The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin ␣v␤3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood- IntroductionAngiogenesis, the formation of new vessels from pre-existing ones, occurs physiologically in specific circumstances such as wound healing and the menstrual cycle. Dysregulated angiogenesis contributes to the pathogenesis of many disorders, including diabetes, cancer, and macular degeneration (reviewed in Carmeliet 1 ). Angiogenic factors such as vascular endothelial growth factor (VEGF) and the angiopoietins (Ang) orchestrate signaling pathways that promote endothelial cell (EC) migration, proliferation, and ultimately the formation of a new vessel. VEGF-A is a major regulator of angiogenesis (reviewed in Grothey and Galanis 2 ) and acts on ECs mainly through VEGF receptor-2 (VEGFR-2), a tyrosine kinase receptor (reviewed in Olsson 3 ), to promote endothelial proliferation, migration, and sprouting of tip cells in the early stages of angiogenesis (reviewed in Gerhardt 4 ). Ang-1 and Ang-2, which bind to the endothelial Tie-2 receptor, act in the later stages of blood vessel formation and are essential for the maturation of a stable vascular network and for the maintenance of endothelial integrity (reviewed in Thomas and Augustin 5 ). Ang-1 and Ang-2 were originally identified as agonist and antagonist of Tie-2 signaling, respectively, with Ang-1 supporting EC survival and endothelial integrity 6 and Ang-2 promoting blood vessel destabilization and regression. 7 However, recent data suggest a more complex picture that includes cross-talk between the VEGF and the Ang pathways. 8 Growth factor signaling pathways are influenced by surface adhesion molecules that mediate cell-cell or cell-matrix interactions, particularly by members of the integrin superfamily. The integrin that has received most attention in ECs is ␣v␤3 (reviewed in Hodivala-Dilke 9 ), which mediates binding to several extracellular matrix proteins and growth factor receptors including VEGFR-2, thus influencing VEGFR-2 signaling (reviewed in Somanath et al 10 ). ␣v␤3 plays a complex role in angiogenesis. Although the origina...

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Protein S is a cofactor for tissue factor pathway inhibitor (TFPI) that critically reduces the inhibition constant for FXa to below the plasma concentration of TFPI. TFPI Kunitz domain 3 is required for this enhancement to occur. To delineate the molecular mechanism underlying enhancement of TFPI function, in the present study, we produced a panel of Kunitz domain 3 variants of TFPI encompassing all 12 surface-exposed charged residues. Thrombin-generation assays in TFPIdepleted plasma identified a novel variant, TFPI E226Q, which exhibited minimal enhancement by protein S. This was confirmed in purified FXa inhibition assays in which no protein S enhancement of TFPI E226Q was detected. Surface plasmon resonance demonstrated concentrationdependent binding of protein S to wildtype TFPI, but almost no binding to TFPI E226Q. We conclude that the TFPI Kunitz domain 3 residue Glu226 is essential for TFPI enhancement by protein S. (Blood. 2012;120(25):5059-5062) IntroductionTissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor consisting of an acidic aminoterminal polypeptide, followed by 3 tandem Kunitz-type domains (Kunitz domains 1, 2, and 3) and a basic carboxyterminal tail. 1,2 TFPI exerts its anticoagulant function by inhibiting tissue factor (TF)-induced coagulation in the blood. [3][4][5] Purified assays have shown that FXa inhibition by TFPI occurs in a 2-step process that can be described by the inhibition constants K i and K i *, 6 respectively, in the following equation:In 2006, Hackeng et al identified protein S as a cofactor for TFPI that is capable of reducing the K i for FXa inhibition by TFPI by 10-fold. 7 More recently, Ndonwi et al showed that protein S enhancement of TFPI is dependent on the TFPI Kunitz domain 3. 8 A TFPI R199L variant showed partial loss of protein S cofactor function compared with wild-type (WT) TFPI. 8 The present study is an investigation of the role of all surface-exposed charged residues of TFPI Kunitz domain 3. Methods Generation, expression, and purification of TFPI variantsTen composite and individual point mutations were generated: D194Q/ R195Q/R199Q, E202Q/R204Q/K218Q, K213Q/R215Q/K232Q, E226Q/ E234Q/R237Q, D194Q, R195Q, R199Q, E226Q, E234Q, and R237Q (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Details of vector construction, protein expression, purification, and quantification can be found in the supplemental Methods. Thrombin-generation assay determined by CATCalibrated automated thrombogram (CAT) was performed in normal or TFPI-depleted plasma (supplemental Methods), as described previously. 9,10 Purified or WT TFPI and TFPI variants in concentrated conditioned medium (0-1.5nM) were added to the plasma. Purification status did not influence inhibitory function. FXa inhibition assayFXa (0.5nM) activity was monitored by the cleavage of the chromogenic substrate S-2765 (Chromogenix) in the presence of absence of TFPI (0-4nM) and protein S (0-320nM) essentially as descr...

We examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion followed by lectin analysis demonstrated that more than 90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF had an approximately 4-fold increased affinity for ADAMTS13 compared with control VWF. PNG-VWF was cleaved by ADAMTS13 faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the N-linked glycan sites at N1515 and N1574 and their presentation of ABO(H) blood group sugars were confirmed with an isolated tryptic fragment. Recombinant VWF was mutated to prevent glycosylation at these sites. Mutation of N1515 did not alter ADAMTS13 binding or increase rate of ADAMTS13 proteolysis. Mutation of N1574 increased the susceptibility of VWF to ADAMTS13 proteolysis and allowed cleavage in the absence of urea. Mutation of N1574 in the isolated recombinant VWF-A2 domain also increased binding and ADAMTS13 proteolysis. These data demonstrate that the N-linked glycans of VWF have a modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but steric hindrance may also be important. IntroductionVon Willebrand factor (VWF) is a large multimeric plasma glycoprotein essential to normal hemostasis, first acting as the carrier molecule for procoagulant factor VIII (FVIII), extending its half-life within the circulation by protecting it from proteolytic degradation, and second, supporting platelet adhesion to thrombogenic surfaces at sites of vascular injury. 1,2 Synthesis of VWF is limited to megakaryocytes and endothelial cells. 3 The pre-pro-VWF molecule comprises a 22-amino acid signal peptide, a 741-amino acid propeptide, and the 2050-amino acid mature subunit. The pro-VWF monomer is composed of 4 types of domains (A-D) arranged as follows: NH 2 -D1-D2-DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK-COOH. VWF multimers are formed by C-and N-terminal intermolecular disulphide bonds, with the largest multimers exceeding 2 ϫ 10 4 kDa and being the most hemostatically active. Within the circulation, the multimeric size of VWF is controlled by the plasma metalloprotease ADAMTS13, which cleaves VWF at the Y1605-M1606 bond within the A2 domain, reducing multimeric size and thus regulating its adhesive function. 4 During synthesis, VWF undergoes extensive posttranslational modification resulting in the addition of 12 N-linked and 10 O-linked glycosylation sites per mature monomer. 5 The overall structural composition of the glycans has been determined, but their exact functional significance is poorly understood. There is some evidence to suggest they protect the molecule from proteolytic degradation and are required for dimerization and subsequent multimerization. [6][7][8] Significantly, a small proportion of the N-linked glycans on VWF present the ABO(H) blood group sugars, 9,10 and the importance of this is highlighted by the well-established ...

We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13. (Blood.