Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

Introduction: Hematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment. We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. Methods: This is a single-centre, case-control, non-interventionist, prospective study. During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed: Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway. Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software. TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA). The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. Results: Those patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests. Table 1 shows the results obtained. Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI. Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI. Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. Conclusions: UBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient. Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes. TFPI inhibition might emerge as a good therapy for some of these patients. Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field. This work was supported by Novo Nordisk Pharma S.A. Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LT: lagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. Figure 1 Figure 1. Disclosures Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

Protective Effects of Kininogen-1 Gene Deficiency in Mouse Models of Venous Thrombosis

Background- High molecular weight Kininogen (HK) is a nonenzymatic co-factor of the contact activation system. HK binds prekallikrein (PK) and FXI to surfaces in proximity to FXII, amplifying PK activation by FXIIa and the reciprocal activation of FXII by activated PK (PKa), as well as FXI activation of FXIIa. PKa cleavage of HK also liberates bradykinin-a proinflammatory and vasoactive nanopeptide. The aim of this study was to define the pro-thrombotic role of kininogen in venous thrombosis (VT) and to use in vivo serial analysis of thrombus development to understand the recruitment and retention of platelets in the growing thrombus in the absence and presence of kininogen. Methods- The development of VT in mice deficient in kininogen (mKng1-/-) was compared to that in their wild-type littermates. A femoral-saphenous stasis VT model was prepared by ligating both saphenous and femoral veins. Next VT formation, growth, and dissolution (n=3 for each group) was monitored using intravital microscopy (IVM) via a multichannel epifluorescence microscope (Nikon Eclipse 90i). To induce stasis VT, FITC-dextran (10 mg/kg, ex/em 488/520 nm) was injected retro-orbitally, and then continuous light irradiation (20x objective, 475nm/35nm) of the saphenous vein was applied for 5 minutes. FITC-dextran fluorescence angiography monitored thrombus formation and dissolution. Immediately after VT formation, platelet accumulation at the thrombus site was monitored in the Cy5 channel (630/38 nm) via injection of a GPIbβ antibody conjugated with Dylight-649 (150nmol/kg), over time. All images were identically windowed in each channel, and thrombus area was measured using NIH ImageJ software. To corroborate IVM studies, we also evaluated a complete stasis model of inferior vena cava (IVC) ligation (n=7-8 per group). Thrombi were harvested after 48 hours and thrombus weight and length were measured to estimate thrombus mass. FXI circulates in blood as a homodimer along with HK. We determined the effect of kininogen deficiency on FXI activity. FXI activity assay used a combination of inhibitors, serially, to monitor the cleavage of substrate specific to activated FXI and release of chromogen, as a function of FXI activity. Finally, to determine the effects of Kng1 deficiency on bleeding, tail vein bleeding times were also determined (n=8 per group). Results- In femoral-saphenous stasis VT, thrombus developed in both groups immediately following FITC-channel light irradiation. However, thrombus size was smaller in Kng1-/- as compared to WT (Figure 1). Results from serial IVM of VT indicated faster thrombus dissolution in the Kng1-/- group. Lower platelet signals, as shown at 2 and 6 hours in the Kng1-/- mice may be consistent with this hypothesis. Thrombus area analysis suggested decreased thrombus formation in the Kng1-/- animals, and temporal analysis indicated faster dissolution by 6 hours (Figure 2). IVC ligation results corroborated the findings of femoral-saphenous DVT model, demonstrating that thrombus weight was significantly lower in Kng1-/- mice as compared to WT (p<0.001, Figure 3). FXI activity was also decreased in the Kng1-/- group (p<0.10). Tail vein bleeding times, however, showed no increased bleeding in Kng1-/- mice. Conclusion- These initial results suggest a pro-thrombotic role of kininogen and a protective role of kininogen deficiency in two murine venous thrombosis models, without incurring a bleeding penalty. Thrombus dissolution was faster and platelet accumulation was inhibited in Kng1-/- mice. These findings suggest that targeting kininogen may provide a new approach to prevent and treat venous thrombosis. Figure 1 Figure 1. Disclosures McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria. Jaffer: Mercator, Inc.: Other: Sponsred research.

DNA Mediated Blood Coagulation: Extracellular DNA Accelerates Fibrinogen Polymerization By Thrombin Independent of Contact Pathway

Introduction- Several in vivo studies and clinical studies have demonstrated extracellular DNA as a mediator of coagulation and this effect was reversed by the administration of DNA degrading enzyme DNase I. However, there is no clear understanding of the mechanism by which extracellular DNA activates coagulation in vitro. Conventionally, it was thought to be the activator of the contact pathway. But recent studies have shown that extracellular DNA isolated without contaminants like silica particles is a weak activator of the contact pathway. In this study, we have investigated the mechanism by which extracellular DNA is contributing to coagulation. Corroborating with recent results, we show that extracellular genomic DNA is a weak activator contact pathway. We determined that extracellular DNA accelerate fibrinogen polymerization by thrombin via a possible template mechanism. Our biophysical studies corroborate the interaction of DNA, thrombin, and fibrinogen. Understanding the mechanism of DNA induced blood coagulation will help address the gaps in the literature as well as develop inhibitors against DNA- mediated thrombosis. Methods- Silica-free extracellular DNA was purified with the PAXgene™ Blood DNA Kit. Contact activation in plasma was measured by monitoring the cleavage of the substrate S2302. To study the contact independent activation of plasma clotting by extracellular DNA, 1.5 µM Corn Trypsin Inhibitor (CTI) was applied to the plasma. Next, acceleration of fibrinogen polymerization by thrombin in presence of extracellular DNA was measured by monitoring the absorbance of 350 nm. Interaction of DNA with fibrinogen and thrombin in phosphate buffer was determined by CD spectroscopy. Results- Our results show that silica-free extracellular genomic DNA is a weak activator of the contact pathway of coagulation [Fig-A]. Moreover, genomic DNA accelerated the plasma clotting even when the contact pathway was inhibited with CTI indicating a contact independent mechanism of the procoagulant activity of extracellular DNA. Interestingly, the presence of extracellular DNA accelerated the polymerization of fibrinogen in presence of thrombin [Fig-B]. A bell-shaped dose-response curve for extracellular DNA indicates a likely template mechanism in which both thrombin and fibrinogen could assemble on the DNA molecule. These results are supported by the results from the CD spectroscopy studies where an alteration of the structure of fibrinogen and thrombin can be noticed in presence of extracellular DNA. Confocal studies further corroborate this observation. Our results also show different nucleic acids activate coagulation via different pathways. Significance- Procoagulant activity of extracellular DNA is demonstrated in several mouse models. However, a clear understanding of the mechanism of procoagulant activity of DNA in vitro has been challenging due to the caveats in the isolation of extracellular DNA where it is often contaminated with silica particles. Here we show a novel procoagulant mechanism of cell- free DNA where it augments the polymerization of fibrinogen by thrombin. These results provide insights into the mechanism of procoagulant activity of DNA which is key to develop therapeutics against procoagulant DNA. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Optimization of Thrombin Generation in Hemophilia a

Background: Hemophilia A (HA) is a bleeding disorder characterized by decreased or absent FVIII. Clinical analysis of coagulation potential in this patient population is classically based on APTT based FVIII assays. Although both the one-stage FVIII assay and the chromogenic FVIII assay can measure FVIII concentrations reliably these types of assays only give insight on the initiation of coagulation. Global coagulation assays, like thrombin generation (TG), can be used to measure the full coagulation spectrum of initiation, amplification and propagation. However the frequently used commercially available TG kits lack sensitivity for measurements of hemophilia plasma within the lower FVIII ranges which are essential in explaining differences in bleeding phenotype. Aim: We aim to optimize the sensitivity of the TG-assay for measurements in hemophilia A patients, especially in the lower FVIII ranges. Methods: In order to minimize patient specific sensitivity a hemophilia A pool plasma (HAPP) was created. Analysis of the influence of pre-analytical variables, like contact activation inhibitors, on the assay was performed. Initiation of coagulation by different reagents was compared for sensitivity towards factor FVIII titrations in patient plasma. Other assay variables like phospholipids and temperature were adjusted to increase sensitivity even further. Results: Commonly used tissue factor (TF) initiated TG at varying concentrations was unable to significantly differentiate in FVIII levels below 20%. In contrast, TG activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. Additionally, a representative baseline TG-curve in severe HA plasma could only be produced using this dual TF/FXIa-activation. There was a value in the addition of contact activation inhibitors in the assay. Higher phospholipid concentrations seem to benefit this assay setup compared to a TF only setup. Conclusion: TF/FXIa dual activation thrombin generation increased assay sensitivity in severe hemophilia plasma, allows for dose-dependent measurements in low FVIII ranges and provides a solid baseline curve that can be used for further clinical evaluation of coagulation potential and possibly therapeutic monitoring in hemophilia A. Figure 1 Figure 1. Disclosures Hackeng: ACS Biomarker BV: Current Employment, Current equity holder in publicly-traded company; Coagulation Profile BV: Current Employment, Current equity holder in publicly-traded company.

In Covid-19 Infection, Plasma Extracellular Vesicle Tissue Factor Activity Does Not Correlate with D-Dimer Levels

Objective: Identify a plasma-based activity, or biomarker, that defines the mechanism(s) by which Covid-19 disease triggers excessive coagulation. Introduction: While acute respiratory syndrome is the fundamental feature of severe Covid-19 disease, having a high level of the coagulation biomarker D-dimer upon admission is associated with increased thrombosis and mortality. As such, hospitalized patients are often placed on anticoagulant heparins. How Covid-19 triggers excessive coagulation is unresolved. Sars-CoV-2 infection could expose existing tissue factor (TF) to blood or, via cytokines, induce TF expression on cells that are in direct contact with blood. Extracellular vesicles (EV) are lipid bound microparticles released by all types of healthy and damaged cell and Covid-19 patient plasma EV TF activity has been recently reported. Cellular activation and damage due to SARS-CoV-2 could also release polyanionic nucleic acids and polyphosphates and generate neutrophil extracellular traps as contact surfaces for clot formation. Methods: Study 1. We attempted to identify excessive coagulation pathway activities in Covid-19 plasma-based, Ca++-induced thrombin generation assays. Assays were performed in the absence and presence of selective extrinsic (TF) and intrinsic (contact activation) pathway inhibitors (n=296 plasma samples). D-dimer levels were also determined. In a smaller study, Covid-19 patient samples were collected directly into citrate or citrate plus corn trypsin inhibitor, then processed for analysis. Study 2. We conducted studies to evaluate the extent to which EV TF activity contributes to the Covid-19-associated coagulopathies. Plasma EVs were isolated and EV TF activity determined by the difference in FXa activity in the absence vs presence of anti-TF antibody. D-dimer and tissue factor pathway inhibitor a (TFPIa) antigen levels were measured. Data from 232 samples collected from 96 Covid-19 positive patients and 18 samples from 14 healthy controls were analyzed. For each study analysis, patient samples were organized into groups based on the disease severity outcomes as follows: hospitalization (Hospitalization; n=37); intensive care (ICU; n=16); mechanical ventilation (Ventilation; n=22); or fatality (Deceased; n=22). Result: Study 1. Covid-19 samples showed considerable thrombin generation variability with some samples failing to generate thrombin; pathway selective inhibitors reduced thrombin generation while heparinase treatment increased thrombin generation. Upon analysis, thrombin generation parameters showed no significant correlations to either D-dimer levels or disease severity. Instead, plasma prepared from blood collected directly into corn trypsin inhibitor revealed that contact activation that occurred post-sample collection dominates procoagulant activity. Study 2. Figure 1, shows EV TF activities, D-dimer and TFPIα levels obtained for Covid-19 samples, with data segregated based on disease severity outcomes. Statistically significant difference versus the Hospitalized group are shown. TFPIa levels were highest in heparin IV patients (24.4+1.5 nM) vs Heparin-SQ (12.8+0.9 nM) vs enoxaparin (10.8 +0.7 nM) (p value:<0.0001). It is known that heparin treatment increases circulating TFPIα, however an increase in TFPIα might also further increase circulating TF/FVIIa/XaTFPI inhibitory complex, which would dissociate in citrated plasma, and might account for the increase in EV TF in other studies. Conclusions: Contact activation that occurs post-sample collection is sufficient to obscure endogenous triggers of coagulation, if present, in Covid-19 patients' plasma. D-dimer and TFPIα strongly correlate with disease severity although the latter is likely affected by heparin treatment. The most severe Covid-19 patients with high D-dimer did not show detectible plasma EV TF activity. Plasma EV TF activity does not appear to adequately represent the mechanism responsible for elevated D-dimer levels in Covid-19 cases. Figure 1 Figure 1. Disclosures Di Paola: CSL Behring: Consultancy, Honoraria.

Immune Checkpoint Inhibitors (ICI) Enhance Cancer Associated Thrombosis (CAT) in Murine Model of Colon Carcinoma

Abstract Background: Venous thromboembolism (VTE) is an important immune-related adverse event (irAE) associated with immune checkpoint inhibitors (ICI) cancer therapy. To better understand the pathogenesis of ICI-induced VTE during ICI treatment, we utilized a murine model of cancer associated thrombosis (CAT) to examine the impact of ICI treatment on the development of flow restriction-induced thrombosis. Methods A syngeneic colon carcinoma murine model (CT26) on BALB/c background was utilized to evaluate venous thrombosis following inferior vena cava (IVC) ligation. Non-tumor-bearing and tumor-bearing animals were treated with therapeutic doses of ICI: anti-programmed cell death receptor 1 antibody (anti-PD1) and cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4) or isotype control antibodies. Mice underwent surgical treatment for IVC ligation followed by surgical retrieval of thrombus. Western blot analysis was performed on plasma cleaved high molecular weight kininogen (cHK), and citrullinated histone H3 (CitH3). Results Tumor-bearing mice undergoing IVC ligation after anti-PD1 and anti-CTLA4 infusion developed larger thrombi compared to mice treated with isotype control IgG. Thrombus weights in CT26 tumor-bearing mice treated with ICI (19.56±4.41 mg) were significantly increased compared to those in mice treated with isotype control IgG (14.67±2.76 mg) (P=0.043). The weight of thrombi in non-tumor-bearing mice was not affected by ICI treatment (9.25±2.22 mg with control IgG vs 9.33±1.15 mg with ICI). In addition, there was a significant increase in thrombus length in mice treated with ICI (9±1.02 mm vs 7.61±0.99 mm with IgG, P=0.039). Cleaved high molecular weight kininogen (cHK), an indicator of contact activation was increased in plasma pre-IVC occlusion from ICI-treated mice compared to IgG-treated mice (68% vs 38% in HK cleavage). Citrullinated histone H3 (CitH3), a NETosis marker, was elevated in plasma (and in thrombus) post-IVC occlusion from ICI-treated mice compared to that of IgG-treated mice. Conclusions ICI treatment of tumor-bearing mice undergoing flow restriction-induced thrombosis resulted in enhanced clot formation. Larger thrombi in ICI treated mice was accompanied with enhanced contact activation and NETosis marker CitH3. Figure 1 Figure 1. Disclosures Khorana: Halozyme: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Anthos: Consultancy, Honoraria. McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria.

Predictive Value for Increased Factor XIa and Plasma Kallikrein Activity in Acute Venous Thromboembolism

Venous thromboembolism (VTE) is associated with increased coagulation activity, which in part can be attributed to the contact pathway of coagulation. Evidence from pre-clinical and epidemiological studies suggests that deficiency in factors of contact activation (e.g. coagulation factors (F) XI and FXII) protects against VTE. However, limited information exists regarding the activation of the contact system in the setting of acute VTE. In the current study, patients with confirmed VTE events (n=321) from the VTEval study and controls (n=300) from the population-based PREVENT-it pilot study were included. Plasma samples were collected from patients after confirmed VTE events or controls upon inclusion in the study. FXI as well as FXIa and plasma kallikrein (PKa) levels were assessed in plasma samples from all subjects using an activated thromboplastin time-based assay (FXI:c), a thrombin generation-based assay (CAT:FXIa) and by measuring inhibitory complexes (FXIa:antithrombin (AT), FXIa:alpha-1-antitrypsin (α1AT), FXIa:C1 esterase inhibitor (C1Inh) and PKa:C1Inh) using enzyme-linked immunoassay (ELISA). After a 2-year follow up period, a composite endpoint of recurrent VTE or death was determined. Increased FXI:c levels were determined in VTE patients compared to control individuals (124.08 ± 37.48% vs. 113.55 ± 27.99%), whereas CAT:FXIa levels were reduced in VTE patients (0 pM [IQR, 0-0.56] vs 0.56 pM [IQR, 0-0.88]). Levels of FXIa:α1AT and FXIa:AT inhibitory complexes were increased in VTE patients compared to controls (median[IQR]; 311.8 pM [238.2-424.0] vs. 202.5 pM [143.7 - 287.5] and 29.1 pM [23.4-38.3] vs 23.2 pM [19.7-29.8], respectively). Considering that 86% of the VTE patients were already on anticoagulant treatment (Table 1), investigation of their possible effect on the biomarkers revealed that only the CAT:FXIa was influenced by the presence of anticoagulants. Logistic regression models revealed a good discriminatory value for FXI:c and FXIa:α1AT (AUC=0.64 [0.6/0.69] and AUC=0.67 [0.62/0.71], respectively) to distinguish VTE from controls, whereas the other biomarkers were not able to distinguish between groups. The outcome recurrent VTE or death could be predicted by the inhibitory complexes, but not by the FXI(a) levels (Figure 1). Only the FXIa:α1AT complexes were able to both detect the presence of VTE (OR per SD [95%CI]: 1.28 [1.01-1.63], p=0.04) and predict recurrent VTE or death (HR per SD [95%CI]: 1.40 [1.2-1.62], p<0.0001). In summary, acute VTE is associated with both elevated FXI:c levels and increased activation of FXI and plasma prekallikrein, the latter specifically indicating contact activation. The generation of FXIa during acute VTE and its association with recurrent VTE suggests an important risk contribution of FXI activation. This study has added evidence favouring the utility of FXIa inhibition in the setting of acute VTE. Figure 1 Figure 1. Disclosures Knoeck: Bayer AG: Consultancy. ten Cate: Bayer AG: Other; Pfizer: Other; LEO Pharma: Other; Gideon Pharmaceuticals: Other; Alveron Pharma: Other. Wild: Bayer AG: Other, Research Funding; Boehringer Ingelheim: Other, Research Funding; Novartis Pharma: Other, Research Funding; Sanofi-Aventis: Other, Research Funding; Astra Zeneca: Other, Research Funding; Daiichi Sankyo Europe: Other, Research Funding.

Genetic Profile of Endotoxemia Reveals an Association With Thromboembolism and Stroke

Background Translocation of lipopolysaccharide from gram‐negative bacteria into the systemic circulation results in endotoxemia. In addition to acute infections, endotoxemia is detected in cardiometabolic disorders, such as cardiovascular diseases and obesity. Methods and Results We performed a genome‐wide association study of serum lipopolysaccharide activity in 11 296 individuals from 6 different Finnish study cohorts. Endotoxemia was measured by limulus amebocyte lysate assay in the whole population and by 2 other techniques (Endolisa and high‐performance liquid chromatography/tandem mass spectrometry) in subpopulations. The associations of the composed genetic risk score of endotoxemia and thrombosis‐related clinical end points for 195 170 participants were analyzed in FinnGen. Lipopolysaccharide activity had a genome‐wide significant association with 741 single‐nucleotide polymorphisms in 5 independent loci, which were mainly located at genes affecting the contact activation of the coagulation cascade and lipoprotein metabolism and explained 1.5% to 9.2% of the variability in lipopolysaccharide activity levels. The closest genes included KNG1 , KLKB1 , F12 , SLC34A1 , YPEL4 , CLP1 , ZDHHC5 , SERPING1 , CBX5 , and LIPC . The genetic risk score of endotoxemia was associated with deep vein thrombosis, pulmonary embolism, pulmonary heart disease, and venous thromboembolism. Conclusions The biological activity of lipopolysaccharide in the circulation (ie, endotoxemia) has a small but highly significant genetic component. Endotoxemia is associated with genetic variation in the contact activation pathway, vasoactivity, and lipoprotein metabolism, which play important roles in host defense, lipopolysaccharide neutralization, and thrombosis, and thereby thromboembolism and stroke.

Polyphosphate Expression by Cancer Cell Extracellular Vesicles Mediates Binding of Factor XII and Contact Activation

Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EV are elevated in cancer, and studies suggest that EV may stimulate thrombosis in cancer patients through expression of tissue factor. However, limited data also implicates EV in activation of the contact pathway of coagulation through activation of factor XII (FXII) to factor XIIa (FXIIa). To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with activation of FXII, EV induced the cleavage of high molecular weight kininogen to cleaved kininogen. We also observed that EV from cancer patients stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or E. coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EV and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by deficiency of FXII, HK, or prekallikrein. Moreover, pre-treatment of EV with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates binding of contact factors to EV, and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.

Mechanism, Functions, and Diagnostic Relevance of FXII Activation by Foreign Surfaces

Factor XII (FXII) is a serine protease zymogen produced by hepatocytes and secreted into plasma. The highly glycosylated coagulation protein consists of six domains and a proline-rich region that regulate activation and function. Activation of FXII results from a conformational change induced by binding (“contact”) with negatively charged surfaces. The activated serine protease FXIIa drives both the proinflammatory kallikrein–kinin pathway and the procoagulant intrinsic coagulation cascade, respectively. Deficiency in FXII is associated with a prolonged activated partial thromboplastin time (aPTT) but not with an increased bleeding tendency. However, genetic or pharmacological deficiency impairs both arterial and venous thrombosis in experimental models. This review summarizes current knowledge of FXII structure, mechanisms of FXII contact activation, and the importance of FXII for diagnostic coagulation testing and thrombosis.