Arhgef1 Deletion Reduces Agonist\Induced Integrin IIb3 Activation Activation of integrin GPIIb\IIIa (IIb3) is required for platelet aggregation49 and involves conformational changes of the latter upon agonist activation

Arhgef1 Deletion Reduces Agonist\Induced Integrin IIb3 Activation Activation of integrin GPIIb\IIIa (IIb3) is required for platelet aggregation49 and involves conformational changes of the latter upon agonist activation.50 Since aggregation was defective in the Arhgef1 deletion platelets, we investigated whether there is a commensurate defect in integrin IIb3 activation. demonstrate, for the first time, that Arhgef1 plays a critical role in platelet function, in?vitro and in?vivo. for 15?minutes) at room temperature (RT), and the platelet\rich plasma was then collected. Platelets were counted with the HEMAVET 950FS Multispecies Hematology System, and the counts were adjusted to 7107 platelets per mL before each experiment. Washed Platelets Preparation Mouse blood was collected as discussed above, mixed with phosphate\buffered saline, pH 7.4, Climbazole and incubated with prostaglandin I2 (10?ng/mL; 5?minutes), followed by centrifugation at 237for 10?minutes at RT. Platelet\rich plasma was?recovered and platelets were pelleted at 483for 10?minutes at RT. The pellets were resuspended in HEPES/Tyrode buffer (20?mmol/L HEPES/potassium hydroxide, pH 6.5, 128?mmol/L NaCl, 2.8?mmol/L KCl, 1?mmol/L MgCl2, 0.4?mmol/L NaH2PO4, 12?mmol/L NaHCO3, 5?mmol/L d\glucose) supplemented with 1?mmol/L EGTA, 0.37?U/mL apyrase and 10?ng/mL prostaglandin?I2. Platelets were then washed and resuspended in HEPES/Tyrode (pH 7.4) without EGTA, apyrase, or prostaglandin I2. Platelets were counted using the HEMAVET 950FS Multispecies Hematology System and adjusted to the indicated concentrations. In Vitro Platelet Aggregation Platelets from both Arhgef1?/? and WT mice were activated with the thromboxane receptor agonist U46619 (2.5?mol/L), thrombin (0.05C0.1?U/mL), or collagen (5?g/mL) and Climbazole their aggregation response was measured by the turbidometric method using model 700 aggregometer (Chrono\Log Corporation). Each experiment was repeated 3 times using 3 separate groups, with blood pooled from 8 mice per group. Comparison is based on difference in maximal aggregation. ATP Release This assay was performed as previously described.39, 40, 41 Platelets were prepared as described above (250?L; 7107/mL) before being placed into siliconized cuvettes and stirred for 5?minutes at 37C. The luciferase substrate/luciferase mixture (12.5?L, Chrono\Log) was then added, followed by the addition of the agonists U46619 (2.5?mol/L), thrombin (0.05C0.1?U/mL), or collagen (5?g/mL). Each experiment was repeated 3 times using 3 separate groups, with blood pooled from PIK3R5 8 mice per group. Comparison is based on difference in maximal secretion. Flow Cytometric Analysis Flow cytometry analysis was performed as previously described.39, 40, 41 Briefly, platelets (2107/mL) from Arhgef1?/? and WT mice were stimulated with U46619 (2.5?mol/L), thrombin (0.1?U/mL), or collagen (5?g/mL) for 5 minutes. Platelets were then fixed with 2% formaldehyde for 30?minutes at RT and incubated with fluorescein isothiocyanateCconjugated CD62P (P\selectin) or PE\conjugated rat anti\mouse integrin IIb3 (active form) JON/A antibodies at RT for 30?minutes in the dark. The platelet (105?platelets/100?L) fluorescent intensities were measured using an Accuri C6 Flow Cytometer (BD Biosciences). Results were analyzed using C\Flow Plus (BD Biosciences). Each experiment was repeated 3 times using 3 separate groups, with blood pooled from 8 mice per group. Comparison is based on difference in mean fluorescent intensity. Fibrin Clot Retraction Assay Clot retraction assay was performed as previously described.42 Briefly, whole blood was collected and washed platelets were isolated as discussed above. CaCl2 was added extemporaneously, at a final concentration of 1 1?mmol/L. The glass tubes that were used for aggregation (Chrono\Log Corporation) were employed for retraction assays. The washed platelets were resuspended at 1108/mL in HEPES\Tyrode buffer (pH 7.4). Fibrinogen (500?g/mL) was added in 0.5\mL platelets aliquots, and clot retraction was initiated by quickly adding thrombin (0.1?U/mL). The reaction was transferred to the glass tube and the reaction was set at RT. Pictures were taken at time intervals of 5?minutes up to half an hour using a digital camera. This experiment was repeated at least 3 times, with blood pooled from a group of Climbazole 8 mice each time. Comparison is based on difference in clot size. Platelet Spreading The spreading of the Arhgef1?/? and WT platelets after stimulation with thrombin (0.1?U/mL) was examined as previously described.43 Briefly, sterile glass coverslips were coated with 0.2?g/mL of fibrinogen for 30?minutes at RT. Washed platelets were placed onto these fibrinogen\coated coverslips for 5, 30, and 45?minutes before they were fixed with 3.7% (vol/vol) formaldehyde for 15?minutes and quenched with 50?mmol/L ammonium chloride. Cells were rinsed with PBS and incubated with tetramethylrhodamine\conjugated phalloidin (1?g/mL) in 10% fetal bovine serum/PBS with 0.2% saponin. Coverslips were mounted and examined and imaged using.