Medicine for regulating platelet activity, screening method and application of medicine
A platelet and drug technology, applied in the field of hematology and biomanufacturing, to achieve broad industrialization prospects, easy synthesis and purification, and low immunogenicity
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Embodiment 1
[0051] Example 1 Hypoxia promotes FUNDC1-dependent mitophagy in platelets in vivo
[0052] We obtained Fundc1 gene knockout mice (F1KO mice for short) from the Sanger Institute (Cambridge, UK), and carried out corresponding identification to prove its Fundc1 gene knockout ability, etc. ( figure 1 a-b). We found that Fundc1 knockout mice also had normal blood and spleen phenotypes ( figure 1 c-f). Next, we exposed the mice to a hypoxic environment with 8% oxygen content for 3 consecutive days, and then isolated platelets from Fundc1 knockout mice and wild-type mice, and detected the expression of mitochondria-related proteins by immunoblotting. Expression level (Tim23 is a molecular marker of mitochondrial outer membrane, Tom20 is a molecular marker of mitochondrial inner membrane), p62 expression level and LC3-II expression level (p62 and LC3-II are biochemical markers of autophagy). We found that in platelets isolated from wild-type mice, the levels of mitochondria-related...
Embodiment 2
[0055] Example 2 Autophagy caused by hypoxia determines the function and quality of mitochondria in vivo
[0056] We synthesized a membrane-permeable peptide that mimics the dephosphorylation and phosphorylation of tyrosine 18 in the LIR domain of FUNDC1. Indeed, a dephosphorylated synthetic peptide (P1: GRKKRRQRRRPQDYESDDESYEVLDLTE) could effectively prevent hypoxia-induced degradation of mitochondrial proteins and p62, and could prevent the conversion of LC-3I to LC3-II in wild-type platelets, while phosphorylated peptide (P2: GRKKRRQRRRPQDYESDDESpYEVLDLTE) does not have this function ( image 3 b). The results of co-immunoprecipitation experiments showed that the P1 peptide effectively inhibited the interaction between FUNDC1 and LC3 in wild-type platelets treated under hypoxic conditions, while the P2 peptide did not ( image 3 a). The above experiments preliminarily indicated that the phosphorylated synthetic peptide P1 could prevent the autophagy of platelet mitochond...
Embodiment 3
[0058] Example 3 Autophagy involved in FUNDC1 can regulate the activity of platelets
[0059] Hypoxia significantly reduced platelet aggregation in wild-type platelets treated with thrombin and ADP, whereas treatment with P1 peptide, but not P2 peptide, reversed this trend ( Figure 4 a-d). On the other hand, platelets derived from Fundc1 knockout mice were treated with ADP and thrombin, and the aggregation ability of platelets was significantly reduced, which may be due to the damage of mitochondrial number ( Figure 4 a-d). Likewise, the ability of ADP- and thrombin-induced platelets to spread on VWF-coated coverslips was inhibited both in hypoxia-treated wild-type platelets and in untreated Fundc1-deficient platelets ( Figure 4 e-h). But the expressions of ADP and thrombin receptors P2Y12 and Par2 and the downstream signaling platelet integrin protein receptor IIbIIIa did not change significantly. Fundc1 insufficiency did not promote apoptosis of platelets (treated wit...
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