Low Tissue Factor Research Articles

Characteristics of Venous Thrombi in Animal Models of Obesity, Diabetes and Hypertension

Introduction: Deep venous thrombosis (DVT) is defined as a vessel occlusion of the venous system and the pathophysiology is based on the change in blood flow, the vascular wall and/or blood elements. Obesity, diabetes and hypertension are being investigated as risk factors for DVT, since some clinical studies have suggested a correlation between DVT and arterial disease.Objective: In this study we evaluated the size and components of venous thrombus in animals with risk factors for arterial disease.Methods: The study was approved by the ethics committee on animal use of the Unicamp. Forty-five male Wistar and 15 spontaneous hypertensive rats (SHR) were separated into 4 groups of 15 animals. The control group (CTL) consisted of Wistar rats fed with standard chow diet; the obese group consisted of Wistar rats fed with a hyperlipidemic diet for 8 weeks; the diabetic group consisted of Wistar rats with diabetes induced by streptozotocin and were fed with chow diet; and the hypertension group consisted of SHR rats fed chow diet. All groups had DVT-induced stasis at 12 weeks old. After 3 hours, each animal was sacrified and the thrombus was removed. The weight of the thrombus was evaluated. Tissue factor (TF), and the presence of macrophages (M1 and M2) and endothelial cells were evaluated in thrombi using immunohistochemistry (IHC) and Western Blot technique.Results: The phenotype of obesity characterized by an increase in body weight was confirmed only in the group fed with hyperlipidemic diet (CTL = 420 g ± 46 g vs Obese = 557 g ± 62 g vs Diabetes = 399 ± 104 vs Hypertension 259 ± 21; p ≤0.001) An increase in epididymal fat was observed only in the obese group (CTL = 6.5 ± 2.3 vs Obese = 10 ± 3.3 vs Diabetes = 4.9 ± 2.4 vs Hypertension 2.6 ± 0.3; p ≤0.001). Increased blood fast glucose was observed in diabetic group (CTL = 107 ± 19 vs Obese = 147 ± 12 vs Diabetes = 442 ± 91 vs Hypertension 149 ± 30; p ≤0.001). There was no difference between thrombus weight between control and experimental groups (CTL = 0.06 ± 0.01 vs Obese = 0.024 ± 0.08 vs Diabetes = 0.0073 ± 0.01 vs Hypertension = 0.031 ± 0.02; p =0.228). No differences were observed in the presence of macrophages, and endothelial cells were not detected in our samples. Western blot showed lower TF expression in all experimental groups (obese, diabetes and hypertension) compared to controls (CTL = 31 ± 2 vs Obese = 14 ± 0.4 vs Diabetes = 2.1 ± 0.9 vs Hypertension = 2.5 ± 1; p =0,002), which was consistent with the qualitative IHC analysis. No additional qualitative differences were observed by IHC regarding P-selectin, macrophages and endothelial cells.Conclusion: Obesity, diabetes and hypertension have been recognized as risk factors for DVT. However, no differences could be observed in thrombus weight and in the presence of endothelial cells and macrophages within the thrombus in the well-established venous stasis rat model in young rats at this time point. The lower expression of TF observed in our study could be due to differences in the composition of other cellular components of the thrombi, or in the source of TF in these different models. Additional studies are warranted to investigate these hypotheses at other time points and older rats. DisclosuresNo relevant conflicts of interest to declare.

Mutant IDH1 and thrombosis in gliomas.

Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30% of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0%). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90% in wild-type versus 2-6% in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

Abstract 4771: PET imaging of tissue factor using 64Cu-labeled active site-inhibited factor VII: A potential companion diagnostic for tissue factor targeted cancer therapies

Abstract Introduction: The transmembrane glycoprotein tissue factor (TF) is the primary initiator of coagulation. In addition to its physiological role, TF is associated with a variety of pathophysiological processes including tumor growth, tumor angiogenesis and metastasis. Increased TF expression has been reported in 53-89% of all pancreatic adenocarcinomas and clinically correlates with advanced stage and poor survival. Non-invasive imaging of tumor TF status holds great clinical potential as a companion diagnostic for anti-cancer therapy targeting TF. The goal of our study was to develop and evaluate a PET tracer for imaging of TF expression in pancreatic cancer by utilizing the natural ligand for TF, coagulation factor VII. Experimental procedures: Active site-inhibited factor VII (FVIIai) was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) followed by radiolabeling with 64Cu (64Cu-NOTA-FVIIai). Mice bearing subcutaneous pancreatic adenocarcinoma tumors (BxPC-3) were subjected to PET imaging 1, 4, 15 and 36 hours post-injection of 64Cu-NOTA-FVIIai for longitudinal assessment. The in vivo specificity of 64Cu-NOTA-FVIIai towards TF was evaluated in a competition blocking experiment and in a panel of subcutaneous pancreatic tumor models exhibiting low, medium and high TF expression. Additionally, the ability of 64Cu-NOTA-FVIIai to visualize TF expression in orthotopically implanted BxPC-3 tumors was evaluated by PET/MRI. Ex vivo biodistribution, immunohistochemistry and flow cytometry was performed to verify the in vivo imaging data. Results: PET imaging at 1, 4, 15 and 36 hours after injection of 64Cu-NOTA-FVIIai revealed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3 and 2.4 ± 0.3% injected dose per gram (%ID/g), respectively. A graduate increase in image contrast (tumor to background ratio) was observed over the imaging time course with no further increase beyond 15 hours. Competition with excess unlabeled FVIIai significantly reduced tumor uptake of 64Cu-NOTA-FVIIai (p< 0.001) in subcutaneous BxPC-3 tumors, while the uptake was unaffected in other organs. The ex vivo biodistribution confirmed the data obtained by in vivo PET imaging. In pancreatic cancer models with different levels of TF expression, the tumor uptake was found to be significantly different from each other (p < 0.001). This was in agreement with the TF level evaluated by immunohistochemistry and flow cytometry. Furthermore orthotopic BxPC-3 tumors were visualized and distinguishable by PET/MRI using 64Cu-NOTA-FVIIai as the PET tracer. Conclusions: 64Cu-NOTA-FVIIai is well suited for specific PET imaging of TF in pancreatic cancer and its uptake is related to the tumor TF expression level. The data supports further development of 64Cu-NOTA-FVIIai as a companion diagnostic and theranostic agent. Citation Format: Lotte K. Kristensen, Carsten H. Nielsen, Troels E. Jeppesen, Mette M. Jensen, Jacob Madsen, Bo Wiinberg, Lars C. Petersen, Andreas Kjaer. PET imaging of tissue factor using 64Cu-labeled active site-inhibited factor VII: A potential companion diagnostic for tissue factor targeted cancer therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4771.

Comparative biochemical studies of fresh frozen plasma and pooled solvent/detergent-treated plasma (octaplasLG® ) with focus on protein S and its impact in different thrombin generation assay set-ups.

The solvent/detergent treatment enables effective and robust inactivation of all lipid-enveloped viruses, but also inactivates partly sensitive plasma proteins such as protein S. The aim of this study was to investigate the thrombin generation capacity of octaplasLG® , in particular focusing on the function of protein S in thrombin generation assay and the impact of assay settings. Sixteen octaplasLG® batches and 32 units of single donor fresh frozen plasma (FFP) were investigated. For protein S, both functional activity and free antigen levels were measured. Thrombin generation assay was performed using two fluorogenic tests with different triggers. Finally, rotational thromboelastometry was performed. Mean protein S levels were lower in octaplasLG® , but a wider range of values was found for FFP. Clotting parameters and thrombin generation capacities overlapped between the two plasma groups as demonstrated using both thrombin generation assays and different triggers. Spiking studies with protein S-depleted plasma, human purified protein S or antibodies against protein S confirmed a correlation between protein S and thrombin generation capacity under specific assay conditions, especially in an assay with low tissue factor concentration. Correlation between protein S and thrombin generation capacity was demonstrated in the TGA. Due to higher variability in protein S content in the FFP group, overlapping haemostatic potentials of the two plasma groups were found.

Abstract 534: Deficiency of Protease-activated Receptor 2 Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm Independent of Vascular Smooth Muscle Cell Tissue Factor

Objective: Tissue factor (TF) is constitutively expressed in subendothelial cells, including vascular smooth muscle cells (VSMCs), and maintains hemostasis. The TF/factor VIIa complex as well as factor Xa have been shown to activate protease-activated receptor 2 (PAR-2). We and others have shown that TF/FVIIa-PAR-2 signaling induces migration of VSMCs in vitro and this is reduced in TF and PAR-2 deficient cells. We previously demonstrated C57BL/6 mice with 1% of normal TF levels (Low TF mice) had increased suprarenal abdominal aortic diameter and incidence of abdominal aortic aneurysm (AAA) compared to littermate controls. However, the source of cellular TF and whether signaling occurred through PAR-2 has not been examined. Our objective was to determine the importance of VSMC-specific deletion of TF and PAR-2 deficiency on the etiology of AAA. Methods and Results: VSMC-specific TF deficiency was evaluated on a low-density lipoprotein receptor deficient (Ldlr -/- ) background. Male mice were fed a fat-enriched diet (21% milk fat) and infused with angiotensin II (AngII; 1,000 ng/kg/min) for 28 days. Ldlr -/- /TF flox/flox SM22αCre + mice had increased abdominal aortic luminal diameters (Cre + : 1.92 ± 0.16 (n = 12); Cre - : 1.22 ± 0.03 mm (n = 10); P = 0.001) and incidence of AAA (100% vs. 50%; P = 0.01) compared to Cre - littermates. Next, we determined the effects of PAR-2 deficiency in AAA. Ldlr -/- /PAR-2 +/+ (n = 12) and Ldlr -/- /PAR-2 -/- (n = 15) male mice were fed a fat-enriched diet and infused with AngII for 28 days. PAR-2 deficiency attenuated abdominal aortic luminal diameters (PAR-2 +/+ : 1.78 ± 0.06 mm; PAR2 -/- : 1.17 ± 0.03 mm; P = 0.001) and incidence of AAA (84% versus 40%; P = 0.048) when compared to littermate controls. Total plasma cholesterol concentrations, lipoprotein cholesterol distributions, or increased systolic blood pressure was not different between groups. Conclusion: These results suggest that TF limits AAA growth and incidence via VSMCs in a PAR-2-independent manner. To verify this hypothesis, studies are currently underway to determine the effect of Factor Xa (FXa) inhibitors in PAR-2 deficient mice.

Abstract 526: 4-hydroxy-2-nonenal (HNE), a Lipid Peroxidation Product, Exerts Both Pro- and Anti-thrombotic Effects on Vascular Cells

Background: Oxidative stress and generation of lipid peroxidation (LPO) products are detrimental in the pathogenesis of atherosclerosis and associated acute thrombotic events. However, recent studies suggest that moderate oxidative stress and low levels of LPO products can induce adaptive immune responses and exert beneficial effects. Tissue factor (TF) is a critical initiator of coagulation and aberrant TF expression on vascular cells under inflammation triggers intravascular thrombosis. HNE, a highly reactive LPO product and TF have been shown to be associated with atherosclerosis. Recently, we demonstrated that HNE decrypts procoagulant activity of pre-existing TF on activated monocytes and endothelial cells and generates TF+ microparticles. Here, we investigated the effect of HNE on induction of TF and cell adhesion molecules in monocytes and endothelial cells that were not perturbed earlier. Methods and results: THP-1 monocytic cells and endothelial cells (HUVEC) were stimulated with LPS and TNF-α/IL1-β, respectively, in the presence of a control vehicle or varying concentrations of HNE that are pathophysiologically relevant. TF induction was measured at mRNA (by qRT-PCR), protein (by immunoblotting) and activity levels (in factor X activation assay). Pre-treating cells with HNE inhibited TNF-α/IL1-β- or LPS-stimulated TF procoagulant activity in a dose-dependent manner. THP-1 and HUVEC varied in their sensitivities to HNE (THP-1> HUVEC). HNE-mediated inhibition of TF activity correlated with lower TF mRNA and protein levels. Our results demonstrate that HNE prevents TNF-α/IL1-β- and LPS-induced IKKβ degradation and thereby inhibits NFκβ activation. In addition to inhibiting TF expression, HNE significantly reduced monocyte adhesion to endothelial cells through downregulating TNF-α/IL1-β-induced expression of endothelial adhesion molecules VCAM-1 and ICAM-1. Conclusion: HNE may play a dual role in regulating TF activity in atherosclerosis. HNE could act as a prothrombotic mediator by increasing coagulant activity of pre-existing TF through decryption process. HNE can also elicit anti-thrombotic and anti-inflammatory effect by inhibiting TF and adhesion molecules in response to stimulus by impairing the NF-ĸB pathway.

Role of tissue factor expression in thrombin generation by canine tumor cells.

To measure thrombin generation by high and low tissue factor (TF)-expressing canine cancer cell lines. Canine cell lines CMT25 (high TF-expressing mammary gland tumor cell line) and HMPOS (low TF-expressing osteosarcoma cell line). Thrombin generation by cancer cells was measured in pooled normal canine plasma by use of calibrated automated thrombography without added trigger reagents. Results were expressed as lag time, time to peak thrombin concentration, peak thrombin concentration, and total thrombin concentration or thrombin generation potential. Corn trypsin inhibitor, hirudin, and annexin V were used to inhibit contact activation, thrombin formation, and phosphatidylserine activity, respectively. Pooled normal human plasma deficient in coagulation factors VII, VIII, IX, X, XI, or XII was used to assess the role of individual coagulation factors on thrombin generation. CMT25 generated significantly more thrombin than did HMPOS (mean ± SD, 3,555 ± 604 nM thrombin•min and 636 ± 440 nM thrombin•min, respectively). Thrombin generation of CMT25 was dependent on factor VII and phosphatidylserine and was independent of contact activation. In contrast, thrombin generation of HMPOS was attributed to contact activation. High TF-expressing canine mammary cancer cells generated thrombin in a plasma milieu in vitro in a factor VII- and phosphatidylserine-dependent manner. These findings support a role for TF in hypercoagulability detected in dogs with mammary gland tumors and potentially for other tumors that strongly express TF.

PO-52 - Effect of tissue factor expression by colorectal cancer on cancer stem cell activity

Abnormal Tissue factor (TF) expression occurs in many cancers including colorectal and is associated with a poor prognosis. TF expression promotes cancer stem cell (CSC) activity in breast and squamous cell carcinoma (SCC). The aim of the research is to determine the effect of TF expression by colorectal cancer cell lines on colorectal CSC activity. Colorectal cancer cell lines with high (DLD-1) and low (SW620) TF expression were stably transduced to knock down (TF-ve) and over express (TF+ve) TF. CSC activity was assessed using the cancer sphere assay (sphere forming efficiency (SFE)=spheres formed / cells plated) and by ALDH1 expression (by florescent activated cell sorting). When comparing transduced cells to their control the ALDH1 expression was normalised to their control. The naturally high TF expressing cell line (DLD-1) compared to the low TF expressing cell line (SW620) had a reduced SFE (0.7 (SD 0.39) vs 1.38 (SD 0.68), p<0.05) and less cells expressed ALDH1 (15.3% (SD 1.2) vs 26.9% (SD 2.1), p=0.02). DLD-1 TF+ve compared to its control had a reduced SFE (0.33 (SD 0.14) vs 0.66 (SD 0.42), p<0.01) and less cells expressed ALDH1 (0.72 (SD 0.069) p=0.001) (normalised to negative control). There was no difference in SFE (1.21 (SD 0.56) vs 1.29 (SD 0.50) p=0.4) and ALDH1 expression (0.81 (SD 1.00) p=0.5) between SW620 TF+ve and its control. DLD-1 TF-ve and SW620 TF-ve had increased SFE compared to their controls (DLD-1: 0.63 (SD 0.27) vs 0.41 (SD 0.35) p<0.01; SW620: 2.03 (SD 0.86) vs 1.21 (SD 0.70) p<0.01), and increased ALDH expression (DLD-1: TF-ve 1.23 (SD 0.74)p=0.04 and SW620: TF-ve 1.31 (SD 0.08) p<0.001 normalised to controls). Unlike in breast and SCC, in colorectal cancer cell lines TF inhibits CSC activity. This has significance if anti-tissue factor treatments are considered as an anti-cancer treatment as this may increase the cancer stem cell activity.

Platelet-Targeted rFVIIa-Xten Improves Thrombin Generation and Fibrin Formation Compared to Recombinant FVIIa

The development of inhibitors to replacement plasma factors in hemophilia is an ongoing clinical complication. Bypass therapies, such as recombinant factor VIIa (rFVIIa), have emerged as important alternative on-demand strategies for hemophilic patients with inhibitors to treat spontaneous bleeds and prevent bleeding during surgery. However, general prophylaxis strategies for hemophilia inhibitor patients are lacking. An attractive approach for effective prophylaxis and on-demand treatment includes engineering potent rFVIIa variants with prolonged pharmacokinetics. Because the primary mechanism of action of rFVIIa is thought to occur on the platelet surface, we combined rFVIIa with platelet-targeting and XTEN half-life extension technologies to improve its pharmacokinetic profile and procoagulant activity. Platelet-targeting was achieved by recombinant fusion of an antibody fragment which binds the human alpha IIb integrin. Half-life extension was achieved by fusion of an XTEN polypeptide which increases the hydrodynamic radius, and therefore half-life, of rFVIIa. We have shown that these rFVIIa modifications improve the pharmacokinetics and efficacy of rFVIIa in vivo in humanized alpha IIb transgenic hemophilia A mice. The goal of the current study was to evaluate and compare thrombin generation and fibrin formation kinetics in hemophilic platelet-rich plasma in the presence of platelet-targeted rFVIIa-XTEN or rFVIIa. To achieve this, platelet-rich plasma from normal human donors was treated with an inhibitory factor VIII antibody to model hemophilia A and spiked with doses of platelet-targeted rFVIIa-XTEN or rFVIIa. Reactions were triggered with low tissue factor and recalcification. Thrombin generation (n=9) was monitored by calibrated automated thrombography and fibrin formation (n=7) was monitored optically on a plate reader. Non-linear regression analysis of dose responses was used to determine EC50 values for each parameter for each donor. Platelet-targeted rFVIIa-XTEN increases the rate and peak of thrombin generation with 2- to 6-fold lower EC50 values (peak and rate, respectively) than rFVIIa. These data were confirmed by thrombin generation in platelet-rich plasma from 1 hemophilia A donor and 1 hemophilia B donor which demonstrated similar responses to platelet-targeted rFVIIa-XTEN with 2.5- to 5-fold lower EC50 values (peak and rate, respectively) than rFVIIa. Notably, platelet count-dependent changes in thrombin generation activity were similar between platelet-targeted rFVIIa-XTEN and rFVIIa. Fibrin formation analysis indicated platelet-targeted rFVIIa-XTEN increases the rate and time to plateau of fibrin formation with 5- to 13-fold lower EC50 values (rate and time to plateau, respectively) than rFVIIa. Analysis of fibrin network structure by confocal microscopy indicated platelet-targeted rFVIIa-XTEN increases fibrin network density in platelet-rich plasma clots. Together, these data show platelet-targeted rFVIIa-XTEN has more procoagulant activity than rFVIIa by supporting more thrombin generation and faster fibrin formation and suggest our approach has the potential to be an effective alternative for the treatment and prevention of bleeds in hemophilia patients with inhibitors. DisclosuresAleman:Biogen: Employment, Equity Ownership. Kistanova:Biogen: Employment, Equity Ownership. Moore:Biogen: Employment, Equity Ownership. Schellenberger:Amunix Operating Inc: Employment. Peters:Biogen: Employment. Salas:Biogen: Employment, Equity Ownership.

Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury

Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury. We measured levels of TF activity in the liver, white blood cells and circulating microparticles, and a marker of activation of coagulation (thrombin-antithrombin complexes (TATc)) in the plasma of mice subjected to bile duct ligation for 12days. We used wild-type mice, mice with a global TF deficiency (low TF mice), and mice deficient for TF in either myeloid cells (TF(flox/flox),LysMCre mice) or in hepatocytes (TF(flox/flox),AlbCre). Wild-type mice with liver injury had increased levels of white blood cell, microparticle TF activity and TATc compared to sham mice. Low TF mice and mice lacking TF in hepatocytes had reduced levels of TF in the liver and in microparticles and exhibited reduced activation of coagulation without a change in liver fibrosis. In contrast, mice lacking TF in myeloid cells had reduced white blood cell TF but no change in microparticle TF activity or TATc. Hepatocyte TF activates coagulation in a mouse model of chronic liver injury. TF may contribute to the hypercoagulable state associated with chronic liver diseases in patients.

Enhanced antitumor effect of anti-tissue factor antibody-conjugated epirubicin-incorporating micelles in xenograft models.

For the creation of a successful antibody–drug conjugate (ADC), both scientific and clinical evidence has indicated that highly toxic anticancer agents (ACA) should be conjugated to a monoclonal antibody (mAb) to administer a reasonable amount of ADC to patients without compromising the affinity of the mAb. For ordinary ACA, the conjugation of a mAb to ACA-loaded micellar nanoparticles is clinically applicable. Tissue factor (TF) is often overexpressed in various cancer cells and tumor vascular endothelium. Accordingly, anti-TF-NC-6300, consisting of epirubicin-incorporating micelles (NC-6300) conjugated with the F(ab')2 of anti-TF mAb was developed. The in vitro and in vivo efficacy and pharmacokinetics of anti-TF-NC-6300 were compared to NC-6300 using two human pancreatic cancer cell lines, BxPC3 (high TF expression) and SUIT2 (low TF expression), and a gastric cancer cell line, 44As3 (high TF expression). The intracellular uptake of epirubicin was faster and greater in BxPC3 cells treated with anti-TF-NC-6300, compared with NC-6300. Anti-TF-NC-6300 showed a superior antitumor activity in BxPC3 and 44As3 xenografts, compared with NC-6300, while the activities of both micelles were similar in the SUIT2 xenograft. A higher tumor accumulation of anti-TF-NC-6300 compared to NC-6300 was seen, regardless of the TF expression levels. However, anti-TF-NC-6300 appeared to be localized to the tumor cells with high TF expression. These results indicated that the enhanced antitumor effect of anti-TF-NC6300 may be independent of the tumor accumulation but may depend on the selective intratumor localization and the preferential internalization of anti-TF-NC-6300 into high TF tumor cells.

Antitumor effect of antitissue factor antibody-MMAE conjugate in human pancreatic tumor xenografts.

Tissue factor (TF) triggers the extrinsic blood coagulation cascade and is highly expressed in various types of cancer. In this study, we investigated the antitumor effect of an antibody–drug conjugate (ADC) consisting of an anti‐TF monoclonal antibody and monomethyl auristatin E (MMAE). MMAE was conjugated to an anti‐human TF or anti‐mouse TF antibody using a valine‐citrulline linker that could be potentially hydrolyzed by cathepsin B in the acidic environment of the lysosome. The cytotoxic and antitumor effects of the ADCs against four pancreatic cancer cell lines were analyzed. Both the ADC with the anti‐human TF antibody and that with the anti‐mouse TF antibody were stable under physiological conditions. The anti‐human ADC was internalized in TF‐expressing human tumor cell lines, followed by effective MMAE release. The half maximal inhibitory concentration (IC50) of MMAE was approximately 1 nM for all of the cell lines used. Meanwhile, the IC50 of anti‐human ADC was 1.15 nM in the cell lines showing high TF expression, while exceeding 100 nM in the cells showing low TF expression levels. Anti‐human ADC with passive and active targeting ability exerted significant suppression of tumor growth as compared to that observed in the saline group (p < 0.01). Also significant tumor growth suppressions were seen at the anti‐mouse ADC and control ADC groups compared to the saline group (p < 0.01) due to EPR effect. Because various clinical human cancers express highly amount of TF, this new anti‐TF ADC may deserve a clinical evaluation.

Contact Activation Inhibition and Platelet Rich Plasma Are Required in Order to Differentiate Bleeders from Non-Bleeders in FXI Deficiency Using the Thrombin Generation Assay

Introduction: FXI deficiency is characterised by a variable bleeding tendency. No clear correlation exists between bleeding and either FXI:C level or genotype. Some patients with major FXI deficiency (FXI:C <15 IU/dL) do not exhibit excessive bleeding, even after surgery or trauma, while others with partial deficiency (FXI:C 20-60 IU/dL) report significant haemorrhagic symptoms. The variation in bleeding tendency is observed between individuals with the same FXI:C level and also between individuals within families who share the same F11gene mutation. The thrombin generation (TG) assay quantifies the overall haemostatic potential of an individual through the measurement of thrombin production. While reduced TG has been observed in some FXI deficient individuals, there are conflicting reports about the ability of the TG assay to distinguish between patients with a history of bleeding (bleeders) and those without (non- bleeders).Aims: To identify the optimal TG assay conditions which discriminate between normal controls and individuals with different levels of FXI deficiency. To investigate which conditions allow the TG assay to differentiate between bleeders and non-bleeders.Methods: 97 adults with FXI deficiency were studied together with 50 controls. Bleeding histories were taken from each individual and citrated blood samples, both with addition of corn trypsin inhibitor (+ CTI) and without (- CTI). Platelet rich (PRP) and platelet poor plasmas (PPP) were prepared. TG was performed using the CAT method with tissue factor (TF) concentrations of 5pM, 1pM and 0.5pM in the following samples: PPP + CTI, PPP - CTI, PRP + CTI, PRP - CTI. TG results were compared between controls and 3 patient groups: partial FXI deficiency (FXI:C 20-60 IU/dL) (n=78), major low level FXI deficiency (FXI:C 3-15 IU/dL) (n=10) and major very low level FXI deficiency (FXI:C ≤2IU/dL) (n=9) using unpaired t-test or Mann Whitney U tests.A subgroup of 74 patients was divided into bleeders and non-bleeders based solely on their experience of tonsillectomy and /or dental extraction prior to diagnosis of FXI deficiency. Those with excessive bleeding (requiring blood product transfusion or return to theatre/dentist for re-suturing or packing) were classified as bleeders (n=24) and those without, as non-bleeders (n=50). The optimal TG assay conditions to differentiate between bleeders and non-bleeders groups were identified with Receiver Operator Curves (ROC) using Area Under Curve (AUC) values.Results: TG performed in PPP with CTI was unable to differentiate between controls and all of the 3 patient groups. In all other sample types the best discrimination between controls and FXI deficient patients was seen at low TF (0.5pM) with a sequential reduction in peak height and ETP measurements observed as FXI:C levels decreased across the 3 patient groups (see Figure 1).When TG results were compared between the bleeder (n=24) and non-bleeder (n=50) groups, peak height and ETP measured in PRP with CTI were best able to differentiate between the two groups (peak height ROC AUC = 0.9362, P<0.0001) (ETP ROC AUC=0.9362, P<0.0001). PPP samples and those without CTI were unable to segregate bleeders from non-bleeders as effectively.Conclusions: In the presence of contact activation inhibition, FXI:C levels have minimal impact on thrombin generation in PPP but do influence thrombin generation in the presence of platelets. TG measured at low TF in PPP and PRP samples without CTI (with in vitro contact activation of FXI possible) was able to differentiate between controls and individuals with different levels of FXI deficiency but not between bleeders and non-bleeders.TG performed at low TF in PRP with CTI samples demonstrated the greatest differentiation between bleeders and non-bleeders in FXI deficiency providing further support for the clinical importance of platelet interaction with FXI in this disorder and suggesting that these test conditions may best mimic those in vivo.Acknowledgments: This work is supported by a Fellowship Project Award (Bayer Hemophilia Awards Program), an unrestricted grant (LFB Biotechnologies) and a Wycherley Fellowship grant. [Display omitted] [Display omitted] [Display omitted] DisclosuresPike:Bayer: Honoraria, Research Funding; LFB Biotechnologies: Honoraria, Research Funding. Bolton-Maggs:Bio-Products Laboratory (U.K.): Consultancy; LFB Biotechnologies: Consultancy.

Molecular Characterization of the Synergistic Effect on TFPI Inhibition By Fusion of Two Inhibitory Peptides

IntroductionTissue factor pathway inhibitor (TFPI) is a three-Kunitz domain (KD1-3) protease inhibitor that downregulates the extrinsic coagulation pathway. TFPI has a double inhibitory effect; it inactivates factor Xa (FXa) by 1:1 binding via its KD2, and it prevents further FX activation by binding the tissue factor (TF) – factor VIIa (FVIIa) complex via its KD1 and the formation of a quaternary complex. Recently, we demonstrated the crystal structure of a linear TFPI inhibitory peptide composed of 20 amino acids, bound to a TFPI protein composed of N-terminus and KD1. On the other hand, a cyclic TFPI inhibitory peptide of 23 amino acids was shown to co-crystallize with TFPI KD1-KD2. Molecular fusion of the linear and cyclic peptide by an optimized linker sequence would thus target two independent epitopes and combine the antagonistic properties of the two peptides.MethodsThe binding properties of simultaneous interaction of the linear and cyclic peptide with TFPI were studied in Biacore experiments using immobilized human TFPI 1-160 on a CM5 chip. Measurements with the linear or cyclic peptide were done with and without prior saturation of TFPI with the linear peptide and the fusion peptide. The results were confirmed by native-PAGE analysis of peptide/KD1-KD2 mixtures, where the TFPI fragment KD1-KD2 had been incubated with either linear or cyclic peptide or both.The TFPI inhibitory effect of the linear, cyclic, and fusion peptide was assessed in several TFPI sensitive assays including inhibition of FXa, FX activation by TF/FVIIa, and thrombin generation. Calibrated automated thrombography (CAT) was performed in human hemophilia plasma triggered with low tissue factor. To model a situation of elevated plasma levels of TFPI, the assay was carried out at TFPI concentrations up to 10 nM, which is 40-fold higher than the physiological TFPI plasma concentration.ResultsBiacore binding studies demonstrated that binding kinetics of the cyclic peptide to TFPI 1-160 were not influenced by prior saturation of immobilized TFPI with the linear peptide and vice versa. Prior saturation of immobilized TFPI with the fusion peptide prohibited the linear and cyclic peptide from binding to TFPI, clearly demonstrating the independent binding of the two peptides to different epitopes. By native-PAGE, the linear peptide shifted the KD1-KD2 band completely, whereas the cyclic peptide shifted it only partially. In the presence of both peptides, KD1-KD2 shifted to the highest MW to charge ratio, indicating the formation of a ternary complex consisting of K1-K2, cyclic, and linear peptide.Although the linear and cyclic peptide inhibited TFPI in functional assays, fusion of the two molecular entities provided the most efficient inhibition of TFPI. This was most evident in assays involving multiple epitopes of TFPI to provide functions such as inhibition of extrinsic FX activation complex and thrombin generation, or at high TFPI concentrations. Thrombin generation assays using of 5- to 40-fold elevated TFPI showed that, separately, the two monomeric peptides are only partial inhibitors, and that a mixture of these peptides led to an improved response. However, molecular fusion of the two entities resulted in the most efficient TFPI neutralization. Thus, a synergistic effect is achieved by linking both peptides. Importantly, thrombin generation compromised by a 40-fold of normal TFPI level is normalized by fusion peptide concentrations as low as 50 nM.SummaryBased on structural information, we developed a peptide inhibitor composed of two TFPI inhibitory entities. Binding studies support an independent binding mode to non-overlapping binding sites without allosteric cross-talk between binding sites. This introduces synergistic improvement of binding and functional inhibition by bivalent interaction with TFPI. This optimized fusion peptide facilitates efficient TFPI neutralization and resistance to highly increased TFPI levels. Our results further support the use of a fusion peptide in the development of subcutaneous treatment for patients with hemophilia including those with inhibitors. DisclosuresDockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Redl:Baxter Innovations GmbH, Vienna, Austria: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Kammlander:Baxter Innovations GmbH: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Reineke:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Brandstetter:Department of Molecular Biology, University of Salzburg, Salzburg, Austria: Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

A TFPI Inhibitory Half Life Extended Fusion Peptide Proves Efficacy in FVIII Knock out Mice and Marmoset Monkeys

IntroductionTissue factor pathway inhibitor (TFPI) is an important inhibitor of the extrinsic coagulation pathway as it inhibits factor Xa (FXa) and the tissue factor (TF) – FVIIa complex. Inhibition of TFPI with blocking antibodies, aptamers, or peptide inhibitors improves hemostasis and may become an option to treat patients with hemophilia including those with inhibitors. We developed a TFPI-antagonistic fusion peptide (FP) consisting of a linear and a cyclic peptide connected by a linker. The two peptide entities bind to different epitopes on TFPI and together synergistically inhibit TFPI. The FP was further improved by half-life extending (HL) non-covalent albumin binding. HL-FP was characterized for in vitroinhibition of TFPI, pharmacokinetics, and improvement of coagulation in animal models of hemophilia.MethodsHL-FP was characterized in a set of in vitro assays for binding to and inhibition of TFPI. Interaction with immobilized TFPI was studied by BiaCore. Functional inhibition was analyzed in model assay systems such as inhibition of FXa and FX activation by TF/FVIIa and plasma assays according to the calibrated automated thrombography (CAT) protocol at low TF in hemophilia plasma. Addition of TFPI simulated conditions of potentially elevated TFPI plasma concentrations. In a single dose PK study, mice (n=6 per time point) received 400 nmol/kg of the HL-FP intravenously (i.v.) or subcutaneously (s.c.). Plasma was sampled up to 38 h after dosing and HL-FP level quantified by a compound specific LC-MS protocol. To provide an ex vivo activity measure, FVIII inhibitory antibodies were added to mouse plasma to mimic a hemophilic condition and then analyzed by calibrated automated thrombography (CAT). A 2-week repeated i.v. dose study in mice investigated TFPI accumulation due to HL-FP. HL-FP was dosed at 40, 400, and 2000 nmol/kg and mouse plasma TFPI levels determined by ELISA. The efficacy of the HL-FP was studied in a hemophilia A mouse tail cut model and in a marmoset monkey model of ex vivoimprovement of coagulation. FVIII knockout mice (n=16 per group) were dosed i.v. with 12-400 nmol/kg HL-FP in the presence of a sub-therapeutic level of recombinant FVIII (10 U/kg) and blood loss (mg) was assessed. Marmoset monkeys (N=4) received 400 nmol/kg HL-FP i.v. and plasma samples obtained 1 h after dosing were analyzed by CAT in the presence of FVIII inhibitory antibodies.ResultsHL-FP bound to and efficiently inhibited TFPI as demonstrated in several in vitro test systems. Binding affinity of < 1nM correlated well with functional inhibition of TFPI in model assays, resulting in IC50s of ~0.7nM. The HL-fusion peptide (HL-FP) efficiently inhibited plasma TFPI, which resulted in an improvement of all thrombin generation parameters in plasma of hemophilia A and B patients, with EC50s ranging from 6 to 20nM. HL-FP increased peak thrombin levels of hemophilia plasma to or slightly above a range established for individual normal plasma. Non-covalent binding to albumin substantially increased the half-life to ~4 h with ~ 50% s.c. bioavailability in mice. The ex vivo procoagulant activity determined by CAT correlated well with HL-FP plasma concentrations. In the repeated dose study, the HL-FP was well tolerated and did not accumulate TFPI, which strongly indicates that HL-FP did not interfere with TFPI clearance receptor interactions. HL-FP significantly reduced bleeding in the hemophilia mouse tail cut bleeding model at a dose as low as 40 nmol/kg. In marmoset monkeys, HL-FP efficiently improved ex vivo plasma thrombin generation, even at low peptide plasma concentrations (25- 55 nM).SummaryWe developed a TFPI inhibitor composed of two TFPI antagonistic peptides that completely inhibits TFPI. Introduction of an entity non-covalently bound to albumin provides intermediate half-life extension and s.c. bioavailability. This HL-FP improved coagulation and hemostasis in animal models of hemophilia and did not interfere with TFPI clearance receptor interactions. TFPI-antagonistic peptides with a prolonged half-life, resistance to elevated TFPI, and minimal interference with TFPI clearance. Our HL-FP appears to be useful in preventing bleeding in hemophilia and provides a FVIII and FIX independent approach for non-i.v. treatment. DisclosuresDockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Reineke:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Schiviz:Baxter Innovations GmbH, Vienna, Austria: Employment. Hoellriegl:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Role of Tissue Factor in Mycobacterium Tuberculosis-induced Inflammation and Disease Pathogenesis

Tuberculosis, a chronic lung infection caused by Mycobacterium tuberculosis (M.tb), affects nearly one third of the world’s population. Clinical manifestations of TB include hypercoagulable states and thrombotic complications particularly disseminated intravascular coagulation and deep vein thrombosis. Tissue factor (TF) plays an important role in the initiation of inflammation-induced coagulation. Various bacterial infections induce aberrant expression of TF on vascular cells, which contributes to intravascular coagulation and exacerbation of inflammation. Studies have shown that either a genetic deficiency of TF or blockade of TF functional activity reduces coagulopathy, proinflammatory cytokine release and infection-associated mortality. In contrast, TF-dependent coagulation activation and fibrin deposition may be protective in host-defense against certain bacterial infections via reducing pathogen burden and limiting their dissemination. In vitro M.tb infection markedly upregulates TF expression and increases procoagulant activity of macrophages. However, it is not yet known whether TF expression has any functional significance in TB pathogenesis. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth and containment of infection. Wild-type C57BL6 (WT) and transgenic mice that express either very low levels of human TF (low TF, ~1% of WT) or high levels of human TF (HTF, ~100% of WT) in place of murine TF were infected with aerosol exposure of M.tb H37Rv. Mice were sacrificed 2 and 8 weeks post-infection. An evaluation of in vivo TF expression, coagulation activation, proinflammatory cytokines and tissue bacterial burden was performed. M.tb infection did not significantly alter overall TF expression and procoagulant activity in lungs of WT and HTF mice. Although not statistically significant, M.tb infection increased TF activity substantially in the lung homogenates in low TF mice. Nonetheless, TF expression levels in lungs of low TF mice, both uninfected and M.tb.-infected, was negligible as compared to WT and HTF mice. M.tb infection markedly increased TF expression in localized areas within the granulomas of WT and HTF mice. Interestingly, these intensely stained TF positive patches were also present in the granulomas of low TF mice after M.tb infection. The increased localized expression of TF in low TF mice may be responsible for the increased TF activity in the lung homogenates in low TF mice. M.tb infection was not accompanied by systemic and pulmonary activation of coagulation in WT and HTF mice. There was no change in the plasma thrombin-anti-thrombin complexes (TATc) upon M.tb infection in all three genotypes. Although, the bronchoalveolar lavage (BAL) TATc significantly increased (10-fold) after M.tb infection in the low TF mice, still the level was 15-50 folds lower than those in HTF and WT mice. The levels of TNF-α, IFN-γ, IL-6 and IL1-β increased upon M.tb infection but no significant differences in the cytokine profiles of BAL and total lung homogenates were observed among the genotypes. Higher expression of TF in the granuloma of WT and HTF correlated with the presence of small discrete regions of fibrin islands especially extending toward outer margin of the granuloma whereas little fibrin staining was seen in the granuloma of low TF mice. Despite, marked differences in fibrin generation in the granuloma, there were no significant differences in either lung bacterial burden or dissemination to liver and spleen. In summary, our data suggest that TF-mediated coagulation and/or signaling does not appear to contribute to host defense during experimental tuberculosis. However, it is difficult to completely eliminate a role for TF in M.tb. pathogenesis since M.tb. induced significant amount of TF expression in localized areas in the granuloma even in low TF mice. It is possible that this small amount of TF expressed within the granuloma may be sufficient to mediate local coagulant and signaling functions to facilitate M.tb. growth and dissemination. DisclosuresNo relevant conflicts of interest to declare.

Preferential Stimulation of Contact Pathway Factors By Red Cell-Derived Microparticles: New Perspective on Hemostasis

BACKGROUND / OBJECTIVES. Cell-derived microparticles (MP) are known to promote thrombin generation, and more recently have been shown to contribute substantially to hemostasis. This activity derives mainly from platelet MP (PMP) but recent evidence implicates also red cell MP (RMP). Traditionally, MP-mediated procoagulant activity has been ascribed to presentation of negatively charged surfaces for the assembly of tenase and prothrombinase. However, the mechanism of this activity is becoming more complicated as it is emerging that MP-mediated procoagulant activity includes augmentation of the contact pathway [van der Meijden et al,J Thromb. Haemost., 2012; 10:1355-62, Rubin et al, Transfusion, 2013; 53:1744-54] and of ADP-induced platelet aggregation [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Accordingly, we undertook examination of interactions of RMP with specific elements of the contact factor pathway, and related issues such as whether or not RMP can initiate contact pathway by activating FXII directly.METHODS. Plasmas depleted in each of 8 factors (F’s II, V, VII, VIII, IX, X, XI, XII), obtained from commercial sources, were mixed with pooled normal plasma to produce depletion levels of 75% to 95%, i.e., from 25% to 5% of normal. These plasmas were then assayed by thromboelastography (TEG) with / without addition of RMP. The RMP was prepared by high-pressure extrusion of washed RBC [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Blood was drawn into citrated Vacutainers in presence / absence of 140 µg/mL corn trypsin inhibitor (CTI) and run in TEG. Controls were obtained from healthy staff volunteers (n=8) to establish baseline values, +/- addition of RMP. Tissue factor (TF) at 20 or 200 pg/mL was added to standardize extrinsic pathway activation. Anti-FVIIa (Seksui Diagnostics) was added in certain experiments to abolish the TF pathway. Finally, when contact factor activation was fully blocked (no fibrin generation within 2 hrs), kaolin was added in presence / absence of RMP.RESULTS.(1) In the factor depletion experiments, we found that RMP most strongly potentiated depletion of factors of the contact pathway, namely FVIII, FIX, FXI, and FXII, as judged by shortening of R (lag time), p<0.01 to p<0.05, at depletions as low as 5% normal. For example, FVIII at 15% of normal level responded to RMP by 50% reduction in R time. In contrast, RMP had little or no augmenting effect on plasmas depleted of F’s II, V, X. This suggests that RMP promote the contact pathway preferentially.(2) RMP were capable of enhancing the rate of thrombin generation at low concentration of CTI (40 µg/mL), but at high concentration (140 µg/mL) this effect was completely abolished. When kaolin was added to the high concentration CTI-treated blood, it initiated thrombin generation after long delay (~30 min). Upon addition of RMP, the rate of thrombin generation was potentiated considerably (by ~50%). These data indicate that unlike kaolin, RMP are not capable of activating FXII directly but RMP can potentiate FXIIa-mediated thrombin generation.(3) In whole blood treated with low concentration TF (20 pg/mL) and high concentration CTI, RMP increased rate of thrombin generation by 25% compared to controls (p< 0.05). When blood was treated with anti-FVIIa, RMP retained the ability to enhance thrombin generation by roughly 30% (p<0.05). This demonstrates that RMP can potentiate thrombin generation via the TF pathway, although not as robustly as by contact pathway.CONCLUSIONS. First, these results demonstrate highly selective stimulation of elements of the contact pathway by RMP, as opposed to non-specific augmentation of coagulation. However, we do not have evidence of direct activation of FXII to FXIIa by RMP. Instead, reslults can be explained by marked potentiation of small amounts of activated enzyme. Second, it is shown that this activity is entirely independent of the TF pathway. Third, results support the potential value of RMP for use as an infusible hemostatic agent [Jy et al, Thrombosis & Haemostais, 110:751-60 (2013)], for treating a wide variety of clinical conditions involving excessive bleeding. Finally, in view of new evidence showing that MP-mediated procoagulant activity is diverse in action, new avenues of MP-mediated hemostasis are open to exploration. DisclosuresNo relevant conflicts of interest to declare.

Role of tissue factor in Mycobacterium tuberculosis-induced inflammation and disease pathogenesis.

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.

Enhanced thrombin generation and reduced intact protein S in processed solvent detergent plasma

BackgroundStandardized solvent/detergent (S/D)-treated plasma has been developed as an improved alternative to fresh frozen-plasma (FFP) in the management of severe bleeds. This study aimed at exploring compositional modifications that may influence the general applicability of S/D-treated plasma. Materials and methodsS/D-treated plasma and FFP were compared in procoagulant microparticles and concentration of coagulation factors and inhibitors. Compositional differences were correlated with hemostatic and fibrinolytic characteristics as measured by PT, APTT, thrombin generation and thromboelastography. ResultsProcoagulant microparticles were absent in S/D-treated plasma. Procoagulant factors were within the normal range. Antithrombin, TFPI and protein S antigen may be normal or slightly reduced depending on the duration of the S/D-treatment, but S/D-treated plasmas had only 12-14% intact functional protein S. Thrombin generation was subsequently increased, especially at low tissue factor concentration (1 pM). Plasma coagulation times in PT and APTT were normal, but 1.5-fold reduced in thromboelastography at low TF (1 pM). α2-antiplasmin was reduced with a concomitant 3-4 fold shortened clot lysis time measured by thromboelastography in the presence of TF (10 pM) and tissue-type plasminogen activator (0.2μg/ml). Enhanced fibrin degradation could be normalised with tranexamic acid. ConclusionsS/D-treatment seems to induce a procoagulant phenotype that results from a strongly reduced level of intact single chain protein S. Whether this may correct the apparent hemostatic imbalance as suggested from the increased fibrinolysis remains to be established. Our findings may bear implications in patients with deficiencies of natural anticoagulants. Co-administration of tranexamic acid appears beneficial to control enhanced fibrinolysis.

The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood

Corn trypsin inhibitor (CTI), an inhibitor of FXIIa, is used to prevent plasma coagulation by contact activation, to specifically investigate tissue factor (TF)-initiated coagulation. In the present work the specificity of CTI for factor (F) XIIa is questioned. In the commercial available plasma coagulation assays CTI was found to double activated partial thromboplastin time (APTT) at a plasma concentration of 7.3±1.5μm CTI (assay concentration 2.4μm). No effect was found on the prothrombin time (PT) when high TF concentrations were used. Also, with specific antibodies for FXIIa and for FXIa only APTT was found to be extended but not PT. With specific enzyme assays using chromogenic substrates CTI was shown to be a strong inhibitor of FXIIa and a competitive inhibitor of FXIa with Ki =8.1±0.3μm, without effect on the coagulation factors FVIIa, FIXa, FXa and thrombin. In thrombin generation and coagulation (free oscillation rheometry, FOR) assays, initiated with low TF concentrations, no effect of CTI (plasma concentrations of 4.4 and 13.6μm CTI, 25 resp. 100mgL(-1) in blood) was found with ≥1pm TF. At ≤0.1pm TF in the FOR whole blood assay the coagulation time (CT) concentration dependently increased while the plasma CT became longer than the observation time. To avoid inhibition of FXIa and the thrombin feedback loop we recommend that for coagulation assays the concentration of CTI in blood should be below 20mgL(-1) (1.6μm) and in plasma below 3μm.