Heparin potentiates platelet activation in suspension and on surfaces by activating signal amplification pathways. (A) Heparin synergizes with ADP to augment aggregation and granule release. Human platelets (2.5 × 10⁸/mL) in citrate-anticoagulated plasma were stimulated with 2μM ADP in the presence (UFH, 0.2 unit/mL) or absence (NaCl) of UFH at 37°C under stirring conditions. Although heparin alone did not stimulate aggregation (top panel) or granule secretion (bottom panel), platelets exposed to both ADP and heparin underwent irreversible aggregation and low-level granule release. Washed platelets in the presence of 200 μg/mL of exogenously added fibrinogen showed a similar synergistic aggregation response (not shown). (B) Platelets spread on immobilized heparin. Human platelets were added to 8-chamber glass tissue culture slides that had been coated with BSA (1%), fibrinogen (100 μg/mL), or heparin (10 units/mL), and allowed to spread for 45 minutes at 37°C. Note that platelets bound to and spread on immobilized heparin, although with slightly different morphology than on fibrinogen. (C) Akt and GSK3-β become phosphorylated in platelets exposed to heparin. Washed human platelets (2.5 × 10⁸/mL) were placed in an aggregometer cuvette at 37°C in the presence or absence of heparin (1 unit/mL) for 2 minutes, lysed in SDS sample buffer, and subjected to immunoblot analysis using the indicated antibodies. Note that exposure to heparin alone was sufficient to initiate the Akt → GSK3-β signal amplification pathway. (D) Akt and FAK become phosphorylated in platelets exposed to immobilized heparin. Platelets bound to immobilized fibrinogen or heparin for 45 minutes, or those nonadherent to BSA, were subjected to SDS-PAGE/immunoblot analysis using the indicated antibodies. Note that both Akt and FAK, reporters of integrin-mediated outside-in signaling, were phosphorylated in platelets bound to heparin.