Abnormal von Willebrand factor secretion, factor VIII stabilization and thrombus dynamics in type 2N von Willebrand disease mice.

Essentials Type 2N von Willebrand disease involves impaired von Willebrand factor to factor VIII binding. Type 2N von Willebrand disease mutations exhibit qualitative and mild quantitative deficiencies. Type 2N von Willebrand disease mice exhibit unstable venous hemostatic thrombi. The factor VIII-binding ability of von Willebrand factor regulates arteriole thrombosis dynamics. SUMMARY: Background von Willebrand factor (VWF) and factor VIII (FVIII) circulate as a non-covalent complex, with VWF serving as the carrier for FVIII. VWF indirectly influences secondary hemostasis by stabilizing FVIII and transporting it to the site of primary hemostasis. Type 2N von Willebrand disease involves impaired binding of VWF to FVIII, resulting in decreased plasma levels of FVIII. Objectives In these studies, we characterize the impact of three type 2N VWD variants (R763A, R854Q, R816W) on VWF secretion, FVIII stabilization and thrombus formation in a murine model. Methods Type 2N VWD mice were generated by hydrodynamic injections of mutant murine VWF cDNAs and the influence of these variants on VWF secretion and FVIII binding was evaluated. In vivo hemostasis and the dynamics of thrombus formation and embolization were assessed using a murine tail vein transection hemostasis model and an intravital thrombosis model in the cremaster arterioles. Results Type 2N VWD variants were associated with decreased VWF secretion using cell and animal-based models. FVIII-binding to type 2N variants was impaired in vitro and was variably stabilized in vivo by expressed or infused 2N variant VWF protein. Both transgenic type 2N VWD and FVIII knockout (KO) mice demonstrated impaired thrombus formation associated with decreased thrombus stability. Conclusions The type 2N VWD phenotype can be recapitulated in a murine model and is associated with both quantitative and qualitative VWF deficiencies and impaired thrombus formation. Patients with type 2N VWD may have normal primary hemostasis formation but decreased thrombus stability related to ineffective secondary hemostasis.

Histones link inflammation and thrombosis through the induction of Weibel-Palade body exocytosis.

Essentials Dysregulated DNA and histone release can promote pathological immunothrombosis. Weibel-Palade bodies (WPBs) are sentinel-like organelles that respond to proinflammatory stimuli. Histones induce WPB exocytosis in a caspase, calcium and charge-dependent mechanism. A targetable axis may exist between DNA/histones and WPBs in inflammation and immunothrombosis. SUMMARY: Background Damage-associated molecular patterns (DAMPs), including molecules such as DNA and histones, are released into the blood following cell death. DAMPs promote a procoagulant phenotype through enhancement of thrombin generation and platelet activation, thereby contributing to immunothrombosis. Weibel-Palade bodies (WPBs) are dynamic endothelial cell organelles that contain procoagulant and proinflammatory mediators, such as von Willebrand factor (VWF), and are released in response to cell stresses. VWF mediates platelet adhesion and aggregation, and has been implicated as a procoagulant component of the innate immune response. Objective To determine the influence of histones and DNA on WPB release, and characterize their association in models of inflammation. Methods We treated C57BL/6J mice and cultured endothelial cells with histones (unfractionated, lysine-rich or arginine-rich) and DNA, and measured WPB exocytosis. We used inhibitors to determine a mechanism of histone-induced WPB release in vitro. We characterized the release of DAMPs and WPBs in response to acute and chronic inflammation in human and murine models. Results and conclusions Histones, but not DNA, induced the release of VWF (1.46-fold) from WBPs and caused thrombocytopenia (0.74-fold), which impaired arterial thrombus formation in mice. Histones induced WPB release from endothelial cells in a caspase-dependent, calcium-dependent and charge-dependent manner, and promoted platelet capture in a flow chamber model of VWF-platelet string formation. The levels of DAMPs and WPB-released proteins were elevated during inflammation, and were positively correlated in chronic inflammation. These studies showed that DAMPs can regulate the function and level of VWF by inducing its release from endothelial WPBs. This DAMP-WPB axis may propagate immunothrombosis associated with inflammation.

In vitro and in vivo evaluation of the effect of elevated factor VIII on the thrombogenic process.

Factor VIII (FVIII), a procoagulant cofactor, plays a crucial role in the intrinsic coagulation cascade. A causal association between elevated FVIII levels and venous thrombosis incidence has been established; no such association has been confirmed with arterial thrombosis. The independent role of elevated FVIII levels in arteriolar thrombosis was evaluated in a mouse model to determine the thrombogenic potential of elevated levels of FVIII. The in vitro thrombogenic effect of elevated FVIII levels was examined using thrombin-antithrombin (TAT) complex generation and thromboelastography (TEG) assays. The thrombogenic potential of acute and extended elevation of circulating FVIII levels was assessed using ferric chloride induced injury of the cremaster arterioles. The rate of TAT complex formation, and the final concentration of TAT complexes, significantly increased as FVIII levels were elevated from 100% to 400% FVIII activity. TEG analysis of fibrin and clot formation showed that as FVIII levels were elevated, the time to initial fibrin formation decreased and the rate of fibrin formation increased. The acute elevation of circulating FVIII to 400% FVIII activity resulted in significantly decreased times to vessel occlusion. Prolonged elevation of FVIII activity did not significantly affect time to vessel occlusion. In conclusion, acute elevations in FVIII levels result in a non-linear thrombogenic effect, with non-significant increases in thrombogenic risk within the physiological range (FVIII levels up to 200%). Prolonged elevation of plasma FVIII did not further increase the thrombogenic potential of elevated FVIII levels.

Vascular defects in gain-of-function fps/fes transgenic mice correlate with PDGF- and VEGF-induced activation of mutant Fps/Fes kinase in endothelial cells.

BACKGROUND: Fps/Fes is a cytoplasmic tyrosine kinase that is abundantly expressed in the myeloid, endothelial, epithelial, neuronal and platelet lineages. Genetic manipulation in mice has uncovered potential roles for this kinase in hematopoiesis, innate immunity, inflammation and angiogenesis. OBJECTIVE: We have utilized a genetic approach to explore the role of Fps/Fes in angiogenesis. METHODS: A hypervascular line of mice generated by expression of a 'gain-of-function' human fps/fes transgene (fps(MF)) encoding a myristoylated variant of Fps (MFps) was used in these studies. The hypervascular phenotype of this line was extensively characterized by intravital microscopy and biochemical approaches. RESULTS: fps(MF) mice exhibited 1.6-1.7-fold increases in vascularity which was attributable to increases in the number of secondary vessels. Vessels were larger, exhibited varicosities and disorganized patterning, and were found to have defects in histamine-induced permeability. Biochemical characterization of endothelial cell (EC) lines derived from fps(MF) mice revealed that MFps was hypersensitive to activation by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). CONCLUSIONS: MFps mediates enhanced sensitization to VEGF and PDGF signaling in ECs. We propose that this hypersensitization contributes to excessive angiogenic signaling and that this underlies the observed hypervascular phenotype of fps(MF) mice. These phenotypes recapitulate important aspects of the vascular defects observed in both VEGF and angiopoietin-1 transgenic mice. The fps/fes proto-oncogene product therefore represents a novel player in the regulation of angiogenesis, and the fps(MF) line of mice constitutes a unique new murine model for the study of this process.

Hemodynamic and proinflammatory actions of endothelin-1 in guinea pig small intestine submucosal microcirculation.

The hemodynamic and proinflammatory effects of endothelin-1 (ET-1) in proximal (1st/2nd order) and terminal (3rd/4th order) arterioles and venules were examined in small intestine submucosa of anesthetized guinea pigs. Vessel diameter (D), red blood cell velocity, and blood flow (Q) were determined in eight proximal and eight terminal microvessels before and at 20 min of ET-1 suffusion (10(-10), 10(-9), and 10(-8) M) and then with endothelin-A (ET(A))-receptor blockade with BQ-123 (10(-5) M). This protocol was repeated with platelet-activating factor (PAF) inhibition (WEB-2086, 1.0 mg/kg iv; n = 16). The ET-1-mediated microvascular responses were also examined with endothelin-B (ET(B))-receptor blockade using BQ-788 (10(-5) M; n = 11) alone or with ET(A+B)-receptor blockade with BQ-123 + BQ-788 (n = 10). Microvascular permeability was assessed by FITC-albumin (25 mg/kg iv) extravasation in seven series: 1) buffered modified Krebs solution suffusion (n = 6), 2) histamine suffusion (HIS; 10(-3) M, n = 5), 3) ET-1 suffusion (10(-8) M, n = 5), 4) BQ-123 (10(-5) M) plus ET-1 suffusion (n = 5), 5) PAF inhibition before ET-1 suffusion (n = 5), 6) histamine-1 (H1)-receptor blockade (diphenhydramine, 20 mg/kg iv) before ET-1 suffusion (n = 5), and 7) ET(B)-receptor blockade before (BQ-788 10(-5) M; n = 3) or with ET-1 suffusion (n = 3). D and Q decreased at 10(-8) M ET-1 and returned to control values with BQ-123 and BQ-123+BQ788 but not with BQ-788 in proximal microvessels. D did not change in terminal microvessels with ET-1 (10(-8) M) but decreased with BQ-788 and increased with BQ-123. PAF inhibition did not affect the D and Q responses of proximal microvessels to ET-1 but prevented the fall in Q in terminal microvessels with ET-1. ET-1 increased vascular permeability to approximately 1/3 of that with HIS; this response was prevented with BQ-123 and WEB-2086 but not with H1-receptor blockade. This is the first evidence that submucosal terminal microvessel flow is reduced with ET-1 independent of vessel diameter changes and that this response is associated with increased microvascular permeability mediated via ET(A)-receptor stimulation and PAF activation.

Endothelial modulation of skeletal muscle blood flow and VO(2) during low- and high-intensity contractions.

In the present study, we determined whether endothelin (ET)-1 contributed to the observed reduction in muscle blood flow (Q) during contractions with nitric oxide synthase (NOS) inhibition and whether muscle O(2) uptake (VO(2)) would be affected by the decrease in muscle Q with NOS inhibition at different contraction intensities. Muscle Q, VO(2), O(2) extraction ratio (OER), and tension development (TD) were studied in the in situ gastrocnemius muscle preparation in anesthetized dogs. A decrease in the VO(2)-to-TD ratio (VO(2)/TD) was used as an indicator of O(2) limitation. Three contraction protocols were used: 1) isometric twitch contractions at 2 twitches (tw)/s, 2) the same contractions at 4 tw/s, and 3) pretreatment with an ET(A)-receptor antagonist (BQ-123) before 2 tw/s contractions. The muscle was stimulated to contract, and measures were obtained at steady state (approximately 5-8 min). NOS inhibition (N(omega)-nitro-L-arginine methyl ester) was then induced, and measures were repeated at 2, 5, 10, and 15 min. During 2 tw/s contractions, NOS inhibition reduced Q with and without ET(A)-receptor blockade. In both groups, OER increased in response to the fall in Q, with the result being no change in VO(2)/TD. NOS inhibition also decreased Q during 4 tw/s contractions, but OER did not increase, resulting in a reduction in VO(2)/TD 5 and 15 min after N(omega)-nitro-L-arginine methyl ester. These data indicated that 1) a reciprocal increase in ET-1 during NOS inhibition does not influence active hyperemia in skeletal muscle, and 2) during 4 tw/s contractions, the ischemia with NOS inhibition was associated with either an O(2) limitation or an alteration in the efficiency of muscle contractions.

Receptor-mediated vascular and metabolic actions of endothelin-1 in canine small intestine.

The effects of endothelin-1 (ET-1) infusion on blood flow (QG) and O2 uptake (VO2G) were examined in the small intestine of anesthetized dogs (n = 10). Arterial and venous flows of a gut segment were isolated, and the segment was perfused at constant pressure. Arterial and gut venous blood samples were taken, gut perfusion pressure and QG were measured, and O2 extraction ratio (OERG) and VO2G were calculated. ET-1 was infused (0.118 microgram. kg-1. min-1 ia) throughout the experiment. In group 1 (n = 5), ETA receptors were blocked using BQ-123 (0.143 mg. kg-1. min-1 ia) followed by blockade of ETB receptors with BQ-788 (0.145 mg. kg-1. min-1 ia). The order of ETA and ETB receptor blockade was reversed in group 2 (n = 5). In group 1, the decrease in QG observed with ET-1 infusion was partially reversed with BQ-123; no further change occurred after BQ-788 administration. In group 2, addition of BQ-788 to the infusate further decreased QG, whereas addition of BQ-123 returned QG to a value not different from that with ET-1 infusion alone. These data indicated that ET-1-induced vasoconstriction in the gut was mediated via ETA receptors and that this constriction was buffered by activation of ETB receptors. VO2G decreased in proportion to the decrease in QG with ET-1, decreased further with ET-1 plus ETB receptor blockade (group 2), and increased in proportion to the increases in QG with ETA receptor blockade (both groups). No changes in OERG occurred during ETA and ETB receptor antagonism in either group. This study is the first to demonstrate that a flow-limited decrease in gut VO2G occurred with infusion of ET-1 in gut vasculature. An intriguing and novel finding was that, during O2 limitation, OERG was only 50% of that normally associated with ischemia in this tissue.

Understanding perioperative nursing.

Perioperative nursing is a complex arena that involves various roles and procedures. The authors here argue that the key to success is good multidisciplinary communication.

Endothelial modulation of neural sympathetic vascular tone in canine skeletal muscle.

The effect of nitric oxide synthase (NOS) inhibition and endothelin-A (ETA)-receptor blockade on neural sympathetic control of vascular tone in the gastrocnemius muscle was examined in anesthetized dogs under conditions of constant flow. Muscle perfusion pressure (MPP) was measured before and after NOS inhibition (Nomega-nitro-L-arginine methyl ester; L-NAME) and ETA-receptor blockade [cyclo-(D-Trp-d-Asp-Pro-D-Val-Leu); BQ-123]. Zero and maximum sympathetic nerve activities were achieved by sciatic nerve cold block and stimulation, respectively. In group 1 (n = 6), MPP was measured 1) before nerve cold block, 2) during nerve cold block, and 3) during nerve stimulation. Measurements under these conditions were repeated after L-NAME and then BQ-123. The same protocol was followed in group 2 (n = 6) except that the order of L-NAME and BQ-123 was reversed. MPP and muscle vascular resistance (MVR) increased after L-NAME and then decreased to control values after BQ-123. MVR decreased after BQ-123 alone and, with the addition of L-NAME, increased to a level not different from that observed during the control period. MVR fell during nerve cold block. This response was not affected by administration of L-NAME followed by BQ-123, but it was attenuated by administration of BQ-123 before L-NAME. The constrictor response during sympathetic nerve stimulation was enhanced by L-NAME; no further effect was observed with BQ-123, nor was the response affected when BQ-123 was given first. These findings indicate that endothelin contributes to 1) basal vascular tone in skeletal muscle and 2) the increase in skeletal muscle vascular resistance after NOS inhibition. Finally, nitric oxide "buffers" the degree of constriction in skeletal muscle vasculature during maximal sympathetic stimulation.

Role of endothelial factors in active hyperemic responses in contracting canine muscle.

We investigated whether endothelium-derived relaxing factor (EDRF) and prostaglandins, which may be released under conditions of increased blood flow, contribute to the active hyperemia in contracting muscle of anesthetized dogs. The venous outflow from the left gastrocnemius muscle was isolated and measured. The tendon was cut and placed in a force transducer. One group served as a control (Con; n = 9); EDRF synthesis was inhibited using N omega-nitro-L-arginine methyl ester (L-NAME) in a second group (n = 9), and a third group (n = 7) received L-NAME and indomethacin (L-NAME+Indo) to inhibit prostaglandin synthesis. After resting measurements, the distal end of the cut sciatic nerve was stimulated to produce isometric contractions at 1, 2, 4, and 6 twitches/s for 6-8 min, separated by 25-min recovery periods. Blood flow and O2 uptake increased linearly from resting values of 11.8 +/- 2.4 and 0.3 +/- 0.05 ml.100 g-1.min-1, respectively, to maximal values of 84.2 +/- 5.1 and 11.1 +/- 0.7 ml.100 g-1.min-1 in the Con group; neither these values nor those for tension development were different from values observed at comparable contraction frequencies in the L-NAME and L-NAME+Indo groups. At rest, resistance was greater (P < 0.05) in both the L-NAME and L-NAME+Indo groups compared with Con, the highest value (P < 0.05) occurring in the L-NAME+Indo group. Muscle resistance decreased (P < 0.05) in all groups at all contraction frequencies; the values were not different among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Canine hindlimb blood flow and O2 uptake after inhibition of EDRF/NO synthesis.

The nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) was used to determine whether the decrease in canine hindlimb blood flow (QL) with NOS inhibition would limit skeletal muscle O2 uptake (VO2). Arterial inflow and venous outflow from the hindlimb were isolated, and the paw was excluded from the circulation. Pump perfusion from the right femoral artery kept the hindlimb perfusion pressure near the auto-perfused level. Six anesthetized dogs received L-NAME (20 mg/kg i.v.), whereas another group of five dogs received the stereospecific enantiomer N omega-nitro-D-arginine methyl ester (D-NAME 20 mg/kg i.v.). Efficacy of NOS inhibition was tested with intra-arterial boluses of acetylcholine. QL was measured continuously, and whole body and hindlimb VO2 were measured 60 and 120 min after L-NAME or D-NAME. Whole body VO2 remained at control levels, but cardiac output decreased from 117 +/- 17 to 57 +/- 7 ml.kg-1.min-1 60 min after L-NAME (P < 0.05) and remained at that level for the duration of the experiment. Cardiac output was significantly higher in the D-NAME group than in the L-NAME group at 60 min. After L-NAME, QL fell 24% but VO2 increased from 5.2 +/- 0.4 to 7.4 +/- 0.6 ml.kg-1.min-1 (P < 0.05). No change in QL or VO2 occurred after D-NAME. NOS inhibition did not limit hindlimb VO2, despite decreases in blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)