P2Y12 antibody inhibits platelet activity and protects against thrombogenesis

Silva‐Espinoza

et al. 2018

JAHA

Self Cite

100

BackgroundCardiovascular disease is the main cause of death in the United States, with smoking being the primary preventable cause of premature death, and thrombosis being the main mechanism of cardiovascular mortality in smokers. Due to the perception that electronic/e‐cigarettes are “safer/less harmful” than conventional cigarettes, their usage—among a variety of ages—has increased tremendously during the past decade. Notably, there are limited studies regarding the negative effects of e‐cigarettes on the cardiovascular system, which is also the subject of significant debate.Methods and ResultsWe employed a passive e‐VapeTM vapor inhalation system and developed an in vivo whole‐body e‐cigarette mouse exposure protocol that mimics real‐life human exposure scenarios/conditions and investigated the effects of e‐cigarettes and clean air on platelet function and thrombogenesis. Our results show that platelets from e‐cigarette–exposed mice are hyperactive, with enhanced aggregation, dense and α granule secretion, activation of the αIIbβ3 integrin, phosphatidylserine expression, and Akt and ERK activation, when compared with clean air–exposed platelets. E‐cigarette–exposed platelets were also found to be resistant to inhibition by prostacyclin, relative to clean air. Furthermore, the e‐cigarette–exposed mice exhibited a shortened thrombosis occlusion and bleeding times.ConclusionsTaken together, our data demonstrate for the first time that e‐cigarettes alter physiological hemostasis and increase the risk of thrombogenic events. This is attributable, at least in part, to the hyperactive state of platelets. Thus, the negative health consequences of e‐cigarette exposure should not be underestimated and warrant further investigation.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Short‐Term E‐Cigarette Exposure Increases the Risk of Thrombogenesis and Enhances Platelet Function in Mice

Qasim

Silva‐Espinoza

et al. 2018

JAHA

Self Cite

100

“…Platelets (with counts adjusted, as previously described42, 43, 44) were stimulated with 1 μmol/L U46619, 5 μmol/L ADP, or 80 μmol/L TRAP4, and aggregation was measured. Each experiment was repeated at least 3 times, with blood pooled from at least 3 separate groups of 6 to 8 immunized mice.…”

Section: Methodsmentioning

confidence: 99%

“…These experiments were performed, as we previously described 42, 43, 44. Platelets from vaccinated mice (C‐EL2, C‐EL2r, and KLH) were prepared, as previously described (250 μL; 2.5×10 8 /mL), before being placed into siliconized cuvettes and stirred for 5 minutes at 37°C at 237 × g .…”

Section: Methodsmentioning

confidence: 99%

“…Flow cytometric analysis was performed on platelets from vaccinated mice (C‐EL2, C‐EL2r, and KLH), as previously discussed 42, 43, 44. Briefly, platelets (2×10 8 ) were fixed with 2% formaldehyde for 30 minutes at room temperature, before incubation with anti‐mouse glycoprotein IIb‐IIIa, anti–glycoprotein Ib, or anti–glycoprotein VI antibodies at room temperature for 30 minutes in the dark.…”

Section: Methodsmentioning

confidence: 99%

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Investigation of a Thromboxane A ₂ Receptor–Based Vaccine for Managing Thrombogenesis

Alshbool

Espinosa

et al. 2018

JAHA

Self Cite

BackgroundDespite the well‐established role for the thromboxane A2 receptor (TPR) in the development of thrombotic disorders, none of the antagonists developed to date has been approved for clinical use. To this end, we have previously shown that an antibody targeted against TPR's ligand‐binding domain inhibits platelet activation and thrombus formation, without exerting any effects on hemostasis. Thus, the goal of the present studies is to design a novel TPR‐based vaccine, demonstrate its ability to trigger an immune response, and characterize its antiplatelet and antithrombotic activity.Methods and ResultsWe used a mouse keyhole limpet hemocyanin/peptide‐based vaccination approach rationalized over the TPR ligand‐binding domain (ie, the C‐terminus of the second extracellular loop). The biological activity of this vaccine was assessed in the context of platelets and thrombotic diseases, and using a host of in vitro and in vivo platelet function experiments. Our results revealed that the TPR C‐terminus of the second extracellular loop vaccine, in mice: (1) triggered an immune response, which resulted in the development of a C‐terminus of the second extracellular loop antibody; (2) did not affect expression of major platelet integrins (eg, glycoprotein IIb‐IIIa); (3) selectively inhibited TPR‐mediated platelet aggregation, platelet‐leukocyte aggregation, integrin glycoprotein IIb‐IIIa activation, as well as dense and α granule release; (4) significantly prolonged thrombus formation; and (5) did so without impairing physiological hemostasis.ConclusionsCollectively, our findings shed light on TPR's structural biological features, and demonstrate that the C‐terminus of the second extracellular loop domain may define a new therapeutic target and a TPR vaccine‐based approach that should have therapeutic applications.

Mouse transient receptor potential channel type 6 selectively regulates agonist-induced platelet function

Espinosa

Lin

Biochemistry and Biophysics Reports

et al. 2019

Self Cite

While changes in intracellular calcium levels is a central step in platelet activation and thrombus formation, the contribution and mechanism of receptor-operated calcium entry (ROCE) via transient receptor potential channels (TRPCs) in platelets remains poorly defined. In previous studies, we have shown that TRPC6 regulates hemostasis and thrombosis, in mice. In the present studies, we employed a knockout mouse model system to characterize the role of TRPC6 in ROCE and platelet activation. It was observed that the TRPC6 deletion ( Trpc6 −/− ) platelets displayed impaired elevation of intracellular calcium, i.e., defective ROCE. Moreover, these platelets also exhibited defects in a host of functional responses, namely aggregation, granule secretion, and integrin αIIbβ3. Interestingly, the aforementioned defects were specific to the thromboxane receptor (TPR), as no impaired responses were observed in response to ADP or the thrombin receptor-activating peptide 4 (TRAP4). The defect in ROCE in the Trpc6 −/− was also observed with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Finally, our studies also revealed that TRPC6 regulates clot retraction. Taken together, our findings demonstrate that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Thus, TRPC6 may serve as a novel target for the therapeutic management of thrombotic diseases.