Laminin G1 residues of protein S mediate its TFPI cofactor function and are competitively regulated by C4BP.

Protein S is a cofactor in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. It enhances TFPIα-mediated inhibition of factor (F)Xa activity and generation. The enhancement is dependent on a TFPIα-protein S interaction involving TFPIα Kunitz 3 and protein S laminin G-type (LG)-1. C4b binding protein (C4BP), which binds to protein S LG1, almost completely abolishes its TFPI cofactor function. However, neither the amino acids involved in TFPIα enhancement nor the mechanisms underlying the reduced TFPI cofactor function of C4BP-bound protein S are known. To screen for functionally important regions within protein S LG1, we generated 7 variants with inserted N-linked glycosylation attachment sites. Protein S D253T and Q427N/K429T displayed severely reduced TFPI cofactor function while showing normal activated protein C (APC) cofactor function and C4BP binding. Based on these results, we designed 4 protein S variants in which 4 to 6 surface-exposed charged residues were substituted for alanine. One variant, protein S K255A/E257A/D287A/R410A/K423A/E424A, exhibited either abolished or severely reduced TFPI cofactor function in plasma and FXa inhibition assays, both in the presence or absence of FV-short, but retained normal APC cofactor function and high-affinity C4BP binding. The C4BP ß-chain was expressed to determine the mechanisms behind the reduced TFPI cofactor function of C4BP-bound protein S. Like C4BP-bound protein S, C4BP ß-chain-bound protein S had severely reduced TFPI cofactor function. These results show that protein S Lys255, Glu257, Asp287, Arg410, Lys423, and Glu424 are critical for protein S-mediated enhancement of TFPIα and that binding of the C4BP ß-chain blocks this function.

Partial rescue of naturally occurring active site factor X variants through decreased inhibition by tissue factor pathway inhibitor and antithrombin.

BACKGROUND: Activated coagulation factor X (FXa) is the serine protease component of prothrombinase, the physiological activator of prothrombin. Factor X Nottingham (A404T) and Taunton (R405G) are two naturally occurring mutations, identified in families with a bleeding phenotype. OBJECTIVE: To characterize these FX variants functionally. METHODS: The activity and inhibition of recombinant FX variants were quantified in plasma-based and pure component assays. RESULTS: The prothrombin times in FX-depleted plasma supplemented with FX Nottingham and Taunton were greatly increased compared to that of wild-type (WT) FX. Kinetic investigations of activated variants in the prothrombinase complex showed kcat /Km reduced ~50-fold and ~5-fold, respectively, explaining the prolonged prothrombin time (PT). The substituted residues are located in the protease domain Na+ -binding loop, important for the activity of FXa, as well as its inhibition. Both FXa Nottingham and Taunton showed reduced affinity for Na+ . Plasma-based thrombin generation assays triggered with 1 pmol/L tissue factor (TF) demonstrated only small differences in activities compared to WT FX, but large reductions at 10 pmol/L TF. Severely reduced inhibition of both FXa Nottingham and Taunton by tissue factor pathway inhibitor (TFPI) and antithrombin (AT), was shown in pure-component FXa inhibition assays. Factor Xa Nottingham and Taunton produced higher amounts of thrombin than WT FXa in pure-component prothrombinase assays in the presence of TFPI and AT, explaining the results from the plasma-based assay. CONCLUSIONS: Factor X Nottingham and Taunton both display decreased proteolytic activity. However, their reduced activity in plasma triggered by low TF can be rescued by decreased inhibition by the natural FXa inhibitors, TFPI and AT.

The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex.

BACKGROUND: Activated protein C (APC)-mediated inactivation of factor (F)Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex among FVa, APC, and protein S must form on the phospholipid membrane. However, direct demonstration of complex formation has proven elusive. OBJECTIVES: To elucidate the nature of the phospholipid-dependent interactions among APC, protein S, and FVa. METHODS: We evaluated binding of active site blocked APC to phospholipid-coated magnetic beads in the presence and absence of protein S and/or FVa. The importance of protein S and FV residues were evaluated functionally. RESULTS: Activated protein C alone bound weakly to phospholipids. Protein S mildly enhanced APC binding to phospholipid surfaces, whereas FVa did not. However, FVa together with protein S enhanced APC binding (>14-fold), demonstrating formation of an APC/protein S/FVa complex. C4b binding protein-bound protein S failed to enhance APC binding, agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A) with reduced APC cofactor function exhibited essentially normal augmentation of APC binding to phospholipids, but diminished APC/protein S/FVa complex formation, suggesting involvement in interactions dependent upon FVa. Similarly, FVaNara (W1920R), an APC-resistant FV variant, also did not efficiently incorporate into the trimolecular complex as efficiently as wild-type FVa. FVa inactivation assays suggested that the mutation impairs its affinity for phospholipid membranes and with protein S within the complex. CONCLUSIONS: FVa plays a central role in the formation of its inactivation complex. Furthermore, membrane proximal interactions among FVa, APC, and protein S are essential for its cofactor function.

Defective fibrin deposition and thrombus stability in Bambi-/- mice are mediated by elevated anticoagulant function.

BACKGROUND: Bone morphogenetic and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein related to the type I transforming growth factor- ß (TGF-ß) receptor family that is present on both platelets and endothelial cells (ECs). Bambi-deficient mice exhibit reduced hemostatic function and thrombus stability characterized by an increased embolization. OBJECTIVE: We aimed to delineate how BAMBI influences endothelial function and thrombus stability. METHODS: Bambi-deficient mice were subjected to the laser-induced thrombosis model where platelet and fibrin accumulation was evaluated. Expression of thrombomodulin and tissue factor pathway inhibitor (TFPI) was also assessed in these mice. RESULTS: Thrombus instability in Bambi-/- mice was associated with a profound defect in fibrin deposition. Injection of hirudin into Bambi+/+ mice prior to thrombus formation recapitulated the Bambi-/- thrombus instability phenotype. In contrast, hirudin had no additional effect upon thrombus formation in Bambi-/- mice. Deletion of Bambi in ECs resulted in mice with defective thrombus stability caused by decreased fibrin accumulation. Increased levels of the anticoagulant proteins TFPI and thrombomodulin were detected in Bambi-/- mouse lung homogenates. Endothelial cells isolated from Bambi-/- mouse lungs exhibited enhanced ability to activate protein C due to elevated thrombomodulin levels. Blocking thrombomodulin and TFPI in vivo fully restored fibrin accumulation and thrombus stability in Bambi-/- mice. CONCLUSIONS: We demonstrate that endothelial BAMBI influences fibrin generation and thrombus stability by modulating thrombomodulin and TFPI anticoagulant function of the endothelium; we also highlight the importance of these anticoagulant proteins in the laser-induced thrombosis model.

Exosites in Hypervariable Loops of ADAMTS Spacer Domains control Substrate Recognition and Proteolysis.

ADAMTS (A Disintegrin-like and Metalloproteinase domain with Thrombospondin type 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating proteoglycans including versican and aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major versican cleavage fragment was used to determine, for the first time, kinetic constants for versican proteolysis. ADAMTS-5 (kcat/Km 35 × 105 M-1 s-1) is a more potent (~18-fold) versicanase than ADAMTS-4 (kcat/Km 1.86 × 105 M-1 sec-1), whereas ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of ADAMTS-5 19-fold and that of ADAMTS-4 167-fold. Co-deletion of the ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of ADAMTS-5 (residues 739-744 and 837-844) and ADAMTS-4 (residues 717-724 and 788-795) with those of ADAMTS-13, which does not cleave proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of versican. The hypervariable loops of ADAMTS-5 are shown to be important also for its aggrecanase activity. Together with previous work on ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of ADAMTS proteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.

The ADAMTS13-VWF axis is dysregulated in chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is an important consequence of pulmonary embolism that is associated with abnormalities in haemostasis. We investigated the ADAMTS13-von Willebrand factor (VWF) axis in CTEPH, including its relationship with disease severity, inflammation, ABO groups and ADAMTS13 genetic variants.ADAMTS13 and VWF plasma antigen levels were measured in patients with CTEPH (n=208), chronic thromboembolic disease without pulmonary hypertension (CTED) (n=35), resolved pulmonary embolism (n=28), idiopathic pulmonary arterial hypertension (n=30) and healthy controls (n=68). CTEPH genetic ABO associations and protein quantitative trait loci were investigated. ADAMTS13-VWF axis abnormalities were assessed in CTEPH and healthy control subsets by measuring ADAMTS13 activity, D-dimers and VWF multimeric size.Patients with CTEPH had decreased ADAMTS13 (adjusted ß -23.4%, 95% CI -30.9- -15.1%, p<0.001) and increased VWF levels (ß +75.5%, 95% CI 44.8-113%, p<0.001) compared to healthy controls. ADAMTS13 levels remained low after reversal of pulmonary hypertension by pulmonary endarterectomy surgery and were equally reduced in CTED. We identified a genetic variant near the ADAMTS13 gene associated with ADAMTS13 protein that accounted for ∼8% of the variation in levels.The ADAMTS13-VWF axis is dysregulated in CTEPH. This is unrelated to pulmonary hypertension, disease severity or markers of systemic inflammation and implicates the ADAMTS13-VWF axis in CTEPH pathobiology.

A common mechanism by which type 2A von Willebrand disease mutations enhance ADAMTS13 proteolysis revealed with a von Willebrand factor A2 domain FRET construct.

Rheological forces in the blood trigger the unfolding of von Willebrand factor (VWF) and its A2 domain, exposing the scissile bond for proteolysis by ADAMTS13. Under quiescent conditions, the scissile bond is hidden by the folded structure due to the stabilisation provided by the structural specialisations of the VWF A2 domain, a vicinal disulphide bond, a calcium binding site and a N1574-glycan.The reduced circulating high MW multimers of VWF in patients with type 2A von Willebrand disease (VWD) may be associated with mutations within the VWF A2 domain and this is attributed to enhanced ADAMTS13 proteolysis. We investigated 11 VWF A2 domain variants identified in patients with type 2A VWD. In recombinant full-length VWF, enhanced ADAMTS13 proteolysis was detected for all of the expressed variants in the presence of urea-induced denaturation. A subset of the FLVWF variants displayed enhanced proteolysis in the absence of urea. The mechanism of enhancement was investigated using a novel VWF A2 domain FRET construct. In the absence of induced unfolding, 7/8 of the expressed mutants exhibited a disrupted domain fold, causing spatial separation of the N- and C- termini. Three of the type 2A mutants were not secreted when studied within the VWF A2 domain FRET construct. Urea denaturation revealed for all 8 secreted mutants reduced unfolding cooperativity and stability of the VWF A2 domain. As folding stability was progressively disrupted, proteolysis by ADAMTS13 increased. Due to the range of folding stabilities and wide distribution of VWF A2 domain mutations studied, we conclude that these mutations disrupt regulated folding of the VWF A2 domain. They enhance unfolding by inducing separation of N- and C-termini, thereby promoting a more open conformation that reveals its binding sites for ADAMTS13 and the scissile bond.

Factor V has an anticoagulant cofactor activity that targets the early phase of coagulation.

Tissue factor pathway inhibitor (TFPI), the main inhibitor of initiation of coagulation, exerts an important anticoagulant role through the factor Xa (FXa)-dependent inhibition of tissue factor/factor VIIa. Protein S is a TFPI cofactor, enhancing the efficiency of FXa inhibition. TFPI can also inhibit prothrombinase assembly by directly interacting with coagulation factor V (FV), which has been activated by FXa. Because full-length TFPI associates with FV in plasma, we hypothesized that FV may influence TFPI inhibitory function. Using pure component FXa inhibition assays, we found that although FV alone did not influence TFPI-mediated FXa inhibition, it further enhanced TFPI in the presence of protein S, resulting in an ∼8-fold reduction in Ki compared with TFPI alone. A FV variant (R709Q/R1018Q/R1545Q, FVΔIIa) that cannot be cleaved/activated by thrombin or FXa also enhanced TFPI-mediated inhibition of FXa ∼12-fold in the presence of protein S. In contrast, neither activated FV nor recombinant B-domain-deleted FV could enhance TFPI-mediated inhibition of FXa in the presence of protein S, suggesting a functional contribution of the B domain. Using TFPI and protein S variants, we show further that the enhancement of TFPI-mediated FXa inhibition by protein S and FV depends on a direct protein S/TFPI interaction and that the TFPI C-terminal tail is not essential for this enhancement. In FXa-catalyzed prothrombin activation assays, both FV and FVΔIIa (but not activated FV) enhanced TFPI function in the presence of protein S. These results demonstrate a new anticoagulant (cofactor) function of FV that targets the early phase of coagulation before prothrombinase assembly.

Third-harmonic-generation of a diode laser for quantum control of beryllium ions.

We generate coherent ultraviolet radiation at 313 nm as the third harmonic of an external-cavity diode laser. We use this radiation for laser cooling of trapped beryllium atomic ions and sympathetic cooling of co-trapped beryllium-hydride molecular ions. An LBO crystal in an enhancement cavity generates the second harmonic, and a BBO crystal in a doubly resonant enhancement cavity mixes this second harmonic with the fundamental to produce the third harmonic. Each enhancement cavity is preceded by a tapered amplifier to increase the fundamental light. The 36-mW output power of this all-semiconductor-gain system will enable quantum control of the beryllium ions' motion.

A model for the conformational activation of the structurally quiescent metalloprotease ADAMTS13 by von Willebrand factor.

Blood loss is prevented by the multidomain glycoprotein von Willebrand factor (VWF), which binds exposed collagen at damaged vessels and captures platelets. VWF is regulated by the metalloprotease ADAMTS13, which in turn is conformationally activated by VWF. To delineate the structural requirements for VWF-mediated conformational activation of ADAMTS13, we performed binding and functional studies with a panel of truncated ADAMTS13 variants. We demonstrate that both the isolated CUB1 and CUB2 domains in ADAMTS13 bind to the spacer domain exosite of a truncated ADAMTS13 variant, MDTCS (KD of 135 ± 1 0.1 nm and 86.9 ± 9.0 nm, respectively). However, only the CUB1 domain inhibited proteolytic activity of MDTCS. Moreover, ADAMTS13ΔCUB2, unlike ADAMTS13ΔCUB1-2, exhibited activity similar to wild-type ADAMTS13 and could be activated by VWF D4-CK. The CUB2 domain is, therefore, not essential for maintaining the inactive conformation of ADAMTS13. Both CUB domains could bind to the VWF D4-CK domain fragment (KD of 53.7 ± 2.1 nm and 84.3 ± 2.0 nm, respectively). However, deletion of both CUB domains did not prevent VWF D4-CK binding, suggesting that competition for CUB-domain binding to the spacer domain is not the dominant mechanism behind the conformational activation. ADAMTS13ΔTSP8-CUB2 could no longer bind to VWF D4-CK, and deletion of TSP8 abrogated ADAMTS13 conformational activation. These findings support an ADAMTS13 activation model in which VWF D4-CK engages the TSP8-CUB2 domains, inducing the conformational change that disrupts the CUB1-spacer domain interaction and thereby activates ADAMTS13.

Innovation cascades: artefacts, organization and attributions.

Innovation cascades inextricably link the introduction of new artefacts, transformations in social organization, and the emergence of new functionalities and new needs. This paper describes a positive feedback dynamic, exaptive bootstrapping, through which these cascades proceed, and the characteristics of the relationships in which the new attributions that drive this dynamic are generated. It concludes by arguing that the exaptive bootstrapping dynamic is the principal driver of our current Innovation Society.

N-linked glycan stabilization of the VWF A2 domain.

Shear forces in the blood trigger a conformational transition in the von Willebrand factor (VWF) A2 domain, from its native folded to an unfolded state, in which the cryptic scissile bond (Y1605-M1606) is exposed and can then be proteolysed by ADAMTS13. The conformational transition depends upon a Ca(2+)binding site and a vicinal cysteine disulfide bond. Glycosylation at N1574 has previously been suggested to modulate VWF A2 domain interaction with ADAMTS13 through steric hindrance by the bulky carbohydrate structure. We investigated how the N-linked glycans of the VWF A2 domain affect thermostability and regulate both the exposure of the ADAMTS13 binding sites and the scissile bond. We show by differential scanning fluorimetry that the N-linked glycans thermodynamically stabilize the VWF A2 domain. The essential component of the glycan structure is the first sugar residue (GlcNAc) at the N1574 attachment site. From its crystal structures, N1574-GlcNAc is predicted to form stabilizing intradomain interactions with Y1544 and nearby residues. Substitution of the surface-exposed Y1544 to aspartic acid is able to stabilize the domain in the absence of glycosylation and protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilization of the VWF A2 domain acts together with the Ca(2+)binding site and vicinal cysteine disulfide bond to control unfolding and ADAMTS13 proteolysis.

Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke.

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke.

The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis.

ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain-charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.

Conformational activation of ADAMTS13.

A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼ 2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit ADAMTS13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼ 2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/F592Y/R660K/Y661F/Y665F) was ∼ 2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a "closed" conformation of WT ADAMTS13 and suggested a more "open" conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition.

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain.

Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrest-specific 6 chimeras, with either the whole sex hormone-binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest-specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane.

Vessel wall BAMBI contributes to hemostasis and thrombus stability.

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein related to the transforming growth factor-ß superfamily, and is highly expressed in platelets and endothelial cells. We previously demonstrated its positive role in thrombus formation using a zebrafish thrombosis model. In the present study, we used Bambi-deficient mice and radiation chimeras to evaluate the function of this receptor in the regulation of both hemostasis and thrombosis. We show that Bambi(-/-) and Bambi(+/-) mice exhibit mildly prolonged bleeding times compared with Bambi(+/+) littermates. In addition, using 2 in vivo thrombosis models in mesenterium or cremaster muscle arterioles, we demonstrate that Bambi-deficient mice form unstable thrombi compared with Bambi(+/+) mice. No defects in thrombin generation in Bambi(-/-) mouse plasma could be detected ex vivo. Moreover, the absence of BAMBI had no effect on platelet counts, platelet activation, aggregation, or platelet procoagulant function. Similar to Bambi(-/-) mice, Bambi(-/-) transplanted with Bambi(+/+) bone marrow formed unstable thrombi in the laser-induced thrombosis model that receded more rapidly than thrombi that formed in Bambi(+/+) mice receiving Bambi(-/-) bone marrow transplants. Taken together, these results provide strong evidence for an important role of endothelium rather than platelet BAMBI as a positive regulator of both thrombus formation and stability.