Hemostatic Potential Research Articles

The use of thromboelastography and the functional tests with double local hypoxia of the upper limb to assess the risk of thromboembolism in patients undergoing surgery

The use of thromboelastography and the functional tests with double local hypoxia of the upper limb to assess the risk of thromboembolism in patients undergoing surgery

NXT007-mediated hemostatic potential is suppressed by activated protein C-catalyzed inactivation of activated factor V

BackgroundActivated protein C (APC) inactivates activated factor (F) V (FVa) and FVIIIa. NXT007, an emicizumab-based engineered therapeutic bispecific antibody, enhances the coagulation potential of FVIII-deficient plasma (FVIIIdef-plasma) to near normal levels. However, little is known about the effect of APC-induced inactivation in NXT007-mediated hemostatic function. ObjectivesTo investigate the contribution of APC-mediated reactions to NXT007-driven hemostasis. MethodsIn pooled normal plasma (PNP) or FVIIIdef-plasma spiked with NXT007 (10 μg/mL), effects of APC (0-16 nM) were measured using a thrombin generation assay (TGA). The direct effects of APC on cofactor activity of NXT007 or FVIIIa in a FXa generation assay were also measured. The FVdef-plasma and FV Leiden plasma (FVLeiden plasma) were preincubated with 2 anti-FVIII monoclonal antibodies (termed FVIII-depleted), and the APC effect in the presence of NXT007 in FVIII-depleted FVdef-plasma with the addition of exogenous FV (7.5-30 nM) or FVIII-depleted FVLeiden plasma was investigated. ResultsThe APC dose-dependent suppression effect in TGA of FVIIIdef-plasma spiked with NXT007 was similar to that of PNP. FXa generation with NXT007 was not impaired by the addition of APC, suggesting that the APC-induced reaction in TGA with NXT007 was attributed to the direct inactivation of FVa. The addition of APC to FVIII-depleted FVdef-plasma, along with NXT007 and various FV concentrations, showed a similar attenuation to PNP. The NXT007-driven thrombin generation in FVIII-depleted FVLeiden plasma was suppressed by APC, similar to the reaction in native FVLeiden plasma. ConclusionNXT007 did not impair APC-mediated downregulation of FVa in FVIIIdef-plasmas, regardless of the presence of FV mutation with APC resistance.

Factor VIIa releases phosphatidylserine-enriched extracellular vesicles from endothelial cells by activating acid sphingomyelinase

BackgroundOur recent studies showed that activated factor (F) VII (FVIIa) releases extracellular vesicles (EVs) from the endothelium. FVIIa-released EVs were found to be enriched with phosphatidylserine (PS) and contribute to the hemostatic effect of FVIIa in thrombocytopenia and hemophilia. ObjectiveTo investigate mechanisms by which FVIIa induces EV biogenesis and enriches EVs with PS. MethodsFVIIa activation of acid sphingomyelinase (aSMase) was evaluated by its translocation to the cell surface. The role of aSMase in the biogenesis of FVIIa-induced EVs and their enrichment with PS was investigated using specific siRNAs and inhibitors of aSMase and its downstream metabolites. Wild-type and aSMase-/- mice were injected with a control vehicle or FVIIa. EVs released into circulation were quantified by nanoparticle tracking analysis. EVs hemostatic potential was assessed in a murine thrombocytopenia model. ResultsFVIIa activation of aSMase is responsible for both the externalization of PS and the release of EVs in endothelial cells. FVIIa-induced aSMase activation led to ceramide generation and de novo expression of transmembrane protein 16F. Inhibitors of ceramidases, sphingosine kinase, or sphingosine-1-phosphate receptor modulator blocked FVIIa-induced expression of transmembrane protein 16F and PS externalization without interfering with FVIIa release of EVs. In vivo, FVIIa release of EVs was markedly impaired in aSMase-/- mice compared with wild-type mice. Administration of a low dose of FVIIa, sufficient to induce EVs release, corrected bleeding associated with thrombocytopenia in wild-type mice but not in aSMase-/- mice. ConclusionOur study identifies a novel mechanism by which FVIIa induces PS externalization and releases PS-enriched EVs.

Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis

AbstractElevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga−/−) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen–platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga−/− mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

Extracellular vesicles in plasma and cerebrospinal fluid in patients with COVID-19 and neurological symptoms.

Increased levels of extracellular vesicles (EVs) are associated with haemostatic disturbances in various clinical settings. However, their role in COVID-19 patients is still not fully clear. In the present study we investigated EVs in plasma from patients with COVID-19 and neurological symptoms in relation to the activation of coagulation. Nineteen COVID-19 patients with neurological symptoms and twenty-three aged-matched healthy individuals were included. Global coagulation assays were performed and levels of EVs were determined by flow-cytometry in plasma and cerebrospinal fluid (CSF). A procoagulant state characterized by significantly increased overall coagulation- (OCP) and overall haemostatic potential (OHP), diminished overall fibrinolytic potential (OFP) together with a denser fibrin structure was found in patients with COVID-19. Flow cytometry revealed elevated levels of plasma circulating EVs derived from neutrophils (MPO+) and platelets (CD61+), as well as EVs expressing phosphatidylserine (PS+) and complement component C5b-9 (TCC+) in patients with COVID-19 compared with controls. The concentrations of PS+, CD61+ and TCC+ EVs were positively correlated with OCP and OHP in COVID-19 patients. Moreover, we identified CD61+, MPO+ and endothelial cell-derived EVs, as well as EVs exposing PS and TCC in the CSF of patients suffering from neurological symptoms during COVID-19. The unique finding in this study was the presence of EVs in the CSF of COVID-19 patients with neurologic manifestations as well as higher expression of complement protein on circulating plasma EVs. EVs may indicate blood-brain barrier damage during SARS-COV-2 infection.

Prognostic Factors for Restenosis of Superficial Femoral Artery after Endovascular Treatment.

High incidence of superficial femoral artery (SFA) restenosis after percutaneous transluminal angioplasty (PTA) poses a persistent challenge in peripheral arterial disease (PAD) treatment. We studied how the patients' and lesions' characteristics, thrombin generation, overall haemostatic potential (OHP), and single nucleotide polymorphisms (SNPs) of the NR4A2 and PECAM1 genes affected the likelihood of restenosis. In total, 206 consecutive PAD patients with limiting intermittent claudication due to SFA stenosis who were treated with balloon angioplasty with bailout stenting when necessary were included. Patients' clinical status and patency of the treated arterial segment on ultrasound examination were assessed 1, 6, and 12 months after the procedure. Restenosis occurred in 45% of patients, with less than 20% of all patients experiencing symptoms. In the multivariate analysis, predictors of restenosis proved to be poor infrapopliteal runoff, higher lesion complexity, absence of treated arterial hypertension, delayed lag phase in thrombin generation, and higher contribution of plasma extracellular vesicles to thrombin concentration. Poor infrapopliteal runoff increased the risk of restenosis in the first 6 months, but not later. The negative effect of poor infrapopliteal runoff on SFA patency opens questions about the potential benefits of simultaneous revascularisation of below-knee arteries along with SFA revascularisation.

Potential hemostatic compounds targeting urokinase plasminogen activator explored from three Euphorbiaceae species: Euphorbia maculata, Euphorbia humifusa, and Acalypha australis, with bio-affinity ultrafiltration UPLC-MS.

Numerous species of the Euphorbiaceae family, including Euphorbia maculata, Euphorbia humifusa, and Acalypha australis, have been used to manage bleeding disorders. However, few investigations have demonstrated their hemostatic potential, and their procoagulant compounds remain elusive. This study aimed to determine the most active procoagulant extracts from the three species' crude extract (CE) and fractions in order to screen out the active compounds and to analyze their possible mechanisms of action. An integrative approach, comprising prothrombin time and activated partial thromboplastin time evaluations and urokinase-type plasminogen activator (uPA) inhibitory assessment, followed by bio-affinity ultrafiltration paired with UPLC/QTOF-MS targeting uPA and docking simulations, was used. The extracts with highest procoagulant activity were the CE for both E. maculata (EMCE) and E. humifusa (EHCE) and the n-butanol fraction (NB) for A. australis (AANB). The most promising ligands, namely, isoquercetin, orientin, rutin, and brevifolin carboxylic acid, were selected from these lead extracts. All of these compounds exhibited pronounced specific binding values to the uPA target and showed tight intercalation with the crucial side chains forming the uPA active pocket, which may explain their mode of action. The activity validation substantiated their hemostatic effectivity in inhibiting uPA as they had better inhibition constant (Ki) values than the reference drug tranexamic acid. Collectively, the integrative strategy applied to these three species allowed the elucidation of the mechanisms underlying their therapeutic effects on bleeding disorders, resulting in the fast detection of four potential hemostatic compounds and their mode of action.

Effect of Paclitaxel on the Hemostatic Potential of the Blood in the Experiment.

The duration and severity of prothrombotic effects of the maximum tolerated dose of paclitaxel (40 mg/kg) were evaluated in intact outbred mice. Hemostasis was assessed before and on days 1, 2, 5, 7, 10, 15, 20, and 30 after a single injection of paclitaxel using standard coagulation tests (activated partial thromboplastin time, prothrombin time, fibrinogen concentration, and antithrombin III) and a "global" method, low-frequency piezothromboelastography. A pronounced prothrombotic effect of paclitaxel was revealed starting from the first day postinjection that consisted in intensification of fibrinogenesis up to the 7th day in parallel with activation of the anticoagulant mechanisms. On days 7-30 after paclitaxel administration, decompensation of its anticoagulant activity due to paclitaxel-induced damage to the endothelium was observed with the formation of a procoagulant status of the hemostatic potential of the blood. A single administration of the maximum tolerated dose of paclitaxel forms a powerful thrombogenic stimulus during the first week and provides a long-term/trace procoagulant shift in the hemostasis system (days 10-30).

Physico chemical characterisation of pectin incorporated gelatin sponge and its functional evaluation in modulating contact activation haemostasis.

Even though it is a common occurrence in practice, maintaining haemostasis can sometimes become a challenging issue in case of trauma, perioperative period, coagulation disorders, cancers, etc. Hemostatic materials are extensively used to assist in the cessation of bleeding. However, the definition of efficiency of haemostasis varies between intended procedures. This paper explores the feasibility of incorporating agents to increase the efficiency of local haemostasis. Pectin or β -D galacto hexopyranuronic acid/β Gal A, a structural polysaccharide widely present in terrestrial plants having an intrinsic hemostatic potential, is blended with gelatin and is explored in modulating passive haemostasis. The sponges are physico chemically characterized, and their hemostatic efficiency is evaluated in vitro using various assays. Biocompatibility evaluation is done by in vitro cytotoxicity assay. The results suggest that this biopolymer combination is a promising candidate for hemostatic control.

An assessment of the safety, hemostatic efficacy, and clinical impact of low-titer group O whole blood in children and adolescents.

Low-titer group O whole blood (LTOWB) use has been associated with improved survival and less blood transfusions in adult trauma patients. Its use in pediatric trauma has been shown to be safe when using leukoreduced, LTOWB with anti-A, anti-B antibody titers of <1:50. We set out to evaluate the safety, hemostatic potential, and impact on pediatric outcomes at a center using non-leukoreduced, LTOWB with anti-A, anti-B antibody titers of <1:200. Patients younger than 18 years, who received emergency-release, uncrossed matched blood, and presented to our trauma center from November 2017 to April 2021 were included. Patients were divided into those receiving any LTOWB and those receiving only RBC and or plasma (COMP). Primary outcome was 30-day survival. One hundred sixty-four patients received emergency release blood products. Of these, 73 received at least one unit of LTOWB. The LTOWB group were younger (14 years vs. 13 years), more likely to be male (87% vs. 49%), and to have sustained penetrating trauma (44% vs. 23%); all p < 0.05. Low-titer group O whole blood patients received more blood than their COMP counterparts prior to arrival. Serial hemolysis panels (K+, bilirubin, LDH, haptoglobin) obtained at 24 hours, 48 hours, and 72 hours were similar between groups; all p > 0.05. There was no difference in survival by univariate analysis but after adjusting for inverse probability of treatment weights there was an observed association between WB administration and improved survival, with an odds ratio of 2.48 (1.15-5.47). Non-leukoreduced, LTOWB in anti-A/anti-B antibody titers of <1:200 appear safe in children and adolescents. While patients receiving LTOWB had more evidence of shock, higher torso injury severity, and received more prehospital blood products, there may be a mortality benefit with whole blood. Larger, multicenter studies are needed. Therapeutic/Care Management; Level IV.

Relationship between systemic inflammatory response and hypercoagulation in patients with immuno-inflammatory rheumatic diseases

The relationship between the processes of coagulation and inflammation protects the organism from potentially dangerous biological agents. However, hyperinflammation leads to an increase in the procoagulation potential, and activation of hemostasis factors maintains the inflammatory process. This phenomenon is called “immunothrombosis” or “ thromboinflammation”. The study of thromboinflammatory mechanisms is an actual problem of modern medicine, because in the future it will help to improve the therapy of diseases, in the pathogenesis of which thromboinflammation plays a significant role. The aim: to carry out a comparative analysis of the severity of the systemic inflammatory response in patients with immuno- inflammatory rheumatic diseases depending on the manifestations of hypercoagulation.To achieve the aim, a comparative analysis of proinflammatory markers (IL-6, IL-8, IL-10, TNFα, sIL-2R, CRP, ECP, β2-microglobulin) in the blood of patients with immune-inflammatory rheumatic diseases (systemic lupus erythematosus, rheumatoid arthritis, reactive arthritis, ankylosing spondylitis, psoriatic arthritis, rheumatic heart disease) was performed. Based on these inflammatory markers according to the authors' original methodology, the integral index of systemic inflammatory response (SIR) — Reactivity Level (RL) — was calculated. The cohort was divided into 2 groups: with the presence of signs of hypercoagulation and without signs of hypercoagulation according to the presence of elevated D-dimer level (&gt; 500 ng/mL). Control group — healthy blood donors.The results of the study showed that SIR develops in patients with immuno-inflammatory rheumatic diseases regardless of the blood hemostatic potential. Patients with signs of hypercoagulation were characterized by higher values of most proinflammatory molecular markers, as well as increased integral level of SIR, which indicates a strong relationship between coagulation processes and inflammation at the systemic level. In addition, the probability of hypercoagulation increases with increasing severity of SIR (assessed by means of the integral index — RL). Thus, there is a transition of quantitatively more pronounced signs to a new qualitative level of pathological process development.The pathogenesis of immuno-inflammatory rheumatic diseases is characterized by the development of SIR (hypercytokinemia, acute phase response, intravascular leukocyte activation), the severity of which is closely related to intravascular microthrombosis.

Efficacy of Flowable Collagen Hemostat Evaluated in Preclinical Models of Liver Injury and Spinal Cord Exposure.

Excessive bleeding in trauma and surgical settings leads to increased operative time, reoperation rates, and overall healthcare costs. A wide range of hemostatic agents have been developed to control bleeding that can vary considerably in type of hemostatic action, ease of application, cost, risk of infection, and dependence on patient coagulation. Microfibrillar collagen-based hemostatic materials (MCH) have yielded beneficial results in a variety of applications. A new flowable collagen product, containing a modified MCH flour, but in a more convenient flowable delivery system, was evaluated for hemostatic efficacy in preclinical models of solid organ injury and spinal cord exposure. The primary objective of this study was to compare the hemostatic potential and local tissue responses to this novel, flowable collagen-based hemostatic agent to the original flour formulation to confirm that the new method of delivery did not interfere with the hemostatic properties of the MCH flour. When observed visually, the flowable MCH flour mixed with saline (FL) provided more precise application and uniform coverage to injured tissues compared to the dry MCH flour alone (F0). All of the treatments (FL, F0, and gauze) exhibited comparable Lewis bleed grade at all three time points evaluated in the capsular resection liver injury model (bleed grade: 1.0-1.3; p> 0.05 in all cases). FL and F0 exhibited comparable 100% acute hemostatic efficacy and similar long-term histomorphological properties (up to 120 days) in a capsular resection liver injury in pigs, while gauze resulted in significantly lower rates of acute hemostatic efficacy (8-42%, p<0.05 in all cases). In an ovine model of dorsal laminectomy and durotomy, FL and F0 again exhibited comparable results without any neurological effects. Flowable microfibrillar collagen was shown to yield favorable short- and long-term outcomes in two representative applications where hemostatic efficacy is critical to surgical success.

Changes in the blood coagulation system in patients with chronic heart failure

The article provides a review of scientifi c publications dedicated to the study of the hemostatic system in patients with chronic heart failure (CHF). Russian and foreign authors evaluated the risks of bleeding and thrombosis in patients with CHF, as well as changes in the content of individual markers and factors of the coagulation system. The review suggests that the results obtained are quite contradictory. On the one hand, data is presented on an increased risk of thrombosis, changes in the content of some endothelial markers that promote endothelial thrombogenicity, an increase in the content of platelet aggregation markers, and elevated levels of fi brinogen and thrombin. On the other hand, there is data on an increased risk of bleeding in patients with CHF, an increase in some endothelial markers that enhance the anticoagulant properties of the endothelium. From the analysis conducted, it can be concluded that this topic is not suffi ciently studied, and it is diffi cult to judge the hemostatic potential in patients with CHF at present. The use of integrated methods such as thromboelastography (TEG) and low-frequency piezothromboelastography (LF-PTEG) are a promising direction for studying this problem. The results obtained using these methods will likely allow for a more precise understanding of the features of the coagulation system functioning in patients with CHF.

In vitro comparison of cold-stored whole blood and reconstituted whole blood.

Cold-stored whole blood (CSWB) is increasingly used in damage control resuscitation. Haemostatic function of CSWB seems superior to that of reconstituted whole blood, and it is sufficiently preserved for 14-21 days. To provide evidence for a yet insufficiently studied aspect of prehospital CSWB use, we compared in vitro haemostatic properties of CSWB and currently used in-hospital and prehospital blood component therapies. Blood was obtained from 24 O RhD positive male donors. Three products were prepared: CSWB, in-hospital component therapy (red blood cells [RBCs], OctaplasLG and platelets 1:1:1) and prehospital component therapy (RBCs and lyophilized plasma 1:1). Samples were drawn on days 1 and 14 of CSWB or RBC cold storage. On day 14, platelet concentrates at their expiry (5 days) were used for 1:1:1 mixing. Conventional clotting assays, rotational thromboelastometry, thrombin formation and platelet function were assessed. Haemoglobin, platelet count, fibrinogen and coagulation factor levels remained closest to physiological in CSWB. Factor VIII activity decreased markedly by day 14 in CSWB. The decline in platelet function was prominent in CSWB. However, CSWB on day 14 yielded physiological EXTEM MCF, suggesting haemostatically sufficient platelet function. Despite haemodilution and lower coagulation factor levels, in-hospital component therapy was haemostatically adequate. Prehospital component therapy formed the weakest clots. Thrombin formation potential remained comparable and stable in all groups. Current prehospital component therapy fails to offer the clotting potential that CSWB does. CSWB and current in-hospital 1:1:1 component therapy show similar haemostatic potential until 14 days of storage.

Abstract 143: Differential Suppression Of Arterial Versus Venous Thrombosis But Preserved Hemostasis In Mice Expressing Fibrinogen That Cannot Support Fibrin Formation

Elevated circulating fibrinogen correlates with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. However, the precise contribution of fibrin matrix to occlusive arterial and venous thrombus formation in vivo remains largely undefined. To determine the contribution of fibrinogen versus fibrin to arterial thrombosis, venous thrombosis, and hemostatic potential in vivo , arterial and venous thrombotic challenges as well as hemostatic tests were performed in wildtype ( Fga WT ) and Fga EK mice that express normal levels of fibrinogen “locked” in the monomeric form. In a model arterial thrombosis, a 10% FeCl 3 injury to the carotid artery resulted in similar times to occlusion between Fga WT/WT , Fga WT/EK and Fga EK/EK mice, while Fga -/- mice were completely protected. However, when injured with 5% FeCl 3 , Fga EK/EK mice were partially protected from occlusion but had increased embolic events. These results suggest that fibrinogen can contribute to arterial thrombus formation independent of fibrin formation. Consistently, fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content in Fga WT/WT , Fga WT/EK and Fga EK/EK mice were similar. In an inferior vena cava stasis model of venous thrombosis, Fga EK/EK had near complete protection from thrombus formation. Intriguingly, Fga WT/EK mice also displayed significant reduction in thrombus frequency and mass relative to Fga WT/WT mice. Plasma from Fga WT/EK mice produced prolonged prothrombin and thrombin times with reduced fibrin formation based on turbidity assay. In bleeding models, Fga WT/EK and, Fga EK/EK mice had preserved hemostasis in contrast to Fga -/- mice that presented with significantly impaired hemostasis. These findings show that selective prevention of fibrin polymerization significantly reduces venous thrombosis and suppresses arterial thrombosis based on the severity of injury with minimal disruption to hemostasis. Moreover, the data suggests that administration of non-polymerizable fibrinogen may be a therapeutic strategy against thrombosis.

Abstract 513: Localised Anticoagulation Via Gene Therapy: Towards Prevention Of Cardioembolic Stroke

Introduction: The coagulation cascade thrives as the haematological gatekeeper ensuring equilibrium between blood flow and thrombosis. Under pathological conditions, the positive-feedback loops compound upon one another, disrupting this equilibrium and increasing the risk of thrombosis and embolic events. Cardiac thromboembolism persists as the culprit behind most ischemic strokes; a majority arising from the left atrial appendage. In order to combat this disease and overcome the limitations of systemic oral anticoagulants, we developed a novel anticoagulant viral vector to focally target cardiac regions of increased thrombotic potential. Hypothesis: Our novel recombinant lentiviral and adeno-associated viral vectors expressing thrombomodulin will induce a significant cell autonomous anticoagulant effect when expressed by endothelial cells of the left atrial appendage. Methods: The anticoagulant phenotype of these vectors was functionally assessed through in vitro and ex vivo platforms. For in vitro testing, a novel, cell-based version of the calibrated automated thrombogram assay, coupled with the overall haemostatic potential assay were utilised to determine thrombokinetics in cell culture models. For ex vivo testing, freshly isolated and characterized porcine left atrial appendage endothelial cells were used. These cells were used to endothelialise microfluidic devices, which were stimulated with TNF-α prior to perfusion with human whole blood. Changes in fibrin, platelets and neutrophils were investigated in the presence and absence of our novel anticoagulant viral vectors. Results: Experimental findings from our in vitro and ex vivo work showed a significant decrease in the endogenous thrombin potential of vector-transduced cells, coupled with a significant decrease in the velocity of thrombogenesis. Furthermore, transduced cells had a marked decrease in the peak rate of thrombin generation and a significant increase in the time taken to achieve peak thrombin generation. Conclusion: These findings show the impact of our anticoagulant vectors through inhibiting thrombosis and guide the development of focal gene therapy strategies to target thrombosis at its point of origin in the prevention of cardioembolic-stroke.

Global coagulation assays in patients with chronic kidney disease and their role in predicting thrombotic risk

BackgroundDespite cardiovascular diseases and thrombosis being major causes of death in patients with chronic kidney disease (CKD), there remains no effective biomarker to predict thrombotic risk in this population. ObjectiveTo evaluate global coagulation assays in patients with CKD and correlate the biomarkers to clinical outcomes. Material and methodsPatients with eGFR<30 mL/min/1.73m2 were recruited (n = 90) in this prospective observational study. Blood samples were collected for global coagulation assays, including thromboelastography, calibrated automated thrombogram (CAT), overall hemostatic potential (OHP) and tissue factor pathway inhibitor (TFPI). ResultsFollowing adjustment for age and gender, CKD subjects (mean age 66 years, 36 % female) had increased maximum amplitude on thromboelastography (70.1 vs 60.2 mm, p < 0.001), higher peak thrombin (233.2 vs 219.7 mm, p = 0.030) and increased OHP (16.1 vs 6.4 units, p < 0.001) compared to healthy controls (n = 153). TFPI was also increased in CKD patients (36.4 vs 14.5 ng/mL, p < 0.001). Compared to hemodialysis patients (n = 43), peritoneal-dialysis patients (n = 25) had more hypercoagulable parameters. Thirty-five CKD patients reported thrombotic complications – key predictors included dialysis, higher fibrinogen, reduced endogenous thrombin potential, elevated D-dimer and increased TFPI. Using the dialysis cohort, the predictive risk model based on the key predictors performed better than Framingham heart score and number of cardiovascular risk factors (Harrell's C-stat 0.862 vs 0.585 vs 0.565). ConclusionCKD appears to confer a hypercoagulable state compared to healthy controls. Interestingly, reduced thrombin generation and raised TFPI was paradoxically associated with increased thrombotic risks, highlighting possible complex compensatory mechanisms within the coagulation system, which may be important in predicting clinical outcomes.

Factor VIII mutated with Lys1813Ala within the factor IXa-binding region enhances intrinsic coagulation potential.

Factor VIII (FVIII) functions as a cofactor of FIXa for FX activation in the intrinsic tenase complex. The 1811-1818 region in the FVIII A3 domain was observed to contribute to FIXa binding, and the K1813A/K1818A mutant increased the binding affinity for FIXa. The current study aims to identify mutated FVIII protein(s) that increase FVIIIa cofactor activity in the 1811-1818 region. FVIII mutants with K1813A, K1818A, and K1813A/K1818A were expressed in baby hamster kidney cells and were followed by assessments using purified and global coagulation assays for mouse models with hemophilia A (HA). A surface plasmon resonance-based assay revealed that the Kd value of FVIII-K1813A for FIXa interaction was lower than that of the wild-type (WT) (3.9±0.7/6.3±0.3 nM). However, the Km value of FVIII-K1813A for FIXa on tenase activity was comparable with that of the WT, whereas the kcat of this mutant was significantly greater than that of the WT. Thrombin-catalyzed FVIII-K1813A activation was ∼1.3-fold more enhanced than that of the WT, and the spontaneous decay of activated FVIII-K1813A was ∼2.5-fold slower than that of WT. The heat stability assay revealed that the decay rate of FVIII-K1813A was ∼2.5-fold slower than that of WT. Thrombin generation assay and rotational thromboelastometry using blood samples from patients with HA demonstrated that the addition of FVIII-K1813A (0.5 nM) exhibited a coagulation potential compatible with that of WT (1 nM). In the tail clip assay of HA mice, FVIII-K1813A showed a two- to fourfold higher hemostatic potential than that of the WT. FVIII-K1813A, with higher a FIXa binding affinity, enhances the global coagulation potential because of the stability of FVIII/FVIIIa molecules.

An evaluation of von Willebrand factor (recombinant) therapy for adult patients living with severe type 3 von Willebrand disease

ABSTRACT Introduction Von Willebrand Factor (VWF) containing concentrates have been used for the treatment of von Willebrand Disease (VWD) for many years. Recently, however, a novel recombinant VWF (rVWF or vonicog alpha, VONVENDI [US], VEYVONDI [Europe]) has arrived to the market for the treatment of VWD. Initially, rVWF was approved by the U.S. Food and Drug Administration (FDA) for the on-demand treatment and control of bleeding episodes and for the perioperative management of bleeding for patients with VWD. More recently, however, the FDA has approved rVWF for routine prophylaxis to prevent bleeding episodes for those patients with severe type 3 VWD receiving on-demand therapy. Areas Covered This review will focus on recent phase III trial results from NCT02973087 regarding the use of long-term routine twice weekly prophylaxis with rVWF for the prevention of bleed events in patients with severe type 3 VWD. Expert Opinion A novel rVWF concentrate may have greater hemostatic potential over prior plasma-derived VWF concentrates and is now FDA approved for use in routine prophylaxis for patients with severe type 3 VWD in the United States. This greater hemostatic potential may be due to the presence of ultra-large VWF multimers and a more favorable high-molecular-weight multimer pattern compared to prior pdVWF concentrates.

Altered Storage and Function of von Willebrand Factor in Human Cardiac Microvascular Endothelial Cells Isolated from Recipient Transplant Hearts.

The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel-Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMECD), or from nominally healthy donors (controls; HCMECC). Using fluorescence microscopy, WPBs in HCMECC (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMECD (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMECD revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMECD WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMECD were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.