Effect of dominant negative SNAP-23 expression on platelet function.

BACKGROUND: The protein SNAP-23 is part of the secretory pathway in platelets. It is, however, not entirely clear to what extent this protein contributes to the secretory function of platelets. Therefore, we overexpressed a dominant negative mutant with a novel technology that allows the creation of intact transgene-expressing platetets. RESULTS: Overexpression of a dominant negative SNAP-23 mutant that inhibited the binding of the native protein to the docking site within the secretory machinery resulted in significant suppression of the agonist-dependent surface recruitment of P-selectin and CD40L. Simultaneously, release from dense granules was clearly suppressed in the presence of this construct. Also agonist-dependent surface expression of fibrinogen receptor markers CD41 and CD61 was reduced, and agonist-triggered aggregation was inhibited. CONCLUSION: The dominant negative inhibition of SNAP-23 resulted in clear effects on platelet functions. The novel method using recombinant culture-derived platelets allowed the rapid clarification of the functional importance of this protein in intact platelets.

Blocking caspase-activated apoptosis improves contractility in failing myocardium.

Cardiac myocyte apoptosis has been demonstrated in end-stage failing human hearts. The therapeutic utility of blocking apoptosis in congestive heart failure (CHF) has not been elucidated. This study investigated the role of caspase activation in cardiac contractility and sarcomere organization in the development of CHF. In a rabbit model of heart failure obtained by rapid ventricular pacing, we demonstrate, using in vivo transcoronary adenovirus-mediated gene delivery of the potent caspase inhibitor p35, that caspase activation is associated with a reduction in contractile force of failing myocytes by destroying sarcomeric structure. In this animal model gene transfer of p35 prevented the rise in caspase 3 activity and DNA-histone formation. Genetically manipulated hearts expressing p35 had a significant improvement in left ventricular pressure rise (+dp/dt), decreased end-diastolic chamber pressure (LVEDP), and the development of heart failure was delayed. To better understand this benefit, we examined the effects of caspase 3 on cardiomyocyte dysfunction in vitro. Microinjection of activated caspase 3 into the cytoplasm of intact myocytes induced sarcomeric disorganization and reduced contractility of the cells. These results demonstrate a direct impact of caspases on cardiac function and may lead to novel therapeutic strategies via antiapoptotic regimens.