis in cirrhotic patients with gastroesophageal varices.

Cirrhotic patients with gastroesophageal varices and non-tumoral portal vein thrombosis have a higher risk of re-bleeding and poor prognosis. This study aimed to analyze inflammatory biomarkers and thromboelastography in cirrhotic patients with portal vein thrombosis. A total of 385 consecutive cirrhotic patients with gastroesophageal varices were prospectively enrolled between 1 December 2016, and 31 August 2017. Of these, 231 were eligible for analysis and were divided into portal vein thrombosis (n = 103) and non-portal vein thrombosis (n = 128) groups based on computerized tomography angiography findings. Patients with portal vein thrombosis generally had higher Child-Pugh scores than those without portal vein thrombosis (6.38 ± 0.12 vs. 5.81 ± 0.09, P < 0.001). The serum albumin levels were significantly lower in patients with portal vein thrombosis (35.90 ± 0.52 vs. 38.52 ± 0.43, P < 0.001). The portal vein thrombosis group had significant higher serum levels of interleukin 6 [4.85 (3.15-6.99) vs. 3.09 (2.06-5.20) pg/ml, P < 0.001] and tumor necrosis factor alpha [10.70 (7.60-15.20) vs. 9.07 (7.03-11.60) pg/ml, P = 0.020]. The interleukin 6 level was 2.5-fold higher in patients with portal vein thrombosis (adjusted odds ratio: 2.574; 95% confidential interval: 1.248-5.310). Thromboelastography showed that TEG-R, the reaction time, was significantly lower in the portal vein thrombosis group [5.20 (4.80-6.30) vs. 6.00 (5.20-6.95), P = 0.009], indicating enhanced coagulation activity. This study confirmed the important role of systemic inflammation in portal vein thrombosis. Interleukin 6, an important inflammatory cytokine, is independently associated with portal vein thrombosis. The correlation between the interleukin 6 level and portal vein thrombosis requires further investigation.

Role of Protease Exosites for In Vivo Hemostatic Activity and Clearance of FIX

Introduction:FIX(a) binds rapidly and reversibly to vascular endothelium and extravascular matrix, in part mediated by the interaction of specific Gla domain residues with collagen IV. Both anticoagulant heparan sulphate and collagen IV localize predominantly to the basement membrane and supramolecular structure analysis suggests that heparan sulphate chains are integrated into collagen IV-containing networks. Previous work has demonstrated the contribution of the heparin and antithrombin (AT)-binding exosites in the protease domain to the regulation of human FIXa. Here we evaluate the contribution of these protease exosites to FIX hemostatic function and clearance in the hemophilia B (HB) mouse.Methods:Recombinant human FIX variants expressed in HEK293 cells with substitutions in the heparin- (K126A/K132A or R170A) or AT-binding (R150A) exosites (chymotrypsinogen #) were purified to homogeneity. In vivohemostasis was assessed in a saphenous vein bleeding model in HB mice. Anesthetized adult mice were injected with a weight-based dose predicted to achieve 30% FIX plasma concentrations and the exposed left saphenous vein was punctured with a 29 gauge needle. Clot formation at the injury site was repeatedly disrupted over 30 min to determine the average time to clot formation and total #clots/30 min. FIX pharmacokinetics (PK) in HB mice were determined with a weight-based dose injected into the tail vein. Serial retro-orbital blood samples were collected, plasma isolated and FIX content determined with a human FIX specific ELISA. FIX concentrations were plotted versus time and initially fit to the equation: [IX]t = [IX]max*(C1*e(-k1*t)+ C2*e(-k2*t)), where k1 and k2 are rate constants for the initial and terminal elimination phases, respectively. As the 4-parameter fit resulted in some large error estimates, the two phases were fit separately to estimate the respective parameters.Results:Previous characterization of APTT-based coagulant activities for these FIX variants (% WT ± SEM) were as follows: FIX WT 100 ± 1.4, R150A 73.6 ± 1.4, K126A/K132A 20.6 ± 9.2, and R170A 609.2 ± 37. In the saphenous vein bleeding assay, HB mice had no clot formation over 30 min, while wild type mice demonstrated 11 clots/30 min. When HB mice were injected with increasing concentrations of FIX WT (20%-100%), a dose dependent increase was observed from 4.3 to 16.5 clots/30 min (Table 1). When injected with sufficient FIX to achieve 30% plasma levels, the AT-binding exosite variant FIX R150A and heparin binding site variant K126A/K132A demonstrated similar hemostatic activity to FIX WT in the HB mouse. FIX R170A (30%) trended towards enhanced activity relative to WT, while 10% FIX R170A levels demonstrated similar activity to 30% FIX WT (not shown).The PK (5 min to 25 hr) of FIX was examined in HB and wild type mice. FIX WT demonstrated approximately ~30% recovery (initial peak) of predicted levels. FIX K126A/K132A had markedly enhanced recovery (2.2-fold higher than FIX WT), while FIX R150A and R170A demonstrated similar recovery to FIX WT (Table 1, Figure 1). FIX K126A/K132A demonstrated ~70% of predicted recovery. A rapid decrease in FIX levels was observed over the ensuing 4-6 hours with similar k1 values for all proteins. In contrast, terminal phase k2 values trended lower and predicted half-lives longer for the heparin-binding exosite mutants (FIX K126A/K132A and R170A). Consistent with occupation of extravascular sites by endogenous FIX, PK studies in wild type mice demonstrated ~20% enhancement of FIX recovery relative to HB mice (not shown).Conclusions:In contrast to in vitrocoagulant and thrombin generation activity, FIX K126A/K132A demonstrated similar in vivohemostatic activity to FIX WT, markedly enhanced recovery and modestly slower terminal clearance. The enhanced in vivo hemostatic activity of this variant relative to coagulant activity likely results from the markedly enhanced plasma FIX recovery. In contrast, FIX R150 demonstrates similar in vivo hemostatic activity and zymogen clearance to FIX WT. FIX R170A demonstrated ~3-fold enhanced hemostatic potency and similar zymogen clearance compared to FIX WT. This hemostatic potency is consistent with the enhanced coagulant activity of this FIX Padua (R170L) related variant. In addition to the collagen IV binding site, the FIX(a) heparin binding exosite is likely to play a substantial role in FIX localization to the extravascular compartment. [Display omitted] DisclosuresSheehan:Pfizer: Research Funding; Bayer: Consultancy, Research Funding.

Early posttraumatic changes in coagulation and fibrinolysis systems in isolated severe traumatic brain injury patients and its influence on immediate outcome

Objective/backgroundEarly coagulopathy in isolated severe traumatic brain injury occurs despite the lack of severe bleeding, shock, and fluid administration. We aimed to correlate coagulation activation/inhibition, thrombin generation and fibrinolysis with the development of acute trauma induced coagulopathy (TIC) and its effects on early mortality in isolated severe traumatic brain injury (iSTBI) patients. MethodsA prospective screening of iSTBI patients was done for two years. History of anticoagulants, liver disease, hypotension, extracranial injuries, transfusion, brain death were excluded. TIC was defined as international normalized ratio (INR) ≥ 1.27 and/or prothrombin time (PT) ≥ 16.7 seconds and/or activated partial thromboplastin Time (aPTT) ≥ 28.8 seconds on admission following iSTBI. Analysis of tissue factor (TF), tissue factor pathway inhibitor (TFPI), protein C (PC), protein S (PS), thrombin/antithrombin complex (TAT), soluble fibrin monomer (sFM), tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) was done. Cases were categorized as presence or absence of TIC and 20 healthy controls participants were included. ResultsA total of 120 cases met the inclusion criteria, aged 35.7 ± 12.12 years, 96% males. TIC was identified in 50 (41.6%). TIC occurred independently of age, sex, Glasgow coma scale (GCS) but was associated with acidosis (60%; p = .01). Following iSTBI significant decline was seen in coagulation activation. Thrombin generation and fibrinolysis were markedly increased. TF, TFPI, PC and PS were low in TIC compared with control. Significant depletion of PS was seen in TIC versus No-TIC. TBI patients with depleted PS had an odds ratio (OR) of 7.10 (1.61–31.2) for TIC. Receiver operating characteristic curve (ROC) analysis depicted area under the curve (AUC) of 0.73 (95% confidence interval [CI] 0.63–0.84) with a cut-off of ≥74 of PS (specificity 63.9%, sensitivity 72.7%). In-hospital mortality was higher in TIC group (44%) compared with no-TIC (20%) with OR of 4.73 (95% CI 1.68–13.3) and hazard ratio [HR] of 2.8 (95 % CI 1.2–6.4). ConclusionIncidence of TIC in iSTBI is 41.6%, with 4.7 times odds for mortality. Traumatic brain injury causes enhanced coagulation activation, inadequate inhibition, exacerbation of thrombin generation, and subsequent increased fibrinolysis. ROC curve analysis revealed a cut-off of PS ≤ 74 with specificity 63.8%, sensitivity 72.7% for development of TIC.

Shiga toxin 2a binds antithrombin and heparin, but does not directly activate platelets

Escherichia coli-induced hemolytic uremic syndrome (eHUS) is a life-threatening complication of infection with Shiga toxin (Stx), in particular Stx2a-producing Escherichia coli. Enhanced coagulation activation with formation of microthrombi seems to be a key event in development of eHUS. Platelet activation has been postulated as a possible, but controversially debated mechanism.The present study investigated the effect of Stx2a on plasmatic coagulation and platelets. Binding studies were initially performed with ELISA and co-immunoprecipitation and supported by quartz crystal microbalance with dissipation monitoring (QCM-D). Antithrombin (AT) activity was measured using the automated BCS XP® system. ROTEM® was used for functional coagulation testing. Platelet binding and activation was studied with FACS and light-transmission aggregometry.We found binding of Stx2a to AT, an important inhibitor of blood coagulation, but only a mild albeit significant reduction of AT activity against FXa in the presence of Stx2a. QCM-D analysis also showed binding of Stx2a to heparin and an impaired binding of AT to Stx2a-bound heparin. ROTEM® using Stx2a-treated platelet-poor plasma revealed a significant, but only moderate shortening of clotting time. Neither binding nor activation of platelets by Stx2a could be demonstrated.In summary, data of this study suggest that Stx2a binds to AT, but does not induce major effects on plasmatic coagulation. In addition, no interaction with platelets occurred. The well-known non-beneficial administration of heparin in eHUS patients could be explained by the interaction of Stx2a with heparin.

AB102. P076. Preoperative panel of CA 19-9, coagulation FVIII, fibrin turnover marker D-dimer and thrombin time predicts postoperative survival in pancreatic ductal adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-associated death worldwide. It is often diagnosed at a late stage, but even after surgery survival is poor. CA 19-9 is the most used tumor marker for PDAC, and it correlates with survival. PDAC associates with enhanced coagulation activity. The aim of this study was to explore whether a combination of CA 19-9, FVIII, D-dimer and thrombin time (TT) predicts outcome after surgery better than CA 19-9 alone.

The Host Response in Patients with Sepsis Developing Intensive Care Unit-acquired Secondary Infections.

Sepsis can be complicated by secondary infections. We explored the possibility that patients with sepsis developing a secondary infection while in the intensive care unit (ICU) display sustained inflammatory, vascular, and procoagulant responses. To compare systemic proinflammatory host responses in patients with sepsis who acquire a new infection with those who do not. Consecutive patients with sepsis with a length of ICU stay greater than 48 hours were prospectively analyzed for the development of ICU-acquired infections. Twenty host response biomarkers reflective of key pathways implicated in sepsis pathogenesis were measured during the first 4 days after ICU admission and at the day of an ICU-acquired infection or noninfectious complication. Of 1,237 admissions for sepsis (1,089 patients), 178 (14.4%) admissions were complicated by ICU-acquired infections (at Day 10 [6-13], median with interquartile range). Patients who developed a secondary infection showed higher disease severity scores and higher mortality up to 1 year than those who did not. Analyses of biomarkers in patients who later went on to develop secondary infections revealed a more dysregulated host response during the first 4 days after admission, as reflected by enhanced inflammation, stronger endothelial cell activation, a more disturbed vascular integrity, and evidence for enhanced coagulation activation. Host response reactions were similar at the time of ICU-acquired infectious or noninfectious complications. Patients with sepsis who developed an ICU-acquired infection showed a more dysregulated proinflammatory and vascular host response during the first 4 days of ICU admission than those who did not develop a secondary infection.

A Novel Beneficial Role for Macitentan in Patients at High Thrombosis Risk: Attenuation of Platelet-Mediated Derangements of Blood Coagulation in Eisenmenger Syndrome

IntroductionEisenmenger syndrome is a rare manifestation of congenital cardiac disease with pulmonary hypertension. Potentially dangerous thrombotic and haemorrhagic complications frequently occur in patients with ES and raise significant clinical dilemmas. Underlying mechanisms are unknown but are thought to be linked to the underlying pulmonary vascular disease. Endothelin-1 (ET-1) signalling in the pulmonary vasculature is implicated in the pathogenesis of ES and Macitentan (a novel dual ET-1 receptor antagonist) has been shown to improve clinical outcomes. However the effect of Macitentan on reversing abnormal blood coagulation is unknown.We aimed to characterise derangements in physiological procoagulant and anticoagulant pathways in ES and to assess the effect of Macitentan therapy in modulating haemostasis in this disorder. We hypothesized that an enhanced understanding of pro-and anticoagulant mechanisms in these patients has the potential to improve risk stratification and targeting of antithrombotic therapy to patients at high risk of thrombosis.MethodsBlood was collected from subjects with ES and age/gender-matched healthy controls into citrated tubes, some containing corn trypsin inhibitor (CTI, a contact pathway inhibitor; final concentration 50µg/mL).Parameters of plasma thrombin generation in platelet-rich and platelet-poor plasma were assessed by calibrated automated thrombography (CAT) in the presence or absence of exogenous tissue factor (TF), anionic phospholipid (PL) and activated protein C (APC).ResultsPlatelet-dependent blood coagulation measured by CAT was enhanced in ES platelet-rich plasma (PRP) relative to healthy volunteers despite lower platelet counts: peak thrombin generation (TG) was significantly higher in ES subjects v controls in PRP (230.6±10.9 vs 188.3±13.1 nM; p=0.025) and lagtime to TG (6.1±0.3 vs 7.8±0.5 mins, p=0.005), time to peak TG (11.5±0.5 vs 15.2±0.6 mins, p=0.0003) and TG velocity index (43.9±3.7 vs 26.3±2.4 nM/min, p=0.001) were significantly accelerated. No significant differences in TG parameters in PPP were observed between groups suggesting a critical mechanistic role for platelet activity in supporting hypercoagulability in ES. TG in PRP was also found to be significantly enhanced in ES in the presence of CTI, suggesting that in vitro or in vivocontact pathway activation does not contribute to the enhanced coagulation activation observed in this disorder.The anticoagulant activity of APC is PL dependent and in healthy PPP incubated with exogenous PL (as a substitute for platelet PL), TG was potently inhibited. Conversely, in healthy PRP (in the absence of exogenous PL), APC activity was attenuated. Remarkably, this effect was not observed among the ES subjects, where APC anticoagulant activity was significantly increased in PRP, with significantly lower residual peak plasma thrombin generation and endogenous thrombin potential relative to that observed in healthy volunteer PRP. APC anticoagulant activity in PPP (both in the presence and absence of exogenous PL) was similar between groups. These findings suggest that platelets also modulate the activity of the APC pathway in ES, but in a manner which could promote bleeding.Among the 7 subjects who commenced macitentan therapy, at 6 months peak TG in PRP was significantly lower (234.5±9.8 vs 204.6±14.1 nM, p=0.002) and the enhanced sensitivity to APC in PRP was also attenuated. No significant difference in any parameter of TG was observed in PPP following macitentan therapy.ConclusionPlatelet activity appears to contribute significantly to hypercoagulability in ES but also appears to modulate the activity of endogenous anticoagulant pathways reflecting the clinical phenotype where bleeding and thrombotic risks frequently co-exist. Our findings suggest that targeted pharmacological agents such as the dual endothelin-1 antagonists (which crucially do not confer a bleeding risk) may attenuate platelet-mediated derangements in blood coagulation. Given the significant clinical dilemmas posed by the presence of competing thrombotic and haemorrhagic tendencies in ES, these finding are likely to be of significant translational relevance. DisclosuresWalsh:Actelion UK: Research Funding. Maguire:Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

NF-κB-dependent increase in tissue factor expression is responsible for hypoxic podocyte injury.

Fibrin deposition within glomeruli is commonly seen in kidney biopsy specimens, suggesting enhanced coagulant activity. Tissue factor (TF) is a coagulation factor which is also related to various biological effects, and TF is upregulated by hypoxia in cancer cells. Recently, hypoxic podocyte injury has been proposed, therefore, we investigated TF expression in hypoxia. Conditionally immortalized human podocytes were differentiated and treated under hypoxic or normoxic conditions. mRNA expressions of TF and tissue factor pathway inhibitor (TFPI) were analyzed by quantitative RT-PCR. Protein levels of TF and TFPI were tested by enzyme-linked immunosorbent assay. We employed small interfering RNA (siRNA) to temporary knockdown early growth response protein 1 (Egr-1), hypoxia-inducible factor-1α (HIF-1α) and TF. The expression of CD2-associated protein (CD2AP) mRNA and phalloidin staining was examined to assess podocyte injury. Hypoxia increased mRNA expression of TF (6h: 2.3±0.05 fold, p<0.001, 24h: 5.6±2.4 fold, p<0.05) and suppressed TFPI (6h: 0.54±0.04 fold, p<0.05, 24h: 0.24±0.06 fold, p<0.001) compared with normoxia. Similarly, protein levels of TF were increased and TFPI were decreased. Egr-1 siRNA did not change TF mRNA expression. Pyrrolidine dithiocarbamate (PDTC), a nuclear factor kappa B (NF-κB) inhibitor, significantly reduced hypoxia induced TF expression, and HIF-1α knockdown further increased TF. Hypoxia resulted in decreased CD2AP and actin reorganization in podocytes, and these changes were attenuated by TF siRNA. Hypoxia increased the expression of TF in human podocytes NF-κB dependently. TF may have a critical role in the hypoxic podocyte injury.

Galectin-3 deficiency exacerbates hyperglycemia and the endothelial response to diabetes.

BackgroundDiabetes promotes maladaptive changes in the endothelium that lead to its dysfunction and contribute to the vascular pathology of diabetes. We have previously reported the up-regulation of galectin-3, a β-galactoside-binding lectin, in the endothelium and sera of diabetic mice, implicating this molecule in diabetic vasculopathy and suggesting its potential as a biomarker of the disease. Therefore, we sought to assess the role of galectin-3 in the vascular pathology of diabetes.MethodsGalectin-3 knockout mice (KO) and wild-type mice (WT) were fed either a high-fat diet (HFD) (60 % fat calories) to produce insulin resistant diabetes, or standard chow (12 % fat calories), and their metabolic and endothelial responses were measured. After 8 weeks, the aortic and skeletal muscle endothelia were isolated by fluorescence sorting of CD105+/CD45− cells and comprehensive transcriptional analyses were performed. Transcripts differentially dysregulated by HFD in KO endothelium compared to WT were confirmed by semi-quantitative RT-PCR, and protein expression was determined by immunofluorescence of aortic and muscle tissue. Ingenuity® Pathway Analysis was used to identify pathways up-regulated by HFD in the KO, such as the coagulation cascade, and measurements of blood clotting activity were performed to confirm these results.ResultsKO mice demonstrate greater hyperglycemia and impaired glucose tolerance but lower insulin levels on HFD compared to WT. KO mice demonstrate a more robust transcriptional response to HFD in the vascular endothelium compared to WT. Transcripts dysregulated in the KO endothelium after HFD are involved in glucose uptake and insulin signaling, vasoregulation, coagulation, and atherogenesis. One of the most down-regulated transcripts in the endothelium of the KO after HFD was the glucose transporter, Glut4/Slc2a4. GLUT4 immunofluorescence confirmed lower protein abundance in the endothelium and muscle of the HFD-fed KO. Prothrombin time was decreased in the diabetic KO indicating increased coagulation activity.ConclusionsGalectin-3 deficiency leads to exacerbated metabolic derangement and endothelial dysfunction. The impaired tissue uptake of glucose in KO mice can be attributed to the reduced expression of GLUT4. Enhanced coagulation activity in the diabetic KO suggests a protective role for galectin-3 against thrombosis. These studies demonstrate that galectin-3 deficiency contributes both to the pathogenesis of diabetes and the associated vasculopathy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-015-0230-3) contains supplementary material, which is available to authorized users.

Thrombocytopenia impairs host defense during murine Streptococcus pneumoniae pneumonia.

Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. In patients, thrombocytopenia is correlated with an adverse outcome of pneumonia. Platelets can modulate the host response to infection in several ways, that is, by facilitating clot formation, production of antimicrobial proteins, and interaction with neutrophils. We studied the effect of thrombocytopenia during murine pneumococcal pneumonia. Animal study. University research laboratory. Mice. Pneumonia was induced by intranasal inoculation of S. pneumoniae. Platelets were depleted by anti-mouse thrombocyte serum; controls received nonimmunogenic serum. In separate studies, mice were treated with the platelet P2Y12 receptor inhibitor clopidogrel or placebo. Thrombocytopenic mice (platelet counts < 1% of uninfected controls) showed a reduced survival during pneumococcal pneumonia (27% vs 75% among controls; p = 0.003), which was associated with higher bacterial loads in lungs, spleen, and blood. Thrombocytopenic mice showed enhanced coagulation activation (thrombin-antithrombin complexes) in plasma. Proinflammatory cytokine levels were higher in plasma but not in lungs of thrombocytopenic mice. Although clopidogrel treatment strongly prolonged the bleeding time, it did not impact on bacterial loads during pneumococcal pneumonia. Platelets play a protective role during pneumococcal pneumonia independent of their aggregation.

An Immunodeficient Mouse Model With Circulating Human Platelets To Evaluate The Platelet Clearance and Pharmacokinetics Of Platelet-Targeted Coagulation Factor VIIa

Treatment of bleeding episodes in hemophilia patients with inhibitory antibodies to factor VIII or factor IX by recombinant activated factor VII (rFVIIa, NovoSeven) has been sub-optimal partly due to its low affinity to activated platelets and its short half-life in circulation. To develop a rFVIIa variant with enhanced coagulant activity, we have targeted rFVIIa to platelets by monoclonal antibodies that recognize the human platelet receptor αIIbβ3. However, the assessment of pharmacokinetic parameters in mice is limited by the lack of recognition of mouse αIIbβ3 by antibodies to human αIIbβ3. The present study addresses the need to develop appropriate in vivo models to study this new class of bypass therapeutics.First, we evaluated the survival of human platelets in hemophilia A, NOD/SCID, and NOD/SCID/gamma (NSG) mice. Platelet concentrates were prepared from normal human donors and transfused retro-orbitally into mice. The whole blood from dosed mice was then collected via tail vein laceration at various times and the human platelet counts in blood was determined by flow cytometry after staining with fluorescently labeled antibodies against human CD42b, mouse CD61, human FVII to visualize the human platelets, mouse platelets, and the FVIIa candidate that bound to human platelets, respectively. The half-life of human platelets in NOD/scid/gamma (NSG) mice was approximately 4 hours, which is considerably longer than the 0.8 hour half-life observed in hemophilia A mice.The effect of platelet-targeted FVIIa variants on the clearance of human platelets was then investigated in NSG mice. The FVIIa candidates were pre-selected for their inactivity toward human platelet activation and aggregation, as determined in a battery of in vitro assays. In agreement with the in vitro results, all of these selected candidates did not affect the clearance of the transfused human platelets when dosed in NSG mice at 5 nmol/kg. In contrast, a control antibody fusion protein that is known to activate platelets and cause thrombocytopenia in vivo led to rapid platelet clearance in NSG mice. Similar results were observed for these proteins in cynomolgous monkeys when dosed at 2 nmol/kg.The NSG mice with circulating human platelets were also explored to evaluate the clearance of FVIIa candidates that remain platelet-associated in vivo. To improve the pharmacokinetics, we have fused XTEN, a hydrophilic peptide that increases the dynamic radius of payload proteins, to the platelet-targeted FVIIa candidates. When tested in NSG mice model for the clearance of platelet-associated protein, addition of XTEN markedly reduced the clearance rate, resulting in several fold increase in exposure.Together these data indicate that NSG mice with circulating human platelets can be used to assess the safety and pharmacokinetics of the platelet-targeted FVIIa variants, and the method can be adapted to evaluate other agents designed to utilize platelet-targeting approaches. Disclosures:Tan:Biogen Idec: Employment, Equity Ownership. Chen:Biogen Idec: Employment, Equity Ownership. Salas:Biogen Idec: Employment, Equity Ownership. Peters:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment; Biogen Idec: Equity Ownership. Jiang:Biogen Idec: Employment; Biogen Idec: Equity Ownership.

Depressive symptoms, perceived social support, and prothrombotic measures in patients with venous thromboembolism

IntroductionPsychosocial factors have been associated with both a prothrombotic state and an increased risk of venous thromboembolism (VTE). We examined the relation of depressive symptoms and social support with D-dimer, an integrative measure of enhanced coagulation activity, and several additional prothrombotic measures in patients with VTE. MethodsWe studied 173 patients with a previous deep venous thrombosis and/or pulmonary embolism (mean age±SD 45±14years, 55% men). Clinical and lab assessments took place ≥3months after VTE and ≥1month after discontinuation of oral anticoagulants. The patients rated depressive symptoms and social support by validated questionnaires. ResultsAfter adjusting for sociodemographic and medical covariates, interactions emerged between depressive symptoms and social support for D-dimer (p=0.012) and aPTT (p=0.002). As opposed to patients with high levels of social support, those with low levels of social support showed a direct association of depressive symptoms with D-dimer (r=0.19, p=0.014) and an inverse relationship with aPTT (r=-0.14, p<0.09). Depressive symptoms were associated with levels of thrombin-antithrombin complex (r=0.19, p=0.016). Greater social support was associated with prolonged aPTT (r=0.16, p=0.046). There were no significant associations of depressive symptoms and social support with D-dimer, fibrinogen, FII:C, FV:C, FVII:C, FVIII:C, FX:C, INR, and thrombin time. ConclusionsDepressive symptoms are associated with enhanced coagulation activity in patients with VTE, particularly so in those who perceive low levels of social support. Conversely, high levels of social support may contribute directly and through buffering the effect of depressive symptoms to attenuated clotting activity in VTE patients.

Low but sustained coagulation activation ameliorates glucose-induced podocyte apoptosis: protective effect of factor V Leiden in diabetic nephropathy

AbstractWhereas it is generally perceived to be harmful, enhanced coagulation activation can also convey salutary effects. The high prevalence of the prothrombotic factor V Leiden (FVL) mutation in whites has been attributed to a positive selection pressure (eg, resulting from reduced blood loss or improved survival in sepsis). The consequences of enhanced coagulation activation, as observed in FVL carriers, on microvascular diabetic complications remain unknown. We therefore investigated the role of FVL in diabetic nephropathy. In heterozygous or homozygous diabetic FVL mice, albuminuria and indices of diabetic nephropathy were reduced compared with diabetic wild-type mice. This was associated with reduced glomerular apoptosis and preservation of podocytes in diabetic FVL-positive mice. In vitro, low-dose thrombin (50pM) prevented, whereas high-dose thrombin (20nM) aggravated, glucose-induced apoptosis in podocytes. In diabetic patients, the FVL mutation, but not the plasminogen activator inhibitor-1 4G/5G polymorphism, is associated with reduced albuminuria, which is consistent with a nephroprotective role of low but sustained thrombin generation. Consistently, anticoagulation of diabetic FVL-positive mice with hirudin abolished the nephroprotective effect. These results identify a nephroprotective function of low but sustained thrombin levels in FVL carriers, supporting a dual, context-dependent function of thrombin in chronic diseases.

Eosinophilia and Thrombosis in Parasitic Diseases: An Overview

It is known that peripheral blood eosinophilia (PBE) is a normal hematopoietic response to several parasitic diseases, but it is less known that PBE promotes a hypercoagulable state that may favor thrombosis. Scope of this article is to explore which parasitic infestations are most likely to be complicated by thrombosis and to highlight the pathogenetic contribution of PBE to vascular occlusions in this setting. A review of the world literature revealed 18 cases in which PBE was associated with vascular occlusion though no specific surveys were dedicated to this topic. The eosinophil exerts its thrombogenic potential by inhibition of the natural anticoagulant pathways and release of tissue factor with enhanced coagulation activation leading to vascular occlusion. It is hoped that this review contributes to the awareness of the link between PBE and thrombosis in parasitic disorders to foster research in this area.

Impact of Decreased Renal Function on Coagulation and Fibrinolysis in Patients With Non-Valvular Atrial Fibrillation

The prothrombotic state in non-valvular atrial fibrillation (NVAF) increases the risk of thromboembolic events. It is also well known that a prothrombotic state exists in renal failure. Hemostatic disorders play a role in thromboembolic mortality in both NVAF and renal failure. However, little is known about the influence of renal function on hemostasis in patients with NVAF. A total of 190 consecutive outpatients with NVAF who were not receiving anticoagulant therapy were enrolled in the present study. Patients were stratified in groups based on estimated glomerular filtration rate (eGFR). Plasma concentrations of thrombin-antithrombin complex (TAT) and D-dimer were measured and the influence of renal function on coagulation and fibrinolysis was assessed. A relatively weak and inverse relationship of eGFR to TAT and D-dimer was observed (r=0.28, P=0.0001; r=0.30, P<0.0001). Both mean TAT and D-dimer concentrations in groups gradually and significantly increased as eGFR decreased (P<0.0001). On multivariate regression analysis, decreased eGFR concentration was a significant predictor for elevation of TAT (P<0.05) and D-dimer (P<0.01) in patients with NVAF. The enhanced coagulation activation appeared to be related to a reduction in residual renal function in patients with NVAF. This suggests that decreased renal function might be a candidate predictor of thromboembolic events in patients with NVAF.

Blood rheology at term in normal pregnancy and in patients with adverse outcome events

Plasma volume expansion of more than 1.5 l and sustainable activation of the hemostatic system that results in a steady rise of the fibrinogen/fibrin turnover are contemporary physiological events during normal pregnancy. In contrast, adverse outcome of pregnancy i.e. pre-eclampsia commonly coincide with hemo concentration and over activation of blood coagulation both of which alter blood rheology. On the basis of 4,985 consecutively recorded singleton pregnancies values range of blood rheological parameters in women with normal and complicated outcome of pregnancy at the time of their delivery were compared. Plasma viscosity (pv) was determined using KSPV 1 Fresenius and RBC aggregation (stasis: E0 and low shear: E1) using MA1-Aggregometer; Myrenne. Seventy-nine point four percent (n=3,959) had normal pregnancy outcome and 1,026 with adverse outcome of pregnancy had pre-eclampsia (8.4%; n=423), had newborn with a birth-weight < 2,500 g (9.5%; n=473), had early-birth before week 37 (9.3%; n=464), and/or were diagnosed with intra uterine growth retardation (IUGR) (5.0%; n=250). In women with normal pregnancy outcome mean (+/-SD) of pv was 1.31+/-0.09 mPa s, of E0 was 21.6+/-5.3, and of E1 was 38.4+/-7.9 while in women with adverse outcome means for rheological parameters were statistically significantly different i.e. pv: 1.32+/-0.08 mPa s; p=0.006, E0: 22.1+/-5.5; p=0.002 and E1: 39.5+/-8.5; p=0.0006. Subgroup analysis revealed statistical significant lower pv in women who either had pre term delivery or a low birth-weight child (p<0.005) as compared to women who had normal pregnancy outcome while patients with pre-eclampsia had markedly higher low shear and stasis RBC aggregation (p<0.0001). None of the rheological results at term were correlated with either maternal age (r<0.04), BMI (r<0.09), maternal weight gain until delivery (r<0.04), or fetal outcome such as APGAR-score (r<0.09) art. pH in the umbilical cord (-0.05<r<0.04), fetal birth-weight (r<0.04). Women who did vs. did not receive iron supplementation during pregnancy had lower pv (p<0.0001) and higher E0 (p<0.01) while smokers vs. non-smokers had lower E0 (p=0.0002). There was a strong correlation between hemoglobin concentrations and pv at term (spearman r=0.15; p<0.0001). To our knowledge this is the largest trial that consecutively assessed blood rheological parameters in pregnant women at term. Distribution of pv and RBC aggregation was studied in women with normal and complicated pregnancy. We found lower pv and increased RBC aggregation in patients with different adverse outcome of pregnancy compared to normal pregnancy. Interestingly, in pre-eclampsia hemo concentration and increased fibrinogen turnover due to enhanced coagulation activation are weighty co factors of pv but were associated with lower pv in patients with pre-eclampsia. However, coincidental increased RBC aggregation and hemo concentration may potentially derogate blood flow in the materno-fetal unit that is commonly traceable using vessel duplex ultra sound in pre-eclampsia.

Endogenous β-Adrenergic Receptors Inhibit Lipopolysaccharide-Induced Pulmonary Cytokine Release and Coagulation

Beta2-adrenergic receptors are expressed on different cell types in the lung, including respiratory epithelial cells, smooth muscle cells, and macrophages. The aim of the current study was to determine the role of beta-adrenergic receptors in the regulation of lung inflammation induced by instillation via the airways of lipopolysaccharide (LPS) (a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA) (a component of the gram-positive bacterial cell wall). Mice inhaled the beta-adrenergic antagonist propranolol or saline 30 minutes before and 3 hours after intranasal LPS or LTA administration. LPS and LTA induced a profound inflammatory response in the lungs as reflected by an influx of neutrophils and the release of proinflammatory cytokines and chemokines into bronchoalveolar lavage fluid (BALF). Propranolol inhalation resulted in enhanced LPS-induced lung inflammation, which was reflected by a stronger secretion of TNF-alpha, IL-6, and monocyte chemoattractant protein-1 into BALF and by enhanced coagulation activation (thrombin-antithrombin complexes). In LTA-induced lung inflammation, propranolol did not influence cytokine release but potentiated activation of coagulation. Propranolol did not alter neutrophil recruitment in either model. This study suggests that beta-adrenergic receptors, which are widely expressed in the lungs, serve as negative regulators of pulmonary cytokine release and coagulation induced by LPS and less so during LTA-induced pulmonary inflammation.

Unfractionated Heparin but Not Enoxaparin Causes Delayed Plasma PAI-1 Depletion in Hemodialysis Patients: A Prospective Study

Heparin modulates function of vascular endothelial cells. We studied the effects of unfractionated heparin (UFH) versus low-molecular-weight heparin (LMWH), enoxaparin, used as anticoagulants during hemodialysis (HD) on plasma levels of circulating endothelium-derived molecules: thrombomodulin (TM), von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1), cell surface adhesion molecules (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and prothrombin fragment 1+2 (PF 1+2). Twenty-five patients undergoing enoxaparin-anticoagulated HD were randomly assigned either to continue enoxaparin (n = 13) or receive UFH (n = 12) during HD and followed prospectively for 12 weeks. Plasma immunoreactive TM, vWF, PAI-1, ICAM-1, E-selectin, and PF 1+2 were measured before the randomization procedure and after 12 weeks, at the start, after 10 and 180 minutes of HD. The switch from LMWH to UFH resulted in decreased pre-HD levels of PAI-1 and TM and increased PF 1+2 concentrations. Predialysis PAI-1 negatively correlated with the total dose of UFH/kg/HD. No significant differences were observed in the other variables before and during HD after switching from LMWH to UFH. During enoxaparin-anticoagulated HD, only plasma PAI-1 levels decreased by 32% after 180 minutes compared with the baseline values. This percentage decrease positively correlated with predialysis PAI-1 levels and marginally with PF 1+2 concentrations after 180 minutes of enoxaparin-anticoagulated HD. Enoxaparin-anticoagulated HD is related to transient plasma PAI-1 decrease, whereas UFH anticoagulation may be the cause of delayed PAI-1 and TM depletion, which is an untoward consequence of enhanced coagulation activity in chronic HD patients.

Coagulation and Fibrinolysis in Preeclampsia and Neonates

Coagulation and fibrinolysis were determined in 67 Indonesian women admitted to the University Hospitals for delivery in Medan. They were diagnosed to be at term gestation (mean 39.3 +/- 1.1 weeks) with moderate and severe preeclampsia (n=32) and in labor, and 8 had preterm labor (gestation mean 33.5 +/- 2.6 weeks). Twenty-seven normal pregnant women in labor (gestation mean 39.7 +/- 1.0 weeks) served as controls. Cord blood from 23 neonates from normal pregnancy and 31 neonates from preeclampsia was also evaluated. Preeclamptic women in labor showed further enhanced coagulation activation (F(1+2)) with raised urokinase-like plasminogen activator (u-PA) activity and reduced plasminogen activator inhibitor-2 (PAI-2) levels. In preterm preeclampsia, significantly reduced antithrombin III (ATIII) and PAI-2 levels with further elevated tissue-type PA (t-PA) antigen and plasminogen activator inhibitor-1 (PAI-1) antigen were seen compared to normal pregnancy. These would suggest a state of enhanced thrombin generation with elevated fibrinolytic/inhibitor proteins in preterm preeclampsia. The reduced PAI-2 levels seen in preeclampsia have been suggested to be associated with reduced placental function. Neonates born to mothers of either normal pregnancy or preeclampsia at term showed similar hemostatic changes with reduced fibrinogen, ATIII, t-PA, u-PA antigen, PAI-1 levels, and coagulation activation compared to their respective maternal plasma levels. No significant differences in hemostatic parameters studied between the neonates of both cohorts were seen, and this would suggest that the neonates were protected from the adverse effects of preeclampsia and their hemostatic system was physiologically balanced.