Effects of Heparin and Bivalirudin on Thrombin-Induced Platelet Activation: Differential Modulation of PAR Signaling Drives Divergent Prothrombotic Responses

Figure 1. Heparin preferentially inhibits thrombin-induced platelet activation via PAR4. Intracellular calcium mobilization, platelet aggregation, and P-selectin exposure was measured in suspensions of washed platelets supplemented with 1 U/mL antithrombin after addition of the PAR1-inhibitor vorapaxar (5 μmol/L), the PAR4-inhibitor BMS-986120 (100 nmol/L), or heparin (0.2 U/mL, unless specified as 1 U/mL). Platelets were activated with 4 U/mL α-thrombin. (A–C) show the inhibitory effects of PAR-inhibitors alone and in combination. Calcium profiles generated with PAR1-AP (15 μM) and PAR4-AP (150 μM) are presented for comparison. (D–F) show the effects of heparin ± PAR inhibitors. Results represent the mean ± SEM (Calcium measurements: Control, VPX, n = 12; BMS, BMS+VPX, n = 8; UFH, n = 11; UFH+BMS, n = 7; UFH+VPX, n = 6; PAR1- and PAR4-AP, n = 3. LTA: In (C) Control, n = 10; BMS, n = 6; VPX, n = 9; BMS+VPX, n = 5. In (F) UFH, n = 8; UFH+BMS, UFH+VPX, n = 4. Flow cytometry: Control, n = 12; BMS, VPX, UFH, n = 10; BMS+VPX, n = 9; UFH+BMS, UFH+VPX, n = 4). (B,E) show representative data. **P < 0.01; ***P < 0.001; not significant (ns). VPX, vorapaxar; BMS, BMS-986120; UFH, unfractionated…

Figure 2. PAR4 activation triggers a delayed prothrombotic response in bivalirudin-treated platelets exposed to thrombin which can be suppressed by sub-therapeutic doses of heparin. Suspensions of washed platelets supplemented with 1 U/mL antithrombin were incubated for 10 min with vorapaxar (5 μmol/L), BMS-986120 (100 nmol/L), heparin (0.2 U/mL), or bivalirudin (1 μg/mL) as indicated, followed by exposure to 4 U/mL α-thrombin. In (A,B,J,K), intracellular calcium mobilization was monitored continuously for 30 min using the calcium probe Fluo-4. Data is presented as a fraction of the maximum fluorescence intensity registered after α-thrombin stimulation (4 U/mL). In (C,D,L) aliquots of platelets were incubated after thrombin treatment as indicated and then analyzed with flow cytometry for P-selectin exposure to assess α-granule release. Data is presented as mean fluorescence intensity (MFI). In (E,F) platelet aggregation was monitored continuously for 20 min using LTA. In (G–I) platelets stained with a fluorescent platelet marker (red) were allowed to spread on fibrinogen-coated wells for 15 min before treatment as indicated, and then monitored for Annexin V-binding (green) as a marker for…

Figure 3. Different effects of heparin and bivalirudin on thrombin-induced platelet activation via PAR1 and PAR4. Schematic drawing illustrating the early and late inhibitory effects of heparin/antithrombin and bivalirudin on thrombin-induced platelet activation. AT, antithrombin; PAR, protease activated receptor; Thr, thrombin.