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NLRP3 regulates platelet function and thrombosis
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Figure 2. Platelet aggregation and activation. (A) Platelet aggregation was induced by addition of collagen, CRP, thrombin or ADP to platelets from wild-type (WT) or NLRP3-/- mice. Representative aggregation traces using platelets from WT (green) or NLRP3-/- (red) mice are shown together with combined data (mean ± SE) for three mice (Student t-test). (B) Platelet P-selectin expression and (C) activated aIIbβ3 (JON/A binding) were assessed in platelets before and after treatment with the indi- cated concentrations of collagen, CRP, ADP or thrombin for 10 min by flow cytometry using phycoerythrin-conjugated anti-P-selectin antibody (Ebioscience) or JON/A antibody (emfret ANALYTICS) (mean ± SE, n = 3-6). *P<0.05.
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and reduced prothrombin times of 8.54 ± 0.30 s (n = 8) compared with the wild-type control values of 63.43 ± 26.94 s (n = 7) and 9.07 ± 0.37 s (n = 10), respectively, as well as elevated levels of coagulation factors VIII and IX (data not shown), platelets from wild-type or NLRP3-/- mice were injected into thrombocytopenic or wild-type mice to eliminate non-platelet hemostatic or thrombotic factor variations. The platelet numbers in mice receiving infusion of wild-type or NLRP3-/- platelets either before, after injec- tion of anti-aIIb antibody or after infusion were compara- ble (Online Supplementary Figure S1). In addition, injection of anti-aIIb antibody did not affect the function of donor platelets as the tail bleeding time of thrombocytopenic mice receiving donor platelets (9 h after antibody) was sim- ilar to that of wild-type mice (Figure 1D-i). A significantly prolonged tail bleeding time (P<0.001) (Figure 1D-ii), delayed arterial thrombus formation (Figure 1E-i) as well as reduced platelet accumulation (relative fluorescence at the site of vascular injury) (P<0.0001) (Figure 1E-ii and iii) were detected in mice with NLRP3-/- platelets, suggesting that platelet NLRP3 deficiency significantly impairs hemostasis and arterial thrombus formation in vivo.
NLRP3-/- platelets display mildly reduced platelet aggrega- tion and normal degranulation and aIIbβ3 activation
As platelet aggregation plays an important role in platelet function, we further investigated the effect of NLRP3 on platelet aggregation and activation. As shown in Figure 2A, platelet aggregation in response to a low dose of collagen (0.5 μg/mL), thrombin (0.05 U/mL) and ADP (10 μM) was
significantly reduced in NLRP3-/- platelets compared with that of wide-type platelets (P<0.05), which was consistent with the findings of a previous study showing that NLRP3 deficiency affects platelet aggregation and activation.15 However, no differences of platelet aggregation were found in response to a relatively high dose of collagen (1 μg/mL), thrombin (0.1 U/mL), ADP (20 μM) as well as collagen-relat- ed peptide (CRP: 0.5 and 1 μg/mL) (Figure 2A). As platelet granule secretion induced by agonist stimulation plays criti- cal roles in the amplification of platelet signaling and subse- quent activation and aggregation, we also measured platelet degranulation, represented by P-selectin expression and aIIbβ3 activation by flow cytometry. Interestingly, P-selectin upregulation (Figure 2B) and aIIbβ3 activation (Figure 2C) were normal in NLRP3-/- platelets after stimulation by colla- gen, CRP, thrombin or ADP. This is in contrast to the find- ings of Murthy and colleagues15 and could be related to sub- tle differences in platelet preparations and experimental con- ditions. A direct link between NLRP3 and a granule release has not been described, and differences in rate and extent of P-selectin exposure are consistent with studies that have demonstrated agonist-related differential kinetics of platelet degranulation and secretion.23-25
NLRP3 deficiency in platelets affects integrin aIIbβ3 outside-in signaling
Platelet spreading and clot retraction, two processes reg- ulated by early- and late-aIIbβ3 outside-in signaling, respectively,26 were evaluated. Spreading of NLRP3-defi- cient platelets (Figure 3A) but not platelet adhesion (Figure
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