Heparin modulates integrin function in human platelets.
OBJECTIVE
Heparin binds to human platelets and can cause activation and aggregation, although the mechanisms are unknown. To determine how heparin alters platelet function, we identified platelet-binding sites for heparin and measured heparin's influence on the function of platelet integrin alpha(IIb)beta(3) (glycoprotein IIb/IIIa).


METHODS
Photoaffinity cross-linking and affinity chromatography experiments were performed to identify platelet membrane proteins that bind heparin. Heparin's effect on fibrinogen binding to platelets was measured with a radioligand-binding assay. The translocation to the cytoskeleton of Rap2, a guanosine triphosphate-binding protein, was measured from platelets aggregating in response to heparin and other agonists.


RESULTS
Cross-linking and affinity chromatographic experiments positively identified the integrin alpha(IIb)beta(3) as a heparin-binding site. Heparin aggregation was calcium dependent. Low concentrations of unfractionated porcine mucosal heparin (2-5 U/mL) significantly increased fibrinogen I 125 binding to activated platelets, whereas higher doses did not. Heparin-mediated platelet aggregation was completely blocked by GRGDS peptide (5 mmol/L), a competitive inhibitor of fibrinogen binding, and was blocked by EDTA (2 mmol/L), which dissociates the functional integrin complex. Aggregation was associated with Rap2 translocation to the cytoskeleton, a sign of outside-in signaling.


CONCLUSIONS
Heparin binds to the alpha(IIb)beta(3) integrin in vitro and ex vivo, and heparin increases fibrinogen binding to the integrin. Heparin-mediated aggregation requires an intact integrin and ligand and leads to Rap2 translocation to the cytoskeleton-an outside-in signal of ligand engagement. Heparin may directly modulate platelet integrin function, most likely through direct binding and modulation of integrin function.
Sobel
M
M
Department of Surgery, Section of Vascular Surgery, Syracuse Veterans Affairs Medical Center and Upstate Medical University, State University of New York, USA. Michael.Sobel@med.va.gov
Fish
W R
WR
Toma
N
N
Luo
S
S
Bird
K
K
Mori
K
K
Kusumoto
S
S
Blystone
S D
SD
Suda
Y
Y
D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects*; D002478:Cells, Cultured; D005340:Fibrinogen / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism
D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011956:Receptors, Cell Surface; C037680:heparin receptor; D005340:Fibrinogen; D006493:Heparin