Tyrosine phosphorylation of Pyk2 is mediated by SFKs and is required for shear-resistant PMN attachment to platelets. (A) Untreated PMNs or PMNs pretreated with DMSO, 10 μM PP2, or 10 μM PP3 were stirred for 3 minutes with activated, fixed platelets (ratio 5:1) in the presence or in the absence of 5 μM EGTA. Pyk2 was immunoprecipitated from total cell lysates, and immune complexes were analyzed for phospho-Pyk2 by immunoblotting with PY99 and then reprobed with anti-Pyk2 antibody to detect total Pyk2. The bars report the ratio between optical density of PY99 and PYK2 (ie, phospho-Pyk2/total Pyk2). The figure is representative of results obtained in 3 different experiments. (B) PMNs from wild-type, double hck^−/−fgr^−/−, or triple hck^−/−fgr^−/−lyn^−/− mice were stirred in the absence or in the presence of activated, fixed wild-type platelets and processed as described in panel A. The bars indicate the ratio between optical density of PY99 and PYK2 (phospho-Pyk2/total Pyk2). The figure is representative of results obtained in 2 different experiments performed with PMNs isolated from pooled bone marrow obtained from 5 wild-type or 5 knock-out mice. (C) Human PMNs pretreated with DMSO or the indicated concentrations of the Pyk2 inhibitor tyrphostin A9 (AG17) were stirred for 3 minutes with activated, fixed platelets (ratio 5:1). Samples were immediately fixed with an equal volume of 2% PFA and the formation of mixed platelet-PMN conjugates was analyzed by flow cytometry, as described in “Materials and methods.” The number of platelets recruited by 100 PMNs and the percentage of PMNs with attached platelets are graphed (mean ± SEM, n = 3).