Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM + HDL contained S1P, whereas ApoM − HDL did not. Moreover, HDL in Apom −/− mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P-human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM + HDL induced S1P 1 receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM − HDL did not. Importantly, lack of S1P in the HDL fraction of Apom −/− mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P 1 receptor on endothelial cells, is a vasculoprotective constituent of HDL.endothelial function | crystal structure | sphingolipids | vascular permeability | atherosclerosis S phingosine-1-phosphate (S1P), the phosphorylated metabolite of D-sphingosine, binds to five G protein-coupled receptors (S1P 1 -S1P 5 ) and regulates a plethora of biological actions (1-6). In particular, the prototypical S1P 1 receptor is essential for vascular maturation during development and promotes endothelial cell migration, angiogenesis, and barrier functions (7-9). Thus, S1P is required for maintenance of the barrier property of the lung endothelium (10). Plasma S1P, which is derived from several cellular sources (11,12), is associated with HDL (∼65%) and albumin (∼35%) (3, 5). HDLinduced vasorelaxation as well as barrier-promoting and prosurvival actions on the endothelium have been attributed to S1P signaling (2, 4, 13). Hence, much of the endothelium-protective actions of HDL may result from the actions of S1P on the endothelial S1P receptors. However, the molecular nature of the S1P binding to HDL and interaction with S1P receptors has not been characterized.Apolipoprotein M (apoM) is a lipocalin that resides mainly in the plasma HDL fraction (14). The retained hydrophobic NH 2 -terminal signal peptide anchors apoM in the phospholipid layer of the lipoprotein and prevents filtration of the ∼22-kDa protein in the kidney (15). The biological functions of apoM are understood only partly. Studies in apoM gene-modified mice suggest that apoM has antiatherogenic effects, possibly related in part to apoM's ability to increase cholesterol efflux from macrophage foam cells, to increased preβ-HDL formation, and to antioxidative effects (16)(17)(18). The recent elucidation of the crystal structure of human recombinant apoM (r-apoM) demonstrated a typical lipocalin fold characterized by an eightstranded antiparallel β-barrel enclosing an internal binding pocket that probably facilitates binding of small lipophilic ligands (19).Indeed, r-apoM expressed in Escherichia coli was found to co...
The primary aim of this study was to investigate the possible relationship between coagulation factor level and bleeding frequency during prophylactic treatment of haemophilia after stratification of the patients according to joint scores. The secondary aim was to obtain a systematic overview of the doses of coagulation factors prescribed for prophylaxis at the Malmo haemophilia treatment centre during a 6-year period. A retrospective survey of medical records for the years 1997-2002 and pharmacokinetic study results from the 1990s was complemented by collection of blood samples for coagulation factor assay when needed. Information on the dosing and plasma levels of factor VIII or factor IX, joint scores and incidence of bleedings (joint bleeds and 'other bleeds') was compiled. The patients were stratified by age (0-6, 7-12, 13-18, 19-36 and >36 years) and joint score (0, 1-6 and >6). Individual pharmacokinetic parameters of plasma coagulation factor activities (FVIII:C and FIX:C) were estimated. Trough levels during the treatment were calculated, as well as the number of hours per week of treatment during which plasma FVIII:C/FIX:C fell below a 1, 2 or 3% target level. Fifty-one patients with haemophilia A (two moderate, 49 severe) and 13 with haemophilia B (all severe) were included, yielding data for 364 patient-years of treatment. There was a wide range of dosing schedules, the most common ones being three times a week or every other day for FVIII and twice a week or every third day for FIX. The overall relationship between FVIII:C/FIX:C levels and incidence of joint bleeding was very weak, even after stratification of the patients according to joint score. There was no relationship between coagulation factor level and incidence of other bleeds. In this cohort of patients on high-dose prophylactic treatment, dosing was based more on clinical outcome in terms of bleeding frequency than on the aim to maintain a 1% target level of FVIII:C/FIX:C. Some patients did not bleed in spite of a trough level of <1% and others did in spite of trough levels >3%. The practical implication of our findings is that dosing in prophylactic treatment of haemophilia should be individualized. Thus, proposed standard regimens should be implemented only after careful clinical consideration, with a high readiness for re-assessment and individual dose tailoring.
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...
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 ( k cat / K m 35 × 10 5 M −1 s −1 ) is a more potent (~18-fold) versicanase than ADAMTS-4 ( k cat / K m 1.86 × 10 5 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.
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