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Our previous studies revealed that interaction of fibrin with the very low density lipoprotein (VLDL) receptor plays a prominent role in transendothelial migration of leukocytes and thereby inflammation. The major goal of our subsequent studies is to establish the structural basis for this interaction. As the first step toward this goal, we localized the fibrin-binding sites within cysteine-rich (CR) domains 2-4 of the VLDL receptor. In this study, we have made a next step toward this goal by establishing the nuclear magnetic resonance solution structure of the recombinant VLDLR(2-4) fragment containing all three fibrin-binding CR domains of this receptor. The structure revealed that all three CR domains have a similar general fold. Each domain contains a calcium-binding loop, and the loop in the CR3 domain has a unique conformation relative to the other two. Domains CR2 and CR3 interact with each other, while CR4 is flexible relative to the other two domains. In addition, analysis of the electrostatic potential surface of VLDLR(2-4) revealed extended negatively charged regions in each of its CR domains. The presence of these regions suggests that they may interact with three positively charged clusters of the fibrin βN domain whose involvement in interaction with the VLDL receptor was demonstrated earlier. Altogether, these findings provide a solid background for our next step toward establishing the structural basis for fibrin-VLDL receptor interaction.

Our recent study revealed that fibrin interacts with the very low density lipoprotein receptor (VLDLR) on endothelial cells through its βN domains, and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major aims of this study were to further characterize this interaction and localize the fibrin-binding site in the VLDLR. To localize the fibrin-binding site, we expressed a soluble extracellular portion of this receptor, sVLDLRHT, its N- and C-terminal regions, VLDLR(1–8)HT and des(1–8)VLDLRHT, respectively, and a number of VLDLR fragments containing various combinations of CR domains and confirmed their proper folding by fluorescence spectroscopy. Interaction of these fragments with the (β15–66)2 fragment corresponding to a pair of VLDLR-binding βN domains of fibrin was tested by different methods. Our experiments performed by an enzyme-linked immunosorbent assay and surface plasmon resonance revealed that the VLDLR(1–8)HT fragment containing eight CR domains...

Our previous studies revealed that in fibrinogen the αC-domains are not reactive with their ligands, suggesting that their binding sites are cryptic and become exposed upon its conversion to fibrin, in which these domains form αC polymers. On the basis of this finding, we hypothesized that polymerization of the αC-domains in fibrin results in the exposure of their binding sites and that these domains adopt the physiologically active conformation only in αC-domain polymers. To test this hypothesis, we prepared a recombinant αC region (residues Aα221-610) including the αC-domain (Aα392-610), demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and stabilized such oligomers by covalently cross-linking them with factor XIIIa. Cross-linked Aα221-610 oligomers were stable in solution and appeared as ordered linear, branching filaments when analyzed by electron microscopy. Spectral studies revealed that the αC-domains in such oligomers were folded into compact structures of high thermal stability with a significant amount of β-sheets. These findings indicate that cross-linked Aα221-610 oligomers are highly ordered and mimic the structure of fibrin αC polymers. The oligomers also exhibited functional properties of polymeric fibrin because, in contrast to the monomeric αC-domain, they bound tPA and plasminogen and stimulated activation of the latter by the former. Altogether, the results obtained with cross-linked Aα221-610 oligomers clarify the structure of the αC-domains in fibrin αC polymers and confirm our hypothesis that their binding sites are exposed upon polymerization. Such oligomers represent a stable, soluble model of fibrin αC polymers that can be used for further structure-function studies of fibrin αC-domains.

The carboxyl-terminal regions of the fibrinogen Aalpha chains (alphaC regions) form compact alphaC-domains tethered to the bulk of the molecule with flexible alphaC-connectors. It was hypothesized that in fibrinogen two alphaC-domains interact intramolecularly with each other and with the central E region preferentially through its N-termini of Bbeta chains and that removal of fibrinopeptides A and B upon fibrin assembly results in dissociation of the alphaC regions and their switch to intermolecular interactions. To test this hypothesis, we studied the interactions of the recombinant alphaC region (Aalpha221-610 fragment) and its subfragments, alphaC-connector (Aalpha221-391) and alphaC-domain (Aalpha392-610), between each other and with the recombinant (Bbeta1-66)2 and (beta15-66)2 fragments and NDSK corresponding to the fibrin(ogen) central E region, using laser tweezers-based force spectroscopy. The alphaC-domain, but not the alphaC-connector, bound to NDSK, which contains fibrinopeptides A and B, and less frequently to desA-NDSK and (Bbeta1-66)2 containing only fibrinopeptides B; it was poorly reactive with desAB-NDSK and (beta15-66)2 both lacking fibrinopeptide B. The interactions of the alphaC-domains with each other and with the alphaC-connector were also observed, although they were weaker and heterogeneous in strength. These results provide the first direct evidence for the interaction between the alphaC-domains and the central E region through fibrinopeptide B, in agreement with the hypothesis given above, and indicate that fibrinopeptide A is also involved. They also confirm the hypothesized homomeric interactions between the alphaC-domains and display their interaction with the alphaC-connectors, which may contribute to covalent cross-linking of alpha polymers in fibrin.

According to the existing hypothesis, in fibrinogen, the COOH-terminal portions of two Aalpha chains are folded into compact alphaC-domains that interact intramolecularly with each other and with the central region of the molecule; in fibrin, the alphaC-domains switch to an intermolecular interaction resulting in alphaC-polymers. In agreement, our recent NMR study identified within the bovine fibrinogen Aalpha374-538 alphaC-domain fragment an ordered compact structure including a beta-hairpin restricted at the base by a 423-453 disulfide linkage. To establish the complete structure of the alphaC-domain and to further test the hypothesis, we expressed a shorter alphaC-fragment, Aalpha406-483, and performed detailed analysis of its structure, stability, and interactions. NMR experiments on the Aalpha406-483 fragment identified a second loose beta-hairpin formed by residues 459-476, yielding a structure consisting of an intrinsically unstable mixed parallel/antiparallel beta-sheet. Size-exclusion chromatography and sedimentation velocity experiments revealed that the Aalpha406-483 fragment forms soluble oligomers whose fraction increases with an increase in concentration. This was confirmed by sedimentation equilibrium analysis, which also revealed that the addition of each monomer to an assembling alphaC-oligomer substantially increases its stabilizing free energy. In agreement, unfolding experiments monitored by CD established that oligomerization of Aalpha406-483 results in increased thermal stability. Altogether, these experiments establish the complete NMR solution structure of the Aalpha406-483 alphaC-domain fragment, provide direct evidence for the intra- and intermolecular interactions between the alphaC-domains, and confirm that these interactions are thermodynamically driven.

Interaction of lipoprotein(a) with fibrin associated with atherosclerotic lesions promotes its accumulation in the lesions, thereby contributing to the development of atherothrombosis. Numerous studies revealed that this interaction occurs through the apolipoprotein(a) [apo(a)] component of lipoprotein(a) and COOH-terminal Lys residues generated by partial degradation of fibrin with plasmin (a COOH-Lys-dependent mechanism). At the same time, the mechanism of the interaction of apo(a) with intact fibrin(ogen) remained unclear. Our recent study identified the Lys-independent apo(a)-binding sites within the fibrin(ogen) alphaC domains which contribute to an alternative Lys-independent mechanism. In this study, we performed direct measurements of the interaction between apo(a) and various fibrin(ogen) fragments representing the whole fibrin(ogen) molecule except the alphaC regions. The experiments revealed that the apo(a)-binding site, identified previously within fibrinogen gamma chain residues 207-235 [Klose, R., et al. (2000) J. Biol. Chem. 275, 38206-38212], is a high-affinity site and mainly Lys-independent, suggesting that it should also contribute to the Lys-independent mechanism. The experiments also identified a novel Lys-dependent high-affinity apo(a)-binding site within the sequence of gamma chain residues 287-411. This site may provide interaction of apo(a) with intact fibrin(ogen) through another alternative mechanism, which depends on internal Lys residues. Thus, apo(a) may interact with intact fibrin through the Lys-independent and Lys-dependent mechanisms, while the COOH-Lys-dependent mechanism may prevail in the presence of fibrinolytic activity.

Nonsubstrate interaction of thrombin with fibrinogen promotes sequential cleavage of fibrinopeptides A and B (fpA and fpB, respectively) from the latter, resulting in its conversion into fibrin. The recently established crystal structure of human thrombin in complex with the central part of human fibrin clarified the mechanism of this interaction. Here, we reveal new details of the structure and present the results of molecular modeling of the fpA- and fpB-containing portions of the Aalpha and Bbeta chains, not identified in the complex, in both fibrinogen and protofibrils. The analysis of the results reveals that in fibrinogen the fpA-containing portions are in a more favorable position to bind in the active site cleft of bound thrombin. Surface plasmon resonance experiments establish that the fpB-containing portions interact with the fibrin-derived dimeric D-D fragment, suggesting that in protofibrils they bind to the newly formed DD regions bringing fpB into the vicinity of bound thrombin. These findings provide a coherent rationale for the preferential removal of fpA from fibrinogen at the first stage of fibrin assembly and the accelerated cleavage of fpB from protofibrils and/or fibrils at the second stage.

The fibrinogen gamma-module sequences, gamma190-202 or P1, and gamma377-395 or P2, were implicated in interaction with the alpha(M)I-domain of the leukocyte receptor alpha(M)beta(2). P1 is an integral part of the gamma-module central domain, while P2 is inserted into this domain forming an antiparallel beta-strand with P1. We hypothesized earlier that separation of P2 from P1 may regulate interaction of fibrin(ogen) with leukocytes during the inflammatory response. To test the relative contributions of these sequences to the interaction and the effect of their separation, we prepared the recombinant gamma-module (gamma148-411) and its halves, gamma148-286 and gamma287-411 fragments containing P1 and P2, respectively, and evaluated their affinities for the recombinant alpha(M)I-domain. In a solid-phase binding assay, the immobilized gamma-module exhibited high affinity for alpha(M)I (K(d) = 22 nM), while the affinities of the isolated gamma148-286 and gamma287-411 halves were much lower (K(d)'s = 521 and 194 nM, respectively), indicating that both halves contribute to the interaction in a synergistic manner. This is consistent with the above hypothesis. Further, we prepared the recombinant gamma148-191 and gamma192-286 fragments corresponding to the NH(2)-terminal and central domains, respectively, as well as gamma148-226 containing P1, and tested their interaction with alpha(M)I. The immobilized gamma192-286 fragment bound to alpha(M)I with K(d) = 559 nM, while both gamma148-191 and gamma148-226 failed to bind suggesting that P1 does not contribute substantially to the binding and that the binding occurs mainly through the gamma227-286 region. To further localize a putative binding sequence, we cleaved gamma192-286 and analyzed the resulting peptides. The only alpha(M)I-binding activity was associated with the gamma228-253 peptide, indicating that this region of the central domain contains a novel alpha(M)beta(2)-binding sequence.

The beta chain 15-42 sequence of the fibrin(ogen) E region was implicated in heparin binding [Odrljin et al. (1996) Blood 88, 2050-2061]; whether heparin binds to other fibrin(ogen) regions remains to be clarified. To address this question, we studied the interaction of heparin with fibrinogen, fibrin, and their major fragments D(1), D-D, E(1), E(3), and alphaC, which together cover the entire structure of the molecule, by ligand blotting, surface plasmon resonance, and fluorescence. All three techniques revealed that at physiological ionic conditions only fibrin(ogen) and the E(1) fragment bind heparin, indicating that the only physiologically relevant heparin-binding site of fibrin(ogen) is located in its E region. To test whether the beta15-42 sequence is sufficient to form this site or some additional sequences are also involved, we tested the interaction of heparin with a number of beta15-42-containing fragments. The synthetic beta15-42 peptide bound heparin weakly (K(d) = 44.5 microM) while the recombinant beta15-57 and beta15-64 fragments exhibited almost 7-fold higher affinity (K(d) = 6.4 and 7.1 microM, respectively), indicating that the beta43-57 region is also important for heparin binding. At the same time the recombinant dimeric disulfide-linked (beta15-66)(2) fragment which mimics the dimeric arrangement of the beta chains in fibrin bound heparin with high affinity (K(d) = 66 nM), almost 100-fold higher than that for the monomeric fragments. This affinity was similar to those determined for fibrin and the E(1) fragment (K(d) = 72 and 70 nM, respectively) suggesting that (beta15-66)(2) mimics well the heparin-binding properties of the latter two. Altogether, these results indicate that the only heparin-binding site in fibrin(ogen) is formed by NH(2)-terminal portions of the beta chains, including residues beta15-57, and that dimerization is essential for high-affinity binding.

The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) alphaC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D-D:E(1) complex and a recombinant alphaC fragment (residues Aalpha221-610) corresponding to the alphaC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D-D:E(1) complex or the immobilized alphaC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the alphaC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the alphaC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH(2) terminal connector region of the alphaC domain since in ELISA, the immobilized recombinant Aalpha221-391 sub-fragment bound fibronectin well while the immobilized recombinant Aalpha392-610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aalpha221-391 region of the fibrinogen alphaC domain that is not accessible in fibrinogen but becomes exposed in fibrin.

We hypothesized that the alpha C-domain of human fibrinogen (residues hA alpha 221-610) and of other species consists of a compact COOH-terminal region (hA alpha 392-610) and a flexible NH(2)-terminal connector region (hA alpha 221-391) which may contain some regular structure [Weisel and Medved (2001) Ann. N.Y. Acad. Sci. 936, 312-327]. To test this hypothesis, we expressed in E. coli recombinant fragments corresponding to the full-length human alpha C-domain and its NH(2)- and COOH-terminal regions as well as their bovine counterparts, bA alpha 224-568, bA alpha 224-373, and bA alpha 374-568(538), respectively, and tested their folding status by fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC). All three methods revealed heat-induced unfolding transitions in the full-length bA alpha 224-568 and its two COOH-terminal fragments, indicating that the COOH-terminal portion of the bovine alpha C-domain is folded into a compact cooperative structure. Similar results were obtained by CD and DSC with the full-length and the COOH-terminal h392-610 human fragments. The NH(2)-terminal fragments of both species, b224-373 and h221-392, did not exhibit any sign of a compact structure. However, their heat capacity functions, CD spectra, and temperature dependence of ellipticity at 222 nm were typical for peptides in the extended helical poly(L-proline) type II conformation (PPII), suggesting that they contain this type of regular structure. This is consistent with the presence of proline-rich tandem repeats in the sequence of both bovine and human connector regions. These results indicate that both bovine and human fibrinogen alpha C-domains consist of a compact globular cooperative unit attached to the bulk of the molecule by an extended NH(2)-terminal connector region with a PPII conformation.

Conversion of fibrinogen into fibrin results in the exposure of cryptic interaction sites and modulation of various activities. To elucidate the mechanism of this exposure, we tested the accessibility of the Aalpha148-160 and gamma312-324 fibrin-specific epitopes that are involved in binding of plasminogen and its activator tPA, in several fragments derived from fibrinogen (fragment D and its subfragments) and fibrin (cross-linked D-D fragment and its noncovalent complex with the E(1) fragment, D-D. E(1)). Neither D nor D-D bound tPA, plasminogen, or anti-Aalpha148-160 and anti-gamma312-324 monoclonal antibodies, indicating that their fibrin-specific epitopes were inaccessible. The Aalpha148-160 epitope became exposed only upon proteolytic removal of the beta- and gamma-modules from D. At the same time, both epitopes were accessible in the D-D.E(1) complex, indicating that the DD.E interaction resulted in their exposure. This exposure was reversible since the dissociation of the D-D.E(1) complex made the sites unavailable, while reconstitution of the complex made them exposed. The results indicate that upon fibrin assembly, driven primarily by the interaction between complementary sites of the D and E regions, the D regions undergo conformational changes that cause the exposure of their plasminogen- and tPA-binding sites. These changes may be involved in the regulation of fibrin assembly and fibrinolysis.

Integrin alpha v beta 3, a widely distributed fibrinogen receptor, recognizes the RGD572-574 motif in the alpha chain of human fibrinogen. However, this motif is not conserved in other species, nor is it required for alpha v beta 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other alpha v beta 3 binding sites. Fibrinogen has conserved C-terminal domains in its alpha (E variant), beta, and gamma chains (designated alpha EC, beta C, and gamma C, respectively), but their function in cell adhesion is not known, except that alpha IIb beta 3, a platelet fibrinogen receptor, binds to the gamma C HHLGGAKQAGDV400-411 sequence. Here we used mammalian cells expressing recombinant alpha v beta 3 to show that recombinant alpha EC and gamma C domains expressed in bacteria specifically bind to alpha v beta 3. Interaction between alpha v beta 3 and gamma C or alpha EC is blocked by LM609, a function-blocking anti-alpha v beta 3 mAb, and by RGD peptides. alpha v beta 3 does not require the HHLGGAKQAGDV400-411 sequence of gamma C for binding, and alpha EC does not have such a sequence, indicating that the alpha v beta 3 binding sites are distinct from those of alpha IIb beta 3. A small fragment of gamma C (residues 148-226) supports alpha v beta 3 adhesion, suggesting that an alpha v beta 3 binding site is located within the gamma chain 148-226 region. We have reported that the CYDMKTTC sequence of beta 3 is responsible for the ligand specificity of alpha v beta 3. gamma C and alpha EC do not bind to wild-type alpha v beta 1, but do bind to the alpha v beta 1 mutant (alpha v beta 1-3-1), in which the CYDMKTTC sequence of beta 3 is substituted for the corresponding beta 1 sequence CTSEQNC. This suggests that gamma C and alpha EC contain determinants for fibrinogen's specificity to alpha v beta 3. These results suggest that fibrinogen has potentially significant novel alpha v beta 3 binding sites in gamma C and alpha EC.

Human fibrinogen gamma-module comprising residues gamma148-411 was expressed in Escherichia coli and refolded in vitro. Differential scanning calorimetry revealed that in addition to the two previously identified independently folded thermolabile domains, one in each half of the module, the gamma-module also contains one or two thermostable domains that melt above 65 degrees C. To localize the latter, an NH2-terminal 6-kDa fragment was prepared by limited proteolysis of the recombinant gamma-module. It melted at high temperature, indicating that this portion is folded into a compact structure that represents a thermostable domain, also identified in the proteolytic fibrinogen fragment D1 which contains the natural gamma-module. Thus the NH2-terminal half of the gamma-module forms two domains, a thermostable one and a thermolabile one, leaving the rest of the module to be responsible for the formation of the other one or two domains. The thermal stability of some domains was lower in the recombinant gamma-module than in its natural counterpart in D1, reflecting most probably the loss of interactions with neighboring domains; however, the major functional sites were essentially preserved. The module bound Ca2+ and was stabilized by it against denaturation and proteolysis. It inhibited fibrin polymerization and was efficiently cross-linked by factor XIIIa. The gamma-module supported adhesion of platelets via their GP IIbIIIa (alpha(IIb)beta3) receptor in the same manner as D1 fragment. It also supported the adhesion of alpha(M)beta2- (Mac-1-) transfected cells and in the fluid phase was more effective than D1 as an inhibitor of that adhesion, suggesting that the Mac-1 binding site is better exposed.

The Ca(2+)-induced interaction between the Gla and EGF domains of human factor IX was investigated by means of three fragments: 6-kDa Gla, 19-kDa (EGF)2, and 25-kDa Gla-(EGF)2. Size-exclusion chromatography and spectroscopic measurements revealed that the Gla-EGF interaction is rather strong; it can be reconstituted by mixing the 6-kDa and 19-kDa fragments which form a stable 1:1 heterocomplex in the presence of Ca2+. By itself, the 6-kDa Gla self-associates in these conditions. The Gla-EGF interaction can be disrupted in 5 M urea where the compact structure of both domains is preserved. Binding of Ca2+ to 19-kDa (EGF)2 occurred with a Kd of 71 microM in the absence and 108 microM in the presence of 5 M urea and stabilized the first EGF domain, increasing its Tm by 12 degrees C. Addition of Ca2+ to the 6-kDa and 25-kDa fragments produced biphasic changes in their fluorescence; the intensity increased slightly at low Ca2+ concentration and then decreased in a monotonic manner. In 5 M urea, only the decrease occurred, with apparent Kds of 0.33 and 0.30 mM for 6-kDa Gla and 25-kDa Gla-(EGF)2, respectively. Thus, in 5 M urea in the presence of Ca2+, the isolated Gla domain has a compact structure and Ca2+ binding properties similar to those in the 25-kDa fragment. In the absence of urea, the Gla domain interacts either with itself, when isolated, or with the first EGF domain when present, as in the 6-kDa/19-kDa heterocomplex, in the 25-kDa fragment and presumably intact factor IX.

Out of 29 disulfide bonds in human fibrinogen, 7 were cleaved during limited reduction under nondenaturing conditions in calcium-free buffer: 2 A alpha 442Cys-A alpha 472Cys and 2 gamma 326Cys-gamma 339Cys intrachain disulfide bonds in the carboxy-terminal ends of the A alpha- and gamma-chains and the symmetrical disulfide bonds at gamma 8Cys, gamma 9Cys, and A alpha 28Cys. We studied the loss of thrombin clottability that followed limited reduction and the increase in the susceptibility of the fibrinogen A alpha 19-A alpha 20 bond to hydrolysis by thrombin. Using differential scanning calorimetry, we show that the extent of unfolding and denaturation of specific domains following limited reduction is small. Heat absorption peaks corresponding to the melting of the major regions of compact structure give high calorimetric enthalpies, as in untreated nonreduced fibrinogen, indicating that substantial regions of native structure are still present in partially reduced fibrinogen. Thrombin releases fibrinopeptide A at an identical rate as in nonreduced fibrinogen while fibrinopeptide B release is slower. Sedimentation velocity studies show that thrombin treatment leads to complex formation; however, gelation does not occur. Amino-terminal analysis indicates that the second thrombin cleavage in the A alpha-chain at A alpha 19-A alpha 20 takes place only after fibrinopeptide A release. Thus, the loss of clottability appears to result from perturbation of carboxy-terminal polymerization sites, probably a consequence of gamma 326Cys-gamma 339Cys intrachain disulfide bond cleavage. The thrombin-treated partially reduced fibrinogen remains soluble in buffered saline and fully expresses at least one epitope, B beta 15-21, unique to fibrin. Furthermore, this nonclottable form accelerates the tissue plasminogen activator dependent conversion of plasminogen to plasmin.

Cl-s is a multidomain serine protease that participates in Ca2+-dependent protein-protein interactions with other subcomponents of Cl, the first component of human complement. Proteolytically derived fragments that retain some of the functional properties of the parent protein have been isolated, and their thermal stability has been investigated by differential scanning calorimetry. Three endothermic transitions are observed in whole Cl-s near 37, 49, and 60 degrees C in 0.05 M Tris-HCl, pH 7.2, containing 0.22 M NaCl and 0.1 mM EDTA. The first (37 degrees C) and third (60 degrees C) transitions are also seen in Cl-s-A, a derivative comprised mainly of the intact nonenzymatic A chain. The second (49 degrees C) and third transitions are seen in Cl-s-gamma B, a fragment comprised of the intact B chain, disulfide linked to the C-terminal gamma region of the A chain. Thus, the first transition, which is alone stabilized by Ca2+, corresponds to the melting of the N-terminal alpha beta region of the A chain, the second to the melting of the catalytic B chain domain, and the third to the gamma region. The gamma region is comprised of two homologous short consensus repeat (SCR) motifs that are also found in several other complement and coagulation proteins. A new 24-kDa fragment, Cl-s-gamma, which contains these two SCRs, was isolated from plasmic and chymotryptic digests of Cl-s-A. Cl-s-gamma exhibits a reversible transition near 60 degrees C corresponding to the highest temperature peak in whole Cl-s and Cl-s-A.(ABSTRACT TRUNCATED AT 250 WORDS)