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Fibrinogen is rather inert in the circulation, however, after conversion into fibrin it participates in various physiological processes including fibrinolysis. Initiation of fibrinolysis occurs through a number of orchestrated interactions between fibrin, plasminogen and its activator tPA which result in generation of plasmin. Numerous studies localized a set of specific low affinity tPA- and plasminogen-binding sites in each D region of fibrin(ogen). The tPA-binding site includes residues gamma312-324 and the plasminogen-binding site includes residues Aalpha148-160; they bind tPA and plasminogen with a K(d) of about 1 micro M. Another set of high affinity tPA- and plasminogen-binding sites (K(d)s = 16-33 nM) was identified in the compact portion of each fibrin(ogen) alphaC-domain within residues Aalpha392-610. All these sites are cryptic in fibrinogen and become exposed in fibrin. Recent studies with recombinant and proteolytic fibrin(ogen) fragments clarified the molecular mechanisms by which these sites become exposed. Namely, upon fibrin assembly, the interaction between the D and E regions causes conformational changes in the former that expose the low affinity binding sites. The exposure of the high affinity binding sites in the alphaC-domains is connected most probably with their switch from an intramolecular interaction in fibrinogen to an intermolecular one in fibrin. These mechanisms serve to minimize degradation of circulating fibrinogen and confine fibrinolysis to places of fibrin deposition.

We prospectively evaluated the diagnostic performance of a new soluble fibrin assay in 303 consecutive patients with suspected pulmonary embolism and examined potentially useful cut-off levels at which this disease can be safely excluded. In addition, the diagnostic accuracy was calculated in the subgroups of in- and outpatients. The ROC curve of the assay in the total study cohort had an area under the curve of 0.69. The cut-off level associated with a sensitivity and negative predictive value of 100% was 20 ng/ml, but the specificity was only 4%. The cut-off level with a sensitivity of 90% was 30 ng/ml, which corresponded with a specificity and negative predictive value of 27% and 86%. respectively. The diagnostic performance was comparable in the subgroups of in- and outpatients. We conclude that the soluble fibrin assay has a low diagnostic accuracy and seems unsuitable as a screening test for the exclusion of pulmonary embolism.

We prospectively evaluated the diagnostic accuracy of the Fibrinostika FbDP assay in 304 consecutive patients with suspected pulmonary embolism and examined potentially useful cut-off points at which the disease can be excluded. The prevalence of pulmonary embolism was 31%. The assay generated an area under the Receiver Operating Characteristic curve of 0.79 (95% CI 0.73-0.84). A cut-off point of 0.05 microg/ml yielded a sensitivity, specificity, negative predictive value and an exclusion efficiency of 100% (95% CI 96-100), 5% (95% CI 2-9), 100% (95% CI 69-100) and 3% (95% CI 2-6), respectively. A clinically useful cut-off point seems to be 0.11 microg/ml which corresponded with a sensitivity, specificity, negative predictive value and an exclusion efficiency of 96% (95% CI 90-99), 27% (95% CI 24-28), 93% (95% CI 84-98) and 20% (95% CI 16-25), respectively. We conclude that the assay has potential clinical utility for the exclusion of pulmonary embolism, but it cannot be used as a sole test.

D-dimer levels are used in clinical practice as markers for the presence or exclusion of venous thrombosis. Therefore, it is important that the performance of the D-dimer tests is well controlled. One of the components of a laboratory quality program is external quality assessment (EQA). The main aim of EQA is to identify the degree of agreement among laboratories. In addition to identifying the individual laboratory performance, it also identifies the characteristics of the different reagents and methods that are used in different laboratories. Recently, the D-dimer test was included in the ECAT External Quality Assessment program. Eighty-one laboratories in four rounds applied semiquantitative and quantitative D-dimer assays to three samples (concentrations normal, slightly elevated, and elevated). Although the reported values varied widely, the classification of the samples was mostly correct for the normal and the elevated samples. The slightly elevated sample was classified as normal by all laboratories using semiquantitative methods and by 60% of the laboratories using quantitative methods, and this varied between 45% and 86% for the different methods. In conclusion, D-dimer methods show a large variation, and the quality of the D-dimer assessment could be improved by standardization and by using cut-off ranges.

Fibrin formation is a multistep process initiated by thrombin. At first thrombin converts fibrinogen to fibrin molecules which in vivo form soluble complexes with fibrinogen. Soluble fibrin is considered to be an early biochemical marker for intravascular fibrin formation and impending thrombotic events, such as deep venous thrombosis (DVT), pulmonary embolism (PE) and disseminated intravascular coagulopathy (DIC). A new enzyme immunoassay (EIA) was developed on the basis of a monoclonal antibody directed against a fibrin specific neo-epitope located on the gamma-chain of fibrinogen; gamma-(312-324). In addition, it was possible to prepare a lyophilized reference material of thrombin-generated soluble fibrin, that allowed for full antigen recovery after reconstitution with buffer. Assay conditions, e.g. solid phase-Ig concentration and buffer composition, sample and conjugate dilution, and incubation times were optimised. The present assay was found to be specific (no interference of homologous antigens) and reproducible (intra-assay CV 4-8%, interassay CV 4-9%), and therefore highly suited for measuring soluble fibrin levels in a plasma milieu. The median normal value for soluble fibrin was determined in plasma samples obtained from apparently healthy volunteers (n = 81) and found to be 0.040 microg/ml, with a range (10-90 percentiles) of 0.026-0.059 microg/ml. A retrospective study showed that soluble fibrin levels were highly significantly increased in patients with a confirmed diagnosis of DIC (median 1.042 microg FEU/ml, range 0.160-2.319 microg/ml, n = 21, P<0.0001 vs. normal). PE (median 0.527 microg FEU/ml, range 0.084-1.234 microg/ml, n = 29, P<0.0001 vs normal) and DVT (median 0.126 microg FEU/ml, range 0.059-0.878 microg/ml, n = 36, P<0.0001 vs. normal), as determined by the Mann-Whitney U-Test.

Polymerized fibrin strongly enhances tissue plasminogen activator (tPA)-mediated plasminogen activation, concomitant with exposure of 'fibrin-specific' epitopes at 'Aalpha148-160' and 'gamma312-324'. To investigate which aspects of polymerization are involved in these activities, we explored the fibrin polymerization process by evaluating the ability of factor XIIIa-crosslinked fibrinogen polymers to expose 'fibrin-specific' epitopes and enhance plasminogen activation. Crosslinked normal fibrinogen, fibrinogen with deficient [des Bbeta1-42] or defective [Birmingham (AalphaR16H)] fibrin 'D:E' assembly sites ('E(A)'), or with defective end-to-end self-association sites ('D:D') [Cedar Rapids (gammaR275C)], exposed both 'fibrin-specific' epitopes and enhanced tPA-dependent plasminogen activation, whereas non-crosslinked fibrinogens showed minimal or no such activities. Epitope expression in crosslinked fibrinogen was retained in the presence of the fibrin E(A) site peptide homolog, gly-pro-arg-pro (GPRP), which inhibits fibrin D:E association, except for the Aalpha148-160 epitope in des Bbeta1-42 fibrinogen, which was not expressed. Fibrin prepared from crosslinked normal or abnormal fibrinogen, except for the des Bbeta1-42 fibrin epitopes, which were reduced or absent, expressed 'fibrin-specific' epitopes even in the presence of GPRP, which otherwise impairs such expression in non-crosslinked fibrin. Epitope exposure in fibrin prepared from non-crosslinked fibrinogen was nearly normal in Cedar Rapids fibrin (heterozygous D:D defect), but reduced in Birmingham fibrin (heterozygous E(A) defect), nil in des Bbeta1-42 fibrin (E(A) deficient), and absent in all cases in the presence of GPRP. In contrast, plasminogen activation stimulatory activity that had been exposed in crosslinked normal fibrinogen or in crosslinked des Bbeta1-42 or Cedar Rapids fibrin, was preserved to a large extent in the presence of GPRP, suggesting that once enhanced stimulatory activity and epitopes are exposed, they are not completely reversible. The findings indicate that end-to-end intermolecular associations (D:D) are not critical for 'fibrin-specific' epitope exposure, but that polymerization brought about in fibrinogen through factor XIIIa crosslinking, or in fibrin through 'D:E' interactions, is necessary for 'fibrin-specific' (more correctly, 'polymerization-specific') epitope exposure and enhancement of plasminogen activation.

The term "D-dimer assay" suggests, that these assays report the concentrations of the end-stage degradation product of crosslinked fibrin. This hardly occurs in patients. Degradation products of cross-linked fibrin rather occur with a wide range of molecular weights, and comprise various numbers of the D-dimer motif. Moreover, the numerical values obtained with different "D-dimer" assays vary widely. The variations are probably due to differences in reactivity of the various monoclonal antibodies used with the various D-dimer containing degradation products as they occur in patients; and to the various calibrators with a value assigned by the manufacturers. For the reasons indicated above a calibrator in the strict sense (e.g., pure D-dimer) is not feasible. This study shows that: it appears feasible to generate a conversion factor for each of the "D-dimer" assays studied, which will make the widely varying results obtained with these kits comparable; that the conversion factors can be based on a pool of real patient samples; and that the conversion factors are pool-independent. The next and final step in this study is to prepare and make available an international reference material for use by manufacturers and others facing a comparison problem. This is being carried out, in collaboration with the NIBSC (Dr. P. Gaffney), and the material is expected to be available in early 1997.

The present paper shows that conformationally changed fibrinogen can expose the sites A alpha-(148-160) and gamma-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5 degrees C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, has exposed the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes A alpha-(148-160) and gamma-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is not longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during self-association.

Pre- and postoperative plasma levels of Prothrombin Fragment 1 + 2 (F1 + 2), Thrombin-antithrombin III complex (TAT), Fibrinopeptide A (FpA), Fibrin and Fibrinogen Degradation Products (FbDP, FgDP) and Soluble Fibrin (SF) were measured in 40 patients undergoing gastric surgery in order to compare patients operated for benign (n = 21) and malignant (n = 19) disease. Plasma levels of F1 + 2, TAT, FbDP and SF on the first postoperative day were significantly higher than before operation. F1 + 2 and FbDP levels were further increased one week after surgery, at which time FgDP levels were also higher than preoperatively. A significant postoperative increase in FpA levels was found only in patients with malignant disease. When age was taken into consideration, significant differences between patients with and without malignancy were found only in the late postoperative period, as cancer patients had higher FpA and FbDP levels one week after surgery and higher FbDP levels one month after discharge from hospital.

In 12 patients treated with 100 mg rt-PA/3 h for acute myocardial infarction (AMI), serial fibrinogen levels were measured with the Clauss clotting rate assay ("functional fibrinogen") and with a new enzyme immunoassay for immunologically intact fibrinogen ("intact fibrinogen"). Levels of functional and "intact fibrinogen" were strikingly different: functional levels were higher at baseline; showed a more pronounced breakdown during rt-PA therapy; and a rebound phenomenon which was not seen for "intact fibrinogen". The ratio of functional to "intact fibrinogen" was calculated for each individual patient and each time point. The mean ratio (n = 12) was 1.6 at baseline, 1.0 at 90 min, and increased markedly between 8 and 24 h to a maximum of 2.1 (p < 0.01), indicating that functionality of circulating fibrinogen changes during AMI and subsequent thrombolytic therapy. The increased ratio of functional to "intact fibrinogen" seems to reflect a more functional fibrinogen at baseline and following rt-PA infusion. This is in keeping with data that the relative amount of fast clotting "intact HMW fibrinogen" of total fibrinogen is increased in initial phase of AMI. The data suggest that about 20% of HMW fibrinogen are converted to partly degraded fibrinogen during rt-PA infusion. The rebound phenomenon exhibited by functional fibrinogen may result from newly synthesized fibrinogen with a high proportion of HMW fibrinogen with its known higher degree of phosphorylation. Fibrinogen- and fibrin degradation products were within normal range at baseline. Upon infusion of the thrombolytic agent, maximum median levels of 5.88 micrograms/ml and 5.28 micrograms/ml, respectively, were measured at 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble fibrin is considered as a molecular marker for intravascular fibrin formation, and impending thrombotic events. Most of the existing assays are less suitable for routine clinical applications and their specificity may be limited. We have developed a sandwich-type EIA with a fibrin-specific MoAb described by us before (Proc Natl Acad Sci 1989; 86: 8951) as the capture antibody. An other MoAb (G8; Thromb Haemostas 1988; 60: 145) with an epitope in the carboxyl-terminal sections of the fibrin alpha-chains, was labeled with peroxidase and used as the tagging antibody. The EIA is calibrated against plasma spiked with known concentrations of soluble fibrin. The time-to-result of the EIA is only 1.5 h. Concentrations as low as 0.5 micrograms soluble fibrin/ml plasma are readily measureable. Heparin has no effect on the results. Fibrinogen and fibrin(ogen) degradation products are not detected. The values of fibrinopeptide A and soluble fibrin values found with the "COA-SET soluble fibrin" assay correlated well with the soluble fibrin values found with our EIA i.e. r = 0.998 and 0.984, respectively. The run-to-run variabilities were 7.9% and 6.6% for samples with low and high soluble fibrin concentrations, respectively. The within-run variabilities were 2.5, 1.8, 4.0 and 4.6% for samples with 1, 0.5, 0.25 and 0.125 micrograms soluble fibrin/ml, respectively. The sensitivity, specificity, accuracy and short time-to-result make our EIA suitable for routine clinical applications and the monitoring of the effectivity of heparinization.

Antifibrin monoclonal antibody Y22, of IgG1-subclass, has its epitope in the D-domain of fibrin. In a thrombin time assay, Y22 and its F(ab)2 fragments interfere with clotting of citrated plasma. Transmission and scanning electronmicroscopic studies show that clotting of citrated blood or plasma in the presence of Y22 results in formation of thin, short fibrin fibres. The (smaller) Fab fragments of Y22 did not have an anti-clotting effect. This suggests that the anticoagulant effect of Y22 is due to steric hindrance of the association of fibrin monomers. A control antibody and its F(ab)2 and Fab fragments have no effect on fibrin formation. In a parabolic rate assay, Y22 Fab fragments interfered strongly with the fibrin-induced enhancement of the t-PA-catalyzed plasminogen activation, whereas intact Y22 and a control antibody did not. In contrast with their effects on the fibrin assembly, the effects of Y22, Y22-F(ab)2 and Y22-Fab on the capacity of fibrin to act as a rate-enhancer in the plasminogen activation by t-PA appears to decrease with the size of the immunoreactive entity. As is discussed, this may be due to the differential accessibility of sites involved in stimulation and polymerization which are located in the fibrin D-domain.

We have developed a two-step enzyme immunoassay (EIA) that allows the quantitation of degradation products derived from fibrinogen (FbgDP) and that does not detect degradation products derived from cross-linked (XDP) or noncrosslinked fibrin (fdp). The EIA is based on two monoclonal antibodies (FDP-14 and Y-18), developed in our institute. FDP-14 is used as catching antibody. It complexes exclusively with degradation products, irrespective whether these are derived from fibrinogen or from fibrin. It does not complex with intact fibrinogen or fibrin. Y-18 is reactive with fibrinogen and fibrinopeptide A-comprising fibrinogen fragments. It is used, conjugated with horse-radish peroxidase, as tagging antibody. The FbgDP-EIA is highly specific, accurate and sensitive. The coefficient of variation is between 3 and 8%; the lower detection limit is less than 0.025 micrograms/ml. The assay has been applied to plasma from patients with suspected disseminated intravascular coagulation (DIC), to plasma from patients undergoing streptokinase (SK) therapy for acute myocardial infarction and to plasma from newborn babies. DIC patients had no or very low levels of FbgDP, but high levels of other degradation products, SK-treated patients showed high levels of degradation products two hours after termination of the SK infusion. A considerable fraction of these degradation products was shown to be FbgDP. Plasma from newborn babies contained elevated levels of FbgDP associated with prolonged prothrombin times.

The most commonly used fibrinogen assays in the clinic are clotting rate assays, e.g. the Clauss method. Such functional assays may be disturbed by e.g. heparin, anticoagulant fibrinogen degradation products (FgDP) and in the case of a dysfibrinogenemia. Immunological methods would not suffer from these interferences. However, immunological assays for fibrinogen, which do not measure FgDPs, do not exist. To set up such an enzyme immunoassay (EIA) we developed two monoclonal antibodies. The first monoclonal antibody (G8) has its epitope in the carboxyl-terminal 150 amino acid stretches of the fibrinogen A alpha-chains. G8 is used to coat the wells of microtitration plates, and is the capture antibody in this EIA. The second antibody (Y18) has been described by us previously (Blood 1985; 66: 503). It is directed against fibrinopeptide A, covalently bound to the alpha-chains i.e. against the amino-terminal stretches of the A alpha-chains. Y18 is conjugated with horse-radish peroxidase, and used as tagging antibody. The EIA does not react with, and is not interfered by FgDP such as purified fragments X and Y, up to a concentration of 800 micrograms/ml. An FgDP mixture such as generated by Streptokinase treatment of plasma does not respond. Fibrin degradation products (whole blood lysate) up to 800 micrograms/ml do not interfere nor do heparin, EDTA or oxalate. The time-to-result of the EIA is only 45 minutes. Some patient plasmas yielded dose-response curves which are not parallel with the calibration curve of the EIA. An explanation for this phenomenon could not be given.(ABSTRACT TRUNCATED AT 250 WORDS)

At least four molecular forms of plasminogen are known. Two of those forms have glutamic acid at their amino-terminal end, and are designated as glu-plasminogen. The other two have lysine, methionine and/or valine as amino-terminal amino acid and are collectively designated as lys-plasminogen. Two subforms (I and II) each of glu- and lys-plasminogen exist. The I-forms are glycosylated at asn-288 and thr-345, whereas the II-forms are only glycosylated at thr-345. In a previous publication (Thromb Haemostas 1984; 52: 347-349) we have described the separation of the I- and II-forms of plasminogen in lysine-Sepharose in phosphate buffers. Now we have combined those findings with the differential affinity of glu- and lys-plasminogen for aminohexyl-Sepharose through their aminohexyl-sites, recently described by Christensen (Biochem J 1984; 223: 431-421). Acid/urea electrophoresis, end-group determination and carbohydrate analysis show that the combination of affinity chromatography on lysine-Sepharose in phosphate buffers, and on aminohexyl-Sepharose provides an efficient procedure to separate the four molecular forms of plasminogen.

We have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes. The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the B beta-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i.e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products. We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

The anticoagulant properties of fibrinogen digestion products change with stage of digestion. On digestion with leukocyte elastase, in the presence of calcium ions, the anticoagulant potency of fibrinogen digests first increases, then decreases sharply, and in late stages increases again. This is different from plasmin digestion where only an increase in anticoagulant activity is seen followed by a slow decrease. From SDS-gel electrophoresis it appears that both the early rise and the decrease in anticoagulant activity are associated with the stage of elastase-produced X-like fragments. This is confirmed with pure fragments: X-like fragments (purified from elastase digests of fibrinogen of different stages by ammonium sulphate precipitation and ion-exchange chromatography) give an increase and decrease in anticlotting activity which correlates very well with that of the potency of the digest from which they are purified. As expected, and in contrast with (late) plasmic X-fragments, late elastase X-like fragments have a low anticoagulant potency. The molecular basis for the gain and loss in anticoagulant activity going from early to late X-like fragments is obscure. Immunological tests, calcium-binding experiments and affinity chromatography on immobilized thrombin-activated NDSK suggest that the changes in anticoagulant activity are not due to a proteolytic change in the carboxyl-terminal part of the gamma-chain in the D moiety of the molecule. Our data suggest a correlation with the stage of digestion of the A alpha-chain in the X-like fragments.

Previously, we demonstrated that in vivo regulation of liver fibrinogen synthesis occurs via the fibrinogen mRNA level. However, the molecular regulatory mechanism of fibrinogen synthesis is still not well understood. Fibrinogen or fibrin degradation products might play an important role in regulating fibrinogen synthesis. In our present study, we have injected rats intraperitoneally with purified homologous fragments and measured the liver content of mRNA specific coding for fibrinogen. Increased levels of fibrinogen mRNA and elevated plasma fibrinogen concentrations were observed in rats after administration of fibrinogen degradation products X, Y, DEGTA, Dcate or E. Fragment E or E' has a less stimulatory effect than X, Y or Dcate, whereas cross-linked fibrin degradation product D dimer does not increase fibrinogen synthesis. This article reports for the first time a stimulatory effect of the high molecular weight fibrinogen degradation products on fibrinogen synthesis.

The blood clearance of purified plasmin degradation products (X, Y, D-dimer, Dcate, D EGTA, E-fibrin, Ecate and E EGTA) of rat fibrin(ogen) has been studied in a homologous system. Transcapillary flux rate constants, fractional catabolic rate constants and the ratios of intravascular and extravascular pools have been calculated. There are considerable differences in these constants between the two early fibrinogen degradation products X and Y. Also the differences between fibrinogen D and E fragments, derived from different parts of the fibrinogen molecule, are considerable. However, the differences between fibrin fragment D-dimer and fibrinogen fragment Dcate are relatively small. Also the differences between fragments E-fibrin, Ecate and E EGTA are small. The main fraction of all E-fragments is extravascular.

Native human plasminogen, the proenzyme of plasmin (E.C. 3.4.21.7) occurs in blood in two well defined forms, affinity forms I and II. In this paper, the feasibility of separating these forms of human native plasminogen by affinity chromatography, is shown to be dependent on two factors: 1) the ionic composition of the buffer containing the displacing agent: buffers of varying contents of sodium, Tris, phosphate and chloride ions were compared, and 2) the type of adsorbent. Two adsorbents were compared: Sepharose-lysine and Sepharose-bisoxirane-lysine. Only in the phosphate containing buffers, irrespective of the type of adsorbent, the affinity forms can be separated. The influence of the adsorbent can be accounted for by a large difference in dissociation constants of the complex between plasminogen and the immobilized lysine.

The abnormal fibrinogen Haifa is characterized by the fact that calcium present during enzymatic digestion by plasmin does not protect the Haifa D gamma chain against further plasmin attack as it does in normal molecules. Since calcium binding to fibrinogen, ADP--platelet aggregation cofactor activity and gamma dimerization process induced by factor XIIIa are normal for fibrinogen Haifa, the corresponding sequences in the gamma chain are not involved. It seems rather that the anomaly resides near the gamma 302 plasmin cleavage site that is protected when calcium is bound to the gamma chain and that this affects the availability of the polymerization site located in the C terminal part of the chain.