Role of ‘B‐b’ knob‐hole interactions in fibrin binding to adsorbed fibrinogen

Geer, Carri B.; Tripathy, Ashutosh; Lord, Susan T.; Gorkun, Oleg V.

doi:10.1111/j.1538-7836.2007.02774.x

Cited by 26 publications

(23 citation statements)

References 43 publications

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“…38 The investigators reported K D s of 5.8M and 3.7M for desA-NDSK and desAB-NDSK, respectively. The slightly higher affinity of desAB-NDSK was attributed to B:b interactions because coinjection of knob B peptides with desA-NDSK or desAB-NDSK hindered only the desAB-NDSK interaction with fibrinogen.…”

Section: Discussionmentioning

confidence: 99%

“…The slightly higher affinity of desAB-NDSK was attributed to B:b interactions because coinjection of knob B peptides with desA-NDSK or desAB-NDSK hindered only the desAB-NDSK interaction with fibrinogen. 38 Even though this elegant study established the presence of B:b binding interactions, these SPR experiments were performed under equilibrium conditions (ie, low flow rates) and determined only a single equilibrium binding constant. 26 Nonetheless, dynamic off-rates of desA-NDSK (8.6 ϫ 10 Ϫ4 s Ϫ1 ) and desAB-NDSK (1.35 ϫ 10 Ϫ3 s Ϫ1 ) from fibrinogen have been calculated from bond-strength measurements recorded with laser tweezers-based force spectroscopy.…”

Section: Discussionmentioning

confidence: 99%

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

Stabenfeldt

Gossett

Barker

2010

Blood

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Stabenfeldt

Gossett

Barker

2010

Blood

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“…9 This suggests that in fibrin, the binding site corresponding to knob 'A' is not limited to the GPR sequence and therefore has substantially higher affinity.…”

Section: Knob-hole Interactionsmentioning

confidence: 99%

Mechanisms of fibrin polymerization and clinical implications

Weisel

Litvinov

2013

Blood

401

363

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Research on all stages of fibrin polymerization, using a variety of approaches including naturally occurring and recombinant variants of fibrinogen, x-ray crystallography, electron and light microscopy, and other biophysical approaches, has revealed aspects of the molecular mechanisms involved. The ordered sequence of fibrinopeptide release is essential for the knob-hole interactions that initiate oligomer formation and the subsequent formation of 2-stranded protofibrils. Calcium ions bound both strongly and weakly to fibrin(ogen) have been localized, and some aspects of their roles are beginning to be discovered. Much less is known about the mechanisms of the lateral aggregation of protofibrils and the subsequent branching to yield a 3-dimensional network, although the aC region and B:b knob-hole binding seem to enhance lateral aggregation. Much information now exists about variations in clot structure and properties because of genetic and acquired molecular variants, environmental factors, effects of various intravascular and extravascular cells, hydrodynamic flow, and some functional consequences. The mechanical and chemical stability of clots and thrombi are affected by both the structure of the fibrin network and cross-linking by plasma transglutaminase. There are important clinical consequences to all of these new findings that are relevant for the pathogenesis of diseases, prophylaxis, diagnosis, and treatment. (Blood. 2013;121(10):1712-1719 IntroductionFibrin polymer is an end product of the enzymatic cascade of blood clotting. In vivo formation of the polymeric fibrin network, along with platelet adhesion and aggregation, are the key events in salutary stopping of bleeding at the site of injury (hemostasis) as well as in pathological vascular occlusion (thrombosis). Fibrin polymerization comprises a number of consecutive reactions, each affecting the ultimate structure and properties of the fibrin scaffold. These properties determine the development and outcomes of various diseases, such as heart attack, ischemic stroke, cancers, trauma, surgical and obstetrical complications, hereditary and acquired coagulopathies, and thrombocytopathies. In addition to providing a better understanding of the pathogenesis of such disorders, the knowledge of the molecular mechanisms of fibrin formation provides a foundation for new diagnostic tools and therapeutic approaches. Fibrinopeptide releaseFibrinogen is 45 nm-long and made up of 6 paired polypeptide chains (Aa Bb g) 2 held together by 29 disulfide bonds. There are now crystal structures of large parts of fibrinogen, including the g-and b-nodules, the coiled coil, and the central nodule.1 Still missing in the crystal structures are the flexible NH 2 -terminal (N-terminal) ends of the Aa-chains and the carboxyl-terminal (C-terminal) portion of the Aa-chain, but modern simulation techniques make possible partial computational reconstructions of these parts of fibrin(ogen) (Figure 1).Fibrin polymerization is initiated by the thrombin cleavage of fibrinopeptides A (...

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“…[41][42][43][44] B:b interactions are reported to be exceptionally weak as characterized by high affinity constants and a low strength force to rupture the bonds. 43,45 Prior studies suggest any assembly based on cleavage of FpB alone would be restricted to nonphysiologic conditions of low salt concentrations and low temperatures. 29,39,46 Fib AEK mice and fibrinogen AEK derived from these animals provide novel tools and reagents for more comprehensive studies exploring the consequences of FpA release, FpB release, or both to polymer formation and clot structure both in vitro and in vivo.…”

Section: Arg16cysmentioning

confidence: 99%