Genetic duplication of tissue factor reveals subfunctionalization in venous and arterial hemostasis.

Tissue factor (TF) is an evolutionarily conserved protein necessary for initiation of hemostasis. Zebrafish have two copies of the tissue factor gene (f3a and f3b) as the result of an ancestral teleost fish duplication event (so called ohnologs). In vivo physiologic studies of TF function have been difficult given early lethality of TF knockout in the mouse. We used genome editing to produce knockouts of both f3a and f3b in zebrafish. Since ohnologs arose through sub- or neofunctionalization, they can unmask unknown functions of non-teleost genes and could reveal whether mammalian TF has developmental functions distinct from coagulation. Here we show that a single copy of either f3a or f3b is necessary and sufficient for normal lifespan. Complete loss of TF results in lethal hemorrhage by 2-4 months despite normal embryonic and vascular development. Larval vascular endothelial injury reveals predominant roles for TFa in venous circulation and TFb in arterial circulation. Finally, we demonstrate that loss of TF predisposes to a stress-induced cardiac tamponade independent of its role in fibrin formation. Overall, our data suggest partial subfunctionalization of TFa and TFb. This multigenic zebrafish model has the potential to facilitate study of the role of TF in different vascular beds.

Phosphatidylthreonine is a procoagulant lipid detected in human blood and elevated in coronary artery disease.

Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography-tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.

Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo.

Platelets play a central role in thrombosis, hemostasis, and inflammation. We show that activated platelets release inorganic polyphosphate (polyP), a polymer of 60-100 phosphate residues that directly bound to and activated the plasma protease factor XII. PolyP-driven factor XII activation triggered release of the inflammatory mediator bradykinin by plasma kallikrein-mediated kininogen processing. PolyP increased vascular permeability and induced fluid extravasation in skin microvessels of mice. Mice deficient in factor XII or bradykinin receptors were resistant to polyP-induced leakage. PolyP initiated clotting of plasma via the contact pathway. Ablation of intrinsic coagulation pathway proteases factor XII and factor XI protected mice from polyP-triggered lethal pulmonary embolism. Targeting polyP with phosphatases interfered with procoagulant activity of activated platelets and blocked platelet-induced thrombosis in mice. Addition of polyP restored defective plasma clotting of Hermansky-Pudlak Syndrome patients, who lack platelet polyP. The data identify polyP as a new class of mediator having fundamental roles in platelet-driven proinflammatory and procoagulant disorders.

Lactadherin's Multistate Binding Predicts Stable Membrane-Bound Conformations of Factors V and VIII's C Domains.

Protein binding to negatively charged lipids is essential for maintaining numerous vital cellular processes where its dysfunction can lead to various diseases. One such protein that plays a crucial role in this process is lactadherin, which competes with coagulation factors for membrane binding sites to regulate blood clotting. Despite identifying key binding regions of these proteins through structural and biochemical studies, models incorporating membrane dynamics are still lacking. In this study, we report on the multimodal binding of lactadherin and use it to gain insight into the binding mechanisms of its C domain homologs, factor V and factor VIII. Molecular dynamics simulations enhanced with the highly mobile mimetic model enabled the determination of lactadherin's multimodal binding on membranes that revealed critical interacting residues consistent with prior NMR and mutagenesis data. The binding occurred primarily via two dynamic structural ensembles: an inserted state and an unreported, highly conserved side-lying state driven by a cationic patch. We utilized these findings to analyze the membrane binding domains of coagulation factors V and VIII and identified their preferred membrane-bound conformations. Specifically, factor V's C domains maintained an inserted state, while factor VIII preferred a tilted, side-lying state that permitted antibody binding. Insight into lactadherin's atomistically resolved membrane interactions from a multistate perspective can guide new therapeutic opportunities in treating diseases related to blood coagulation.

Inhibitors of Polyphosphate and Neutrophil Extracellular Traps.

The contact pathway of blood clotting has received intense interest in recent years as studies have linked it to thrombosis, inflammation, and innate immunity. Because the contact pathway plays little to no role in normal hemostasis, it has emerged as a potential target for safer thromboprotection, relative to currently approved antithrombotic drugs which all target the final common pathway of blood clotting. Research since the mid-2000s has identified polyphosphate, DNA, and RNA as important triggers of the contact pathway with roles in thrombosis, although these molecules also modulate blood clotting and inflammation via mechanisms other than the contact pathway of the clotting cascade. The most significant source of extracellular DNA in many disease settings is in the form of neutrophil extracellular traps (NETs), which have been shown to contribute to incidence and severity of thrombosis. This review summarizes known roles of extracellular polyphosphate and nucleic acids in thrombosis, with an emphasis on novel agents under current development that target the prothrombotic activities of polyphosphate and NETs.

Molecular View into Preferential Binding of the Factor VII Gla Domain to Phosphatidic Acid.

Factor VII (FVII) is a serine protease with a key role in initiating the coagulation cascade. It is part of a family of vitamin K-dependent clotting proteins, which require vitamin K for formation of their specialized membrane-binding domains (Gla domains). Membrane binding of the FVII Gla domain is critical to the activity of FVII, mediating the formation of its complex with other clotting factors. While Gla domains among coagulation factors are highly conserved in terms of amino acid sequence and structure, they demonstrate differential binding specificity toward anionic lipids. Although most Gla domain-containing clotting proteins display a strong preference for phosphatidylserine (PS), it has been demonstrated that FVII and protein C instead bind preferentially to phosphatidic acid (PA). We have developed the first model of the FVII Gla domain bound to PA lipids in membranes containing PA, the highly mobile membrane mimetic model, which accelerates slow diffusion of lipids in molecular dynamics simulations and therefore facilitates the membrane binding process and enhances sampling of lipid interactions. Simulations were performed using atomic level molecular dynamics, requiring a fixed charge to all atoms. The overall charge assigned to each PA lipid for this study was -1. We also developed an additional model of the FVII Gla domain bound to a 1:1 PS/PC membrane and compared the modes of binding of PS and PA lipids to FVII, allowing us to identify potential PA-specific binding sites.

Influence of Steric Shield on Biocompatibility and Antithrombotic Activity of Dendritic Polyphosphate Inhibitor.

The polyanion, inorganic polyphosphate (polyP), is a procoagulant molecule which has become a promising therapeutic target in the development of antithrombotics. Neutralizing polyP's prothrombotic activity using polycationic inhibitors is one of the viable strategies to design new polyP inhibitors. However, in this approach, a fine balance between the electrostatic interaction of polyP and the inhibitor is needed. Any unprotected polycations are known to interact with negatively charged blood components, potentially resulting in platelet activation, cellular toxicity, and bleeding. Thus, designing potent polycationic polyP inhibitors with good biocompatibility is a major challenge. Building on our previous research on universal heparin reversal agent (UHRA), we report polyP inhibitors with a modified steric shield design. The molecular weight, number of cationic binding groups, and the length of the polyethylene glycol (PEG) chains were varied to arrive at the desired inhibitor. We studied two different PEG lengths (mPEG-750 versus mPEG-350) on the polyglycerol scaffold and investigated their influence on biocompatibility and polyP neutralization activity. The polyP inhibitor with mPEG-750 brush layer, mPEG750 UHRA-10, showed superior biocompatibility compared to its mPEG-350 analogs by a number of measured parameters without losing its neutralization activity. An increase in cationic binding groups (25 groups in mPEG750 UHRA-8 and 32 in mPEG750 UHRA-10 [HC]) did not alter the neutralization activity, which suggested that the mPEG-750 shield layer provides significant protection of cationic binding groups and thus helps to minimize unwanted nonspecific interactions. Furthermore, these modified polyP inhibitors are highly biocompatible compared to conventional polycations that have been previously used as polyP inhibitors (e.g., PAMAM dendrimers and polyethylenimine). Through this study, we demonstrated the importance of the design of steric shield toward highly biocompatible polyP inhibitors. This approach can be exploited in the design of highly biocompatible macromolecular inhibitors.

Biotechnological synthesis of water-soluble food-grade polyphosphate with Saccharomyces cerevisiae.

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2-40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO3 , in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, 31 P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP.

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids.

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Alteration of blood clotting and lung damage by protamine are avoided using the heparin and polyphosphate inhibitor UHRA.

Anticoagulant therapy-associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis.

Factor XII Activation Promotes Platelet Consumption in the Presence of Bacterial-Type Long-Chain Polyphosphate In Vitro and In Vivo.

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose100 Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.

Calcium-Induced Lipid Nanocluster Structures: Sculpturing of the Plasma Membrane.

The plasma membrane of the cell is a complex, tightly regulated, heterogeneous environment shaped by proteins, lipids, and small molecules. Ca2+ ions are important cellular messengers, spatially separated from anionic lipids. After cell injury, disease, or apoptotic events, anionic lipids are externalized to the outer leaflet of the plasma membrane and encounter Ca2+, resulting in dramatic changes in the plasma membrane structure and initiation of signaling cascades. Despite the high chemical and biological significance, the structures of lipid-Ca2+ nanoclusters are still not known. Previously, we demonstrated by solid-state nuclear magnetic resonance (NMR) spectroscopy that upon binding to Ca2+, individual phosphatidylserine lipids populate two distinct yet-to-be-characterized structural environments. Here, we concurrently employ extensive all-atom molecular dynamics (MD) simulations with our accelerated membrane mimetic and detailed NMR measurements to identify lipid-Ca2+ nanocluster conformations. We find that major structural characteristics of these nanoclusters, including interlipid pair distances and chemical shifts, agree with observable NMR parameters. Simulations reveal that lipid-ion nanoclusters are shaped by two characteristic, long-lived lipid structures induced by divalent Ca2+. Using ab initio quantum mechanical calculations of chemical shifts on MD-captured lipid-ion complexes, we show that computationally observed conformations are validated by experimental NMR data. Both NMR measurements of diluted specifically labeled lipids and MD simulations reveal that the basic structural unit that reshapes the membrane is a Ca2+-coordinated phosphatidylserine tetramer. Our combined computational and experimental approach presented here can be applied to other complex systems in which charged membrane-active molecular agents leave structural signatures on lipids.

Polyphosphate and RNA Differentially Modulate the Contact Pathway of Blood Clotting.

The contact pathway of the plasma clotting cascade is dispensable for normal hemostasis, but contributes to thrombosis and serves as a bridge between inflammation and coagulation. This pathway is triggered upon exposure of plasma to certain anionic polymers and artificial surfaces. Recently, extracellular nucleic acids and inorganic polyphosphate (polyP) have been implicated as being important (patho)physiologically relevant activators of this pathway. However, mechanistic details regarding how nucleic acids or polyP modulate the individual reactions of the contact pathway have been lacking. In this study, we investigate the ability of RNA homopolymers and polyP to bind the primary constituents of the contact pathway: factor XIa, factor XIIa, and plasma kallikrein, in the presence and absence of high molecular weight kininogen (HK), an important cofactor in this pathway. We examine seven proteolytic activation reactions within the contact pathway and report that polyP greatly enhances the rate of all seven, while RNA is effective in supporting only a subset of these reactions. HK both enhances and suppresses these proteolytic activation reactions, depending on the specific reaction evaluated. Overall, we find that polyP is a potent mediator of contact pathway activation reactions in general, that RNA secondary structure may be important to its procoagulant activity, and that nucleic acids versus polyP may differentially modulate specific enzyme activation events within the contact pathway.

Multiplexed silicon photonic sensor arrays enable facile characterization of coagulation protein binding to nanodiscs with variable lipid content.

Interactions of soluble proteins with the cell membrane are critical within the blood coagulation cascade. Of particular interest are the interactions of γ-carboxyglutamic acid-rich domain-containing clotting proteins with lipids. Variability among conventional analytical methods presents challenges for comparing clotting protein-lipid interactions. Most previous studies have investigated only a single clotting protein and lipid composition and have yielded widely different binding constants. Herein, we demonstrate that a combination of lipid bilayer nanodiscs and a multiplexed silicon photonic analysis technology enables high-throughput probing of many protein-lipid interactions among blood-clotting proteins. This approach allowed direct comparison of the binding constants of prothrombin, factor X, activated factor VII, and activated protein C to seven different binary lipid compositions. In a single experiment, the binding constants of one protein interacting with all lipid compositions were simultaneously determined. A simple surface regeneration then facilitated similar binding measurements for three other coagulation proteins. As expected, our results indicated that all proteins exhibit tighter binding (lower Kd ) as the proportion of anionic lipid increases. Interestingly, at high proportions of phosphatidylserine, the Kd values of all four proteins began to converge. We also found that although koff values for all four proteins followed trends similar to those observed for the Kd values, the variation among the proteins was much lower, indicating that much of the variation came from the kinetic binding (kon) of the proteins. These findings indicate that the combination of silicon photonic microring resonator arrays and nanodiscs enables rapid interrogation of biomolecular binding interactions at model cell membrane interfaces.

Deeper understanding of carboxylase.

In this issue of Blood, Tie et al report the development of a cleverly engineered, cell-based system for studying mutations in γ-glutamyl carboxylase (GGCX), the enzyme responsible for converting glutamate residues in certain proteins to γ-carboxyglutamate (Gla). They use this cell-based assay system to help explain the clinical manifestations of some otherwise puzzling GGCX gene mutations in humans that cause phenotypes ranging from severe bleeding to Keutel syndrome.

Polyphosphate is a novel cofactor for regulation of complement by a serpin, C1 inhibitor.

The complement system plays a key role in innate immunity, inflammation, and coagulation. The system is delicately balanced by negative regulatory mechanisms that modulate the host response to pathogen invasion and injury. The serpin, C1-esterase inhibitor (C1-INH), is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. Like other serpin-protease partners, C1-INH interaction with C1s is accelerated by polyanions such as heparin. Polyphosphate (polyP) is a naturally occurring polyanion with effects on coagulation and complement. We recently found that polyP binds to C1-INH, prompting us to consider whether polyP acts as a cofactor for C1-INH interactions with its target proteases. We show that polyP dampens C1s-mediated activation of the classical pathway in a polymer length- and concentration-dependent manner by accelerating C1-INH neutralization of C1s cleavage of C4 and C2. PolyP significantly increases the rate of interaction between C1s and C1-INH, to an extent comparable to heparin, with an exosite on the serine protease domain of the enzyme playing a major role in this interaction. In a serum-based cell culture system, polyP significantly suppressed C4d deposition on endothelial cells, generated via the classical and lectin pathways. Moreover, polyP and C1-INH colocalize in activated platelets, suggesting that their interactions are physiologically relevant. In summary, like heparin, polyP is a naturally occurring cofactor for the C1s:C1-INH interaction and thus an important regulator of complement activation. The findings may provide novel insights into mechanisms underlying inflammatory diseases and the development of new therapies.

DNA ladders can be used to size polyphosphate resolved by polyacrylamide gel electrophoresis.

PAGE is often used to resolve inorganic polyphosphates (polyP), but unfortunately polyP size ladders are not commercially available. Since several dyes that are commonly used to detect nucleic acids in gels also stain polyP, we examined the utility of commercially available DNA size ladders for estimating polyP polymer lengths by gel electrophoresis. Narrow size fractions of polyP were prepared and their polymer lengths were quantified using NMR. Commercially available DNA ladders and these polyP fractions were then subjected to PAGE to determine the relationship between migration of DNA vs polyP, which was found to be: log10 (dsDNA length in bp) = 1.66 × log10 (polyP length in phosphate units) - 1.97. This relationship between DNA and polyP size held for a variety of different polyacrylamide concentrations, indicating that DNA size ladders can readily be employed to estimate polyP polymer lengths by PAGE.

Design of Polyphosphate Inhibitors: A Molecular Dynamics Investigation on Polyethylene Glycol-Linked Cationic Binding Groups.

Inorganic polyphosphate (polyP) released by human platelets has recently been shown to activate blood clotting and identified as a potential target for the development of novel antithrombotics. Recent studies have shown that polymers with cationic binding groups (CBGs) inhibit polyP and attenuate thrombosis. However, a good molecular-level understanding of the binding mechanism is lacking for further drug development. While molecular dynamics (MD) simulation can provide molecule-level information, the time scale required to simulate these large biomacromolecules makes classical MD simulation impractical. To overcome this challenge, we employed metadynamics simulations with both all-atom and coarse-grained force fields. The force field parameters for polyethylene glycol (PEG) conjugated CBGs and polyP were developed to carry out coarse-grained MD simulations, which enabled simulations of these large biomacromolecules in a reasonable time scale. We found that the length of the PEG tail does not impact the interaction between the (PEG) n-CBG and polyP. As expected, increasing the number of the charged tertiary amine groups in the head group strengthens its binding to polyP. Our simulation shows that (PEG) n-CBG initially form aggregates, mostly with the PEG in the core and the hydrophilic CBG groups pointing toward water; then the aggregates approach the polyP and sandwich the polyP to form a complex. We found that the binding of (PEG) n-CBG remains intact against various lengths of polyP. Binding thermodynamics for two of the (PEG) n-CBG/polyP systems simulated were measured by isothermal titration calorimetry to confirm the key finding of the simulations that the length PEG tail does not influence ligand binding to polyP.

How it all starts: Initiation of the clotting cascade.

The plasma coagulation system in mammalian blood consists of a cascade of enzyme activation events in which serine proteases activate the proteins (proenzymes and procofactors) in the next step of the cascade via limited proteolysis. The ultimate outcome is the polymerization of fibrin and the activation of platelets, leading to a blood clot. This process is protective, as it prevents excessive blood loss following injury (normal hemostasis). Unfortunately, the blood clotting system can also lead to unwanted blood clots inside blood vessels (pathologic thrombosis), which is a leading cause of disability and death in the developed world. There are two main mechanisms for triggering the blood clotting, termed the tissue factor pathway and the contact pathway. Only one of these pathways (the tissue factor pathway) functions in normal hemostasis. Both pathways, however, are thought to contribute to thrombosis. An emerging concept is that the contact pathway functions in host pathogen defenses. This review focuses on how the initiation phase of the blood clotting cascade is regulated in both pathways, with a discussion of the contributions of these pathways to hemostasis versus thrombosis.