Low Tissue Factor Research Articles

Mathematical modeling identifies clotting factor combinations that modify thrombin generation in normal and factor VIII-, IX-, or XI-deficient blood

BackgroundIn healthy individuals, plasma levels of clotting proteins naturally vary within a range of 50% to 150% of their mean values. We do not know how these variations modify thrombin generation. ObjectivesTo assess the impact of protein level variations on simulated thrombin generation in normal and factor (F)VIII-, FIX-, or FXI-deficient blood. MethodsWe used a mathematical model of flow-mediated coagulation to simulate thrombin generation with all possible combinations of clotting protein variations within the normal range and for various tissue factor levels. We selected, analyzed, and ranked combinations that enhanced thrombin generation compared with baseline. ResultsProtein variations most strongly affected thrombin generation at intermediate tissue factor levels. Low tissue factor levels prevented coagulation initiation, while high tissue factor levels always triggered thrombin generation. At intermediate levels, we identified protein variations that substantially modified thrombin generation. Low-normal FV shortened lag times and increased thrombin generation, whereas high-normal FV lengthened lag times and reduced thrombin generation. With severe FVIII and FIX deficiencies, low-normal tissue factor pathway inhibitor α and antithrombin amplified the effect of low-normal FV. For moderate FVIII and FIX deficiencies, high-normal tissue factor pathway inhibitor α and antithrombin enhanced the impact of high-normal FV in reducing thrombin production. In normal and FXI-deficient blood, high-normal FVIII and FIX significantly boosted thrombin generation. ConclusionOur mathematical model predicted how variations in clotting protein levels, within the normal range, could contribute to the variability of bleeding phenotypes observed with clotting factor deficiencies. Our study generated experimentally testable hypotheses that could aid in developing new therapies toward normal hemostasis.

The procoagulant signature of cancer cells drives fibrin network formation in tumor microenvironment and impacts its quality. Implications in cancer cell migration and the resistance to anticancer agents

IntroductionCancer cells induce hypercoagulability in the tumoral microenvironment by expressing Tissue Factor (TF). We aimed to study the impact of the procoagulant signature of cancer cells on the quality and structure of fibrin network. We also studied the impact of fibrin clot shield (FCS) on the efficiency of anticancer agents and the migration of cancer cells. Materials and methodsPancreatic cancer cells BXPC3 and breast cancer cells MDA-MB231 and MCF7, were cultured in the presence of normal Platelet Poor Plasma (PPP), diluted 10 % in conditioning media. Their potential to induce thrombin generation and their fibrinolytic activity were assessed. The structure of fibrin network was analyzed with Scanning Electron Microscopy (SEM). Cancer cells' mobility with fibrin clot and their interactions with fibrin were observed. Cancer cells were treated with paclitaxel (PTX) or 4-hydroxy-tamoxifen (4OHTam) in the presence or absence of FCS. ResultsCancer cells, in presence of PPP, induced fibrin network formation. High TF-expressing cancer cells (BXPC3 and MDA-MB23 cells), led to dense fibrin network with fine fibers. Low TF expressing cells MCF7 led to thick fibers. Exogenous TF enhanced the density of fibrin network formed by MCF7 cells. Cancer cells through their inherent profibrinolytic potential migrated within the fiber scaffold. The BXPC3 and MCF7 cells moved in clusters whereas the MDA-MB231 cells moved individually within the fibrin network. FCS decreased the efficiency of PTX and 4OHTam on the viability of cancer cells. ConclusionsThe procoagulant signature of cancer cells is determinant for the quality and structure of fibrin network in the microenvironment. Original SEM images show the architecture of “bird's nest”-like fibrin network being in touch with the cell membranes and surrounding cancer cells. Fibrin network constructed by triggering thrombin generation by cancer cells, provides a scaffold for cell migration. Fibrin clot shields protect cancer cells against PTX and 4OHTam.

Clot Waveform Analysis: From Hypercoagulability to Hypocoagulability - A Review.

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are coagulative screening tests used for the diagnosis of several pathologic conditions, such as liver failure, coagulation factor deficiencies, anti-phospholipid antibodies (lupus anticoagulant), and factor VIII inhibitors. A new test was developed several years ago to detect the amount of thrombin generated during plasma clotting, using low tissue factor concentrations and fluorogenic substrates, and it has since been used successfully in conditions ranging from hypocoagulable to hypercoagulable states. However, the test is expensive and difficult to perform in nonspecialized laboratories, and efforts have thus been made to find an economic and easily implementable test suitable for routine use, even in nonspecialist laboratories. To evaluate clot waveform analysis (CWA) of PT and aPTT, aiming to show the dynamics of clot formation; that is, the "hidden" features of both tests. CWA can be implemented by using an automated coagulometer with dedicated software. The aim of this review was to evaluate whether CWA is able to detect both hypercoagulative and hypocoagulative states. Using MedLine, we searched and retrieved articles relating to CWA. We only considered articles published in English, but with no limits in terms of article type, publication year, or geography. CWA was shown to be a reliable test in patients with both hypercoagulable and hypocoagulable states. It represents a simple and inexpensive global test that can easily provide information on the behavior of the coagulation system. Both the first and second derivatives are computed by using dedicated software implemented with an on-board algorithm in a routine automated coagulometer.

Thrombin generation and all-cause mortality in The Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)

Introduction: Thrombin generation assay (TGA) is a laboratory method that provides the global evaluation of hemostasis. The association between thrombin generation and all-cause mortality is poorly investigated and results are contradictory. This study evaluated whether TGA parameters are associated with all-cause mortality in a prospective cohort. Methods: This study was conducted in 2,588 participants enrolled at baseline of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). TGA was performed using the Calibrated Automated Thrombogram (CAT) method, and its parameters lagtime, time-to-peak, peak, Endogenous Thrombin Potential (ETP) and normalized ETP (nETP) were evaluated according to the reference interval (RI). The association between TGA parameters and all-cause mortality was estimated by Cox regression and adjusted for confounders. Results: The mean follow-up time was 6.6 ± 2.7 years and 85 deaths occurred. After adjustment, time-to-peak values above the RI at low and high tissue factor (TF) concentrations were associated with higher risk of death [HR = 2.45 (95 % CI: 1.17–5.13) and HR = 2.24 (95 % CI: 1.02–4.93), respectively] and nETP and peak values below RI at high TF concentration were associated with higher risk of death [HR = 3.85 (95 % CI: 1.39–10.68) and HR = 2.56 (95 % CI: 1.17–5.61), respectively]. Conclusions: Delayed thrombin generation was associated with higher risk of all-cause mortality.

Design of a Superior Factor VIIIa Mimetic By Coupling a Membrane Binding Domain to a Factor IXa Binding Antibody Fragment

Emicizumab is a bispecific antibody that augments Factor X (FX) activation by factor IXa (FIXa) in the absence of factor VIIIa (FVIIIa) by binding and approximating the substrate FX and protease FIXa. Clinical use of this FVIIIa mimetic significantly reduces bleeding episodes in Hemophilia A (HA) patients. Membrane dependent assembly of FVIIIa-FIXa-FX is an absolute requirement for the intrinsic tenase activity. In contrast, emicizumab lacks any membrane binding ability itself and exhibits significant FXa generation even in the absence of membranes. This major distinction likely allows FVIIIa to function at sub-nanomolar concentrations to greatly accelerate the activation of FX while only much lower enhancements of rate are achieved at several 100-fold higher concentrations of emicizumab. We sought to test whether the addition of a membrane binding feature in the monospecific FIXa binding fragment of emicizumab could alone approximate the cofactor activity obtained with the bispecific antibody. We first generated a single chain antibody fragment (scFv) of the FIXa binding arm of emicizumab by joining the variable heavy chain (V H) with the variable light chain (V L) to produce V H9V L. In line with previous observations that FIXa one arm antibodies of emicizumab that can only bind FIXa and impart a modest cofactor mimetic activity, V H9V L, facilitated FIXa-mediated FX activation at a much lower rate in comparison to bispecific emicizumab. The scFv, V H9V L, was used as a template to introduce membrane binding properties. The Factor V C2-domain, which binds membrane, was fused at the C-terminus of V H9V L via a repeating Gly 4Ser flexible linker of ~ 5.7 nm to allow the V H and V L domains of the scFv to potentially orient correctly at the membrane surface. The resulting construct V H9V LC2 showed a substantial amplification in cofactor mimetic activity as compared to V H9V L. In vitro kinetic analyses with purified proteins and synthetic phospholipid vesicles containing phosphatidylcholine (PC) and phosphatidylserine (PS) (PC:PS, 75:25 %) showed approximately 22-fold faster activation of FX by V H9V LC2 as compared to V H9V L. Remarkably, despite its inability to bind FX, cofactor mimetic activity of V H9V LC2 was about two-fold higher than that of emicizumab. Potent enhancement of FX activation by V H9V LC2 observed with purified proteins was also reflected in thrombin generation assays (TGA). V H9V LC2 added at 100 nM brought thrombin generation into the reference range in congenital FVIII-deficient HA plasma supplemented with 4 µM PC:PS and triggered with either low tissue factor (0.1 pM), or factor XIa (0.1 nM). The role of C2 domain-mediated increase in cofactor activity of V H9V LC2 was further assessed by using an antibody fragment (E 9scFv) that inhibits the binding of factor V to membranes. Increasing concentrations of E 9scFv caused a dose-dependent reduction in the rate of FXa formation catalyzed by the complex of V H9V LC2: FIXa (20nM:25nM). Inhibition saturated at ~ 85% at 100 nM E 9scFv, indicating a substantial contribution of membrane binding to the cofactor mimetic function of V H9V LC2. To evaluate whether V H9V LC2 can restore clotting in HA plasma with FVIII inhibitors, normal pooled plasma was supplemented with a FVIII neutralizing antibody to mimic FVIII inhibitor plasma. Addition of V H9V LC2 to this plasma restored clotting time to the levels shown by normal pooled plasma, suggesting that V H9V LC2 can bypass FVIII inhibitor activity. The engineered scFv with a factor V C2 domain fusion, V H9V LC2, demonstrates that mere addition of a membrane binding feature to the FIXa arm of the emicizumab surpasses the mimetic activity achieved by a bispecific antibody that exploits both bridging and allostery to be an effective cofactor. Such a construct also avoids the burden of productive assembly from a combination of three separate polypeptide chains, as in a functional bispecific antibody. Replicating the membrane binding feature into bispecific FVIIIa mimetic antibodies could further amplify function and more closely resemble FVIIIa in both activity and membrane dependent regulation. Our results provide surprising insights into how a single chain, monospecific, membrane-anchored FVIIIa mimetic enhances the catalytic activity of FIXa.

Multicenter Evaluation of the Hemostatic Activity of Emicizumab in Patients with Severe Hemophilia a Using Thrombin Generation Assay

Background: Emicizumab has been approved for the prophylaxis of patients with hemophilia A with or without inhibitors. However, the follow-up of these patients is poorly defined and makes difficult the management of those presenting breakthrough bleeds while on prophylaxis with emicizumab. Objectives: The aim of this study was to evaluate the value of thrombin generation assay (TGA) as a surrogate marker of the hemostatic efficacy of emicizumab. The secondary objective was to study the correlation between TGA and the methods used to measure the blood concentration of emicizumab. Methods: TGA was modified to enhance its sensitivity to emicizumab through the use of a reagent combining a low amount of tissue factor (TF) and activated factor XI (FXIa). Quantification of emicizumab was performed by three different methods, this modified one-step assay, and two methods based on liquid chromatography and mass spectrometry (LC-MS). Results: Emicizumab procoagulant activity is of note highly dependent on FIX activity, while FIX itself is activated by FXIa, the latter depending on thrombin and platelets. Consistent with these findings, TGA measured in platelet rich plasma (PRP) showed a significantly higher coagulation capacity compared to platelet poor plasma (PPP) in patients receiving prophylaxis with emicizumab. These results emphasize the need for platelets to capture the full hemostatic effect of emicizumab. Of course, using fresh PRP is not classical in real life hospital settings. Although this appears as the best assessment measurement, this condition could be a major barrier to the deployment of PRP TGA in hospital laboratories to monitor emicizumab therapy and guide combined therapy with emicizumab and bypassing agents. In order to overcome this issue, we propose a modified TGA PPP assay using added traces of FXIa to the mixture of low TF and phospholipids. This provides a better activation of FIX, required for the procoagulant activity of emicizumab. Using FXIa/TF-triggered TGA, a relationship was observed between the area under the TG curve (ETP) and the clinical response of patients to emicizumab. Patients who experienced breakthrough bleeds while they were on emicizumab, had significantly lower ETP than others without bleeding episodes. The ultrastructure of fibrin clots was consistent with TGA results and showed that the hemostatic activity of emicizumab is similar to Factor VIII activity between 20 and 30 IU/dL. Finally, pharmacokinetic/pharmacodynamic analyses showed no correlation between ETP and LC-MS nor with modified one-stage activated partial thromboplastin time, but a statistically significant correlation was observed between the LC-MS methods and the time to peak results of TGA. Conclusion: The modified TGA used here can segregate patients with good clinical response to emicizumab from those bleeding on emicizumab. This approach can be of valuable interest for the management of patients treated with emicizumab

Measuring Thrombin Generation in Platelet Rich Plasma with the ST Genesia

Background: Thrombin generation (TG) is nowadays used more and more for diagnosing and monitoring of patients. The ST Genesia is a fully automated device for measuring TG in platelet poor plasma (PPP). As the samples are not being inverted prior to the measurement once loaded in the device, it was not known whether it was possible to measure TG in platelet rich plasma (PRP). Platelets can sediment in time possibly affecting TG results. The goal of this study was to evaluate the use of the ST Genesia for measuring TG in PRP in time and to investigate whether the assay is sensitive to platelet antagonists. Methods: Blood was taken from 6 healthy donors and PRP was immediately prepared by centrifuging the blood once for 15 minutes at 220g. The PRP was divided into 14 tubes: 11 tubes for measuring TG in time; i.e. after 0, 15, 30, 45, 60, 75, 90, 105, 120, 180, 240 minutes after the samples were loaded on board; 1 tube for platelet count measurement, 1 tube for TG with the Calibrated Automated Thrombogram (CAT) assay and 1 tube for freezing the PRP. The PRP reagent of the CAT assay was used for triggering TG that contains a low tissue factor concentration. Tukey's multiple comparisons test was used for statistical analysis. Results: The platelet count remained stable in time even without inverting the vial prior to the measurement up to 3 hours. After 4 hours, platelet count was significantly lower (p<0.01). TG in PRP remained stable in time up to 3 hours, after that lagtime and time-to-peak were extended by an average of 48% and 16%, respectively (p-values <0.0001 and 0.0121, respectively), while ETP and peak height remained stable (with low CV's of <6.0% and <7.3%, respectively). The ST Genesia proved to be sensitive to platelet antagonists aspirin (100µM) and Cangrelor (500nM) as peak height decreased with 22% and 24%, respectively. TG measured with the CAT and the ST Genesia was comparable. Measuring TG in PRP after being frozen at -80°C resulted TG kinetics similar to TG in PPP, with a shorter lagtime (-67%) and time-to-peak (-64%), with an increased peak height (+82%), and velocity index (+383%), and an unaffected ETP. Conclusion: The ST Genesia can be used for the reproducible measurement of TG in PRP up to two hours after loading the samples in the device.

Effect of factor XI inhibition on tumor cell-induced coagulation activation

BackgroundCancer-associated thrombosis is a frequent complication in patients with malignancies. While factor XI (FXI)/FXIa inhibition is efficacious in preventing postoperative venous thromboembolism, its role in tumor cell-induced coagulation is less defined. ObjectivesWe thus aimed to provide mechanistic insights into FXI/FXIa inhibition in tumor cell-induced coagulation activation. MethodsProcoagulant activity (PCA) of 4 different tissue factor (TF) expressing tumor cell lines was analyzed by single-stage clotting and thrombin generation assay in the presence of a FXIa inhibitor, BMS-262084 (BMS), an inhibitory FXI antibody (anti-FXI), or peak and trough concentrations of rivaroxaban or tinzaparin. Further, tumor cell-induced platelet aggregation was recorded. Recombinant human TF served as positive control. ResultsAlthough BMS and anti-FXI potently inhibited FXIa amidolytic activity, both inhibitors efficiently mitigated recombinant human TF- and tumor cell-induced fibrin clot formation and platelet aggregation only in the presence of low TF PCA. The anticoagulant effects showed an inverse correlation with the magnitude of cellular TF PCA expression. Similarly, BMS markedly interfered with tumor cell-induced thrombin generation, with the most prominent effects on peak and total thrombin. In addition, anticoagulant effects of FXIa inhibition by 10 μM BMS were in a similar range to those obtained by 600 nM rivaroxaban and 1.6 μM tinzaparin at low TF PCA levels. However, rivaroxaban and tinzaparin also exerted marked anticoagulant activity at high TF PCA levels. ConclusionOur findings indicate that FXI/FXIa inhibition interferes with tumor cell-induced coagulation activation only at low TF PCA expression levels, a finding with potential implications for future in vivo studies.

How to assess hypercoagulability in heparin-induced thrombocytopenia? Biomarkers of potential value to support therapeutic intensity of non-heparin anticoagulation

BackgroundIn case of heparin-induced thrombocytopenia (HIT), the switch to a non-heparin anticoagulant is mandatory, at a therapeutic dose. Such a treatment has limitations though, especially for patients with renal and/or hepatic failure. Candidate laboratory tests could detect the more coagulable HIT patients, for whom therapeutic anticoagulation would be the more justified.Patients and methodsThis was a monocentre observational prospective study in which 111 patients with suspected HIT were included. Nineteen were diagnosed with HIT (ELISA and platelet activation assay), among whom 10 were classified as HITT + when a thrombotic event was present at diagnosis or during the first following week. Two plasma prethrombotic biomarkers of in vivo activation of the haemostasis system, procoagulant phospholipids (ProcoagPPL) associated with extracellular vesicles and fibrin monomers (FM test), as well as in vitro thrombin potential (ST Genesia; low picomolar tissue factor) after heparin neutralization (heparinase), were studied. The results were primarily compared between HITT + and HITT- patients.ResultsThose HIT + patients with thrombotic events in acute phase or shortly after (referred as HITT+) had a more coagulable phenotype than HIT + patients without thrombotic events since: (i) clotting times related to plasma procoagulant phospholipids tended to be shorter; (ii) fibrin monomers levels were statistically significantly higher (p = 0.0483); (iii) thrombin potential values were statistically significantly higher (p = 0.0404). Of note, among all patients suspected of suffering from HIT, we did not evidence a hypercoagulable phenotype in patients diagnosed with HIT compared to patients for whom the diagnosis of HIT was ruled out.ConclusionThe three tests could help identify those HIT patients the most prone to thrombosis.

Characterization of CRISPR/Cas9-edited human placental allogenic stromal cells with low tissue factor expression and reduced thrombotic effects

The tissue factor (TF/CD142) expressed by mesenchymal stromal cells (MSCs) has been regarded as a safety concern in clinical applications as it may trigger thrombosis when MSCs administered intravenously. Human placental allogenic stromal cells (ASCs) are culture-expanded, undifferentiated MSC-like cells derived from full-term postpartum placenta and possess immunomodulatory and pro-angiogenic activities, however, express TF. Here we performed CRISPR/Cas9-mediated TF gene knock out (TFKO) in ASCs, leading to significantly lower TF expression, activity and thrombotic effects. ASCs’ characteristics including expansion, expression of phenotypic markers and secretory profile remained unchanged in edited cells, and their immunomodulatory activities, which are functionally relevant to therapeutic applications, were not affected upon TFKO. Taken together, this study provides a feasible strategy which could improve the clinical safety features of MSC-based cell therapy by CRISRP/Cas9-mediated TF gene knock out.

Targeting the contact system in a rabbit model of extracorporeal membrane oxygenation.

Previous studies suggested that contact pathway factors drive thrombosis in mechanical circulation. We used a rabbit model of veno-arterial extracorporeal circulation (VA-ECMO) to evaluate the role of factors XI and XII in ECMO-associated thrombosis andorgan damage. Factors XI and XII (FXI, FXII) were depleted using established antisense oligonucleotides before placement on a blood-primed VA-ECMO circuit. Decreasing FXII or FXI to< 5% of baseline activity significantly prolonged ECMO circuit lifespan, limited the development of coagulopathy, and prevented fibrinogen consumption. Histological analysis suggested that FXII depletion mitigated interstitial pulmonary edema and hemorrhage whereas heparin and FXI depletion did not. Neither FXI nor FXII depletion was associated with significant hemorrhage in other organs. Invitro analysis showed that membrane oxygenator fibers (MOFs) alone are capable of driving significant thrombin generation in a FXII- and FXI-dependent manner. MOFs also augment thrombin generation triggered by low (1 pM) or high (5 pM) tissue factor concentrations. However, only FXI elimination completely prevented the increase in thrombin generation driven by MOFs, suggesting MOFs augment thrombin-mediated FXI activation. Together, these results suggest that therapies targeting FXII or FXI limit thromboembolic complications associated with ECMO. Further studies are needed to determine the contexts wherein targeting FXI and FXII, either alone or in combination, would be most beneficial in ECMO. Moreover, studies are also needed to determine the potential mechanisms coupling FXII to end-organ damage in ECMO.

The effect of a next generation factor VIII mimetic bispecific antibody (Mim8) on assays of factor VIII activity and thrombin generation

BackgroundMim8 is a next generation bispecific antibody developed for the prophylactic treatment of hemophilia A. The sensitivity of activated plasma thromboplastin time (APTT), assays measuring factor VIII activity (FVIII:C) and thrombin generation to plasma containing Mim8 was assessed. MethodsCongenital severe hemophilia A plasma was spiked with Mim8 at 0 μg/mL to 20 μg/mL. APTT was measured with 4 reagents, one-stage FVIII with 9 reagents, and chromogenic FVIII with 6 assays. Thrombin generation was assessed in a low tissue factor system. ResultsAt 1-μg/mL Mim8, the APTT was shortened to within normal limits and one-stage FVIII:C was >1.00 IU/mL with all APTT reagents. Modified one-stage assays calibrated with Mim8 reference material calibrated recovered within 20% of the target Mim8 concentration with most APTT reagents. Factor VIII:C of bovine only chromogenic assays was <0.04 IU/mL at all Mim8 concentrations. Factor VIII:C of human only chromogenic assay was >4.00 IU/mL at 20-μg/mL Mim8. Factor VIII:C of hybrid bovine FX, human FIXa chromogenic assays ranged from 0.076 to >3.00 IU/mL at 20-μg/mL Mim8. Normal thrombin generation was restored at 5-μg/mL Mim8. ConclusionsAPTT-based assays are sensitive to Mim8 and should not be performed in the presence of the drug. Chromogenic assays containing human proteins or hybrid human/bovine proteins demonstrated variable sensitivity to Mim8. Bovine only chromogenic assays were largely insensitive to the presence of Mim8. Thrombin generation normalized at increased Mim8 concentrations. Modified one-stage and chromogenic assays could be used to quantify the Mim8 concentration in plasma.

Beyond uterine atony: characterizing postpartum hemorrhage coagulopathy

Postpartum hemorrhage is a leading cause of morbidity and mortality worldwide, yet the associated early coagulopathy is not well defined. We hypothesized that women who develop postpartum hemorrhage have a distinct derangement of thrombin generation and coagulation factors compared with postpartum women without postpartum hemorrhage. This prospective study of pregnant patients with postpartum hemorrhage was completed at a single urban hospital. Blood was drawn on postpartum hemorrhage diagnosis and 2 and 4 hours later. Assays of patients with postpartum hemorrhage included thrombelastography, whole blood thrombin generation, coagulation factor activity, tissue factor levels and activity, and tissue factor pathway inhibitor levels, which were compared with that of patients without postpartum hemorrhage. A total of 81 patients were included in this study. Of those patients, 66 had postpartum hemorrhage, and 15 served as controls. Compared with patients without PPH, patients with postpartum hemorrhage had lower fibrinogen levels (469.0 mg/dL vs 411.0 mg/dL; P=.02), increased tissue plasminogen activator resistance (fibrinolysis 30 minutes after maximal clot strength: 8.7% vs 4.2%; P=.02), decreased peak thrombin concentration (150.2 nM vs 40.7 nM; P=.01), and decreased maximal rate of thrombin generation (60.1 nM/minute vs 2.8 nM/minute; P=.02). Furthermore, compared with patients without postpartum hemorrhage, patients with postpartum hemorrhage had decreased tissue factor levels (444.3 pg/mL vs 267.1 pg/mL; P=.02) and increased tissue factor pathway inhibitor levels (0.6 U/mL vs 0.8 U/mL; P=.04), with decreased tissue factor pathway inhibitor ratios (624 vs 299; P=.01). PPH is not only an issue of uterine tone and mechanical bleeding but also a distinct coagulopathy that is characterized by decreased fibrinogen level, clot breakdown resistance, and markedly low thrombin generation. This pathology seemed to be driven by low tissue factor and high tissue factor pathway inhibitor levels.

Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis.

Our recent studies suggest that sphingomyelin levels in the plasma membrane influence TF (tissue factor) procoagulant activity. The current study was performed to investigate how alterations to sphingomyelin metabolic pathway would affect TF procoagulant activity and thereby affect hemostatic and thrombotic processes. Macrophages and endothelial cells were transfected with specific siRNAs or infected with adenoviral vectors to alter sphingomyelin levels in the membrane. TF activity was measured in factor X activation assay. Saphenous vein incision-induced bleeding and the inferior vena cava ligation-induced flow restriction mouse models were used to evaluate hemostasis and thrombosis, respectively. Overexpression of SMS (sphingomyelin synthase) 1 or SMS2 in human monocyte-derived macrophages suppresses ATP-stimulated TF procoagulant activity, whereas silencing SMS1 or SMS2 increases the basal cell surface TF activity to the same level as of ATP-decrypted TF activity. Consistent with the concept that sphingomyelin metabolism influences TF procoagulant activity, silencing of acid sphingomyelinase or neutral sphingomyelinase 2 or 3 attenuates ATP-induced enhanced TF procoagulant activity in macrophages and endothelial cells. Niemann-Pick disease fibroblasts with a higher concentration of sphingomyelin exhibited lower TF activity compared with wild-type fibroblasts. In vivo studies revealed that LPS+ATP-induced TF activity and thrombin generation were attenuated in ASMase-/- mice, while their levels were increased in SMS2-/- mice. Further studies revealed that acid sphingomyelinase deficiency leads to impaired hemostasis, whereas SMS2 deficiency increases thrombotic risk. Overall, our data indicate that alterations in sphingomyelin metabolism would influence TF procoagulant activity and affect hemostatic and thrombotic processes.

An Antibody Targeting Human FV Promotes Thrombin Generation and Reduces Bleeding in a Hemophilic Mouse Model

Hemophilia is a bleeding disorder that results from a deficiency of factor VIII (Hemophilia A; HA) or factor IX (Hemophilia B; HB). Protein replacement therapy ameliorates bleeding but can result in the development of neutralizing antibodies. Innovative nonfactor therapies are in development for hemophilia which target anticoagulant proteins such as tissue factor pathway inhibitor (TFPI), antithrombin, and activated protein C (APC). While these approaches show promise, they influence multiple coagulation factors and pathways which could raise safety concerns. Considering its essential role in coagulation in enhancing thrombin generation (TG), we hypothesized that altering the functional properties of FV/FVa could be an attractive approach for HA and HB treatment. Activated FV (FVa) assembles with FXa on activated cellular surfaces and enhances the kinetics of TG by several orders of magnitude. Here we assess whether monoclonal antibodies targeting all forms of FV are effective in promoting TG in hemophilia using both in vitro and in vivo models in hemophilic mice. Using an initial TG assay screen, we identified a procoagulant antibody (GB5) that binds with high affinity (Kd <0.5 nM) to human (h)FV, FVa, and FV-short and investigated its mechanism of action. Using proteolytic fragments of FV and western blotting experiments, we found that GB5 likely binds in the middle of the light chain region. To evaluate the functional effects of GB5 in vitro, we first employed a plasma-based TG assay using HA or HB plasma triggered with low (0.2 pM) tissue factor. The presence of saturating GB5 increased TG about 3-fold in HA plasma. Similar results were observed with HB plasma. Interestingly, when TG was essentially eliminated in HA and HB plasma with addition of exogenous APC, GB5 restored TG. These results suggest that the procoagulant effect of GB5 neutralizes the anticoagulant effect of APC and enhanced TG in hemophilic plasma To begin to assess the mechanism, the effect of GB5 on FV activation in the TG assay was evaluated. TG assay samples were removed over time, quenched, and analyzed by immunoblotting using labeled FV specific antibodies. In the presence of GB5, ~95% of the starting full-length FV was converted to FVa within 3 min compared to 5 min in the absence of GB5. These results show that FV is activated faster in the presence of GB5 in the TG assay. To extend these data, we next assessed the influence of GB5 in the TG assays using FV-deficient plasma supplemented with either FV or FVa and TFPI⍺. The data show that GB5 had essentially no effect on the TG assay when FV deficient plasma was reconstituted with FVa, but enhanced TG at least 3-fold when supplemented with FV. Consistent with this, the kinetics of prothrombin activation with FXa-FVa-membranes in the presence or absence of GB5 were the same. These data suggest that GB5 impacts reactions that influence FV, with no direct effect on FVa procoagulant function. To evaluate whether GB5 has a procoagulant effect in vivo, a tail vein transection (TVT) model was employed in HA mice. In this model, one lateral tail vein is transected, and blood is collected over 40 min with clots at the vessel wall disrupted every 10 min as needed. Since GB5 interacts with human and not mouse FV, plasma derived human FV was infused into HA mice. We found that blood loss in the TVT model using HA mice infused with human FV alone (5 μg/mouse) or GB5 alone (20 μg/mouse) was no different than mice infused with PBS (n = 3-5 mice/group except GB5 alone where n = 1: ~600 μl blood loss). In contrast to these data, when human FV and GB5 were co-administered, blood loss was reduced ~3-fold (~200 μl) which was statistically significant relative to the controls but did not reach the level of wild-type mice (blood loss ~50 μl). Overall, the results show that GB5 is a procoagulant monoclonal antibody that binds with high affinity to human FV and influences reactions that lead to its activation and inactivation. We hypothesize that the net effect of GB5 is the enhanced production of FVa which has a major impact on TG. In plasma-based systems with and without the protein C system and an in vivo hemophilic model, GB5 is procoagulant and can reduce bleeding. Our work shows that targeting the common coagulation pathway at the level of FV has benefit in the context of hemophilia and may be a viable and safe therapeutic approach.

Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.

Antithrombotic potential of Lippia alba: A mechanistic approach

Ethnopharmacological relevanceLippia alba (Mill.) N.E.Br. ex Britton & P. Wilson is traditionally used in Brazil as an adjunct in the relief of mild anxiety, as an antispasmodic, and as an antidyspeptic. This medicinal species was included in the Phytotherapeutic Form of the Brazilian Pharmacopeia 2nd edition (2021) and has already been described as the most used medicinal plant in a study with patients from an Anticoagulation Clinic in Brazil. Meanwhile, no studies were found that support the safety of the use of L. alba in patients using anticoagulants, a drug with several safety limitations. Aim of the studyProvide scientific evidence to ensure the safety of the concomitant use of L. alba and warfarin and support the management of these patients by evaluating its in vitro anticoagulant effect and chemical composition. And, as a timely complementation, evaluate the potential of this medicinal species in the development of new antithrombotics. MethodsThe chemical profile of L. alba derivatives was analyzed by chromatographic methods such as Ultra-Performance Liquid Chromatography (UPLC) coupled with electrospray ionization mass spectrometry (ESI-MS), qualitative UPLC using Diode-Array Detection, and Thin Layer Chromatography. The anticoagulant activity was evaluated by the innovative Thrombin Generation Assay by Calibrated Automated Thrombogram method and using traditional coagulometric tests: prothrombin time, activated partial thromboplastin time, and plasma fibrinogen measurement. ResultsExtracts and fractions prolonged the coagulation time in all the tests and reduced thrombin formation in thrombin generation assay. Coagulation times with the addition of ethanloic extract (2.26 mg/mL) was 17.78s, 46.43s and 14.25s respectively in prothrombin time, activated partial thromboplastin time and fibrinogren plasma measurement. In thrombin generation test, this same extract showed ETP as 323 nM/min compared to control (815 nM/min) with high tissue factor and 582 nM/min compared to control (1147 nM/min) using low tissue factor. Presence of flavonoids, phenylpropanoids, and triterpenes were confirmed by chromatographic methods and 13 compounds were identified by UPLC-ESI-MS. Based on these results and on the scientific literature, it is possible to propose that phenylpropanoids and flavonoids are related to the anticoagulant activity observed. ConclusionThe results demonstrate the in vitro anticoagulant activity of L. alba, probably due to the activation of intrinsic and extrinsic pathways. It is concluded, then, that there is a potential for interaction, which needs to be further studied, between L. alba and warfarin. Also, this medicinal species shows a great potential for use in the development of new antithrombotics.

Thromboelastography and thrombin generation assessments for pediatric severe hemophilia A patients are highly variable and not predictive of clinical phenotypes

BackgroundSevere hemophilia A (SHA) patients vary in severity of bleeding, arthropathy, and requirements for replacement factor VIII (FVIII). Baseline hemostatic activity assays using calibrated automated thrombography (CAT) and thromboelastography (TEG) may offer insights into the physiological basis of clinical heterogeneity. ObjectivesUse CAT and TEG to measure baseline hemostatic activity in a cohort of 30 pediatric SHA patients with available clinical data. Determine effect of contact activation inhibition with corn trypsin inhibitor (CTI). Assess heterogeneity among patients for baseline hemostatic activity and examine correlations between assay results and clinical parameters including FVIII dosing regimen, von Willebrand factor level, and Pettersson arthropathy score. MethodsSHA blood after FVIII washout was subjected to TEG, and platelet‐rich (PRP) and platelet‐poor plasma was used for CAT assays. Varying concentrations of tissue factor (TF) were used. Statistical analysis examined relationships between assay results, and clinical parameters. ResultsCTI treatment was required to obtain TEG and CAT results representative of baseline hemostatic activity. Weak activity was observed in assays with low TF concentrations (0.5–2 pM), and most but not all samples approached normal activity levels at high TF concentrations (10–20 pM). A significant positive correlation was observed between results of TEG and CAT‐PRP assays. Correlations were not detected between hemostatic assay results and clinical parameters. ConclusionsIn vitro hemostatic assay results of samples containing platelets showed concordance. Assay results were not predictive of FVIII requirements or correlated with other clinical parameters. SHA patient heterogeneity is influenced by factors other than baseline hemostatic activity.

Performance and reference intervals of thrombin generation test: results from the Brazilian longitudinal study of adult health (ELSA-Brasil). A cross-sectional study.

The thrombin generation test (TGT) has shown promise for investigation of hemorrhagic and thrombotic diseases. However, despite its potential, it still needs standardization. Moreover, few studies have established reference values for TGT parameters. In Brazil, these values have not yet been established. To determine TGT performance and reference intervals for TGT parameters in healthy individuals. Cross-sectional study conducted among participants in the Brazilian Longitudinal Study of Adult Health (Estudo Longitudinal de Saúde do Adulto, ELSA-Brasil). The reference sample consisted of 620 healthy individuals. The calibrated automated thrombogram (CAT) method, under low and high tissue factor (TF) conditions, was used to assess thrombin generation. Test performance was analyzed using intra and interassay coefficients of variation (CV) and reference intervals were calculated using the nonparametric method proposed by the International Federation of Clinical Chemistry and the Clinical and Laboratory Standards Institute. The intraassay CV ranged from 1.4% to 2.2% and the interassay CV, 6.8% to 14.7%. The reference intervals for TGT parameters under low and high TF conditions were, respectively: lagtime: 3.0-10.3 and 1.4-3.7 min; endogenous thrombin potential (ETP): 1134.6-2517.9 and 1413.6-2658.0 nM.min; normalized ETP: 0.6-1.3 and 0.7-1.4; peak: 103.2-397.7 and 256.4-479.0 nM; normalized peak: 0.3-1.3 and 0.7-1.2; and time-to-peak: 5.6-16.0 and 3.4-6.7 min. These parameters were categorized relative to sex. TGT performance was adequate and the proposed reference intervals were similar to those of other studies. Our findings may be useful for consolidating the TGT, through contributing to its standardization and validation.

PO-31: The small-molecule FXIa inhibitor, BMS-26208, prevents tumor cell-induced coagulation activation predominantly in tumor entities with lower tissue factor (TF) expression

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