Adamts-18 Is a Novel Candidate Gene Of Vascular Development That Is Related To Aggravated Thrombosis: Evidence From a Adamts-18 Knock Out Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 31-31
Author(s):  
Wei Zhang ◽  
Dawei Bu ◽  
Suying Dang ◽  
Tao Hong ◽  
Thomas Wisniewski
Keyword(s):  
Blood Flow ◽  
Carotid Artery ◽  
Middle Cerebral Artery ◽  
Cerebral Artery ◽  
Common Carotid Artery ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Surface Display

Abstract HIV-ITP patients have a unique antibody (Ab) against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). The search for a physiologic ligand that could induce this reaction was undertaken by panning the GPIIIa49-66 peptide with a phage surface display 7-mer peptide library. From 20 positive clones, 1 had 70% identity with a C-terminal region of ADAMTS-18 (a disintegrin and metalloproteinase with thrombospondin (TSR)-like motif 18), which is secreted by endothelial cell (EC). The recombinant C-terminal fragment of ADAMTS-18 can completely dissolve platelet aggregates formed in vitro. Moreover, this fragment lyses thrombi formed in the cerebral artery of mice and reduces infarction and neurologic impairment in murine ischemic stroke model (Blood 113: 6051, 2009). However, whether ADAMTS-18 represents the dominant physiologic mechanism controlling thrombus dissolution in vivo remains to be clarified. Here, we used ADAMTS-18-deficient (ADAMTS-18-/-) mice to study the contributions of ADAMTS-18 to thrombus formation in vivo. To investigate possible functional differences between WT and ADAMTS-18-/- platelets, we tested WT and ADAMTS-18-/- platelets in a model of pulmonary thromboembolism induced by infusion of a mixture of platelet agonist collagen (25 µg per mouse) and epinephrine (1 µg per mouse). In lung tissue Hematoxylin and eosin-stained (HE) slides, the mean number of thrombi per lung was same in the ADAMTS-18-/- group compared with WT group (163.7 ±14.38 vs 174.9 ±11.73, n=30/group, P=0.5480). In vitro, there is no difference between WT and ADAMTS-18-/- platelet aggregation trace and activation initiated by various platelet agonists ADP (10 µM) or collagen (2 µg/mL). No difference was noted on WT and ADAMTS-18-/- platelet adhesion on immobilized ligand (fibrinogen). These results indicated ADAMTS-18 had no effect on platelet function. We next evaluate the effect of ADAMTS-18 on thrombus formation in a second well-established carotid artery thrombosis model, which is induced by 10% FeCl3 patch. In the process of surgical operation, we unexpectedly observed that all ADAMTS-18-/- mice have premature common carotid artery bifurcation compared with WT mice. A Doppler flow monitor showed ADAMTS-18-/- mice exhibited significantly reduced carotid artery blood flow than WT mice (ADAMTS-18-/- vs WT, 0.5 ± 0.11 vs 0.75 ± 0.21 mL/min, n=7/group, P=0.0298), which results in shortened time of thrombus formation (ADAMTS-18-/- vs WT, 452.17 ± 68.88 vs 611.43 ± 92.02 sec, n=7/group, P=0.0005 ). Immunohistochemistry staining showed that the common carotid artery of ADAMTS-18-/- mice had increased adventitial collagen deposition compared with WT mice. In vivo matrigel plug assay demonstrated that ADAMTS-18-/- mice had significantly lower density of blood vessels compared to the WT mice. Since the middle cerebral artery arises from the internal carotid artery, we conjecture that ADAMTS-18-/- mice would have aggravated brain infarction for the less cerebral blood flow supplying. This proved to be true. In transient middle cerebral artery occlusion (tMCAO) model, the infarction size in ADAMTS-18-/- mice was significantly larger than in WT mice (mean infarction %, 25.68 ± 4.13 vs 17.41 ± 3.24, n=8, P=0.0012). Taken together, these observations suggest vasculature is the potential site of action of ADAMTS-18. To our knowledge, this is the first validation study of linkage and association of ADAMTS-18 as a pro-vasculature gene that is related to aggravated thrombosis. Disclosures: No relevant conflicts of interest to declare.

CHANGES IN BLOOD FLOW OF COMMON CAROTID ARTERY AND MIDDLE CEREBRAL ARTERY DURING EXERCISE

1999 ◽  
Vol 31 (Supplement) ◽  
pp. S194
Author(s):  
N. Murase ◽  
T. Katsumura ◽  
T. Osada ◽  
T. Hamaoka ◽  
Y. Yamamoto ◽  
...  
Keyword(s):  
Blood Flow ◽  
Carotid Artery ◽  
Middle Cerebral Artery ◽  
Cerebral Artery ◽  
Common Carotid Artery

scholarly journals Thrombus Imaging Using 3D Printed Middle Cerebral Artery Model and Preclinical Imaging Techniques: Application to Thrombus Targeting and Thrombolytic Studies

Pharmaceutics ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1207
Author(s):  
Andrea Vítečková Wünschová ◽  
Adam Novobilský ◽  
Jana Hložková ◽  
Peter Scheer ◽  
Hana Petroková ◽  
...  
Keyword(s):  
Middle Cerebral Artery ◽  
Cerebral Artery ◽  
Imaging Techniques ◽  
Barium Sulphate ◽  
Flow Conditions ◽  
Preclinical Imaging ◽  
Blood Clots ◽  
3D Printed

Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque®) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.

scholarly journals DAKS1, a Kunitz Scaffold Peptide from the Venom Gland of Deinagkistrodon acutus Prevents Carotid-Artery and Middle-Cerebral-Artery Thrombosis via Targeting Factor XIa

2021 ◽  
Vol 14 (10) ◽  
pp. 966
Author(s):  
Zhiping Jia ◽  
Yunyang Liu ◽  
Xiaoru Ji ◽  
Yizheng Zheng ◽  
Zhengyang Li ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Middle Cerebral Artery ◽  
Cerebral Artery ◽  
Inhibitory Activity ◽  
Bleeding Risk ◽  
Venom Gland ◽  
Artery Occlusion ◽  
Factor Xia ◽  
Deinagkistrodon Acutus

Scaffold-based peptides (SBPs) are fragments of large proteins that are characterized by potent bioactivity, high thermostability, and low immunogenicity. Some SBPs have been approved by the FDA for human use. In the present study, we developed SBPs from the venom gland of Deinagkistrodon acutus (D. acutus) by combining transcriptome sequencing and Pfam annotation. To that end, 10 Kunitz peptides were discovered from the venom gland of D. acutus, and most of which peptides exhibited Factor XIa (FXIa) inhibitory activity. One of those, DAKS1, exhibiting strongest inhibitory activity against FXIa, was further evaluated for its anticoagulant and antithrombotic activity. DAKS1 prolonged twofold APTT at a concentration of 15 μM in vitro. DAKS1 potently inhibited thrombosis in a ferric chloride-induced carotid-artery injury model in mice at a dose of 1.3 mg/kg. Furthermore, DAKS1 prevented stroke in a transient middle cerebral-artery occlusion (tMCAO) model in mice at a dose of 2.6 mg/kg. Additionally, DAKS1 did not show significant bleeding risk at a dose of 6.5 mg/kg. Together, our results indicated that DAKS1 is a promising candidate for drug development for the treatment of thrombosis and stroke disorders.

Abstract W P93: MiR-122 Improves Stroke Outcomes after Middle Cerebral Artery Occlusion in Rats

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
DaZhi Liu ◽  
Glen C Jickling ◽  
Bradley P Ander ◽  
Heather Hull ◽  
Xinhua Zhan ◽  
...  
Keyword(s):  
Middle Cerebral Artery ◽  
Cerebral Artery ◽  
Messenger Rna ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Neurological Impairment ◽  
Artery Occlusion ◽  
Neuroprotective Effects ◽  
Genomic Studies

MicroRNA (miRNA) are recently discovered small (~22 nucleotides), non-coding RNA that regulate translation of messenger RNA (mRNA) to protein. Though there are only hundreds of miRNAs, each of them can potentially regulate hundreds of target genes, via base-pairing with complementary sequences in mRNA. This provides one approach that targets a single miRNA to have effects on multiple genes. Our previous genomic studies have demonstrated that miR-122 decreased significantly in blood of experimental strokes produced by middle cerebral artery (MCA) occlusion in rats as well as in blood of patients with ischemic strokes. Therefore, we hypothesized that elevating blood miR-122 has the potential for treating stroke. Using the newly developed in vivo polyethylene glycol-liposome based miRNA transfection system and rat suture MCAO occlusion model, we show that injection of chemically modified mimic miR-122 (600ug/rat, i.v.) through tail vein immediately after MCAO occlusion significantly decreases the neurological impairment and significantly attenuates brain infarct volumes. Ongoing studies are identifying the target genes that are associated with the neuroprotective effects of miR-122 following stroke. Acknowledgements: This study was supported by NIH grant R01NS066845 (FRS). There were no conflicts of interest.

STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 485-485
Author(s):  
Firdos Ahmad ◽  
Lucia Stefanini ◽  
Timothy Daniel Ouellette ◽  
Teshell K Greene ◽  
Stefan Feske ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Critical Role ◽  
Phosphatidyl Serine ◽  
Flow Chamber ◽  
Procoagulant Activity ◽  
Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

A Locally Administered Novel Heparin Complex Attenuates Collagen-Induced Platelet Activation and Thrombosis In Vivo,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3361-3361
Author(s):  
Riitta Lassila ◽  
Annukka Jouppila ◽  
Ulla M Marzec ◽  
Stephen R Hanson
Keyword(s):  
Blood Flow ◽  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Ptfe Graft ◽  
Thrombin Time ◽  
Thrombosis Model ◽  
Baboon Model ◽  
Induced Aggregation

Abstract Abstract 3361 We have developed a semi-synthetic antithrombotic heparin complex, APL001, to mimic mast cell-derived natural heparin proteoglycans (HepPG). HepPG attenuate platelet-collagen interactions under blood flow by inhibiting VWF- and GPIIb/IIIa -mediated platelet aggregation. In addition, rat-derived HepPG arrest platelet thrombus growth on collagen surfaces or at vascular injury sites, both in vitro and in vivo (Lassila et al.ATVB 1997, Kauhanen et al. ATVB 2000, Olsson et al. Thromb Haemost 2002). Our objective was to study the inhibitory capacity of APL001 for preventing human platelet aggregation in vitro and acute thrombosis in a baboon model in vivo. The effects of unfractionated heparin (UFH) and APL001 were compared in relevant coagulation assays (APTT, PT, thrombin time, anti-FXa activity, fibrinogen, FVIII:C and VWF activity (VWF:RCo) and antigen). Additionally, agonist-induced (collagen, ristocetin and ADP) platelet aggregation in citrate or hirudin-anticoagulated whole blood (Multiplate®) (n=10 healthy subjects), and platelet function analysis (PFA100®) in citrated platelet rich plasma (PRP) were assessed. In a well-established baboon thrombosis model a collagen-coated PTFE graft (length 2 cm, lumen 4 mm) was placed in an arterio-venous shunt. Prior to blood contact the thrombogenic surface was treated for 10 min with UFH or APL001 (both at 4 mg/mL). Thrombus formation was initiated by exposing the surface to blood flow (100 mL/min, shear rate 265−1), and the deposition of 111-In-labeled platelets and of fibrin was quantified continuously over 1h. Fibrin thrombus accumulation was assessed from the incorporation of circulating 125-I-fibrinogen. In the heparin-relevant coagulation tests APL001 was comparable or 20–30% more potent than UFH while FVIII, fibrinogen and VWF variables remained unaltered. In contrast to UFH, APL001 (300 μg/mL) consistently inhibited collagen- and ristocetin-induced platelet aggregation, whereas UFH had only a modest effect in comparison with PBS control (Table). ADP-induced aggregation was unaffected. Comparable results were observed in the PRP aggregation assay. PFA100 testing also demonstrated inhibitory effects. In the in vivo thrombosis model (n=4) APL001 reduced platelet deposition on collagen (vs. the results with UFH) by 34% (p=0.01), while platelet accumulation in distal propagated thrombus was reduced by 61% (p=0.16). APL001-treated surfaces accumulated 45% less fibrin than the UFH-treated surfaces (p=0.008). In conclusion, when compared with UFH APL001 inhibited both collagen- and ristocetin-induced platelet aggregation in human blood, while anticoagulant properties were comparable. In the absence of systemic antithrombotic drugs, exposure of APL001 to a highly thrombogenic collagen surface arrested thrombus formation in an in vivo baboon model. This finding suggests that locally administered APL001 alone, due to its dual antiplatelet and anticoagulant effects, may limit the growth and size of thrombus and thereby prevent subsequent thrombo-occlusion.TableAnticoagulantInhibition-% of platelet aggregation ± SDConc. 300 μg/mLnColl (3.2 μg/mL)Ristocetin (0.77 mg/mL)ADP (6.4 μM)CitrateAPL0011033 ± 1543 ± 166 ± 24UFH1011 ± 1323 ± 153 ± 7p value0.0030.0100.700HirudinAPL0011032 ± 1043 ± 178 ± 10UFH108 ± 1116 ± 166 ± 9p value0.0000.0020.600 Disclosures: Lassila: Aplagon: Chief Scientific Advisor.

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