Activated platelets in heparinized shed blood: the "second hit" of acute lung injury in trauma/hemorrhagic shock models.

The return of heparinized shed blood (SB) in trauma/hemorrhagic shock (T/HS) models remains controversial because of potential anti-inflammatory properties. Although ubiquitous as an anticoagulant, heparin is ineffective on cellular coagulation as an antithrombotic agent. Therefore, we hypothesized that returning heparinized SB would paradoxically enhance acute lung injury (ALI) after T/HS because of the infusion of activated platelets. Sprague-Dawley rats, anesthetized with pentobarbital, underwent laparotomy and hemorrhage-induced shock (MAP of 30 mmHg × 45 min). Animals were resuscitated with a combination of normal saline and returned SB. Shed blood was collected in either 80 U/kg of heparin, 800 U/kg of heparin, or citrate or diluted 1:8 with normal saline. An additional group of animals were pretreated with a platelet P2Y12 receptor antagonist (clopidogrel) before T/HS. Bronchoalveolar lavage, lung myeloperoxidase assays, pulmonary immunofluorescence, and blood smears were conducted. Bronchoalveolar lavage protein increased in animals resuscitated with heparinized SB (T/HS + 80 U/kg Hep 1.62 ± 0.29, T/HS + 800 U/kg Hep 1.30 ± 0.15 vs. T/SS 0.51 ± 0.16 and T/HS Citrate 0.7 ± 0.09) (P < 0.0001). Blood smears and platelet function assays revealed platelet aggregates and increased platelet activation. Animals pretreated with a platelet P2Y12 receptor antagonist were protected from postinjury ALI (P < 0.0001). Animals with return of SB had increased pulmonary polymorphonuclear leukocyte sequestration (P < 0.0001). Pulmonary immunofluorescence demonstrated microthrombi only in the T/HS group receiving heparinized SB (P < 0.0001). The return of heparinized SB functions as a "second hit" to enhance ALI, with activated platelets propagating microthrombi and pulmonary polymorphonuclear leukocyte recruitment.

Inhibition of Mer and Axl receptor tyrosine kinases in astrocytoma cells leads to increased apoptosis and improved chemosensitivity.

Astrocytomas account for the majority of malignant brain tumors diagnosed in both adult and pediatric patients. The therapies available to treat these neoplasms are limited, and the prognosis associated with high-grade lesions is extremely poor. Mer (MerTK) and Axl receptor tyrosine kinases (RTK) are expressed at abnormally high levels in a variety of malignancies, and these receptors are known to activate strong antiapoptotic signaling pathways that promote oncogenesis. In this study, we found that Mer and Axl mRNA transcript and protein expression were elevated in astrocytic patient samples and cell lines. shRNA-mediated knockdown of Mer and Axl RTK expression led to an increase in apoptosis in astrocytoma cells. Apoptotic signaling pathways including Akt and extracellular signal-regulated kinase 1/2, which have been shown to be activated in resistant astrocytomas, were downregulated with Mer and Axl inhibition whereas poly(ADP-ribose) polymerase cleavage was increased. Furthermore, Mer and Axl shRNA knockdown led to a profound decrease of astrocytoma cell proliferation in soft agar and a significant increase in chemosensitivity in response to temozolomide, carboplatin, and vincristine treatment. Our results suggest Mer and Axl RTK inhibition as a novel method to improve apoptotic response and chemosensitivity in astrocytoma and provide support for these oncogenes as attractive biological targets for astrocytoma drug development.