Journal: Diabetes

Article Title: Platelet Tissue Factor Synthesis in Type 2 Diabetic Patients Is Resistant to Inhibition by Insulin

doi: 10.2337/db09-1008

Figure Lengend Snippet: Effect of inhibitors of signaling pathways on TF synthesis in platelets. A : Platelets were preincubated with a mimetic of active clopidogrel metabolite (AR-C69931 MX, AR, 250 nmol/l), the aspirin-like inhibitor indomethacin (Indo, 100 μmol/l) or the combination (15 min, 37°C). AR-C69931 MX and indomethacin did not affect platelet adhesion ( insert ) but reduced surface-induced TF synthesis (4 h, 37°C). B : Platelets were preincubated with the adenylyl cyclase activator forskolin (40 μmol/l), the stable prostacyclin mimetic iloprost (1 μg/ml), the Ca 2+ quencher BAPTA/AM (25 μmol/l), ionophore A23187 (12 μmol/l) in the absence and presence of CaCl 2 (1 mmol/l), the PI3-K/PKB inhibitors wortmannin (0.5 μmol/l) and LY294002 (10 μmol/l), the Akt inhibitor 1701-1 (2 μmol/l), and ML-9 (100 μmol/l) (15 min, 37°C). The inhibitors reduced the surface-induced TF synthesis (4 h, 37°C). Addition of vehicle alone did not change surface-induced TF levels (as shown in C ). C : Platelets in the presence of excess of ADP and the TxA 2 mimetic U-44619 were preincubated with iloprost, BAPTA/AM, wortmannin, LY294002, the Akt inhibitor 1701-1, and ML-9 before platelets were adhered to collagen III (4 h, 37°C) and TF activity was determined. D : Platelets were adhered to fibrinogen (4 h, 37°C). The blot shows the 60-kDa band (in the presence of DTT) of monocyte (mono) TF, surface-induced platelet TF protein, the inhibition by puromycin (puro), and by the cAMP-raising agent iloprost (ilo). A , insert and D : Representative example for n = 3. A–C : TF activity in collagen III–adhered platelets was expressed as 100%. Data are means ± SE. n = 3–4. *Denotes a significant difference, P < 0.05. ns, not significant.

Article Snippet: We obtained human recombinant insulin (solubilized according to the recommendations of the manufacturer in 10 mmol/l acetic acid, 100 mmol/l NaCl, and 0.01% BSA to a stock concentration of 100 μmol/l); wortmannin; 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9); LPS ( E. coli 0111:B4); indomethacin; puromycin; collagen types I and III; ADP from Sigma (St. Louis, MO); Akt inhibitor 1701-1 from Biovision (Mountain View, CA); iloprost from Schering (Berlin, Germany); forskolin, BAPTA(AM), ionophore A23187, Tg003, and cantharidin from Calbiochem (La Jolla, CA); LY294002 from Biomol (Plymouth Meeting, PA, USA); cycloheximide from MP Biochemicals (Santa Ana, CA, USA); collagen reagent Horm from Nycomed Pharma; a mixture of 95% type I and 5% type III collagen (Munich, Germany); thrombin and fibrinogen from Enzyme Research Laboratories (South Bend, IN); and U-44619 from Cayman Chemicals (Ann Arbor, MI).

Techniques: Activity Assay, Inhibition

Journal: Diabetes

Article Title: Platelet Tissue Factor Synthesis in Type 2 Diabetic Patients Is Resistant to Inhibition by Insulin

doi: 10.2337/db09-1008

Figure Lengend Snippet: Insulin inhibits synthesis of platelet TF. A : Platelets (plts) were preincubated with 100 nmol/l insulin (5 min, 37°C) and adhered to surface-coated horm collagen (horm), thrombin (thr), collagen I (col I), fibrinogen (fg), collagen III (col III), and VWF (4 h, 37°C), and TF activity was measured. B : To the platelet suspension excess ADP and U-46619 were added and followed by preincubation with insulin (5 min, 37°C) before platelets were adhered to collagen III (4 h, 37°C), and TF activity was determined. C : Dose-dependent inhibition by insulin (ins) (5 min, 37°C) of TF synthesis by collagen III–adhered platelets. D : Splicing was demonstrated by the presence of either premRNA or mature mRNA in platelets that were untreated or preincubated with insulin and allowed to adhere to collagen I (30 min, 37°C). A representative example of n = 4 is shown. E : Platelets from eight type 2 diabetic patients and eight matched control subjects were preincubated with insulin (100 nmol/l, 5 min, 37°C) and allowed to adhere to collagen III (4 h, 37°C), and TF activity was measured. TF activity in adhered platelets without insulin from control subjects and type 2 diabetic patients were not significant different (0.17 ± 0.05 vs. 0.28 ± 0.13 ng TF/2.0 × 10 8 platelets, respectively). A–C and E : F activity in fibrinogen or collagen III–adhered platelets was set at 100%. Data are means ± SE. n = 3–8. mono, monocytes; ns, not significant. *Denotes a significant difference, P < 0.05.

Article Snippet: We obtained human recombinant insulin (solubilized according to the recommendations of the manufacturer in 10 mmol/l acetic acid, 100 mmol/l NaCl, and 0.01% BSA to a stock concentration of 100 μmol/l); wortmannin; 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9); LPS ( E. coli 0111:B4); indomethacin; puromycin; collagen types I and III; ADP from Sigma (St. Louis, MO); Akt inhibitor 1701-1 from Biovision (Mountain View, CA); iloprost from Schering (Berlin, Germany); forskolin, BAPTA(AM), ionophore A23187, Tg003, and cantharidin from Calbiochem (La Jolla, CA); LY294002 from Biomol (Plymouth Meeting, PA, USA); cycloheximide from MP Biochemicals (Santa Ana, CA, USA); collagen reagent Horm from Nycomed Pharma; a mixture of 95% type I and 5% type III collagen (Munich, Germany); thrombin and fibrinogen from Enzyme Research Laboratories (South Bend, IN); and U-44619 from Cayman Chemicals (Ann Arbor, MI).

Techniques: Activity Assay, Inhibition