Smaller Multimers Research Articles

Multimerin: A Series of Large Disulfide-Linked Multimeric Proteins Within Platelets

Platelets contain proteins with biochemical properties that are well adapted to promoting hemostasis. One important adhesive protein is von Willebrand factor (vWF), which is a very large protein comprised of a series of multimers, ranging from 860,000 to over 10 million daltons. In this report we describe a second platelet protein, p-155, which has a similar unique multimeric composition. Using agarose-acrylamide gel electrophoresis, platelet p-155 was shown to be composed of multimers ranging from less than 450 Kd to many million daltons. Based on this unique structure, we propose that the native molecule be designated as multimerin. Comparison with vWF showed that multimerin contained less of the very high molecular weight multimers. Differential reduction demonstrated that the smallest multimer is a trimer, composed of three 155-Kd subunits. Platelet releasate was demonstrated to contain mainly the smaller multimers, suggesting that the larger multimers bind to the platelet surface. Other studies indicate that multimerin and vWF are the two largest platelet proteins and the only two platelet proteins exhibiting a complex, disulfide-linked multimeric composition with variability in multimer size.

The Effect of an Anthracycline Agent Daunorubicin on von Willebrand Factor in Plasma

The effect of daunorubicin (DNR) on von Willebrand factor (vWF) in plasma was studied. Ristocetin induced-platelet aggregation (RIPA) in five acute myeloid leukemia (AML) patients was decreased after administration of DNR (p<0.05). And then plasma vWF antigen (vWF: Ag) and ristocetin cofactor (RCof) in seven AML patients were measured. After administration of DNR, vWF: Ag was increased (p<0.05), whereas RCof was decreased (p<0.01). RCof was decreased significantly (p<0.005) in the DNR-treated plasma (DTP) of normal subjects, in which DNR was added and incubated at 37°C for 30min, in comparison with the control in which DNR was not added. Further more, RCof was decreased significantly (p<0.01) even in the DTP from which DNR was removed by gel filtration. Change of the multimeric composition of vWF were investigated by crossed immunoelectrophoresis (CIE) and SDS-agarose gel electrophoresis. In the plasma of AML patients after administration of DNR and DTP, the components corresponding to the small multimer increased. These results suggested that DNR could induce the molecular abnormality of vWF.

Multimeric Composition of Endothelial Cell-Derived von Willebrand Factor

Abstract The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC- conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.

Multimeric composition of endothelial cell-derived von Willebrand factor

The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC-conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.

Molecular association of pentanols in n-heptane II: Viscosities as a function of temperature covering low concentration range

Abstract Viscosities of binary mixtures of isomeric pentanols in n-heptane at various mole fractions, F (F = 0.025 to 0.600) have been measured in the low temperature range (T = 233 to 293 K) with an automatic viscometer operating in a closed system. The temperature dependence of viscosity has been found to follow a simple relation, thereby allowing for the calculation of Ev, the activation energy for viscous flow. The latter is very sensitive to changes in molecular associates, especially at low concentrations. The results show that at low concentrations small multimers dominate for both 1-pentanol and 2-pentanol. At high concentrations, while the former may associate to give large linear multimers; the latter may result in the formation of large cyclic multimers with a low dipole moment. For 3-pentanol and tert-pentanol, small multimers are likely to dominate at low concentrations and there is a change in the dominating multimers from a linear type to a cyclic one, with increasing concentration in the limit F

Effect of fibronectin and von willebrand factor on the adhesion of human fixed washed platelets to collagen immobilized beads

The adhesion of human fixed washed platelets(FWP) to collagen was measured using collagen immobilized beads. The addition of normal plasma or severe von Willebrand disease (VWD) plasma to FWP decreased the adhesion, suggesting the presence of some inhibitors of platelet adhesion in human plasma. Although the adhesion of FWP in severe VWD plasma was not different from that of FWP in normal plasma, the addition of purified von Willebrand factor(vWF, 1–2 u/ml ristocetin cofactor) to FWP in buffer increased the FWP adhesion at higher flow rates, and the percent of adhesion in the absence of vWF was 10 %(collagen 500 ug) and 30% (collagen 1,000 ug) of that in the presence of vWF at 10 ml/min. The enhancing effect of the vWF on FWP adhesion was also observed by pretreatment of the collagen column with vWF suggesting the important role of bound vWF to the collagen; adhesion 72% to the collagen column(1,600 ug) treated with vWF and 16 % to the collagen column without the pretreatment at 10 ml/min. The promoting effect of vWF was also present in some commercial factor VIII preparations which had no large or intermediate multimers of vWF antigen. The adhesion of FWP was inhibited by fibronectin(FN) and the binding of ristocetin cofactor(vWF:RGb) to collagen fiber was also inhibited by FN; bound vWF:RCo to 50 ug/ml collagen in the absence or presence of 125 ug/ml FN were 60% and 8% respectively. It is suggested that vWF, even small multimer of vWF:Ag, is involved in the initial platelet-collagen interaction at high flow rates, while plasma FN acts as one of anti-adhesion factor.

Multimeric pattern discrepancy between platelet and plasma von Willebrand factor in type IIC von Willebrand disease.

von Willebrand factor (vWF) from platelet lysate and plasma, collected in the presence of protease inhibitors, was studied in two patients with type IIC von Willebrand disease (vWD). Platelet and plasma vWF showed the smallest multimer increased, but the latter had a repeating single band whereas the former had a repeating "doublet." This platelet-plasma discrepancy observed for the first time in these patients suggests that the repeating "doublet" or single band described in other type IIC patients represent minor subgroups of type IIC vWD.

Von Willebrand disease type IIC with different abnormalities of von Willebrand factor in the same sibship.

A family with von Willebrand disease has been identified in which different members of the same sibship exhibit different abnormalities of von Willebrand factor (vWF). The two most severely affected sibs (bleeding time over 20 min) had abnormalities of vWF similar to those seen in type IIC. The smallest detectable multimer was increased and the triplet structure of individual multimers was replaced with a single band. The largest multimers could not be detected and there were relatively more small multimers than intermediate sized forms. vWF antigen (vWF:Ag) was decreased to 12.5-17% by electroimmunoassay (EIA) and to 3.2-5.5% by immunoradiometric assay (IRMA). In the less severely affected sibling (bleeding time 12.5 min) there was a similar relative increase in the smallest detectable multimer. However, the larger multimers were present and the relative concentration of large to small multimers was similar to normal. The triplet structure was altered in that the relative proportion of satellite bands to the central predominant band was decreased. vWF:Ag concentrations were moderately decreased (40-80% by EIA and 25-35% by IRMA). The father and grandfather showed a vWF multimeric pattern similar to the less severely affected sibling but there was no decrease in vWF:Ag concentration and their bleeding times were normal. These observations suggest that the interplay of several genetic factors is responsible for the expression of von Willebrand disease in this family.

Visualization of von Willebrand Factor Multimers by Immunoenzymatic Stain Using Avidin-Biotin Peroxidase Complex

A technique for the detection of von Willebrand factor multimers separated by discontinuous SDS agarose electrophoresis has been developed using non-radioactive compounds. The multimeric patterns were visualized by monospecific anti-human vWF:Ag followed by incubation with biotinylated antibody. After addition of avidin-biotin-peroxidase complex, the peroxidase activity was detected by 4-chloro-1-naphthol, giving sharp bands with a clear background. By this method, the differences of vWF:Ag multimers could be easily observed between normal plasma and the plasmas from variant type vWD (IIA, IIB, platelet-type). Large and intermediate multimers were absent in the plasma with vWD type IIA, while only large multimers were absent in the plasma with vWD IIB and platelet-type. The absence of large multimers was also observed in two commercial FVIII preparations having the ratio of vWF/vWF:Ag 0.18 and 0.63. The preparation with the ratio of 0.63 showed the presence of larger intermediate multimers. Electrophoresis in SDS 1.5% agarose gel revealed triplet structure of each small multimer, and a relative increase of the smallest subband was observed in vWD IIA plasma, platelet-type vWD plasma and commercial FVIII preparations. The procedures described are easy and safe to perform and are useful for screening or classifying cases with vWD in general laboratories.

Substructure of human von Willebrand factor.

Using electron microscopy, we have visualized the substructure of human von Willebrand factor (vWf) purified by two different approaches. vWf multimers, which appear as flexible strands varying in length up to 2 micron, consist of dimeric units (protomers) polymerized linearly in an end-to-end fashion through disulfide bonds. Examination of small multimers (e.g., one-mers, two-mers, and three-mers) suggests that each protomer consists of two large globular end domains (22 X 6.5 nm) connected to a small central node (6.4 X 3.4 nm) by two flexible rod domains each approximately 34 nm long and approximately 2 nm in diameter. The protomer is 120 nm in length when fully extended. These same structural features are seen both in vWf molecules that were rapidly purified from fresh plasma by a new two-step procedure and in those purified from lyophilized intermediate-purity Factor VIII/vWf concentrates. The 240,000-mol wt subunit observed by gel electrophoresis upon complete reduction of vWf apparently contains both a rod domain and a globular domain and corresponds to one half of the protomer. Two subunits are disulfide-linked, probably near their carboxyl termini, to form the protomer; disulfide bonds in the amino-terminal globular ends link promoters to form vWf multimers. The vWf multimer strands have at least two morphologically distinct types of ends, which may result from proteolytic cleavage in the globular domains after formation of large linear polymers. In addition to releasing fragments that were similar in size and shape to the repeating protomeric unit, plasmic degradation of either preparation of vWf reduced the size of multimers, but had no detectable effect on the substructure of internal protomers.

Effects of plasmin on von Willebrand factor multimers. Degradation in vitro and stimulation of release in vivo.

von Willebrand factor (vWF), a multimeric protein that mediates platelet adhesion, circulates in association with the procoagulant Factor VIII (FVIII). In previous reports, plasmin was shown in vitro to inactivate FVIII and cleave the vWF subunit extensively, but to cause only a modest decrease in vWF platelet-agglutinating activity. In the present study, the digestion of vWF multimers by plasmin was analyzed by sodium dodecyl sulfate-agarose gel electrophoresis and radioimmunoblotting. In vitro, plasmin degraded the large vWF multimers to smaller forms that could be distinguished from the small multimers present before digestion only by a slightly increased electrophoretic mobility. These plasmin-cleaved "multimers" were composed of disulfide-linked fragments with no intact vWF subunits. Thus, many plasmin cleavages occur within disulfide loops. The slight increase in mobility of plasmin-digested vWF is in part explained by the early cleavage from the multimers of a 34,000-mol wt peptide, which was purified and partially sequenced. The amino-terminal sequence (33 residues) agrees with the previously reported sequence (15 residues) for the amino terminus of the intact vWF subunit. Analysis of plasmin-digested vWF allowed deduction of a model for the native vWF structure, including the approximate location of the interprotomer disulfide bond(s). To determine whether plasmin would digest vWF in vivo, plasmas from 12 patients and 2 normal volunteers who received intravenous streptokinase (SK) were analyzed. Rather than vWF digestion, a two- to threefold rise in vWF antigen and platelet-agglutinating activity occurred within 2 h after a single SK dose, and the increase was greatest among the largest multimers. In contrast, FVIII clotting activity dropped to 10-20% of pre-SK levels. Thus, although plasmin destroys FVIII, a pharmacologically induced fibrinolytic state is associated with significant release of vWF from endothelial cells, platelets, or some other storage pool.

新しい中間型第ＶＩＩＩ因子製剤「乾燥低フィブリノゲン抗血友病人グロブリン，ＲＣＧ‐５日赤」中の第ＶＩＩＩ因子／ｖｏｎ Ｗｉｌｌｅｂｒａｎｄ因子

We have studied in vitro properties of a new intermediate type of factor VIII (FVIII) concentrate, lyophilized anti-hemophilic globulin concentrate with low content of fibrinogen (RCG-5, Japan Red Cross), and compared it with those of commercial cryoprecipitate (cryo) and FVIII concentrates with special reference to FVIII/vWF activities. RCG-5 was prepared from cryoprecipitation and subsequent defibrination with heat treatment at 56°C for 5min.RCG-5 was found to contain 19.5u/ml of FVIII clotting activity (VIII: C) on the average, 28.0u/ml of FVIII antigen (VIII: Ag), 29.0u/ml of ristocetin co-factor (RCof), 30.7u/ml of vWF antigen (vWF: Ag) and 10.4mg/ml (0.5mg/u. of VIII: C) of fibrinogen. The ratio of VIII: Ag to VIII: C and vWF: Ag to RCof in RCG-5 was 1.4 and 1.1, respectively. They were very close to those in cryo. These suggest that the inactivation of biological activities of VIII: C and RCof, and/or denaturation of antigenicity of FVIII and vWF little occurred during production procedure.RCG-5 has a remarkable correcting effect on the defective ristocetin induced platelet aggregation (RIPA) in the patient with von Willebrand disease (vWD). It also showed large, intermediate and small multimer of vWF just similar to normal plasma or cryo. These results may lead to a conclusion that RCG-5 is expected to be effective for good hemostasis not only in the patients with hemophilia A but vWD.

Effect of Carbohydrate Modifications of Factor VIII/ von Willebrand Factor on Binding to Platelets

This study compares the ability of unmodified and carbohydrate-modified forms of factor VIII/von Willebrand factor (FVIII/vWF) protein to bind to platelets in the presence of ristocetin or thrombin. Treatment of intact FVIII/vWF with alpha-D-neuraminidase results in more than 95% desialylation. Asialo FVIII/vWF retains total activity in ristocetin- and thrombin-mediated binding to platelets as demonstrated by direct and competitive binding assays. Examination of its multimeric pattern by sodium dodecyl sulfate-agarose electrophoresis reveals a normal multimeric structure. Treatment of intact FVIII/vWF with beta-D-galactosidase results in the removal of 20% of galactose (agalacto FVIII/vWF) whereas 55% of galactose is released from asialo FVIII/vWF (asialo agalacto FVIII/vWF). Agalacto and asialo-agalacto FVIII/vWF are both unable to bind to platelets in the presence of ristocetin. In contrast, they still bind to thrombin-stimulated human (except thrombasthenic) platelets. Removal of either ultimate (agalacto FVIII/vWF) or ultimate and penultimate (asialo-agalacto FVIII/vWF) galactose results in the same loss of the larger molecular weight multimers and in an increase of smaller multimers. These results suggest (1) that sialic acid does not play a significant role in ristocetin- or thrombin-mediated FVIII/vWF-platelets interactions and multimeric structure of FVIII/vWF (2) that ultimate beta-linked galactose residues are essential for the maintenance of a normal multimer organization (3) that ristocetin- and thrombin-mediated binding of FVIII/vWF to platelets differ in FVIII/vWF galactose requirement.

Role of carbohydrate in multimeric structure of factor VIII/von Willebrand factor protein.

The carbohydrate moiety of the factor VIII/von Willebrand (vW) factor protein is important in the expression of vW factor activity and the intravascular survival of the protein. Studies of normal human factor VIII/vW factor protein indicate that there is a requirement of a full complement of penultimate galactose for the maintenance of a normal multimeric structure. Release of penultimate galactose by beta-galactosidase or modification by galactose oxidase results in loss of the largest molecular weight multimers and increased numbers of intermediate and smaller multimers. In contrast, terminal galactose on the factor VIII/vW factor protein does not appear to play a significant role in the maintenance of the multimer organization. The abnormalities in multimeric structure and molecular size were demonstrated by NaDodSO4/polyacrylamide/agarose gel electrophoresis, NaDodSO4/glyoxyl-agarose electrophoresis, and sucrose density ultracentrifugation. These studies indicate that the penultimate galactose plays a role in the maintenance of the largest multimers of the factor VIII/vW factor protein. This may explain why, in some patients with variant forms of vW disease, a carbohydrate abnormality also may affect the multimeric structure of the plasma factor VIII/vW factor protein.

An electroblotting technique for the detection of factor VIII/von Willebrand factor multimers in plasma.

A new, relatively simple technique has been developed in order to study the multimers of factor VIII/von Willebrand factor (VIII/vWF). It involves electrophoresis on SDS agarose gels and electrophoretic transfer (electroblotting) of the separated protein bands onto nitrocellulose membranes, to which they are non-covalently bound. VIII/vWF multimers are then detected by 125I-labelled antibodies to VIII/vWF, and autoradiography. Optimum electrophoretic transfer occurred at 0.5 A, for 18 h, on 0.8% agarose gels, thus enabling detection of the multimeric profile of VIII/vWF in 5 microliters of normal plasma. The multimeric profile for haemophilia A patients was identical to that for normal plasma. In plasma from patients with von Willebrand's disease (vWd), various patterns were seen, with a preponderance of smaller multimers in type II (atypical) vWd, similar to those seen in cryosupernatant. Heterogeneity within a particular type of vWd was also evident. Investigation of commercial factor VIII concentrates showed the presence of 'doublets' of VIII/vWF. Unlike other reported techniques, the rapid transfer and fixing of the protein bands to the nitrocellulose, minimizes loss of resolution, and handling of the paper is easier. It is possible to cut a sample electrophoresis strip into several areas, for incubation with different antibodies. Preliminary experiments also suggest that double antibody techniques are possible, or even removal of a first radiolabelled antibody by low pH, and then incubation of the separated proteins with a second, unrelated antibody.

Botrocetin (venom coagglutinin): reaction with a broad spectrum of multimeric forms of factor VIII macromolecular complex.

Botrocetin, originally called venom coagglutinin, is a Bothrops factor that causes aggregation of blood platelets in the presence of the von Willebrand component of the factor VIII macromolecular complex. The complex consists of a series of multimers with a molecular weight of about 1-20 x 10(6). Ristocetin, another agent that causes platelet aggregation dependent on von Willebrand factor, reacts with only the higher molecular weight multimers. We report on the reactivity of botrocetin in relation to the multimeric structure of the factor VIII complex. Several plasmas or plasma fractions with abnormal distribution of the multimeric sizes were examined, including variant von Willebrand disease type IIA with lack of the higher molecular weight forms, commercial antihemophilic factor concentrates with a preponderance of lower molecular weight forms, cryoprecipitate-free plasma containing mainly the smaller multimers, and a chromatographic fraction of plasma containing only the highest molecular weight polymers. Factor VIII-related antigen content was adjusted to 25-100%. All of the preparations lacking the high molecular weight forms caused prompt platelet aggregation with botrocetin, but none of them caused aggregation in the ristocetin test made isochronal with the botrocetin test. The very high molecular weight polymers were equally effective with botrocetin and ristocetin. These findings indicate that the Bothrops factor is reactive with a broad spectrum of high to low molecular weight forms of the factor VIII complex, suggesting that bioassays of von Willebrand factor with botrocetin should correlate better with immunoassays for factor VIII-related antigen and could reflect better the full platelet-aggregating function of the complex than do ristocetin determinations.

Variant von Willebrand's disease: characterization of two subtypes by analysis of multimeric composition of factor VIII/von Willebrand factor in plasma and platelets.

We have examined the multimeric composition of factor VIII/von Willebrand factor in plasma and platelet lysates by means of sodium dodecyl sulfate agarose electrophoresis followed by staining with (125)I-labeled affinity-purified antibody. In normal plasma and platelet lysates, factor VIII/von Willebrand factor displayed 10 distinct multimers that ranged in apparent molecular weight from 0.86 to 9.9 x 10(6). The molecular weight difference between adjacent bands was 0.8-1.1 x 10(6). Larger material, not resolved into discrete bands, was also present with an average M(r) of 14.5 x 10(6). Though the dimer (apparent M(r) = 0.48 x 10(6)) and the monomer (apparent M(r) = 0.28 x 10(6)) generated by reduction of disulfide bonds were readily identified in this system, they were not detected in normal plasma or platelets. No differences were observed between fresh plasma prepared without anticoagulant and fresh or frozen plasma anticoagulated with either citrate or heparin. "Variant" (type II) von Willebrand's disease could be divided into two subtypes. In subtype IIA, factor VIII/von Willebrand factor in plasma consisted predominantly of the five smaller multimers with traces of the sixth and seventh (M(r) up to 4.5 x 10(6)). In subtype IIB, all these multimers were easily detected and, in addition, bands of intermediate size (M(r) = 8.5 x 10(6) and smaller) were present. In contrast, the multimeric composition of IIB platelet factor VIII/von Willebrand factor was identical to normal, whereas in subtype IIA the larger multimers were absent from platelets as well as from plasma. In subtype IIB, binding of factor VIII/von Willebrand factor to platelets occurred at lower concentrations of ristocetin than required for normal and multimers of smaller size than in normal bound. On the contrary, in subtype IIA, binding was minimal, as was true of normal factor VIII/von Willebrand factor of equivalent size. Thus, physical as well as functional differences in the two subtypes of variant von Willebrand's disease described suggest that different pathogenetic mechanisms underlie the factor VIII/von Willebrand factor abnormalities in these patients.

Microwave Absorption and Molecular Structure in Liquids. LXXIX. Dielectric Behavior and Association of Some Normal Alcohols in n-Heptane Solution

Dielectric constants and losses at 25°C of 1-butanol, 1-hexanol, 1-heptanol, and 1-decanol, each at six concentrations from 0.03 to 0.3 mole fraction in n-heptane, have been measured at wavelengths from 2 mm to 600 m. At the lower concentrations, the dielectric absorption may be resolved into two dispersion regions, characterized by relaxation times τ3 (≈ 3 psec and almost concentration independent) and τ2 (ranging from 25 to 100 psec and concentration dependent). The latter dispersion increases its contribution relative to the former with increasing alcohol concentration. At the higher concentrations a final process with a long relaxation time, τ1 (strongly concentration dependent), appears. This third process corresponds to the principal low-frequency dispersion already well characterized in pure primary normal aliphatic alcohols. The high-frequency process, because of its very short relaxation time, is attributed to rotation of the hydroxyl group about its C–O bond, the intermediate process is related to the reorientation of the monomer and possibly small multimers or a polymer chain segment, such as an -OR group rotating about its bond, while the low-frequency process involves hydrogen-bonded structures of some considerable size.

Non-racemic mixture model: a computational approach.

The behavior of a slight chiral bias in favor of l-amino acids over d-amino acids was studied in an evolutionary mathematical model generating mixed chiral peptide hexamers. The simulations aimed to reproduce a very generalized prebiotic scenario involving a specified couple of amino acid enantiomers and a possible asymmetric amplification through autocatalytic peptide self-replication while forming small multimers of a defined length. Our simplified model allowed the observation of a small ascending but not conclusive tendency in the l-amino acid over the d-amino acid profile for the resulting mixed chiral hexamers in computer simulations of 100 peptide generations. This simulation was carried out by changing the chiral bias from 1% to 3%, in three stages of 15, 50 and 100 generations to observe any alteration that could mean a drastic change in behavior. So far, our simulations lead to the assumption that under the exposure of very slight non-racemic conditions, a significant bias between l- and d-amino acids, as present in our biosphere, was unlikely generated under prebiotic conditions if autocatalytic peptide self-replication was the main or the only driving force of chiral auto-amplification.