Identification of Peptide Antagonists to Glycoprotein Ibα That Selectively Inhibit von Willebrand Factor Dependent Platelet Aggregation

Abstract

GPIbalpha is an integral membrane protein of the GPIb-IX-V complex found on the platelet surface that interacts with the A1 domain of von Willebrand factor (vWF-A1). The interaction of GPIbalpha with vWF-A1 under conditions of high shear stress is the first step in platelet-driven thrombus formation. Phage display was used to identify peptide antagonists of the GPIbalpha-vWF-A1 interaction. Two nine amino acid cysteine-constrained phage display libraries were screened against GPIbalpha revealing peptides that formed a consensus sequence. A peptide with sequence most representative of the consensus, designated PS-4, was used as the basis for an optimized library. The optimized selection identified additional GPIbalpha binding peptides with sequences nearly identical to the parent peptide. Surface plasmon resonance of the PS-4 parent and two optimized synthetic peptides, OS-1 and OS-2, determined their equilibrium dissociation GPIbalpha binding constants ( K Ds) of 64, 0.74, and 31 nM, respectively. Isothermal calorimetry corroborated the K D of peptide PS-4 with a resulting affinity value of 68 nM. An ELISA demonstrated that peptides PS-4, OS-1, and OS-2 competitively inhibited the interaction between the vWF-A1 domain and GPIbalpha-Fc in a concentration-dependent manner. All three peptides inhibited GPIbalpha-vWF-mediated platelet aggregation induced under high shear conditions using the platelet function analyzer (PFA-100) with full blockade observed at 150 nM for OS-1. In addition, OS-1 blocked ristocetin-induced platelet agglutination of human platelets in plasma with no influence on platelet aggregation induced by several agonists of alternative platelet aggregation pathways, demonstrating that this peptide specifically disrupted the GPIbalpha-vWF-A1 interaction.

The proof is in the crystal

“…The patients who presented with ICH as the first manifestation of the ITP were excluded from treatment/cost analysis. As in previous studies, 2,5,6 head trauma and hemorrhagic manifestations (beyond mild cutaneous) were significant risk factors for ICH and, in the case of more bleeding manifestations, increased mortality. In contrast to other studies, the authors found no relationship between concurrent nonsteroidal anti-inflammatory drug use, wet purpura, and gastrointestinal or vaginal bleeding, but this may be due in part to the limitations of retrospective reporting in a survey study.…”

“…5 Now, the riddle of OS-1's mechanism of action has been solved, and the solution appears in this issue of Blood. McEwan et al determined the crystal structure of OS-1 in complex with GPIb␣ and found that, unlike the situation with RGD-or KGDbased inhibitors of ␣ IIb ␤ 3 , OS-1 does not mimic the binding of VWF to GPIb␣.…”

Novel Antiplatelet Activity of Protocatechuic Acid through the Inhibition of High Shear Stress-Induced Platelet Aggregation

“…Because PCA significantly inhibited ristocetin-induced platelet aggregation, which occurs in a vWF A1-GP Ib␣-dependent manner in the absence of high shear stress, the third possibility may be excluded. Incidentally, blockers of vWF A1-GP Ib␣ interaction that have been reported were mostly antibodies or peptide mimetic (Benard et al, 2008;Clemetson and Clemetson, 2008;Kiefer and Becker, 2009). Few studies have been published on small molecules that have anti-SIPA or vWF-GP Ib blocking effects to our knowledge.…”

Current State and Novel Approaches of Antiplatelet Therapy

“…As proof of this concept, a short cyclic peptide, CTERMALHNLC, which binds GPIbα, potently inhibits vWF binding not through competitive inhibition but by preventing formation of a ligandreceptive structural conformation. 83,84 An alternative strategy targeting the ADP-P2Y1/P2Y12 activation pathway is the use of a soluble form of CD39 (solCD39), an ectonucleoside triphosphate diphosphohydrolase. 85 This approach uses the ability of CD39 to hydrolyze ADP, dissimilar to the current ADP-P2Y12 drugs clopidogrel, prasugrel, and ticagrelor that block ADP-mediated platelet activation by binding to the receptor directly.…”