The protein C activator AB002 rapidly interrupts thrombus development in baboons

Abstract

Although thrombin is a key enzyme in the coagulation cascade and is required for both normal hemostasis and pathologic thrombogenesis, it also participates in its own negative feedback via activation of protein C, which downregulates thrombin generation by enzymatically inactivating factors Va and VIIIa. Our group and others have previously shown that thrombin's procoagulant and anticoagulant activities can be effectively disassociated to varying extents through site-directed mutagenesis. The thrombin mutant W215A/E217A (WE thrombin) has been one of the best characterized constructs with selective activity toward protein C. Although animal studies have demonstrated that WE thrombin acts as an anticoagulant through activated protein C (APC) generation, the observed limited systemic anticoagulation does not fully explain the antithrombotic potency of this or other thrombin mutants. AB002 (E-WE thrombin) is an investigational protein C activator thrombin analog in phase 2 clinical development (clinicaltrials.gov NCT03963895). Here, we demonstrate that this molecule is a potent enzyme that is able to rapidly interrupt arterial-type thrombus propagation at exceedingly low doses (<2 µg/kg, IV), yet without substantial systemic anticoagulation in baboons. We demonstrate that AB002 produces APC on platelet aggregates and competitively inhibits thrombin-activatable fibrinolysis inhibitor (carboxypeptidase B2) activation in vitro, which may contribute to the observed in vivo efficacy. We also describe its safety and activity in a phase 1 first-in-human clinical trial. Together, these results support further clinical evaluation of AB002 as a potentially safe and effective new approach for treating or preventing acute thrombotic and thromboembolic conditions. This trial was registered at www.clinicaltrials.gov as #NCT03453060.

Conflict of interest statement

Conflict-of-interest disclosure: E.I.T., A.G., B.D.M., M.W., C.U.L., and N.G.V. are employees of Aronora, Inc. J.J.S. reports receiving consulting fees from Aronora Inc. A.G., O.J.T.M., N.G.V., E.I.T., and Oregon Health & Science University (OHSU) have a financial interest in Aronora, Inc. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee. The remaining authors declare no competing financial interests.

© 2020 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

AB002 interrupts thrombus formation in a primate thrombosis model. (A-B) Real-time platelet accumulation within collagen-coated synthetic vascular grafts (4-mm i.d.; 20 mm length) (A) and the tail region where the thrombus elongated distal to the graft (B). These devices were temporarily inserted into the femoral arteriovenous shunt of juvenile baboons. Blood flow through the device was adjusted to 100 mL/min, producing an initial shear rate of 265 s−1. Thrombi were allowed to grow for 30 minutes prior to intervention (black arrow). The average thrombus size at the time of intervention was set to 100%. Control experiments (n = 15) received no intervention or saline, whereas treatments were either tPA (n = 8) or increasing doses of E-WE thrombin (n = 4-7 per group). Posttreatment platelet accumulation was monitored for an additional 60 minutes. (C) Total fibrin deposition within the grafts was assessed at the end of each experiment. (D) aPTT was measured in platelet-poor plasma samples collected 10 minutes posttreatment. (E) Real-time γ-camera images of the developing thrombi taken at 30, 60, and 90 minutes. Images were recorded at 5-minute intervals and quantified in panels A and B. Each row of images shows representative images at each time point for control, AB002- (2 µg/mL), tPA-, and enoxaparin-treated baboons. Dimensions used to quantify graft and tail thrombus are indicated above each column. All data are expressed as mean plus or minus SEM. Asterisks denote significant differences to control (*P < .05; **P < .01; ***P < .001), pound symbols indicate significant differences to tPA (#P < .05; ##P < .01; ###P < .001). For panel B, all treatment groups, including tPA, are significantly different from control (P < .001), but not different from each other. Real-time platelet deposition was analyzed by 2-way ANOVA, and terminal fibrin and aPTT were analyzed using 1-way ANOVA.

Figure 2.

AB002 interrupts occlusive thrombus formation. (A) Real-time platelet accumulation within small diameter collagen-coated synthetic vascular grafts (2-mm i.d.; 20 mm length). Blood flow through the device was adjusted to 100 mL/min, producing an initial wall shear rate of 2120 s−1. Thrombi were allowed to grow for 15 minutes before treatment (black arrow). E-WE thrombin was given as an IV bolus (n = 3-6 per treatment group); controls (n = 5) received no treatment. Data are means plus or minus SEM. (B) Time to occlusion is shown. Horizontal bars denote mean of all data points. All control grafts occluded within 21 to 33 minutes. AB002 treatment significantly increased the time to occlusion (*P < .05; Gehan-Breslow-Wilcoxon test), with 8 of 12 grafts remaining patent for the entire 60 minutes.

Figure 3.

AB002 catalytically converts protein C to APC on activated platelet aggregates and inhibits thrombin-mediated TAFI activation. (A) Washed human platelets were activated and aggregated by collagen and treated with 50 nM AB002 or S195A-WE and 100 nM protein C. Platelets treated with protein C only and thrombin mutants incubated with protein C in the absence of platelets were prepared at the same time. Supernatants were collected after 1 hour and APC measured. Data are shown as the mean ± 1 standard deviation for 4 independent experiments (n = 4 donors). *P < .05 compared with platelet-only control and #P < .05 compared with platelet-free AB002-only control (repeated measures ANOVA and Holm-Sidak 2-tailed post hoc tests). (B) Washed human platelets were activated by collagen and pretreated with blocking reagents for 1 hour: 40 µg/mL anti-TM or anti-endothelial protein C receptor (EPCR), or 20 µg/mL recombinant human receptor-associated protein (rhRAP). Anti-CD31 (40 µg/mL) was used as a negative control for antibody blocking. Pretreated platelets were incubated with 50 nM AB002 and 100 nM protein C or protein C alone, as indicated. Supernatants were collected after 1 hour and APC measured. Data are shown as the mean ± 1 standard deviation for 4 independent experiments (n = 4 donors). *P < .05 compared with platelet-only control and #P < .05 compared with platelets with AB002 control (repeated measures ANOVA and Holm-Sidak 2-tailed post hoc tests). (C) WT α-thrombin (WT) or AB002 were added to HEPES buffered saline (HBS) with or without TM, purified TAFI was added, and samples incubated at room temperature for the indicated times before evaluating the activation of TAFI. Samples containing 500 nM purified TAFI and 150 nM WT with or without 15 nM TM (lanes 1-4) or 150 nM AB002 with or without 15 nM TM (lanes 5-12) were subjected to automated western blot analysis. (D) AB002 was added to 5 nM WT and 5 nM TM in increasing concentrations from 5 to 200 nM and TAFI activity measured. The activity of WT in the absence of AB002 was set to 100%. (E) A standard aPTT reaction was performed in a KC4 coagulometer using either healthy or TAFI-deficient platelet-poor plasma; at the end of the incubation time, 2 µg/mL tPA with or without 10 µg/mL AB002 was added, and the reaction was recalcified to initiate clot formation. aPTT was measured and time to complete lysis of the clot was monitored. Horizontal bars denote mean of all data points plus or minus SEM. *P < .05 (independent Student t test). (F) Putative mechanism by which low doses of AB002 can rapidly interrupt developing thrombi through its ability to locally activate protein C on the platelet-rich thrombus luminal surface and by competitive inhibition of TAFI activation (TAFIa).

Figure 4.

Dose-escalation schedule for AB002 phase 1 clinical study. Subjects were screened and those who met the eligibility criteria were randomized to receive either AB002 or placebo at a 4:1 ratio, except for cohort 1, in which the ratio was 4:2 to include 2 sentinel subjects (1 active and 1 placebo). Each cohort was dosed sequentially. Prior to dose escalation, all safety data were collected from each subject through day 14 and reviewed by the safety review committee during a dose-escalation safety review meeting.

Figure 5.

Coagulation parameters from all subjects from the AB002 phase 1 clinical study. Four cohorts were administered a single dose of AB002 (cohort 1, 0.5 µg/kg; cohort 2, 1.0 µg/kg; cohort 3, 2.0 µg/kg; cohort 4, 4.0 µg/kg [n = 4 each dose level]) or placebo (all placebo-dosed subjects are grouped together [n = 5]). Plasma samples were collected at specific intervals to observe the effect of drug administration on (A) aPTT, (B) APC-PCI complex formation, (C) prothrombin time (PT) and (D) protein C levels. For aPTT and PT, samples were analyzed at 0, 5, 15, 30 minutes, 1, 2, 24 hours, and 14 and 28 days. For APC-PCI, samples were analyzed at 0, 5, 15, 30 minutes, and 1, 2, 4, 24 hours, and, for protein C, samples were analyzed at 0, 1, 2, 4, and 24 hours. There were no measurable levels of APC-PCI complex in placebo-dosed subjects, thus data are not shown. Black dashed lines indicate laboratory reference ranges and red dashed lines indicate clinically significant ranges. All data are expressed as mean plus or minus SEM.