Summary
The HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the γ chains and the RGD sequences in the Aα
chains of human fibrinogen are potential recognition sites for the binding of soluble
fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus,
addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human
platelets. In contrast, we reported previously that RGDS had relatively little inhibitory
effect on these functions of rabbit platelets. In the present study, we found that
H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent
manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide
to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated
that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent
manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside
extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen
γ chain sequence. Proteins of ∼135 kDa and ∼95 kDa were specifically eluted by soluble
H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody
against human GPIIIa. In control samples, no detectable protein from rabbit platelet
lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results
demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting
with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as
a suitable thrombosis model for testing the efficacy of peptide mimetics derived from
H12.
Keywords
Rabbit platelets - fibrinogen - GPIIb-IIIa - gamma chain