Mitochondrially mediated integrin αIIbβ3 protein inactivation limits thrombus growth

Abstract

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin αIIbβ3 or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin αIIbβ3 and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin αIIbβ3 epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin αIIbβ3 function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β3 cytoplasmic domain, coincided closely with integrin αIIbβ3 inactivation. Calpain inhibition blocked integrin β3 cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β3 and associated regulatory proteins, resulting in integrin αIIbβ3 inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.

Keywords: Calpain; Integrin; Mitochondria; Platelets; Thrombosis.

FIGURE 1.

Dependence of platelet shape change and integrin α_Iibβ₃ epitope modulation on mPTP formation. A, platelets were stimulated with thrombin (0.5 units/ml) and convulxin (100 ng/ml) and visualized continuously. Merged fluorescent (JON/A) and differential interference contrast (DIC) images were merged and are shown at the indicated time points. Arrows indicate platelets that assume a rounded morphology. B–D, platelet diameter (B), JON/A-positive (C), and TMRM-positive (D) platelets were assessed in CypD^+/+ and CypD^−/− platelets stimulated with Thr/Cvx. Platelet diameter was quantified relative to initial diameter. Up to 500 platelet diameters were counted in each condition. TMRM-positive platelets were quantified relative to the number present immediately prior to stimulation. JON/A+ platelets were quantified relative to peak levels (1 min after stimulation). E, platelet morphology and TMRM fluorescence were assessed at the indicated time points in Thr/Cvx-stimulated CypD^+/+ and CypD^−/− platelets. Arrows point to rounded platelets. Scale bar = 5 μm. *, p < 0.05. n = 3–6 mice in each experiment. The dashed line connecting two asterisks indicates same significance level in between these observations.

FIGURE 2.

Mitochondrial-dependent events limit platelet aggregation and adhesion. A, assessment of platelet recruitment by TMRM(+) and TMRM(-) platelets. Recalcified CTI/apyrase-treated whole blood from β-actin GFP transgenic mice was perfused for 5 min at 100 s over washed adherent platelets stimulated previously with Thr/Cvx. The percentage of adherent platelets with stably associated GFP+ platelets was determined. *, p < 0.05. n = 3. B, aggregation of CypD^+/+ and CypD^−/− platelets in the presence of the indicated agonist(s). *, p < 0.05. n = 5–11 mice. C, adherence of CypD^+/+ and CypD^−/− platelets to type I collagen. Recalcified CTI/apyrase-treated whole blood was perfused over the collagen surface for 10 min at 1500/s shear rates. PE-CD61 was used to label platelets. JON/A was used at a 1:40 dilution. Scale bars = 20 μm. D, platelet coverage following perfusion was compared relative to CypD^+/+. *, p < .01 relative to CypD^+/+; #, p < 0.01 relative to CypD^−/−. n = 3–6 mice.

FIGURE 3.

Thrombotic and hemostatic response in mice with megakaryocyte/platelet-specific deficiency of CypD. A, thrombosis was initiated within the mesenteric artery by photochemical injury, and time to stable occlusion was determined in littermate CypD^plt+/− and CypD^plt−/− mice. Stable occlusion occurred within 1200 s in 9 of 20 CypD^plt+/− mice and 2 of 14 CypD^plt−/− mice. *, p < 0.05 by Mann-Whitney U test. B, tail bleeding time. Shown is the time to first and final cessation of bleeding in CypD^plt+/− and CypD^plt−/− mice. p = 0.14, n = 17.

FIGURE 4.

Calpain-mediated cleavage of the cytoplasmic domain of integrin β₃ is dependent on CypD. A and B, washed CypD^+/+ or CypD^−/− platelets were stimulated for 5 min with the indicated agonist(s) with or without MDL28170 (50 μm) or Q-VD-Oph (QVD) (50 μm), and cleavage of the integrin β₃ cytoplasmic tail (C-20) and talin was assessed by Western blot analysis (0.5 units/ml Thr, 100 ng/ml Cvx, and 1 μm Iono). B, quantification of integrin β₃ cleavage. Intensity of C-20 binding was assessed relative to unstimulated platelets. *, p < 0.05 compared with untreated platelets; #, p < 0.05 to relative to CypD^+/+. n = 3. C, washed CypD^+/+ or CypD^−/− platelets were stimulated for 5 min with the indicated agonist(s) ± MDL and assessed by Western blot analysis using antibodies specific to the integrin β₃ cytoplasmic tail (C-20) and integrin β₃ extracellular domain (N-20). *, p < 0.05 relative to unstimulated, #, p < 0.05. D, kinetics of integrin β₃ cleavage in CypD^+/+ and CypD^−/− platelets stimulated with thrombin and convulxin. *, p < 0.05. n = 3. Intensity of C-20 binding was assessed relative to unstimulated platelets.

FIGURE 5.

Calpain acts downstream of mPTP formation to mediate secondary integrin α_Iibβ₃ inactivation. A–C, fibrinogen-adherent platelets that were untreated (W/O MDL) or MDL-treated were stimulated with Thr/Cvx. A, morphology and TMRM fluorescence of control and MDL-treated platelets 1 and 10 min after stimulation. Scale bar = 5 μm. B and C, platelet rounding (B) and JON/A binding (C) in control and MDL-treated fibrinogen-adherent platelets stimulated with Thr/Cvx. n = 3. D, platelet aggregation in control and MDL-treated platelets. *, p < 0.05, n = 6. E, adherence of control and MDL-treated CypD^+/+ and CypD^−/− platelets in flow conditions. CTI/apyrase-treated whole blood was perfused at 1500 s over type I collagen in a microfluidics assay. Representative of n = 3. F, effect of MDL treatment on mPTP formation and PS exposure in control and MDL-treated cells assessed using TMRM and annexin V, respectively. n = 3 in each condition.

FIGURE 6.

CypD-dependent cytoplasmic alkalinization is associated with enhanced calpain activation and integrin α_Iibβ₃ inactivation. A and B, intracellular pH was measured by flow cytometry assay using SNARF-1 AM as a pH indicator. pH_i was determined by the ratio of fluorescent emission at 640 nm and 580 nm. A, pH_i was assessed in CypD^+/+ and CypD^−/− platelets, respectively, after platelets were treated with the indicated agonists. n = 4. *, p < 0.05 compared with unstimulated (Unstim.) platelets; #, p < 0.05 to relative to CypD^+/+ platelets. B, pH_i and integrin α_IIbβ₃ activation in strongly stimulated platelets. PAC-1 was used to evaluate integrin α_IIbβ₃ in human platelets. The dark peak indicates PAC-1(-) platelets and the light gray peak PAC-1(+) platelets. A pH_i increase using the SNARF-1 AM indicator is associated with an increase in the 640/580 nm fluorescence ratio. The drawn gate encompasses the fluorescent ratio associated with a pH_i of 7.2 (left panel) to 7.4 (right panel). n = 3.