Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking.

Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size.

Does topology drive fiber polymerization?

We have developed new procedures to examine the early steps in fibrin polymerization. First, we isolated fibrinogen monomers from plasma fibrinogen by gel filtration. Polymerization of fibrinogen monomers differed from that of plasma fibrinogen. The formation of protofibrils was slower and the transformation of protofibrils to fibers faster for the fibrinogen monomers. Second, we used formaldehyde to terminate the polymerization reactions. The formaldehyde-fixed products obtained at each time point were examined by dynamic light scattering and transmission electron microscopy (TEM). The data showed the formaldehyde-fixed products were stable and representative of the reaction intermediates. TEM images showed monomers, short oligomers, protofibrils, and thin fibers. The amount and length of these species varied with time. Short oligomers were less than 5% of the molecules at all times. Third, we developed models that recapitulate the TEM images. Fibrin monomer models were assembled into protofibrils, and protofibrils were assembled into two-strand fibers using Chimera software. Monomers were based on fibrinogen crystal structures, and the end-to-end interactions between monomers were based on D-dimer crystal structures. Protofibrils assembled from S-shaped monomers through asymmetric D:D interactions were ordered helical structures. Fibers were modeled by duplicating a protofibril and rotating the duplicate 120° around its long axis. No specific interactions were presumed. The two protofibrils simply twisted around one another to form a fiber. This model suggests that the conformation of the protofibril per se promotes the assembly into fibers. These findings introduce a novel mechanism for fibrin assembly that may be relevant to other biopolymers.

Submillisecond elastic recoil reveals molecular origins of fibrin fiber mechanics.

Fibrin fibers form the structural scaffold of blood clots. Thus, their mechanical properties are of central importance to understanding hemostasis and thrombotic disease. Recent studies have revealed that fibrin fibers are elastomeric despite their high degree of molecular ordering. These results have inspired a variety of molecular models for fibrin's elasticity, ranging from reversible protein unfolding to rubber-like elasticity. An important property that has not been explored is the timescale of elastic recoil, a parameter that is critical for fibrin's mechanical function and places a temporal constraint on molecular models of fiber elasticity. Using high-frame-rate imaging and atomic force microscopy-based nanomanipulation, we measured the recoil dynamics of individual fibrin fibers and found that the recoil was orders of magnitude faster than anticipated from models involving protein refolding. We also performed steered discrete molecular-dynamics simulations to investigate the molecular origins of the observed recoil. Our results point to the unstructured αC regions of the otherwise structured fibrin molecule as being responsible for the elastic recoil of the fibers.

Hydrodynamic characterization of recombinant human fibrinogen species.

INTRODUCTION: Fibrinogen is a key component of the blood coagulation system and plays important, diverse roles in several relevant pathologies such as thrombosis, hemorrhage, and cancer. It is a large glycoprotein whose three-dimensional molecular structure is not fully known. Furthermore, circulating fibrinogen is highly heterogeneous, mainly due to proteolytic degradation and alternative mRNA processing. Recombinant production of human fibrinogen allows investigating the impact on the three-dimensional structure of specific changes in the primary structure. METHODS: We performed analytical ultracentrifugation analyses of a full-length recombinant human fibrinogen, its counterpart purified from human plasma, and a recombinant human fibrinogen with both Aα chains truncated at amino acid 251, thus missing their last 359 amino acid residues. RESULTS: We have accurately determined the translational diffusion and sedimentation coefficients (Dt(20,w)(0), s(20,w)(0)) of all three species. This was confirmed by derived molecular weights within 1% for the full length species, and 5% for the truncated species, as assessed by comparison with SDS-PAGE/Western blot analyses and primary structure data. No significant differences in the values of Dt(20,w)(0) and s(20,w)(0) were found between the recombinant and purified full length human fibrinogens, while slightly lower and higher values, respectively, resulted for the recombinant truncated human fibrinogen compared to a previously characterized purified human fibrinogen fragment X obtained by plasmin digestion. CONCLUSIONS: Full-length recombinant fibrinogen is less polydisperse but hydrodynamically indistinguishable from its counterpart purified from human plasma. Recombinant Aα251-truncated human fibrinogen instead behaves differently from fragment X, suggesting a role for the Bß residues 1-52 in inter-molecular interactions. Overall, these new hydrodynamic data will constitute a reliable benchmark against which models of fibrinogen species could be compared.

The isolation of fibrinogen monomer dramatically influences fibrin polymerization.

Fibrin polymerization begins with the thrombin-catalyzed cleavage of fibrinopeptides from fibrinogen and proceeds through several assembly steps to form an insoluble fibrin clot. Using dynamic light scattering (DLS), we found that purified fibrinogens are polydisperse, containing small amounts of fibrinogen complexes. In order to characterize the impact of these complexes, we used gel filtration chromatography to isolate monomers from three fibrinogens: plasma, recombinant, and recombinant variant Aα251. SDS-PAGE analysis showed that the polypeptides in the monomers were indistinguishable from those in the initial fibrinogen. DLS showed the fibrinogen monomers were monodisperse. We used turbidity to follow polymerization and found the polymerization of fibrinogen monomers was markedly different from the polymerization of the initial fibrinogen; the final optical density (OD) was significantly higher for monomers. Moreover, the polymerization curve for fibrinogen monomers was independent of the polymerization curves of the fibrinogen samples without gel filtration. For example, monomers isolated from two recombinant fibrinogen preparations polymerized similarly even though the final OD increased 2-fold for one preparation and 3-fold for the other. Scanning electron microscopy of the fibrin clots verified the turbidity data; monomer clots had thicker fibers. We conclude that fibrinogen complexes alter the kinetics of polymerization and impair the assembly of monomers into protofibrils and fibers.

An engineered fibrinogen variant AαQ328,366P does not polymerise normally, but retains the ability to form α cross-links.

A fibrin clot is stabilised through the formation of factor XIIIa-catalysed intermolecular ε-lysyl-γ-glutamyl covalent cross-links between α chains to form α polymers and between γ chains to form γ dimers. In a previous study we characterised fibrinogen Seoul II, a heterozygous dysfibrinogen in which a cross-linking acceptor site in Aα chain, Gln328, was replaced with Pro (AαQ328P). Following on the previous study, we investigated whether the alteration of Gln residues Aα328 and Aα366 affects fibrin polymerisation and α chain cross-linking. We have expressed three recombinant fibrinogens: AαQ328P, AαQ366P, and AαQ328,366P in Chinese hamster ovary cells, purified these fibrinogens from the culture media and performed biochemical tests to see how the introduced changes affect fibrin polymerisation and α chain cross-linking. Thrombin-catalysed fibrin polymerisation of all variants was impaired with the double mutation being the most impaired. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed α polymer formation with all three engineered proteins. This study demonstrates that AαQ328 and AαQ366 are important for normal fibrin clot formation and in the absence of residues AαQ328 and AαQ366, other Gln residues in the α chain can support FXIIIa-catalysed fibrin cross-linking.

The assembly of nonadhesive fibrinogen matrices depends on the αC regions of the fibrinogen molecule.

Adsorption of fibrinogen on fibrin clots and other surfaces strongly reduces integrin-mediated adhesion of platelets and leukocytes with implications for the surface-mediated control of thrombus growth and blood compatibility of biomaterials. The underlying mechanism of this process is surface-induced aggregation of fibrinogen, resulting in the assembly of a nanoscale multilayered matrix. The matrix is extensible, which makes it incapable of transducing strong mechanical forces via cellular integrins, resulting in insufficient intracellular signaling and weak cell adhesion. To determine the mechanism of the multilayer formation, the physical and adhesive properties of fibrinogen matrices prepared from human plasma fibrinogen (hFg), recombinant normal (rFg), and fibrinogen with the truncated αC regions (FgAα251) were compared. Using atomic force microscopy and force spectroscopy, we show that whereas hFg and rFg generated the matrices with a thickness of ∼8 nm consisting of 7-8 molecular layers, the deposition of FgAα251 was terminated at two layers, indicating that the αC regions are essential for the multilayer formation. The extensibility of the matrix prepared from FgAα251 was 2-fold lower than that formed from hFg and rFg. In agreement with previous findings that cell adhesion inversely correlates with the extensibility of the fibrinogen matrix, the less extensible FgAα251 matrix and matrices generated from human fibrinogen variants lacking the αC regions supported sustained adhesion of leukocytes and platelets. The persistent adhesiveness of matrices formed from fibrinogen derivatives without the αC regions may have implications for conditions in which elevated levels of these molecules are found, including vascular pathologies, diabetes, thrombolytic therapy, and dysfibrinogenemia.

Fibrinogen residue γAla341 is necessary for calcium binding and 'A-a' interactions.

The fibrinogen γ-module has several important sites relating to fibrinogen function, which include the high affinity calcium binding site, hole 'a' that binds with knob 'A', and the D:D interface. Residue γAla341, which is located in the vicinity of these sites, is altered in three variant fibrinogens: fibrinogen Seoul (γAla341Asp), Tolaga Bay (γAla341Val), and Lyon III (γAla341Thr). In order to investigate the impaired polymerisation of fibrinogens γAla341Asp and γAla341Val to understand the role of γAla341 in fibrin polymerisation and fibrinogen synthesis, we have expressed γAla341Asp and γAla341Val in Chinese hamster ovary (CHO) cells, purified these fibrinogens from the culture media and performed biochemical tests to elucidate their function. Expression in CHO cells was similar for these variants. For both variants the kinetics of thrombin-catalysed FpA release was not different from normal fibrinogen, while FpB release was slower than that of normal. Thrombin-catalysed polymerisation of both variants was dependent on the calcium concentration. At physiologic calcium (1 mM) the variants showed impaired polymerisation with a longer lag period and a slower Vmax than normal fibrinogen. Scanning electron micrographs showed the clots were less organised than normal, having thicker and more twisted fibers, and larger pores. Analysis by SDS-PAGE showed that factor XIIIa-catalysed γ and α chain cross-linking was delayed, and plasmin-catalysed lysis was not reduced by the presence of 5 mM calcium or 5 mM GPRP (Gly-Pro-Arg-Pro). Our data indicate that fibrinogen residue γAla341 is important for the proper conformation of the γ-module, maintaining calcium-binding site and 'A-a' interactions.

Substitution of the human αC region with the analogous chicken domain generates a fibrinogen with severely impaired lateral aggregation: fibrin monomers assemble into protofibrils but protofibrils do not assemble into fibers.

Fibrin polymerization occurs in two steps: the assembly of fibrin monomers into protofibrils and the lateral aggregation of protofibrils into fibers. Here we describe a novel fibrinogen that apparently impairs only lateral aggregation. This variant is a hybrid, where the human αC region has been replaced with the homologous chicken region. Several experiments indicate this hybrid human-chicken (HC) fibrinogen has an overall structure similar to normal. Thrombin-catalyzed fibrinopeptide release from HC fibrinogen was normal. Plasmin digests of HC fibrinogen produced fragments that were similar to normal D and E; further, as with normal fibrinogen, the knob 'A' peptide, GPRP, reversed the plasmin cleavage associated with addition of EDTA. Dynamic light scattering and turbidity studies with HC fibrinogen showed polymerization was not normal. Whereas early small increases in hydrodynamic radius and absorbance paralleled the increases seen during the assembly of normal protofibrils, HC fibrinogen showed no dramatic increase in scattering as observed with normal lateral aggregation. To determine whether HC and normal fibrinogen could form a copolymer, we examined mixtures of these. Polymerization of normal fibrinogen was markedly changed by HC fibrinogen, as expected for mixed polymers. When the mixture contained 0.45 µM normal and 0.15 µM HC fibrinogen, the initiation of lateral aggregation was delayed and the final fiber size was reduced relative to normal fibrinogen at 0.45 µM. Considered altogether, our data suggest that HC fibrin monomers can assemble into protofibrils or protofibril-like structures, but these either cannot assemble into fibers or assemble into very thin fibers.

Molecular mechanisms affecting fibrin structure and stability.

Fibrin structure and stability have been linked to many thrombotic diseases, including venous thromboembolism. Analysis of the molecular mechanisms that affect fibrin structure and stability became possible when the crystal structure of fibrinogen was solved. Biochemical studies of natural and recombinant variant fibrinogens have examined the interactions that mediate the conversion of soluble fibrinogen to the insoluble fibrin network. These studies identified intermolecular interactions that control fibrin structure, although some critical events remain ambiguous. Studies show that fibrin structure modulates the enzymatic lysis of the fibrin network, so the molecular mechanisms that control structure also control stability. Studies show that the mechanical stability of the fibrin clot depends on the properties of the fibrin monomer, leading investigators to explore the molecular basis of the monomer's mechanical properties. The work summarized here provides insights that might allow the development of pharmaceuticals and treatments to modulate fibrin structure and stability in vivo and thereby prevent or limit thrombotic disease.

Evidence that αC region is origin of low modulus, high extensibility, and strain stiffening in fibrin fibers.

Fibrin fibers form the structural scaffold of blood clots and perform the mechanical task of stemming blood flow. Several decades of investigation of fibrin fiber networks using macroscopic techniques have revealed remarkable mechanical properties. More recently, the microscopic origins of fibrin's mechanics have been probed through direct measurements on single fibrin fibers and individual fibrinogen molecules. Using a nanomanipulation system, we investigated the mechanical properties of individual fibrin fibers. The fibers were stretched with the atomic force microscope, and stress-versus-strain data was collected for fibers formed with and without ligation by the activated transglutaminase factor XIII (FXIIIa). We observed that ligation with FXIIIa nearly doubled the stiffness of the fibers. The stress-versus-strain behavior indicates that fibrin fibers exhibit properties similar to other elastomeric biopolymers. We propose a mechanical model that fits our observed force extension data, is consistent with the results of the ligation data, and suggests that the large observed extensibility in fibrin fibers is mediated by the natively unfolded regions of the molecule. Although some models attribute fibrin's force-versus-extension behavior to unfolding of structured regions within the monomer, our analysis argues that these models are inconsistent with the measured extensibility and elastic modulus.

The molecular origins of the mechanical properties of fibrin.

When normal blood circulation is compromised by damage to vessel walls, clots are formed at the site of injury. These clots prevent bleeding and support wound healing. To sustain such physiological functions, clots are remarkably extensible and elastic. Fibrin fibers provide the supporting framework of blood clots, and the properties of these fibers underlie the mechanical properties of clots. Recent studies, which examined individual fibrin fibers or cylindrical fibrin clots, have shown that the mechanical properties of fibrin depend on the mechanical properties of the individual fibrin monomers. Within the fibrin monomer, three structures could contribute to these properties: the coiled-coil connectors the folded globular nodules and the relatively unstructured αC regions. Experimental data suggest that each of these structures contributes. Here we review the recent work with a focus on the molecular origins of the remarkable biomechanical properties of fibrin clots.

Increased thrombosis susceptibility and altered fibrin formation in STAT5-deficient mice.

To explore the effect(s) of growth hormone signaling on thrombosis, we studied signal transduction and transcription factor 5 (STAT5)-deficient mice and found markedly reduced survival in an in vivo thrombosis model. These findings were not explained by a compensatory increase in growth hormone secretion. There was a modest increase in the activity of several procoagulant factors, but there was no difference in the rate or magnitude of thrombin generation in STAT5-deficient mice relative to control. However, thrombin-triggered clot times were markedly shorter, and fibrin polymerization occurred more rapidly in plasma from STAT5-deficient mice. Fibrinogen depletion and mixing studies indicated that the effect on fibrin polymerization was not due to intrinsic changes in fibrinogen, but resulted from changes in the concentration of a circulating plasma inhibitor. While thrombin-triggered clot times were significantly shorter in STAT5-deficient animals, reptilase-triggered clot times were unchanged. Accordingly, while the rate of thrombin-catalyzed release of fibrinopeptide A was similar, the release of fibrinopeptide B was accelerated in STAT5-deficient plasma versus control. Taken together, these studies demonstrated that the loss of STAT5 resulted in a decrease in the concentration of a plasma inhibitor affecting thrombin-triggered cleavage of fibrinopeptide B. This ultimately resulted in accelerated fibrin polymerization and greater thrombosis susceptibility in STAT5-deficient animals.

Dynamic regulation of fibrinogen: integrin αIIbß3 binding.

This study demonstrates that two orthogonal events regulate integrin αIIbß3's interactions with fibrinogen, its primary physiological ligand: (1) conformational changes at the αIIb-ß3 interface and (2) flexibility in the carboxy terminus of fibrinogen's γ-module. The first postulate was tested by capturing αIIbß3 on a biosensor and measuring binding by surface plasmon resonance. Binding of fibrinogen to eptifibatide-primed αIIbß3 was characterized by a k(on) of ~2 × 10(4) L mol(-1) s(-1) and a k(off) of ~8 × 10(-5) s(-1) at 37 °C. In contrast, even at 150 nM fibrinogen, no binding was detected with resting αIIbß3. Eptifibatide competitively inhibited fibrinogen's interactions with primed αIIbß3 (K(i) ~0.4 nM), while a synthetic γ-module peptide (HHLGGAKQAGDV) was only weakly inhibitory (K(i) > 10 µM). The second postulate was tested by measuring αIIbß3's interactions with recombinant fibrinogen, both normal (rFgn) and a deletion mutant lacking the γ-chain AGDV sites (rFgn γΔ408-411). Normal rFgn bound rapidly, tightly, and specifically to primed αIIbß3; no interaction was detected with rFgn γΔ408-411. Equilibrium and transition-state thermodynamic data indicated that binding of fibrinogen to primed αIIbß3, while enthalpy-favorable, must overcome an entropy-dominated activation energy barrier. The hypothesis that fibrinogen binding is enthalpy-driven fits with structural data showing that its γ-C peptide and eptifibatide exhibit comparable electrostatic contacts with αIIbß3's ectodomain. The concept that fibrinogen's αIIbß3 targeting sequence is intrinsically disordered may explain the entropy penalty that limits its binding rate. In the hemostatic milieu, platelet-platelet interactions may be localized to vascular injury sites because integrins must be activated before they can bind their most abundant ligand.

Stiffening of individual fibrin fibers equitably distributes strain and strengthens networks.

As the structural backbone of blood clots, fibrin networks carry out the mechanical task of stemming blood flow at sites of vascular injury. These networks exhibit a rich set of remarkable mechanical properties, but a detailed picture relating the microscopic mechanics of the individual fibers to the overall network properties has not been fully developed. In particular, how the high strain and failure characteristics of single fibers affect the overall strength of the network is not known. Using a combined fluorescence/atomic force microscope nanomanipulation system, we stretched 2-D fibrin networks to the point of failure, while recording the strain of individual fibers. Our results were compared to a pair of model networks: one composed of linearly responding elements and a second of nonlinear, strain-stiffening elements. We find that strain-stiffening of the individual fibers is necessary to explain the pattern of strain propagation throughout the network that we observe in our experiments. Fiber strain-stiffening acts to distribute strain more equitably within the network, reduce strain maxima, and increase network strength. Along with its physiological implications, a detailed understanding of this strengthening mechanism may lead to new design strategies for engineered polymeric materials.

Impaired protofibril formation in fibrinogen gamma N308K is due to altered D:D and "A:a" interactions.

"A:a" knob-hole interactions and D:D interfacial interactions are important for fibrin polymerization. Previous studies with recombinant gammaN308K fibrinogen, a substitution at the D:D interface, showed impaired polymerization. We examined the molecular basis for this loss of function by solving the crystal structure of gammaN308K fragment D. In contrast to previous fragment D crystals, the gammaN308K crystals belonged to a tetragonal space group with an unusually long unit cell (a = b = 95 A, c = 448.3 A). Alignment of the normal and gammaN308K structures showed the global structure of the variant was not changed and the knob "A" peptide GPRP was bound as usual to hole "a". The substitution introduced an elongated positively charged patch in the D:D region. The structure showed novel, symmetric D:D crystal contacts between gammaN308K molecules, indicating the normal asymmetric D:D interface in fibrin would be unstable in this variant. We examined GPRP binding to gammaN308K in solution by plasmin protection assay. The results showed weaker peptide binding, suggesting that "A:a" interactions were altered. We examined fibrin network structures by scanning electron microscopy and found the variant fibers were thicker and more heterogeneous than normal fibers. Considered together, our structural and biochemical studies indicate both "A:a" and D:D interactions are weaker. We conclude that stable protofibrils cannot assemble from gammaN308K monomers, leading to impaired polymerization.

The presence of gamma' chain impairs fibrin polymerization.

INTRODUCTION: A fraction of fibrinogen molecules contain an alternatively spliced variant chain called gamma'. Plasma levels of this variant have been associated with both myocardial infarction and venous thrombosis. Because clot structure has been associated with cardiovascular risk, we examined the effect of gamma' chain on clot structure. MATERIALS AND METHODS: We expressed three fibrinogen variants in Chinese hamster ovary (CHO) cells: gamma/gamma homodimer, gamma/gamma' heterodimer, and gamma'/gamma' homodimer. We observed thrombin-catalyzed fibrinopeptide release by HPLC, fibrin polymerization by turbidity, and clot structure by scanning electron microscopy. We characterized post-translational modifications by mass spectrometry. RESULTS: Fibrinopeptide A was released at the same rate for all three fibrinogens, while fibrinopeptide B was released faster from the gamma'/gamma' homodimer. The rise in turbidity was slower and final absorbance was lower during polymerization of gamma'-containing fibrinogens than for gamma/gamma fibrinogen. Micrographs showed that gamma'/gamma' fibrin clots are composed of very thin fibers, while the diameter of gamma/gamma' fibers is similar to gamma/gamma fibers. Further, the fiber networks formed from gamma'-containing samples were non-uniform. Mass spectrometry showed heterogeneous addition of N-glycans and tyrosine sulfation in the gamma' chain. CONCLUSIONS: The presence of gamma' chains slows lateral aggregation and alters fibrin structure. We suggest these changes are likely due to charge-charge repulsion, such that polymerization of the gamma'/gamma' homodimer is more impaired than the heterodimer since these repulsions are partially offset by incorporation of gamma chains in the gamma/gamma' heterodimer.

Fibrinogen variant BbetaD432A has normal polymerization but does not bind knob "B".

Fibrinogen residue Bbeta432Asp is part of hole "b" that interacts with knob "B," whose sequence starts with Gly-His-Arg-Pro-amide (GHRP). Because previous studies showed BbetaD432A has normal polymerization, we hypothesized that Bbeta432Asp is not critical for knob "B" binding and that new knob-hole interactions would compensate for the loss of this Asp residue. To test this hypothesis, we solved the crystal structure of fragment D from BbetaD432A. Surprisingly, the structure (rfD-BbetaD432A+GH) showed the peptide GHRP was not bound to hole "b." We then re-evaluated the polymerization of this variant by examining clot turbidity, clot structure, and the rate of FXIIIa cross-linking. The turbidity and the rate of gamma-gamma dimer formation for BbetaD432A were indistinguishable compared with normal fibrinogen. Scanning electron microscopy showed no significant differences between the clots of BbetaD432A and normal, but the thrombin-derived clots had thicker fibers than clots obtained from batroxobin, suggesting that cleavage of FpB is more important than "B:b" interactions. We conclude that hole "b" and "B:b" knob-hole binding per se have no influence on fibrin polymerization.