JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

Abstract

Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin α(IIb)β(3). Once platelet activation has occurred, integrin α(IIb)β(3) stabilizes thrombus formation by providing agonist-independent "outside-in" signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A-deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

Figure 1

Ablation of Jam-A results in prothrombotic phenotype. (A) Western blot analysis of proteins from Jam-A^wt/wt and Jam-A^gt/gt platelets. Human platelet lysate was used as a positive control. (B) Tail-bleeding time of Jam-A^wt/wt and Jam-A^gt/gt mice before genotyping (n = 32). Mean bleeding time is denoted by a horizontal line. (C) Carotid artery blood flow in Jam-A^wt/wt and Jam-A^gt/gt mice following exposure to 7.5% FeCl₃. Shown is the representative flow trace of Jam-A^wt/wt and Jam-A^gt/gt mice (n = 8). Duration of injury is denoted by no. 1 and the time to occlude is denoted by no. 2. (D) Quantitation of occlusion time from panel C.

Figure 2

In vivo thrombosis is enhanced in the absence of Jam-A. (A) Fluorescence images of cremaster muscle of Jam-A^wt/wt and Jam-A^gt/gt mice on in vivo laser-induced injury. Quantitation of peak fluorescence and the thrombus size was analyzed using Intelligent Imaging software (n = 40 injuries). (B) In vivo laser-induced injury assay as in panel A using chimeric mice generated by lethally irradiated Jam-A^wt/wt mice receiving BM transplantation from Jam-A^gt/gt mice (n = 22 injuries). Quantitation of the data analyzed using Intelligent Imaging software.

Figure 3

Absence of Jam-A accelarate pulmonary thromboembolism. (A) Survival curve of Jam-A^wt/wt and Jam-A^gt/gt mice after induction of pulmonary thromboembolism (n = 18). (B) Representative images of lung isolated from Jam-A^wt/wt and Jam-A^gt/gt injected with PBS or PBS and Evans blue or a mixture of collagen and epinephrine solution. (C) H&E-stained sections of lungs.

Figure 4

Ablation of Jam-A results in hyperaggregability. (A) Representative aggregation tracings of platelets from Jam-A^wt/wt and Jam-A^gt/gt mice induced by AYPGQV (PAR 4 peptide), ADP, or collagen as indicated. Experiments were performed at least 3 times independently. (B) Surface expression of integrin α_IIbβ₃ on Jam-A^wt/wt and Jam-A^gt/gt platelets as analyzed by flow cytometry. (C) FITC-labeled Fg binding to Jam-A^wt/wt and Jam-A^gt/gt platelets stimulated with 100μM AYPGKF and analyzed by flow cytometry. (D) Representative flow cytometric histogram of JON/A binding to Jam-A^wt/wt and Jam-A^gt/gt platelets stimulated with AYPGKF. Quantitation of mean fluorescence intensity of JON/A binding on stimulation with various concentrations of AYPGKF from 3 independent experiments.

Figure 5

The granular secretion is normal in Jam-A^gt/gt platelets. (A) ATP secretion from Jam-A^wt/wt and Jam-A^gt/gt platelets stimulated by various concentrations of AYPGKF. Quantitation of experiments performed 3 times independently. (B) Flow cytometric histogram of P-selectin exposure on Jam-A^wt/wt and Jam-A^gt/gt platelets stimulated with 100μM AYPGKF for 10 minutes. Quantitation of mean fluorescence intensity normalized over control IgG from at least 3 independent experiments. (C) Quantitation of thrombin (0.1 U/mL)–induced TxA2 generation in Jam-A^wt/wt and Jam-A^gt/gt platelets (n = 3). (D) Flow cytometric histogram of PECAM-1 expression on Jam-A^wt/wt and Jam-A^gt/gt platelets. Quantitation of mean fluorescence intensity was normalized over control IgG from at least 3 independent experiments.

Figure 6

Absence of Jam-A promotes platelet spreading on immobilized Fg. (A) Representative DIC images of Jam-A^wt/wt and Jam-A^gt/gt platelets spread on immobilized Fg. BSA was used as a control. Zoomed views are indicated with arrowheads. (B) Quantification of the percentage of fully spread platelet on immobilized Fg compared with BSA. At least 100 individual platelets per view in triplicate for each time point were analyzed. Data shown are the quantification from at least 3 independent experiments. (C) Platelet surface area from panel A. Quantitation of > 50 platelets in a given view in triplicate were analyzed from at least 3 independent experiments.

Figure 7

Ablation of Jam-A results in enhanced integrin outside-in signaling. (A) Photographs of clot retraction using washed Jam-A^wt/wt and Jam-A^gt/gt platelets. Quantitation of the percentage of clot retraction from 3 independent experiments. (B) Western blots of protein lysates from Jam-A^wt/wt and Jam-A^gt/gt platelets exposed for 1 hour to immobilized Fg or BSA and probed with anti-phospho-specific p38 or anti-p38, or anti-phospho-specific Erk2 or anti-Erk2, or anti-phospho–specific myosin light chain or anti–myosin light chain. Quantitation of normalized optical density of 3 independent experiments. (C) Western blots of protein lysates from panel A probed using anti-phospho–specific β₃Y⁷⁷³ or anti-β₃. Quantitation of normalized optical density of 3 independent experiments.