Meghan T. Walsh

ADAMTS13: a new link between thrombosis and inflammation.

von Willebrand factor (VWF) levels are elevated and a disintegrin-like and metalloprotease with thrombospondin type I repeats–13 (ADAMTS13) activity is decreased in both acute and chronic inflammation. We hypothesized that by cleaving hyperactive ultralarge VWF (ULVWF) multimers, ADAMTS13 down-regulates both thrombosis and inflammation. Using intravital microscopy, we show that ADAMTS13 deficiency results in increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in inflamed veins. Both processes were dependent on the presence of VWF. Depletion of platelets in Adamts13−/− mice reduced leukocyte rolling, suggesting that platelet interaction with ULVWF contributes to this process. Increased levels of endothelial P-selectin and plasma VWF in Adamts13−/− compared with wild-type (WT) mice indicated an elevated release of Weibel-Palade bodies. ULVWF multimers released upon stimulation with histamine, a secretagogue of Weibel-Palade bodies, slowed down leukocyte rolling in Adamts13−/− but not in WT mice. Furthermore, in inflammatory models, ADAMTS13 deficiency resulted in enhanced extravasation of neutrophils, and this process was also dependent on VWF. Our findings reveal an important role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade body secretion, and in the regulation of leukocyte adhesion and extravasation during inflammation.

Differential stimulation of monocytic cells results in distinct populations of microparticles

Background: Microparticles (MPs), small vesicles shed from stimulated cells, permit cross‐talk between cells within a particular environment. Their composition is thought to reflect their cell of origin, and differs according to whether they are produced by stimulation or by apoptosis. Whether MP properties vary according to stimulus is not yet known. Methods: We studied the characteristics of MPs produced from monocytic THP‐1 cells upon stimulation with lipopolysaccharide or a soluble P‐selectin chimera, using proteomics, flow cytometry, western blotting, and electron microscopy. Results: Utilizing a novel criterion of calcein‐AM staining to define MPs, we found that MP populations were similar with respect to size, presence and organization of cytoskeleton, and expression of certain antigens. The MPs shared the same level of procoagulant activity. We found that MPs also have distinct characteristics, depending on stimuli. These include differences in phosphatidylserine expression and expression of proteins from specific subcellular locations such as the mitochondria, and of unique antigens such as leukocyte‐associated immunoglobin‐like‐receptor (LAIR)‐1, which was found only upon stimulation with the soluble P‐selectin chimera. Conclusion: We found that the properties of MPs depend on the stimulus that produced them. This supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.

Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion

AIMS Oxidative stress accompanies inflammatory and vascular diseases. The objective of this study was to explore whether reactive oxygen species can activate shedding of platelet receptors and thus suppress platelet function. METHODS AND RESULTS Hydrogen peroxide and glucose oxidase were chosen to model oxidative stress in vitro. We demonstrate that oxidative damage activated tumour necrosis factor-alpha-converting enzyme (TACE) and induced shedding of its targets, glycoprotein (GP) Ibalpha and GPV, in murine and human platelets. Also, 12-HpETE, a peroxide synthesized in the platelet lipoxygenase pathway, induced TACE-mediated receptor cleavage. The TACE activation was independent of platelet activation, as alpha-granule secretion, activation of alphaIIbbeta3, or phosphatidylserine expression was not observed. TACE activation induced by hydrogen peroxide was dependent on p38 mitogen-activated protein kinase signalling, whereas protein kinase C, phosphoinositide 3-kinase, and caspases were not involved. Inhibition of p38 cytoplasmic targets, phospholipase A(2) and heat shock protein 27, did not prevent shedding, whereas blocking 12-lipoxygenase or Src kinase slightly inhibited TACE activation. The loss of the GPIbalpha receptor induced by oxidative stress rendered platelets unable to incorporate into a growing thrombus in vivo. CONCLUSION Oxidative stress can render platelets functionally less active by shedding key adhesion receptors via the activation of p38. This suggests that oxidative injury of platelets may attenuate their function.

Prothrombotic Effects of Fibronectin Isoforms Containing the EDA Domain

Objective—Fibronectin (FN) plays an important role in the formation of stable arterial thrombi at the site of vascular injury. FN containing Extra Domain A (EDA+FN) is absent from normal plasma, but elevated plasma levels of EDA+FN are found in several pathological conditions. We hypothesized that EDA+FN plays a special role in thrombosis. Methods and Results—We used mouse strains constitutively including (EDA+/+) or excluding (EDA−/−) the EDA domain in all tissues and plasma. Using a flow chamber and the ferric-chloride injury model we found that EDA+FN accelerates thrombosis both in vitro and in vivo at arterial shear rates. In EDA+/+ mice thrombi (>30 &mgr;m) grew faster when compared with EDAWT/WT (6.6±0.2 minutes versus 8.3±0.6 minutes, P<0.05) and the mean vessel occlusion time was shorter (9.9±0.4 minutes versus 14.6±1.7 minutes, P<0.05). However, the presence of EDA+FN affected neither single platelet adhesion to subendothelium nor thrombosis in veins. In addition, the mortality rate of EDA+/+ mice after collagen/epinephrine infusion was twice that of EDAWT/WT or EDA−/− mice. Conclusions—Our findings reveal that EDA+FN has prothrombotic activity, and its presence in plasma may worsen pathological conditions in which this form is elevated.

The effect of C1 inhibitor on intestinal ischemia and reperfusion injury

Complement activation and neutrophil stimulation are two major components in events leading to ischemia and reperfusion (IR) injury. C1 inhibitor (C1INH) inhibits activation of each of the three pathways of complement activation and of the contact system. It is also endowed with anti-inflammatory properties that are independent of protease inhibition. The goal of these studies was to investigate the role and mechanism of C1INH in alleviating IR-induced intestinal injury. C57BL/6, C1INH-deficient (C1INH(-/-)), bradykinin type 2 receptor-deficient (Bk2R(-/-)), and C3-deficient mice (C3(-/-)) were randomized into three groups: sham operated control, IR, and IR + C1INH-treated groups. Ischemia was generated by occlusion of the superior mesenteric artery followed by reperfusion. C1INH or reactive center-cleaved inactive C1INH (iC1INH) was injected intravenously before reperfusion. IR resulted in intestinal injury in C57BL/6, C1INH(-/-), Bk2R(-/-), and C3(-/-) mice with significantly increased neutrophil infiltration into intestinal tissue. In each mouse strain, C1INH treatment reduced intestinal tissue injury and attenuated leukocyte infiltration compared with the untreated IR group. C1INH inhibited leukocyte rolling in the mesenteric veins of both C57BL/6 and C3-deficient mice subjected to IR. C1INH treatment also improved the survival rate of C57BL/6 and C1INH(-/-) mice following IR. Similar findings were observed in the IR animals treated with iC1INH. These studies emphasize the therapeutic benefit of C1INH in preventing intestinal injury caused by IR. In addition to the protective activities mediated via inhibition of the complement system, these studies indicate that C1INH also plays a direct role in suppression of leukocyte transmigration into reperfused tissue.

Absence Of Adamts13 On A Vwf Deficient Background Is No Longer Prothrombotic In A Murine Model Of Arterial Thrombosis

ISTH07A1_P-S-278: Contact View [P-S-278] ABSENCE OF ADAMTS13 ON A VWF DEFICIENT BACKGROUND IS NO LONGER PROTHROMBOTIC IN A MURINE MODEL OF ARTERIAL THROMBOSIS A.K. Chauhan, M.T. Walsh, D.G. Motto, D. Ginsburg, D.D. Wagner. Department of Pathology, Harvard Medical School, CBR Institute for Biomedical Research, Boston; Department of Internal Medicine, University of Iowa, Iowa; Department of Internal Medicine, University of Michigan, Michigan, United States Introduction: In circulation Ultra-Large von Willebrand factor (UL-VWF) is cleaved by ADAMTS13 into a series of smaller multimers. We have shown that ADAMTS13 deficiency in the mouse results in accelerated thrombosis in injured arterioles. We reasoned that if VWF is the only ADAMTS13 substrate relevant for this model, then VWF deficiency would result in the abrogation of the effect of ADAMTS13 deficiency. Alternatively, if we observe an effect of ADAMTS13 deficiency on thrombus formation in the absence of VWF, this would suggest the existence of an ADAMTS13 substrate (s) in addition to VWF. Methods: We crossed Adamts13-/mice on a VWF-/genetic background. Using the ferric chloride injury model we compared experimental thrombosis in arterioles of Adamts13-/-/VWF-/mice with VWF -/mice by intravital microscopy. Results: Since Adamts13-/mice of mixed genetic background were used in early studies, we first confirmed our previous findings of accelerated thrombus formation, in Adamts13 -/on C57BL6/J background. In the Adamts13-/mice, thrombi >20 μm were seen at 5.7 ± 0.5 min as compared to 7.9 ± 0.4 min in wild-type (WT) mice (P 20 μm in Adamts13-/-/ VWF-/(10.7 ± 0.4 min) was similar to VWF-/mice (10.6 ± 0.7 min). The rate of individual thrombus growth in Adamts13-/-/VWF-/was also similar to VWF-/thrombi. After 40 min of observation, thrombi failed to grow to occlusive size in either the Adamts13-/-/VWF-/or VWF-/mice. Conclusions: The absence of ADAMTS13 in the setting of VWF-deficiency is no longer prothrombotic in arterial thrombosis, suggesting that at least in this model system, VWF is the only ADAMTS13 substrate relevant to thrombus growth and stability at arterial shear. Chauhan AK, Walsh MT, Motto DG, Ginsburg D, Wagner DD. ABSENCE OF ADAMTS13 ON A VWF DEFICIENT BACKGROUND IS NO LONGER PROTHROMBOTIC IN A MURINE MODEL OF ARTERIAL THROMBOSIS. J Thromb Haemost 2007; 5 Supplement 2: P-S-278 Date: Sunday, July 8, 2007 Session Info: Poster: TTP/HUS and ADAMTS 13 Room: Exhibition Area file:///E|/working/jaymehra/Poster%20TTPHUS%20and%...3/Abstract%20ISTH07A1_P-S-278%20Contact%20View.htm (1 of 2) [1/16/2014 7:07:02 PM]