Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.     

Fast Track by physician assistants shortens waiting and turnaround times of trauma patients in an emergency department

Purpose

We sought to determine whether the introduction of a separate patient flow comprising patients with simple, non-complex health issues [Fast Track (FT)] in a Dutch emergency department setting (ED), without the introduction of additional staff, and treated by a physician assistant, would have favourable effects on waiting and turnaround times without deleterious effects for patients with a higher urgency.

Study design

We used a prospective comparative intervention design for our study.

Methods

The waiting times and length of stay for surgical and orthopaedic patients in the ED were measured and compared 3 months before and 3 months after the introduction of FT.

Results

During the study period, 1,289 patients were treated before, and 1,393 after the introduction of FT. After the introduction of FT, we observed a decrease of 12 min (13 %) in the median length of stay for the total group. The median waiting time decreased by 41 min (69 %). The group comprising patients with low to moderate urgency levels showed a median reduction of 12 min in length of stay, whereas the length of stay for urgent patients was reduced by 19 min. The waiting time for the low to moderate urgency patients decreased by 68 min, while the urgent patient group showed a reduction of 32 min.

Conclusions

The introduction of FT performed by a physician assistant resulted in a significant drop in waiting time and length of stay in a Dutch ED setting. This reduction was realised without the allocation of additional staff and even reduced waiting and turnaround times for the patients with a high urgency.     

Differential effects of combined Ad5- delta 24RGD and radiation therapy in in vitro versus in vivo models of malignant glioma.

PURPOSE: The integrin-targeted conditionally replicating adenovirus Ad5-delta 24RGD has been shown to possess strong oncolytic activity in experimental tumors and is currently being developed toward phase I clinical evaluation for ovarian cancer and malignant glioma. Previously, we reported that combination therapy of Ad5-delta 24RGD with irradiation led to synergistic antitumor activity in s.c. glioma xenografts. In the current study, the underlying mechanism of action to this synergy was studied and the effects of combined therapy were assessed in an orthotopic glioma model. EXPERIMENTAL DESIGN AND RESULTS: Sequencing studies in U-87 monolayers showed that delivery of irradiation before Ad5-delta 24RGD infection led to a greater oncolytic effect than simultaneous delivery or infection before irradiation. This effect was not due to enhanced virus production or release. Experiments using a luciferase-encoding vector revealed a small increase in transgene expression in irradiated cells. In tumor spheroids, combination therapy was more effective than Ad5-delta 24RGD or irradiation alone. Staining of spheroid sections showed improved penetration of virus to the core of irradiated spheroids. Mice bearing intracranial tumors received a combination of Ad5-delta 24RGD with 1 x 5 Gy total body irradiation or with 2 x 6 Gy whole brain irradiation. In contrast to the in vitro data and reported results in s.c. tumors, addition of radiotherapy did not significantly enhance the antitumor effect of Ad5-delta 24RGD. CONCLUSIONS: Combined treatment with Ad5-delta 24RGD and irradiation shows enhanced antitumor activity in vitro and in s.c. tumors, but not in an orthotopic glioma model. These differential results underscore the significance of the selected tumor model in assessing the effects of combination therapies with oncolytic adenoviruses.     

EXEL-7647 inhibits mutant forms of ErbB2 associated with lapatinib resistance and neoplastic transformation.

PURPOSE: Mutations associated with resistance to kinase inhibition are an important mechanism of intrinsic or acquired loss of clinical efficacy for kinase-targeted therapeutics. We report the prospective discovery of ErbB2 mutations that confer resistance to the small-molecule inhibitor lapatinib. EXPERIMENTAL DESIGN: We did in vitro screening using a randomly mutagenized ErbB2 expression library in Ba/F3 cells, which were dependent on ErbB2 activity for survival and growth. RESULTS: Lapatinib resistance screens identified mutations at 16 different ErbB2 amino acid residues, with 12 mutated amino acids mapping to the kinase domain. Mutations conferring the greatest lapatinib resistance cluster in the NH2-terminal kinase lobe and hinge region. Structural computer modeling studies suggest that lapatinib resistance is caused by multiple mechanisms; including direct steric interference and restriction of conformational flexibility (the inactive state required for lapatinib binding is energetically unfavorable). ErbB2 T798I imparts the strongest lapatinib resistance effect and is analogous to the epidermal growth factor receptor T790M, ABL T315I, and cKIT T670I gatekeeper mutations that are associated with clinical drug resistance. ErbB2 mutants associated with lapatinib resistance transformed NIH-3T3 cells, including L755S and T733I mutations known to occur in human breast and gastric carcinomas, supporting a direct mechanism for lapatinib resistance in ErbB2-driven human cancers. The epidermal growth factor receptor/ErbB2/vascular endothelial growth factor receptor inhibitor EXEL-7647 was found to inhibit almost all lapatinib resistance-associated mutations. Furthermore, no ErbB2 mutations were found to be associated with EXEL-7647 resistance and lapatinib sensitivity. CONCLUSIONS: Taken together, these data suggest potential target-based mechanisms of resistance to lapatinib and suggest that EXEL-7647 may be able to circumvent these effects.