grgds integrin-blocking peptide  (Millipore)

Journal: Thrombosis Research

Article Title: Involvement of Src kinases and PLCγ2 in clot retraction

doi: 10.1016/j.thromres.2006.09.003

Figure Lengend Snippet: The Src kinase inhibitors and α IIb β 3 blockade by GRGDS peptide reduced time course of clot retraction and inhibited protein tyrosine phosphorylation during clot retraction. (A) Human washed platelets (5 × 10 8 /ml) were preincubated with DMSO, 50 μM PP2, 40 μM PD173952. Clot retraction assays were started by adding 250 μl of 2 U/ml thrombin to 250 μl of platelets in the presence of 2 mg/ml fibrinogen and 2 mM CaCl 2 (final concentrations: 2.5 × 10 8 /ml of platelets, 1 U/ml of thrombin, 1 mg/ml of fibrinogen, 1 mM CaCl 2 ). The volume of remaining fluid was measured to assay the degree of clot retraction. The volume was expressed as mean ± SE ( n = 5–18 from 2–5 experiments). Results were analyzed using unpaired Student's t -test. One asterisk denotes p < 0.05 and two denote p < 0.005 between control and PP2/PD173952. (B) Human washed platelets in the presence or absence of 1 mM GRGDS peptide were stimulated by 250 μl of 2 U/ml thrombin as described. Reactions were terminated by addition of 2× lysis buffer. Samples were sonicated 3 periods of 15 s and insoluble debris removed by centrifugation at 15,000 g for 10 min. The supernatant was solubilized by addition of 4× SDS sample buffer. The platelet proteins were separated by SDS-PAGE and blotted with anti-phosphotyrosine antibody (4G10). (C) Human washed platelets (5 × 10 8 /ml) were preincubated with 50 μM PP3 or PP2 before starting assays of clot retraction. Protein tyrosine phosphorylation was analyzed as described above.

Article Snippet: Integrin α IIb β 3 blocking peptide GRGDS was from Peptide Institute (Osaka, Japan).

Techniques: Phospho-proteomics, Control, Lysis, Sonication, Centrifugation, SDS Page

Journal: Thrombosis Research

Article Title: Involvement of Src kinases and PLCγ2 in clot retraction

doi: 10.1016/j.thromres.2006.09.003

Figure Lengend Snippet: α IIb β 3 outside-in regulation of MLC phosphorylation was inhibited by Src inhibitor or PLC inhibitor. 0.4 ml of human washed platelets at 3 × 10 8 /ml pretreated with 10 μM indomethacin and 3 U/ml of apyrase followed by being incubated without (A) or with 50 μM PP2, PP3 (B), 10 μM U73122, or U73323 (C), and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. Bound and unbound platelets were solubilized by 4× Laemmli sample buffer, followed by immediate sonication for three periods of 5 s. Platelet proteins were separated by SDS-PAGE on 15% gels, electrotransferred, and then phospho-MLC or total MLC were blotted using anti-phospho-MLC mAb or anti-MLC pAb. (D) 0.4 ml of washed platelets at 3 × 10 8 /ml from PLCγ2+/+ and PLCγ2-/- mice were pretreated with 10 μM indomethacin and 3 U/ml of apyrase and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. The following procedure is the same as that for human platelets. The data are representatives of at least two experiments.

Article Snippet: Integrin α IIb β 3 blocking peptide GRGDS was from Peptide Institute (Osaka, Japan).

Techniques: Phospho-proteomics, Incubation, Sonication, SDS Page

Journal: Thrombosis Research

Article Title: Involvement of Src kinases and PLCγ2 in clot retraction

doi: 10.1016/j.thromres.2006.09.003

Figure Lengend Snippet: Multiple mechanisms of regulation of the contractile apparatus underlie clot retraction. Thrombin receptors are able to regulate clot retraction through activation of PLCβ and Rho kinase. α IIb β 3 outside-in signalling is required for optimal clot retraction through bifurcating signals, namely activation of PLCγ2 and phosphorylation of the diY motif, which combine to mediate clot retraction. This interaction could occur by recruitment of myosin to the phosphorylated diY motif in combination of activation of MLC kinase downstream of PLCγ2.

Article Snippet: Integrin α IIb β 3 blocking peptide GRGDS was from Peptide Institute (Osaka, Japan).

Techniques: Activation Assay, Phospho-proteomics