The small guanine-nucleotide-binding protein Rap1 plays a key role in platelet aggregation and hemostasis, and we recently identified Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. In search of Rap1GAP2-associated proteins, we performed yeast-2-hybrid screening and found synaptotagmin-like protein 1 (Slp1) as a new binding partner. We confirmed the interaction of Rap1GAP2 and Slp1 in transfected COS-1 and HeLa cells and at endogenous level in human platelets. Mapping studies showed that Rap1GAP2 binds through amino acids T524-K525-X-T527 within its C-terminus to the C2A domain of Slp1. IntroductionBlood platelets are essential for hemostasis and play an important role in the development of thrombosis in the vasculature. 1 During primary hemostasis, platelets adhere to sites of endothelial damage and the initial platelet coat is soon reinforced by additional platelets forming a stable aggregate. At the same time, platelets secrete their intracellular granules containing substances that further activate platelets in an autocrine loop and affect local coagulation and endothelial and smooth muscle cell functions. 2 Two main types of secretory granules have been described in platelets, dense granules, and ␣-granules. Dense granules contain small molecules, including adenosine diphosphate, adenosine triphosphate, and 5-hydroxytryptamine (5-HT, serotonin), whereas ␣-granules store many proteins, such as fibrinogen, von Willebrand factor, various cytokines, and growth factors. 3,4 Dense granule secretion is regulated by the small GTPase Rab27 that is localized at the granule membrane. 5 Rab27 probably mediates its action by promoting granule motility or by enhancing tethering and fusion of granules with the plasma membrane. 6,7 For these functions, Rab27 needs to interact with effector proteins, and Munc13-4 was identified as the first Rab27-binding protein in platelets. 8 Other proteins involved in dense granule secretion are the small GTPase Ral 9 and membrane proteins of the soluble NSF attachment protein receptor family either bound to the granule, such as 10 or to the plasma membrane, such as SNAP-23 and syntaxin 2. 11 Granule secretion is accompanied by the development of tight cellular interactions between platelets. This aggregation of platelets is mediated by integrins and other adhesion receptors. 2 Integrin activation is tightly regulated by the small guanine-nucleotidebinding protein Rap1, and deletion of the platelet isoform Rap1b in mouse platelets results in a bleeding phenotype. 12 Rap1 activity is controlled by GTPase-activating proteins (GAPs) and guaninenucleotide exchange factors (GEFs). We recently discovered Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. 13 Rap1GAP2 contains a conserved central catalytic GAP domain, an N-terminal 14-3-3-binding site, and a large C-terminal region of unknown function. Platelet activation results in phosphorylation of Rap1GAP2 on serine 9, binding of 14-3-3 protein, and probably inhibition of GAP function. 14 In parallel, ...
BackgroundGeneric drugs and generic substitution belong to the tools by which healthcare costs may be reduced. However, low awareness and reluctance among healthcare professionals towards generic drugs may negatively affect the rational use of generic substitution.MethodsThe study aimed to analyze opinions and attitudes towards generic drugs and generic substitution among Czech physicians including their understanding of generic substitution legislative rules and the physicians´ previous experience in this field. Using random allocation, 1551 physicians practicing in the Czech Republic were asked to participate in the sociological representative survey conducted from November to December 2016, through face-to-face structured interviews comprising 19 items. Factor analysis as well as reliability analysis of items focused on legal rules in the context of physicians’ awareness were applied with p-value of < 0.05 as statistically significant.ResultsOf a total of 1237 (79.8%) physicians (43.7% males; mean age 47.5 ± 11.6 years, 46.3% general practitioners) 24.8% considered generic drugs to be less safe, especially those with specialized qualification (p < 0.01). However, only 4.4% of the physicians noticed any drug-related problems, including adverse drug reactions associated with generic substitution. The majority of physicians felt neutrally about performing generic substitution in pharmacies, nor they expressed any opinion on characteristics of generics, even though a better understanding of the legislation and higher need of accordance of substituted drugs were associated with more positive attitudes towards generic substitution (p < 0.05). Physicians showed low knowledge score of legislative rules (mean 3.9 ± 1.6 from maximum 9), nevertheless they overestimated the law, as they considered some rules valid, even if the law does not require them. Cronbach alpha of all legislative rules that regulate generic substitution increased from 0.318 to 0.553 if two optional rules (physician consent and strength equivalence) would be taken into account.ConclusionsThere is no sufficient awareness of generic drugs and generic substitution related issues among Czech physicians, although a deeper knowledge of legislation improves their perception about providing generic substitution.
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