Safe Interruption of Ongoing Thrombosis by the Protein C Activator Thrombin Analog, W215A/E217A; a Comparison to Enoxaparin in Primates.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 909-909 ◽  
Author(s):  
Ulla M. Marzec ◽  
Leslie Bush ◽  
Erik Tucker ◽  
Andras Gruber ◽  
Stephen R. Hanson ◽  
...  
Keyword(s):  
Group Treatment ◽  
Protein C ◽  
Double Mutant ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Bleeding Risk ◽  
Expansion Chamber ◽  
Platelet Deposition ◽  
Type Flow ◽  
Human Thrombin

Abstract Antithrombotic agents are effective but pose significant bleeding risk, thus safer new agents are needed. Since the infused double mutant human thrombin (W215A/E217A; WE) binds endothelial thrombomodulin, activates protein C locally in the intraluminal boundary layer, and prevents thrombus formation without hemostasis impairment, we investigated whether WE could also interrupt the growth of existing thrombi. WE was compared to standard interventional doses of the low molecular weight heparin, enoxaparin (Sanofi) in awake baboons. Thrombosis was initiated by interposing a two part device consisting of a 2 cm, 4 mm ID polyethylene terephthalate vascular graft (DVG) followed by a 2 cm silicone rubber expansion chamber, 9 mmID. Upon initiation of blood flow at 100 mL/min, wall shear rate of 265 sec-1 in the 4 mm ID segment (arterial type flow) and 29 sec-1 in the expansion chamber (venous type flow), platelet thrombus was monitored by gamma camera imaging of autologous 111In-labelled platelets for 60 min or 90 min. Fibrin accumulation was quantified by homologous 125I-labelled fibrinogen. Hemostasis was assessed as template bleeding time prolongation. APTT was also monitored. Treatments started 25 min after the initiation of thrombosis by enoxaparin, 0.5 mg/kg (LD) and 1 mg/kg (HD) IV bolus or WE, 7.5 μg/kg/hr (LD) and 15 μg/kg/hr (HD) infusion (1/3 dose given as a bolus followed by IV infusion over 60min) and compared to saline infused controls. All interventions were given systemically, downstream from the device and 5–6 studies comprised each study group. Treatment with enoxaparin at 25 min after initiation of thrombus growth resulted in a reduction of platelet deposition of 19 % with the LD and 76 % with the HD after 35 min of therapy in the expanded venous type segment. WE reduced venous thrombosis by 45 % and 65 % at the two doses studied and was further reduced to 65 % and 80 % following 65 min of WE infusion as compared to controls. Arterial type thrombosis was reduced by WE by 34 % and 39 % respectively for the two doses studied. HD enoxaparin reduced arterial type thrombosis by 37 %. Fibrin accumulation paralleled the platelet deposition. APTT was prolonged 1.57- and 1.87-fold respectively for the WE doses without any bleeding time prolongation. Enoxaparin prolonged the APTT 1.4- and 1.75-fold, similar to WE but with a 1.13- and 1.75-fold prolongation of the bleeding time. In conclusion, the double mutant human thrombin, WE, effectively interrupted both venous and arterial type growing thrombi at very low doses without detectable compromise to hemostasis. Likewise, enoxaparin at the doses tested had similar efficacy but with a significantly prolonged bleeding time at the 1 mg/kg bolus dose. WE might provide a safer alternative to heparins in the treatment of acute progressive thrombosis.

Comparison of Enoxaparin to the Thrombin Variant Protein C Activator, W215A/E217A, in a Primate Model of Thrombosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1868-1868
Author(s):  
Ulla M. Marzec ◽  
Leslie Bush ◽  
Andras Gruber ◽  
Stephen R. Hanson ◽  
Enrico Di Cera
Keyword(s):  
Protein C ◽  
Bleeding Time ◽  
Activated Protein C ◽  
Expansion Chamber ◽  
Primate Model ◽  
Endothelial Surface ◽  
Endogenous Protein ◽  
Platelet Deposition ◽  
Type Flow

Abstract Antithrombotic therapy by thrombomodulin-dependent activation of endogenous protein C might be safer than currently used therapies, since endogenous activated protein C (APC) is generated and probably concentrated on the endothelial surface and not in blood that escapes the blood vessels during injury. We compared the antithrombotic and antihemostatic activities of a protein C selective recombinant double mutant human thrombin (W215A/E217A; WE) that activates endogenous protein C on the endothelial surface, in vivo, with a low molecular weight heparin, enoxaparin in awake non anticoagulated baboons. Thrombosis was initiated by interposing a two part device consisting of a 2cm, 4mm ID, polyethylene terephthalate vascular graft (DVG) followed by a 2cm silicone tubing chamber, 9mm ID, into a chronic femoral arteriovenous shunt in baboons. Upon initiation of blood flow at 100mL/min, wall shear rate of 265sec−1 in the 4mm ID segment (arterial type flow) and 29sec−1 in the expansion chamber (venous type flow), platelet thrombus growth was monitored by gamma camera imaging of autologous 111In-labeled platelets for 1 hour. Fibrin accumulation was quantified by homologous 125I-labeled fibrinogen. Bleeding time and aPTT were monitored. WE was administered as a loading IV bolus (1/3 dose) 10 minutes before thrombus initiation followed by maintenance infusion (2/3 dose) for the duration of the study at 3 dose levels: 1.8 μg/kg (dose-1), 3.75 μg/kg (dose-2) and 7.5 μg/kg (dose-3). Results were compared to controls given normal saline and to 3 doses of IV enoxaparin (1/2 dose loading + 1/2 dose maintenance): 0.3 mg/kg (dose-1), 0.6 mg/kg (dose-2) and 1.2mg/kg (dose-3) (3–5 animals in each study group). WE inhibition of platelet deposition in the chamber was dose dependent and reduced by 57%, 76% and 95% for dose 1, 2 and 3 respectively, compared to controls (p < 0.05 each). Enoxaparin likewise reduced thrombosis in the chamber by 49%, 83% and 91% (p< 0.05 each) respectively at the 3 doses tested. Fibrin accumulation paralleled the platelet deposition data in the chamber. Thrombus growth on DVG was only reduced at dose level 3 for both WE and enoxaparin by 18% and 47%, respectively as determined by platelet deposition. Fibrin accumulation was reduced by 35% by WE and 51% for enoxaparin at dose 3 on the arterial thrombosis segment. Bleeding times did not increase significantly with any of the treatments except for dose-3 of enoxaparin which prolonged the bleeding time from 3.5±1.3 minutes to 5.3 ± 1.4 minutes. Prolongation of APTT was 1.14, 1.33, and 2.04 fold for WE doses 1, 2 and 3, respectively and enoxaparin prolonged the APTT by 1.25, 1.6 and 1.9 fold at dose 1, 2 and 3, respectively. We conclude that low dose WE infusion is safe and about two orders of magnitude more antithrombotic than enoxaparin infusion in the baboon model of thrombosis.

scholarly journals A Short Half-Life αIIbβ3 Antagonist ANTP266 Reduces Thrombus Formation

2018 ◽  
Vol 19 (8) ◽  
pp. 2306 ◽  
Author(s):  
Tong-Dan Liu ◽  
Shen-Hong Ren ◽  
Xue Ding ◽  
Zhou-Ling Xie ◽  
Yi Kong
Keyword(s):  
Half Life ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Bleeding Risk ◽  
Effective Dosage ◽  
Enzyme Linked Immunosorbent Assay ◽  
Severe Bleeding ◽  
Antithrombotic Agent ◽  
Integrin Αiibβ3 ◽  
Plasma Half Life

Integrin αIIbβ3 plays a pivotal role in platelet aggregation. Three αIIbβ3 antagonists have been approved by the Food and Drug Administration (FDA) for the treatment of cardiovascular diseases. Unfortunately, all of these three drugs can cause the side effect of severe bleeding. Therefore, developing a new αIIbβ3 antagonist with low bleeding was needed. In the present study, we screened compounds by using a fibrinogen/integrin αIIbβ3 enzyme-linked immunosorbent assay (ELISA), and a novel αIIbβ3 antagonist ANTP266 was attained. The antithrombotic effects of ANTP266 were estimated by using two animal models, the bleeding risk was estimated by using a mice tail cutting assay, and the plasma half-life time was tested by LC-MS/MS. The results showed that ANTP266 potently decreased thrombosis formation, while not prolonging bleeding time at its effective dosage. The bleeding of ANTP266 reduced rapidly as time went on from 5 to 60 min, but tirofiban produced high bleeding continuously. The plasma half-life of ANTP266 in rats was 10.8 min. Taken together, ANTP266 is an effective antithrombotic agent with a low bleeding risk. The shorter bleeding time benefits from its short plasma half-life. ANTP266 could be a candidate for developing the αIIbβ3 antagonist of rapid elimination for a patient undergoing percutaneous coronary intervention.

scholarly journals Rutin-Loaded Silver Nanoparticles With Antithrombotic Function

2020 ◽  
Vol 8 ◽  
Author(s):  
Haitao Wu ◽  
Manlin Su ◽  
Hui Jin ◽  
Xinyu Li ◽  
Puyu Wang ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Aqueous Solubility ◽  
Bleeding Risk ◽  
Activated Partial Thromboplastin Time ◽  
Coagulation Parameter ◽  
Mice Model ◽  
Serum Stability ◽  
Hemorrhage Risk

In this paper, we fabricated rutin-loaded silver nanoparticles (Rutin@AgNPs) as the nano-anticoagulant with antithrombotic function. The serum stability, anticoagulation activity, and bleeding risk of Rutin@AgNPs were evaluated. The results showed Rutin@AgNPs had good serum stability, hemocompatibility, and cytocompatibility. The anticoagulation activity of rutin was maintained, and its stability and aqueous solubility were improved. The Rutin@AgNPs could provide a sustained release to prolong the half-life of rutin. The results of the coagulation parameter assay and thrombus formation test in mice model showed that the activated partial thromboplastin time and prothrombin time were prolonged, and Rutin@AgNPs inhibited the thrombosis in the 48 h period. Moreover, the limited bleeding time indicated that the Rutin@AgNPs significantly minimized the hemorrhage risk of rutin. This Rutin@AgNPs is a potential anticoagulant for antithrombotic therapy.

scholarly journals Antithrombotic activity of protein S infused without activated protein C in a baboon thrombosis model

2012 ◽  
Vol 107 (04) ◽  
pp. 690-698 ◽  
Author(s):  
Ulla Marzec ◽  
Andras Gruber ◽  
Stephen R. Hanson ◽  
Mary J. Heeb
Keyword(s):  
Blood Flow ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Fibrin Deposition ◽  
Thrombosis Model ◽  
Platelet Deposition ◽  
Type Flow

SummaryProtein S (ProS) is an essential plasma protein that enhances the anticoagulant activity of activated protein C (APC). In vitro, purified native human Zn2+-containing ProS also exerts direct anticoagulant activity by inhibiting prothrombinase and extrinsic FXase activities independently of APC. We investigated antithrombotic effects of ProS infused without APC in a baboon shunt model of thrombogenesis that employs a device consisting of arterial and venous shear flow segments. In in vitro experiments, the Zn2+-containing human ProS used for the studies displayed >10-fold higher prothrombinase inhibitory activity and anticoagulant activity in tissue factor-stimulated plasma, and four-fold higher inhibition of the intrinsic pathway than the Zn2+-deficient ProS used. In the thrombosis model, ProS (33 μg/minute for 1 hour) or saline was infused locally; platelet and fibrin deposition in the shunt were measured over 2 hours. During experiments performed at 50 ml/minute blood flow, Zn2+-containing ProS inhibited platelet deposition 73–96% in arterialtype flow segments and 90–99% in venous-type flow segments; Zn2+-deficient ProS inhibited platelet deposition 52% in arterial-type flow segments and 65–73% in venous-type flow segments. At 100 ml/min blood flow rate, Zn2+-containing ProS inhibited platelet deposition by 39% and 73% in the respective segments; Zn2+-deficient ProS inhibited platelet deposition by 5% and 0% in the respective segments. Zn2+-containing ProS suppressed fibrin deposition by 67–90%. Systemic APC-independent ProS activity was significantly increased and thrombin-antithrombin complex levels were significantly decreased after infusion of ProS. Thus, infused human Zn2+-containing ProS is antithrombotic in primates, and may have therapeutic potential even in protein C-deficient human patients.These studies were presented in part in abstract form at an oral presentation at the XXIth Congress of the International Society on Thrombosis and Haemo -stasis, Geneva, Switzerland, August 2007.

Engineering activated protein C to maximize therapeutic efficacy

2015 ◽  
Vol 43 (4) ◽  
pp. 691-695 ◽  
Author(s):  
Louise M. Quinn ◽  
Clive Drakeford ◽  
James S. O’Donnell ◽  
Roger J.S. Preston
Keyword(s):  
Clinical Trials ◽  
Protein Engineering ◽  
Inflammatory Disease ◽  
Therapeutic Efficacy ◽  
Protein C ◽  
Thrombus Formation ◽  
Activated Protein C ◽  
Bleeding Risk ◽  
Protective Role ◽  
Chronic Inflammatory Disease

The anticoagulant-activated protein C (APC) acts not solely as a crucial regulator of thrombus formation following vascular injury, but also as a potent signalling enzyme with important functions in the control of both acute and chronic inflammatory disease. These properties have been exploited to therapeutic effect in diverse animal models of inflammatory disease, wherein recombinant APC administration has proven to effectively limit disease progression. Subsequent clinical trials led to the use of recombinant APC (Xigris) for the treatment of severe sepsis. Although originally deemed successful, Xigris was ultimately withdrawn due to lack of efficacy and an unacceptable bleeding risk. Despite this apparent failure, the problems that beset Xigris usage may be tractable using protein engineering approaches. In this review, we detail the protein engineering approaches that have been utilized to improve the therapeutic characteristics of recombinant APC, from early studies in which the distinct anti-coagulant and signalling activities of APC were separated to reduce bleeding risk, to current attempts to enhance APC cytoprotective signalling output for increased therapeutic efficacy at lower APC dosage. These novel engineered variants represent the next stage in the development of safer, more efficacious APC therapy in disease settings in which APC plays a protective role.

scholarly journals Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 639-642 ◽  
Author(s):  
A Gruber ◽  
JH Griffin ◽  
LA Harker ◽  
SR Hanson
Keyword(s):  
Protein C ◽  
Thrombus Formation ◽  
Activated Protein C ◽  
Graft Patency ◽  
Scintillation Camera ◽  
Primate Model ◽  
Baboon Model ◽  
Platelet Deposition ◽  
Antithrombotic Effects

The in vivo antithrombotic properties of human plasma activated protein C (APC), a natural anticoagulant enzyme, were investigated in a baboon model of thrombus formation on prosthetic vascular grafts. Infusion of 0.25 to 1.1 mg/kg/h purified, human, APC inhibited blood clotting, as measured by the activated partial thromboplastin time (APTT), and reduced vascular graft platelet deposition by 40% to 70%, as determined by the real-time scintillation camera imaging of 111In-labeled platelet deposition. APC infusion also preserved graft patency. Hemostatic plug formation remained normal, as measured by the template bleeding times. These results suggest that APC administration may produce immediate antithrombotic effects under arterial flow conditions.

scholarly journals Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 639-642 ◽  
Author(s):  
A Gruber ◽  
JH Griffin ◽  
LA Harker ◽  
SR Hanson
Keyword(s):  
Protein C ◽  
Thrombus Formation ◽  
Activated Protein C ◽  
Graft Patency ◽  
Scintillation Camera ◽  
Primate Model ◽  
Baboon Model ◽  
Platelet Deposition ◽  
Antithrombotic Effects

Abstract The in vivo antithrombotic properties of human plasma activated protein C (APC), a natural anticoagulant enzyme, were investigated in a baboon model of thrombus formation on prosthetic vascular grafts. Infusion of 0.25 to 1.1 mg/kg/h purified, human, APC inhibited blood clotting, as measured by the activated partial thromboplastin time (APTT), and reduced vascular graft platelet deposition by 40% to 70%, as determined by the real-time scintillation camera imaging of 111In-labeled platelet deposition. APC infusion also preserved graft patency. Hemostatic plug formation remained normal, as measured by the template bleeding times. These results suggest that APC administration may produce immediate antithrombotic effects under arterial flow conditions.

Varicose vein surgery under anti-platelet therapy

Phlebologie ◽  
2008 ◽  
Vol 37 (06) ◽  
pp. 287-297 ◽  
Author(s):  
P.-M. Baier ◽  
Z. T. Miszczak
Keyword(s):  
Varicose Vein ◽  
Bleeding Time ◽  
Varicose Veins ◽  
Bleeding Risk ◽  
Bleeding Complications ◽  
Review Of The Literature ◽  
Varicose Vein Surgery ◽  
Anti Platelet Therapy ◽  
Male Patients ◽  
Acute Atherothrombosis

Summary Background: Platelet function inhibitors (PFI) are used for prophylaxis of atherothrombosis. These drugs cause a prolongation of the bleeding time and should eventually be stopped before an elective operation. However, there is a risk that a perioperative pause of PFI lead to acute atherothrombosis. Objective: Our aim was to study whether a discontinuation of PFI therapy is necessary to avoid bleeding complications in patients undergoing varicose vein surgery. Methods: Selective review of the literature and retrospective analysis of clinical data of our own patients. Results: In the years 2002 to 2007 a total of 10 827 patients have been operated on varicose veins, 673 (6.2%) of these aged 32–86 years (67 ± 7.9) receiving permanent PFI therapy: 256 male patients (38.0%) and 417 female (62.0%), 39.1% categorized as ASA III patients: male 11.6%, female 27.5%. 38 patients who continued PFI therapy did not demonstrate haemorrhagic complications and none of those pausing anti-platelet medication experienced thromboembolic complications. The literature survey confirmed our finding that it is not necessary to suspend PFI medication for varicose vein surgery as the bleeding risk can be controlled for by technical means. Conclusion: Discontinuation of PFI therapy prior to interventions on varicose veins does not seem to be necessary, further studies are essential though.

ADP Plays a Key Role in Thrombogenesis in Rats

1988 ◽  
Vol 59 (02) ◽  
pp. 225-230 ◽  
Author(s):  
J P Maffrand ◽  
A Bernat ◽  
D Delebassée ◽  
G Defreyn ◽  
J P Cazenave ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Vascular Wall ◽  
Cyclooxygenase Inhibitors ◽  
High Dose ◽  
Silk Thread ◽  
Dense Granules ◽  
Prolonged Bleeding Time ◽  
Venous Shunt

SummaryThe relative importance of ADP, arachidonic acid metabolites and serotonin as thrombogenic factors was evaluated in rats by comparing, after oral administration, the effects of two inhibitors of ADP-induced platelet aggregation (ticlopidine and PCR 4099), three cyclo-oxygenase inhibitors (aspirin, triflusal and indobufen) and a selective serotonin 5HT2 receptor antagonist (ketanserin) on platelet aggregation, in four platelet-dependent thrombosis models and on bleeding time. Platelet aggregation induced by ADP and collagen was completely inhibited by ticlopidine and PCR 4099 whereas only the collagen aggregation was reduced by the cyclo-oxygenase inhibitors. Ketanserin or a depletion of platelet serotonin by reserpine did not affect platelet aggregation. Ticlopidine and PCR 4099 greatly prolonged rat tail transection bleeding time. This is probably related to their known ability to inhibit ADP-mediated platelet aggregation. In contrast, the cyclooxygenase inhibitors did not affect bleeding time at all. Reserpine and ketanserin prolonged bleeding time by interfering with the action of serotonin on the vascular wall. Ticlopidine and PCR4099 were very potent antithrombotics in all the models. Aspirin, only at a high dose, inhibited poorly thrombus formation on a silk thread in an arterio-venous shunt, suggesting that the inhibition of cyclo-oxygenase was not responsible. Triflusal was inactive in all models while indobufen slightly reduced thrombus formation in the silk thread and metallic coil models. Ketanserin and reserpine reduced thrombus only in the metallic coil model. Thrombus formation was greatly reduced in fawn-hooded rats, which lack ADP in their platelet dense granules because of a genetic storage pool deficiency. Taken together, the results obtained with the drugs and with the fawn-hooded rats support the concept that ADP plays a key role in thrombogenesis in rats.

Antithrombotic Effects and Bleeding Time Prolongation with Synthetic Platelet GPIIb/llla Inhibitors in Animal Models of Platelet-Mediated Thrombosis

1994 ◽  
Vol 71 (01) ◽  
pp. 095-102 ◽  
Author(s):  
Désiré Collen ◽  
Hua Rong Lu ◽  
Jean-Marie Stassen ◽  
Ingrid Vreys ◽  
Tsunehiro Yasuda ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Bolus Injection ◽  
Cell Injury ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Femoral Vein ◽  
Thrombotic Occlusion ◽  
Antithrombotic Effects

SummaryCyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-a-aspartyl-cyclic (1→5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-prolyl-L-arginyl-glycyl-L-α-aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1→9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor.The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 μg/kg, ex vivo ADP-induccd platelet aggregation with ID50 of 10 μg/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 ± 9 to 1,100 ± 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 μg/kg, ex vivo platelet aggregation with an ID50 of 50 μg/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 μg/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induccd ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 ± 0.4 to 12 ± 2 min. Infusion of 300 μg/kg of TP9201 over 60 min completely prevented thrombotic occlusion, inhibited ex vivo platelet aggregation, but was not associated with prolongation of the template bleeding time.TP9201, unlike G4120, inhibits in vivo platelet-mediated thrombus formation without associated prolongation of the template bleeding time.

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