Exosome Polyphosphate Mediates the Activation of FXII By Cancer Cell-Derived Exosomes

Introduction Cancer-associated thrombosis (CAT) affects up to 20% of patients with cancer, and causes substantial morbidity and mortality. Although tissue factor has been most studied, several other mechanisms contribute to the development of CAT. Recently, a role for the contact activation pathway in pathologic thrombosis and in CAT has been recognized. We have observed that the cleaved form of high molecular weight kininogen (cHK), an a priori demonstration of contact activation, circulates in plasma in many patients with active cancer. Contact activation is initiated by the activation of factor XII (FXII) to FXIIa, and our studies as well as those of others suggest that circulating extracellular vesicles (EV) in cancer patients may mediate FXII activation. Polyphosphate (PP) is a known FXII activator, although other biological material such as RNA and DNA may also stimulate contact activation. Therefore, to better define the mechanisms of cancer EV-mediated FXII activation, we characterized the ability of exosomes, defined as EV of < 150 nm in size, to stimulate FXII activation in normal human plasma. Methods Exosomes were isolated using a multistep ultrafiltration method from HDF (human dermal fibroblast), LC3.6 (pancreatic cancer), HT29 (colorectal cancer), H1975 (non-small cell lung cancer), and U937 (monocytic lymphoma) cell lines. The quality of the exosome preparation was assessed using electron microscopy (Fig. A). Exosomes were quantified by measuring protein concentration (DC™ Protein Assay Kit II, Bio-Rad 5000112), and equal amounts of exosome, as judged by protein content, were added to citrated normal human plasma. FXIIa generation in plasma was quantified colorimetrically using the substrate H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroaniline dihydrochloride (S-2302). Dextran sulfate was used as a positive control to induce FXII activation. Briefly, different amounts of exosomes, as determined by protein content (100 ng to 1 microgram), were added normal human plasma containing S-2302, after which the A405 was recorded. A standard curve was prepared by adding known amounts of FXIIa to the same plasma in parallel. Formed FXIIa activity induced by the exosomes was characterized by the relative purified FXIIa activity in the standard curve. Additionally, the generation of cHK also was assessed in exosome-activated plasma. Result Exosomes derived from cancer cell lines activated FXII in a manner concordant with the relative prothrombotic risk of these tumors, i.e. LC3.6 > HT-29 > H1975 > U937 = HDF (Fig. B). Coincident with the activation of FXII to FXIIa, exosomes induced the cleavage of high molecular weight kininogen to cHK in a dose-dependent manner (Fig. C). Since the mechanisms by which exosomes induce FXII activation have not been characterized, we assessed the roles of polyphosphate (PP), RNA and DNA expressed on the exosome surface. Pretreatment of exosomes from cancer cells with calf intestinal alkaline phosphatase (CIP), but not RNase or DNase, inhibited the ability of these exosomes to initiate FXII activation in a dose-dependent manner (Fig D). The maximal effect occurred at a concentration of CIP of 20 U/ml, and the relative reduction in FXII activating activity by exosomes from a specific cellular source was proportional to the maximal activity of the exosome preparation before treatment. A decrease in exosome PP content lead to a parallel reduction in FXII activating ability. (Fig.E). Conclusion These studies demonstrate that exosomes derived from both primary, non-transformed cells (HDF), as well as cancer cell lines have the ability to support FXII autoactivation in human plasma. Their ability to do is proportional to the relative risk of thrombosis associated with cancers derived from their parental cell lines. Treatment of exosomes with CIP substantially reduces the FXII activating capacity of those derived from the tested cancer cell lines and HDF. Human plasma exosomal PP is a constitutive potential source for contact activation that may be increased in cancer patients. Its relative contribution to thrombin generation via FXII activation versus that of tissue factor remains to be determined in individuals and in cancer types. However, neutralization of exosomal PP provides an additional pathway for prevention of cancer-associated thrombosis. Disclosures Schmaier: Biomotiv: Consultancy; Alnylam: Research Funding; Enzyme Research Laboratories: Honoraria; Shire: Consultancy, Honoraria, Research Funding; Temple University: Patents & Royalties; Cleveland Clinic Foundation: Research Funding. Khorana:Pfizer: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Bayer: Consultancy.

The Serpins C1-Inhibitor and Antithrombin Synergistically Regulate the Catalytic Life of Activated Factor XI in Healthy Individuals and Thrombophilic Patients

Background: The prothrombotic potential of coagulation factor XI (FXI) is demonstrated by the hypercoagulable state associated with increased plasma levels of FXI and by the risk of thromboembolic events of FXI concentrates. Although inhibition of activated FXI (FXIa) by antithrombotic drugs is emerging as a new approach in anticoagulation, the knowledge about physiological inhibitory mechanisms, which are involved in the control of the activity of FXIa, is limited. Methods: To evaluate the impact of the SERPINs antithrombin (AT) and C1-inhibitor (C1-INH) on the catalytic life of FXIa, we analyzed inactivation patterns of exogenously added FXIa in normal human plasma, in plasma deficient for the FXIa inhibitors C1-INH and AT, and in a purified system containing different levels of these inhibitors. FXIa was added to the respective sample matrix with a final concentration of 10 ng/mL and endogenous FXIa inactivation stopped by addition of benzamidine. Subsequently, the residual amount of FXIa was quantified by an enzyme capture assay using a monoclonal antibody to capture FXIa. To further evaluate the clinical impact of FXIa inhibitor levels on thrombotic risk, plasma levels of AT, C1-INH, and of the FXIa inhibitors α1-antitrypsin and α2-antiplasmin were measured in a cohort of 98 thrombophilic patients and compared with matched healthy controls. Results: The FXIa inhibition assay demonstrated coefficients of intra- and inter-assay variation of 13.2% and 15.2%, respectively. The catalytic half-life of FXIa in normal human plasma was 133.8 ± 18.8 s (mean ± SD). FXIa half-life times prolonged to 251.6 ± 29.4 s in plasma with decreased activity levels of C1-INH of 25% and to 175.7 ± 3.7 s in AT deficient plasma. After addition of plasma purified C1-INH or AT, FXIa half-life times shortened in a concentration-dependent manner. The dependence of the FXIa inactivation rate on levels of C1-INH and AT was further demonstrated in a purified system as shown in Fig. 1. The observation that AT and C1-INH additively control the catalytic life of FXIa prompted us to measure FXIa inhibitor levels in thrombophilic patients, as a combination of critically low inhibitor levels might constitute a thrombotic risk factor. However, plasma levels of more than one inhibitor below the normal range were not observed in the thrombophilic patients of our study population. Recently, the prothrombotic potential of FXIa was further supported by the detection of residual amounts of FXIa in therapeutic immunoglobulin preparations (IVIG) associated with thromboembolic events. In our system even the presence of high concentrations of IVIG did not affect FXIa inactivation kinetics, making it most unlikely that the prothrombotic potential of IVIG results from impaired FXIa clearance. Conclusion: The approach utilized in this study allows sensitive and reproducible monitoring of the catalytic life of FXIa in biological sample matrices. The data obtained demonstrate the significant and synergistic contribution of AT and C1-INH to FXIa inactivation in plasma. Critically decreased FXIa inhibitor levels are not a frequent finding in thrombophilic patients. Fig. 1 C1-INH- and AT-dependence of FXIa inactivation rates. Inhibitor levels equivalent to physiological levels in human plasma are referred to as 100%. Fig. 1. C1-INH- and AT-dependence of FXIa inactivation rates. Inhibitor levels equivalent to physiological levels in human plasma are referred to as 100%. Disclosures Rühl: Bayer, CSL-Behring: Honoraria, Research Funding. Oldenburg:Baxter, Bayer, Biogen Idec, Biotest, CSL-Behring, Grifols, Novo Nordisk, Octapharma, Swedish Orphan Biovitrum and Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

A Biomarker Assay For The Detection Of Contact System Activation

The contact system is composed of circulating plasma proteins that can both initiate coagulation through the intrinsic pathway and promote inflammation through the generation of bradykinin. Activation of the contact system is mediated by the assembly of factor XII (FXII), high molecular weight kininogen (HMWK), and prekallikrein (inactive form of the serine protease plasma kallikrein [pKal]) on a negatively charged surface. Bradykinin is produced from contact system activation by the proteolytic action of active pKal on its substrate HMWK. In vivo, vascular tissue injury or other unknown precipitants may expose negatively charged surfaces capable of supporting contact system activation. In vitro, the contact system is activated following exposure of plasma to substances such as kaolin, dextran sulfate, phospholipids, or surfaces such as glass. We developed a Western blot assay to detect the presence of both intact and cleaved HMWK in human and cynomolgus monkey plasma samples. Using this assay we showed that endogenous HMWK in normal human plasma was converted to cleaved kininogen following the addition of contact system activators, such as kaolin, dextran sulfate, or ellagic acid. HMWK was also shown to be cleaved by the addition of trace amounts of either FXIIa or pKal to normal human plasma. The utility of the Western blot assay was further demonstrated using plasma samples from patients with hereditary angioedema with C1 inhibitor (C1-INH) deficiency (HAE-C1-INH). HAE-C1-INH is a rare, autosomal dominant disease caused by mutations in one of the two alleles of the C1-INH gene (SERPING1). C1-INH is a serine protease inhibitor that blocks the activity of FXIIa and pKal in the contact system. HAE-C1-INH patients suffer from recurrent, localised angioedema attacks due to episodic activation of the contact system arising from unregulated pKal activity, which cleaves HMWK to release bradykinin, a potent vasoactive peptide mediating edema formation. Our results demonstrate that the method could detect significant contact system activation in HAE patient samples collected between attacks (i.e. quiescent disease samples) as compared to normal human plasma. Plasma samples collected during an HAE-C1-INH attack exhibited further elevations in cleaved kininogen. We also measured contact activation in plasma samples from cynomolgus monkeys that were dosed subcutaneously (SC) with DX-2930, a potent and specific monoclonal antibody inhibitor of plasma kallikrein being developed for HAE-C1-INH. DX-2930 blocked the cleavage of HMWK induced by kaolin-mediated contact system activation in a dose-dependent manner (5, 25, or 50 mg/Kg DX-2930, SC). In summary, this study presents a simple method that can be used both to identify disease states associated with contact system activation, and to serve as a pharmacodynamic biomarker for the development of DX-2930 as a treatment for HAE-C1-INH. Disclosures: Faucette: Dyax Corp: Employment. Cicardi:Dyax Corp: Consultancy. Kenniston:Dyax Corp: Employment. Sexton:Dyax Corp: Employment. Adelman:Dyax Corp: Employment.

Zymogen-Like FXa Is An Effective Pro-Hemostatic To Reverse The Anticoagulant Effects Of Direct FXa Inhibitors

Oral anticoagulants are the mainstay of treatment for prothrombotic disorders. The emerging oral factor Xa (FXa) inhibitors, which include rivaroxaban and apixaban, have been shown to be highly effective anticoagulants in several clinical scenarios, including venous thromboembolism and non-valvular atrial fibrillation. Compared to warfarin, direct FXa inhibitors have less variable pharmacokinetics, may not require routine monitoring of coagulation parameters, and have comparable to a somewhat lower bleeding risk. Despite these advantages, no approved strategy has been developed to reverse the anticoagulant effects of these drugs in the event of life-threatening bleeding or emergent need for surgery. This represents an urgent unmet clinical need. Our group has recently developed a panel of FXa mutants that are more zymogen-like than wild-type (wt)-FXa. These “zymogen-like” FXa variants have lower activity in in vitro assays compared to wt-FXa due to impaired active site maturation. Furthermore, the variants have longer plasma half-lives (>30 minutes) in vitro compared to wt-FXa (1-2 minutes) due to diminished reactivity with antithrombin III (ATIII) and tissue factor pathway inhibitor (TFPI). Remarkably however, binding to FVa rescues the activity of these zymogen-like FXa variants and as a result they are highly effective procoagulants in vivo in the setting of hemophilia (Nat. Biotech; 2011, 29:1028-33). We hypothesized that these variants could also be effective procoagulants to overcome the effects of direct FXa inhibitors. Furthermore, since direct FXa inhibitors bind the FXa active site, we expect them to compete with ATIII and TFPI for FXa binding and prolong their half-lives. We tested both of these hypotheses in in vitro coagulation studies and in vivo hemostasis models. Rivaroxaban dose-dependently inhibited thrombin generation in thrombin generation assays (TGA) when added to normal human plasma. Specifically, 500 nM rivaroxaban, the expected therapeutic steady-state plasma concentration, decreased peak thrombin generation to ∼10% of normal, and addition of 3 nM of the FXa zymogen-like variant FXaI16L restored peak thrombin generation to 105% of normal. Higher concentrations of rivaroxaban (2.5 µM) completely abrogated thrombin generation in this assay, but 10 nM FXaI16L restored thrombin generation to 72% of normal under these conditions. We compared these data to results obtained with other proposed reversal strategies. Gla-domainless, catalytically inactive FXa (GD-FXaS195A), which has been shown to reverse the effects of rivaroxaban by scavenging the inhibitor, restored thrombin generation in the presence of 500 nM rivaroxaban, but required high concentrations (1 µM; >300-fold greater than FXaI16L) to be effective. In addition, activated prothrombin complex concentrates (FEIBA), which have been shown to have some ex vivo efficacy, were ineffective under our assay conditions. In tail-clip hemostasis studies in mice, rivaroxaban dose-dependently increased blood loss, with 50 mg/kg rivaroxaban resulting in 217% of normal blood loss. Addition of FXaI16L (200 mg/kg) reduced rivaroxaban-induced blood loss to 141% of normal. To examine the effect of rivaroxaban on the half-life of FXa, we pre-incubated FXaI16L or wt-FXa with or without rivaroxaban in normal human plasma and then performed TGA experiments after various incubation times. When wt-FXa or FXaI16L were pre-incubated in plasma in the absence of rivaroxaban, their half-lives were 4.6 minutes and 1.37 hours, respectively. Remarkably, when wt-FXa or FXaI16L were incubated in plasma in the presence of 500 nM rivaroxaban, their respective half-lives were prolonged to 9.4 hours (123-fold increase) and 18.1 hours (13.2-fold increase). These results suggest that a zymogen-like FXa variant, FXaI16L, can reverse the effects of rivaroxaban in vitro and in vivo. Furthermore, FXaI16L is a bypassing agent that only requires catalytic amounts of protein, in contrast to scavengers or “true” antidotes like GD-FXaS195A that require stoichiometric concentrations. This indicates that much lower quantities of FXaI16L may be effective in vivo. We also showed that rivaroxaban dramatically prolongs the half-life of FXa in plasma, possibly by competing with ATIII and TFPI for FXa binding. This work provides a starting point for the development of a long half-life reversal strategy for the emerging FXa inhibitors. Disclosures: Patel-Hett: Pfizer: Employment. Jasuja:Pfizer: Employment. Fruebis:Pfizer: Employment. Pittman:Pfizer: Employment. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; Alnylam: Consultancy.

Different Effects of Plasma from Patients with Acute on Chronic Liver Failure on the Proliferation and Biotransformation Function of C3A Cells

Objective To investigate the effects of plasma from patients with acute on chronic liver failure on the proliferation and biotransformation function of C3A cells in vitro, and provide experimental data for C3A cells to be efficiently used in the bioartificial liver system. Methods C3A cells were incubated in 100% normal human plasma (NHP) and 100% abnormal plasma (AP) from patients with acute on chronic liver failure. Growth morphology of the two groups were observed under inverted microscope and scanning electron microscope. The method of methyl thiazolyl tetrazolium (MTT) was conducted for the proliferation activities of C3A cells. The cellular apoptosis rates were assessed by the flow cytometer. The biotransformation function of cells was evaluated through diazepam metabolic amount assay. The concentrations of epithelial growth factor (EGF), transforming growth factor-α (TGF-α) and interleukin-1 (IL-1) were detected in plasma of the two groups. Results A: The proliferation activities of C3A cells incubated in 100% AP for 24, 48, 72, 96 and 120 hours were significantly higher than that in 100% NHP (P < 0.01). B: Observation under the inverted microscope indicated that the cells in 100% AP were growing faster than those in 100% NHP after cells attached to the plastic at 24 and 48 hours. The same phenomena was observed under the scanning electronic microscope. C: The C3A cells cultured in both groups of plasma showed the same apoptosis rate at 48 hours and there was no statistical difference. D: The diazepam metabolic value of C3A cells incubated in 100% AP for 24, 72 and 120 hours were lower than that in 100% NHP and were statistically different (P < 0.01). E: The concentrations of TGF-α, EGF and IL-1 in AP were significantly higher than that in NHP (P < 0.01). Conclusions Compared with normal human plasma, the plasma from patients with acute on chronic liver failure has more obvious effect to facilitate the proliferation of C3A cells, but also decreases partial biotransformation function of C3A cells.

Thrombin inhibition profiles in healthy individuals and thrombophilic patients

SummaryInhibition of thrombin by endogenous inhibitors plays a central role in the spatiotemporal control of clot formation. A failure to adequately inactivate thrombin such as in antithrombin deficiency generates a strong prothrombotic phenotype. To study if and to what extent delayed thrombin inactivation rates beyond antithrombin deficiency contribute to the prothrombotic phenotype we measured thrombin inhibition profiles in plasma samples obtained from 16 healthy individuals and 39 thrombophilic patients, including 17 patients diagnosed positive for anti-prothrombin/phospholipid antibodies. To test thrombin inhibition, thrombin was added to plasma, and endogenous thrombin inhibition stopped by addition of the reversible thrombin inhibitor argatroban. Subsequently, the amount of argatroban-complexed thrombin was measured using an oligonucleotide-based enzyme capture assay. In normal human plasma thrombin at concentrations up to 4 ng/ml (109 pM) became inactivated with an average half-life time of 56.4 ± 4.7 seconds (s). In antithrombin-deficient plasma the thrombin half-life was prolonged to 168.2 ± 14.9 s. Among the thrombophilic patients, only one with mild antithrombin deficiency showed impaired thrombin inactivation rates, whereas all other patients including the antiphospholipid positive patients showed thrombin inhibiting capacities within the normal range. We conclude that thrombin added to normal human plasma at subthreshold levels of ∼100 pM or below becomes inactivated with a half-life time below 1 minute. Antiphospholipid antibodies do not prolong thrombin half-life times, making it unlikely that delayed thrombin inactivation contributes to the thrombotic phenotype of the antiphospholipid syndrome. In contrast, plasma levels of antithrombin falling below 80% of normal markedly prolong the thrombin half-life.