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Blood, 1 January 2007, Vol. 109, No. 1, pp. 130-138. Prepublished online as a Blood First Edition Paper on August 29, 2006; DOI 10.1182/blood-2006-07-033910.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Polymerization of fibrin: direct observation and quantification of individual B:b knob-hole interactions1 Department of Cell and Developmental Biology and 2 Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC; 3 Departments of Pathology and Medicine, School of Medicine, State University of New York, Stony Brook, NY; and 4 Center for Vascular and Inflammatory Diseases and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 5 Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA;
The polymerization of fibrin occurs primarily through interactions between N-terminal A- and B-knobs, which are exposed by the cleavage of fibrinopeptides A and B, respectively, and between corresponding a- and b-holes in the  - and ß-modules. Of the potential knob-hole interactions—A:a, B:b, A:b, and B:a—the first has been shown to be critical for fibrin formation, but the roles of the others have remained elusive. Using laser tweezers–based force spectroscopy, we observed and quantified individual B:b and A:b interactions. Both desA-fibrin with exposed A-knobs and desB-fibrin bearing B-knobs interacted with fragment D from the D364H fibrinogen containing b-holes but no functional a-holes. The strength of single B:b interactions was found to be 15 to 20 pN, approximately 6-fold weaker than A:a interactions. B:b binding was abrogated by B-knob mimetic peptide, the (ß15-66)2 fragment containing 2 B-knobs, and a monoclonal antibody against the ß15-21 sequence. The interaction of desB-fibrin with fragment D containing a- and b-holes produced the same forces that were insensitive to A-knob mimetic peptide, suggesting that B:a interactions were absent. These results directly demonstrate for the first time B:b binding mediated by natural B-knobs exposed in a fibrin monomer.
The fibrin clot is a branched protein polymer that provides the three-dimensional (3-D) scaffold for a thrombus in vertebrates. Fibrin is formed from a soluble precursor, fibrinogen, upon the action of thrombin, a blood enzyme. Thrombin normally cleaves 4 relatively small peptides from fibrinogen, producing fibrin monomers capable of spontaneous self-assembly into 2-stranded oligomeric structures called protofibrils. Once the protofibrils reach a critical size (approximately10 nm thick and 600-800 nm long), they aggregate laterally to form fibers, often 80 to 120 nm in diameter, organized into the branched network called a fibrin clot.1-3
Fibrinogen is a plasma protein composed of 3 pairs of polypeptides designated A
The self-assembly of fibrin monomers into a fibrin clot requires the exposure by thrombin action of new polymerization sites that are masked in fibrinogen. Thrombin initially cleaves the Arg16-Gly17 bond in the A  -chains of human fibrinogen, releasing fibrinopeptide A (FpA). The cleavage leads to exposure of the N-terminal sequence Gly17-Pro18-Arg19 in the fibrin -chains (ie, fibrinogen A-chains without the FpA), which is called A-knob (Figure 1B).7 A-Knobs are complementary to a-holes located in the -modules of another fibrin molecule, and their interaction is termed A:a binding. Cleavage of FpA and exposure of A-knobs, making desA-fibrin, are necessary and sufficient to form fibrin clots. Fibrin a-holes are also necessary for clot formation; if they are blocked by A-knob mimetic Gly-Pro-Arg-Pro (GPRP) peptide8 or impaired by a point mutation of residue Asp364,9 thrombin-induced fibrin polymerization is prevented. Together, these data suggest that A:a interactions are the driving force of fibrin polymerization and clot formation. Recently, using laser tweezers–based force spectroscopy, we observed the interactions between A-knobs and a-holes at the single-molecule level and found the A:a bonds to be strong and stable.10 The laser tweezers technique that enables quantification of individual knob-hole interactions is based on the ability of the optical system to measure the binding strength of 2 surface-bound protein molecules.11,12 B-knob becomes exposed after thrombin cleaves the Arg14-Gly15 bond in the Bß-chain; this is followed by the release of fibrinopeptide B (FpB). The sequence of the newly exposed N-terminal motif in the remaining ß-chain is Gly15-His16-Arg17-Pro18, which is considered the working part of B-knob. Importantly, thrombin cleavage of FpB is much slower than that of FpA so that most, if not all, the FpB removal and exposure of B-knobs occur after fibrin polymers have already formed.13,14 Consistent with this, the Gly-His-Arg-Pro (GHRP) peptide, a B-knob mimetic, does not prevent fibrin clot formation,15 suggesting that B-knob–mediated interactions are not necessary for protofibril formation or for their lateral aggregation. On the other hand, the surmise about the existence of B:b interactions and their importance for lateral aggregation is based on a great deal of indirect, but consistent, data that can be roughly segregated into 3 groups: (1) differences in clot formation dynamics and structure after selective release of FpA or FpB16-23; (2) binding of GHRP or its derivatives to fibrin(ogen) and their fragments and effects of the peptide(s) on fibrin formation8,15,24-29; (3) consequences of naturally occurring or recombinant mutations of B-knobs or b-holes for fibrinogen conversion to fibrin.30-32 Perhaps the most significant, though not absolute, argument in favor of the occurrence of B:b and other B-knob-mediated interactions during fibrin polymerization is the formation of clots, at 15°C or lower, from desB-fibrin.33,34 The desB-fibrin, with exclusively or predominantly exposed B-knobs (Figure 1C), is produced upon selective cleavage of FpB over FpA and can be formed using variant fibrinogens with impaired release of FpA, such as fibrinogen Metz,34-36 Schwarzach,37 or Frankfurt XIII,38 or specific snake venom enzymes.20,33,39 Notwithstanding the evidence that the interactions mediated by B-knob and b-hole contribute to fibrin polymerization, no experimental approach has directly shown the existence of B:b knob-hole interactions using protein molecules rather than mimetic peptides. In our preceding work, we were also unable to detect B:b, B:a, or A:b binding in the presence of strong dominant A:a interactions.10 In this study, for the first time, direct evidence is provided for binding between b-holes and natural A- or B-knobs exposed in a fibrin monomer. The B:b and A:b bonds were found to be much weaker than the A:a bonds, suggesting that b-holes are involved in subsidiary knob-hole binding reactions that reinforce fibrin polymerization.
Expression and purification of fibrinogens
Two recombinant fibrinogens, normal and variant
Preparation of D fragments from normal and
Fragment D from normal fibrinogen was purified as previously described.42 Trypsin digestion of Expression and preparation of the recombinant fibrin (ß15-66)2 fragment Recombinant (Bß1-66)2 fragment mimicking the dimeric arrangement of the Bß-chain in fibrinogen, which forms 2 BßN-domains, was produced in Escherichia coli and purified, as has been described elsewhere.43 To produce the activated (ß15-66)2 fragment corresponding to the fibrin ßN-domain with 2 exposed B-knobs (Figure 1H), (Bß1-66)2 was treated with thrombin and purified as described earlier.43 Purification of fibrin DD fragment
The DD fragment (Figure 1G) was purified from a trypsin digest of fully cross-linked fibrin based on a modification of a published procedure.5 Briefly, human fibrinogen (ERL, South Bend, IN) was clotted in the presence of CaCl2 and L-cysteine-HCl by the addition of Coating surfaces with desB- or desA-fibrin or recombinant (ß15-66)2
Surfaces coated with the interacting proteins were prepared as described previously,10,44 with some important modifications. desB-Fibrin (Figure 1C) was formed from the naturally occurring homodimer fibrinogen variant Frankfurt XIII with impaired cleavage of FpA because of the A Coating surfaces with the fragment D
Fragment D from normal fibrinogen or fibrinogen Model system to study knob-hole interactions
We used a laser tweezers–based model system to study interactions between 2 surface-bound proteins.10,12,44 A laser tweezers or gradient optical trap is formed by focusing a laser beam with a microscope objective to a spot in the specimen plane.45 This system permits the measurement of discrete rupture forces produced by surface-bound molecular pairs during repeated intermittent contact.12,44 To study particular pairs of knob-hole interactions, combinations of fibrin(ogen) molecules and their fragments, bearing either knobs or holes, were bound to pedestals and beads. For these studies, desB-fibrin or the (ß15-66)2 fragment (both containing B-knobs) or desA-fibrin (containing A-knobs) were covalently bound to stationary pedestals anchored to the inner surface of a flow chamber. Suspension of latex beads (107/mL) coated covalently with fragments D (containing a- and b-holes) or D- Measurement of binding strength, data processing, and data analysis The position of the optical trap and, hence, a fragment D–coated latex bead was oscillated in a triangular waveform at 0.5 Hz with a pulling velocity of 1.8 µm/s, which corresponded to a loading rate of 400 pN/s. All experiments were conducted at a trap stiffness of 0.22 ± 0.01 pN/nm, as computed from the bandwidth of Brownian motion. Contact duration between interacting surfaces varied from 10 to 200 milliseconds. Rupture forces were collected at 2000 scans/s (0.5-ms resolution). Results of many experiments under similar conditions were averaged so that each rupture force histogram represented 103 to 104 repeated contacts of more than 10 different bead–pedestal pairs. Individual forces measured during each contact–detachment cycle were collected into 5 pN–wide bins. The number of events in each bin was plotted against the average force for that bin after normalizing for the total number of interaction cycles. The percentage of events in a particular force range (bin) represented the probability of rupture events at that tension. Optical artifacts observed with or without trapped latex beads produced signals that appeared as forces smaller than 10 pN. Accordingly, rupture forces in this range were not considered when the data were analyzed. Rupture force histograms were fit with multimodal Gaussian curves (Origin 7.5; OriginLab, Northampton, MA) to determine the position of a peak that corresponded to the most probable rupture force and the area of each peak that reflected cumulative binding probability for the underlying interactions.12
Comparison of A:a and B:b interactions
The basic model system to study the B:b interactions consisted of 2 surfaces, one coated with desB-fibrin from homodimeric dysfibrinogen Frankfurt XIII with the mutation A
Interactions between B-knobs and b-holes To prove the specificity of observed B:b interactions, the rupture force measurement was repeated in the presence of increasing amounts of the GHRPam peptide, a B-knob mimetic (Figure 3B-D), or GPRPam peptide, a A-knob mimetic.8,15,46,47 Cumulative probability of interactions with rupture forces greater than 10 pN decreased monotonically with increasing GHRPam (Table 1). At 5 mM GHRPam, probability dropped to 34.5% ± 5.5%, and at 10 mM, cumulative probability decreased approximately10-fold to 7.2% ± 1.3% (Table 1). The inhibitory effect of the GHRPam peptide was reversible inasmuch as the rupture force profile (Figure 3F) was restored to the original profile after the peptide was removed, as was the cumulative binding probability (Table 1). For comparison, 10 mM GPRPam peptide had a much smaller effect than the same molar amount of GHRPam on force distribution (Figure 3G) and cumulative binding probability (Table 1), suggesting that GPRPam does not compete well for B:b interactions.
Although the inhibitory effect of GHRPam on the overall probability of interactions between desB-fibrin and D- Interactions between B-knobs and a-holes
To further probe B-knob interactions, the force spectrum for desB-fibrin and fragment D from normal fibrinogen (Figure 1E) or D-dimer obtained from cross-linked normal fibrin (Figure 1G) was measured. Rupture force profiles of the interaction of desB-fibrin with fragment D (Figure 4A) and with D-dimer (Figure 4C) were nearly the same as the interaction of desB-fibrin with D-
To test this hypothesis further, several competitive inhibition experiments were performed. To inhibit B:b interactions of desB-fibrin with fragment D, B-knob was blocked with 100 µg/mL mAb T2G1,48,49 an antibody against the ß15-21 portion of fibrin comprising the B-knob (Figure 4D), and b-hole was blocked with either 10 mM GHRPam (Figure 4E) or 37 µM (400 µg/mL) recombinant (ß15-66)2 fragment (Figure 1H) comprising the fibrin ßN-domain with 2 B-knobs (Figure 4F). Force spectra and cumulative probability for interactions (Table 1) in all 3 cases were nearly fully suppressed. To block B:a interactions specifically, the 2-mM GPRPam peptide was added, but it caused no changes in the force distribution (Figure 4B) or the cumulative probability (Table 1). These data together suggest that B:a interactions do not contribute to fibrin binding. To reduce the possibility that desB-fibrin–fragment D binding is caused by portions of the fibrin molecule other than B-knob, the interactions of (ß15-66)2 (Figure 1H) with fragment D (Figure 1E) were measured. Based on the observed ability of the (ß15-66)2 fragment to compete with desB-fibrin for b-holes (Figure 4F), (ß15-66)2 was expected to bind like a functional fibrin ßN-domain containing B-knobs. Accordingly, the rupture force spectrum for (ß15-66)2 and fragment D had 3 peaks at 15 ± 1 pN, 31 ± 4 pN, and 52 ± 10 pN (Figure 5A), similar to the force profile obtained for desB-fibrin and fragment D (Figure 4A). The interaction of (ß15-66)2 with fragment D was insensitive to GPRPam (Figure 5B) and highly sensitive to GHRPam (Figure 5C), and the latter effect was reversible (Figure 5D). Thus, B:b interactions had the same characteristics irrespective of whether B-knob was a part of a fibrin molecule or recombinant isolated ßN-domain.
Interactions between A-knobs and b-holes
The strength of A:b interactions was measured using desA-fibrin as the source of A-knobs and fragment D-
It has been proposed that fibrin polymerization is mediated by the interactions of A- and B-knobs in central regions, with complementary a- and b-holes in distal regions, of fibrin monomers. Using an optical trap–based system to measure knob-hole interactions,44 we previously uncovered only strong A:a bonds. Neither B:b nor hypothetical A:b and B:a bonds were detected.10 The current experiments were designed so that the strong A:a bonds, which might have obscured weaker or less probable knob-hole interactions, were eliminated. The key experimental variation was to use only B-knob–bearing molecules represented by surface-bound thrombin-treated fibrinogen variant A R16C with no release of FpA (desB-fibrin monomer) or the recombinant (ß15-66)2 fragment so that A-knobs of fibrin were unavailable for interaction. To reduce the likelihood of interactions other than those mediated by knobs and holes, the other surface was coated with D fragments obtained from normal fibrinogen or fibrinogen variant D364H, in which a-holes were not active.9 The lack of A:a interactions at these interfaces was confirmed by the absence of rupture forces stronger than 100 pN, which were typical for the A:a pairs (Figure 2),10 and the incapacity of GPRPam, the a-hole blocker, to abrogate interactions between desB-fibrin and fragment D (Figure 4B). With the A:a bonding suppressed, we found that fibrin fragments could interact through B:b and A:b, but not B:a, bonds.
The results indicated that a-holes, when they are exposed, do not bind to B-knobs. First, no difference was observed in rupture force profiles when desB-fibrin interacted with either D-
In contrast to the absence of measurable B:a binding, A-knobs in desA-fibrin displayed specific reactivity with b-holes in D-
The surface-bound desB-fibrin monomer readily reacted with D-
Several indirect arguments support the idea that the 3 decreasing peaks of rupture force histograms in Figures 3 to 5 are indicative of single, double, and triple B:b binding, respectively. First, the maximum values of the weak (15-20 pN), intermediate (30-40 pN), and strong (50-60 pN) force peaks are roughly quantized, as would be predicted if they represented multiples of the bimolecular interactions.52 Second, when the single-molecule binding probability was approximately 50%, the double-molecule interactions were statistically expected to occur in 25% (0.52) of the binding events, and the probability of triple interactions was 12.5% (0.53), which approximately corresponded to most of the experimentally observed peak areas (Table 1). Third, the stronger forces were more susceptible to the inhibitory effect of GHRPam (Figure 3E), which is consistent with the assumption that the stronger forces reflected multiple interactions and, therefore, disappeared first. Fourth, the high incidence of multiple intermolecular interactions was confirmed by the relatively common occurrence (up to 20%) of stepwise detachment (Figure 2D) of the interacting surfaces. Based on these considerations, the binding strength of the individual B:b interactions represented by the weakest peaks in the force spectra was estimated to be approximately 15 to 20 pN. Given that the experimental conditions were the same as those used to study the A:a interactions (125-130 pN),10 the B:b bonds seemed to be approximately 6- to 8-fold weaker than the A:a bonds, which is remarkably consistent with the estimation that We found that the inhibitory effect of the GHRPam peptide on the B:b interactions was fully pronounced only at 10 mM, whereas the GPRPam peptide effectively inhibited the A:a interactions at 1 mM.10 This finding is in good agreement with the approximately 1 order of magnitude difference in the affinities of the GPRP and GHRP peptides toward human fibrinogen, which were equal to 4 x 104 M-1 and 7 x 103 M-1, respectively.15 Importantly, in our experiments, the inhibitory effect of the GHRPam peptide resulted from specific and selective binding to b-hole and could not be attributable to nonspecific electrostatic shielding because 10 mM GPRPam, unlike the GHRPam peptide, induced only moderate suppression of the B:b interactions (Table 1), perhaps because of its ability to bind not only to a-holes25 but also to b-holes46 (O.V.G., unpublished data, April 1, 2006). Surprisingly, in the previous work, we were unable to reveal the B:b interactions when desAB-fibrin was exposed to fibrinogen or fragment D despite the presence of B-knobs and b-holes on the touching surfaces.10 One likely explanation is that the weak B:b interaction could be masked by nonspecific forces up to 40 pN that were excluded from data analysis. Even if the A:a and B:b interactions occurred simultaneously, the probability of the B:b binding seemed to be substantially smaller, which was in line with the absolute prevalence of the A:a binding during earlier stages of fibrin polymerization. The alternative possibility is that the B:b binding could not occur concurrently with the much stronger (and perhaps faster) A:a interactions. If the latter assumption was true, it might have explained why the cleavage of FpB and the subsequent B:b interactions occurred only in the later stages of fibrin formation, when the A:a bonding was completed. The presumption about mutual exclusiveness of A:a and B:b interactions is consistent with a recently proposed mechanism for delayed FpB cleavage.54 At least 2 conceivable mechanisms explain the late B:b bonding by formation and/or activation of b-holes upon fibrin polymerization. The first implies assembly of a new B-knob-binding site by alignment of the D regions on 2 fibrin monomers on polymerization. Proposed more than 2 decades ago,55 this mechanism was disproved in the subsequent work56 and seems inconsistent with the crystallographic data showing 2 structurally and functionally independent b-holes in the D-dimer.25 The second mechanism presumes conformational rearrangement of the ß-module, similar to the changes induced by GHRPam binding,26 followed by increased affinity of b-hole to B-knob. To retest these possibilities, desB-fibrin was exposed to the D-dimer (Figure 4C). However, neither the strength nor the binding probability of the interactions between desB-fibrin and fragment DD were different compared with the monomeric fragment D. Therefore, our result suggests that b-holes act independently and that polymerization by itself does not change the strength of B:b binding. This result agrees with the recent hypothesis that each B-knob first binds a single b-hole, and then locks the ß-module in a different conformation that changes the properties of an entire clot.28
In conclusion, our data for the first time directly demonstrate the existence of B:b knob-hole interactions between fibrin molecules. We also observed A:b interactions but did not detect B:a binding. Although formation of the B:b bonds is in agreement with current notions of fibrin polymerization, particularly lateral aggregation of protofibrils, physiologic relevance of the A:b binding remains unclear. Two possibilities may explain how B:b interactions can promote lateral aggregation. The first is that B:b binding occurs between the protofibrils because of long and highly flexible B-knobs that can bind b-holes of the neighboring protofibrils without substantial conformational rearrangements (Figure 7A-B). Alternatively, B:b binding may occur within protofibrils (Figure 7C) promoting their lateral aggregation indirectly by a number of secondary mechanisms. First, the FpB cleavage has been shown to cause exposure of
This work was supported by National Institutes of Health grants HL-30954 (J.W.W.), HL-31048 and HL-56051 (L.M.) and by American Heart Association grant 0365340U (O.V.G.). O.V.G. was supported by the grant HL-31048 awarded to Susan T. Lord. We thank Dr Susan T. Lord (Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC) for her enthusiastic support and help with this research and Dr Inge Scharrer (Zentrum der inneren Medizin, J.W. Goethe Universität, Frankfurt, Germany) for identifying the patient and plasma samples of fibrinogen Frankfurt XIII.
Submitted July 6, 2006; accepted August 9, 2006.
Prepublished online as Blood First Edition Paper, August 29, 2006
DOI: 10.1182/blood-2006-07-033910
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
Conflict-of-interest disclosure: the authors declare no competing financial interests.
Correspondence: Rustem I. Litvinov,Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 1040 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104-6058; e-mail: litvinov{at}mail.med.upenn.edu.
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Abstract
Introduction
His, the substitution discovered in a heterozygous individual. J Biol Chem 1997; 272:29596–29601.