Thrombosomes Show Dose-Dependent Increase in Thrombus Formation in a Model of Deep Arterial Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2319-2319
Author(s):  
Nikhil Vilas Joshi ◽  
Jennifer Raftis ◽  
AJ Lucking ◽  
Narendra Tandon ◽  
M Fitzpatrick ◽  
...  
Keyword(s):  
Whole Blood ◽  
Immunohistochemical Staining ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Arterial Injury ◽  
Dependent Manner ◽  
Flow Conditions ◽  
Structural And Functional Properties ◽  
Dose Dependent

Abstract Abstract 2319 Introduction Thrombosomes are novel lyophilized human platelet derivatives that retain a number of key platelet structural and functional properties. Building on preliminary in vitro studies, we here sought to investigate whether Thrombosomes, suspended in platelet free plasma (PFP), would enhance and be incorporated in thrombus generated under flow conditions within a validated and well-characterised model of deep arterial injury. Methods PFP was obtained by centrifuging citrated whole blood from six healthy non-smoking volunteers and filtering with a 0.22μm filter. Thrombosomes were suspended in 60 mL of PFP to generate final concentrations of 20 and 200 × 106Thrombosomes /mL. Immediately prior to use, 1.2 mL of 1M CaCl2 was added to permit fibrin generation. Thrombus formation was assessed using the Badimon Chamber at low (212 s−1) and high (1690 s−1) shear rates with porcine aortic tunica media as the thrombogenic substrate. Total thrombus area and platelet-rich area were measured histomorphometrically using conventional and immunohistochemical staining respectively. Fluorescent labeled Thrombosomes were added to the extracorporeal circuit in the Badimon chamber to study the ex vivo thrombus generation in the whole blood. Electron microscopy of Thrombosomes and platelets was undertaken. Results Thrombosomes contributed towards thrombus formation in whole human blood as evidenced by incorporation of fluorescent-labeled Thrombosomes into the thrombus. Immunohistochemical staining of the glycoprotein IIb/IIIa receptor confirmed incorporation of Thrombosomes into the thrombus. In the high shear chamber, mean thrombus area increased in a dose-dependent manner following the addition of Thrombosomes (704 μm2 [95% CI, 224 – 1184 μm2], 1511 μm2 [95% CI, 687 – 2336 μm2] and 2378 μm2 [95% CI, 1567 – 3189 μm2] for PFP and Thrombosomes at concentrations of 20 and 200 × 106/mL respectively [P= 0.003]). In the low shear chamber, total thrombus area for the PFP was 4962 μm2 (95% CI, 2489 – 7434 μm2). The addition of Thrombosomes at concentrations of 20 and 200 × 106/mL led to a numerical increase in mean thrombus area to 6170 μm2 (95% CI, 3944 – 8397 μm2) and 7504 μm2 (95% CI, 3864 – 11144 μm2) respectively, although this was not statistically significant (P= 0.2969). Conclusions Thrombosomes suspended in PFP and exposed to injured arterial tissue under physiologically relevant flow conditions are incorporated into thrombus and enhance thrombus formation in a dose dependent manner. These findings act as impetus to undertake clinical studies of this rehydrated, lyophilized infusible hemostatic platelet product. Disclosures: Fitzpatrick: Cellphire Inc.: Employment, Equity Ownership. Feuerstein:Cellphire: Consultancy. Newby:Cellphire: Research Funding.

Antithrombotic Activity of SR 46349, a Novel, Potent and Selective 5-HT2 Receptor Antagonist

1993 ◽  
Vol 69 (03) ◽  
pp. 262-267 ◽  
Author(s):  
J M Herbert ◽  
A Bernat ◽  
G Barthelemy ◽  
F Dol ◽  
M Rinaldi
Keyword(s):  
Oral Administration ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Venous Shunt ◽  
Induced Aggregation ◽  
Dose Dependent

SummarySR 46349 (trans-4-[(3Z)3-(2-dimethylaminoethyl)oxyimino-3(2-fluorophenyl)propen-1-yl] phenol, hemifumarate) is the first member of a newly-developed 5-HT2 antagonist series. SR 46349 potently inhibited serotonin-induced aggregation of rabbit and human platelets (IC50 = 1 and 3.9 nM respectively) but had no effect on the action of other platelet aggregating agents. SR 46349 was 118 and 25 times more potent than ketanserin against 5-HT + epinephrine-induced aggregation of rabbit and human platelets respectively.A single per os administration of SR 46349 (1 mg/kg) resulted in a strong inhibition of 5-HT + epinephrine-induced platelet aggregation in the rabbit as measured ex vivo (67% inhibition, 6 h after the administration). Intravenous or oral administration of SR 46346 inhibited in a dose-dependent manner venous thrombosis induced by ligature of the jugular vein of rabbits whose blood was made hypercoagulable by i.v. administration of tissue thromboplastin. The doses of SR 46349 which inhibited 50% of thrombus formation were 1.5 ± 0.8 mg/kg and 17 ± 0.5 mg/kg after i.v. or oral administration respectively. When given i.v. to rabbits, SR 46349 exhibited a dose-dependent antithrombotic effect in an arterio-venous shunt model. Significant increase of the bleeding time was observed after the i.v. administration of 5 mg/kg of SR 46349 (3-fold increase). In dogs, SR 46349 inhibited cyclic coronary artery blood flow variations, complete abolition of CFVs being achieved after the i.v. administration of 0.5 mg/kg.In conclusion, SR 46349 is a highly potent, selective antagonist of serotonin in vitro and is to be considered as a potent, orally active antithrombotic agent.

Lyophilised reconstituted human platelets increase thrombus formation in a clinical ex vivo model of deep arterial injury

2012 ◽  
Vol 108 (07) ◽  
pp. 176-182 ◽  
Author(s):  
Jennifer Raftis ◽  
Andrew Lucking ◽  
Amanda Hunter ◽  
Mike Millar ◽  
Mike Fitzpatrick ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Morphological Characteristics ◽  
Principal Component ◽  
Arterial Injury ◽  
Human Platelets ◽  
Dependent Manner ◽  
Ex Vivo Model ◽  
Electron Microscopic

SummaryPlatelets are the principal component of the innate haemostatic system that protect from traumatic bleeding. We investigated whether lyophilised human platelets (LHPs) could enhance clot formation within platelet-free and whole blood environments using an ex vivo model of deep arterial injury. Lyophilised human platelets were produced from stored human platelets and characterised using conventional, fluorescent and electron microscopic techniques. LHPs were resuspended in platelet-free plasma (PFP) obtained from citrated whole human blood to form final concentrations of 0,20 and 200 × 109 LHPs/L. LHPs with recalcified PFP or whole blood were perfused through the chamber at low (212 s-1) and high (1,690 s-1) shear rates with porcine aortic tunica media as thrombogenic substrate. LHPs shared morphological characteristics with native human platelets and were incorporated into clot generated from PFP or whole blood. Histomorphometrically measured mean thrombus area increased in a dose-dependent manner following the addition of LHPs to PFP under conditions of high shear [704 μm2 ± 186 μm2 (mean ± SEM), 1,511 μm2 ± 320 μm2 and 2,378 μm2 ± 315 μm2, for LHPs at 0, 20 and 200 × 109 /l, respectively (p= 0.012)]. Lyophil-ised human platelets retain haemostatic properties when reconstituted in both PFP and whole blood, and enhance thrombus formation in a model of deep arterial injury. These data suggest that LHPs have the potential to serve as a therapeutic intervention during haemorrhage under circumstances of trauma, and platelet depletion or dysfunction.

Platelet ADP receptors contribute to the initiation of intravascular coagulation

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 594-600 ◽  
Author(s):  
Catherine Leon ◽  
Meike Alex ◽  
Antje Klocke ◽  
Eberhard Morgenstern ◽  
Christine Moosbauer ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Intravascular Coagulation ◽  
Adp Receptors ◽  
Adp Receptor ◽  
Deficient Mice ◽  
Storage Pools

Abstract While the adenosine 5′-diphosphate (ADP) pathway is known to enhance thrombus formation by recruiting platelets and leukocytes to the primary layer of collagen-adhering platelets, its role for the initiation of coagulation has not been revealed. Ex vivo inhibition of the P2Y12 ADP receptor by clopidogrel administration diminished the rapid exposure of tissue factor (TF), the major initiator of coagulation, in conjugates of platelets with leukocytes established by the contact of whole blood with fibrillar collagen. Under in vitro conditions, the P2Y12 and P2Y1 ADP receptors were both found to be implicated in the exposure of TF in collagen-activated whole blood. Immunoelectron-microscopy revealed that collagen elicited the release of TF from its storage pools within the platelets. Functional activation of the intravascular TF was reduced by inhibition of the ADP receptors, partially due to the disruption of the platelet-neutrophil adhesions. Injection of collagen into the venous system of mice increased the number of thrombin-antithrombin complexes, indicative for the formation of thrombin in vivo. In P2Y1-deficient mice, the ability of collagen to enhance the generation of thrombin was impaired. In conclusion, the platelet ADP pathway supports the initiation of intravascular coagulation, which is likely to contribute to the concomitant formation of fibrin at the site of the growing thrombus.

Platelet Deposition Induced by Severely Damaged Vessel Wall Is Inhibited by a Boroarginine Synthetic Peptide with Antithrombin Activity

1994 ◽  
Vol 71 (04) ◽  
pp. 511-516 ◽  
Author(s):  
J J Badimon ◽  
D Weng ◽  
J H Chesebro ◽  
V Fuster ◽  
L Badimon
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Synthetic Peptide ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Blood Platelet ◽  
Flow Conditions ◽  
Local Flow ◽  
Platelet Deposition ◽  
Damaged Vessel

SummaryThrombin plays a key role in platelet activation and thrombosis. Specific inhibition of thrombin appears to be one of the best approaches to prevent thrombus formation. We have studied the effects of a synthetic a-aminoboronic acid derivative - [Ac, (D) Phe-Pro-Boro-Arg-Hydrocloric acid] - on platelet deposition on severely damaged arterial wall. Platelet deposition was evaluated under well characterized rheological conditions in an original perfusion chamber and detected by autologous mIn-labeled platelets. The study was performed “in vivo” in a porcine model of arterial thrombosis triggered by severely damaged vessel wall at blood flow conditions mimicking mild stenosis (1690 s−1) and patent (212 s−1) vessels. In addition, ex-vivo platelet aggregation activity was evaluated by whole blood impedance aggregometry using collagen, ADP and thrombin as agonists. The synthetic a-aminoboronic peptide was intravenously administered as a bolus followed by continuous infusion. Ex vivo thrombin-induced whole blood platelet aggregation was totally abolished, while ADP- and Collagen-induced whole blood platelet aggregation was not modified. The effects of the synthetic antithrombin on platelet deposition were evaluated in native blood (non-anticoagulated) conditions and in combination with heparin. Under both experimental conditions, the synthetic peptide significantly inhibited platelet deposition at local flow conditions of both high (1690 s−1) and low (212s−1) shear rates. Our results suggest that specific inhibition of locally generated thrombin might be a good strategy to prevent platelet dependent arterial thrombus formation independently of the local flow shear rate of the area at risk.

Are ovine Leydig cells able to aromatize androgens?

1997 ◽  
Vol 9 (2) ◽  
pp. 193 ◽  
Author(s):  
Barbara Biliska ◽  
Marcin Le´sniak ◽  
Barbara Schmalz
Keyword(s):  
Aromatase Inhibitor ◽  
Incubation Period ◽  
Immunohistochemical Staining ◽  
Leydig Cells ◽  
Culture Medium ◽  
Aromatase Activity ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Testosterone Synthesis

The conversion of testosterone into oestradiol by ovine Leydig cells culturedin vitrowas studied using the non-steroidal aromatase inhibitor CGS 16949A. Additionally, aromatase activity was detected by immunohistochemical staining of cultured Leydig cells or cryosections. The cells were obtained from testes of Polish Mountain rams 5–6 months old (immature) or 12–15 months old (mature). Leydig cells were cultured alone (controls) or incubated for 6 h in the presence of testosterone. Aromatase inhibitor was then added to the cultures which were incubated for a further 18 h. After a 24-h incubation period, testosterone and oestradiol secretion were determined by testing the culture medium using radioimmunological methods. The addition of testosterone to the culture medium enhanced oestradiol synthesis, suggesting that exogenous testosterone could also be aromatized to oestradiol by ovine Leydig cells in vitro. In the presence of CGS 16949A, the conversion of testosterone to oestradiol was significantly suppressed in a dose-dependent manner. All Leydig cells obtained from testes of mature rams and stained immunohistochemically were positive for aromatase, whereas Leydig cells from immature males were negative. The localization of immunoreactive aromatase appeared to be dependent on the age of the donor ram. It is suggested therefore, that mature Leydig cells in the ram are not only the site for testosterone synthesis, they are also capable of converting androgens into oestrogens.

In Vitro Evaluation of the Effect of rFVIIa on Anti Coagulated Human Whole Blood Treated with Abciximab Using Thromboelastography (TEG).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1114-1114
Author(s):  
Dorthe Viuff ◽  
Soeren Andersen ◽  
Brian Lauritzen ◽  
Mirella Ezban
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Ex Vivo ◽  
Maximum Amplitude ◽  
Bleeding Complications ◽  
Clot Lysis ◽  
Anova Model ◽  
Vitro Effect ◽  
Dose Dependent

Abstract Abciximab inhibits platelet aggregation by binding to GPIIb/IIIa. Thus, Abciximab is effective in prevention and treatment of thromboembolic events. Although major bleeding complications are rare, Abciximab has the potential to increase bleeding which often is managed by temporary discontinuation of administration of drug. However, an effective and instant haemostatic intervention may be needed in some cases of acute serious bleeding. The aim of this study was to investigate 1) the effect of in vitro addition of Abciximab to whole blood (WB) from 8 healthy donors, 2) to determine the haemostatic potential of ex vivo addition of 10, 25 (25nM≈90ug/kg), and 100nM rFVIIa (NovoSeven®, Novo Nordisk) to WB treated with 4μg/ml of Abciximab (ReoPro®, Lilly) and 3) to compare the response of rFVIIa in TEG assays containing tissue factor (TF, Innovin 1:42500) or Kaolin (standard initiator provided by Haemsoscope) with or without tPA (0.75nM). TEG Platelet Mapping was performed on all Abciximab-treated blood samples before conducting TEG demonstrating 70–100% inhibition of ADP-induced platelet aggregation. The following TEG parameters were used: Reaction time (R-time), Maximum Thrombin generation (MTG), Maximum Amplitude (MA) and Area under the TEG fibrinolysis curve calculated from MA (AUC lysis). The statistical analysis was performed by a two-way ANOVA model. A significant effect of adding rFVIIa (10 nM) to Abciximab-treated blood was observed for all clot formation parameters and with both TF and Kaolin as initiator. rFVIIa demonstrated a significant dose-dependent improvement of the TEG parameters in the assay containing kaolin, in kaolin+tPA (except MA) and in TF (except for MTG). rFVIIa showed significant dose-dependent protection against clot lysis (AUC lysis) in the assay containing kaolin+tPA, whereas no significant effect on clot lysis was observed in the assay containing TF. In conclusion, addition of rFVIIa to WB treated with Abciximab significantly improves most of the TEG parameters. Comparison of Abciximab+rFVIIa TEG parameters with TEG parameters obtained in normal WB (n=59) showed normalization of the R-value, however, none of the other TEG parameters reached normal values although a significant effect was observed after addition of rFVIIa. The TEG assay using kaolin as initiator showed significant dose-dependent effect of rFVIIa on all TEG parameters. In contrast, in the TEG assay initiated with TF the dose-dependent response of rFVIIa was only significant for some of the parameters. This may be due to a strong initiating effect of TF in the assay thus lowering the sensibility to rFVIIa. In contrast, in the assay initiated with Kaolin only TF-independent effects of rFVIIa are measured. The design of a TEG assay therefore seems to be important for evaluating the in vitro effect of rFVIIa. The clinical relevance of these findings is currently unknown and needs to be evaluated further. TEG values (±SD) using the kaolin assay R (sec) MTG (mm×100/sec) MA (mm) Lysis AUC (+tPA) (mm2) *: Significant effect compared to the Abciximab treated blood sample. P<0.05 Normal value 359±63 14.4±2.2 60±6 31707 Abciximab treated 527±56 7.6±2.3 33±10 14725±5110 rFVIIa 10nM 458±76* 8.3±2.1* 35±10* 15837±4634* rFVIIa 25nM 413±42* 9.2±2.7* 37±11* 16120±4969* rFVIIa 100nM 383±41* 9.0±2.2* 37±10* 18658±5693*

scholarly journals EXTH-62. PRECLINICAL EFFICACY OF THE IMIPRIDONE ONC-206 AGAINST MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii100-ii101
Author(s):  
Tobey MacDonald ◽  
Anshu Malhotra ◽  
Jingbo Liu ◽  
Hongying Zhang ◽  
Matthew Schneiderjan ◽  
...  
Keyword(s):  
Cell Death ◽  
Malignant Glioma ◽  
Ex Vivo ◽  
Clinical Results ◽  
Normal Brain ◽  
Dependent Manner ◽  
Group 3 ◽  
Dose Dependent

Abstract Treatment for medulloblastoma (MB) is typically ineffective for MYC amplified or metastatic SHH, Group 3 and 4 subgroups. Promising preclinical and clinical results have been obtained for adult and pediatric malignant glioma treated with ONC-201, a selective antagonist of DRD2, a G-protein coupled receptor that regulates prosurvival pathways. Herein, we report the activity of ONC-201 and ONC-206, which has increased non-competitive antagonism of DRD2, against MB. We treated three different MB cell types representative of SHH- and Group 3-like cells, with varied levels of DRD2 expression, and consistently observed increased cell death in a dose-dependent manner at lower doses of ONC-206 compared to ONC-201. We also evaluated ClpP as an additional drug target in MB. ClpP is a mitochondrial protease that has been shown to directly bind and be activated by ONC 201, and is highly expressed at the protein level across pediatric MB, malignant glioma and ATRT, but not normal brain. We observed that similar to ONC-201, ONC-206 treatment of MB cells induces the restoration of mitochondrial membrane potential to the non-proliferative state, degradation of the mitochondrial substrate SDHB, reduction in survivin and elevation in ATF4 (integrated stress response). Importantly, ONC-206 treatment induced significant cell death of patient-derived SHH, WNT, and Group 3 tumors ex vivo and Group 4 cells in vitro, while having no observable toxicity in normal brain. ONC-206 treatment of a transgenic mouse model of Shh MB in vivo significantly reduces tumor growth and doubles survival time in a dose-dependent manner following 2 weeks of therapy. Additional in vivo data will be reported in preparation for a planned Phase I study of ONC-206 in children with malignant brain tumors.

Malignant transformation in melanocytes is associated with increased production of procoagulant microvesicles

2011 ◽  
Vol 106 (10) ◽  
pp. 712-723 ◽  
Author(s):  
Luize Lima ◽  
Andreia Oliveira ◽  
Luiza Campos ◽  
Martin Bonamino ◽  
Roger Chammas ◽  
...  
Keyword(s):  
Malignant Transformation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Biological Effects ◽  
Flow Cytometric Analysis ◽  
Dependent Manner ◽  
Initiator Protein ◽  
Prothrombinase Complex ◽  
Thrombosis Model

SummaryShedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte- derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation- related protumoural responses.

scholarly journals Novel Structurally-Modified Allosteric Effectors of Hemoglobin Exhibit Superior Antisickling Properties

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 218-218
Author(s):  
Osheiza Abdulmalik ◽  
Tanvi Deshpande ◽  
Mohini Ghatge ◽  
Yan Zhang ◽  
Jurgen Venitz ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Phase I ◽  
Sickle Cell ◽  
Clinical Studies ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Cell Disease ◽  
Hb S ◽  
Dose Dependent

Abstract Sickle cell disease (SCD) continues to cause significant morbidity, mortality and healthcare disparities. Despite considerable progress in understanding the underlying pathophysiology and investigating various therapeutic strategies, novel pharmacologic approaches to ameliorate SCD continue to hold immense potential and promise, especially for patients in developing countries. Our group and others have recently renewed and refocused attention to candidate drugs that directly bind to hemoglobin (Hb) and increase oxygen (O2) affinity, preventing the fundamental pathophysiology of the disease, i.e., sickle Hb (Hb S) polymerization and red blood cell (RBC) sickling. While several candidate drugs have shown biological activity in-vitro, ex-vivo and in animal studies, their ultimate success in clinical studies was hampered by toxicity concerns and/or low oral bioavailability. Recent promising reports from a phase I/II study on 5-HMF renews optimism for this therapeutic approach. We reasoned that modifications of vanillin--a previously reported antisickling agent and food constituent without known toxicities--to enhance its efficacy, would represent a feasible approach in rationally developing clinically useful candidate drugs. Consequently, we designed and synthesized two classes of compounds: INN and TD series. The former are pyridyl derivatives of vanillin, rationalized to stereospecifically inhibit deoxy-Hb S polymer formation while increasing the fraction of the soluble oxy-Hb S in regions of low O2 tension. The TD compounds represent further modification of corresponding INN compounds (with a methoxyl group on the pyridine ring), rationalized to exhibit similar dual antisickling effects, but with enhanced direct polymer destabilization properties. We subjected a prototypical compound from each class (INN-270 and TD-7) to our battery of exploratory in-vitro assays, specifically: 1) rates of Hb S binding/modification, 2) corresponding change in O2 affinity, 3) direct inhibition of Hb S polymerization, and 4) inhibition of RBC sickling under hypoxia. We incubated 0.5, 1, or 2 mM of either INN-270 or TD-7 with RBCs from patients with homozygous SCD, under hypoxia (4% O2/96% N2 gas mixture) in a shaker-incubator at 37 ˚C for 3 h. Assays were conducted in at least three replicates utilizing different samples on different days. At the conclusion of each assay, aliquot samples (~ 10 μl each) were drawn into a fixing solution under hypoxia to preserve RBC morphology for analyses. Residual RBC suspensions were washed, hemolyzed, and subjected to: cation-exchange HPLC (to determine Hb modification); P50 analyses to establish change in O2 affinity; and temperature-dependent delay time studies to establish a delay in Hb S polymerization. Our results show that both compounds permeated RBC membranes without causing hemolysis, bound to and modified intracellular Hb at high levels in a dose dependent manner, increased O2 affinity significantly, and inhibited sickling of RBCs under hypoxia. TD-7 modified Hb S in a dose-dependent manner (to 92.3 ± 5.2 %, n=4 at 2 mM), shifted O2 equilibrium to the left (Δp50 = 45.6 ± 8.2 %, n=3 at 2 mM), and inhibited RBC sickling (by 95 -100 %, n=4). Preliminary delay time analyses also showed that at 2 mM, TD-7 increased the Hb S polymerization times from 18.1 ± 1.0 min to 24.5 ± 0.5 min. INN-270 showed a similar profile, however with a lower efficacy (at 2 mM) for Hb S modification (to ~ 75 %), Δp50 of 40.3 %, sickling inhibition by ~ 70 %, and increased delay times from 15.6 ± 0.5 min to 19.7 ± 1.0 min. We have elucidated the dual antisickling mechanism of action of INN-270 and TD-7 by X-ray crystallography. Two molecules of each compound bind to Hb via Schiff-base, and a series of hydrogen-bond/hydrophobic interactions that favor a high-O2-affinity Hb state. Importantly, the methoxyl group on the pyridine ring of TD-7 forms hydrogen-bond interactions with the surface-located αF-helix, resulting in a conformational change, possibly explaining the improved potency. Based on our results, both TD7 and INN 270 exhibited greater than a 40- and 3-fold superiority in efficacy compared to vanillin and 5-HMF, respectively. We conclude that our findings justify a prospective, structure-based approach to designing novel antisickling agents with enhanced potency. In-vitro/ex-vivo murine and human PK/PD studies are currently ongoing to help guide planned in-vivo PK/PD studies in mice. Disclosures Venitz: Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: Consultancy. Safo:Baxter and AesRx companies have licensed our patented antisickling compounds. Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: #7160910; #7119208 Patents & Royalties, Consultancy, Research Funding.

Functional imaging of shear-dependent activity of ADAMTS13 in regulating mural thrombus growth under whole blood flow conditions

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1295-1298 ◽  
Author(s):  
Yasuaki Shida ◽  
Kenji Nishio ◽  
Mitsuhiko Sugimoto ◽  
Tomohiro Mizuno ◽  
Masaaki Hamada ◽  
...  
Keyword(s):  
Blood Flow ◽  
Shear Rate ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Arterial Occlusion ◽  
Generation Process ◽  
Flow Conditions ◽  
Normal Hemostasis

Abstract The metalloprotease ADAMTS13 is assumed to regulate the functional levels of von Willebrand factor (VWF) appropriate for normal hemostasis in vivo by reducing VWF multimer size, which directly represents the thrombogenic activity of this factor. Using an in vitro perfusion chamber system, we studied the mechanisms of ADAMTS13 action during platelet thrombus formation on a collagen surface under whole blood flow conditions. Inhibition studies with a function-blocking anti-ADAMTS13 antibody, combined with immunostaining of thrombi with an anti-VWF monoclonal antibody that specifically reflects the VWF-cleaving activity of ADAMTS13, provided visual evidence for a shear rate–dependent action of ADAMTS13 that limits thrombus growth directly at the site of the ongoing thrombus generation process. Our results identify an exquisitely specific regulatory mechanism that prevents arterial occlusion under high shear rate conditions during mural thrombogenesis.

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