In Vivo Porcine Aged Deep Vein Thrombosis Model for Testing Ultrasound-based Thrombolysis Techniques

SummaryA quantitative and non-occlusive deep vein thrombosis model was developed in rabbits. We used this model to test the antithrombotic activity of the prothrombinase complex inhibitors factor rXai and its chemical analog glutamyl-glycyl-arginyl chloromethyl ketone inactivated human factor Xa (EGR-Xai), along with the thrombin inhibitors D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) and heparin. Dose dependent effects of the inhibitors during constant infusion were monitored. Measurements included thrombus weights, hemostatic parameters and both cuticle and ear bleeding times. In this model, factor rXai and EGR-Xai had comparable in-vivo efficacy, and showed 80%-93% inhibition at plasma levels of 6.5 nM (rXai) and 8 nM (EGR-Xai). Effects on ex-vivo clotting times varied among the inhibitors. At 80-100% thrombus inhibition, factor rXai and EGR-Xai had no statistically significant effect, while PPACK extended thrombin clotting time (TCT) times 2.3-fold, and heparin prolonged both activated partial thromboplastin time (APTT), prothrombin time (PT) and TCT ex-vivo clotting times 6.9-, 1.2-, and 7-fold respectively. At these dosages, cuticle and ear bleeding times were prolonged for all inhibitors and showed increases of 177%-389% (cuticle) and 45%-129% (ear). Our results demonstrate that direct inhibition of prothrombinase complex assembly is effective in arresting venous thrombosis.

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Inhibition of Prothrombinase and Not Soluble Factor Xa (fXa) Predicts Efficacy of fXa Inhibitors in an In Vivo Model of Deep Vein Thrombosis.

Blood ◽

10.1182/blood.v108.11.902.902 ◽

2006 ◽

Vol 108 (11) ◽

pp. 902-902

Author(s):

Suzanne Delaney ◽

Ann Arfsten ◽

Sherin Halfon ◽

Gail Siu ◽

John Malinowski ◽

...

Keyword(s):

Deep Vein Thrombosis ◽

Plasma Concentration ◽

Plasma Concentrations ◽

Factor Xa ◽

Vein Thrombosis ◽

Prothrombinase Complex ◽

Antithrombotic Efficacy ◽

Deep Vein

Abstract Factor Xa (fXa) inhibitors are being tested in the clinic for the prevention and treatment of deep vein thrombosis (DVT) following orthopedic surgery. The antithrombotic efficacy of these drug candidates has traditionally been established in animal models as it is not known whether fXa amidolytic activity, activated partial thromboplastin time (aPTT) or prothrombin time (PT) predict efficacious doses. The present study was designed to test the hypothesis that the potency of fXa inhibitors against fXa incorporated into the prothrombinase complex would predict in vivo antithrombotic efficacy. Eight fXa inhibitors from four structurally distinct chemical series with a range of activities against fXa were tested for their ability to inhibit the prothrombinase complex in human plasma. Thrombin generation and subsequent cleavage of a specific thrombin substrate was used as a measure of prothrombinase activity, inhibitory activity being defined by the concentration of inhibitor required to produce a 2-fold extension in the time to maximal thrombin production (2x lag). In vitro rabbit PTs were also determined. Inhibition in the rabbit DVT model was assessed as previously described (Thromb Haemost1994; 71:357) and related to plasma concentrations of drug. Agent fXa IC50 (nM) Prothrombinase 2x lag (μM) Plasma concentration in DVT (μM) Thrombosis inhibition (%) Rabbit PT 2x change (μM) PRT50034 0.5 0.18 0.06 94 7.0 PRT54681 1.3 0.22 1.14 37 2.7 PRT54676 0.7 0.24 1.65 47 1.7 PRT54004 0.4 0.25 1.04 47 1.0 PRT54456 0.8 0.34 3.39 41 1.5 PRT56848 4.4 0.92 5.2 11 4.7 PRT54955 3.5 1.35 4.6 19 8.8 PRT57106 8.2 1.66 9.2 0 64 All compounds inhibited soluble fXa by 50 % at concentrations less than 10 nM. However, the rank order of potencies for inhibition of soluble fXa differed from that required to inhibit the prothrombinase complex. There was also poor correlation between the 2x lag value for prothrombinase inhibition and the concentration required to achieve a 2x change in rabbit PT (r2 = 0.57). Neither the activities of fXa inhibition nor the change in rabbit PT predicted activity in the DVT model. In contrast, compounds could be broadly divided into 3 levels of efficacy for inhibition of in vivo thrombus growth depending on their potency in the in vitro prothrombinase assay. PRT50034 had the lowest 2x lag value of 0.18 μM and was the most potent inhibitor of in vivo thrombosis with 94 % inhibition at a plasma concentration of 65 nM. The second group of compounds, with 2x lag values in the prothrombinase assay ranging from 0.22 to 0.34 μM, inhibited in vivo thrombus formation by 37 to 47 % at plasma concentrations ranging from 1.04 to 3.39 μM. Compounds in the third category were the least potent prothrombinase inhibitors (2x lag values greater than 0.92 μM) and were unable to significantly inhibit in vivo thrombosis even at plasma concentrations of 9.2 μM. These data show that the 2x lag value obtained in the prothrombinase assay, and not inhibition of soluble fXa or extension of rabbit PT, is capable of predicting fXa inhibitor efficacy in the in vivo rabbit DVT model.

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

Blood ◽

10.1182/blood.v126.23.2277.2277 ◽

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.

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