Building better fibrin knob mimics: an investigation of synthetic fibrin knob peptide structures in solution and their dynamic binding with fibrinogen/fibrin holes

Abstract

Fibrin polymerizes via noncovalent and dynamic association of thrombin-exposed "knobs" with complementary "holes." Synthetic knob peptides have received significant interest as a means for understanding fibrin assembly mechanisms and inhibiting fibrin polymerization. Nevertheless, the inability to crystallize short peptides significantly limits our understanding of knob peptide structural features that regulate dynamic knob:hole interactions. In this study, we used molecular simulations to generate the first predicted structure(s) of synthetic knobs in solution before fibrin hole engagement. Combining surface plasmon resonance (SPR), we explored the role of structural and electrostatic properties of knob "A" mimics in regulating knob:hole binding kinetics. SPR results showed that association rates were most profoundly affected by the presence of both additional prolines as well as charged residues in the sixth to seventh positions. Importantly, analyzing the structural dynamics of the peptides through simulation indicated that the 3Arg side chain orientation and peptide backbone stability each contribute significantly to functional binding. These findings provide insights into early fibrin protofibril assembly dynamics as well as establishing essential design parameters for high-affinity knob mimics that more efficiently compete for hole occupancy, parameters realized here through a novel knob mimic displaying a 10-fold higher association rate than current mimics. IntroductionThe activation and polymerization of the blood-circulating protein fibrinogen, a 340-kDa glycoprotein with 6 polypeptide chains (A␣B␤␥) 2 , is the primary homeostatic mechanism preventing excessive blood loss after vascular injury. This process is initiated by the activated serine protease thrombin, which specifically cleaves 4 N-terminal arginyl-glycine motifs on the 2 adjacent A␣ and B␤ chains of fibrinogen, releasing 2 sets of fibrinopeptides A and B (FpA and FpB) and exposing cryptic fibrin polymerization knobs "A" and "B," respectively. 1-5 The newly exposed fibrin knobs noncovalently interact with complementary "holes"' within the 2 distal C-terminal regions of the ␥ and ␤ chains (complementary holes "a" and "b," respectively) to initiate fibrin protofibril assembly. Understanding the fundamentals of this dynamic and noncovalent knob:hole interaction will lead to both a more thorough understanding of fibrin assembly mechanisms and the establishment of design criteria for superior anticoagulants with high polymerization hole affinity to inhibit fibrin assembly.Evidence for fibrin knob:hole interactions was first conclusively shown when fibrin polymerization was inhibited by synthetic knob A tripeptides (Gly-Pro-Arg) competing for fibrin holes. 6,7 Characterization of the equilibrium binding affinities of both knob A and B peptide variants to fibrinogen showed that the knob A peptides (ie, GPRV and GPRP) have higher affinities to fibrinogen than knob B peptides (ie, GHRP and AHRP) under calcium-free conditions. [6][7][8] In the presenc...

Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat

“…Briefly, integrins (R & D Systems, Minneapolis, MN) were covalently immobilized to gold-coated SPR sensor chips via self-assembled monolayer surface chemistry to generate a nonfouling surface with a controlled density of reactive carboxylic acid groups. Mixed self-assembled monolayers were generated on goldcoated chips as previously described (37,38) by incubating with a 10:1 mixture of 1 mM of tri(ethylene glycol)-terminated alkanethiols (HS-(CH2)11-(OCH2CH2)3-OH (ProChimia, Gdansk, Poland) and carboxylic acid-terminated alkanethiols (HS-(CH2)11-(OCH2CH2)6-OCH2COOH) overnight. The senor chip was then loaded into the Biacore 2000, and the carboxylic acid-terminated alkanethiol surface was activated by flowing 200 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma-Aldrich) and 50 mM N-hydroxysuccinimide (Sigma-Aldrich; 5 l/min for 10 min).…”

Molecular interference of fibrin's divalent polymerization mechanism enables modulation of multiscale material properties

“…Binding to fragment D was only observed for the native non-binding peptide and the PEGylated version at high (mM) concentrations, which is likely due to nonspecific interactions. Previous reports characterizing the affinity of knob A for fragment D also reported a 2-site binding model [25], presumably because knob A binds both hole a and hole b . The binding affinities of the knob B have not been previously reported, however, knob B is thought to only bind hole b .…”

“…Fragment D was isolated by incubating the plasmin-digested fibrinogen with GPRPAA beads at room temperature for 30 minutes, with occasional agitation [24, 25]. The unbound proteins and protein fragments were removed with excessive washing with HEPES + CaCl 2 buffer.…”

“…Briefly, Fragment D was covalently immobilized to gold-coated SPR sensor chips via self-assembled monolayer surface chemistry to generate a nonfouling surface with a controlled density of reactive carboxylic acid groups. Mixed self-assembled monolayers were generated on gold-coated chips by incubating with a 1-mM mixture of tri(ethylene glycol)–terminated alkanethiols (HS-(CH2)11–(OCH2CH2)3–OH; ProChimia) and carboxylic acid– terminated alkanethiols (HS-(CH2)11–(OCH2CH2)6–OCH2COOH) overnight [25]. On loading the senor chip into the Biacore 2000, the carboxylic acid–terminated alkanethiols in all 4 flow cells was activated by flowing 200 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma- Aldrich) and 50 mM N-hydroxysuccinimide (Sigma-Aldrich; 5 μL/minute for 10 minutes).…”

Fibrin Network Changes in Neonates after Cardiopulmonary Bypass: Erratum

“…Neonatal fibrinogen was isolated from PPP samples through affinity chromatography using glycyl-L-prolyl-L-arginyl-L-proline (GPRP)-sepharose beads 15 . Following a one-hour incubation period, fibrinogen was dissociated from the beads using 1M sodium borate, 50mM sodium acetate, pH 5.4.…”