Differential proteomic analysis of platelets suggested target-related proteins in rabbit platelets treated with Rhizoma Corydalis

Figures & data

Figure 1. The 2-DE proteome images of control (A) and RC-treated (B) platelets. The differentially expressed protein spots were shown by the arrows.

Figure 2. The differentially expressed protein spots in Figure 1.

Table 1. The results of proteins identification of differentially expressed proteins using MALDI-TOF-MS/MS.

Spot no.	Protein name	NCBI acc. no.	MW/PI	Protein score	Sequence coverage	Fold changed	Function
cytoskeletal
2	Gamma non-muscle actin	gi|1703	42.1/5.30	188	8%	0.33	Involved in cell mobility, exchanges nucleotide faster than α skeletal muscle actin and also bounds profilin with greater affinity
4	Lamin isoform X1	gi|655846237	74.3/6.73	240	8%	3.21	Involved in cell mobility, keeps the nucleus shape and strength
16	Prohibitin	gi|291405834	29.8/5.57	574	31%	0.35	Binds to monomeric actin and phophatidyl inositol phosphates, increases the rate of nucleotide exchange on actin
17	Alpha-smooth muscle actin	gi|1701	42.3/5.23	53	4%	2.19	Involved in cell mobility, activates and dedifferentiates mesangial cells
21	Tropomodulin-3	gi|291402996	39.6/4.86	204	13%	1.91	The only Tmod isoform detected in platelets and megakaryocytes (MKs), caps actin filament (F-actin) pointed ends and binds tropomyosins (TMs), regulates actin polymerization and stability
35	Alpha-smooth muscle actin	gi|1701	42.3/5.23	75	4%	0.15	Involved in cell mobility, activates and dedifferentiates mesangial cells
40	Low quality protein: zyxin	gi|655815444	62.8/6.04	132	8%	0.27	A actin-binding protein, interacts with α-actinin and enables activated phosphoprotein
43	WD repeat-containing protein 1	gi|655604311	77.0/6.02	146	12%	4.35	Regulates the components of multi-protein complexes involved in transcriptional activation or repression, signal transduction
44	WD repeat-containing protein 1	gi|655604311	66.9/6.02	500	19%	95.68	Regulates the components of multi-protein complexes involved in transcriptional activation or repression, signal transduction, etc.
46	Actin-related protein 3	gi|291391454	47.7/5.61	167	11%	3.08	Major constituent of the ARP2/3 complex, generates new barbed ends by stimulating nucleation, required for actin assembly
47	Alpha-smooth muscle actin	gi|1701	42.4/5.23	34	4%	3.06	Involved in cell mobility, activates and dedifferentiates mesangial cells
49	Cullin-2 isoform X1	gi|291409897	87.5/6.45	30	2%	0.21	The help of F-box or SOCS box proteins attaches to the substrate
Cell signalling
6	Ras-related protein Rap-1b	gi|291389525	21.0/6.65	99	7%	0.35	Rap 1 isoform and the most abundant Ras family member, regulates crosstalk between platelet integrin α2β1 and integrin αIIbβ3
12	Annexin A4	gi|655606395	36.1/5.30	163	18%	0.27	A cytosolic calcium-binding protein with four repeat domains, interacts with the phospholipids membrane to regulate the signal transduction
15	Guanine nucleotide-binding protein G(i) subunit alpha-2	gi|655834605	40.9/5.35	293	20%	0.49	Involved in platelet activation signal pathways, such as binds P2Y12 receptor to active the downstream transferring signal.
26	ADP-ribosylation factor 3	gi|291389073	20.6/6.84	113	23%	0.55	The member of Ras GTP, binds G protein to regulate transferring signal
28	Synaptotagmin-7	gi|655882206	71.2/9.63	33	2%	0.13	Calcium signalling
30	Transient receptor potential cation channel subfamily M member 3	gi|291383356	197.5/7.56	36	1%	5.95	Calcium binding and signalling
48	Ribosomal protein S6 kinase alpha-2	gi|655895951	73.2/7.33	39	2%	43.84	The member of MAPK signalling pathway
Molecular chaperones
9	Heat shock cognate 71 kDa protein	gi|291383777	71.1/5.37	423	6%	0.3	May be involved in protecting the centrosome and intermediating filaments during heat shock
39	Protein disulphide isomerase-associated 3 precursor	gi|217030873	56.7/5.98	146	11%	0.32	Catalyzes disulphide exchange reaction, mediates platelet aggregation and secretion
42	Chaperonin-containing T-complex polypeptide beta subunit	gi|209981451	57.8/6.01	79	10%	3.1	Mediates the folding of proteins and folding pathway from denatured state to correctly folded product; assembles various newly synthesized polypeptides
Directly related to the platelet function
11	Fibrinogen beta chain	gi|291401109	56.7/8.42	113	6%	2.09	Polymerizes into fibrin; acts as cofactor in aggregation
19	Zinc-finger protein 64 homologue, isoforms	gi|655881403	78.8/8.88	43	2%	4.24	Transcription/transcription regulation, involved in various cellular process
23	Peroxiredoxin-6	gi|291397244	25.0/5.73	435	27%	0.22	Oxidase activity (direct oxidation of H2O2)
34	Fibrinogen beta chain	gi|291401109	56.7/8.42	44	4%	0.24	Polymerizes into fibrin; acts as cofactor in aggregation
37	Serum albumin precursor	gi|29653363	70.8/5.85	295	18%	3.78	Transports and depots protein for numerous endogenous and exogenous compounds in the circulation system, maintains the osmotic pressure of plasma and scavenges oxygen free radicals (platelet function: associated with α granules)
45	Fibrinogen gamma chain isoform X1	gi|291401113	51.0/5.68	185	17%	0.26	Polymerizes into fibrin; acts as cofactor in aggregation
Enzyme
3	Succinyl-CoA:3-ketoacid coenzyme A transferase 1	gi|291395294	56.5/8.62	213	8%	3.65	Activates acetoacetate by transferring the CoA group to produce acetoacetyl-CoA and succinate
5	Isocitrate dehydrogenase [NADP] cytoplasmic	gi|291392139	47.1/6.61	137	13%	0.3	An essential cofactor for many enzymatic reactions including glutathione metabolism and cholesterol biosynthesis
8	V-type proton ATPase subunit B, brain isoform	gi|291401067	56.7/5.57	376	13%	6.93	ATPase, couples ATP hydrolysis to proton transport, acidizes a variety of intracellular compartments in eukaryotic cells
14	Pyruvate carboxylase	gi|655899056	35.2/9.60	149	15%	4.15	Anaplerosis influences insulin secretion in beta cells; catalyzes the ATP-dependent carboxylation of pyruvate to form oxaloacetate
18	Medium-chain specific acyl-CoA dehydrogenase	gi|291398674	46.5/8.65	178	6%	0.19	Catalyzes the rate-determining step of fatty acid β-oxidation in mitochondria
20	V-type proton ATPase catalytic subunit A	gi|655854861	68.8/5.36	202	6%	3.12	ATPase, responsible for acidifying a variety of intracellular compartments in eukaryotic cells, may participate in maintenance of cytoplasmic Ca^2+ homeostasis.
22	Methylcrotonoyl-CoA carboxylase subunit alpha	gi|655853635	80.8/6.80	90	4%	0.13	Catalyzes the fourth step in the leucine catabolic pathway
25	Acyl-coenzyme A synthetase ACSM3	gi|291390708	66.0/8.76	43	2%	0.32	Esterifies free fatty acids to CoA to form acyl-CoAs
29	Guanylate cyclase soluble subunit beta-1	gi|655856074	71.2/5.23	98	4%	0.35	Converts GTP into cGMP
31	Protein monoglycylase TTLL8	gi|655894895	108.4/9.35	33	2%	3.21	Modifies both tubulin and non- tubulin proteins to generate side chains of glycine on the gamma- carboxyl groups of specific glutamate residues of target proteins
32	arf-GAP with GTPase, ANK repeat and PH domain-containing protein 3	gi|655844868	93.8/7/86	39	1%	3.43	GTPase-activating protein for ARF1, involved in ARF GTPase activator activity, regulates the adapter protein 3 (AP-3)-dependent trafficking of proteins in the endosomal-lysosomal system
33	Beta beta enolase	gi|14141143	47.3/7.63	64	4%	0.32	Catalyzes the production of phosphoenolpyruvic acid
38	Arachidonate 12-lipoxygenase, 12S-type	gi|291405193	76.3/5.82	55	5%	0.29	Shows broad substrate specificity and blocks platelets aggregation induced by agonists
50	V-type proton ATPase subunit B, brain isoform	gi|291401067	56.7/5.57	108	10%	2.53	ATPase, acidulated cellular organelles
51	Succinyl-CoA:3-ketoacid coenzyme A transferase 1	gi|291395294	56.5/8.62	142	3%	46.03	Rate-limiting enzyme, converts GAP into GTP and succinic acid
52	Mitochondrial ATP synthase, H + transporting F1 complex beta subunit, partial	gi|89574025	45.6/5.21	356	10%	103.83	Catalyzes ATP synthesis,
Unclassified
1	Mitochondrial inner membrane protein isoform X1	gi|655606040	84.0/5.92	170	5%	0.16	Synthesizes ATP and provides energy, eliminates oxygen free radicals
10	Ubiquinone biosynthesis protein COQ9	gi|655788788	44.0/9.07	95	6%	2.35	Involved in biosynthesis of COQ10
13	Elongation factor 1 delta	gi|1134985	31.1/5.06	81	22%	2.35	EF-Tu: catalyzes the production of GDP
24	Initiation factor 4AI	gi|227238	46.3/5.32	324	23%	0.2	Participate in initiation process of eukaryotic translation
27	Elongation factor Tu	gi|291390870	49.7/6.94	196	17%	0.3	Controls protein synthesis and translation
36	Transcription factor HIVEP3	gi|655850235	257.8/8.5	47	20%	3.96	Regulates gene transcription
41	NSFL1 cofactor p47 isoform X2	gi|291388803	40.6/5.03	231	14%	0.19	Determines the reaction type

Figure 3. Effect of RC on the platelet intracellular calcium [Ca²⁺]_i concentration [A, mean ± SD (n = 3), ###p < .001, vs. resting platelet group, **p < .01, ***p < .001, ##p < .01, vs. THR activated group], P-selectin [B, mean ± SD (n = 3), #p < .05, vs. resting platelet group, *p < .05, vs. THR-activated group], cGMP [C, mean ± SD (n = 3), ##p < .01, vs. resting platelet group, *p < .05, vs. THR-activated group] and 5-HT [D, mean ± SD (n = 3)].

Figure 4. The possible signal pathways for RC antiplatelet function.