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

Abstract

Heparin and bivalirudin are widely used as anticoagulants in the setting of acute thrombosis. In this study, we investigated how these drugs affect the ability of thrombin to generate a prothrombotic platelet response via activation of the protease-activated receptors (PARs) 1 and 4. We examined the effects of heparin/antithrombin and bivalirudin on PAR1- and PAR4-mediated intracellular calcium mobilization, aggregation, α-granule release, and procoagulant membrane exposure in platelets exposed to thrombin concentrations likely to be encountered in the thrombus microenvironment during thrombosis. At physiological antithrombin levels, heparin treatment resulted in complete and sustained inhibition of thrombin-induced PAR4-mediated platelet activation, but transient PAR1 signaling was sufficient to elicit significant α-granule release and platelet aggregation. In contrast, bivalirudin treatment resulted in rapid and profound inhibition of signaling from both PAR receptors, followed by a delayed phase of PAR4-mediated platelet activation, resulting in a robust prothrombotic response. Combination treatment with bivalirudin and subtherapeutic concentrations of heparin completely inhibited the residual platelet activation observed with single drug treatment at all time-points. Our results show that heparin and bivalirudin have different and complementary inhibitory effects on the activation of PAR1 and PAR4 by thrombin.

Keywords: PAR1; PAR4; bivalirudin; heparin; thrombin.

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 heparin.

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 procoagulant membrane exposure using confocal microscopy. Pictures are taken after 45 min. Scalebar represents 50 μm. Results show the mean ± SEM (Calcium measurements: Control, BIV, n = 21; BIV+VPX, n = 18; BIV+BMS, n = 13; UFH, BIV+UFH, n = 11. LTA: Control, BIV+VPX, BIV+UFH, n = 8; BIV, n = 13; BIV+BMS, n = 9. Flow cytometry: Control, BIV, BIV+VPX, BIV+BMS, BIV+UFH, n = 12; UFH, n = 10. Microscopy: THR, BIV, n = 4. UFH, n = 3). (A,E,G,J) show representative data. *P < 0.05; **P < 0.01; ***P < 0.001; not significant (ns). THR, thrombin; VPX, vorapaxar; BMS, BMS-986120; UFH, unfractionated heparin; BIV, bivalirudin.

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.