Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I

Abstract

The involvement of exosite I in α-thrombin (FIIa) binding to platelet glycoprotein Ibα (GPIbα), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIbα amino terminal domain (GPIbα-N) fully sulfated on three tyrosine residues and solved the structure of its complex with FIIa. We found that sulfotyrosine (Tys) 278 enhances the interaction mainly by establishing contacts with exosite I. We then evaluated how substituting tyrosine with phenylalanine, which cannot be sulfated, affects FIIa binding to soluble or surface-immobilized GPIbα-N. Mutating Tyr 276 , which mostly contacts exosite II residues, markedly reduced FIIa interaction with both soluble and immobilized GPIbα-N; mutating Tyr 278 or Tyr 279 , which mostly contact exosite I residues, reduced FIIa complexing in solution by 0-20% but affinity for immobilized GPIbα-N 2 to 6-fold, respectively. Moreover, three exosite I ligands-aptamer HD1, hirugen, and lepirudin-did not interfere with soluble FIIa complexing to GPIbα-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired FIIa binding to immobilized GPIbα-N and platelet GPIb nearly as much as aptamer HD22 and heparin, both exosite II ligands. Bound HD1 and hirugen alter Trp 148 orientation in a loop near exosite I preventing contacts with the sulfate oxygen atoms of Tys 279 . These results support a mechanism in which binding occurs when the two exosites of one FIIa molecule independently interact with two immobilized GPIbα molecules. Through exosite engagement, GPIbα may influence FIIa-dependent processes relevant to hemostasis and thrombosis.tyrosine sulfation | tyrosylprotein sulfotransferase-2 | protease-activated receptor | platelet activation | platelet aggregation

Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4

“…PPACK-a-thrombin was produced by treating human (h) a-thrombin (Haematologic Technologies Inc.) with a 25-fold molar excess of PPACK for 8 h at 22-25°C and 3 days at 4°C as previously described 22 . Residual thrombin proteolytic activity was assessed by measuring the clotting time of a 4-mg ml À 1 fibrinogen solution and confirmed to be o1:1,000,000 of untreated a-thrombin 22 .…”

“…Biotin-PPACK (B-PPACK) and human a-thrombin (FIIa; Haematologic Technologies Inc.) were mixed to block the active site of the protease (B-PPACK-a-thrombin) and characterized 22 . B-PPACK-a-thrombin at 2 or 20 nM was added to 2 Â 10 7 ml À 1 PGE 1 -treated washed mouse platelets and incubated for 15 min at room temperature (22-25°C) in the presence of 0.1% fatty-acid-free bovine serum albumin (BSA-FAF; Sigma Aldrich, St Louis, MO, USA).…”

“…Exosite II is the major heparin-binding site 20 , but can also interact with highly sulfated polysaccharides and g 0 -fibrinogen 21 . These exosites are also important for a-thrombin binding to GPIba, the principal thrombin-binding site on platelets [22][23][24] . The importance of the GPIba/exosite I and II interaction in promoting thrombin binding to GPIba has been well defined 22,25,26 .…”

“…These exosites are also important for a-thrombin binding to GPIba, the principal thrombin-binding site on platelets [22][23][24] . The importance of the GPIba/exosite I and II interaction in promoting thrombin binding to GPIba has been well defined 22,25,26 . However, despite more than 30 years of investigation and two published crystal structures 23,24 , the precise pathophysiological function of the thrombin-GPIba interaction remains unclear.…”

The maternal immune response to fetal platelet GPIbα causes frequent miscarriage in mice that can be prevented by intravenous IgG and anti-FcRn therapies

“…In addition, the anti-GPIbα polyclonal antibodies generated in our antiGPIbα FNIT model may block thrombin binding to platelet GPIbα (42,43), which may increase free thrombin, further enhancing the conversion of fibrinogen to fibrin. To test this, we purified IgG from anti-GPIbα serum and preimmune GPIbα serum.…”