Circulating Coagulation Factors Research Articles

Mechanisms of Hypercoagulability Driving Stroke Risk in Obesity: A Mendelian Randomization Study.

Obesity is hypothesized to induce a hypercoagulable state that increases stroke risk. The molecular mechanisms underlying this association are largely uncharacterized. We aimed to apply mendelian randomization to identify whether the association of genetically proxied body mass index (BMI) with cardioembolic stroke risk is mediated by changes in levels of circulating coagulation factors. Genetic proxies for BMI and levels of circulating coagulation factors were obtained, respectively, from the Genetic Investigation of ANthropometric Traits consortium (n = 694,649) and deCODE cohort (n = 35,559). Genetic associations with cardioembolic stroke risk were obtained from the GIGASTROKE consortium (10,804 cases and 1,234,804 controls). We performed a two-sample mendelian randomization analysis testing the association of genetically proxied BMI with cardioembolic stroke risk, genetically proxied BMI with levels of coagulation factors, and genetically proxied levels of coagulation factors with cardioembolic stroke risk. These estimates were carried forward to mediation and sensitivity analyses. A 1-SD increase in genetically proxied BMI associated with increased cardioembolic stroke risk (OR of cardioembolic stroke per 1-SD of BMI 1.20, 95% CI 1.08-1.33, p = 8.65 × 10-4) with similar findings in statistical sensitivity analyses more robust to the inclusion of pleiotropic variants. Genetically proxied BMI was further associated with increased levels of Factor VII, Factor Xa, Factor XI, and Protein S (all p < 5.9 × 10-6). Of these factors, genetically proxied levels of Factor XI were associated with cardioembolic stroke risk (OR of cardioembolic stroke per 1-SD increase in Factor XI levels 1.32, 1.19-1.46, p = 6.18 × 10-8). The mediated effect of genetically proxied BMI through Factor XI accounted for 26% (6%-49%) of the total effect of BMI on cardioembolic stroke. Human genetic data support increased levels of Factor XI as a mechanistic explanation for how obesity increases cardioembolic stroke risk. The clinical relevance of this association warrants further investigation within ongoing clinical trials of Factor XI inhibition.

Etranacogene Dezaparvovec (Hemgenix)

CADTH recommends that public drug plans reimburse Hemgenix for the treatment of hemophilia B if certain conditions are met. Hemgenix should only be covered to treat patients (≥ 18 years of age) with moderately severe to severe hemophilia B (circulating coagulation factor IX [FIX] ≤ 2%) if their bleeding requires ongoing prophylactic treatment, their titre of the neutralizing antibody to variant adeno-associated virus 5 (AAV5) is below 1:900, they do not have FIX inhibitors, and if they have not previously received gene therapy to treat hemophilia B. Hemgenix should only be reimbursed if it is prescribed by specialists who are experts in treating hemophilia B and the cost of Hemgenix is reduced.

A rare case of acquired hemophilia A

Acquired hemophilia A (AHA) is a rare bleeding disorder characterized by IgG autoantibodies directed against circulating coagulation factor (F) VIII neutralizing its coagulant function. The usual clinical presentation of AHA is spontaneous or provoked bleeding in a person with a negative personal or family history of a coagulopathy and can lead life threatening bleeding especially in older patients. Here we report a case of 54-year-old male patient presenting with blood and passage of clots in urine. He was treated with recombinant factor VII and VIII with steroid as eradication therapy. Underlying cause remained unclear in this case.

Fidanacogene Elaparvovec (Beqvez)

&#x0D; CADTH recommends that Beqvez be reimbursed by public drug plans for the treatment of adults with moderately severe to severe hemophilia B (congenital factor IX deficiency) who are negative for neutralizing antibodies to variant adeno-associated virus (AAV) serotype Rh74 , if certain conditions are met.&#x0D; Beqvez should only be covered to treat adult patients (aged 18 years or older) with circulating coagulation factor IX (FIX:C) activity of 2% or less and bleeding that requires ongoing prophylactic treatment. Beqvez should not be covered if the patient has FIX inhibitors or has previously received gene therapy for hemophilia B.&#x0D; Beqvez should only be reimbursed if prescribed by specialists who have expertise in treating hemophilia B and if the cost of Beqvez is reduced. Beqvez is a 1-time therapy.&#x0D;

A genetic association study of circulating coagulation factor VIII and von Willebrand factor levels

AbstractCoagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10−9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

Functional Assessment of VWD Type 2N Variants Using Mammalian Cell Displayed Surface-Tethered Von Willebrand Factor

Background von Willebrand factor (VWF) is a large plasma protein critical in hemostasis and thrombosis. Defects in VWF cause the inherited autosomal bleeding disorder von Willebrand disease (VWD). One critical function of VWF is to bind circulating coagulation factor VIII (FVIII) and protect it from rapid clearance. Type 2N VWD is a recessive form of VWD characterized by low FVIII levels due to impairment in VWF-FVIII binding. Type 2N VWD clinically is similar to mild-to-moderate hemophilia A, an X-linked bleeding disorder caused by deficiency in FVIII. It is critical to distinguish type 2N VWD from hemophilia A because the treatments are different. VWF genotyping can inform accurate diagnoses, treatment, and testing in relatives. With the rapid expansion of DNA sequencing, a growing number of DNA variants are being identified in the VWF gene. However, the interpretation of significance of these variants is often challenged by insufficient evidence to inform confident classification regarding pathogenicity. In vitro functional assays provide an avenue to develop the evidence needed to accurately assign functional significance to DNA variants. We adapted multiplexed surface tethering of extracellular proteins (MultiSTEP), our mammalian cell surface display system for secreted proteins, to display VWF and test the functional impact of VWF variants on VWF-FVIII binding. Methods We constructed a VWF cDNA of common VWF coding sequences by mutating rare variants to the more common nucleotides. The regions encoding the VWF propeptide were deleted (∆Pro-VWF). ∆Pro-VWF was cloned into our MultiSTEP cassette that provides a C-terminal flexible linker, Strep II tag, and CD28 transmembrane domain. Three VWF missense variants implicated in type 2N VWD (Table 1) that had varying levels of evidence to support classification of pathogenicity were chosen for study - VWF c.2561 G&amp;gt;A (p.R854Q), c.2572 T&amp;gt;A (p.C858S), and c.2635 G&amp;gt;A (p.D879N). Variants were subcloned into our ∆Pro-VWF cassette and then recombined into the genomically integrated MultiSTEP landing pad in HEK293 Freestyle cells. Following induction of expression, VWF-displaying cells were incubated with recombinant FVIII. VWF expression was measured using a polyclonal anti-VWF antibody, and VWF-FVIII binding was detected with two different monoclonal antibodies to FVIII. Cells were incubated with fluorescently-conjugated secondary antibodies and cell surface VWF and FVIII abundance determined by flow cytometry. Results We show that ∆Pro-VWF robustly displays in our MultiSTEP system and binds FVIII. All three VWF missense variants express VWF at levels similar to ∆Pro-VWF. Relative to ∆Pro-VWF all three variants show marked impairment in VWF-FVIII binding (Figure 1), providing strong in vitro evidence for pathogenicity in type 2N VWD useful in variant reinterpretation (bold column in Table 1). Conclusions We have developed an in vitro mammalian cell surface display system for the interrogation of VWF variants and adapted it to study VWF-FVIII binding. The Pathogenic variant p.R854Q shows decreased VWF-FVIII binding in this assay, as expected. The Likely Pathogenic variant p.D879N and the Variant of Uncertain Significance p.C858S both exhibit a profound loss of VWF-FVIII binding in this assay. Our results provide evidence for reclassification of p.C858S to Likely Pathogenic for type 2N VWD. We expect our system can be used at scale to similarly interrogate the functional significance of a multiplexed library of VWF variants and improve variant interpretation in VWD.

Interplay Among Lipoproteins, Endothelial Cells and Platelets

Epidemiological studies have allowed variables related to coagulation to be implicated in the process of atheromatous plaque formation. These include fibrinogen, factors VIII, VII, and von Willebrand, as well as fibrinolytic activity. It is important to note that the reported changes are reversible and there is a reduction in atherosclerotic disease when hyperlipidemia is corrected. The interference that dyslipidemia makes in the hemostasis system is located in three fundamental points: the endothelial cell, platelets and circulating coagulation factors and fibrinolysis. The lumen of the vessels of the entire cardiovascular system is covered by a single layer of juxtaposed endothelial cells, which are in direct contact with the circulating blood. They separate the blood from the subendothelial matrix, where adhesive proteins important for activating coagulation such as collagen, von Willebrand factor and fibronectin are found. The endothelial cell has multiple functions that, as a whole, protect from the activation of coagulation and thrombus formation. The role of hemostasis in the initial phase of atherogenesis is mainly related to platelet activation. They have a lipoprotein cell membrane containing glycoproteins, which function as important receptors for platelet activation represented by the processes of adhesion, shape change, secretion of granules and aggregation for platelet plug formation.

Circulating prekallikrein levels are correlated with lipid levels in the chinese population: a cross-sectional study

BackgroundRecent evidence has revealed that circulating coagulation factor prekallikrein (PK), an important part of the kallikrein-kinin system, regulates cholesterol metabolism, but the association between serum PK and lipid levels is unclear.MethodsThis cross-sectional study included 256 subjects (aged from 1 month to 90 years) who underwent physical examinations at the First People’s Hospital of Huaihua, China. After overnight fasting, serum was collected for PK and lipid testing. Spearman correlation analysis and multivariable logistic regression analysis were used to analyze the association of PK level with lipid levels and the likelihood risk of hyperlipidemia. The possible threshold value of PK was calculated according to the receiver operating characteristic (ROC) curve.ResultsThe median serum PK level was 280.9 µg/mL (IQR 168.0, 377.0), and this level changed with age but not sex. The serum PK level was positively correlated with the serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels. A nonlinear relationship was observed between serum PK and high-density lipoprotein cholesterol (HDL-C) levels. The serum PK level was positively correlated with HDL-C when its level was lower than 240 µg/mL and negatively correlated with HDL-C when its level was higher than 240 µg/mL. The regression analysis demonstrated that an elevated serum PK level was significantly associated with the likelihood risk of hypercholesterolemia and hypertriglyceridemia. The ROC curve showed that the possible threshold values of serum PK for hypercholesterolemia and hypertriglyceridemia occurrences were 344.9 µg/mL and 305.7 µg/mL, respectively.ConclusionsElevated serum PK levels were significantly associated with the likelihood of hypercholesterolemia and hypertriglyceridemia, and the possible threshold values of PK levels were 344.9 µg/mL and 305.70 µg/mL, respectively, suggesting that higher PK levels may be a risk factor for cardiovascular diseases.

Modulation of Extravascular Binding of Recombinant Factor IX Impacts duration of Efficacy in Mouse Models.

There is an emerging concept that in addition to circulating coagulation factor IX (FIX), extravascular FIX contributes to hemostasis. Evaluate efficacy of extravascular FIX using animal models of tail clip bleeding and ferric chloride induced thrombosis. Mutant rFIX proteins with described enhanced (rFIXK5R) or reduced (rFIXK5A) binding to extracellular matrix were generated and characterized using in vitro aPTT, one stage clotting and modified FX assays. Using hemophilia B mice, pharmacokinetic parameters and in vivo efficacy of these proteins was compared against rFIX wild type protein (rFIXWT) in a tail clip bleeding and FeCl3-induced thrombosis model. Respective tissue disposition of FIX was evaluated using immunofluorescence. In vitro characterization demonstrated comparable clotting activity of rFIX proteins. Pharmacokinetic profile showed that rFIXK5A displayed highest plasma exposure compared to rFIXWT and rFIXK5R. Immunofluorescence evaluation of liver tissue showed that rFIXK5R was detectable up to 24h, where as rFIXWT and rFIXK5A were detectable only at 15 minutes. In the tail clip bleeding model, rFIXK5R displayed significant hemostatic protection against bleeding incidence for up to 72h post intravenous administration of 50 IU/kg, whereas the efficacy of rFIXK5A was already reduced at 24h. Similarly, in the mesenteric artery thrombus model, rFIXK5R and rFIXWT demonstrated prolonged efficacy compared to rFIXK5A. Using two different in vivo models of hemostasis and thrombosis, we demonstrate that mutated rFIX protein with enhanced binding (rFIXK5R) to extravascular space confers prolonged hemostatic efficacy in vivo despite lower plasma exposure, while rFIXK5A rapidly lost its efficacy despite higher plasma exposure.

Features of the hemostatic system at combat-caused acoustic injuries

Introduction: Exposure to high-intensity sounds can cause significant damage to the human auditory system. Joint damage to the auditory system by high-intensity sound and shock waves causes acubarotrauma. Such lesions are systemic in nature, affecting not only the auditory system but also the entire human body. It significantly impairs the quality of life and general condition of the body, causes numerous complications. Therefore, the issues of diagnosis and treatment of hearing impairments caused by acutraumas and acubarotraumas received in combat conditions are highly relevant. Aim: to study the hemostatic indices of patients with combat-caused acoustic trauma and identify the most informative ones. Patients and methods: 22 patients with combat acoustic trauma and 10 healthy volunteers were tested. The object of the study was citrate plasma, which was obtained by standard methods. The content of fibrinogen was determined by thrombin-like enzyme Ancistron-H. Partially activated thromboplastin time and prothrombin time were determined by tests of Siemens, USA. The share of functionally inactive forms of prothrombin was determined by Ecamulin-based diagnostic test. The content of protein C activity was determined by cleavage of synthetic chromogenic substrate (Siemens, USA). The concentrations of soluble fibrin and D-dimer were evaluated by immunodiagnostic test systems "DIA-soluble fibrin" and "DIA-D-dimer" (Diaprof-Med, Ukraine). Statistical processing of the results was performed by WinPEPI package for biometric statistical processing with Student's t-test for evaluation of the difference between patients and the control group. Results and discussion: It was found that fibrinogen’s level in 25% of patients remained at the level of control. In 27% of patients this index decreased on the level of the trend, and in 50% - increased 1.6 times. The elevat ion of fibrinogen’s level proves for a risk of thrombotic complications, since this acute phaseprotein is well-known index of hypercoagulability. Partially activated thromboplastin time index in 14% of the tested group remained at the level of control values. In 14% of patients it was probably 1.4 times shorter, and in 73% of patients it was 1.6 times longer than control. Prolonged partially activated thrombin time may prove for the increased level of circulating coagulation factor VIII. In 82% of patients the index of prothrombin time was probably 2.4 times prolonged, where as in 18% of patients was almost at the level of control. It may prove for an imbalance between the components of blood clotting system. The indices of Ecamulin-based test were multidirectional. In 18% of patients it was on the level of control, in 55% of patients it was slightly shorter, and in 27% of patients - probably elongated 1.4 times. The level functionally inactive forms of prothrombin in 50% of patients didn’t differ of control values, in 23% it was tended to decrease, and in 27% it was 1.6 more. The latter proves for the activation of the blood coagulation system. Protein C is a physiological inhibitor of the blood coagulation system. Its’ level in 50% of patients was reduced by 2.2 times in contrast to control, in 14% it remained on the normal level. As well-known, a decrease in protein C level proves for imbalance between coagulation and anticoagulation in the direction of the first one and is associated with thrombosis. The content of D-dimer in 33% of patients was on the level of control, while the level of soluble fibrin was increased 4 times. In 42% there was a 1.4-fold decrease in D-dimer content at a slight increase of soluble fibrin level. In 25% of patients the indices of D-dimer and soluble fibrin were increased in 4 and 7 times, respectively. Elevated level of soluble fibrin is an early prognostic index of the activation of blood clotting system. Comprehensive determination of the content of soluble fibrin and the level of D-dimer allows to estimate the correlation between the accumulation and destruction of the soluble fibrin. Conclusion: From the above data it follows that the determination of the single index is not enough for adequate assess of the risk of thrombosis and the effectiveness of antithrombotic therapy. On this reason the comprehensive definition of the noted indices should be included into the clinical practice a mandatory algorithm for the diagnosis of prothrombotic conditions in patients with acoustic trauma hearing disturbances.

Hemophilia: The Past, the Present, and the Future.

Hemophilia: The Past, the Present, and the Future.

The Effect of Platelets on Thrombin Generation in Cord Blood and Peripheral Neonatal Blood in the Premature Infant; The Event Study

IntroductionInfants born very preterm (<32 weeks) are at increased risk of haemorrhage, particularly intraventricular haemorrhage which can result in short and long term morbidity. Routine clinical laboratory tests such as the prothrombin time and activated partial thromboplastin time are frequently abnormal in this patient group but do not appear to correlate with clinical outcomes. Moreover, while reduced levels of circulating coagulation factors and platelet hypo-reactivity have been reported, plasma thrombin generation (TG), has been reported to be similar or even enhanced in very preterm infants when compared to term infants.Extracellular vesicles, comprising of lipid-bound nanoparticles released from cells (including platelets), may potentially play a role in modulating neonatal haemostasis. We aimed to characterize phospholipid (PL)-dependent plasma thrombin generation in platelet-rich (PRP) and platelet-poor plasma (PPP) in premature infants in both umbilical cord blood & peripheral neonatal blood using calibrated automated thrombography (CAT).MethodsIn this prospective observational study, PRP and PPP was prepared by centrifugation from citrated umbilical cord blood and peripheral neonatal blood collected from premature infants (24 - 31 weeks) and healthy term controls (>37 weeks). No samples were collected from heparinised lines. Parameters of plasma thrombin generation in PRP were assessed using CAT, with thrombin generation stimulated by tissue factor only (TF; final concentration 1pM). CAT was also repeated in PPP using only TF and no exogenous PL (rendering the assay dependent upon the endogenous PL content of plasma).ResultsIn the analysis of umbilical cord blood PRP, plasma thrombin generation was accelerated in the preterm infant group with a significantly shorter time to peak TG observed. The other parameters of TG were similar in both groups (Table 1).In the subset of infants from whom peripheral blood samples were available, there was further evidence of enhanced plasma TG in preterm PRP relative to term infants (Table 2). In this subgroup, the lag time and time to peak thrombin were significantly shorter in the preterm group and peak thrombin was significantly higher.TG was also assessed in both PPP and PRP prepared from umbilical cord blood samples in a subgroup of infants (n=10 term, n=6 preterm) using 1pM TF only, rendering the assay dependent on the phospholipid content of plasma. No difference was observed in any CAT parameters, suggesting that neonatal PPP phospholipid content (potentially from circulating extracellular vesicles) is sufficient to support thrombin generation in the absence of exogenous phospholipid.ConclusionThese preliminary data suggest that neonatal PRP, measured in both umbilical cord blood and peripheral neonatal blood, has similar thrombin generation or may even be hypercoagulable, compared with healthy term controls. Moreover, neonatal plasma phospholipid appears to support thrombin generation in the absence of exogenous phospholipid. This ongoing prospective study aims to further characterize the platelet-dependency of neonatal thrombin generation and evaluate the potential role of extracellular vesicles in neonatal haemostasis. [Display omitted] DisclosuresMaguire: Actelion: Research Funding; Bayer Pharma: Research Funding. Ni Ainle: Leo Pharma: Research Funding; Actelion: Research Funding; Daiichi-Sankyo: Research Funding; Bayer Pharma: Research Funding.

POS-403 VARIATIONS IN THE THROMBIN GENERATION PROFILE AND CLOTTING FACTOR LEVELS IN THE PATIENTS UNDERGOING MAINTENANCE HEMODIALYSIS

Chronic kidney disease (CKD) patients are at high risk of cardiovascular disorders and thrombosis. CKD-V patients undergoing maintenance hemodialysis exhibit varying degrees of hemostatic dysregulation. Endogenous thrombin potential (ETP) is important to the pathogenesis of vaso-occlusive complications. This study investigated ETP and its relevance to circulating coagulation factor levels in CKD-V patients.

Acquired Haemophilia A in Association with Influenza A and Urinary Tract Infection.

The lack of awareness of and experience with acquired haemophilia (AHA) suggests that diagnosis is frequently delayed, resulting in suboptimal treatment.AHA should be suspected in recent-onset abnormal bleeding in patients not receiving anticoagulant treatment, and in the presence of isolated prolonged activated partial thromboplastin time (aPTT).Treatment is based on eradication of the inhibitor, control of the bleeding and identification of underlying conditions.

Meet the First Author.

Meet the First Author.

Genome-Wide Multiple Sclerosis Association Data and Coagulation.

The emerging concept of a crosstalk between hemostasis, inflammation, and immune system prompt recent works on coagulation cascade in multiple sclerosis (MS). Studies on MS pathology identified several coagulation factors since the beginning of the disease pathophysiology: fibrin deposition with breakdown of blood brain barrier, and coagulation factors within active plaques may exert pathogenic role, especially through the innate immune system. Studies on circulating coagulation factors showed complex imbalance involving several components of hemostasis cascade (thrombin, factor X, factor XII). To analyze the role of the coagulation process in connection with other pathogenic pathways, we implemented a systematic matching of genome-wide association studies (GWAS) data with an informative and unbiased network of coagulation pathways. Using MetaCore (version 6.35 build 69300, 2018) we analyzed the connectivity (i.e., direct and indirect interactions among two networks) between the network of the coagulation process and the network resulting from feeding into MetaCore the MS GWAS data. The two networks presented a remarkable over-connectivity: 958 connections vs. 561 expected by chance; z-score = 17.39; p-value < 0.00001. Moreover, genes coding for cluster of differentiation 40 (CD40) and plasminogen activator, urokinase (PLAU) shared both networks, pointed to an integral interplay between coagulation cascade and main pathogenic immune effectors. In fact, CD40 pathways is especially operative in B cells, that are currently a major therapeutic target in MS field. The potential interaction of PLAU with a signal of paramount importance for B cell pathogenicity, such as CD40, suggest new lines of research and pave the way to implement new therapeutic targets.

Edoxaban improves acute venous thromboembolism while preserving protein C and protein S levels

BackgroundIt is well known that warfarin inhibits the synthesis of vitamin K-dependent anticoagulants, including thrombin, protein C and S, and factor Xa, leading, paradoxically, to an initial hypercoagulable state. Edoxaban, a direct inhibitor of activated factor X is widely used for the treatment of acute venous thromboembolism (VTE). However, the effect of edoxaban on circulating coagulation factors, in patients with acute VTE, remains unknown. Methods and resultsWe enrolled 57 patients with acute VTE with/without pulmonary embolism treated with edoxaban (n=37) or warfarin (n=20) in a clinical setting. Before treatment and 2 weeks after treatment, we evaluated thrombotic burden using ultrasound or computed tomography angiography. We also evaluated thrombin generation, represented by prothrombin fragment F1+2; thrombus degradation, represented by D-dimer; and levels of anticoagulants, including protein C, protein S, and antithrombin III. Both edoxaban and warfarin treatment improved thrombotic burden and decreased prothrombin fragment F1+2, and D-dimer. Edoxaban treatment preserved protein C and protein S levels. In contrast, warfarin decreased protein C and protein S levels. Neither treatment affected antithrombin III. ConclusionsEdoxaban improves VTE while preserving protein C and protein S levels, thereby indicating that edoxaban improves thrombotic burden while maintaining levels of anticoagulants.

Lack of Substantial Antithrombotic Effect of Low Circulating Coagulation Factor VII Levels in a Baboon Model of Acute Vascular Graft Thrombosis

Localized pathologic thrombin generation by the tissue factor/factor VII (TF/FVII)-dependent extrinsic pathway of blood coagulation has been suggested to be the key initiator of thrombosis following blood vessel injury and exposure of blood to the subendothelial matrix that contains coagulation/platelet activator matrix proteins, such as collagen and TF. Inherited FVII deficiency (<1% FVII activity) causes severe symptomatic coagulopathy. Yet, there are no data to suggest that it also protects against thrombosis; in fact, the contrary may be true (Giansily-Blaziot et al, Thromb Res. 2012;130:S47). It has been reported that active site-blocked FVIIa (FVIIai) at doses that do not affect the prothrombin time (PT) help restore cyclic flow variations in stenotic carotids of rabbits without affecting clotting times (Cirillo et al., J Thromb Haemost. 2003;1:992). However, in a previous primate study, we could not demonstrate the antithrombotic effect of FVIIai at a dose that caused significant prolongation of the bleeding time in our established baboon model of acute vascular graft thrombosis (Gruber et al., Thromb Res. 2007;119:121-7). The present study was designed to determine the effect of severe yet incomplete experimental FVII deficiency in the same standardized primate model of thrombosis that we used for evaluating the effect of FVIIai. First, baseline (negative control) graft thrombosis was evaluated in baboon studies (N=6), followed by establishing the efficacy of the low molecular weight heparin (LMWH), enoxaparin, at a single 1 mg/kg dose, as positive control. LMWH at this dose was moderately but demonstrably antithrombotic, as assessed by measuring the inhibition of fibrin and platelet deposition in TF- or collagen-coated vascular grafts. Reducing circulating FVII activity levels by 95% to 99% after a 2 week long pre-treatment with a FVII antisense oligonucleotide (FVII ASO) caused significant and sustained (>30 days) systemic anticoagulation, as evidenced by PT increase that reached 2.0-2.6 fold baseline. Surprisingly, fibrin deposition was not affected, and platelet deposition in the thrombogenic devices (N=8) was also not inhibited on collagen-coated grafts, and only marginally inhibited on TF. Treatment with vitamin K antagonists that reduces the level of vitamin K-dependent coagulation enzymes, including FVII, also prolong the PT. Warfarin-induced doubling of the PT is antithrombotic and moderately safe. Yet, in this study where FVII ASO selectively reduced FVII levels, the observed and significant PT prolongation was not associated with anti-fibrin or anti-platelet effects on collagen, and showed an unremarkable anti-platelet effect on TF. We conclude that if the TF/FVII pathway drives the propagation of large vessel thrombi under flow in primates indeed, very low FVII activity (≈1%) must be sufficient to sustain thrombogenesis, supporting the clinical data on deep vein thrombosis, routinely observed in FVII deficiency. Since FVII activity cannot be entirely eliminated without bleeding risks, these data suggest that targeting the TF/FVII pathway with FVII ASO or inhibitors may not be an entirely sound antithrombotic treatment approach. DisclosuresWallisch:Aronora, Inc: Employment. Tucker:Aronora, Inc: Employment, Equity Ownership. Murray:Isis Pharmaceuticals, Inc.: Employment. Crosby:Isis Pharmaceuticals, Inc.: Employment. Monia:Isis Pharmaceuticals, Inc.: Employment. Gruber:Aronora, Inc: Employment, Equity Ownership.

Coagulation in liver toxicity and disease: Role of hepatocyte tissue factor

The liver is the primary source of a number of circulating coagulation factors, and acute liver injury and chronic liver disease are each associated with alterations in blood coagulation. Current views of the connection between liver injury and coagulation extend beyond the impact of liver disease on synthesis of coagulation factors to include a role for coagulation factor activity in the initiation and progression of liver disease. Mechanisms of coagulation initiation in liver disease are not completely understood. Compared to other tissues, liver expresses very low levels of tissue factor (TF). Recent studies indicate that expression of TF by hepatocytes comprises the majority of liver procoagulant activity, and that hepatocyte TF activates coagulation induced by liver injury. This review will briefly cover the expression and regulation of TF by hepatocytes, the role of TF in coagulation triggered by liver toxicity, and the contribution of coagulation activity to the progression of liver disease.

Restraint stress enhances arterial thrombosis in vivo – role of the sympathetic nervous system

Stress is known to correlate with the incidence of acute myocardial infarction. However, the molecular mechanisms underlying this correlation are not known. This study was designed to assess the effect of experimental stress on arterial thrombus formation, the key event in acute myocardial infarction. Mice exposed to 20 h of restraint stress displayed an increased arterial prothrombotic potential as assessed by photochemical injury-induced time to thrombotic occlusion. This increase was prevented by chemical sympathectomy performed through 6-hydroxydopamine (6-OHDA). Blood-born tissue factor (TF) activity was enhanced by stress and this increase could be prevented by 6-OHDA treatment. Vessel wall TF, platelet count, platelet aggregation, coagulation times (PT, aPTT), fibrinolytic system (t-PA and PAI-1) and tail bleeding time remained unaltered. Telemetric analysis revealed only minor hemodynamic changes throughout the stress protocol. Plasma catecholamines remained unaffected after restraint stress. Tumor necrosis factor alpha (TNF-α) plasma levels were unchanged and inhibition of TNF-α had no effect on stress-enhanced thrombosis. These results indicate that restraint stress enhances arterial thrombosis via the sympathetic nervous system. Blood-borne TF contributes, at least in part, to the observed effect whereas vessel wall TF, platelets, circulating coagulation factors, fibrinolysis and inflammation do not appear to play a role. These findings shed new light on the understanding of stress-induced cardiovascular events.