Endothelial inflammation and neutrophil transmigration are modulated by extracellular matrix composition in an inflammation-on-a-chip model.

Inflammatory diseases are often characterised by excessive neutrophil infiltration from the blood stream to the site of inflammation, which damages healthy tissue and prevents resolution of inflammation. Development of anti-inflammatory drugs is hindered by lack of in vitro and in vivo models which accurately represent the disease microenvironment. In this study, we used the OrganoPlate to develop a humanized 3D in vitro inflammation-on-a-chip model to recapitulate neutrophil transmigration across the endothelium and subsequent migration through the extracellular matrix (ECM). Human umbilical vein endothelial cells formed confluent vessels against collagen I and geltrex mix, a mix of basement membrane extract and collagen I. TNF-α-stimulation of vessels upregulated inflammatory cytokine expression and promoted neutrophil transmigration. Intriguingly, major differences were found depending on the composition of the ECM. Neutrophils transmigrated in higher number and further in geltrex mix than collagen I, and did not require an N-formyl-methionyl-leucyl-phenylalanine (fMLP) gradient for transmigration. Inhibition of neutrophil proteases inhibited neutrophil transmigration on geltrex mix, but not collagen I. These findings highlight the important role of the ECM in determining cell phenotype and response to inhibitors. Future work could adapt the ECM composition for individual diseases, producing accurate models for drug development.

Antisense Therapy Attenuates Phospholamban p.(Arg14del) Cardiomyopathy in Mice and Reverses Protein Aggregation.

Inherited cardiomyopathy caused by the p.(Arg14del) pathogenic variant of the phospholamban (PLN) gene is characterized by intracardiomyocyte PLN aggregation and can lead to severe dilated cardiomyopathy. We recently reported that pre-emptive depletion of PLN attenuated heart failure (HF) in several cardiomyopathy models. Here, we investigated if administration of a Pln-targeting antisense oligonucleotide (ASO) could halt or reverse disease progression in mice with advanced PLN-R14del cardiomyopathy. To this aim, homozygous PLN-R14del (PLN-R14 Δ/Δ) mice received PLN-ASO injections starting at 5 or 6 weeks of age, in the presence of moderate or severe HF, respectively. Mice were monitored for another 4 months with echocardiographic analyses at several timepoints, after which cardiac tissues were examined for pathological remodeling. We found that vehicle-treated PLN-R14 Δ/Δ mice continued to develop severe HF, and reached a humane endpoint at 8.1 ± 0.5 weeks of age. Both early and late PLN-ASO administration halted further cardiac remodeling and dysfunction shortly after treatment start, resulting in a life span extension to at least 22 weeks of age. Earlier treatment initiation halted disease development sooner, resulting in better heart function and less remodeling at the study endpoint. PLN-ASO treatment almost completely eliminated PLN aggregates, and normalized levels of autophagic proteins. In conclusion, these findings indicate that PLN-ASO therapy may have beneficial outcomes in PLN-R14del cardiomyopathy when administered after disease onset. Although existing tissue damage was not reversed, further cardiomyopathy progression was stopped, and PLN aggregates were resolved.

Phospholamban antisense oligonucleotides improve cardiac function in murine cardiomyopathy.

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp-/-), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.

Mathematical Model Predicts that Acceleration of Diabetic Wound Healing is Dependent on Spatial Distribution of VEGF-A mRNA (AZD8601).

INTRODUCTION: Pharmacologic approaches for promoting angiogenesis have been utilized to accelerate healing of chronic wounds in diabetic patients with varying degrees of success. We hypothesize that the distribution of proangiogenic drugs in the wound area critically impacts the rate of closure of diabetic wounds. To evaluate this hypothesis, we developed a mathematical model that predicts how spatial distribution of VEGF-A produced by delivery of a modified mRNA (AZD8601) accelerates diabetic wound healing. METHODS: We modified a previously published model of cutaneous wound healing based on coupled partial differential equations that describe the density of sprouting capillary tips, chemoattractant concentration, and density of blood vessels in a circular wound. Key model parameters identified by a sensitivity analysis were fit to data obtained from an in vivo wound healing study performed in the dorsum of diabetic mice, and a pharmacokinetic model was used to simulate mRNA and VEGF-A distribution following injections with AZD8601. Due to the limited availability of data regarding the spatial distribution of AZD8601 in the wound bed, we performed simulations with perturbations to the location of injections and diffusion coefficient of mRNA to understand the impact of these spatial parameters on wound healing. RESULTS: When simulating injections delivered at the wound border, the model predicted that injections delivered on day 0 were more effective in accelerating wound healing than injections delivered at later time points. When the location of the injection was varied throughout the wound space, the model predicted that healing could be accelerated by delivering injections a distance of 1-2 mm inside the wound bed when compared to injections delivered on the same day at the wound border. Perturbations to the diffusivity of mRNA predicted that restricting diffusion of mRNA delayed wound healing by creating an accumulation of VEGF-A at the wound border. Alternatively, a high mRNA diffusivity had no effect on wound healing compared to a simulation with vehicle injection due to the rapid loss of mRNA at the wound border to surrounding tissue. CONCLUSIONS: These findings highlight the critical need to consider the location of drug delivery and diffusivity of the drug, parameters not typically explored in pre-clinical experiments, when designing and testing drugs for treating diabetic wounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00678-9.

The Effect of Platelet-Rich Plasma on Endothelial Progenitor Cell Functionality.

Platelets mediate circulating endothelial progenitor cell (EPC) recruitment and maturation, participating in vascular repair, however the underlying mechanism(s) remain unclear. We investigated the effect of platelet-rich plasma (PRP) on the functionality of CD34+-derived late-outgrowth endothelial cells (OECs) in culture. Confluent OECs were coincubated with PRP under platelet aggregation (with adenosine diphosphate; ADP) and nonaggregation conditions, in the presence/absence of the reversible P2Y12 platelet receptor antagonist ticagrelor. Outgrowth endothelial cell activation was evaluated by determining prostacyclin (PGI2) and monocyte chemoattractant protein-1 (MCP-1) release and intercellular adhesion molecule-1 (ICAM-1) membrane expression. Similar experiments were performed using human umbilical vein endothelial cells (HUVECs). Platelet-rich plasma increased ICAM-1 expression and PGI2 and MCP-1 secretion compared with autologous platelet-poor plasma, whereas ADP-aggregated platelets in PRP did not exhibit any effect. Platelet-rich plasma pretreated with ticagrelor prior to activation with ADP increased all markers to a similar extent as PRP. Similar results were obtained using HUVECs. In conclusion, PRP induces OEC activation, a phenomenon not observed when platelets are aggregated with ADP. Platelet inhibition with ticagrelor restores the PRP capability to activate OECs. Since EPC activation is important for endothelial regeneration and angiogenesis, we suggest that agents inhibiting platelet aggregation, such as ticagrelor, may promote platelet-EPC interaction and EPC function.

Recombinant human prothrombin (MEDI8111) combined with fibrinogen dose-dependently improved survival time and reduced blood loss in a porcine model of dilutional coagulopathy with uncontrolled bleeding.

: Uncontrolled bleeding due to trauma and coagulopathy is an area with high unmet medical need and high mortality rate. Treatment recommendations focus on transfusion of blood components while optimal therapy to improve coagulation remains to be established. The haemostatic effect of 2, 4 and 8 mg/kg recombinant prothrombin (MEDI8111) co-administered with 100 mg/kg fibrinogen (n = 7-8) was investigated in a porcine model of dilutional coagulopathy with uncontrolled bleeding. Vehicle (n = 11), fibrinogen alone (100  mg/kg , n = 15) were included as controls. Dilutional coagulopathy was induced by replacing ∼75% of the blood volume with hydroxyethyl starch and a standardized liver incision was made followed by intravenous administration of study compounds. Survival time and blood loss were determined up to 120 min after liver incision. Rotational thromboelastometry (ROTEM EXTEM), prothrombin time (PT), thrombin--antithrombin complex and thrombin generation were measured at baseline, after dilution and 10, 40, 80 and 120 min after compound administration. Administration of MEDI8111+fibrinogen improved haemostasis, decreased blood loss and dose-dependently improved survival time compared to fibrinogen. All pigs receiving a dose of 8 mg/kg MEDI8111+fibrinogen, which restored normal prothrombin concentration, survived to the end of the experiment with close to normal haemostasis as measured by PT and ROTEM EXTEM CT. Administration of fibrinogen and MEDI8111 was sufficient to improve survival time and haemostasis in severely coagulopathic pigs. The dose-dependent haemostatic improvement observed with MEDI8111 administration suggests that prothrombin concentration was rate limiting for coagulation.

Genetic deletion of ADP-activated P2Y12 receptor ameliorates lithium-induced nephrogenic diabetes insipidus in mice.

AIM: Therapeutic use of lithium in bipolar disorder is limited by the development of nephrogenic diabetes insipidus (NDI). We reported that pharmacological blockade of P2Y12 receptor (R) with clopidogrel or prasugrel significantly ameliorated lithium-induced NDI in rodents. Using mice genetically lacking P2Y12 -R we evaluated whether the observed amelioration is mediated through P2Y12 -R METHODS: P2ry12-/- mouse line (C57/BL6) was rederived from cryopreserved embryos of the knockout (KO) mice generated by Deltagen Inc. Syngeneic wild type (WT) mice obtained by heterozygous crossing were inbred. Groups of adult WT and KO mice were fed lithium-added (40 mmol LiCl/kg food) or regular diet, and euthanized after 2 or 4 weeks. Twenty-four hour urine samples and terminal blood and kidney samples were analyzed. RESULTS: At both time points, lithium-induced polyuria and decrease in aquaporin-2 (AQP2) protein abundance in the kidney medulla were less marked in KO vs WT mice. Immunofluorescence microscopy revealed that lithium-induced alterations in the cellular disposition of AQP2 protein in the medullary collecting ducts of WT mice were blunted in KO mice. Serum lithium, sodium and osmolality were similar in both genotypes after lithium treatment. After 2 weeks, lithium induced marked increases in urinary excretion of Na, K, and arginine vasopressin in WT mice but not in KO mice. CONCLUSION: Taken together, our data show that similar to pharmacological blockade, deletion of P2Y12 -R significantly ameliorates lithium-induced NDI, without reducing serum lithium levels. Hence, targeting P2Y12 -R with currently available drugs in the market offers a novel and safer method for treating NDI.

Modified VEGF-A mRNA induces sustained multifaceted microvascular response and accelerates diabetic wound healing.

Capable of mediating efficient transfection and protein production without eliciting innate immune responses, chemically modified mRNA holds great potential to produce paracrine factors at a physiologically beneficial level, in a spatiotemporally controlled manner, and with low toxicity. Although highly promising in cardiovascular medicine and wound healing, effects of this emerging therapeutic on the microvasculature and its bioactivity in disease settings remain poorly understood. Here, we longitudinally and comprehensively characterize microvascular responses to AZD8601, a modified mRNA encoding vascular endothelial growth factor A (VEGF-A), in vivo. Using multi-parametric photoacoustic microscopy, we show that intradermal injection of AZD8601 formulated in a biocompatible vehicle results in pronounced, sustained and dose-dependent vasodilation, blood flow upregulation, and neovessel formation, in striking contrast to those induced by recombinant human VEGF-A protein, a non-translatable variant of AZD8601, and citrate/saline vehicle. Moreover, we evaluate the bioactivity of AZD8601 in a mouse model of diabetic wound healing in vivo. Using a boron nanoparticle-based tissue oxygen sensor, we show that sequential dosing of AZD8601 improves vascularization and tissue oxygenation of the wound bed, leading to accelerated re-epithelialization during the early phase of diabetic wound healing.

Prothrombin time is predictive of low plasma prothrombin concentration and clinical outcome in patients with trauma hemorrhage: analyses of prospective observational cohort studies.

BACKGROUND: Fibrinogen and prothrombin have been suggested to become rate limiting in trauma associated coagulopathy. Administration of fibrinogen is now recommended, however, the importance of prothrombin to patient outcome is unknown. METHODS: We have utilized two trauma patient databases (database 1 n = 358 and database 2 n = 331) to investigate the relationship of plasma prothrombin concentration on clinical outcome and coagulation status. Database 1 has been used to assess the relationship of plasma prothrombin to administered packed red blood cells (PRBC), clinical outcome and coagulation biomarkers (Prothrombin Time (PT), ROTEM EXTEM Coagulation Time (CT) and Maximum Clot Firmness (MCF)). ROC analyses have been performed to investigate the ability of admission coagulation biomarkers to predict low prothrombin concentration (database 1), massive transfusion and 24 h mortality (database 1 and 2). The importance of prothrombin was further investigated in vitro by PT and ROTEM assays in the presence of a prothrombin neutralizing monoclonal antibody and following step-wise dilution. RESULTS: Patients who survived the first 24 h had higher admission prothrombin levels compared to those who died (94 vs.67 IU/dL). Patients with lower transfusion requirements within the first 24 h (≤10 units of PRBCs) also had higher admission prothrombin levels compared to patients with massive transfusion demands (>10 units of PRBCs) (95 vs.62 IU/dL). Admission PT, in comparison to admission ROTEM EXTEM CT and MCF, was found to be a better predictor of prothrombin concentration <60 IU/dL (AUC 0.94 in database 1), of massive transfusion (AUC 0.92 and 0.81 in database 1 and 2 respectively) and 24 h mortality (AUC 0.90 and 0.78 in database 1 and 2, respectively). In vitro experiments supported a critical role for prothrombin in coagulation and demonstrated that PT and ROTEM EXTEM CT are sensitive methods to measure low prothrombin concentration. DISCUSSION: Our analyses suggest that prothrombin concentration at admission is predictive of mortality and transfusion and indicates that prothrombin and fibrinogen are rate limiting in coagulopathy. CONCLUSIONS: Admission PT is predictive of low prothrombin concentration and clinical outcome. PT could therefore be used as a surrogate for prothrombin concentration and further evaluation of point-of-care devices for faster PT analysis is warranted.

Recombinant human prothrombin reduced blood loss in a porcine model of dilutional coagulopathy with uncontrolled bleeding.

: Uncontrolled bleeding remains one of the leading causes of trauma-induced death. Treatment recommendations focus on fresh frozen plasma and blood cell transfusions, whereas plasma concentrates or single coagulation factors have been studied in recent years. The effect of recombinant human prothrombin factor II (rhFII, 8 mg/kg), activated recombinant human factor VII (rhFVIIa, 300 µg/kg), plasma-derived human fibrinogen (pdhFib) (200 mg/kg), activated prothrombin complex concentrate (aPCC, 40 IU/kg), a three-factor combination intended as a minimal PCC (8 mg/kg rhFII, 640 µg/kg recombinant human factor X (rhFX), and 12 µg/kg rhFVIIa), and vehicle were investigated in a porcine model of dilutional coagulopathy with uncontrolled bleeding. Survival time and blood loss were determined up to 120 min after induction of liver injury. Rotational thromboelastometry EXTEM coagulation time and maximum clot firmness, prothrombin time, thrombin-antithrombin complex (TAT), thrombin generation (endogenous thrombin potential, ETP) were measured at baseline, after dilution, drug administration, and end of experiment. rhFII, the three-factor combination, and aPCC significantly (P < 0.01) decreased blood loss vs. vehicle and rhFII also vs. fibrinogen (P < 0.05). Survival times increased significantly for rhFII, aPCC, rhFVIIa, and pdhFib vs. vehicle (P < 0.05), and, coagulation time, maximum clot firmness, and prothrombin time improved in all groups. TAT and ETP increased transiently for rhFII and three-factor combination, whereas persistently increased for aPCC. PdhFib and rhFVIIa did not increase TAT and ETP. rhFII decreased blood loss and improved hemostatic markers and survival. In vivo, thrombin generation (TAT) and potential to form thrombin (ETP) were transiently elevated by rhFII. Addition of rhFVIIa and rhFX to rhFII did not further improve hemostatic efficacy.

Preclinical evaluation of point-of-care prothrombin time as a biomarker test to guide prothrombin replacement therapy in coagulopathic bleeding.

BACKGROUND: Hemorrhage is still a common cause of death in trauma. Central lab measured prothrombin time (lab PT) is predictive of low prothrombin concentration and clinical outcome in trauma patients, however, treatment guidance is limited by slow turnaround times. Here, we have preclinically evaluated the potential of a point-of-care prothrombin time test (POC PT) as a faster alternative to identify patients with low prothrombin concentration. METHODS: Human whole blood was serially diluted and prothrombin time measured by POC PT (CoaguChek XS Pro, Roche) and lab PT. Recombinant human prothrombin (MEDI8111) was added to human whole blood with or without depletion of prothrombin by pretreatment with prothrombin neutralizing antibodies. RESULTS: There was no observable difference in the sensitivity of either test to dilution at blood volumes of 60-100%. At blood volumes of ≤55% (equivalent to 47 mg/L prothrombin), PT sharply increased, with greater dilutional sensitivity observed in the POC test. Both tests were insensitive to prothrombin up to 194 mg/L added MEDI8111 (equivalent to 328 mg/L prothrombin versus endogenous concentration of 129 mg/L). Depletion of endogenous prothrombin inversely correlated with an increase in PT which returned to baseline following addition of 97 mg/L MEDI8111 or above. Both assays correlated well above 48.5 mg/L added MEDI8111 (65.9 mg/L prothrombin). CONCLUSIONS: Our data supports that POC PT tests, such as the CoaguChek XS Pro, are fit for purpose to confirm a coagulopathic threshold for prothrombin and provide a fast, simple, and mobile method to guide MEDI8111 therapy in bleeding trauma patients.

Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen.

Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aß), the main pathological component of Alzheimer's disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bß-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions.

Heparin and Protamine Titration Does Not Improve Haemostasis after Cardiac Surgery: A Prospective Randomized Study.

BACKGROUND: Bleeding complications are common in cardiac surgery. Perioperative handling of heparin and protamine may influence the haemostasis. We hypothesized that heparin and protamine dosing based on individual titration curves would improve haemostasis in comparison to standard dosing. SUBJECTS AND METHODS: Sixty patients scheduled for first time elective coronary artery bypass grafting or valve surgery were included in a prospective randomized study. The patients were randomized to heparin and protamine dosing with Hepcon HMS Plus device or to standard weight and activated clotting time (ACT) based dosing. Blood samples were collected before and 10 minutes, 2 hours and 4 hours after cardiopulmonary bypass. Primary endpoint was endogenous thrombin potential in plasma 2 hours after surgery as assessed by calibrated automated thrombography. Secondary endpoints included total heparin and protamine doses, whole blood clot formation (thromboelastometry) and post-operative bleeding volume and transfusions. Heparin effect was assessed by measuring anti-Xa activity. RESULTS: Endogenous thrombin potential and clot formation deteriorated in both groups after surgery without statistically significant intergroup difference. There were no significant differences between the groups in total heparin and protamine doses, heparin effect, or postoperative bleeding and transfusions at any time point. Significant inverse correlations between anti-Xa activity and endogenous thrombin potential were observed 10 min (r = -0.43, p = 0.001), 2 hours (r = -0.66, p<0.001) and 4 hours after surgery (r = -0.58, p<0.001). CONCLUSION: In conclusion, the results suggest that perioperative heparin and protamine dosing based on individual titration curves does not improve haemostasis after cardiac surgery. Postoperative thrombin generation capacity correlates to residual heparin effect. TRIAL REGISTRATION: www.isrctn.com ISRCTN14201041.

Characterization of thrombin derived from human recombinant prothrombin.

Thrombin (FIIa) is the key enzyme in haemostasis and acts on several substrates involved in clot formation, platelet activation and feed-back regulation of its own formation. During activation of blood coagulation, FIIa is formed by proteolytic cleavage of prothrombin (FII). In the production of recombinant human FII (rhFII), a key question is whether the thrombin formed has the same properties as endogenous thrombin. We have investigated whether FIIa formed from rhFII and plasma-derived human FII (pdhFII) have the same enzymatic and haemostatic properties against a number of substrates and the same haemostatic capacity in plasma, whole blood and on platelets. Pure FIIa was isolated from rhFII and pdhFII cleaved by recombinant ecarin, and analytical methods were developed to compare the activity of FIIa against different substrates. FIIa derived from rhFII and pdhFII were found to have very similar properties in activating FVIII, FXIII, protein C, platelet aggregation and plasma or whole blood coagulation. Further, the same turnover for S-2366 was found with similar KM. However, activation of FV with rhFIIa was approximately 25% more effective than with pdhFIIa and heparin-enhanced inhibition of rhFIIa by antithrombin was significantly more efficient compared with pdhFIIa with 10% higher inhibition both at steady state and at initial rate conditions. Although differences between the two FIIa preparations using ecarin cleavage were observed, FIIa derived from rhFII administered to human would likely be very similar in activity and function as FIIa formed from endogenous FII.

Addition of prothrombin to plasma can result in a paradoxical increase in activated partial thromboplastin time.

In the activated partial thromboplastin time (APTT) assay, a variety of nonphysiological reagents is used to induce contact activation. The sensitivity of the APTT response for different thrombin inhibitors has previously been found to be dependent on the used reagent. Recently, infusion of prothrombin (FII) has been used in in-vivo coagulopathy models and its effect has been analyzed in different assays. Therefore, we investigated whether the FII plasma concentration might affect APTT using different commercial reagents, applying both turbidimetry and viscometry. We compared both plasma-derived human FII (pd-hFII) and recombinant human FII (r-hFII). Similar results were found for pd-hFII and r-hFII with different APTT reagents. As expected, no effect on APTT was found by increasing the plasma concentration of FII using APTT reagents consisting of ellagic acid (Actin FS or Actin). Although with Pathromtin SL, consisting of SiO2, only a slight increase was found, with most other commercial APTT reagents, consisting of SiO2 or kaolin, APTT dose-dependently increased by increasing concentration of FII. Therefore, both Pathromtin SL and Actin FS were used to compare r-hFII and pd-hFII by determining the KM at 37C using FII-depleted plasma, providing values of 6 ± 0.3 nmol/l FII for both. Thus, at normal plasma concentrations of FII, the maximal initial thrombin generation rate should be reached and no effect on the coagulation time is expected at higher FII concentrations. To completely avoid the paradoxical effect in the APTT assay at FII concentrations higher than normal, Actin or Actin FS is the preferable reagent.

Sustained heparin effect contributes to reduced plasma thrombin generation capacity early after cardiac surgery.

INTRODUCTION: Thrombin is a key component in the coagulation cascade, and impaired thrombin generation has been linked to increased bleeding after surgical procedures. The aim was to evaluate postoperative thrombin generation capacity in plasma after cardiac surgery, and its potential associations to activity of individual coagulation factors and heparin. MATERIAL AND METHODS: Forty-eight coronary artery bypass grafting patients were included in a prospective observational cohort study. Thrombin generation capacity was analysed in plasma with calibrated automated thrombogram with tissue factor as activator before (baseline), and 2 h and 24 h after surgery. In addition, plasma activity of coagulation factors II, V, VII, VIII, IX, X, XI, XIII, were determined. Heparin effect was assessed by anti-Xa activity, APTT and thrombin time. RESULTS: Thrombin generation was markedly reduced 2h after surgery compared to baseline. Peak levels decreased with median 74% (interquartile range 52-90), p<0.001, and endogenous thrombin generation potential decreased with 65% (43-86), p<0.001. Postoperative changes in endogenous thrombin generation potential correlated inversely to changes in anti-Xa activity (r=-0.51, p=0.010) and to changes in thrombin time (r=-0.51, p=0.009), but there were no correlations to changes in individual coagulation factor activity. CONCLUSIONS: A marked reduction in thrombin generation potential was observed in the early postoperative phase after cardiac surgery. The decrease was independent of reductions in individual coagulation factor activity but correlated to heparin effects. The results indicate that a sustained heparin effect contributes to the postoperative reduction in thrombin generation capacity.

Plasma activity of individual coagulation factors, hemodilution and blood loss after cardiac surgery: a prospective observational study.

BACKGROUND: Hemodilution and consumption of coagulation factors during cardiopulmonary bypass has been suggested to contribute to bleeding complications after cardiac surgery. The aim was to describe the activity of individual coagulation factors after CABG in relation to hemodilution and postoperative bleeding. MATERIALS AND METHODS: Plasma concentrations of fibrinogen and plasma activity of FII, FV, FVII, FVIII, FIX, FX, FXI and FXIII adjusted for hemodilution were analysed in 57 CABG patients before, and 2h and 24h after surgery. Postoperative bleeding was registered and correlations to coagulation factor activity were calculated. RESULTS: Adjusted plasma concentration of fibrinogen (-14+/-6%), and plasma activity of FII (-9+/-6%), FV (-13+/-8%), FX (-13+/-7%) and FXIII (-9+/-14%) were reduced two hours after surgery compared to baseline (all p<0.001). FVII (+3+/-12%, p=0.34) and FXI (+1+/-19%, p=0.50) were unchanged, while FVIII (+23+/-44%, p=0.006) and FIX (+23+/-17%, p<0.001) increased. Twenty-four hours after surgery fibrinogen (+45+/-27%), FVIII (+93+/-66%) and FIX (+33+/-26%) were all increased (all p<0.001), while FVII (-37+/-14%, p<0.001), FXI (-4+/-18%, p=0.02) and FXIII (-6+/-15%, p=0.004) were decreased. Median postoperative blood loss was 380 ml/12h. There were significant inverse correlations between postoperative blood loss and fibrinogen concentration 2h after surgery (r=-0.33, p=0.019) and between postoperative blood loss and pre- and postoperative FXIII activity (r=-0.34, p=0.009 and r=-0.41, p=0.003, respectively), but not between blood loss and any of the other factors. CONCLUSIONS: There is a marked dissociation in plasma activity of individual coagulation factors after CABG. Plasma concentration of fibrinogen and factor XIII activity correlates inversely to postoperative blood loss after CABG.

Surface plasmon resonance detection of blood coagulation and platelet adhesion under venous and arterial shear conditions.

A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood.

Whole blood coagulation on protein adsorption-resistant PEG and peptide functionalised PEG-coated titanium surfaces.

The aim of this study was to investigate whole blood coagulation on low blood plasma protein adsorbing surfaces. For this purpose, the polycationic graft copolymer poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), PLL-g-PEG grafted with a cell adhesive peptide containing the amino acid sequence -Arg-Gly-Asp- (RGD), and PLL-g-PEG with a control peptide -Arg-Asp-Gly- (RDG) were adsorbed onto titanium (oxide), forming stable monomolecular adlayers through electrostatic attraction. Free oscillation rheometry and complementary techniques were used to measure the coagulation time (CT) and other interactions of the surfaces with native whole blood, recalcified platelet-rich plasma (PRP), and recalcified citrated platelet-free plasma (PFP). The results show that the uncoated titanium surfaces (reference) activated platelets and quickly triggered the coagulation cascade via the intrinsic pathway, whereas the PLL-g-PEG surfaces displayed a prolonged CT, approximately 2-3 times longer compared to uncoated titanium. We hypothesise that blood coagulates outside the vascular system independent of low protein adsorption to or activation by surfaces, due to the absence of an active down-regulation of procoagulative processes by the vascular endothelium.

Surface plasmon resonance and free oscillation rheometry in combination: a useful approach for studies on haemostasis and interactions between whole blood and artificial surfaces.

In haemostatic and biomaterial research biological processes at surfaces and in the bulk phase of the surface-contacting medium are important. The present work demonstrates the usefulness of the combination of surface plasmon resonance (SPR), sensitive to changes in refractive index at surfaces, and free oscillation rheometry (FOR), sensitive to rheological properties of the bulk, for simultaneous real-time measurements on coagulation and fibrinolysis of blood plasma and coagulation of whole blood. SFLLRN stimulated coagulation of native whole blood presented a higher SPR signal with different appearance than plasma coagulation, while the FOR signals corresponding to plasma and whole blood coagulation were similar. This indicated that the SPR technique was more sensitive to cell-surface interactions than to fibrin formation in whole blood during coagulation, while the FOR technique were equally sensitive to coagulation in whole blood and plasma. Spontaneous coagulation of native whole blood in contact with methyl- and hydroxyl-terminated self-assembled monolayers (SAM) on gold and gold surfaces regenerated after coagulation were also studied. The regenerated gold surfaces displayed the shortest coagulation times, although the contact-activation of blood coagulation for these surfaces was low. The methylated and hydroxylated surfaces were comparable in terms of coagulation activation, while the hydroxylated surfaces presented FOR signals that indicated detaching of the coagulum from the surface. The combination of SPR and FOR is well suited for studies of cell- and protein-surface interactions and simultaneous bulk processes. Possible applications are investigations of blood cell defects in patients and monitoring of native whole blood interactions with artificial surfaces.