scholarly journals Identification of the Protein S Binding Site in the Factor IXa Serine Protease Domain Presents Therapeutic Possibilities

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2274-2274
Author(s):  
William E. Plautz ◽  
Satish Sekhar ◽  
Rinku Majumder ◽  
John P. Sheehan
Keyword(s):  
Conflicts Of Interest ◽  
Dissociation Constants ◽  
Ex Vivo ◽  
Factor Xa ◽  
Protein S ◽  
Heparin Binding ◽  
Binding Interaction ◽  
Serine Protease Domain ◽  
Factor Ixa ◽  
Inhibitory Site

Abstract Protein S (PS) has been shown to inhibit Factor IXa (FIXa); however, its binding sites on FIXa have not been fully elucidated. By structural analysis of FIXa in conjunction with previous studies of FIXa's ability to bind heparin, an FIXa inhibitor, we identified that multiple residues that make up the heparin binding exosite, including Lys126, Asn129, Lys132, Arg150, Arg165, and Arg170 (chymotrypsin numbering), might contribute to binding PS; these residues were substituted with either Ala or Leu in separate FIXa mutants. Factor Xa (FXa) generation assays along with fluorescence analyses provided both the catalytic efficiencies and dissociation constants for the FIXa mutants under conditions of varied PS. Furthermore, anisotropic measurements of the fluorescent DEGR-FIXa's binding interaction with PS, when titrated against each of the FIXa mutants, supported the data found in the previous assays. These studies suggested that multiple residues, which are critical for the binding of heparin, such as Lys126, Lys132, Arg165, and Arg170, are also required in binding PS. To additionally verify this data, further ex vivo studies, including co-immunoprecipitation and flow cytometry of PS and FIXa are underway. Although previous studies have shown that the first EGF-like domain of FIXa is required to bind PS, these novel results have not only localized the main inhibitory site of PS on FIXa, but also provided enough information to propose a binding mechanism, which may rely on the reciprocal association between the head groups of PS and FIXa with the other's EGF-like domains, forming a bridging structure upon the cell membrane. Further studies are required to verify this hypothesis, but when clarified, it could provide immense benefits in the production of therapeutics for thrombosis. Disclosures No relevant conflicts of interest to declare.

Lyso-Sulfatide Binds Factor Xa and Potently Inhibits Thrombin Generation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1130-1130
Author(s):  
Subramanian Yegneswaran ◽  
Yajnavalka Banerjee ◽  
Jose A. Fernandez ◽  
Hiroshi Deguchi ◽  
John H. Griffin
Keyword(s):  
Free Amino ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Coagulation System ◽  
Amino Group ◽  
Thrombin Time ◽  
Binding Studies ◽  
Binding Interaction ◽  
Dependent Inhibition ◽  
Gla Domain

Abstract Abstract 1130 Although phospholipids are well-recognized for their effects on coagulation reactions, little is generally known about the effects of sphingolipids on clotting pathways. Negatively-charged sulfatides can potently initiate the intrinsic pathway of coagulation system by binding and autoactivating factor (f) XII. Sphingosine potently inhibits the ability of factor Xa (fXa) to generate thrombin (fIIa) in the prothrombinase complex (II-ase) (fXa/fVa/phospholipids) by interacting directly with fXa's Gla domain. Here we report that lyso-sulfatide (lyso-SF) (sulfogalactosyl sphingosine), a lipid of minor abundance in plasma that is primarily in HDL particles, exhibits potent anticoagulant activity. Lyso-SF dose-dependently prolonged clotting in fXa-1-stage but not thrombin-time clotting assays. Lyso-SF inhibited II-ase activity by > 90 % in purified reaction mixtures (fXa/fVa/II) in the presence of 6 or 30 μM phospholipids (PL). However, lyso-SF did not inhibit fIIa generation by fXa/fVa in the absence of PL, suggesting the absolute requirement of PL for lyso-SF-dependent inhibition of fIIa generation. Lyso-SF inhibited fIIa generation by fXa/PL in the absence of fVa. Additionally, lyso-SF inhibited fIIa generation by Gla-domainless (gd)-fXa in the presence but not in the absence of fVa and PL. Lyso-SF-dependent inhibition of fIIa generation was also observed for fXa/fVa/PL when gd-II was used as the substrate instead of II. However, no inhibition by lyso-SF was observed when using gd-fXa/PL and gd-II/PL in the presence or absence of fVa. Lyso-SF had no effect on fXa or fIIa amidolytic activity. These data plus other studies suggested that ≥ two components of the II-ase complex needed to be PL-bound for potent inhibition of fIIa generation by lyso-SF. PL surfaces bind and assemble each the II-ase protein components; however, PL's and lyso-SF may also alter the conformations of fXa, fVa and II. To gain mechanistic insights for lyso-SF inhibition of II-ase activity, Surface Plasmon Resonance (SPR) and fluorescence spectroscopy were used to define molecular interactions. Remarkably, SPR binding studies showed that lyso-SF binds to immobilized fXa (KD = 83 μM) and gd-fXa (KD = 36 μM). Controls using SPR showed no binding of lyso-SF to immobilized fVIIa or fIXa whereas SPR confirmed the ability of fXa, fVIIa and fIXa to bind PL's. Fluorescence binding assays confirmed SPR data showing that lyso-SF bound to and altered the dansyl fluorescence of dansyl-GluGlyArg-labeled fXa (DEGR-fXa) both in the presence (KD = 50 μM) and absence (KD = 75 μM) of PL and that this binding required calcium ions. Thus, lyso-SF binds fXa outside the Gla domain. Fluorescence monitoring of fVa binding to DEGR-fXa in the presence of PL showed that lyso-SF inhibited this binding interaction. To characterize structure-activity relationships for lyso-SF inhibition of II-ase, different analogs of lyso-SF were tested for their ability to inhibit fIIa generation by gd-fXa/fVa/PL. Psychosine (galactosyl sphingosine), glucosyl sphingosine and lyso-sphingomyelin each inhibited fIIa generation showing that the sulfate ester moiety and the sugar group in lyso-SF were not essential for the anticoagulant effects of lyso-SF. However, acetylation of the free amino group in lyso-SF ablated its inhibition of fIIa generation showing that the free amino group on carbon 2 is essential for the inhibitory activity of lyso-SF. In conclusion, these findings show that lyso-SF and several of its analogs are potent anticoagulant lipids and that the mechanism for inhibition of fXa by lyso-SF may involve its binding to fXa at sites outside fXa's Gla domain. This suggests that certain sphingolipids may exert allosteric downregulation of fXa activity without inhibiting the enzyme's active site or the binding of the Gla domain to PL surfaces. Disclosures: No relevant conflicts of interest to declare.

Protein S As a Potential Treatment for FIX-Derived Deep Vein Thrombosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2277-2277
Author(s):  
Vijaya Satish Sekhar Pilli ◽  
Willium Plautz ◽  
Rinku Majumder ◽  
Paolo Simioni
Keyword(s):  
Deep Vein Thrombosis ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Protein S ◽  
Factor Ix ◽  
Wild Type ◽  
Vein Thrombosis ◽  
Deep Vein

Abstract Background: Every year, 0.1-0.2% of the USA population experiences deep vein thrombosis (DVT). Two causes of DVT are increased Factor IX (FIX) levels and hyperactivating mutations in FIX (FIX Padua variant- R338L and Malmo variant T148A). In principle, inhibition of activated FIX (FIXa) should alleviate DVT. Previous in vitro studies demonstrated that the anticoagulant Protein S (PS) inhibits the intrinsic pathway mediated by wild type FIXa, making PS an attractive candidate to treat DVT. Aims: To establish Protein S as a remedy for FIX-mediated DVT/Padua/Malmo Methods: Anisotropy, clotting assays, thrombin generation assays, co-localization, co-immunoprecipitation, and bleeding assays. Results: We further explored the physiological relevance of the PS-FIXa interaction and PS-mediated inhibition of FIXa by ex vivo (co-immunoprecipitation) and in vivo (co-localization) studies. Because PS can inhibit FIXa in vivo, we used competitive, direct anisotropy assays and co-immunoprecipitation assays to measure the efficiency PS and hyperactive FIXa (R338L) interaction. Interestingly, the results demonstrated that FIXa R338L has lost its affinity towards PS compared with wild type FIXa. The same finding was obtained by ex vivo thrombin generation assays and FXa generation assays supplemented with various concentrations of PS. Thus, to be inhibited, hyperactive FIX requires a greater amount of PS compared with wild type FIXa. We are further confirming this finding with mouse models. Conclusion: Addition of PS to plasma inhibits both wild type and R338L FIXa and extends clotting time. Previous studies showed that the addition of PS has no significant negative effects. Thus, we conclude that PS supplementation potentially constitutes a novel and effective treatment for FIX-mediated DVT. Disclosures No relevant conflicts of interest to declare.

Deletion of Pleiotrophin in LepR+ Perivascular Cells Depletes Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1491-1491
Author(s):  
Heather A Himburg ◽  
Vivian Y. Chang ◽  
Joshua Sasine ◽  
Jenny Kan ◽  
Liman Zhao ◽  
...  
Keyword(s):  
Steady State ◽  
Stromal Cells ◽  
Leptin Receptor ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Heparin Binding ◽  
Perivascular Cells ◽  
Hematopoietic Stem

Abstract Pleiotrophin (PTN) is a heparin binding growth factor which is expressed by bone marrow vascular endothelial cells (BM ECs) and perivascular stromal cells. Treatment of murine or human HSCs ex vivo promotes HSC expansion (Nat Med. 2010 Apr;16(4):475-82) and constitutive deletion of PTN depletes LT-HSCs in steady state and markedly impairs HSC regeneration following myeloablation (Cell Rep. 2012 Oct 25;2(4):964-75; JCI. 2014;18(7):1123-1129). Here, we sought to determine which BM microenvironment cell is responsible for PTN-mediated maintenance of the HSC pool. Utilizing the Cre-loxP system, we deleted PTN from VE-cadherin+ ECs, leptin receptor+ (lepR+) perivascular stromal cells, osteocalcin+ osteoblasts, and vav1+ hematopoietic cells and examined the effects on hematopoiesis. We observed no differences in steady state hematopoiesis or HSC content as measured by long-term competitive repopulation assays in mice lacking PTN expression in osteocalcin+ cells, vav1+ hematopoietic cells or VE-cadherin+ BM ECs. However, deletion of PTN from lepR+ BM perivascular cells caused a significant decrease in BM c-kit+sca-1+lin- cells (KSL cells) and BM SLAM+KSL HSCs, and colony forming cell (CFC) content compared to PTN+/+ controls (*p = 0.04, 0.04, and 0.001, respectively). Importantly, deletion of PTN in lepR+ cells, caused a significant reduction in long-term HSC content as measured by primary and secondary competitive repopulation assays (*P<0.01 for all time points through 20 weeks). These data suggest that LepR+ BM perivascular cells, rather than VE-cadherin+ ECs are the primary source of PTN in the BM niche which contributes to the maintenance of the HSC pool. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anticoagulant Protein S Targets the Factor IXa Heparin-Binding Exosite to Prevent Thrombosis

2018 ◽  
Vol 38 (4) ◽  
pp. 816-828 ◽  
Author(s):  
William E. Plautz ◽  
Vijaya Satish Sekhar Pilli ◽  
Brian C. Cooley ◽  
Rima Chattopadhyay ◽  
Pamela R. Westmark ◽  
...  
Keyword(s):  
Protein S ◽  
Heparin Binding ◽  
Factor Ixa

Protein S Down-Regulates Factor VIIIa by Competing with Factor IXa.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1714-1714
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Kohei Tatsumi ◽  
Yuri Fujita ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Protein S ◽  
Factor X ◽  
Cleavage Rate ◽  
Physiological Concentration ◽  
C2 Domains ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Km Value

Abstract Factor VIII functions as a cofactor in the factor Xase complex responsible for phospholipid surface-dependent conversion of factor X to factor Xa by factor IXa. Factor VIIIa, activated form by thrombin and factor Xa, is down regulated by activated protein C (APC), and the reaction is enhanced by the presence of protein S, a cofactor for APC. It was previously reported that protein S inactivated directly factor Xa or factor Va, however, the direct regulation of factor VIII by protein S remains to be investigated. In the present study, surface plasmon resonance (SPR)-based assay showed that factor VIII bound directly to immobilized protein S (Kd; 70 nM). The isolated A2 and A3 domains also bound to protein S with similar modest affinity (Kd; 15 and 17 nM, respectively), whilst the isolated A1 and C2 domains failed to bind, suggesting the presence of protein S-binding sites within the A2 and A3 domain. Since it is known that factor IXa also interacts with the A2 and A3 domains in factor VIII, we examined the inhibitory effect of factor IXa on the factor VIII and protein S interaction in a SPR-based assay. Active-site modified (EGR−) factor IXa competitively inhibited the binding of protein S to both the A2 and A3-C1-C2 domains dose-dependently. Furthermore, Western blotting analysis using an anti-A1 monoclonal antibody revealed that Arg336 cleavage in factor VIII by factor IXa in the presence of protein S was slower with an ~1.8-fold lower cleavage rate than that in its absence, supporting that protein S competed the factor IXa interaction with factor VIII. Of interest, the reaction with protein S to factor VIII inhibited the generation of factor Xa dose-dependently in a factor Xa generation assay (IC50; 150 nM). The Km value for factor X obtained with factor Xase complex in the presence of physiological concentration of protein S was 19 nM, which was ~2-fold lower than that in its absence (45 nM). Whilst, the Km value for factor IXa in the presence of protein S was greater than 100 nM, which was ~5000-fold higher than that in its absence (21 pM). We demonstrate that protein S not only contributes to down-regulate factor VIIIa activity as a cofactor for APC, but also impairs the factor Xase complex by competing the binding of factor IXa to factor VIII.

Protein S Regulates Factor IXa/VIIIa Activity Independent of Activated Protein C

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2196-2196
Author(s):  
Rinku Majumder
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Activated Protein C ◽  
Protein S ◽  
Clotting Time ◽  
Independent Manner ◽  
Factor Ixa ◽  
New Findings ◽  
Presence And Absence

Abstract Abstract 2196 Protein S (PS) is a vitamin-K-dependent plasma protein that plays an important role in the feedback regulation of thrombin generation. PS is a well recognized anticoagulant protein, the absence of which causes venous and arterial thrombosis. While PS has been studied for over three decades, the precise role this protein plays in attenuating the hemostatic response is far from clear. PS was initially identified as a cofactor for activated protein C (APC). Interestingly, recent reports suggest PS may function in an APC-independent manner as it has been suggested to inhibit prothrombinase complex formation and act as a stimulator of factor Xa-TFPI (tissue factor pathway inhibitor) complex. Based on these new data, we have begun to examine possible roles of PS outside of the PC pathway which contribute to the regulation of thrombin generation. We recently obtained exciting preliminary data which suggests that PS plays an important role in the inhibition of Xa generation by the intrinsic Xase complex. Our central hypothesis is that PS binds to the enzyme FIXa in the presence of Phosphatidyl serine (PhosSer) containing membranes and inhibits FXa generation in the presence and absence of VIIIa. We did clotting assays with FIX deficient plasma both with and without anti PS antibody. Clotting initiated with 0.1 nM FIXa shows a must faster clotting in the presence of anti PS antibody (57 seconds compared to 107 seconds without the incubation with anti PS antibody) (see Table 1). However, there is no difference in clotting times when the clotting is initiated with FVIIa in the presence and absence of anti PS antibody. Our results showing PS is only able to alter FIXa initiated clotting indicates that PS inhibits the FXa generation by FIXa/FVIIIa in plasma. Table 1: Clotting of FIX deficient plasma initiated with FIXa (A) & initiated with FVIIa (B) (A) Conc of FVIIa nM Clotting time in seconds - AntiPS Antibody Clotting time in seconds + AntiPS Antibody 0.1 107 57 1 46 31 5 31 14.7 10 30 14.4 (B) Conc of FVIIa nM Clotting time in seconds - AntiPS Antibody Clotting time in seconds + AntiPS Antibody 0.1 113 112 1 56 54 5 41 42 10 36 35 We determined the binding of PS to factor IXa by determining the change in anisotropy and fluorescence of DEGR(5 (dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginyl chloromethyl ketone)-IXa. The Kd was ~ 1 nM in both cases suggesting a tight binding of PS with factor IXa. We also determined the activation of FX by FIXa in the presence of FVIIIa and 50 μM Phosphatidylserine membrane in the presence of PS. We used physiological concentrations of FX, PS, 0.1 nM FIXa, 5 nM FVIIIa and monitored the rate of FXa generation by using S2222 (chromogenic substrate). There was ~ 95 % inhibition in the rate of FXa generation by FIXa in the 0.2 presence of FVIIIa. Our results clearly indicate that PS inhibits FXa generation by factors IXa/VIIIa. We believe that these new findings have major implications for better understanding how FXa generation is regulated during the initial phases of coagulation and may shed light on a major outstanding question in the field relating to how a key anticoagulant, PS contributes to the down regulation of the haemostatic response. Disclosures: No relevant conflicts of interest to declare.

SR123781A, a Synthetic Heparin Mimetic

2001 ◽  
Vol 85 (05) ◽  
pp. 852-860 ◽  
Author(s):  
J. P. Hérault ◽  
A. Bernat ◽  
P. Savi ◽  
P. Schaeffer ◽  
P. A. Driguez ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Vena Cava ◽  
Subcutaneous Administration ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Binding Domain ◽  
Heparin Binding ◽  
Venous Shunt

SummarySR123781A, a synthetic hexadecasaccharide comprising an anti-thrombin (AT) binding domain, a thrombin binding domain, and a neutral methylated hexasaccharide sequence, was obtained from glucose through a convergent synthesis. SR123781A showed high affinity for human AT (Kd = 58 ± 22 nM) and was a potent catalyst of its inhibitory effect with regard to factor Xa (IC50 = 77 ± 5 ng/ml – 297 ± 13 U/mg) and thrombin (IC50 = 4.0 ± 0.5 ng/ml – 150 ± 30 U/mg). SR123781A which acted exclusively via AT (no effect via heparin cofactor II at a concentration of 6 g/ml) inhibited thrombin generation occurring via both the extrinsic and intrinsic pathways in vitro in human plasma. SR123781A did not compete for 3H-heparin binding to PF4 and did not activate platelets in the presence of plasma from patients with heparin-induced thrombocytopenia. After intravenous or subcutaneous administration to rats, rabbits or baboons, SR123781A displayed prolonged anti-factor Xa and anti-factor IIa activity ex vivo. After intravenous injection to baboons, decreases of the anti-factor Xa and anti-thrombin activities were parallel and disappeared with the same pharmacodynamics. Intravenous administrations of SR123781A strongly inhibited thrombus formation in an experimental model of thromboplastin-induced venous thrombosis in rats with an ED50 value of 18 ± 0.1 g/kg (vs 77 ± 3 g/kg for heparin). SR123781A inhibited arterial thrombus formation induced on a silk thread in an arterio-venous shunt and in the vena cava (ED50 values of 225 ± 10 and 27 ± 8 g/kg, respectively). Compared to standard and low molecular weight heparin and to presently used drugs, SR123781A exhibited a highly favourable anti-thrombotic/bleeding ratio therefore showing that it might be considered as a promising compound in the treatment and prevention of various thrombotic diseases.

Low Molecular Weight Heparin Modulates the Binding and Reactivity of Factor Xa towards Basic Pancreatic Trypsin Inhibitor: A Surface Plasmon Resonance and Kinetics Study.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1623-1623
Author(s):  
Pierre F. Neuenschwander ◽  
Kimberly J. Baker-Deadmond ◽  
Angela D. Jones
Keyword(s):  
Low Molecular Weight Heparin ◽  
Factor Viia ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
Enhancing Effect ◽  
Factor Ixa ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor ◽  
Carboxymethyl Dextran

Abstract Previous studies by us have shown that the blood coagulation factor IXa is modulated by low molecular weight heparin (LMWH), resulting in greater reactivity of this protease with basic pancreatic trypsin inhibitor (BPTI). Since thrombin, factor Xa and factor VIIa share a high degree of homology with factor IXa and all are capable of binding heparin, we examined the potential ability of LMWH to modulate the reactivity of these coagulation proteases towards BPTI. Recombinant BPTI containing a His6-tag on the amino terminal end was constructed, expressed in bacteria and purified by nickel-chelating affinity chromatography (HisTrap). His6-BPTI had identical inhibitory activity as wild-type BPTI towards both trypsin and factor IXa. Surface plasmon resonance (Biacore 3000) was used to examine the binding and reacitvity of His6-BPTI to various coagulation proteases under different conditions. Initial biacore studies (using factor IXa) suggested that the carboxymethyl dextran moeity of CM5 biacore chips can paritally mimic heparin binding to the coagulation proteases, resulting in all sensed interactions being largely a reflection of the heparin-bound enzyme conformer. Thus, a hydrophobic HPA chip that lacks the carboxymethyl dextran matrix was used to prepare self-assembling monolayer surfaces of 100 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 90 mol% DOPC and 10 mol% of the nickel-chelating phospholipid 1,2 dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} (DOGS-NTA). His6-BPTI was efficiently, specifically and reproducibly captured only onto the surface containing DOGS-NTA. This binding was also dependent on the presence of nickel, and the generated BPTI-phospholipid surface was found to bind trypsin in a BPTI-dependent fashion. Efficient regeneration of the surface was accomplished between cycles by sequential injections of 10 mM EDTA and 10 mM NaOH (to completely remove the enzyme-BPTI complexes) followed by re-loading of the surface with nickel and re-capture of BPTI. Subsequent injections of thrombin, factor IXa, factor Xa, factor VIIa (both in the absence and presence of saturating levels of soluble tissue factor) over the BPTI-phospholipid surface was examined in the absence and presence of 10 μM of the LMWH enoxaparin (15 oligosaccharide units). As previously observed, the binding of factor IXa to BPTI was greatly enhanced by the presence of enoxaparin. Interestingly, the binding of factor Xa to the BPTI surface was also substantially enhanced by enoxaparin, while that of thrombin was only moderately (though consistently) enhanced and that of factor VIIa remained unaffected. Solution-based inhibition studies with factor Xa and BPTI or the isolated second Kunitz-type inhibitor of tissue factor pathway inhibitor (TFPI-K2) confirmed a 2- to 3-fold enhancing effect of enoxaparin on factor Xa reacitivity towards BPTI, with no effect apparent towards TFPI-K2. The enhancing effect of LMWH towards BPTI was also observed with high levels of fondaparinux (100 μM). Although the high levels of fondaparinux required are likely not clinically relevant and presumably reflect the poorer binding of fondaparinux (5 oligosaccharide units) to factor Xa compared with enoxaparin, these results underscore the ability of LMWH to modulate factor Xa reactivity upon occupation of the heparin-binding exosite and may have implications for the rational drug design of specific factor Xa inhibitors.

Pathological Shear Regulates ADAM10 Activity on Circulating Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2194-2194
Author(s):  
Elizabeth E Gardiner ◽  
Jianlin Qiao ◽  
Cheewee Tan ◽  
Jane Frances Arthur ◽  
Mohammad Al-Tamimi ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Factor Xa ◽  
Fold Increase ◽  
Human Platelets ◽  
Calcium Flux ◽  
Short Exposure ◽  
Protective Mechanism

Abstract Abstract 2194 Metalloproteinase-mediated ectodomain shedding of platelet-specific receptors for collagen (GPVI) and von Willebrand factor (GPIbα of the GPIb-IX-V complex) is triggered by either ligand-induced platelet activation-dependent pathways, or by activation-independent pathways mediated by Factor Xa or induced by the thiol-modifying agent, N-ethylmaleimide. We recently reported that shed soluble GPVI (sGPVI) was elevated in plasma of 159 ischaemic stroke patients compared with 159 community-based controls (P=0.0168), and in 29 patients with disseminated intravascular coagulation compared with healthy donors (n=25, P=0.002), consistent with a pathophysiological role for GPVI shedding from human platelets. Our new studies now show that transient exposure of human platelets to arterial or pathological shear rates of 3000–10,000 s−1 for 1–5 min ex vivo in a cone-plate viscometer, in the absence of GPVI ligand or platelet activation, activated sheddases producing a 2- to 3-fold increase in plasma sGPVI and a corresponding loss of surface GPVI from sheared platelets. Shear-induced GPVI shedding was blocked by GM6001 or GI254023, a selective inhibitor of ADAM10. In contrast to shear-induced platelet aggregation, shedding was unaffected by inhibitors of aggregation (VWF-blocking anti-GPIbα mAb, AK2, or the αIIbβ3 antagonist, RGD peptide) or by the absence of VWF in a patient with von Willebrand's disease Type III (VWF antigen levels <1%). Further, shedding of GPVI increased for up to 10 min after cessation of a short exposure of platelets to shear even when signalling, secretion and aggregation was blocked by inhibiting intracellular kinases (PP2, piceatannol), thromboxane generation (aspirin), ADP (apyrase) and calcium flux (BAPTA). Together, the combined results provide the first evidence that receptor sheddase activity can be regulated by hydrodynamic shear stress independent of cellular activation. This may represent a novel protective mechanism for down-regulating platelet reactivity as a response to pathological shear. Disclosures: No relevant conflicts of interest to declare.

Characterization of Antithrombin-Specific RNA Aptamers for Use in Anticoagulant Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4236-4236
Author(s):  
Yolanda Fortenberry
Keyword(s):  
Unfractionated Heparin ◽  
Vascular Injury ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Dependent Manner ◽  
Rna Aptamer ◽  
Rna Molecules ◽  
Factor Ixa

Abstract Historically, unfractionated heparin (a complex polysaccharide classified as a glycosaminoglycan) has been used in the treatment and prevention of thromboembolic disorders. However, due to complications associated with heparin (such as bleeding and heparin-induced thrombocytopenia), low molecular weight heparins (LMWHs) have been developed and are being used in place of unfractionated heparin. LMWHs have proven effective as antithrombotic agents; however, the effects of LMWHs cannot be readily reversed, while unfractionated heparin can be reversed by protamine sulfate. The overall goal of this project was to characterize an antithrombin (AT)-specific RNA aptamer that will combine the safety and efficacy of LMWH with the reversibility of unfractionated heparin. I have developed an RNA molecule (aptamer) that binds to AT. Using Systematic Evolution of Ligands by EXponential enrichment (SELEX) we created a combinatorial library consisting of single stranded RNA molecules with 20-40 randomized positions resulting in approximately 1014 different sequences. The RNA libraries after round 12 consisted of several different aptamers, and there was a progressive decrease in library complexity. We isolated four individual RNA molecules that bind in the nanomolar range to AT. Each displayed the same general properties, they accelerated factor Xa inhibition by AT in a dose-dependent manner. It should be noted that as anticipated, the aptamers did not promote the enhanced inhibition of thrombin by AT. Overall, our results show that these molecules are able to enhance the inhibition of factor Xa by AT. We suspect that these aptamers mimic the action of LMWH by binding to the D-helix of AT. Our previous AT specific RNA aptamer, Aptamer 7-4.16 was shown to prolonged bleeding in vivo in a vascular injury model. These data showed that Aptamer 7-4.16 is as effective as heparin in preventing clotting after vascular injury. We suspect that these new aptamers will have a similar effect in vivo. Proof of the concept was established by the work of Rusconi et al. (Nature, 2002), who developed an RNA aptamer and RNA aptamer antidote directed against coagulation factor IXa. The factor IXa aptamer completely inhibits the activation of factor X. Its anticoagulant action is controlled by the antidote, which is able to reverse the effects of the factor IXa aptamer within seconds. This aptamer/antidote pair is currently being tested clinically. Our data suggesting that antidotes to our AT aptamers have the ability to reverse its activity. If successful, this aptamer/antidote pair will have advantages over LMWHs because not only will it be able to control thrombosis in a fashion similar to LMWHs, but the availability of an antidote will allow for better therapeutic regulation and intervention. Disclosures No relevant conflicts of interest to declare.

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