Development of novel inhibitors of thrombosis for the prevention of cardiovascular events
BACKGROUND:: Thrombotic events driven by platelet aggregation and activation of the coagulation system remain the leading cause of global morbidity and mortality. Antiplatelet and anticoagulant agents have proven effective in the treatment and prevention of these events in at-risk patients. However, bleeding remains a major concern and there is a large unmet clinical need for newer agents that can provide more effective antithrombotic protection. Novel candidates have been identified that may allow for a wider therapeutic index. Among these, platelet protease-activated receptor 4 (PAR4) and exosite 1 of thrombin have emerged as two of the most promising targets for antiplatelet and anticoagulant therapies, respectively. OBJECTIVES:: Given the central role of platelet aggregation to this thesis, we first sought to examine the feasibility of using a newer technique of platelet aggregometry, 96-well plate aggregometry (96-WPA), instead of the current gold standard, light transmission aggregometry (LTA). 96-WPA offers advantages over LTA but as yet there is almost no data regarding test-retest repeatability or agreement between the two techniques. Thereafter, the principal aims of this thesis were to explore the antiplatelet, anticoagulant and antithrombotic effects of PAR4 and exosite 1 thrombin inhibition in humans through a series of in vitro and ex vivo studies. Additional objectives included determination of whether platelet responses to PAR4 signaling are dependent on additional input from other agonist-receptor pathways or vice-versa and establishing the safety and tolerability of intravenous PAR4 agonism in mice in order to facilitate future studies into establishing the wider role of PAR4 within the human vasculature. METHODS AND RESULTS:: Repeatability and agreement between two methods of platelet aggregometry Test-retest repeatability (standard deviation, coefficient of variation (CV) and coefficient of repeatability) and agreement between 96-WPA and LTA were assessed constructing replicate agonist-induced dose-response curves from which peak aggregation and EC50 were determined. The pre-specified selected agonists were adenosine diphosphate (ADP), arachidonic acid (AA) and PAR4-activating peptide (PAR4-AP). Within-day CV for 96-WPA was 3.4%, 3.6% and 3.6% for peak aggregation and 9.6%, 7.1% and 6.9% for EC50 with ADP, AA and PAR4-AP, respectively. Corresponding values for between-day CV were 8.7%, 5.4%, and 6.6% for peak aggregation and 23.4%, 28.5%, and 23.1% for EC50. Overall, results for test-retest repeatability were comparable to LTA, although the latter appeared marginally superior in terms of the between-day CV. With respect to agreement, ADP and PAR4-AP peak aggregation measured consistently higher with 96-WPA whereas the EC50 was consistently lower. No significant measurement bias was observed with AA. Dependence of PAR4 on other agonist-receptor pathways for the activation and aggregation of human platelets All studies were performed in vitro using blood from healthy volunteers with results pooled from a minimum of 6 subjects. Activation of platelets by PAR4-AP (25 M) resulted in strong p-selectin expression (86.5 ± 2.6%), platelet-monocyte binding (85.8 ± 1.5%) and platelet aggregation (88.5 ± 3.4%). Platelet responses to PAR4-AP were completely inhibited by the PAR4 antagonist BMS-986120 but were unaffected by SCH-79797 (PAR1 antagonist), apyrase (ADP scavenger) or indomethacin (cyclooxygenase inhibitor). In contrast, PAR1 platelet activation and aggregation were both partially inhibited by apyrase. BMS-986120 had no effect on platelet activation or aggregation in response to PAR1, ADP or AA stimulation. Tolerability and safety of systemic PAR4 agonism in mice PAR4-agonist peptide (PAR4-AP) AYPGKF-NH2 (target blood concentration 11.25 M and 112.5 M) or control (0.9% saline) was administered intravenously to mice (n=15 total, 5 mice per group). Mice were observed for 24 hours then sacrificed with blood drawn for laboratory analyses and tissue samples prepared for histological examination. AYPGKF-NH2 was well tolerated with no evidence of systemic perturbation, thrombosis or change in any of the haematological, inflammatory, hepatic and renal blood markers examined. No distortion of parenchymal architecture, necrosis or inflammatory cell infiltrate of the lungs, spleen, liver and kidney was observed in any of the samples. Effect of oral PAR4 antagonism with BMS-986120 on platelets and thrombosis Forty volunteers were enrolled into a phase 1 parallel-group prospective randomized open-label blinded endpoint trial. Ex vivo platelet activation, platelet aggregation and thrombus formation were measured at 0, 2 and 24 hours after (a) oral BMS-986120, or (b) oral aspirin followed at 18 hours with oral aspirin and oral clopidogrel. BMS-986120 demonstrated highly selective and reversible inhibition of PAR4-AP stimulated platelet activation and aggregation. Compared to pre-treatment, BMS-986120 reduced total thrombus area at high shear by 29.2% (p<0.001) at 2 hours and by 21.4% (p=0.002) at 24 hours, driven by a decrease in platelet-rich thrombus formation. In contrast to aspirin and aspirin in combination with clopidogrel, BMS-986120 had no effect on thrombus formation at low shear (p=0.08). There were no serious adverse events. Effect of exosite 1 thrombin inhibition with JNJ-9375 on coagulation, platelets and thrombosis Fifteen healthy volunteers were enrolled into a double-blind randomized crossover study of JNJ-9375 (2.5, 25 and 250 μg/mL), bivalirudin (positive control) and matched placebo. JNJ-9375 caused concentration-dependent prolongation of blood coagulation and agonist-selective inhibition of platelet activation. Compared to placebo, JNJ-9375 (250 g/mL) reduced mean total thrombus area by 41.1% (p<0.001) at low shear and 32.3% (p=0.025) at high shear. Under both shear conditions, there was a dose-dependent decrease in fibrin-rich thrombus (p<0.001 for both). In contrast to bivalirudin, JNJ-9375 had no effect on platelet-rich thrombus formation. CONCLUSIONS:: This thesis demonstrates that a) both 96-WPA and LTA demonstrate good and comparable within- and between-day repeatability but owing to a systematic measurement bias the techniques should not be considered interchangeable, b) under conditions designed to replicate clinically relevant levels of thrombin, platelet responses to PAR4 stimulation were not dependent on input from other major agonist-receptor pathways or vice-versa, c) even at suprathreshold blood concentrations, intravenous PAR4 agonism with AYPGKF-NH2 was not associated with adverse effects in mice, d) oral PAR4 antagonism with BMS-986120 inhibits ex vivo human thrombus formation at high shear driven by a reduction in platelet deposition, and e) exosite 1 thrombin antagonism with JNJ-9375 inhibits ex vivo human thrombosis formation driven by a reduction in fibrin deposition. Results from this thesis suggest PAR4 antagonism and exosite 1 thrombin inhibition have major potential as novel antiplatelet and anticoagulant strategies and that further investigation in clinical trials is warranted.