Procoagulant Effects Research Articles

Platelet Apoptosis and PAI-1 are Involved in the Pro-Coagulant State of Immune Thrombocytopaenia Patients Treated with Thrombopoietin Receptor Agonists.

The treatment goal for patients with immune thrombocytopaenia (ITP) is to raise platelet counts to levels that minimize or stop bleeding. Thrombopoietin receptor agonists (TPO-RAs) have been successfully and extensively employed as second-line therapy for ITP. However, TPO-RAs have a small but significant increase in the risk of thrombosis. The aim of this study was to elucidate the mechanisms involved in the pro-coagulant effect of TPO-RAs to take them into account when considering their use in ITP patients with concomitant diseases/conditions that might increase risk of suffering thrombotic events. Eighty-two patients with chronic primary ITP (40 untreated and 42 undergoing TPO-RA therapy) and 112 healthy individuals were recruited. The patients with ITP undergoing TPO-RA therapy presented a pro-coagulant profile due to the formation of a more fibrinolysis-resistant clot because of increased plasminogen activator inhibitor-1 (PAI-1) levels. Increase in platelet content of PAI-1 might be the result of the effect of TPO-RA during megakaryopoiesis, as suggested by experiments performed in MEG-01 cells. Moreover, patients under TPO-RA treatment presented an enhanced pro-coagulant activity associated with microparticles and an increased platelet apoptosis that causes a higher exposure of phosphatidylserine and, consequently, a larger surface for the binding of the prothrombinase complex.

No effect of tranexamic acid on platelet function and thrombin generation (ETAPlaT) in postpartum haemorrhage: a randomised placebo-controlled trial.

Background: Postpartum hemorrhage (PPH) is a leading cause of maternal mortality and morbidity. The WOMAN trial showed that tranexamic acid (TXA) reduces death due to bleeding in women with PPH. To determine whether TXA has pro-thrombotic effects in women with PPH, we measured endogenous thrombin potential (ETP), coagulation factors V, VIII, von Willebrand (vW), fibrinogen, D-Dimers and platelet function. Methods: We conducted a sub-study within the WOMAN trial, an international randomized, parallel-group, double blind, placebo-controlled trial. Women with primary PPH were randomly allocated to receive 1 gram of tranexamic acid or matching placebo. Baseline blood samples were collected just prior to the first dose and a follow up sample was collected 30±15 minutes afterwards. We compared before and after changes in coagulation parameters between treatment groups using repeated measurement ANOVA. Change in ETP was the primary outcome. We did an intention-to-treat analysis using ANCOVA with adjustment for baseline and the time interval between the blood samples. Findings: A total of 187 patients were randomized to receive TXA (n=93) or matching placebo (n=94). Six patients were excluded due to incomplete data. The reduction in ETP from baseline to follow up was 43.2 nM*min (95%CI, -16.6 to 103.1) in the TXA group and 4.6 nM*min (95%CI, -51.4 to 60.6) in the placebo group. The difference was not statistically significant (95%CI, -42.9 to 120). There were no significant effects of TXA treatment on any other parameters (ADPtest, TRAPtest, coagulation factors activity, fibrinogen levels, D-Dimer level). Conclusion: We found no evidence that tranexamic acid treatment for PPH has substantial pro-coagulant effects. However, larger studies are needed to confirm or refute more modest effects. Trial registration: ISRCTN76912190 (initially registered 10/12/2008, WOMAN-ETAPlat included on 28/10/2013) and NCT00872469 (initially registered 31/03/2009, WOMAN-ETAPlat included on 28/10/2013).

Preparation and Properties of Carboxymethyl Chitosan/Alginate/Tranexamic Acid Composite Films.

In this study, the porous composite films of carboxymethyl chitosan/alginate/tranexamic acid were fabricated, with calcium chloride as the crosslinking agent and glycerin as a plasticizer. The composite films were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The properties of the composite films, including water absorption, air permeability, and cumulative release rate, were tested. In addition, their hemostatic performance was evaluated. The results showed that the appearance of the films with good adhesion was smooth and porous. FTIR showed that chemical crosslinking between carboxymethyl chitosan and sodium alginate was successful. The excellent cumulative release of tranexamic acid in the composite films (60–80%) gives the films a significant procoagulant effect. This has good prospects for the development of medical hemostasis materials.

The Effect of Haemostatic Dressing Prototypes for the Emergency Services in the Porcine Haemostatic System.

Coagulopathy can develop when hemostatic dressings are used to stop massive bleeding, even in patients without prior history of clotting disorders. The selection of procoagulants, which effectively control bleeding and prevent disseminated intravascular coagulation (DIC) and thrombosis, is a significant challenge. The aim of this study was to evaluate the effect of two prototypes of haemostatic dressing in the porcine haemostatic system. The total number of animals used in our experiments was 24. Group I: pigs were treated with the developed prototype of sponge dressing, made of Na-Ca chitosan/algal composite of microfibers and nanofibers. Group II: animals were treated with a seton gauze modified with a polymer mixture of Na-Ca chitosan/algal composite of microfibers and nanofibers. Group III: animals were treated with non-hemostatic dressing and this group was the control. Blood was sampled five times to determine changes in the coagulation and fibrinolytic profiles: before injury: i) at 1 h, ii) at 24 h, iii) at 7, and iv) at 14 days following injury. Significant changes were observed in the coagulation parameters, in the total numbers of white blood cells and platelets in groups I and II, compared to controls. The modified haemostatic dressings used in this study produced a strong procoagulant effect in pigs. This, together with high fibrinogen concentrations, which can cause DIC, require further studying.

Investigation of the Effects of Molecular Parameters on the Hemostatic Properties of Chitosan.

Hemorrhea is one of the major problems in war, trauma care, and surgical operation that threaten the life of the injured and patients. As a novel polymeric hemostatic agent, biodegradable chitosan can stop bleeding through a variety of approaches. In this paper, chitosan with various molecular parameters was prepared from chitin as raw material through deacetylation, oxidative degradation, hydrophilic modification, and salt formation reactions. The influence of different polymer parameters on the hemostatic effects of chitosan was investigated by in vitro coagulation time and dynamic coagulation assay. The results showed that when the molecular weights were high (105–106) and approximate, the coagulation effect of chitosan improved with a decrease of the deacetylation degree and achieved a prominent level in a moderate degree of deacetylation (68.36%). With the same degree of deacetylation, the higher the molecular weight of chitosan, the better the procoagulant effect. The substituent derivatives and acid salts of chitosan showed significant procoagulant effects, especially the acid salts of chitosan. In addition, the hemostasis mechanism of chitosan with various parameters was preliminarily explored by analyzing the plasma recalcification time (PRT). The efforts in this paper laid a basis for further study of the structure–activity relationship and the mechanism of chitosan hemostasis.

Platelet Apoptosis and PAI-1 Content Are Involved in the Procoagulant Profile of Immune Thrombocytopenia Patients Responders to Agonists of Thrombopoietin Receptor.

Background: The treatment goal for patients with immune thrombocytopenia (ITP) is to raise platelet counts to levels that minimize or stop bleeding. Thrombopoietin receptor agonists (TPO-RAs) have been successfully and extensively employed as second-line therapy for ITP. TPO-RAs, however, have a small but significant increase in the risk of thrombosis.Aim: The aim of this study was to elucidate the mechanisms involved in the procoagulant effect of TPO-RAs.Methods: This is a prospective, observational and transversal study. Eighty-two patients with chronic primary ITP, 40 without treatment for at least six months (UT-ITP) and 42 responders to TPO-RA therapy (64.3% with eltrombopag and 35.7 % with romiplostim) were recruited. One hundred and twelve healthy participants were also included.ROTEM® (naTEM test: only recalcification) was performed on platelet rich plasma adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until 2 mm of amplitude [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2 mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded.Surface exposure of phosphatidylserine (PS), active caspase-3, -8 or -9 and prothrombinase complex binding to platelets were assessed by flow cytometry.Plasma and platelet levels of PAI-1 were determined by ELISA (eBioscience Ltd., Hatfield, United Kingdom). The effect of TPO and romiplostim on PAI-1 content of MEG-01 cells was evaluated by Western blot. Three MEG-01 cell cultures were initiated simultaneously: control without drugs and treated with either TPO (100 ng/mL) or romiplostim (53 μg/mL). Samples were collected at the start and after 24, 48 and 72 hours to determine the PAI-1 content.The statistical analysis was performed using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA).Results: The ROTEM® studies showed significant differences in the dynamics of clot formation when comparing the control with ITP samples.There was a delay in clot formation in the UT-ITP group, as observed by a prolonged CT [expressed as median (p25-p75): control: 516 (490- 633) s; UT-ITP: 938 (914-1348) s, p<0.001], and a diminished alpha angle (mean±SD; control: 61.7±5.6 degrees; UT-ITP: 49.2±7.3 degrees, p<0.05). Nevertheless, samples from patients with UT-ITP reached the same MCF as those from healthy controls (control: 45.3±2.4 mm; UT-ITP: 46.9±3.7 mm). On the other hand, patients with ITP undergoing TPO-RA therapy presented an initial clot formation similar to that of the control group [expressed as median (p25-p75): CT, 672 (598-928) s; alpha angle, 55.8±5.8 degrees] but achieved a higher MCF (53.1±4.5 mm, p<0.05) and a reduced clot lysis after 60 min (control: 91.8±4.0%; UT-ITP: 93.7±4.0%, TPO-RA ITP: 97.6±1.7, p<0.05).Higher values of MCF observed with platelets from ITP patients treated with TPO-RAs might be a consequence of their augmented apoptosis signs: platelets from this group exposed more PS than controls and this situation was accompanied by an increased activity of caspases-3,7, -8 and -9 (Figure 1 A and B). Moreover, platelets from ITP patients on treatment with TPO-RAs bound more prothrombinase complex than platelets from UT-ITP patients and healthy controls (Figure 1 C).Reduced clot lysis observed in ITP patients treated with TPO-RA was due, at least in part, to increased plasma and platelet levels of PAI-1 (Table 1).Increase in platelet content of PAI-1 might be the result of the effect of TPO-RAs during megakaryopoiesis since treatments of MEG-01 cells with TPO or romiplostim induced a 3-fold increase in their endogenous PAI-1 content after an incubation period of 48 hs.Conclusion: The patients with ITP undergoing TPO-RAs therapy presented a procoagulant profile due to the formation of a more fibrinolysis-resistant clot because of increased platelet and plasma PAI-1 levels.Moreover, platelets from this group of patients showed more signs of apoptosis that causes a higher exposure of PS and, consequently, a larger surface for the binding of the prothrombinase complex.Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresÁlvarez-Roman:SOBI: Consultancy; NovoNordisk: Consultancy; Shire: Consultancy. Jimenez-Yuste:Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Sobi: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.

New insight into antiphospholipid syndrome: antibodies to β2glycoprotein I-domain 5 fail to induce thrombi in rats.

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of β2glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analyzed the in vivo pro-coagulant effect of two groups of 5 sera IgG each reacting selectively with domain 1 or domain 5 in lipopolysaccharide (LPS)-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy, and vascular deposition of β2glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-β2glycoproteinI antibodies served as controls. All the anti-domain 1-positive IgG exhibited potent pro-coagulant activity while the anti-domain 5-positive and the negative control IgG failed to promote blood clot and vessel occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound β2glycoproteinI while being able to interact with fluid-phase β2glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit a thrombogenic effect in vivo, and the interaction of these antibodies with circulating β2glycoproteinI suggests their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining “really at risk” patients for more appropriate treatments to avoid recurrences and disability.

Methyl isocyanate inhalation induces tissue factor-dependent activation of coagulation in rats

Methyl isocyanate (MIC) is a highly toxic industrial chemical causing acute lethality after inhalation. The objective of this study was to determine whether alterations in hemostasis also occur in the immediate hours after exposure. Male rats were exposed to MIC (125–500 ppm) by nose-only vapor inhalation for 30 min. Arterial O2 saturation was monitored prior to exposure, and hourly thereafter. Rats were euthanized at 1, 2, 4, and 8 hr and plasma analyzed for recalcification clotting time, tissue factor (TF) activity, and protein levels. Hypoxemia, as assessed by pulse oximetry, was an early feature of MIC inhalation. In contrast to sham or low (125 ppm) concentrations, 250 and 500 ppm MIC caused significant declines in blood oxygen saturation (% SpO2) at 1 hr, which remained at deficit during the postexposure period. Commensurate with hypoxemia, plasma clotting time was significantly accelerated 1 hr after MIC inhalation (sham treatment: 955 ± 62.8 s; 125 ppm MIC: 790 ± 62 s; 250 ppm: 676 ± 28.0 s; 500 ppm: 581 ± 175 s). This procoagulant effect was transient, with no difference observed between sham and all MIC groups by 8 hr. Similarly, elevated TF activity and protein were detected in plasma 1 hr after MIC inhalation, each of which showed a progressive decline back to control levels at later timepoints. This study demonstrates that MIC inhalation resulted in hypoxemia and transient hypercoagulability of blood. Accelerated clotting occurred rapidly and was likely due to intravascular TF, which initiates the extrinsic coagulation pathway.

Pro-Coagulant and Pro-Thrombotic Effects of Paclitaxel Mediated by Red Blood Cells.

Paclitaxel is one of the most widely used anti-cancer drugs, but numerous case reports of thrombotic events in the cancer patients using paclitaxel raise concern over its pro-thrombotic risk. We investigated whether paclitaxel can elicit pro-thrombotic properties in red blood cells (RBCs) through phosphatidylserine (PS) exposure and microvesicle (MV) release. In freshly isolated human RBCs, paclitaxel induced thrombin generation through PS exposure and MV release, whereas either coagulation factors or platelets were unaffected. Paclitaxel-induced PS exposure in RBC was mediated by scramblase activation which was induced by calcium-independent protein kinase C (PKC)ζ activation. Paclitaxel also increased RBC-endothelial cell adhesion and RBC aggregate formation which can also contribute to thrombosis. Indeed, intravenous administration of paclitaxel to rats induced PS exposure and PKCζ activation in RBCs in vivo which ultimately promoted venous thrombus formation. These results demonstrated that paclitaxel may elicit pro-thrombotic properties in RBCs through PS exposure and MV release, which can ultimately promote thrombus formation.

Visualization of The Procoagulant Effects of Combined Oral Contraceptives (COCs)

It is well established that the use of oral contraceptives increases the risk of venous thromboembolism. Attempts to understand the underlying mechanisms has resulted in documentation of changes in many specific haemostatic factors. The changes related to the thrombosis risk have not been established and prevented validated evaluation of new COCs for thrombosis risk before exposing large groups of women and evaluating clinical effects, i.e. venous thrombotic embolism (VTE) [1].

Differential heme-mediated modulation of Deinagkistrodon, Dispholidus, Protobothrops and Pseudonaja hemotoxic venom activity in human plasma.

Envenomation by vipers with hemotoxic enzymes continues to be a worldwide source of morbidity and mortality. The present work examined the effects of exposure of venom enzymes to carbon monoxide and O-phenylhydroxylamine, agents that modulate the biometal heme, by forming carboxyheme and metheme, respectively. Four venoms obtained from medically important, diverse snake venom found in Africa, Asia and Australia were analyzed. The species that had venom tested in human plasma with thrombelastography and heme modulating agents were Deinagkistrodon acutus, Protobothrops mucrosquamatus, Dispholidus typus and Pseudonaja textilis. These venoms varied four hundred-fold in potency (ng-µg/ml) to exert procoagulant effects on human plasma; further, there was species specific variability in venom inhibition after exposure to carboxyheme or metheme agents. Lastly, using a wide range of carbon monoxide concentrations, it was determined that the factor V component of P. textilis venom was likely inhibited before the factor X component. Further investigation using this thrombelastograph-based, venom "kinetomic" methodology involving heme modulation will demonstrate in time its laboratory and clinical utility.

Clinical Cellular Therapeutics Accelerate Clot Formation.

Clinical cellular therapeutics (CCTs) have shown preliminary efficacy in reducing inflammation after trauma, preserving cardiac function after myocardial infarction, and improving functional recovery after stroke. However, most clinically available cell lines express tissue factor (TF) which stimulates coagulation. We sought to define the degree of procoagulant activity of CCTs as related to TF expression. CCT samples from bone marrow, adipose, amniotic fluid, umbilical cord, multi‐potent adult progenitor cell donors, and bone marrow mononuclear cells were tested. TF expression and phenotype were quantified using flow cytometry. Procoagulant activity of the CCTs was measured in vitro with thromboelastography and calibrated thrombogram. Fluorescence‐activated cell sorting (FACS) separated samples into high‐ and low‐TF expressing populations to isolate the contribution of TF to coagulation. A TF neutralizing antibody was incubated with samples to demonstrate loss of procoagulant function. All CCTs tested expressed procoagulant activity that correlated with expression of tissue factor. Time to clot and thrombin formation decreased with increasing TF expression. High‐TF expressing cells decreased clotting time more than low‐TF expressing cells when isolated from a single donor using FACS. A TF neutralizing antibody restored clotting time to control values in some, but not all, CCT samples. CCTs demonstrate wide variability in procoagulant activity related to TF expression. Time to clot and thrombin formation decreases as TF load increases and this procoagulant effect is neutralized by a TF blocking antibody. Clinical trials using CCTs are in progress and TF expression may emerge as a safety release criterion. Stem Cells Translational Medicine 2018;7:731–739

Nanoparticles in the lungs of old mice: Pulmonary inflammation and oxidative stress without procoagulant effects

Pulmonary exposure to nanoparticles (NPs) has been shown to induce pulmonary as well as cardiovascular toxicity. These effects might be enhanced in elderly subjects as a result of a compromised immunity and/or declined organ functions. To study the adverse in vivo effects of NPs in a model for the elderly, we exposed 18-month-old C75Bl/6 mice to multi-walled carbon nanotubes (MWCNTs) or ZnO NPs by intratracheal instillation once a week during 5 consecutive weeks. Pulmonary and hemostatic toxicity was determined 24 h (T1) and 8 weeks (T2) after the last administration.Both NP types significantly increased the pulmonary macrophages at both time points. The MWCNTs and ZnO NPs also induced a pulmonary influx of neutrophils, which was even larger at T2 compared to T1. All NPs induced only a modest increase of pulmonary IL-1β, IL-6 and KC levels. Both types of NPs also increased blood neutrophils. Red blood cells were not significantly affected. Both NPs significantly increased coagulation factor VIII levels at both time points. Histological analysis revealed the presence of MWCNTs in the alveolar macrophages up to 8 weeks after the last administration and the ZnO NPs induced a pronounced alveolar inflammation.In these 18-month-old mice, NPs caused pulmonary inflammation (without evidence of oxidative stress) accompanied by large increases in coagulation factor VIII up to 8 weeks after the last NP exposure. The persistence of the MWCNTs in the lungs resulted in translocation from the lungs to the left heart and the ZnO NPs induced a fibrosis-like pathology.

Factor IX from prothrombin complex concentrate augments low dose tissue factor-triggered thrombin generation in vitro

BackgroundProthrombin complex concentrate (PCC) is increasingly used to correct acquired coagulopathy in trauma and surgery. Dosing of PCC is guided by the prothrombin time, which only reflects the onset of thrombin generation, but does not account for variations in intrinsic pathway coagulation factors, including factor IX (FIX). We hypothesised that FIX contained in PCC could strongly influence thrombin generation patterns. MethodsPooled normal, FIX-deficient, and warfarinised plasma were used to analyse the effects of FIX contained in PCC. PCC was evaluated at final concentrations of 0.2 and 0.4 IU ml−1 in FIX-deficient and normal plasma, and at 0.6 IU ml−1 in warfarinised plasma with elevated FVIII (1.5 IU ml−1), 40% dilution with saline, or both. The effects on thrombin generation were assessed by measuring both procoagulant and inhibitory segments. ResultsFIX-deficient plasma had lower peak thrombin generation [30.6 (20.5–35.8) nM vs 130.2 (107–168) nM] and endogenous thrombin potential [472 (391–532) nM vs 1096 (958–1190) nM] than normal plasma. PCC addition resulted in significant increases of peak thrombin generation [81.8 (37.3–98.3) nM] and endogenous thrombin potential [808 (472–842) nM] in FIX-deficient plasma. The combination of FVIII and PCC resulted in greater increases relative to each agent alone, restoring normal thrombin generation. After 40% dilution, adding PCC, FVIII, or both, to FIX-deficient plasma increased peak thrombin generation, and prolonged the inhibitory phase of the endogenous thrombin potential. ConclusionsFIX derived from PCC strongly enhances tissue factor-triggered thrombin generation in the presence of elevated FVIII activity. Haemodilution further enhances procoagulant effects of FIX and FVIII by slowing down inhibition of procoagulant enzymes. Dosing of PCC per prothrombin time may underestimate PCC's procoagulant potential because it does not account for intrinsic tenase or antithrombin activity.

Venom proteomics and antivenom neutralization for the Chinese eastern Russell\u2019s viper, Daboia siamensis from Guangxi and Taiwan

The eastern Russell’s viper (Daboia siamensis) causes primarily hemotoxic envenomation. Applying shotgun proteomic approach, the present study unveiled the protein complexity and geographical variation of eastern D. siamensis venoms originated from Guangxi and Taiwan. The snake venoms from the two geographical locales shared comparable expression of major proteins notwithstanding variability in their toxin proteoforms. More than 90% of total venom proteins belong to the toxin families of Kunitz-type serine protease inhibitor, phospholipase A2, C-type lectin/lectin-like protein, serine protease and metalloproteinase. Daboiasiamensis Monovalent Antivenom produced in Taiwan (DsMAV-Taiwan) was immunoreactive toward the Guangxi D. siamensis venom, and effectively neutralized the venom lethality at a potency of 1.41 mg venom per ml antivenom. This was corroborated by the antivenom effective neutralization against the venom procoagulant (ED = 0.044 ± 0.002 µl, 2.03 ± 0.12 mg/ml) and hemorrhagic (ED50 = 0.871 ± 0.159 µl, 7.85 ± 3.70 mg/ml) effects. The hetero-specific Chinese pit viper antivenoms i.e. Deinagkistrodon acutus Monovalent Antivenom and Gloydius brevicaudus Monovalent Antivenom showed negligible immunoreactivity and poor neutralization against the Guangxi D. siamensis venom. The findings suggest the need for improving treatment of D. siamensis envenomation in the region through the production and the use of appropriate antivenom.

Soluble endothelial protein C is associated with blood pressure variability and salt consumption but not mean blood pressure in patients with newly diagnosed primary hypertension

ABSTRACTBackground: Hypertension is a widespread disease involving frequent thrombotic complications. Blood pressure variability (BPV) has recently been shown to be associated with end-organ damage and cardiovascular events. However, the pathogenesis of the relation between BPV and cardiovascular events has not yet been explained. Soluble endothelial protein C (sEPCR) exhibits a procoagulant effect by reducing the anticoagulant and anti-inflammatory effects of protein C and activated protein C. The purpose of this study was to evaluate sEPCR levels in hypertensive individuals and the parameters affecting that level, particularly BPV.Methods: Fifty-one newly diagnosed hypertensive subjects and 31 healthy individuals were included in the study. Twenty-four-hour ambulatory blood pressure monitoring (ABPM) was performed after office control, and simultaneous 24-h urine was collected. BPV was calculated with average real variability (ARV) from ABPM data. Blood specimens were collected under appropriate conditions for sEPCR levels and biochemical tests. sEPCR levels were compared between the patient and healthy groups, after which parameters affecting sEPCR elevation in the hypertensive group were evaluated.Results: sEPCR levels were significantly high in the hypertensive group (p < 0.05). At multivariate regression analysis in the hypertensive group, sEPCR was determined to be independently associated with 24-h systolic ARV (ß = 0.572, p < 0.05) and 24-h urine Na (ß = 0.428, p < 0.05).Conclusion: In our study, sEPCR was high in hypertensive individuals, and this elevation was related to ARV and urine Na excretion independently of mean blood pressure.

Inhibition of Tissue Factor Pathway Inhibitor (TFPI) as a Treatment for Haemophilia: Rationale with Focus on Concizumab.

Replacement therapy with missing factor (F) VIII or IX in haemophilia patients for bleed management and preventative treatment or prophylaxis is standard of care. Restoration of thrombin generation through novel mechanisms has become the focus of innovation to overcome limitations imposed by protein replacement therapy. Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine protease inhibitor that regulates tissue factor (TF)-induced coagulation through a FXa-dependent feedback inhibition of the TF.FVIIa complex in plasma and on endothelial surfaces. Concizumab is a monoclonal, humanised antibody, specific for the second Kunitz domain of TFPI that binds and inhibits FXa, abolishing the inhibitory effect of TFPI. Concizumab restored thrombin generation in FVIII and FIX deficient plasmas and decreased blood loss in a rabbit haemophilia model. Phase 1 single and multiple dose escalation studies in haemophilia patients demonstrated a dose dependent decrease in TFPI levels and a pro-coagulant effect with increasing d-dimers and prothrombin fragment 1 + 2. A dose dependent increase in peak thrombin and endogenous thrombin potential was observed with values in the normal range when plasma TFPI levels were nearly undetectable. A few haemophilia patients in the highest dose cohorts with complete inhibition of plasma TFPI showed a decreased fibrinogen concentration with normal levels of anti-thrombin and platelets and no evidence of thrombosis. Pharmacokinetic parameters were influenced by binding to the target (TFPI), demonstrating target mediated drug disposition. A trend towards decreasing bleeding tendency was observed and this preventative effect is being studied in Phase 2 studies with additional data gathered to improve our understanding of the therapeutic window and potential for thrombosis.

Carbon Monoxide-Releasing Molecule Enhances Coagulation and Decreases Fibrinolysis in Normal Canine Plasma.

The dog is an important companion animal and also purpose-bred for research studies. Coagulopathies in dogs are common, although the availability of blood products for therapy is inconsistent throughout the profession. A pro-coagulant therapeutic that is readily available and easily stored would be useful for the treatment of coagulopathies. Tricarbonyldichlororuthenium (II) dimer [Carbon monoxide-releasing molecule-2 (CORM-2)] acts as a prothrombotic agent in plasma by increasing the velocity of clot formation and clot strength, and by decreasing the clot's vulnerability to fibrinolysis. We sought to test CORM-2's effect on coagulation and fibrinolysis in vitro in canine plasma using thromboelastography. Measures of the rate of clot formation and clot strength in plasma without CORM-2 were highly correlated with fibrinogen concentration. We found that CORM-2 significantly enhanced the rate of clot formation and clot strength and significantly reduced the rate of fibrinolysis and the clot lysis time. The per cent change in rate of clot formation and clot strength was not significantly correlated with fibrinogen concentration, indicating that CORM-2's pro-coagulant effect is not dependent on fibrinogen concentration. This study corroborates studies in other species that show that CORM-2 is pro-coagulant in plasma, and lays the groundwork for developing CORM-2 as a therapeutic agent for canine coagulopathies. Future studies will evaluate the effect of CORM-2 on whole blood both in vitro and in vivo.

Proteomics, functional characterization and antivenom neutralization of the venom of Pakistani Russell's viper (Daboia russelii) from the wild

The venom proteome of wild Pakistani Russell's viper (Daboia russelii) was investigated through nano-ESI-LCMS/MS of the reverse-phase HPLC fractions. A total of 54 venom proteins were identified and clustered into 11 protein families. Phospholipase A2 (PLA2, 63.8%) and Kunitz-type serine protease inhibitor (KSPI, 16.0%) were most abundant, followed by snake venom serine protease (SVSP, 5.5%, mainly Factor V activating enzyme), vascular endothelial growth factor (VEGF, 4.3%), snake venom metalloproteinase (SVMP, 2.5%, mainly Factor X activating enzyme) and phosphodiesterase (PDE, 2.5%). Other minor proteins include cysteine-rich secretory protein (CRiSP), snake venom C-type lectin/lectin-like protein (snaclec), nerve growth factor, L-amino acid oxidase and 5′-nucleotidase. PLA2, KSPI, SVSP, snaclec and SVMP are hemotoxic proteins in the venom. The study indicated substantial venom variation in D. russelii venoms of different locales, including 3 Pakistani specimens kept in the USA. The venom exhibited potent procoagulant activity on human plasma (minimum clotting dose = 14.5 ng/ml) and high lethality (rodent LD50 = 0.19 μg/g) but lacked hemorrhagic effect locally. The Indian VINS Polyvalent Antivenom bound the venom immunologically in a concentration-dependent manner. It moderately neutralized the venom procoagulant and lethal effects (normalized potency against lethality = 2.7 mg venom neutralized per g antivenom). Biological significanceComprehensive venom proteomes of D. russelii from different locales will facilitate better understanding of the geographical variability of the venom in both qualitative and quantitative terms. This is essential to provide scientific basis for the interpretation of differences in the clinical presentation of Russell's viper envenomation. The study revealed a unique venom proteome of the Pakistani D. russelii from the wild (Indus Delta), in which PLA2 predominated (~60% of total venom proteins). The finding unveiled remarkable differences in the venom compositions between the wild (present study) and the captive specimens reported previously. The integration of toxicity tests enabled the correlation of the venom proteome with the envenoming pathophysiology, where the venom showed potent lethality mediated through coagulopathic activity. The Indian VINS Polyvalent Antivenom (VPAV) showed binding activity toward the venom protein antigens; however the immunorecognition of small proteins and PLA2-dominating fractions was low to moderate. Consistently, the antivenom neutralized the toxicity of the wild Pakistani Russell's viper venom at moderate efficacies. Our results suggest that it may be possible to enhance the Indian antivenom potency against the Pakistani viper venom by the inclusion of venoms from a wider geographical range including that from Pakistan into the immunogen formulation.

Temporal phenotyping of circulating microparticles after trauma: a prospective cohort study

BackgroundAfter severe polytrauma the dynamic process of coagulation may deteriorate towards a trauma-induced coagulopathy (TIC) promoting a dramatic increase in morbidity and mortality. Recent evidence suggests that microparticles (MPs) play a pivotal role at the interface between cellular and plasmatic coagulation systems. However, the impact of MPs on functional coagulation has not been clarified yet in the setting of traumatic injuries. We assessed the temporal patterns of circulating MP concentrations including their cellular origin in the context of clinical presentation and global coagulation assays.MethodsBlood samples from 22 consecutive polytrauma patients (ISS ≥16) from 2015 were collected at hospital admission, after 24 and 72 h and compared to those from healthy individuals and minor injured patients with isolated extremity fractures. Flow cytometry (BD Accuri C6; Heidelberg/Germany) was used to determine MP concentrations and cellular origin using cell-specific markers (platelet derived (PDMP): CD42b+, CD61+, CD62p+; endothelial cell derived (EDMP): CD144+, CD62e+, CD144+/62e+). Results were correlated with clinical data and results from viscoelastic testing (ROTEM).ResultsTwenty two polytrauma patients (17 males, agemedian 60 yrs) with a median ISS 26.5 (IQR 14.5) were assessed. PDMP and EDMP concentrations increased significantly in polytrauma patients as compared to healthy individuals and minor injured patients. MP concentrations correlated with injury severity (CD144+: ρsp = 0.79, p < 0.001; CD42b+: ρsp = 0.61, p < 0.001). EDMP displayed a negative correlation with aPTT (CD144/62e+, ρsp = − 0.55, p < 0.05), INR (CD144/62e+, ρsp = − 0.61, p < 0.05) and ROTEM-INTEM CT (CD144/62e+, ρsp = − 0.68, p < 0.05) reflecting increased dynamics of clot formation and an overall procoagulative effect. Additionally, EDMP showed a negative association with FIBTEM values (10 min amplitude, maximum clot firmness) indicating a fibrinolytic potential.DiscussionIn a small cohort, analysing most severly injured patients, the association of increased MP levels and altered coagulation parameters could be demonstrated. However, these findings are based on correlation analysis, which do not enable causel evidence. Therefore, further in-vitro studies are needed analysing the underlying pathomechanisms.ConclusionIn conclusion, this study could demonstrate that PDMP and EDMP levels increase significantly following polytrauma correlating with injury severity. Although severe coagulopathy was not observed, EDMP levels were associated with improved coagulation parameters suggesting their essential role for regulating blood coagulation after trauma.